Effects of lucanthone on the sedimentation properties of DNA from HeLa cells. Exposure of HeLa cells to lucanthone (3 microgram/ml) caused dissociation of a fast-sedimenting duplex DNA complex, as judged by lysis and sedimentation in alkaline sucrose gradients. The effect of lucanthone on the DNA complex resembled that of actinomycin D and ionizing radiation. Protein synthesis inhibitors such as cycloheximide or inhibitors of DNA synthesis such as hydroxyurea did not lead to dissociation of the complex. Lucanthone was more active than were hycanthone and five other closely related thiaxanthenones tested. Lucanthone promoted X-ray-induced denaturation of DNA in intact cells, as judged by their nuclear immunoreactivity to antinucleoside antibodies. Lucanthone did not inhibit repair of X-ray-induced DNA single-strand breaks.


Cell cycle-specific oncogenic transformation of C3H/10T1/2 clone 8 mouse embryo cells by 1-beta-D-arabinofuranosylcytosine. 1-beta-Darabinofuranosylcytosine at concentrations ranging from 10(-3) to 10(-6) M induces oncogenic transformation in the C3H/10T1/2 clone 8 mouse embryo cell line. Cell lines derived from type III transformed foci grew in soft agarose and produced tumors in immunosuppressed syngeneic mice. With cells synchronized by postconfluent inhibition of growth or isoleucine deprivation, transformation was cell cycle dependent. Maximal transformation was seen in cells treated when in S phase, although some transformation was seen in cells treated in G1 phase of the cell cycle.


Stimulation of DNA synthesis by human placental lactogen or insulin in organ cultures of benign human breast tumors. Twenty biopsy specimens of benign human breast tumors obtained from 20 patients were processed into small slices and individually cultured for 2 days in Medium 199. The medium was supplemented with bovine insulin (5.0 microgram/ml), human placental lactogen (HPL) (10.0 microgram/ml), or ovine prolactin (10.0 microgram/ml). Four hr prior to termination, [3H]thymidine was added to the culture medium to determine DNA synthesis. The addition of insulin to the culture medium consistently increased: (a) the mean incorporation of [3H]thymidine into chemically extracted DNA (p less than 0.05); (b) the mean number of [3H]thymidine-radiolabeled epithelial cells (p less than 0.05), and (c) the mean number of epithelial cells bearing mitotic figures. The addition of HPL increased the mean number of [3H]thymidine-radiolabeled epithelial cells (p less than 0.05) and the mean number of epithelial cells bearing mitotic figures (p less than 0.05). [3H]Thymidine incorporation into chemically extracted DNA was also increased when HPL was added to the medium, although this increase did not quite achieve the 5% level of significance. The addition of ovine prolactin to the culture medium did not have any significant effect on DNA synthesis. This study provides evidence that insulin and HPL are direct stimulants of DNA synthesis of the epithelium contained in benign human breast tumors.


DNA damage and repair in L1210 cells exposed to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. The DNA of L1210 cells exposed to low concentrations of 1-(2-chloroethyl)=3-cyclohexyl-1-nitrosourea has been analyzed for the presence of single-strand breaks. DNA from 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea-treated cells both sediments more slowly than control DNA on alkaline sucrose gradients andshows a greater extent of strand separation of the DNA helix in alkali. These effects are a typical result of exposure of cellular DNA to alkylating agents or ionizing radiation. The extent of DNA damage caused by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea has been related to the same amount of damage resulting from exposure of cells to low doses of gamma-irradiation. The rate and extent of repair of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea-induced damage is slow and incomplete, compared with the repair of gamma-irradiation damage to DNA. It is concluded that 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea behaves as a weak alkylating agent, a property that may explain its antitumor properties.


A model for prediction of chemotherapy response to 5-fluorouracil based on the differential distribution of 5-[18F]fluorouracil in sensitive versus resistant lymphocytic leukemia in mice. The distribution of 5-[18F]fluorouracil has been compared in two variants of the same tumor in c57bL X DBA/2 F1 mice: solid L1210 lymphocytic leukemia tumor susceptible to 5-fluorouracil treatment and the same tumor, made resistant to the drug over a 34-generation span. Significant differences in 5-[18F]fluorouracil distribution were observed, most notably in the tumor:blood ratios at 12 hr postinjection. The drug-responsive tumor showed a 20:1 concentration ratio, whereas the drug-resistant tumor only had a 4:1 concentration ratio. We postulate that these differences, observed here in this animal tumor model, may be a reflection of similar ratio differences in humans. This technique may allow, by noninvasive quantification of tumor:blood ratios following administration of 5-[18F]fluorouracil to man, the differentiation of those human tumors that are likely to respond to drug therapy from those in which the response will be minimal or nil.


Metabolism of dimethylnitrosamine and 1,2-dimethylhydrazine in cultured human bronchi. The metabolic activation of several chemical classes of procarcinogens is being studied in cultured human bronchi. Previous studies have shown that carcinogenic polynuclear aromatic hydrocarbons are metabolically activated by the bronchial epithelium. In the study reported here, dimethylnitrosamine and 1,2-dimethylhydrazine were also found to bind to both cellular DNA and protein. Bronchial DNA was methylated in both the O-6 and N-7 positions of guanine. In addition to polynuclear aromatic hydrocarbons, an aliphatic nitrosamine and a methylhydrazine can now be added to the list of xenobiotic chemical carcinogens metabolized by human bronchus.


Uncomplicated starvation versus cancer cachexia. Host starvation is a common accompaniment to the presence of cancer. Diminished intake is a major contributor to this starvation and does not require that the oropharynx or gastrointestinal tract be the primary site. There is suggestive evidence that the normal adaptive mechanisms of the nontumor-bearing host to starvation that result in body protein conservation are not functioning in the tumor-bearing host. Cancer cachexia has some similarity to the metabolic disturbances of host metabolism that are seen in major injury or sepsis. The growing tumor shows little respect for normal constraints of host tissue growth. With the widespread availability of methods of total parenteral nutrition, the interrelationship of nutrition and host-tumor growth assumes greater importance.


Nutritional consequences of radiotherapy. Curative doses of radiotherapy, when directed to any portion of the gastrointestinal tract, may result in serious nutritional consequences from the effects of radiation on the altered function of normal tissues. Symptoms from radiotherapy resulting in nutritional alterations are usually dependent upon dose, time, and fractionation of radiation administered, and the volume included in the treatment field. These effects directly related to radiation may be enhanced by other associated cancer therapy, e.g., surgery or chemotherapy. Careful observation and prompt attention to supportive therapy are mandatory to minimize the nutritional consequences of radiation injury. Well-designed clinical trials are necessary to demonstrate any possible increased tolerance to radiation therapy and the preventative benefits of nutritional support.


The fully automatic ion-exchange and gel-permeation chromatography of neutral monosaccharides and oligosaccharides with a Jeolco JLc-6AH analyser. Experiences with a Jeolco JLC-6AH carbohydrate analyser used to provide a fully automated analysis service for neutral mono- and oligo-saccharides have been summarised. Modifications have been made to the equipment to extend its application, and a method for routine separations of oligosaccharides (d.p. 2--15) has been established. The degree of separation achieved is far superior to that which may be obtained by gel-filtration techniques under gravity- or peristaltic-pump-aided flow. The versatility and adaptability of equipment for use with assays other than the standard orcinol-sulphuric acid reaction shows that it is not too inconvenient to interchange between various assays and separations.


Pulmonary metastases of the lewis lung tumor-cell kinetics and response to cyclophosphamide at different sizes. The response of lung metastases of the Lewis lung tumor to a single dose of cyclophosphamide (300 mg/kg) has been studied as a function of the size of the metastases. Both artificially induced and naturally arising (true) metastases were studied in the absence of a primary tumor. It was found that the artificial metastases exhibited a change in cure probability from 90% to 37% over a size range of a factor of ten. The true metastases had a larger spread in size for a similar change in cure probability but there were large uncertainties in the data and consequently the results are not inconsistent with those for the artificial metastases. Studies of the cell kinetics of the metastases at various sizes in the critical range for curability failed to demonstrate any marked differences. It was concluded that the results of drug treatment could be explained entirely on the basis of an increase in cell number in the larger metastases.


C4'-Branched-chain surgar nucleosides: synthesis of isomers of psicofuranine. Photoamidation of 3-O-acetyl-1,2:5,6-di-O-isopropylidene-alpha-D-erythro-hex-3-enofuranose (1) afforded 3-O-acetyl-4-C-carbamoyl-1,2:5,6-di-O-isopropylidene-alpha-D-gulofuranose (2) and 3-O-acetyl-3-C-carbamoyl-1,2:5,6-di-O-isopropylidene-D-alpha-allofuranose (3) in 65 and 26% yields, respectively (based on consumed 1). Treatment of 2 and 5% hydrochloric acid in methanol yielded the spiro lactone 5, which was deacetylated to yield 7. Reduction of 5 with sodium borohydride afforded 4-C-(hydroxymethyl)-1,2-O-isopropylidene-alpha-D-gulofuranose (9) in 79% yield. Oxidation of 9 with sodium metaperiodate afforded a dialdose that was reduced with sodium borohydride to give 4-C-(hydroxymethyl)-1,2-O-isopropylidene-alpha-D-erythro-pentofuranose (11) in 88% yield. Treatment of the acetate 12, derived from 11, with trifluoroacetic acid, followed by acetylation, afforded the branched-chain sugar acetate 14. Condensation of the glycosyl halide derived from 14 with N6-benzoyl-N6,9-bis-(trimethylsilyl)adenine yielded an equimolar anomeric mixture of protected nucleosides 15 and 16 in 40% yield. Treatment of the latter compounds with sodium methoxide in methanol afforded 9-[4-C-(hydroxymethyl)-beta-D-erythro-pentofuranosyl] adenine (17) and the alpha-D anomer 18. The structure of 3 was determined by correlation with the known 5,3'-hemiacetal of 3-C-(hydroxymethyl)-1,2-O-isopropylidene-alpha,alpha'-D-ribo-pentodialdose (25).


Antileukemic effect of homo-aza-steroidal ester of [p-[bis(2-chloroethyl)amino]phenyl]acetic acid. The homo-aza-steroidal ester of [p-[bis(2-chloroethyl)amino]phenyl]acetic acid, 3beta-hydroxy-13alpha-amino-13,17-seco-5alpha-androstan-17-oic-13,17,-lactam p-bis(2-chloroethyl)aminophenylacetate (ASE), gives a greater than 50% increased lifespan over controls in the treatment of L1210 leukemia by the ip, sc, and oral routes of administration  and a greater than 150% increased lifespan in the treatment of P388 leukemia by the ip route (the only route tested). Both a daily and a Day 1, 5, and 9 treatment schedule are essentially equally effective. In this study, ASE is compared to the parent compound phenesterin which is inactive in both of these tumor lines. To our knowledge ASE is the first steroidal alkylating agent to show activity in the L1210 leukemia.


A novel synthesis of 2-deoxy-alpha-glycosides. The key step of the synthesis involves the reaction of glycals [3,4,6-tri-O-acetyl-D-glucal (1), the new glycal derivative 4-O-acetyl-1,5-anhydro-2,6-dideoxy-3-C-methyl-3-O-methyl-L-ribo-hex-1-enitol (2), and 3-acetamido-4,6-di-O-acetyl-1,5-anhydro-2,3-dideoxy-D-arabino-hex-1-enitol (3)] with 1.5 molar equivalents of several alcohols in the presence of N-bromosuccinimide in acetonitrile to give mainly the corresponding 2-bromo-2-deoxy-alpha-glycopyranosides (4--21). The glycopyranosides (4-8 and 16-21) from 1 and 3 have the alpha-D-manno configuration and those (10--15) from 2 have the alpha-L-altro configuration. The yields are high from 1, virtually quantitative from 2, and moderate from 3. Debromination of the 2-bromo-2-deoxy compounds with tributylstannane and a radical initiator gives the corresponding 2-deoxy-alpha-glycopyranosides (22-38) in quantitative yields. In particular, the branched-chain glycal 2 reacts with alcohols to give exclusively the corresponding alpha-glycopyranosides (27--32) of cladinose in strikingly high overall yields. The stereoselectivity and regiospecificity of the bromination reaction are described. 1,3-Dibromo-5,5-dimethylhydantoin and N-bromoacetamide are also found to be useful for the reaction.


Cyclophosphamide L2 protocol: a combination chemotherapeutic regimen for advanced non-hodgkin's lymphoma. A group of 49 patients with advanced non-Hodgkin's lymphoma were entered in a combination-chemotherapy protocol (cyclophosphamide L2). Of 14 patients with diffuse poorly differentiated lymphocytic lymphoma (DPDL), 64% responded with two partial remissions (PR) and seven complete remissions (CR). Both PRs are stable at 17+ months while six of the CR group are free of disease at 3+-23+ months. Fifty-three percent of 30 patients with diffuse histiocyctic lymphoma (DHL) responded with 23% attaining CR status. Of the nine PR patients, six are stable at 11+-23+ months while six of the seven CR group are without disease at 9+-27+ months. The major toxic effect was bone marrow suppression with two deaths during periods of neutropenia; one of these deaths was definitely drug related. The encouraging results in the DPDL category have led to a continuation of this protocol for patients with this histologic type. In patients with DHL other approaches are being explored.


A fucogalactoxyloglucan from rapeseed hulls. A polysaccharide, isolated from rapeseed hulls by extraction with aqueous sodium hydroxide-sodium tetraborate, contained residues of L-arabinose, L-fucose, D-xylose, D-galactose, and D-glucose in the proportions of 2:8:25:13:52. Acetolysis furnished cellobiose, 6-O-alpha-D-xylopyranosyl-D-glucose, and 2-O-beta-D-galactopyranosyl D-xylose. The cleavage products from the methylated polysaccharide were examined by g.l.c. of the methyl glycosides and g.l.c.-mass spectrometry of the partially methylated, alditol acetates. The results show that the polysaccharide is a member of the xyloglucan group in which additional fucose and galactose residues terminate some of the side-chains. For comparative purposes, aspects of the structures of xyloglucans from nasturtium seeds and suspension-cultured sycamore cells have been re-examined.


Pilot study of two adriamycin-based regimens in patients with advanced malignant lymphomas. Forty-two patients with advanced malignant lymphomas, all of whom had failed at least one prior course of chemotherapy, were treated with one of two new combination chemotherapy regimens to determine patient tolerability. Twenty-seven patients received regimen 1, after which this study was discontinued, and subsequently 15 additional patients were treated with regimen 2. Regimen 1 (BAP) consisted of BCNU and adriamycin administered iv on Day 1 and prednisone administered orally on Days 1--5 with cycles repeated at 21-day intervals. Regimen 2 (VAP) consisted of VP-16-213 given iv on Days 1 and 2, adriamycin given iv on Day 3, and prednisone given on Days 3--7 in a schedule designed to produce cell cycle synchronization; cycles were repeated at 21--28-day intervals. Both regimens were, in general, well tolerated. Reversible bone marrow depression was the major toxic reaction observed. Nine of 27 patients treated with regimen 1 and five of 15 patients treated with regimen 2 experienced objective tumor regressions. Tolerable dosage levels of both regimens have been defined for future clincial trials.


Adenosine-5'-d: rotational conformation of the 5'-carbinol group. A synthesis of adenosine-5'-d (4), and its p.m.r. spectral characteristics, are described. The presence of deuterium in 4 gives rise to a 2:1 mixture of R and S configurations at C-5, thereby permitting specific assignments for the resonances of the residual 5'-protons. From the observed spin-spin coupling between the latter and H-4', an estimate has been made of the rotamer population of the exocyclic 5'-carbinol group. It is shown that the gauche-gauche rotamer is preponderant (approximately 70%) and the gauche-trans one of minor importance (approximately 20%) in aqueous solution, which contrasts markedly with the preference for the latter rotamer exhibited by adenosine in the solid state.


Transformations of cellobiose derivatives into analogues of lactose. The preparation of benzyl beta-cellobioside 2,3,6,2',3'-pentabenzoate (2b) via a 4',6'-benzylidene acetal in 27% overall yield from cellobiose is described. This pentabenzoate has been utilised for conventional syntheses of the 6'-amino-, 4'-amino-, and 4',6'-diamino-analogues of lactose, via the 4',6'-dimesylate. In addition, reaction of the pentabenzoate 2b with sulphuryl chloride initially afforded the 6'-chloro derivative, which was then slowly transformed into a mixture of the 4',6'-dichloro-lactoside and an isomer in which the non-reducing ring of the disaccharide had been transformed into a 3',6'-dideoxy-3',6'-dichloro-gulopyranoside. The latter probably arose by neighbouring-group participation by the 3'-benzoloxy group prior to the introduction of the secondary 3'-chloro substituent. The former dichlorocompound was subsequently converted into 4',6'-dideoxy-4',6'-dichloro-lactose. It was found that the rate of nucleophilic displacement at C-4' of a cellobioside was much lower than that for the corresponding alpha-linked disaccharide, maltose. Furthermore, nucleophilic displacement at C-4' of a lactoside was accompanied by substantial elimination, to give the 4'-ene, together with some cleavage of the interglycosidic bond. tthe origins of these effects are discussed.


Synthesis of 6,1',6'-tri-O-(mesitylenesulfonyl)sucrose, further examination of "tri-O-tosylsucrose", and the chemistry of 3,6:1',4':3',6'-trianhydrosucrose. Selective trimolar mesitylenesulfonylation of sucrose resulted in the formation of a highly crystalline trimesitylenesulfonate (1), which was isolated in greater than 50% yield without recourse to chromatography. As anticipated, the sulfonyl groups in 1 were located at the primary positions, as treatment with alkali afforded 3.6:1',4':3',6'-trianhydrosucrose (4) in high yield. Fractionation of "tri-O-tosyl-sucrose" by high-pressure liquid chromatography effected separation of the minor isomer from the known, preponderant 6,1',6'-isomer 3. 13C-N.m.r. spectroscopy indicated that the minor isomer was 2,6,6'-tri-O-p-tolylsulfonylsucrose (2). The trianhydride 4 was found to be dimorphous and was further characterized as the diacetate (5), the dibenzoate (6), the di-p-toluenesulfonate (7), and the dimethyl ether (8). Considerable differences in the reactivities toward acylation and etherification of the two axial hydroxyl groups in 4 permitted the preparation, in good yields, of the 4-acetate (9) and of the 4-methyl ether (12). Several derivatives of methyl 3,6-anhydro-alpha-D-glucopyranoside (13) were prepared for comparison with corresponding derivatives of 4, and the hydroxyl groups in 13 also showed differences in reactivities analogous with those of 4.


Stereoselective total synthesis of methyl alpha-D and alpha-L-glucopyranosides. Methyl alpha-L- and alpha-D-glucopyranosides have been synthesized from methyl (R)- and (S)-(2-furyl)glycolates (3), respectively. The key intermediates, methyl 6-O-benzyl-2,3-dideoxy-L(and D)-hex-2-enopyranosid-4-uloses (13), were obtained in seven steps from the ester 3, without change of configuration of the asymmetric center, which became C-5 in the sugar molecule. Reduction of the ketone group at C-4 in the glycoside 13 with sodium borohydride afforded the corresponding methyl 6-O-benzyl-2,3-dideoxy-erythro-hex-2-enopyranosides (14). Epoxidation of the double bond in 14, followed by oxirane ring-opening in the anhydro sugar 16, and subsequent catalytic hydrogenolysis of the benzyl group led to the title compounds.


Total synthesis of hexuronic acids.

Total synthesis of hexuronic acids. Six stereoisomeric hexuronic acids (tert-butyl esters of methyl pyranosides) having the altro, manno, gluco, gulo, galacto, and talo configurations were obtained from cis,trans-tert-butyl 2-methoxy-5,6-dihydro-2H-pyran-6-carboxylate (5). The synthesis involved the following successive steps: epoxidation of the double bond in 5, opening of the epoxides with dimethylamine, Cope degradation of the dimethyl-amino derivatives, and hydroxylation of the double bond in the tert-butyl hex-3-enuronates. All compounds were obtained as pure diastereoisomers in racemic form.


The synthesis of D-ribofuranosyl derivatives of methyl propiolate and a study of the activating influence of the ester group in cycloaddition reactions. 2,3,5-Tri-O-benzyl-D-ribofuranosyl bromide (17) has been converted into methyl 3-(2,3,5-tri-O-benzyl-beta-D-ribofuranosyl) propiolate (8) and its alpha anomer 10 in 21 and 42% yields, respectively, by reaction with the silver salt of methyl propiolate. Attempts to prepare 8 from (beta-D-ribofuranosyl)ethyne (1) by standard methods were unsuccessful. The reactions of the esters 8 and 10 and the ethyne 1 with several 1,3-dipoles have been examined. With diazomethane, 8 and 10 gave the pyrazole esters 20 and 28, respectively, whereast the ethyne 1 reacted more slowly to give a mixture of 23 (37%) and 26 (31%). The ester 10 was converted into the triazoles 32 (51%) and 36 (34%) by reaction with benzyl azide. Treatment of the ester 10 with phenylhydrazine gave the pyrazolone 38 in 71% yield. A number of the products of dipolar addition have been converted into new D-ribofuranosyl-pyrazoles and -triazoles by hydrogenolysis.


A protein-bound glycogen component of rat liver. It has long been claimed, but frequently disputed, that part of the glycogen in rat liver is insoluble in 10% trichloroacetic acid, and a physiological significance was ascribed to the existence of the two pools of glycogen, desmo-glycogen, the insoluble form, and lyo-glycogen, the soluble component. Desmo-glycogen was thought to owe its acid insolubility to a covalent binding to protein. Recent claims that glycogen, similarly insoluble in acid, can be synthesized in vitro have renewed the interest in desmo-glycogen. We have obtained trichloroacetic acid-insoluble glycogen from rat liver and find that, despite subjecting the glycogen to proteolysis, peptide material remains in close association with the glycogen through a number of purification procedures and is freed from glycogen only by enzymic decomposition of the latter. The tenacity with which the glycogen and peptide material remain in association with each other is suggestive of the occurrence of protein-bound glycogen.


The preparation of carbohydrate-protein conjugates: cyanuric trichloride coupling of 2-aminoethyl glycosides, and mixed-anhydride coupling of 8-carboxyoctyl glycosides to bovine serum albumin. Preparation of the following glycosides is described: 2-aminoethyl beta-D-glycosides of (A) 2-acetamido-3,4,6-trio-O-acetyl-2-deoxy-D-glucopyranose, (B) 2-acetamido-4-O-(2-acetamido-3,4,6-trio-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-beta-D-glucopyranose (N,N'-diacetylchitobiose pentaacetate), (C) 4-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-2,3,6-trio-O-acetyl-beta-D-glucopyranose (cellobiose heptaacetate); 8-carboxyoctyl glycosides of (D) cellobiose, and (E) N,N'-diacetylchitobiose. Conjugates were prepared from (A), (B), and (C) by coupling to bovine serum albumin by cyanuric trichloride and subsequent deacetylation; (D) and (E) were coupled to bovine serum albumin by the mixed-anhydride reaction. Conjugates (A) and (B) were insoluble; conjugates (C), (D), and (E) functioned as artificial antigens and gave rise to precipitating antibodies in rabbits. Specificities of the antisera were determined by inhibition studies.


The pituitary of Aphanius dispar (Rüppell) from hypersaline marshes and freshwater. II. Ultrastructure of the rostral pars distalis. An ultrastructural study of the rostral pars distalis of the pituitary of Aphanius dispar specimens taken from freshwater or hypersaline marshes revealed significant structural differences which indicate higher activity of the prolactin cells in the hypotonic medium. Prolactin cells from freshwater specimens had larger secretory granules, a higher amount of endoplasmic reticulum, and expanded intercellular spaces with many secretory lakes. These cells contained an unusual cytoplasmic structure, consisting of twisted canals with vesicular lumina, connected to the endoplasmic reticulum of the cell. This structure is about 1-2 micron in diameter. Stellate cells are characterized by extracellular spacing junctions which are particularly noticeable at the confluence of the interstellate cell canaliculi and the pericapillary space.


Effects of the eye enucleation on the activity of monoamine oxidase and acetylcholinesterase in the superior colliculus of the rat. Changes in the activity of monoamine oxidase (MAO) and acetylcholinesterase (AChE) following unilateral eye enucleation were measured for the superior colliculus (SC), lateral geniculate body (LGB), and nucleus suprachiasmaticus (NSC) in the rat by means of microspectrophotometry. The most conspicuous change of high significance was the decrease of MAO activity in the superficial strata of the SC contralateral to the enucleated eye. The other layers of SC, such as the stratum opticum, the intermediate strata, the deep strata and the central gray did not show significant postoperative changes in MAO acitivity. MAO activity of the LGB and the NSC was also not affected to a significant degree. The site of decrease of MAO activity in the superficial strata of the SC sould be degenerating optic nerve terminals. The strong AChE activity in the superficial strata may be derived from the horizontal cells. AChE activity in the SC showed only slight postoperative changes. The possible roles of the monoaminergic and cholinergic systems in the process of visual information are discussed.


On the nurse cell and the spermatozeugma in Littorina sitkana. Nurse cells develop from diploid cells in the testis. Each cell undergoes a reduction division which leaves the nucleus with half the volume of a normal diploid cell. They send out pseudopodia which form desmosome-like junctions with developing spermatids. The nurse cells detach from the testicular wall, their nuclei degenerate and secretion droplets form in the  cytoplasm. The pseudopodia are drawn in as the cytoplasmic secretions swell and the nurse cell becomes spherical. The eupyrene sperm become grouped unilaterally and at this stage are attached to the nurse cell by only the tips of their acrosomes. At maturity the nurse cells with their clumps of attached eupyrene sperm (spermatozeugmata) are released from the testis via ducts into the seminal vesicles,where they are stored prior to copulation. Nurse cells serve similar functions to those of apyrene sperm which are common among the Molluscs. We believe that the nurse cell and apyrene sperm are homologous.


Possible sites of ultrafiltration in Tubifex tubifex Müller (annelida, oligochaeta). The endothelia of Tubifex tubifex  Müller consist of myoendothelial cells, chloragocytes, or podocytes. The latter seem to occur only as windows on the ventral vessel which has an endothelium of myoendothelial cells elsewhere. The podocytes are large cells, with several processes on the inner side which ramify into several pedicels. These are aligned upon the outside of the basement membrane which lines the inside of the endothelium. The gaps between adjacent pedicels are about 40 nm wide. In capillaries fenestrated endothelia occur with irregular spacings measuring up to 0.4-1 micron. A diaphragm in podocytes or capillary fenestrations do not seem to exist. The basement membrane is the only continuous layer lining the blood vessels and capillaries of Tubifex with a rather uniform diameter in the range of 50nm. It is the only permeability barrier between blood and coelomic fluid.


The ultrastructure of the sheep parotid gland. The sheep parotid is a compound tubular gland; its ultrastructure reflects the function of this gland to secrete large amounts of fluid with very little protein. The cells of the secretory tubules possess extensively folded lateral plasma membranes and a fairly large number of mitochondria. Rapid equilibration of water across the epithelium is assured by the close proximity over large areas of intercellular spaces and the wide secretion canaliculi. Numerous long microvilli extend into the latter. Although secretion granules may be quite numerous, there is evidence that many of these granules are not discharged but undergo degradation by lysosomal enzymes. The intercalated ducts are often dilated but excessive distension is probably prevented by bundles of microfilaments in their epithelial cells.


Degeneration of possible serotonergic nerves in the myenteric plexus of Mytilus induced by 5,7-dihydroxytryptamine (5,7-DHT). Initial observations on the effects of 5,7-DHT on the myenteric plexus of Mytilus reveal extensive lesioning of monoamine nerves containing dense-cored vesicles. At least, two types of monoaminergic nerves could be recognized among the degenerating axons. The most affected were profiles with a predominance of large granular vesicles (56-200 nm) which resemble possible indoleamine nerves in the lamprey gut. The other type had small granular vesicles (40-56 nm) resembling catecholamine nerves of vertebrates. These two types of nerves possibly represent serotonergic and dopaminergic nerves, in accordance with pharmacological, biochemical and physiological evidence available in molluscs. There were, however, nerves with a population of medium-sized granular vesicles (56-130 nm) which were also damaged. Two other types of nerve profiles were unaffected by the drug. One conforms to cholinergic nerves while the other resembles "purinergic" nerves in the vertebrate gut. These results confirm previous studies with dihydroxytryptamines on molluscan smooth muscle.


Oogenesis in Acerentomon gallicum Jonescu (protura). Previtellogenic and vitellogenic stages. Late stages of oogenesis in Acerentomon gallicum Jonescu have been studied by means of light and electron microscopy. Each of the two ovaries of this species consists of a single panoistic ovariole. Late previtellogenic and early vitellogenic oocytes are enclosed in an electron opaque layer, the so-called primary sheath. The precursors for this sheath are most likely synthesized by follicle cells. The yolk develops through autosynthesis, with free ribosomes, dictyosomes and lamellar bodies being involved in the process. Mature yolk spheres contain proteins and polysaccharides. Besides the organelles that take part in vitellogenesis, mitochondria and cisternal stacks of the rough endoplasmic reticulum occur in the ooplasm.


Reactive alterations in non-neuronal elements of the degenerating ventrobasal complex of immature and mature rats. An electron microscopic study. Sprague-Dawley albino rats ranging in age from neonate to 60 days postnatal (dpn)were subjected to cortical extirpations encompassing the SmI somato-sensory projection fields of neurons in the ventrobasal (VB) complex. Eectron microscopy of this region reveals degenerative changes in VB neurons, the rate and severity of which is inversely proportional to the age of the animal (Matthews et al., 1977). Numerous, distinctive non-neuronal elements, similar to those infiltrating the perivascular space of some vessels in the area, rapidly accumulate within the zone of degeneration in animals lesioned between 0 and 9 dpn. These display dense, heterochromatin nuclei, concentrations of of free ribosomes and rosettes, and pleomorphic dense bodies which become more evident as further reactive transformations accompany the phagocytic incorporation of degenerating neuronal remnants. Other non-neuronal elements exhibit a euchromatin nucleus, bundles of microtubules, and fewer free ribosomes. Such cells are also capable of phagocytosis and production of dense bodies. Both variants are comparable in appearance to the "M" cells of previous reports (Matthews and Kruger, 1973b). Cortical lesions of older animals result in the appearance of "M" cells in VB; however, the population densities observed in the immature VB are not achieved. Conversely, astrocytic hypertrophy, associated with the increased incidence of degenerating boutons in the more mature animal, represents a prominent response to injury which does not occur to a significant extent in younger animals. Morphological criteria for determining the nature of some "M" cells are given for a discussion of their presumptive derivation from various mesodermal progenitors and a brief consideration of other hypothesized origins.


A quantitative analysis of the incidence of certain cytoplasmic structures in the ovum of the rat during cleavage. The volumetric density of most cytoplasmic organelles in the segmenting ovum of the rat was determined by morphometric techniques described by Weibel, at the 1-, 2-, 4-, and 8-cell stages, and at the early and late blastocyst stages. During the course of cleavage, the volumetric density of mitochondria remained practically unchanged. The volumetric density of the Golgi complex complex was too small to permit assessment of the differences between the individual stages of development. Significant changes were found in both granular and agranular endoplasmic reticulum, secondary lysosomes, multivesicular bodies, lamellar structures and lipid droplets. Granular endoplasmic reticulum was first observed as late as the 8-cell ovum stage and its volumetric density increased further in the early and late blastocysts. The relative volume of agranular endoplasmic reticulum was highest in the 1-cell ovum and decreased during the course of cleavage. The same is true for multivesicular bodies. The volumetric density of secondary lysosomes increased during cleavage, reaching the highest values in the 8-cell ovum. Lamellar structures were the most voluminous part of the cytoplasm of the segmenting ovum at all stages. Their volumetric density, however, decreased during the course of cleavage. Lipid droplets occur in very small quantities in the 1- to 4-cell ova, but at later stages their volumetric density increased. Our findings underline the importance of acquiring quantitative information about changes in cell organelle populations for assessing morphological and functional relationships during the early stages of cleavage of the ovum.


Freeze-fracture analysis of junctional complexes in the nephron of the garter snake, Thamnophis sirtalis. Zonulae occludentes are shown by freeze-fracture to be pleomorphic along the garter snake nephron. In the neck and proximal segments the occluding junctions are moderately complex with frequent discontinuities in their junctional fibrils. Junctional depth and complexity are maximal in the distal and collecting segments and discontinuities in fibrils are absent. Comparison of these results with similar observations on other tissues indicates that the zonulae occludentes in the neck and proximal segments are "intermediate" to "leaky" and that they may be "very light" in the distal and collecting segments. The findings suggest that in the garter snake nephron transepithelial flow of fluid may occur primarily by passive diffusion through the zonulae occludentes in the neck and proximal segments and by cell-mediated osmotic flow in the distal and collecting segments. Gap junctions occur only in the proximal tubule and areprobably involved in low resistance, intercellular movement of ions.


Effects of cytochalasin B on pancreatic acinar cell structure and secretion. The effects of cytochalasin B (CB) on pancreatic structure and amylase release were studied by use of pancreatic fragments, isolated acini and isolated acinar cells. In pancreatic fragments and isolated acini CB caused the disappearance of microfilaments underlying the apical plasma membrane, loss of apical microvilli and luminal swelling, the last of which was greatly enhanced by addition of protein secretagogues. CB had no effect on basal amylase release but inhibited bethanechol-stimulated amylase in both fragments and acini. Isolated acinar cells, while retaining overall polarity, had lost most of the apical specialization including the microfilament and microvillous complex. Cells were still able to release amylase in response to bethanechol but this release was not affected by CB. The only structural effect of CB on isolated cells was margination of zymogen granules against the plasma membrane. This was, however, not accompanied by increased amylase release. It is concluded that microfilaments are important in maintaining the pancreatic acinar structure. Interference with this structure by CB leads to inhibition of bethanechol-stimulated amylase release. Microfilaments, however, may not play a direct role in secretion.


Prevention by vitamin A of the occurrence of permanent vaginal changes in neonatally estrogen-treated mice. An electron microscopic study. Ovary-indepedent (estrogen-independent) irreversible proliferation and cornification on the vaginal epithelium in ovariectomized mice caused by neonatal injections of 20 microng estradiol-17 beta (E2) was suppressed by injections of 200 IU vitamin A acetate (VA) given simultaneously with E2. More than 2 months after the combined treatment were required for the suppression. A new type of cell of low electron density (Light cells) appeared at 40-120 days of age on the basal lamina of the vaginal epithelium in neonatally E2 plus VA-treated ovariectomized mice only at 50 days. Light cells contained small amounts of organelles. Columnar basal cells resembling those in normal estrous mice were also found in the E2 plus VA-treatedmice at 70-120 days. In one of the 120-day-old, E2 plus VA-treated mice, the epithelium consisted of only 2-3 layers of cells. Neither Light cells nor columnar cells were found in the atrophic epithelium.


Outgrowth in vitro of cervical epithelium separated from the uterovaginal anlage of newborn mice. An ultrastructural study. After gently trypsinization, the pseudostratified columnar Müllerian epithelium that lines the uterine cervix of newborn mice could be separated from the enclosing stromal tissue Pure epithelial tubes explanted in vitro and were allowed to grow in a standard medium for 3-4 days forming a confluent colony of rather closely-fitting cells. The cell sheet was studied by a preparatory technique that allows examination of a large number of cells with preserved intercellular spatial orientation. Attempts were made to identify cultured cells according to the morphology of cell types in the cervicovaginal epithelium in vivo. Electron micrographs revealed that, close to the explant, the cultured cell sheet exhibited several features similar to the Müllerian epithelium in vivo. Outside these central areas of the colony was a broad transitional zone consisting of thin platelike cells distinguished by an abundance of microfilaments. At the periphery of the colonies, bulky cells possessing microvilli and a vacuolated cytoplasm tended to overlpa adjoining platelike cells. These bulky cells had a morphology resembling that of the superficial cells seen in the upper vagina and common cervical canal of immature and diestrous animals. The epithelial development in the cultures apparently simulated the transformation iatified epithelium resembling that of the uppermost vagina and common cervical canal of immature animals. Judged by morphological and cytochemical criteria, the Müllerian cells in the out-growth obviously had many changed features. It thus seems questionable wheter the cells grown in vitro are comparable with the corresponding cells in vivo when used for experiments requiring the corresponding of the culture environment.


Axon regeneration across the site of injury in the optic nerve of the newt Triturus pyrrhogaster. The process by which axons regenerate following a freeze injury to the optic nerve of the newt was analyzed by light and electron microscopy. Freezing destroys cellular constituents in a one millimeter segment of the nerve, leaving intact the basal lamina and the blood supply to the eye. No axons are seen at the site of injury one to seven days post lesion. This contrasts with the persistence of normal-appearing but severed unmyelinated axons within the cranial stump which thus give a false appearance of early regeneration. The first axon sprouts traverse the lesion and enter the cranial strump by ten days. The number of regenerating axons increases rapidly thereafter with no signs of random growth at the site of injury. These axon sprouts tend to be somewhat larger than normal unmyelinated axons and contain dense core vesicles and abnormal organelles similar to those in growing axons in tissue culture. The persisting basal lamina inside the optic sheath appears to provide continuity across the site of injury, to orient axon sprouts, and to favor an orderly process of axon regeneration without neuroma formation.


On the fine structure of polyribosomes.

On the fine structure of polyribosomes. The ultrastructural morphology of ribosomes was studied in tissue sections of rat uterus using defferent fixatives (acrolein formaldehyde, acetic acid, methanol-acetic acid, OsO4) after various pre-incubations in solutions of different osmolarity, electrolyte content, and pH. In addition, whole-mount ribosomes of spread cytoplasm of epithelial cells were examined. The results indicate: Ribosomes are "thickenings" in the course of a cytoplasmic RNP-network which extends in three dimensions between nucleus and plasma membranes. This network consists of fibrils which vary in width depending on intrinsic and extrinsic factors. Often the fibril width is approx. 100 A. In places where the fibril is folded up into ribosomal "granules" and strands the width may be 300 A. Each fibril seems to be composed of two elementary filaments, 10 A up to 40 A thick. Estimation of filament length within a ribosomal "granule" allowed the conclusion that only a fraction of a 45 S-RNA filament is folded up into a "ribosome" in cytological preparations. No morphological evidence was found for clefts within ribosomes or single messenger RNA filaments "piercing" ribosomes. Filaments of membrane-bound ribosomes appearently show continuities with fibrils within the cytoplasmic matrix on the one side and with filaments within the cisternae of the endoplasmic reticulum on the other. Ribosomal filaments of stromal cells also seen to have filamentous continuities with extracellular fibrils. This morphological evidence opens new perspectives concerning the role of RNA filaments in protein synthesis.


Intermolecular duplexes in heterogeneous nuclear RNA from HeLa cells. Rapidly sedimenting hnRNA complexes contain regions of stable intermolecular duplex. Disruption of such complexes, as judged by a reduction in sedimentation rate, requires conditions sufficient to denature the duplex regions. Rapidly sedimenting molecules reappear only when the complementary sequences reanneal-that is, the formation of such complexes is dependent upon time and the concentration of homologous RNA. These experiments lead us to the conclusion that rapidly sedimenting hnRNA complexes consist of two or more largely single-stranded RNA molecules held together by short duplex regions. Precisely such structures have been visualized in the electron microscope. Rapidly sedimenting fractions of native nuclear RNA from preparative sucrose gradients consist primarily of large, multi-molecular complexes interconnected by duplex regions averaging 300 base pairs in length. Exposure of the RNA to severely denaturing conditions eliminates such complexes. Reannealing of the RNA reconstitutes complexes which are indistinguishable from those observed in preparations before denaturation.


The definition of a large viral transcription unit late in Ad2 infection of HeLa cells: mapping of nascent RNA molecules labeled in isolated nuclei. Nuclei isolated from HeLa cells 15 hr after infection with Ad2 synthesize an RNA transcript approximately 25 KB long, beginning between 0.2 and 0.3 on the physical map and extending to (or close to) the right end of the genome. The majority (60-92%) of the RNA transcribed from fragments between 0.2 and 1.0 is synthesized as part of this large molecule which represents the major product of late Ad2 RNA synthesis. Smaller transcripts are also formed mainly from the left end of the Ad2 genome.


The definition of a large viral transcription unit late in Ad2 infection of HeLa cells: mapping by effects of ultraviolet irradiation. The synthesis of Ad2-specific nuclear RNA in HeLa cells 15 hr post-infection has been examined after ultraviolet irradiation. The synthesis of RNA complementary to various segments of Ad2 DNA is decreased differentially, with the most sensitive region at the right end of the physical map and the most resistant in the region from 0 up to 0.2-0.3 (20-30% from the left end of the genome). For a given ultraviolet dose, the amount of labeled RNA homologous to various DNA fragments decreased exponentially from map position 0.2-0.3 to 1.0. These results suggest that a major transcriptional unit late in Ad2 infection is a rightward-transcribing unit between 0.0-0.3 and 1.0.


Variation in chromatin structure in two cell types from the same tissue: a short DNA repeat length in cerebral cortex neurons. We have used micrococcal nuclease as a probe of the repeating structure of chromatin in four nuclear populations from three tissues of the rabbit. Neuronal nuclei isolated from the cerebral cortex contain about 160 base pairs of DNA in the chromatin repeat unit, as compared with about 200 base pairs for nonastrocytic glial cell nuclei from the same tissue, neuronal nuclei from the cerebellum and liver nuclei. All four types of nuclei show the same features of nucleosomal organization as other eucaryotic nuclei so far studied: nucleosomes liberated by digestion with micrococcal nuclease give a "core particle" containing 140 base pairs as a metastable intermediate on further digestion and a series of single-strand DNA fragments which are mutiples of 10 bases after digestion with DNAase I. Nuclei from cerebral cortex neurons, which have a short repeat, are distinct from the others in being larger, in having a higher proportion of euchromatin (dispersed chromatin) as judged by microscopy and in being more active in RNA synthesis in vitro.


In vitro traits of adenovirus-transformed cell lines and their relevance to tumorigenicity in nude mice. Six independently isolated adenovirus 2-transformed rat cell lines and one adenovirus 5-transformed human cell line have been examined in vitro for serum growth requirements, saturation density, anchorage-independent growth, proteolytic enzyme activity and the presence of LETS glycoprotein and T antigen. This series of adenovirus-transformed cell lines exhibits an oncogeni spectrum ranging from being tumorigenic in immunocompetent rats through to nontumorigenic in adult nude mice. The relevance of the in vitro findings to growth potential in vivo is discussed.


Testis-determining H-Y antigen in XO males of the mole-vole (Ellobius lutescens). The XO sex chromosome constitution has been found in both sexes of the mole-vole (Ellobius lutescens) belonging to the rodent family Microtinae. This enigmatic species has apparently been enduring a 50% zygotic lethality. The current serological study revealed the presence in XO males and the absence from XO females of H-Y (histocompatibility Y) antigen. In all the mammalian species studied thus far, the expression of H-Y antigen strictly coincided with the presence of testicular tissue and not necessarily with the presence of the Y chromosome. The testis-organizing function of the H-Y gene appears to have been confirmed. In the mole-vole, X linkage of the testis-organizing H-Y gene is favored over its autosomal inheritance. Only X linkage of the H-Y gene creates a compelling evolutionary need to change the female sex chromosome constitution from XX to XO, and to abandon the dosage compensation by an X inactivation mechanism, so that the nonproductive XH-YX zygote can be eliminated as an embryonic lethal. With regard to the electrophoretic mobilities of three X-linked marker enzymes, however, a genetic difference between the male-specific XH-Y and the female-specific X was not detected. This might reflect a relatively recent speciation.


Retinyl acetate modulation of cell growth kinetics and carcinogen--cellular interaction in mouse epidermal cell cultures. Exposure of mouse epidermal cell cultures to beta-retinyl acetate (RA) affects a number of parameters presumed to be important in chemical carcinogenesis. (1) RA alters the course of differentiation of the epidermal cells in culture resulting in a reduced rate of cell death which normally follows cellular maturation during the first two weeks in culture. The extended life span of the cultures appeared due to prolonged survival of cells and not to increased growth rate since RA inhibited the rate of cellular proliferation. This inhibition took place only after completion of a full cell cycle in the presence of RA. (2) DNA repair in response to physical and chemical agents was quantitatively unaffected in the presence of RA. (3) The activity of constitutive aryl hydrocarbon hydroxylase (AHH) was slightly decreased after exposure to RA but the level of enzyme induced by benz[a]anthracene was strongly reduced to 20% of the controls. (4) In the presence of RA, binding of 7,12-dimethylbenz[a]anthracene to epidermal cell DNA was markedly decreased. In contrast, binding to cellular protein was significantly increased by the retinoid.


Interaction of cis- and trans-dichlorodiammine-platinum(II) with synthetic polydeoxyribonucleotides: effect of equilibrium buoyant densities. Equilibrium buoyant densities in CS2SO4 of four synthetic double-stranded polydeoxyribonucleotides were compared following reaction with either cis-Pt(NH3)2Cl2, trans-Pt(NH3)2Cl2 or PtCl4. While both Pt(NH3)2Cl2 isomers caused significant increases in the buoyant densities (rho) of the two G + C polymers, the cis- isomer increased the rho of poly [d(G-C)] and the trans- isomer increased the rho of poly(dG) - poly(dC) more sharply than the corresponding isomer at low molar ratios of metal ion to polymer. Chloroplatinic acid was without effect. Only cis-Pt(NH3)2Cl2 produced substantial increases in the rho of poly[d(A-T)] and poly(dA) - poly(dT) with increasing metal ion concentrations. These increases were accompanied by a proportional decrease in the thermal transition temperature (Tm) of poly(dA)-poly(dT) and a sharp increase in the Tm of poly[d(A-T)]. Our results indicate  that the cis- isomer reacts with all four polymers to produce rho changes clearly distinct from those values observed after reaction with either the trans- isomer or PtCl4. These results help explain the observation by others that the cis- isomer produces greater rho changes in native DNA than the trans- isomer. On the basis of our data and the published results of others, one may rationalize the quantitative differences between the cis- and trans-compounds of the buoyant densities of naturally occurring DNA; however, additional studies are clearly needed.


The accumulation of O6-methylguanine in the liver and kidney DNA of rats treated with dimethylinitrosamine for a short or a long period. A large dose of dimethylnitrosamine was administered to rats by two different dosing regimens, one being eleven intraperitoneal injections of 5 mg/kg body wt. at 12-h intervals (a dosing regimen strongly carcinogenic for the kidney but not the liver), and the other being a continuous dosing over several weeks by adding 8.5 mg of dimethylnitrosamine to each litre of drinking water giving an approximate daily dose of 0.7 mg/kg body wt. This treatment is known to be strongly carcinogenic for the liver but not the kidney. The accumulation in DNA of liver and kidney of the methylated purines 7-methylguanine and O6-methylguanine under each regimen were measured and compared. With the eleven-injection regimen there was a build up of O6-methylguanine in the DNA of the susceptible organ, the kidney, whilst in the liver virtually no accumulation was detected. Under the prolonged, low concentration regimen the liver, in spite of its susceptibility to the carcinogen did not accumulate o6-methylguanine. The results are discussed in terms of the hypothesis that production of o6-methylguanine and its persistence in the DNA of the target organ are responsible for the carcinogenic action of dimethylnitrosamine.


Some observations on the chemical and stereochemical specificity of the de-alkylation of organophosphorus esters by a hepatic glutathione transferase. Hepatic glutathione (GSH) S-methyl transferase from rabbit, pig and dog demethylates dimethyl phosphate triesters. No stereospecificity towards racemic ethyl methyl 2-chloro-1-(2,4-dichlorophenyl)vinyl phosphate could be demonstrated but the enzyme exhibited some selectivity towards the (+) and (-) forms of O-methyl S-methyl 1-naphthyl phosphorothiolate. Pig liver enzyme, purified 30-fold, demethylated th  prochiral substrate dimethyl 1-naphthyl phosphorothionate with 90% stero-selectivity.


Excision of O6-methylguanine from DNA of various mouse tissues following a single injection of N-methyl-Nitrosourea. The persistence of O6-methylguanine produced by a single dose of N-methyl-N-nitrosourea (MNU) was determined in DNA of various murine tissues and compared with the location of tumours induced by MNU and related alkylating carcinogens in this species. A/J and C3HeB/FeJ mice received a single intravenous injection of MNU (10 mg/kg) and were killed at different time intervals ranging from 4 h to 7 days. The rate rate of loss of O6-methylguanine from brain DNA was considerably slower than from liver DNA; tumours have been found in both organs after administration of MNU and other alkylnitrosoureas. There was no difference in the rate of excision from cerebral DNA of A/J and C3HeB/FeJ mice, although these strains differ significantly in their susceptibility to the neurooncogenic effect of MNU and related carcinogens. Excision of O6-methylguanine from hepatic DNA was significantly slower in A/J than in C3HeB/FeJ mice; both strains habe been found to develop hepatic carcinomas following MNU administration. Seven days after the injection of 3H-MNU, O6-methylguanine concentrations were highest in brain and lung DNA, lowest in the liver, and intermediate in kidney, spleen, small intestine and stomach. The lung is a principal target organ for tumour induction by MNU and other carcinogens in mice; however, neural tumours are usually induced at a low incidence. The results obtained do not contradict the hypothesis that O6-alkylation of guanine in DNA is a critical event in the initiation of tumour induction by alkylating agents. However, the location of tumours produced in mice does not seem to depend solely on the formation and persistence of O6-alkylguanine in DNA.


Membrane lipids as intracellular binders of chlorpromazine and related drugs. Equilibrium dialysis studies with chlorpromazine (CPZ) showed affinity and binding capacity values which were not significantly different with the following binders: rat liver microsomes, mitochondria, mitochondrial membranes, brain synaptosomes, myelin vesicles, and red blood cell membranes. There was no binding to cytosol or mitochondrial matrix. The same binding values as above were obtained with protein-free liposomes of lipids extracted from microsomes, mitochondrial and red cell membranes and of pure egg lecithin. The binding values of the two classes of binding sites of all these preparations were K1=2.7+/-1.0-10(4) M-1, K2=3.8+/-1.7-10(3) M-1, C1=580+/-+/-230 and C1+2=1410+/-500 nmole/mg phospholipid. These values were not altered by elimination of the polar head groups of phospholipids with phospholipase C. The results were confirmed by a UV spectroscopic method whereby the strongest binding signals were obtained with CPZ in the presence of fatty acids such as oleate. It is concluded that the major intracellular binders of CPZ and related drugs are the nonpolar moieties of membrane phospholipids, whereby hydrophobic interactions are mainly involved.


Rate-dependent changes in extracellular potassium in the rabbit atrium. We measured levels of potassium ion in the extracellular space of isolated superfused rabbit atria continuously with double-barreled microelectrodes of which on barrel was a K+ liquid ion-exchanger microelectrode and the other a potential-sensing micropipette. Increases in heart rate resulted in transient increases in extracellular potassium ([K+]0). When the quiescent atrium was stimulated the maximal increase was 0.4 mM at rates of 60/min, 0.7 mM at 90/min, 0.9 mM at 120/min, 1.3 mM at 200/min, and 1.8 mM at 300/min. The increase was not sustained during continued stimulation but declined toward prestimulation levels. When the stimulus was terminated the extracellular potassium activity decreased below bathing solution values by 0.2 mM after 60/min, 0.5 mM after 90/min, 0.7 mM ater 120/min, 0.9 mM after 200/min, and 1.0 mM after 300/min and subsequently returned to a value equal to that of the bathing solution. The magnitude of the decline in extracellular potassium activity during prolonged stimulation was markedly decreased when the bathing solution contained either zero potassium, ouabain, LiCl, or a decreased Po2 such that an elevation in [K+]0 persisted during stimulation. Moreover, the reduction in [K+]0 that followed the cessation of stimulation also was inhibited. These results support a role of the Na-K pump in maintaining extracellular potassium activity during changes in cardiac rate.


The aortic bodies supplied by coronary arteries in the dog. Their contribution to the hypertensive response that follows serotonin injection. I assessed the role of the chemoreceptors (aortic bodies) supplied by the coronary arteries in the hypertensive response induced by left atrial injection of (200 microgram serotonin) in adult anesthetized dogs. I compared the pressor response induced by serotonin during normal coronary circulation with that during exclusion of the central coronary segments from which the coronary blood supply to the aortic bodies arises. Exclusion of the central segments reduced the pressor response significantly from the control responses. Exclusion of the coronary blood supply of the aortic bodies resulted in a reduction of the  control response of more than 50% in only two of the 21 dogs. I conclude that although the aortic bodies supplied by the coronary arteries play a significant role in the total hypertensive response to injected serotonin, their role usually is not predominant.


The canine heart as an electrocardiographic generator. Dependence on cardiac cell orientation. Traditionally it is assumed that during cardiac depolarization the macroscopic current generators that produce electrocardiographic voltages can be represented as a uniform double-layer source, coincident with the macroscopic boundary between resting and depolarized cardiac fibers as measured with extracellular electrodes ("uniform" hypothesis). A segment of this boundary is thus considered as a current dipole oriented perpendicular to the boundary. We present evidence that, contrary to the above, the effective dipoles largely parallel the long axes of cardiac fibers ("axial" hypothesis). Calculated potentials in volume conductors differ markedly in the two cases. The magnitudes of rapid local "intrinsic" deflections also differ markedly. In our experiments, potential fields prodlced by stimulation at several cardiac sites and measured magnitudes of intrinsic deflections during normal depolarization and that caused by stimulation support the axial hypothesis and are incompatible with the uniform hypothesis. Our results suggest that axial orientation of sources is sufficiently strong so that predictions assuming the uniform hypothesis would be seriously in error, although the axial theory alone does not exactly describe all the measured potentials. Axial orientation of current generators must be considered in quantitative prediction of electrocardiographic potentials. tfurther study of the geometry of the intracellular depolarization boundary and its relation to fiber direction and to the frequency of lateral intercellular junctions is required to describe the generators exactly.


Hemodynamics of arterial stenoses at elevated flow rates. This study is concerned with the pressure drop that develops across an arterial stenosis, with particular emphasis on the effect of the stenosis at high blood flow rates induced by a locally administered vasodilator drug. Stenoses, ranging in severity from 55.7% to 91.0% reduction in lumen area were artificially induced in the femoral and carotid arteries of large mongrel dogs. Instantaneous flow rates and pressure drops were measured over a wide range of flow conditions. Mean velocities varied from 3.9 to 88.8 cm/sec. Experimental data support the applicability of a relatively simple equation for predicting the pressure drop over this wide range of velocities and stenosis geometries. Results show that blood flow through a particular artery can increase by a large factor, in the range of 4-5, under conditions of vasodilation with a corresponding large decrease in pressure distal to the stenosis. The pressure drop increases in a nonlinear manner with velocity and thereby accentuates the importance of the stenosis at elevated flow rates. We suggest that a critical stenosis be defined in terms of its effect on maximal flow rather than resting flow.


Angiographic determination of arterial patency after percutaneous catheterization in infants and small children. Patency of the femoral artery of infants and children who previously had percutaneous arterial catheterization at a weight of less than 25 kg was studied angiographically. During the study period, 118 patients had repeat arterial catheterization. 48 from the opposite leg. Femoral artery occlusion was found in four patients, while 44 of the 48 studied from the opposite leg had complete patency. At the later study, all four with blockage were asymptomatic and possessed good pedal pulses, while two had decreased femoral pulsations. When events surrounding initial catheterization were retrospectively analyzed, three of four had decreased pedal pulsations beyond six hours. It is concluded that 3-8% of patients weighing less than 25 kg have arterial occlusion after catheterization. This complication may be entirely asymptomatic, but these patients will require continued observation for possible late vascular problems.


The effects of left ventricular load and contractility on mitral regurgitant orifice size and flow in the dog. Acute mitral regurgitation (MR) was produced in 12 dogs by closed chest partial valvulectomy and the relative contributions of MR pressure gradient (MRG), the time for regurgitant flow (VSI), and the MR orifice area (MRA) to mitral regurgitant volume (MRV) assessed. Aortic and left atrial pressures, biplane left ventricular (LV) angiography, forward flow and mitral regurgitant flow (MRF) were measured following MR induction and following augmentation of left ventricular end-diastolic volume (EDV), increased aortic resistance (angiotensin), and in the presence of increased ventricular contractility (calcium or epinephrine). Mitral regurgitation orifice area was determined by calculation and the diameters of the mitral anulus and subvalvular areas measured angiographically. Angiotensin and volume infusion induced a substantial increase in MRF which was largely dependent on an increase in MRA but not MRG, while augmentation of contractility decreased MRF accompanied by a decrease in MRA, relatively independent of MRG. Left ventricular size and shape are major determinants of MRA and resultant MRF in acute mitral regurgitation. These findings may help to explain the effects of such factors as ventricular loading and volume on the clinical course of mitral regurgitation in man.


Cranial and caudal augulation for coronary angiography revisited. Several similar projections utilizing cranial and caudal angulation of the X-ray tube in various obliquities have been described recently for coronary angiography and left ventriculography. These views provide improved visualization of the proximal branches of the left coronary artery, the region of the crux of the right coronary artery, and the left ventricle in the left anterior oblique projection; structures which in the conventional projections are often superimposed on one another or are foreshortened. The confusing and conflicting terms--"oblique clockwise and anticlockwise table base turn," "half-axial," "angulated," "sit up," "caudo-cranial sagittal augulation," "cranio-caudal sagittal angulation," and "lordotic" projections--should be discarded in favor of the terms "cranial angulation projection" and "caudal angulation projection." The appropriate anterior, rather than the posterior obliquity should be used to refer to rotation of the patient, or the X-ray beam around his long axis.


Pulmonary vascular disease complicating the Blalock-Taussig anastomosis. The pulmonary vascular bed was evaluated following a Blalock-Taussig shunt performed in 36 patients for decreased pulmonary blood flow. The time of follow-up ranged from one to 21 years after operation. No patients developed severe pulmonary changes in less than eight years after institution of the shunt. Ten of 20 patients having a shunt eight years or longer developed some degree of pulmonary vascular disease, mainly in the form of intimal fibrosis. This was severe in six of the 20 patients. As the duration of the shunt increased, so did the incidence of pulmonary vascular disease.


The pulsed Doppler coronary artery catheter preliminary report of a new technique for measuring rapid changes in coronary artery flow velocity in man. A new catheter which measures instantaneous changes in coronary artery blood flow velocity is described. The linear relationship between flow velocity is described. The linear relationship between flow velocity measured with the catheter and volume flow through small arteries is documented with a correlation coefficient of 4=0.99. Coronary flow velocity has been measured from the proximal right and left coronary arteries and aortic ostia of saphenous vein bypass grafts in patients undergoing diagnostic coronary arteriography. The increase in coronary flow following injection of contrast media in normal coronary arteries is similar to the increase in coronary flow reported following near-maximum exercise. This increased flow response following injection of contrast media is severly limited by coronary artery stenosis and may provide a useful method for assessing hemodynamically significant stenosis in patients with coronary artery disease.


Correlation of coronary arteriograms and left ventriculograms with postmortem studies. To assess the accuracy of angiographic determinations of disease of coronary arteries and left ventricular myocardium we compared clinical with postmortem coronary arteriograms and left ventriculograms with myocardial pathology in 28 patients, all of whom died postoperatively and within three months of angiography; 19 had ischemic heart disease, four valvular heart disease, and five both. Comparison of pre and postmortem lumenal occlusion in 315 epicardial coronary segments, excluding those operated upon, showed greater than 50% narrowing discrepancies in 21 (7%). Significant coronary artery lesions were overestimated in six and underestimated in 15. Of the six overestimations, three appeared to be due to coronary spasm; of the 15 underestimations, 12 were due to overlapping images; six discrepancies were unexplained. Comparison of wall motion in 140 ventriculogram segments with myocardial pathology, excluding any post-study or perioperative injury, showed good correlation of reduced motion with 48 (34%) infarcted and 10 (7%) aneurysmal segments. However, 58 (41%) other segments had poor or absent ventriculogram motion, with structurally normal myocardium and patent coronary artery supply; 19 were on infarct margins and 39 in dilated or hypertrophied hearts. Thus, premortem coronary arteriographic occlusions generally indicate atherosclerotic narrowing; but decreased or absent segmental wall motion frequently does not indicate a myocardial lesion. It may be attributable to ischemia in the distribution of a critically narrowed coronary artery or it could be due to abnormal ventricular topography.


Sinus node recovery time and calculated sinoatrial conduction time in normal subjects and patients with sinus node dysfunction. In 61 patients sinus node function was tested by programmed (sinoatrial conduction time, SACT) and overdrive atrial pacing (sinus node recovery time, SRT). In the control group (N = 20), mean sinus cycle length was 773 +/- 140.2 msec, mean absolute SRT 1044 +/- 215.8 msec [corrected SRT (CSRT) 270 +/- 112.5 msec; mean +/- SD] and calculated SACT was 82 +/-19.2 msec. The upper limit of normal SACT was defined as 120 msec. In 41 patients with sinus node dysfunction, mean age (55 +/- 14.7 years), mean spontaneous cycle length during the study (1094 +/- 248.0 msec), the lowest heart rate observed (42 +/- 7.5 beats/min), maximal SRT (2110 +/- 1269.1 msec), maximal CSRT (1016 +/- 1182.8 msec), and calculated SACT (126 +/- 47.3 msec) were significantly longer than in the control group. Abnormalities of sinus node function, as evidenced by the degree of spontaneous bradycardia, SRT and calculated SACT were more frequent in patients with bradycardia-tachycardia syndrome or spontaneous sinoatrial block than in those exhibiting isolated sinus bradycardia. We conclude 1) that in patients with sinus node dysfunction both sinus node automaticity and sinoatrial conduction may be abnormal, and 2) that overdrive and programmed premature atrial stimulation can separate patients with sinus node dysfunction according to their clinical presentation.


An unusual variety of atrioventricular nodal re-entry due to retrograde dual atrioventricular nodal pathways. Three patients with paroxysmal supraventricular tachycardia (PSVT) had discontinuous ventriculo-artrial conduction curves (V1-V2, A1-A2), suggesting dual A-V nodal pathways. Ventricular echoes occurred simultaneously with sudden increase of V-A interval. These echoes were characterized by retrograde P waves occurring in front of QRS, suggesting utilization of a slow pathway for retrograde conduction and a fast pathway for antegrade conduction. In case one, atropine improved retrograde slow pathway and antegrade fast pathway conduction and made A-V nodal re-entry sustained, resulting in PSVT (with retrograde P in front of the QRS). In cases 2 and 3, atropine markedly shortened retrograde fast pathway refractory period and slightly improved antegrade slow pathway conduction. The discontinuous V1-V2, A1-A2 curves and echoes were no longer demonstrable. However, with improvement of retrograde fast pathway and antegrade slow pathway conduction, A-V nodal re-entrant echoes and PSVT were observed, utilizing the slow pathway for antegrade conduction and the fast pathway for retrograde conduction (P simultaneous with QRS).


Induction of iatrogenic electrocardiographic patterns during electrophysiologic studies. Indwelling cardiac catheters by producing local mechanical stimulation or trauma can induce electrocardiographic (ECG) patterns which simulate known electrophysiologic phenomena. Catheter-induced ECG patterns were analyzed in 447 consecutive patients undergoing electrophysiologic studies. Iatrogenic nature of these patterns was suggested by 1) absence prior to placement of catheter; 2) sudden appearance with catheter placement and disappearance with catheter repositioning; 3) reoccurrence with remanipulation of catheters; and 4) simulation (in some cases) by programmed electrical stimulation from the catheter. Common catheter-induced patterns were 1) right bundle branch block (RBBB) lasting less than 24 hours occurred in 19 patients; 2) transient third degree atrioventricular block in His-Purkinje system developed in 3/13 patients with pre-existing left BBB; 3) catheter-induced ventricular pre-excitation which simulated ECG patterns of type B Wolff-Parkinson-White syndrome; 4) fortuitous synchronization of right ventricular excitation from the catheter, and left ventricular excitation from sinus beat resulted in normalization of the QRS complexes in 5/68 patients with pre-existing RBBB; 5) premature beats from the atria, right ventricle, and His bundle, which were common, resulted in complex ECG patterns. These iatrogenic ECG patterns must be identified in order to avoid errors in interpretation.


Myocardial imaging with thallium-201 at rest and during exercise. Comparison with coronary arteriography and resting and stress electrocardiography. Myocardial imaging with intravenous thallium-201 (201Tl) was performed at rest and following maximal treadmill exercise in 101 patients with suspected coronary artery disease. Results were interpreted from Polaroid scintiphotos by three independent observers with complete interobserver agreement in 79%. Of 25 patients with no or insignificant coronary artery disease (less than 50% diameter stenosis), one (4%) had a resting 201Tl image defect, one (4%) had an exercise 201Tl defect, none had an ECG Q wave, and four (16%) had exercise ST-segment depression. Among 76 patients with coronary artery disease (greater than or equal to 50% diameter stenosis), 58 (76%) had a defect on either the rest or exercise 201Tl image. The proportion of patients with an exercise image defect (50/76, 66%) was greater than the proportion with exercise ST depression alone (34/76, 45%; P less than 0.02). Overall, 69 of the 76 (91%) patients with coronary artery disease had either a positive rest or exercise myocardial image and/or a positive rest (ECG Q waves) or exercise (ST depression) electrocardiogram. This exceeded the proportion with only rest or exercise electrocardiographic abnormalities (50/76, 65%; P less than 0.001). We conclude that rest and exercise myocardial imaging with 201Tl is easily accomplished with readily available imaging equipment. The image data enhanced the diagnostic sensitivity of stress electrocardiography, and provided spatial identification of the abnormal segment(s) of myocardium.


Location and size of acute transmural myocardial infarction estimated from thallium-201 scintiscans. A clinicopathological study. A clinicopathological study was performed in 23 patients who died from acute transmural myocardial infarction and who had been studied with thallium-201 during the acute phase. Twenty patients died within five days and three later than five days after scintigraphy. The scintigraphic location and the estimated size of infarction in vivo were correlated with postmortem findings. There was good agreement in 91% between scintigraphic and postmortem location of infarction and in 70% between the ECG and postmortem findings. The size of infarction as determined from computer-processed schematic drawings of postmortem slices of the heart correlated well with the size determined from processed schematic drawings of the scintiscans (r = 0.91 for anterior infarction, r = 0.97 for inferior infarction, r = 0.86 for anterior-inferior infarction). It is concluded that thallium-201 scintigraphy provided more precise location of infarction than the ECG and that the size of the scintigraphically abnormal area reflected the extent of necrotic myocardium.


Comparison of the cardiocirculatory effects of exercise and isoproterenol in children with pulmonary or aortic valve stenosis. Isoproterenol infusion during cardiac catheterization as a simulator of exercise was evaluated with hemodynamic studies in children with pulmonary (15) or aortic (8) valve stenosis. Cardiac function was monitored during rest, mild exercise and isoproterenol infusion. Similar heart rates were maintained in both procedures. Peak pulmonary and aortic valve gradients as well as right and left ventricular dp/dt/p were significantly higher and cardiac index significantly lower with isoproterenol as compared to exercise. Isoproterenol induced a significantly greater decrease in systemic vascular resistance, and systemic blood pressure than did exercise. Left ventricular systolic pressure rose to similar levels and this resulted in substantially higher peak aortic gradients with isoproterenol. A consistent correlation was observed for peak valvular gradients measured during exercise and isoproterenol in both groups of patients. Accordingly, regression equations were obtained allowing reasonably accurate prediction of exercise valvular gradients on the basis of the isoproterenol data.


Simulation of laboratory errors and their effects on interlaboratory quality-control programs. The random errors in an analytical method are additive and can be classified into analytical response-dependent and -independent terms. Non-random errors, caused by systematic faults in the analytical procedure, are not always distriguishable from the random errors, but some cases of non-linear assay response and unsuitable standardisation can be studied usefully in models without random error. Interlaboratory quality control programs cannot distinguish systematic and random error until the pattern of results on a number of specimens, or pairs of specimens, can be studied. In this case linear regression analysis is a powerful method for distinguishing different forms of error especially when response-dependent random errors do not predominate. The range of concentrations used for regression whould be as wide as that in which quantitative distinctions are used in clincal diagnosis and treatment. Preliminary reports, of the results on which the regression analysis is based, are most suitably presented on Youden diagrams with paired specimens.


A design for interlaboratory quality-control programs. A design for interlaboratory quality-control programs is described. Speimens are despatched in pairs to participating laboratories. The results returned by laboratories are compared with each other and with reference results, but the frequent brief reports which are prepared are not regarded as the main laboratory assessments, although these reports can alert laboratories to gross imprecision and inaccuracy. When sufficient results have been returned, a linear regression analysis is carried out between results from each laboratory and the reference results. The statistics obtained from the regression data provide a concise source of information about the form of inaccuracy (imprecision and systematic error) present.


A rapid micromethod for measuring theophylline in serum by reverse-phase high-performance liquid chromatography. We describe an assay system for measuring theophylline in 25 microliters of serum. The procedure involves extraction with a 95:5 mixture of chloroform:isopropanol containing beta-hydroxypropyltheophylline as internal standard, and reverse-phase chromatography on a 4 mm x 30 cm column containing "micron Bondapak C18." Theophylline and beta-hydroxypropyltheophylline are eluted with a 90:10 mixture of sodium acetate butter (20 mmoles/litre pH 4.0) and acetonitrile at a flow rate of 1.8 ml/min., are detected by their absorbance at 254 nm, and quantitated by measuring peak areas. Column temperature has not been found to be critical in this analysis. Each analysis requires 9 minutes of chromatography time with a total analysis time of 20 minutes. Analytical recoveries were found to be 71 to 75% for theophylline and 94% for beta-hydroxypropyltheophylline. This difference in recovery is corrected when determining the theophylline concentration in unknown samples. The method has good precision (coefficients of variation between 7.0% and 7.9% for therapeutic and toxic concentrations). The results obtained with this method compare favourably with results obtained by a published cation-exchange high-performance liquid chromatographic method. None of the metabolites of theophylline, common compounds related to theophylline in structure or drugs tested have been found to interfere with the analysis described.


Ornithine methyl ester. An unusual metabolite encountered in the urine of patients with a urea cycle disorder characterized by hyperammonemia, hyperornithinemia and homocitrullinuria. In the urine of six subjects with a urea cycle disorder characterized by hyperammonemia, hyperornithinemia and homocitrullinuria, an unusual ninhydrin-reaction compound was encountered. This unknown on hydrolysis yielded ornithine as the only amino acid and, on dansylation studied, yielded didansyl ornithine. The metabolite from urine has been shown to have the same chromatographic mobility as ornithine methyl ester on paper cellulose thin layer, and ion exchange chromatography. When trimethylsily derivatives were prepared the unknown and the ornithine methyl ester standard had similar mobility on gas chromatography. Identification of the unknown as the ornithine methyl ester was confirmed by gas chromatography-mass spectrometric analysis. In the patients' urines the concentration of methyl ornithine ranged from 70 to 368 microne moles/g creatinine.


Ethanol-host interactions determined by radiorespirometry of 14C glucoses. 1. Theoretical considerations in continuous flow analysis by Walker, Shepherdson and McGowan have been applied to continuous flow radiorespirometry of 14C-glucoses to demonstrate ethanol response differences between water- and ethanol preferring mice. 2. Ethanol dosages in the n mols/kg range stimulated glucose utilization rates more in ethanol-than in water-preferring mice, while intermediate dosages (micron and low mmol/kg) produced equal stimulation but at different dosages. Pharmacological dosages (20-88 mmols/kg) inhibited glucose rates in water-preferring mice. The inhibition was released at 44 mmols/kg in ethanol-preferring mice. 3. Inhibition release was shown to be associated more with glucose carbons other than one, and considered consistent with a sodium-plus potassium-activated ATPase mechanism. 4. Intermediate ethanol dosage changes could be assigned to differences induced in glucose carbon one metabolism with H2O2-catalase and/or microsomal-ethanol-oxidizing systems (MEOS) mechanisms. 5. Our studies suggest that measurements of adenylate deaminase activities might clarify shifts in transaminations (human) and shifts in mononucleotides seen following chronic ethanol ingestion.


Assessment of five quantitative methods for determination of total proteins in urine. 1. We assessed five quantitative methods for the determination of urinary total proteins, three of them with precipitation followed by spectrophotomollowed by spectrophotometry (Tsuchyga/Benedict, Tsuchyga/Biuret and TCA/Biuret); one with gel-filtration (Sephadex G50/Biuret) and a Turbidimetric procedure (SSA). 2. Day-to-day precision was between 3.58 %CV (Tsuchyga/Benedict) and 11.46 %CV (Sephadex/Biuret). The Tsuchyga/Benedict method showed the closest values to controls quantitated by the Kjeldahl method and the lowest detection limit )17.3 mg of total proteins per liter). Recovery studies showed that a level of 100-200 mg/l the Tsuchyga/Benedict recovered over the 96% of the protein added. 3. Comparison studies of 66 24-hour urine samples showed that the best correlation was obtained between the Tsuchyga/Benedict and the Tsuchyga/Biuret methods (r = 0.996). 4. We conclude that the Tsuchyga/Benedict is a sensitive, precise and accurate procedure for the routine quantitation of urinary total proteins.


Colorimetric determination of creatine phosphokinase isoenzymes: detection in tissues and serum, and usefulness as an aid to diagnosis of myocardial infarction. 1. A colorimetric procedure for creatine phosphokinase (E.C.2.7.3.2.) isoenzymes suitable for routine laboratory use its described. The method utilizes a commercial product for visualization of CK isoenzymes in both serum and tissues. 2. The technic has been applied as an aid to the diagnosis of acute myocardial infarction. An intermediate migrating isoenzyme band (CK-MB) was detected in sera of 19 patients with clinical evidence of myocardial infarction and two patients with myocardial ischemia. The CK(MB) was absent in three patients presenting with symptoms suspect of, but not clinically confirmed as having myocardial infarction.


Analysis of estrogens in small samples of non-pregnancy urine. 1. In a widely used procedure for measuring estrogens in non-pregnancy urine(1), 0.01 to 0.05% of a 24hr specimen is taken for analysis. 2. There is an inverse relationship between 24hr urine volume (chi) and creatinine concentration (81 observations on 75 women; r = -0.780; creatinine (g/iota) = 2.34 -0987 chi(iota); P less than 0.001). 3. The data suggest that, when 24hr output is unknown, creatinine levels may be used to select the amount of urine for estrogen analysis. 4. Volumes found to contain 0.01 - 0.05% of a 24hr specimen were: -- (formula: see text).


A simple micromethod for the direct determination of the total carbon dioxide content in biological fluids. A simple method is presented for the determination of the total carbon dioxide content applicable to all biological fluids. This method makes use of a PCO2-electrode, placed in a perspex reaction/measuring chamber, connected with a IL blood gas analyzer, type 413 equiped with an electronic data-ready indicator system. The advantages of the proposed method are: 1. Depending upon the accuracy required for sera, the sample volume can be reduced to 20 or 10 micron1.2. A high degree of accuracy is obtained depending upon the sample volume used; for sera we calculated variation coefficients of 2%, 1% and 0.8% using sample volumes of 10, 20 and 30 micron 1, respectively. 3. The proposed method is simple and requires only two minutes for one determination. 4. In a comparative study an almost perfect relationship was found between the proposed method and the indirect method by calculation with a constant pK1g value.


Creatine phosphokinase isoenzymes: a comparison of a kinetic and electrophoretic method in the diagnosis of myocardial infarction. A comparison has been made of the results of the levels of the MB isoenzyme of creatine phosphokinase by a kinetic and an electrophoretic method performed on patients suspected of having myocardial infarction. Reviewed in depth are those cases in which discrepant results have occurred. On the basis of the combined clinical and laboratory data, the kinetic method is a more sensitive but less specific indicator of myocardial necrosis. Thus, the kinetic method may be used as a screening test, with confirmation of positive results by electrophoresis.


Alteration of membrane proteins during erythrocyte ageing. Separation of Triton X-100-extracted red-cell membrane proteins by linking SDS-electrophoresis in polyacrylamide to isoelectric focusing revealed differences between young and ageing erythrocytes in vitro, which are attributed to progressing conformational changes among membrane proteins.


[Rapid gas chromatographic method for determination of acetate in human plasma and hemodialysis baths (author's transl)]. A rapid and sensitive method for acetate determination in human plasma and in hemodialysis baths (dialysates) is going to become necessary owing to the extensive using of sodium acetate solution in hemodialysis. The gas chromatographic method reported here allows, in about 35 minutes, the precise and reproducible measurement of acetate concentrations ranging from 0.2 to 20 mmol/1. This method can be used to investigate the kinetics of acetate concentration variations in the blood of patients undergoing hemodialysis with sodium acetate solution and to evaluate the amount of acetate absorbed during this treatment.


Estimation of o,p'-DDD in plasma by gas-liquid chromatography. A method is described for the estimation of o,p'-DDD in plasma. To 50 microl plasma, 0.2 ml acetone is added. The mixture is extracted with 2 ml n-heptane containing p,p'-DDD as an internal standard. Water is removed by adding desiccated sodium sulfate. The organic phase can be injected into a gas chromatograph equipped with an electron capture detector. The o,p'-DDD content of the sample can be read from a calibration curve. Recovery of o,p'-DDD added to plasma is over 93%. The same procedure can be used for the estimation of o,p'-DDE.


Interaction of conjugated bile acids and detergents with a radiosorbent assay of vitamin B-12. The effect of conjugated bile acids and detergents on the radiosorbent technique for the determination of vitamin B-12 activity is reported. It is shown that whereas the non-ionic detergent Triton X-100 has no effect on the vitamin B-12-radiosorbent assay, the addition of ionic detergents (e.g. glycocholic acid, taurocholic acid or sodium laurly sulfate) results in a falsely elevated vitamin B-12 activity presumably due to the disruption of the binding of vitamin B-12 to the intrinsic factor-Sephadex complex. This effect may be of importance not only to the radiosorbent assaying of vitamin B-12, but to the in vivo intestinal absorption of vitamin B-12 as well.


Plasma lipoprotein patterns in  patients receiving dialysis therapy for chronic renal failure. A study is reported of the prevalence, nature and possible aetiology of the hyperlipidaemia found in a group of 74 patients receiving maintenance dialysis. Sixty patients were receiving haemodialysis and 14 peritoneal dialysis. Sixty-four per cent of the male and 83% of the female patients had a lipoprotein abnormality which was predominantly of the Type IV pattern, probably due to a combination of increased hepatic triglyceride synthesis and diminished peripheral clearance.


Plasma cortisol, corticosterone and urea in acute myocardial infarction: clinical and biochemical correlations. 22 patients suffering from acute myocardial infarction were subjected to daily blood sampling for up to 6 days after admission to hospital. 5 patients with myocardial ischaemia and chest pain, but no evidence of infarction, were similarly investigated as controls. Daily measurements of plasma cortisol, corticosterone, cardiac enzymes and urea were performed. Plasma cortisol was elevated in all except one of the infarction cases and corticosterone was raised in 13 cases. Control patients had normal levels. Infarction patients were divided into three clinical groups: uncomplicated cases, complicated and fatalities. The latter group exhibited the highest levels of cortisol and corticosterone; no significant difference was seen between the other two groups. Uraemia tended to be associated with the complicated cases, particularly the fatalities, but not with uncomplicated cases. The three groups could be differentiated statistically on the basis of plasma urea concentration.


Seasonal variation of serum 25-hydroxyvitamin D in patients with chronic renal failure treated by regular haemodialysis. Serum 25-hydroxyvitamin D was measured by a competitive protein-binding assay in 44 normal subjects, 60 uraemic patients on regular haemodialysis at different times of the year and in 13 non-dialyzed uraemic patients. The results obtained indicate that uraemic patients on regular haemodialysis have a mean serum 25-hydroxyvitamin D concentration comparable to controls and that they also exhibit a seasonal variation with a significant reduction during the winter months. However, serum 25-hydroxyvitamin D concentration remained essentially within the normal range and did not reflect the increased incidence of osteomalacia in these patients. In the 13 non-dialyzed uraemic patients, serum 25-hydroxyvitamin D concentrations were lower than in the dialyzed patients, but the explanation is not yet clear. This reduction in serum 25-hydroxyvitamin D was not accompanied by any osteomalacia. The results indicate that deficiency of 25-hydroxyvitamin D in our patients on regular haemodialysis is uncommon and clearly not the explanation of dialysis osteomalacia.


The determination of thallium in urine by atomic absorption spectroscopy and emission spectrography. The detection and measurement of thallium in urine samples using atomic absorption spectroscopy and emission spectrography is described. The sensitivity and precision of the methods are shown to be adequate for rapid routine screening in cases of suspected thallotoxicosis.


A new serum alpha-amylase assay of high sensitivity. The use of chemically modified starch substrates for measurement of serum alpha-amylase activity is described. Action of alpha-amylase on such substrates, in the presence of excess fungal glucoamylase, results in the production of glucose in direct proportion to the amount of alpha-amylase present. The glucose produced is measured by a specific enzymic assay. Results obtained by using this new assay correlate well with activities determined by a conventional saccharogenic assay. The new method is of much higher sensitivity, and is less susceptible to interference, than are most other alpha-amylase assay methods.


Analysis of pregnanediol in urine: trental interference. Patients given Trental (pentoxifyllin, Hoechst A.G.) for the treatment of vascular disorders excrete a substance which interferes with the gas chromatographic anlaysis of pregnanediol. The interfering substance appeared in urine within 4-5 h of the ingestion of 100 mg Trental, and was cleared during the next 4-10 h. The administration of 300 mg/24 h to an anovulatory woman caused an apparent increase in her output of pregnanediol of from less than 1 to 65.8 micronmol/24 h.


Fluorimetric determination of plasma unesterified fatty acid. A reliable and simple method is described for the determination of plasma unesterified fatty acid. The method involves extraction by Dole's reagent, washing with dilute sulphuric acid and determination by the decrease of fluorescence of a dilute solution of 7-hydroxy-4-methylcoumarin. Results on rat and human plasma are in good agreement with those obtained by titration.


Fasting hyperglycaemia and relatively unimpaired meal responses in mild diabetes. Plasma glucose, insulin and triglyceride changes in response to a standard breakfast and an oral glucose tolerance test have been studied in normal, obese and diabetic subjects. Mild diabetics with an abnormal oral glucose tolerance test may have normal or near-normal incremental glucose responses to a standard breakfast. A raised fasting plasma glucose is the predominant day-to-day glucose abnormality of mild diabetes. Diabetics have decreased insulin responses to oral glucose compared with the meal, and the deficient insulin response to glucose probably accounts for both the raised fasting plasma glucose levels and the abnormal oral GTT. The initial insulin response to a meal is normal in mild diabetics, and is probably stimulated by secretogogues other than glucose. The oral glucose tolerance test is apposite for the diagnosis of diabetes in view of the impaired insulin response to glucose, but accurate measurement of the basal plasma glucose may be of equal value. The diabetic and obese subjects had normal triglyceride levels, and there was no detectable impairment of disposal of the exogenous triglyceride following the breakfast.


Penile size and growth in children and adolescents with isolated gonadotrophin defiency (IGnD). Penile length and circumference were measured in twenty boys with isolated gonadotrophin deficiency, before and during the administration of androgen therapy. Their age ranged between 3 and 20 years. Considering as normal, measurements above the tenth centile, in all the patients but four the penile length was below normal, and in two it was borderline. Penis circumference was normal in two, borderline in four and subnormal in sixteen. Regular administration of androgen therapy increased penile length in eleven out of fourteen patients with achievement of normal length in four. It is concluded that congenital lack of LH and testicular androgen activation causes small sized penises, even in the prepubertal period. Gonadotrophin deficiency should be looked for in patients with measurements below the tenth centile. Early diagnosis and institution of androgen therapy between 11 and 12 years is likely to increase penile size and prevent the psychological side effects of undersized genitals and delayed puberty.


Glucagon and diabetes. II. Complete suppression of glucagon by insulin in human diabetes. In order to determine whether glucagon levels of diabetic subjects are suppressible, alpha cell responsiveness to acute insulin administration (0.1 units/kg intravenously) was determined in fourteen juvenile onset, healthy diabetic and eight control subjects. In the diabetics, insulin produced a significant but slow fall in blood glucose over 60 min (P less than 0.01). On the other hand, glucagon levels fell dramatically in all diabetics to undetectable levels (P less than 0.001). Only one diabetic became hypoglycaemic and he alone showed a rebound rise of glucagon at 60 min. The rate of fall of blood glucose in the diabetic subjects was not influenced by the basal glucagon level (r=0.13) or the rate of fall of plasma glucagon (r=0.04). The glucose and glucagon responses of control subjects to insulin administration were in sharp contrast to the diabetics: blood glucose levels fell rapidly to hypoglycaemic levels and were associated with a major rise in glucagon levels (mean rise 116 pmol/1, P less than 0.001). We conclude that alpha cell hyperfunction in human diabetes can be completely suppressed by insulin administration and is therefore not autonomous, and that the slow rate of fall of blood glucose following insulin administration in diabetics is not secondary to glucagon excess.


Normal plasma calcitonin: circadian variation and response to stimuli. A simple, reproducible extraction method has been developed, which is capable of measuring calcitonin in normal individuals. Normal calcitonin levels show a circadian variation, with a peak around midday and respond to known stimuli for calcitonin release.