Virus-like particles induced by bromodeoxyuridine in melanoma and neuroblastoma of Xiphophorus. 5-Bromodeoxyuridine (BrdUrd) injected into the muscular tissue of fish bearing melanoma or neuroblastoma induces the production of virus-like particles in these tumors. The particles in the melanoma are morphologically similar to papovaviruses of polyoma-type, those in the neuroblastoma resemble oncornaviruses of B- and C-type.


Cytofluorimetric measurements on the DNA contents of tumor cells in human thyroid gland. The relative DNA contents of thyroid cell nuclei were measured in surgically removed thyroid tissue of 50 patients by means of cytofluorimetry. Smears were prepared immediately after removal of the thyroid nodules according to the classical Feulgen technique. The fluorescence intensities were always compared with those of healthy thyroid tissue prepared in the same way. In each case samples were investigated by the usual histology. The observations indicate that differentiated carcinomas of thyroid gland have an increased DNA content of nuclei, at about the tetraploid level. Among them the follicular carcinomas (11 cases) showed an even higher DNA content of about 250% of the diploid level. Frequency distribution of the cell pools studied revealed a widely scattered aneuploidization of the malignant tumor cells. The benign adenomas displayed only a moderate increase of nuclear DNA content reaching only about 130% of the diploid value. Among the 22 adenomas classified histologically as of benign character, two cases showed very highly increased and widely scattered DNA contents. These latter two cases might be in process of malignant transformation. DNA cytofluorimetry may contribute to a more safe differential diagnosis of the "follicular neoplasia" of the thyroid gland.


Incorporation studies of nucleic acid precursors in gastric cancer: an attempt in individualized chemotherapy. Measurements of the rate of incorporation of radioactively labeled nucleic acid precursors into the DNA and RNA of gastric carcinoma cell suspensions indicated variable rates of proliferation for the tumors. The rate of incorporation generally correlates to the cytological level of differentiation of the carcinoma. Reduced differentiation of the tumors showed a corresponding increase in the rate of proliferation. Knowing the proliferation-dependent effect of most cytostatica, this results in a resistance to cytostatica of highly differentiated gastric cancers. The nucleic acid synthesis of proliferatively active tumors could only be partially inhibited by the cytostatica tested (5-fluorouracil, adriamycin). Carcinomas with metabolic possibility for compensation of the active mechanism of the cytostatica were biochemically resistant. Due to the resulting methodical problems and unaccountable patient-dependent causes of resistance, a conclusive statement about cytostatica-sensitive tumors is difficult to make in incorporation studies.


Hepatic failure in a patient treated with dacarbazine (DTIC) for malignant melanoma. A 55-year-old white woman received chemotherapy with DTIC after surgery for malignant melanoma (stage I, SSM IV, depth of invasion 12 mm). She died suddenly during the second treatment cycle. Autopsy revealed massive necrosis of the liver and thrombosis of the hepatic veins. The cause of the fatal outcome is attributed to the adverse toxic effects of DTIC.


Breast cancer in Israel: laterality and survival. Laterality was examined in 10,702 cases of breast cancer in Israeli Jewish women. The overall left-right ratio was 1.04 and was higher in women over 60. The only population group with a left-right ratio less than 1 was the group of women born in Asian and Middle Eastern countries outside of Israel. There were no differences between the survival curves of women with right- or left-sided breast cancers at any stage. It is concluded that laterality of tumor is not an indicator of survival in breast cancer.


Defect in polyamine metabolism in a BHK cell mutant temperature-sensitive for rRNA maturation. A mutant of BHK cells (ts422E) temperature-sensitive for processing 32S rRNA to 28S rRNA (Toniolo et al., '73) also loses the ability to synthesize polyamines and 5.8S rRNA when shifted to the non-permissive temperature (39 degrees). The activity of several enzymes not involved with polyamine synthesis, methylation of 32S rRNA, and small nuclear RNA production are apparently unaffected after at least 24 hours at 39 degrees. When cultures are returned to the permissive temperature (33 degrees), polyamine synthesizing capacity returns to normal as mature rRNA production resumes.


The effect of protease inhibitors and decreased temperature on the degradation of different classes of proteins in cultured hepatocytes. Leupeptin, chymostatin and antipain inhibited the degradation of long-lived proteins in cultured rat hepatocytes by 20-30%, probably by inhibiting lysosomal proteases: (1) Leupeptin and chymostatin decreased to a similar extent the degradation of an exogenous protein 125I-asialo fetuin, a process known to occur within lysosomes. (2) In extracts of cells treated with leupeptin, cathepsin B activity was inhibited by 35-50%. (3) Leupeptin, chymostatin and antipain inhibited proteolysis by homogenates of liver lysosomes but not by the supernatant fraction. These agents, however, do not appear to rapidly permeate the membrane of isolated lysosomes. Leupeptin, chymostatin and antipain did not inhibit the breakdown of short-lived normal cell proteins, and ones containing amino acid analogs. Even when the amount of abnormal proteins was increased, such that it comprised a large fraction of cell protein, the degradation of these polypeptides was still very rapid and not affected by these inhibitors. The pathway for the degradation of short-lived cell proteins thus appears distinct from that responsible for degradation of long-lived cell proteins. In accord with this conclusion, reduction of the temperature of cultures inhibited the breakdown of long-lived proteins to a much greater extent than it affected the breakdown of short-lived ones. Treatment of cultured hepatocytes with glucagon, or deprivation for serum or amino acids stimulated the degradation of the more stable cell proteins but did not affect the breakdown of 125I-asialo-fetuin. Under these conditions leupeptin and chymostatin inhibited the breakdown of long-lived cell proteins to the same extent as in control cultures. Thus, lysosomal enzymes seem to play an important role in protein breakdown both in fed hepatocytes and in cells where proteolysis is accelerated.


Transmembrane potential and ionic content of rat alveolar macrophages. The cell volume, cell water, intracellular ionic concentrations, and transmembrane potential of rat alveolar macrophages were determined. The measurements were made on cells which had been separated from the medium by centrifugation through dibutyl phthalate in order to greatly reduce the trapped extracellular space. The mean cell volume of the alveolar macrophages is 1,525 cubic microns and 72% of this volume is water. The intracellular fluid is high in Na+ (97 mM) and lower in K+ (50 mM) and the intracellular Cl- concentration in 64 mM. The transmembrane potential, as measured from the equilibrium distribution of tritiated triphenylmethyl phosphonium and by using the fluorescent probe, Di-S-C3(5), is approximately -37 millivolts. Neither Na+, K+, nor Cl- is distributed at equilibrium. However, the K+ permeability of alveolar macrophage membranes appears to be greater than Na+ permeability.


Role of cytoplasmic ATP in the restoration and maintenance of a membrane permeability barrier in transformed mammalian cells. Addition of ATP to medium surrounding intact, transformed 3T3 cells activates the formation of aqueous channels in the plasma membrane. This results in efflux of nucleotide pools and ions and entry into the cytosol of charged, phosphorylated species. In such permeabilized cells, glycolysis is totally dependent on the external addition of glucose, inorganic phosphate, ADP, K+, Mg2+ and NAD+ which restore lactic acid formation to levels found in untreated cells. As expected, such reconstitution of glycolytic activity is found to restore intracellular ATP levels. This is accompanied by sealing of the membrane channels so that efflux of nucleotide pools ceases. Pyruvate, a substrate for mitochondrial ATP synthesis, when provided along with ADP and inorganic phosphate also produces sealing of the membrane channels. On the other hand, reactivation of pentose phosphate shunt activity, which does not lead to ATP synthesis, does not induce restoration of the membrane permeability barrier. Furthermore, compounds which lower the internal ATP pool prevent sealing, and also render the plasma membrane more sensitive to external ATP (Rozengurt and Heppel, '79). Sealing of aqueous channels following restoration of the internal ATP pool is associated with phosphorylation of the inner membrane surface, and is unaffected by inhibitors of protein synthesis, microfilament or microtubular assembly. These results indicate the probable role of intracellular ATP in the restoration and/or maintenance of an active membrane barrier against efflux of small molecules and ions in transformed 3T3 cells.


An X-ray microanalysis survey of the concentration of elements in the cytoplasm of different mammalian cell types. Electron probe energy dispersive X-ray microanalysis was performed on freeze-dried tissue sections. The dry weight concentration of elements (mmole/kg dry weight) was measured in the cytoplasm of several cell types from adult mice and rats. This comparative investigation showed: (1) That the energy dispersive X-ray spectrum of element concentration from the cytoplasm of a specific cell type allows one to distinguish this specific cell type from other cell types with considerable accuracy. (2) That there is a relationship between the concentration of the various elements and the ultrastructural features of the cytoplasmic regions being analyzed. For example, areas rich in ribosomes are also rich in P, K and Mg. (3) These data support the idea that K is directly involved in the control of protein synthesis. The catalog of element concentrations in the cytoplasm of 13 cell types from both mice and rats should be of value to others who seek to answer various questions about these cell types.


Ribosome biosynthesis in Tetrahymena thermophila. IV. Regulation of ribosomal RNA synthesis in growing and growth arrested cells. Three parameters involved in the production of new ribosomal RNA (rRNA) were measured in Tetrahymena thermophilia: (i) the rate of synthesis of the rRNA precursor, (ii) the rate of processing of the RNA precursor and rRNA intermediates and (iii) the efficiency of utilization of the rRNA precursor in producing mature ribosomal RNA. These parameters were measured in cells in exponential growth and in cells starved in a dilute salt solution. Growing cells synthesize rRNA 20 times faster and process rRNA precursors and intermediates 10 to 15 times more rapidly than do starved cells. Both utilize their rRNA precursors with an efficiency of one in converting them to mature rRNA.


Oocyte maturation: aberrant post-fusion responses of the rabbit primary oocyte to penetrating spermatozoa. Primary oocytes cannot be fertilized normally; they begin to develop this capacity as meiosis resumes. To elucidate the changes involved in acquisition of their fertilizability, rabbit primary oocytes displaying a germinal vesicle (GV oocytes) were placed in Fallopian tubes inseminated previously with spermatozoa, recovered 2--5 h later and examined by light and electron microscopy. At least 4 aspects of GV oocyte/sperm interaction were abnormal. Although the vestments and oolemma seem normally receptive to spermatozoa, fusion with the oolemma of the primary oocyte did not elicit exocytosis of cortical granules, and consequently multiple entry of spermatozoa into the ooplasm was common. Secondly, the GV oocyte cortex failed to achieve a normal englufment of the anterior part of the sperm head. It sank into the ooplasm capped by only a small rostral vesicle or left the stable inner acrosomal membrane as a patch in the oolemma. Only rarely then was there significant dispersion of the sperm chromatin, and this remained surrounded by nuclear envelope. The persistence of this envelope constitutes a further aberrant feature, for it disappears immediately in secondary oocytes and was absent in primary oocytes in which germinal vesicle breakdown had occurred. The results are discussed with particular reference to current ideas about male pronucleus formation.


Interaction of lectins with proteins of the endoplasmic reticulum and Golgi system of rat liver. The interaction of glycoproteins of rough and smooth microsomal and Golgi membranes with Sepharose-bound lectins has been studied. One of these lectins was a crude preparation from wheat germ lipase which was found to bind primarily to N-acetyl neuraminic acid. Rough microsomes, smooth microsomes and Golgi membranes contain glycoproteins which bind to Concanavalin A (Con A specific for mannose residues) in decreasing amounts in the order indicated (rough, smooth and Golgi) and to wheat germ agglutinin (WGA, glucosamine-specific) and to the crude lipase preparation in increasing amounts in the order indicated. The small amount of binding of rough microsomes and Golgi membranes to Crotalaria (galactose-specific) increases substantially after neuraminidase treatment. Three submicrosomal particle preparations enriched either in AMPase or in NADH- or NADPH-oxidizing electron-transport enzymes contain glycoproteins which bind Con A and wheat germ agglutinin. The latter binding is sensitive to neuraminidase treatment. Two other submicrosomal particle preparations, both enriched in glucose-6-phosphatase activity, bind preferentially to WGA. This binding is, however, not sensitive to neuraminidase. Prolonged incubation with Ervilia lectin (mannose-specific) inhibits NADH-ferricyanide reductase activity, while the electron-transport chain involving cytochrome b5 is also inhibited by Crotalaria, indicating that both the flavoprotein and the cytochrome b5 are glycoproteins whose oligosaccharide chains have terminal mannose or galactose residues.


A cytochemical and radioautographic study of the ultrastructural organization of puff-like fibrillar structures in plant interphase nuclei (Allium porrum). Loose, fibrillar, spherical structures have been observed during recent years in interphase nuclei of both animal and plant cells. These nuclear formations have been referred to as karyosomes, fibrillar bodies, micropuffs and centromeres. In order to gain further information on the nature of these structures, a cytochemical and radioautographic investigation was undertaken using plant meristematic cells (Allium porrum). For that purpose roots were fixed with either formaldehyde or glutaraldehyde in order to carry out cytochemical tests for DNA, RNA and proteins. Certain of the preparations were also first digested with DNase, RNase or proteinase K and then stained according to different procedures. Other specimens were labelled with thymidine for high-resolution radioautographic observations. Staining with diaminobenzidine (DAB) revealed that these nuclear puff-like formations consisted partly of a loose fibrillar meshwork containing nucleic acids. Part of this fine fibrillar reticulum persisted whether the preparations were digested with DNase or RNase before staining with DAB, thus indicating that these nuclear structures contained both DNA and RNA. The fact that these formations incorporate thymidine furnished additional support for the view that they correspond to specific chromosome segments. Staining with ethanolic phosphotungstic acid or digestion of specimens with proteinase K showed that these loose fibrillar structures also consisted of proteins. Judging from their ultrastructure, their association with the chromatin reticulum as well as from their cytochemical characteristics, these nuclear formations most likely correspond to centromeres. In view of the presence of DNA within these structures, it is possible to distinguish them from other equally spherical nuclear formations, observed in certain plant species, that have generally been referred to as karyosomes or micronucleoli and that appear to consist of ribonucleoproteins.


Rectal papillae in Musca domestica: the cuticle and lateral membranes. The role of specialized regions of insect rectal papillae in the regulation of water and ion uptake is well documented. Although the apparatus for active uptake of water or ions is located in various cell membranes, the absorbed molecules must first pass through the cuticle which lines the rectal epithelium. Most cuticle (e.g. abdominal) has been shown to be permeable only to molecules soluble in wax, and to be impermeable to water and ions. Obviously if such cuticle lined the rectum, absorption of water and ions would be severely restricted. The present freeze-fracture and lanthanum tracer study was undertaken to investigate in more detail both the morphological features of the rectal papillae cuticle which could be responsible for its anomalous permeability and the various cell membranes involved in this transport. It has been suggested from permeability studies that the anomalous permeability of rectal papillae cuticle could be due to the lack of a complete wax layer over the surface of the rectal cuticle. The present study strongly supports this suggestion. Thus, the freeze-fracture micrographs have shown that a surface layer of the cuticle reacts during fracturing like a lipid bilayer. However, in rectal papilla cuticle this surface bilayer is interrupted at each epicuticular depression by areas of different fracturing behaviour. These discontinuities in the surface bilayer probably allow the rectal contents to contact directly the true cuticular matrix. They could, therefore, explain the case with which water and ions penetrate the rectal cuticle and so gain access to the underlying epithelial cells. Although similar discontinuities are present on some of the rectal cuticle surface external to the rectal papillae, they appear to be filled in by plugs of lipid-like material. The lateral plasma membranes of the rectal papillae cells are generally considered to be the main site of active transport. The present lanthanum tracer and freeze-fracture study has shown that the lateral plasma membranes contain 3 distinct differentiations. Septate junctions are present at the apical and basal surfaces of the epithelial layer; a further membrane differentiation is found adjacent to the septate junctions; and thirdly, an array of short, variable length, non-anastomosing linear structures covers most of the lateral plasma membrane surface. These latter structures, unlike known types of cell junctions do not show equivalent arrays in apposing membranes even when the lateral plasma membranes of adjacent cells are closely apposed. The possible function of these structures is discussed.


Scanning electron-microscopic study of the uptake of Leishmania parasites by macrophages. The interaction of promastigotes of the protozoan parasite Leishmania tropica with mouse peritoneal macrophages in vitro was studied by scanning electron microscopy. Motile promastigotes attached to host cells by their flagellar tips to which the macrophages responded by producing rather closely fitting lamellar sheaths and progressively enveloping first the flagellum and then the body of the parasite. Lamellar advance during engulfment was rapid in the first 10 min but much slower later on. Fully engulfed parasites could be seen after 1 h but most parasites associated with host cells remained extracellular even after 4 h. On the other hand, parasites immobilized by fixation adhered by either their flagellar or somatic ends. Engulfment proceeded at a steady rate, and by 4 h most of them were completely engulfed. Both the attachment and engulfment stages of parasite uptake were inhibited by low temperature, cytochalasin D and mild fixation of macrophages. The rheological features of the host cells' response to parasite adherence indicate that invasion by parasites is through phagocytosis rather than penetration.


The effect of cell mass on the cell cycle timing and duration of S-phase in fission yeast. Two isotopic methods for measuring DNA replication in the fission yeast Schizosaccharomyces pombe are described. The first is a method for measuring the total quantity of [3H]uracil incorporated into DNA after pulse labelling. The second is a means of detecting DNA replication in single cells by autoradiography. Both of these techniques have been used to investigate the timing and duration of S-phase in a series of mutant strains whose cell mass at division varies over a 3-fold range. The results support the hypothesis that in S. pombe there are 2 different controls over the timing of S-phase: an attainment of a critical cell mass and a dependency upon the completion of the previous mitosis coupled with a short minimum time in G1. Strains whose cell mass at birth is above this critical level initiate DNA replication almost immediately after septation, that is, very soon after the previous mitosis. Strains whose cell mass at birth is below the critical level do not initiate replication until the critical cell mass is attained. The duration of S-phase has been estimated from the proportion of cells whose nuclei are labelled after a pulse of given duration. S-phase is short in S. pombe, lasting only about 0.1 of a cell cycle in wild type. Cell mass at S-phase does not have any consistent effect on this length. We have also investigated the degree of synchrony of S-phase initiation in daughter cells, and have found that, in a cell cycle 240 min long, their S-phases are initiated within 1--2 min of each other. This result indicates that between sisters variability in the duration of the G1 phase is small compared with variability in the total cell cycle time, and argues against the hypothesis that the rate of cell cycle traverse is determined by a random transition in G1.


Isolation of a residual protein structure from nuclei of the myxomycete Physarum polycephalum. A nuclear framework structure has been obtained from isolated interphase nuclei of Physarum polycephalum by extraction with 2.5 M NaCl and subsequent digestion with DNase. Whole-mount electron micrographs showed a nuclear lamina containing residual pore structures associated with the fibrous internal matrix. The matrix was continuous with fibrillar remnants of the nucleolus. The structure was shown to consist of 2 major polypeptides of 23,000 and 36,500 Daltons as well as 30 to 40 minor polypeptides of various molecular weight classes. The 2 major polypeptides were also prominent in preparations of the residual nucleolar material, suggesting that matrix proteins are common to both structures. The predominance of low-molecular-weight polypeptides in Physarum nuclear matrix suggests that there may be significant differences in composition of nuclear structural proteins between lower and higher eukaryotes.


Immunoelectron-microscopic studies of endoplasmic reticulum-Golgi relationships in the intracellular transport process of lipoprotein particles in rat hepatocytes. Endoplasmic reticulum (ER)-Golgi relationships in the intracellular transport process of secretory proteins in rat hepatocytes have been studied using lipoprotein particles as a marker for the secretory protein and cytochrome P-450 as a marker enzyme for the ER membranes. Ferritin immunoelectron-microscopic observation revealed that, while almost all the microsomal vesicles derived from ER membranes are heavily labelled with ferritin anti-cytochrome P-450 antibody conjugates, labelling of the small peripheral vesicles containing lipoprotein particles, the stacks of Golgi saccules, especially the outermost saccule which is sometimes fenestrated, condensing vacuoles in the trans-Golgi region and the secretion droplets of lipoprotein were scanty and at the control level. Such a characteristic pattern of labelling was especially evident when these structures were prepared from phenobarbital-treated rats. These findings indicate that the membranes of the small peripheral vesicles do not contain cytochrome P-450 and that the cytochrome is probably not transferred to Golgi saccules in the transport process of lipoprotein from ER to Golgi. It is suggested, therefore, that the small peripheral vesicles are formed by budding of the special regions of ER membrane where microsomal marker proteins such as cytochrome P-450 are excluded and the membrane proteins destined to the Golgi complexes are clustered. It is also shown that lysosomal membranes are not labelled with the anti P-450 antibody conjugates.


Temperature effects on anaphase chromosome movement in the spermatocytes of two species of crane flies (Nephrotoma suturalis Loew and Nephrotoma ferruginea Fabricius). Using phase-contrast cinemicrography on living crane fly (Nephrotoma suturalis Loew and Nephrotoma ferruginea Fabricius) spermatocytes, we have studied the effects of a range of temperatures (6--30 degrees C) on the anaphase I chromosome-to-pole movements of both autosomes and sex chromosomes. In contrast to previous work we have been able to study chromosome-to-pole velocities of autosomes without concurrent pole-to-pole elongation. In these cells we found that the higher the temperature, the faster was the autosomal chromosomes movement. From reviewing the literature we find that the general pattern of the effects of temperature on chromosome movement is similar whether or not pole-to-pole elongation occurs simultaneously with the chromosome-to-pole movement. Changes in cellular viscosities calculated from measurements of particulate Brownian movement do not seem to be able to account for the observed velocity differences due to temperature. Temperature effects on muscle contraction speed, flagellar beat frequency, ciliary beat frequency, granule flow in nerves, and chromosome movement have been compared, as have the activation energies for the rate-limiting steps in these motile systems: no distinction between possible mechanisms of force production is possible using these comparisons. The data show that even the different autosomes within single spermatocytes usually move at different speeds. These velocity differences cannot simply be related to chromosome size as the autosomes are visually indistinguishable. The sex chromosomes start their anaphase poleward movement after that of the autosomes, and move more slowly (by a factor of about 4), but their velocities appear to be affected by temperature in the same fashion as those of the autosomes. The interval between the onset of autosome anaphase and sex chromosome anaphase is also affected by temperature: the higher the temperature, the shorter the interval between the 2 stages. We have observed abnormalities in sex chromosome segregation, which may be due to temperature, but have not determined what the exact temperature shift conditions are that cause these abnormalities.


Epithelial-fibroblastic organization in cultures grown from human embryonic kidney: its significance for morphogenesis in vivo. The morphological behaviour of explants of human embryonic kidney has been studied in order to investigate the rules governing interactions between epithelial and fibroblastic cells from the same tissue. When the fragments are cultured, epithelia migrate out first and are followed, a few days later, by cables which grow out from the fragments and which are composed of multilayered fibroblasts. These cables extend through the epithelia to reach the substratum, to which they adhere. The epithelia maintain an upper surface free of spread fibroblasts and are unable to multilayer, although occasional rounded-up cells adhere to this surface. Fibroblasts, however, not only multilayer in the cables but can act as a substratum for epithelial cells which migrate on the cable surface. Fibroblasts and epithelia from kidney thus follow the same behavioural rules that govern the interactions between kidney epithelia and fibroblasts from different tissues. The suggestion that these rules derive from tissue differences and that cells from the same tissue are more tolerant of one another is not borne out. These observations and those reported by others are interpreted in terms of the functional properties of the cells in vivo. It is further pointed out that only those epithelia that maintain a free surface in vivo would be expected to show this property in vitro. Finally, the implications of cells with essentially the same properties generating more than one structure are considered.


Mitochondrial associations with specific microtubular components of the cortex of Tetrahymena thermophila. I. Cortical patterning of mitochondria. Many of the mitochondria of Tetrahymena thermophila are localized in the cell cortex in regular, identifiable patterns. Two different mitochondrial patterns are seen; whether a cell expresses one or the other type apparently depends upon nutrient conditions in the culture and not upon other factors tested. Consistent associations between cortical mitochondria and certain of the cortical microtubular bands are seen at the light-microscopic level. Electron-microscopic observations confirm this and, furthermore, identify ultrastructural associations between cortical microtubules and the cortically located mitochondria. It appears that the cortical microtubular arrays serve as a guide for the localization (and thus patterning) of the cortical mitochondria.


The loss of morphogenetic potential and induction of phenylalanine ammonia-lyase in suspension cultures of Phaseolus vulgaris. The loss of morphogenetic potential in bean suspension cultures has been investigated by measuring the amounts of phenylalanine ammonia-lyase activity induced in the cells when they are transferred from a medium in which they are grown and maintained to an induction medium. The tissue has been grown in 2 types of medium: (1) supplemented with 2,4-dichlorophenoxyacetic acid as the only growth hormone, and (2) supplemented with 2,4-dichlorophenoxyacetic acid and coconut milk. When cells were grown in medium with only 2,4-dichlorophenoxyacetic acid for a period of 5--10 subcultures and samples were transferred to the induction medium at intervals during the subcultures, the amounts of phenylalanine ammonia-lyase activity and the number of xylem elements induced progressively declined. Cells grown in the presence of coconut milk did not lose the ability to induce phenylalanine ammonia-lyase or xylem elements. Cells grown in the presence of coconut milk were cloned and clones capable of producing different amounts of phenylalanine ammonia-lyase when transferred to induction medium were obtained. However, clones producing low amounts of activity did not grow faster in the medium lacking coconut milk and no evidence was obtained to show that selective growth of non-inducible cells was responsible for the loss of morphogenetic potential. In addition to the induction brought about by the presence of naphthylacetic acid and kinetin in the induction medium the cells could also be stimulated to produce phenylalanine ammonia-lyase activity by dilution at subculture. This increase in activity occurred within 10 h of the dilution, whereas that produced by the hormones in the induction medium occurred after 120 h. The induction produced by dilution also occurred in these cells which had lost their ability to respond to the hormonal induction. Thus the mechanism that produced the increase in phenylalanine ammonia-lyase activity was intact but had lost its ability to respond to the hormones of the induction medium. The loss of inducibility was therefore probably not due to a genetic change in the cells brought about by continuous growth in a medium lacking coconut milk, but to reversible changes in the hormonal requirements of the cells necessary for induction.


An unusual cell surface modification: a double plasma membrane. The occurrence of an unusual double plasma membrane structure is reported; it has been studied in conventional thin sections, after lanthanum-impregnation and with freeze-fracturing. This modification of the plasmalemma is found where the luminal cell membrane (I membrane) of gut microvilli in the haematophagous insect, Rhodnius prolixus, is surrounded by a second, outer membrane (O membrane), the 2 separated from one another by a highly regular I-O space of about 10 nm. Lanthanum impregnation reveals the presence of columns inclined at an angle, within this I-O space; as in the continuous junctions which link the lateral borders of these cells, these columns may maintain the very precise I-O distance. From the outer microvillar membranes radiate short spoke-like fibrils or sheets which encounter another more extensive system of myelin-like sheets. Freeze-fracturing reveals that the spoke-like sheets and the other ones which lie like a tube, around and parallel to the microvilli, contain linear ridges composed of particles, lying at random within layers of the myelin-like material which also extends into the lumen of the gut. The microvillar membanes, both O and I, fracture into faces containing rows of either PF particles or EF pits arranged as spiral ridges or grooves around the sides and across the tip of each microbillus. These could be the insertion sites of one or both of the I-O columns and spoke-like sheets while the sheets could represent a variant of peritrophic membrane. The double membrane may be a cellular device to increase the strength of the microvillar layer in these blood-sucking animals, since the cell layer must withstand great pressure owing to a sudden massive extension of the gut during a blood meal.


Rhizoplast and rootlet system of the flagellar apparatus of Chlamydomonas moewusii. The ultrastructure of the flagellar apparatus of Chlamydomonas moewusii was examined in detail. Two rhizoplasts were found associated with the basal bodies of this biflagellate and were observed to extend to the central area of the cell. A segment of smooth endoplasmic reticulum ran parallel to each rhizoplast. These 2 segments anastomose beneath the basal bodies and the tubule proceeds over the top of the distal connecting fibre. A functional role for the smooth endoplasmic reticulum in these locations is discussed. Four sets of rootlet microtubules emanate from a region between the 2 basal bodies and the distal connecting fibre. Two sets have a three-over-one arrangement and the other 2 sets are doublets. Cytoplasmic microtubules were seen associated with possible nucleating sites on the rootlet microtubules. The association of the observed structures are discussed and compared to the flagellar apparatus of C. reinhardtii.


Origin and spatial distribution of maternal messenger RNA during oogenesis of an insect, Oncopeltus fasciatus. In order to investigate the origin and spatial distribution of maternal mRNA during oogenesis, in situ hybridization with [3H]-poly(U) was utilized for the detection of poly(A)-containing RNA [poly(A)+RNA] in histological sections of Oncopeltus fasciatus ovaries. In the germarium poly(A)+RNA was found to accumulate in the trophocyte cytoplasm concomitant with the maturation of these cells. Poly(A)+RNA was also detected in the trophic cores and nutritive tubes suggesting that these channels participate in the transport of trophocyte-derived mRNA to the oocytes. Although large amounts of poly(A)+RNA were also detected in the cytoplasm of the follicle cells, particularly during late vitellogenesis when pseudopod-like processes projected into the ooplasm, no evidence was obtained for the transport of poly(A)+RNA from these processes to the oocytes. The content of poly(A)+RNA in the oocyte cytoplasm continually increased during oogenesis. In stage 2--4 oocytes poly(A)+RNA accumulation occurred in the apparent absence of transcriptional activity in the germinal vesicle nuclei suggesting that most maternal mRNA molecules synthesized during early oogenesis are of trophocyte origin. Poly(A)+RNA also continued to accumulate after chorion formation, when the nutritive tubes are longer active in RNA transport. This implies that other sources of maternal mRNA may exist during late oogenesis. The distribution of poly(A)+RNA molecules in the oocyte cytoplasm appeared to be uniform throughout oogenesis with one exception. During late vitellogenesis poly(A)+RNA activity was significantly enhanced in the anterior and posterior periplasmic cytoplasms relative to the lateral periplasm and the endoplasm. After chorion formation these variations disappeared. The results suggest that maternal mRNA molecules arise from at least 2 sources during oogenesis. During late vitellogenesis these molecules appear to be subject to differential localization in the polar perimeters of the oocyte cytoplasm.


Wall substructure of microtubules polymerized in vitro from tubulin of crayfish nerve cord and fixed with tannic acid. Microtubules of axons of crayfish nerve cord normally have 12 wall protofilaments and microtubules of surrounding glial cells have 13 protofilaments. Tubulin was isolated from such nerve cords and polymerized in vitro; tannic acid staining of sedimented microtubules showed that microtubules with 12 and 13 wall protofilaments were present, suggesting that the ability to form a microtubule with a given number of protofilaments is inherent in the tubulin or its associated proteins. The temperature of polymerization was found to influence the number of protofilaments in resultant microtubules. With assembly at 20 degrees C, for example, most of the complete microtubules had 13 protofilaments, while at 40 degrees C most showed 10 protofilaments. It is suggested that the temperature of in vitro polymerization, among other factors, can influence the angle of binding between adjacent protofilaments and thus alter the number of protofilaments required to complete the circumference of the tubule.


Isolation, identification and quantitation of urinary organic acids. An application of the HISLIB program for the comparison of gas chromatographic-mass spectrometric profiles of urinary organic acids isolated by extraction and ion-exchange methods is described. Ion-exchange methods are clearly superior to solvent extraction in terms of the variety of compounds isolated. However, the former method has practical difficulties which make solvent extraction more attractive for rapid analyses. For the compounds isolated by both methods, the precision of analysis is similar, with standard deviations of relative concentration in the range 10--30% for most compounds.


Analysis of fatty acids from human lipids by gas chromatography. A rapid, quantitative method is described for the analysis of fatty acids from human lipids, namely serum lipids and lipids from adipose tissue biopsies. The method includes extraction of serum lipids with chloroform--methanol, hydrolysis with tetramethylammonium hydroxide, methylation with methyl iodide and N,N-dimethylformamide and gas chromatographic analysis on a Supelcoport SP-2320 column. Fat biopsies are analysed without extraction. Optimal hydrolysis conditions have been investigated.


Corticosteroid analysis in biological fluids by high-performance liquid chromatography. A sensitive, specific, and reproducible high-performance liquid chromatographic assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples are extracted with methylene chloride, washed with sodium hydroxide and then water, and chromatographed on a microparticulate silica gel column with UV detection at 254 nm. Sensitivity was greater than 15 ng for all four steroids. Specificity was supported by use of dual wave-length UV detection and/or radioimmunoassay. The assay has been applied in pharmacokinetic studies and a typical plasma concentration--time profile for the three steroids is presented for one subject who received 50 mg of prednisone.


Determination of free, total, and esterified cholesterol by high-performance liquid chromatography. A method is described for measuring free, total, and esterified cholesterol in blood serum in which reversed-phase liquid chromatography is used and the eluate is monitored at 200 nm. The sample for total cholesterol is prepared according to the Abell-Kendall procedure, and for free cholesterol an extract of serum--isopropanol (1:5, v/v) is used. The column is a muBondapak C18, 10 micrometers, and the mobile phase for total cholesterol is isopropanol--acetonitrile (50:50, v/v); for free cholesterol, it is isopropanol--acetonitrile--water (60:30:10). An approximation of the free cholesterol, triglycerides, and individual cholesteryl esters is obtained from single chromatograms of isopropanol extracts of serum if the first mobile phase is used. In a comparison study with the Abell-Kendall method for total cholesterol, the correlation is excellent and the precision is acceptable.


High-performance liquid chromatographic determination of dopamine sulfoconjugates in urine after L-dopa administration. A procedure was developed for the separation and determination of dopamine-3-O-sulfate (DM-3-S) and dopamine-4-O-sulfate (DM-4-S) in the urine of subjects administered L-DOPA. The method consists of sample preparation using cation- and anion-exchange resins followed by determination of the sulfates by high-performance liquid chromatography. The addition recoveries were 96 +/- 2.9% (S.D.) for DM-3-S and 93 +/- 3.0% (S.D.) for DM-4-S. Twenty samples could be measured per day. When every 2-h urine specimen from normal subjects was analyzed after L-DOPA administration (0.5 g), the maximum excretion of each sulfate was observed in the second 2-h specimen. For the first 6 h 7.5 +/- 1.5% (S.D.) of the administered L-DOPA was excreted as DM-O-sulfates. During this time, the ratio of DM-4-S to the DM-O-sulfates was 11.7 +/- 0.58% (S.D.).


Analytical isotachophoresis of uremic blood samples. Uremic blood samples were analyzed for ionogenic substances using analytical isotachophoresis. Multicomponent separations proved that the uremic state shows significant differences from the normal state, especially with regard to anionic low-molecular-weight substances. As a quantitative parameter the ratio of anionic higher-molecular-weight substances to anionic low-molecular-weight substances is proposed: the HL ratio. Separation patterns and HL ratios were studied during nine weeks for one patient on chronic hemodialysis. The patient showed a low HL ratio due to excess of low-molecular-weight substances. Separation patterns before and after hemodialysis showed clear differences and the HL ratio increased. The method of analysis is neither time- nor sample-consuming and sample preparation is not needed. Experimental procedures are easily standardized and results are reliable.


Rapid separation of DNA constituents, bases, nucleosides and nucleotides, under the same chromatographic conditions using high-performance liquid chromatography with a reversed-phase column. Four components of three sets of DNA constituents, bases, deoxyribonucleosides and deoxyribonucleoside 5'-monophosphate, were sufficiently resolved under one set of chromatographic conditions using high-performance liquid chromatography with a reversed-phase column (Zorbax ODS) and the solvent 0.4 M NH4H2PO4 (pH 3.5). The effect of pH and salt concentration in the solvent on the retention of these compounds in the column was thoroughly investigated. Proportionality of peak height to the content, and reproducibility and recovery of the four bases were satisfactory under appropriate conditions and as little as 1 microgram of DNA could be analysed for its base composition by this method.


Chlorpheniramine. I. Rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine by high-performance liquid chromatography. A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.


Reliable routine method for the determination of plasma amitriptyline and nortriptyline by gas chromatography. A gas chromatographic method has been developed for the determination of amitriptyline and nortriptyline in plasma. OV-17 is used in a 1 m long packed column, with a flame ionization detector and an electronic integrator. Five internal standards are added. The base-specific extraction procedure and the method of calibrating the chromatograph are described in detail. The accuracy, precision and reliability of the method are demonstrated by the results of nearly 700 determinations of each drug, at concentrations ranging from 5 to 400 ng/ml in the plasma. An interlaboratory comparison with a double radioactive isotope derivative assay for nortriptyline has also shown satisfactory agreement.


Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography. A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in combination with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10(3) concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.


A rapid, sensitive method for accurate determination of the lecithin/sphingomyelin ratio by thin-layer chromatography and reflectance spectrofluorometry. A highly reproducible thin-layer chromatographic procedure has been developed for accurate determination of the lecithin/sphingomyelin ratio. Two interfering compounds, phosphatidyl inositol and phosphatidyl serine, have been investigated and eliminated by adsorption onto DEAE-cellulose. A uniform fluorescence staining procedure employing 2',7'-dichlorofluorescein has been developed. Accurate quantitation was performed by direct measurement of the reflected fluorescence intensity of the lecithin and sphingomyelin fluorophore spots with a spectrofluorometer equipped with a thin-layer scanning attachment. Stability and reproducibility studies are reported.


Gas-liquid chromatographic method for the measurement of plasma levels of d-7,8-dimethoxy-3-methyl-phenyl-2,3,4,5-tetrahydro-1h-3-benzazepine acid maleate (SCH-12679) and its major metabolites in aggressive mental retardites. A sensitive gas-liquid chromatographic technique for the quantitative analysis of SCH-12679 (d-7,8-dimethoxy-3-methyl-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine acid maleate) and its major metabolites in plasma of aggressive mental retardates receiving therapeutic doses of the medicament has been developed. The lower limits of detection are 20 ng/ml for SCH-12679, 0.5 ng/ml for 3-desmethyl SCH-12679 and 0.4 ng/ml for 7-desmethyl plus 8-desmethyl SCH-12679. SCH-12679 is estimated with a flame ionization detector. Its metabolites are quantitated using an electron-capture detector after conversion of the compounds to their heptafluorobutyryl derivatives by reaction with the appropriate anhydride. Data on plasma levels of unchanged SCH-12679, 3-desmethyl SCH-12679 and a combination of 7-desmethyl and 8-desmethyl SCH-12679 in fifteen patients treated with the medicament are presented.


Cimetidine assay in human plasma by liquid chromatography. An assay for the determination of cimetidine in human plasma is described. Cimetidine was extracted from alkalized plasma with ethyl acetate, washed once over hydrochloric acid, re-extracted into ethyl acetate, and the organic phase was evaporated to dryness. The residue was dissolved in ethanol and injected into a liquid chromatograph. In vitro sulphoxidation was found to occur in whole blood, for which reason the assay was performed in plasma. The accuracy of the method was found to be within 3% and the lower limit for sensitivity was demonstrated to be 0.1 mg/l using 750 microliters plasma. Five volunteers received 1 g cimetidine perorally per day given in four doses with various intervals. Blood samples were drawn hourly, five dose intervals over two days. The average minimum concentration of plasma cimetidine was found to correlate significantly with the mean value of the area under the time/concentration curve over a period of three dose intervals (r = 0.96).


Estimation of disaccharides in plasma and urine by gas-liquid chromatography. A technique for the estimation of disaccharides in plasma and urine using gas-liquid chromatography is described. The procedure involves the formation of trimethylsilyl derivatives followed by injection of the reaction mixture directly onto the column. The method is precise, linear over a wide range and gives recoveries of 93--99%. The limit of sensitivity is 80 micrograms per 100 ml, but with modification of the volumes used, levels of 40 micrograms per 100 ml may be quantitated.


Charge-transfer and water-mediated adsorption. III. Adsorption on tryptophan-substituted Sephadex and Sepharose. The adsorption of tyrosine, tryptophan and some related compounds on tryptophan and tryptophanyltryptophan gels is similar to but much stronger than that observed with the parent gel Sephadex G-25. The adsorption has been attributed mainly to charge transfer or redistribution of electrons and protons leading to pi-bonding and hydrogen bonding, in addition to hydrophobic adsorption. The participation of organized water surrounding the gel matrix, the ligands and the solute molecules has been considered. The application of this principle of adsorption has been exemplified by chromatographic experiments with a cellulase preparation of tryptophan-Sepharose 4B.


Titration curves of polypeptide chains by combined isoelectric focusing-electrophoresis in 8 M urea. Titration curves of reduced and alkylated polypeptide chains can be successfully performed in 8 M urea-polyacrylamide gel plates by electrophoresis perpendicular to a stationary stack of focused carrier ampholytes. All buffers and thiol reagents with pK values in the range pH 3--10 should be removed, since their pH-dependent ionization affects the migration and apparent pI values of the protein chains. No blurring of the patterns below pH 4.5 is observed, as usually found in titration curves in the absence of urea, thus allowing the direct titration of Glu and Asp residues. It is not possible by the present technique to titrate any group below pH ca. 3 and above pH ca. 10, due to the lack of suitable carrier ampholytes and to a "flooding" phenomenon, with concomitant identical electrophoretic mobility for all protein species, irrespective of their relative pI values and amino acid composition. The "electrophoretic titration curves" thus obtained were well correlated with the overall amino acid composition of the polypeptide chains analyzed.


Simple and rapid method for the determination of progesterone in rat plasma by gas-liquid chromatography with electron-capture detection. A method for estimating progesterone in rat plasma using gas-liquid chromatography (GLC) with electron-capture detection is described. By means of a column chromatographic technique for the extraction of progesterone from plasma, it is possible to isolate the steroid in such a specific way that, contrary to previously published methods, no additional chromatographic purification and separation steps are needed prior to the final GLC analysis. As a derivative for electron-capture detection we used the 3,20-di-O-pentafluorobenzyloxime of progesterone. This derivative is superior to those used hitherto with regard to sensitivity and stability. The high overall recovery (mean value, 89.4%) makes this method especially convenient for research work when only little plasma is available, or when the concentration of progesterone is very low.


Chromatographic isolation of thymic factors impairing neuromuscular transmission. Thymopoietins I and II are chemically related peptides able to inhibit neuromuscular transmission. They were first isolated from bovine thymus by a protein-denaturating method, which may have destroyed other thymic factors displaying the same biological activity. The present investigation, based on a new gentle isolation procedure, suggests that thymopoietins are the only factors impairing neuromuscular transmission that are present in the thymus. The yield of the new procedure is at least twice as high as that of the original method.


Analysis of azanaphthalenes and their enzyme oxidation products by high-performance liquid chromatography, infrared spectroscopy and mass spectrometry. Adsorption and reversed-phase high-performance liquid chromatography (HPLC) have been successfully used to separate metabolites from the parent heterocycles (isoquinoline, 3-methylisoquinoline, phthalazine, quinazoline, quinoxaline and cinnoline). Retention data are reported. The metabolites, hydroxyazanaphthalenes, which arise as a result of aldehyde oxidase catalysed oxidation, could be extracted in microgram quantities from incubation mixtures by n-butanol. Complete identification of the oxidation products was achieved by collecting fractions corresponding to each compound from the HPLC eluate and subjecting these to infrared and mass-spectral analysis.


Quantitative liquid chromatographic determination of cefatrizine in serum and urine by fluorescence detection after post-column derivatization. A fast, specific and sensitive high-performance liquid chromatographic procedure for the determination of cefatrizine, an orally active cephalosporin, in serum and urine is proposed. The drug is determined by the internal standard method, using cephradine as the internal standard. The separation is carried out on a reversed-phase column, filled with octadecylsilane chemically bonded microparticles. The eluent is a mixture of acetonitrile with 0.025 M sodium phosphate buffer (pH 7). Quantitation is effected by fluorescence detection of the fluorophores formed after post-column derivatization with fluorescamine in a packed-bed reactor. The chromatographic conditions and the conditions for the post-column derivatization are discussed. The method has been applied to serum and urine samples, which were analysed after deproteinization with trichloroacetic acid and injection of the clear supernatant. The accuracy and reproducibility of the procedure were investigated by the determination of the cefatrizine content in spiked serum and urine samples.


Class separation of bile lipids by thin-layer chromatography. A thin-layer chromatography technique is described that permits separation of each class of bile lipid, such as cholesterol, free (unconjugated) bile acids, glycine- and taurine-conjugated bile acids and phospholipids, in a single run. The use of silica gel G-aluminium pre-coated sheets facilitates further processing, such as the extraction in situ of each class of separated bile lipids for determination by conventional methods.


Protein binding of coherin and other small molecules by thin-layer gel chromatography. The protein binding stoichiometry of small molecules is here determined on a nanomole scale by a simplified procedure utilizing chromatography on thin layers of cross-linked dextran gels. New data are presented on the thin layer chromatographic properties of representative ligands, including a-amino acids, peptides, dyes and fluorigenic reagents, in relation to their molecular weights, polar characteristics, gel water regain values and denaturants, providing criteria for the general application of this method to studies of ligand binding with large as well as small molecules. By this procedure coherin peptides, A1 and B1--4, respectively, bind to coherin C in the molar ratio, 2:1, with a binding constant of about 10(5) M-1. Coherin C is believed to act as a carrier peptide.


Rapid determination of DDT and related compounds in soils via carbon skeleton gas chromatography-mass spectrometry. The levels of DDT and related compounds in soil samples from an apple orchard have been determined. Extraction of residues was achieved by cyclic steam extraction and by conventional solvent extraction methods allowing comparison of the two methods. Determination of the organochlorine residues present in the extracts was performed by gas-liquid chromatography-electron-capture detection and by carbon skeleton gas chromatograph-mass spectrometry. Excellent agreement between the two determination techniques was achieved. Prior to application of p,p'-DDT, a value of approximately 0.4 ppm was obtained for Delta DDT in the soil. The major component of this total was DDE. After spraying the apple trees with technical grade, p,p'-DDT, Delta DDT for the soil rose to 0.6 ppm. This increase was due to translocation of p,p'-DDT from the trees.


Fingerprinting of carbohydrates of Streptococcus mutans by combined gas-liquid chromatography-mass spectrometry. A modified method is described for the analysis, by gas-liquid chromatography, of various sugars as the trimethylsilyl derivatives of their methyl glycosides. The technique was employed for the analysis of the cellular carbohydrate of Streptococcus mutans NCTC 10832 and provided reproducible fingerprints, consisting of peaks due to glycerol, rhamnose, xylose, galactofuranse, glucose, N-acetylglucosamine and N-acetylmuramic acid. Absolute identification of the latter was by combined gas-liquid chromatography--mass spectrometry.


Determination of morphine and its major metabolite, morphine-3-glucuronide, in blood by high-performance liquid chromatography with electrochemical detection. The construction of an electrochemical detector flow cell for high-performance liquid chromatography from stainless steel, which also serves as the auxiliary electrode, is described. Glassy carbon is used for the working electrode in a novel configuration based on a hybrid of the wall-jet and thin-layer arrangements. Application of the detector to the determination of morphine in small blood samples is described. The metabolite, morphine-3-glucuronide, is also determined by hydrolysis with beta-glucoronidase prior to extraction. The detector is capable of detecting less than 1 ng of morphine injected.


Determination of zomepirac in plasma by high-pressure liquid chromatography. A sensitive, specific and precise high-pressure liquid-chromatographic method for the determination of the analgesic agent, zomepirac, in plasma samples is described. The lowest concentration of zomepirac that can be measured accurately and precisely (coefficient of variation less than 20%) in a 2 ml plasma sample is 10 ng/ml. The standard curve is linear in the concentration range of 10 to 5000 ng/ml. To date, this procedure has been employed successfully in analysing over 10,000 clinical plasma samples.


Paired-ion reversed-phase high-performance liquid chromatography of phenol sulfates in synthetic mixtures, algal extracts and urine. Phenol sulfate esters have been analyzed by paired-ion reversed-phase high-performance liquid chromatography. The method provided direct, rapid chromatography of phenol sulfates in crude extracts of the red alga (Polysiphonia lanosa (2,3-dibromo-4,5-dihydroxybenzyl alcohol 1',4-disulfate), of the brown alga Ascophyllum nodosum (1,2,3,5-tetrahydroxybenzene 2,5-disulfate), and in rat urine (resorcinol mono- and disulfates). Detector response (254 nm) was linear within the approximate range from 30--125 ng to 5--10 microgram. Semipreparative scale chromatography provided sufficient amounts of purified phenol sulfates for further analysis by paper electrophoresis.


Application of a high-performance liquid chromatographic fluorescence method for the rapid determination of alpha-tocopherol in the plasma of cattle and pigs and its comparison with direct fluorescence and high-performance liquid chromatography-ultraviolet detection methods. High-performance liquid chromatography is used to develop a sensitive method for the determination of tocopherol levels in the plasma of cattle and pigs. This method is compared with a similar method using UV detection and one using direct fluorescence determination of tocopherol. Finally a double injection technique used in conjunction with fluorescence detection is shown to enhance the rate of analysis of the tocopherol levels in bovine plasma extracts.


Rapid and simple determination of aflatoxin M1 in milk in the low parts per 10(12) range. A method for extracting aflatoxin M1 from milk is proposed in which the use of disposable Extrelut clean-up columns simplifies the analysis considerably in comparison with existing methods. The quantitative determination is based on one-dimensional thin-layer chromatography and fluorescence densitometric measurement. The detection limit is 5 ppt (parts per 10(12)) in milk and the recovery is 78 +/- 4% at a level of 50 ppt.


Application of pattern recognition and feature extraction techniques to volatile constituent metabolic profiles obtained by capillary gas chromatography. The applicability of threshold logic units, a form of nonparametric pattern recognition, to the processing of metabolic profile data obtained by high-efficiency glass capillary column gas chromatography has been investigated. The test data included profiles of the volatile constituents of urine from normal individuals and from individuals with diabetes mellitus. A feature extraction algorithm allowed for dimensionality reduction and indicated the constituents most important in the normal versus pathological distinction. With an optimum number of dimensions, a normal versus pathological prediction rate of 93.75% was achieved. Gas chromatography-mass spectrometry was utilized to identify important profile constituents.


Quantitative gas-liquid chromatography of neutral sugars in human serum glycoproteins. Fucose, mannose, and galactose as predictors in ovarian and small cell lung carcinoma. A precise and accurate gas-liquid chromatographic (GLC) method has been developed for the quantitative analysis of the neutral sugars L-fucose (6-deoxygalactose), mannose, galactose, and glucose in ethanol precipitates of human serum proteins. The chromatographic conditions and sample preparation resulted in short analysis time (20 min per run) and made routine analyses practicable (twelve samples per day). The alditol acetate derivatization yielded single derivatives for each sugar. Complete separation was achieved on a 2.0 m X 2 mm I.D. column with 2.0% Silar-7CP on Chromosorb W AW 80--100 mesh. The results of hydrolysis showed that the release of fucose and galactose preceded the release of mannose. Hydrolysis with AG 50 W-X8 (H+) ion-exchange resin in 0.5 N HCl at 100 degrees for 7 h optimized glycosidic bond cleavage with only minimal destruction of fucose, mannose and galactose. A combination of strong cation- and anion-exchange resin columns was used to remove chromatographic background of peptides, amino acids, amino sugars, and inorganic ions. An average R.S.D. of less than 4% with recovery of greater than 86% for the three sugars was achieved. The homogeneity of the chromatographic peaks for the neutral sugars of normal human serum glycoproteins was confirmed by GLC--mass spectrometry. Significantly elevated ratios of fucose, galactose, and mannose to serum protein were observed for patients with small cell lung and ovarian carcinomas.


Determination of plasma testosterone by mass fragmentography using testosterone-19-d3 as an internal standard. Comparison with radioimmunoassay. Analytical procedures for the measurement of testosterone by mass fragmentography (MF) using trideuterated testosterone (testosterone-19,19,19-d3) are described. For the calculation of plasma testosterone, peak height ratios were measured by MF performed on the molecular ions of the TFA derivative of testosterone (m/e 480) and testosterone-19,19,19-d3 (m/e 483). The sensitivity of the method was judged from the lower limit of detection of the mass spectrometer which was at 10 pg. For the measurement of the precision, the inter- and intra-assay coefficients of variation (C.V.) were calculated by using a pooled plasma sample; they were 3.15% and 1.79%, respectively. The specificity was investigated by the use of 5 alpha-dihydrotestosterone and the MF method was found to afford a highly selective technique. These results obtained by MF have been compared with the results obtained by a radioimmunoassay method.


Rapid method for the assay of 4-aminobutyric acid (GABA), glutamic acid and aspartic acid in brain tissue and subcellular fractions. The thin-layer electrophoretic separation at pH 4.8 of brain extracts and a procedure for fluorescent staining of the plates with fluorescamine are described for the rapid routine determination of 4-aminobutyric acid (GABA), glutamic acid and aspartic acid in brain extracts and in particulate fractions of brain tissue. Automated sample application, electrophoretic separation using two chambers, and quantitation by in situ fluorescence scanning allows the assay of 280 samples within three working days. The method is reproducible (S.D. less than 8% of the mean) within the range of 0.2--2 nmole per spot. The staining procedure can be applied to a variety of related analytical problems. The method has proved useful for the determination of the specific radioactivities of GABA, glutamic acid and aspartic acid in metabolic studies.


The role of spectral selectivity in fluorescence detection for liquid chromatography. The use of a cut-off filter has been compared with a monochromator for emission energy selection in the fluorescence detector used in the analysis of environmental samples for polycyclic aromatic hydrocarbons by liquid chromatography. The use of a cut-off filter can provide greater sensitivity for compounds that are well separated by the chromatography. However, samples that contain a large number of compounds often result in overlapping peaks, and for such samples, it has been found that the spectral selectivity of monochromatic emission selection reduces the interference due to compounds eluting near the compounds of analytical interest. This spectral selectivity results in greater sensitivity and a smaller quantitation error in the analysis of these complex samples.


A comparison of differential pulse and d.c. amperometric detection modes for the liquid chromatographic determination of oxalic acid. Oxalic acid determinations are made using two modes of electrochemical detection, namely, classical d.c. and differential pulse, after separation by ion-pair, ion exchange high performance liquid chromatography. Peak height ratio plots are constructed and compared for uric acid and oxalic acid mixtures using both electrochemical detection modes. The enhanced selectivity realized by use of the differential pulse electrochemical detection mode is demonstrated.


Use of borohydride reduction in the separation of estrogen carbonyls. The chromatographic behavior of the estrogen carbonyls, equilenin, equilin, and estrone, and their respective sodium borohydride reduction products has been studied. The separation of the 17 alpha-hydroxy reduced compounds is superior to the separations achieved for the parent carbonyls using both reversed-phase and normal-phase systems. The reductions appear quantitative by the chromatographic systems used but other work indicates that a small quantity of 17 alpha-hydroxy isomer is produced. The separations developed here have been used in the identification of 9-dehydroestrone, an impurity in estrone, and in the identification of the estrogens in a commercial aqueous suspension of estrogenic substances.


Determination of chlorinated phenols in surface-treated lumber by HPLC. A solution of internal standard in acetonitrile is used to extract samples of wood from lumber which had been commercially treated with chlorinated phenols. The tetrachlorophenol(TCP) and pentachlorophenol( PCP) were analytically separated from each other and from the other wood extractive compounds using a high performance liquid chromatograph (HPLC). The ultraviolet (UV) absorptions of the TCP, PCP, and internal standard were automatically measured as they eluted. The UV absorption peaks were integrated, and the amounts of TCP and PCP present were calculated with a dedicated microcomputer. Compared with the method previously used, this method is faster (up to 144 samples per 48 hours), has the same accuracy and precision, and it is linear over the concentration range used.


The use of glass capillary columns for food and essential oil analysis. Foods and essential oils represent complex mixtures whose components frequently embrace a wide range of functional groups and volatilities. The analyst concerned with these products may be interested in overall separation or "fingerprinting," in correlating the amount of individual compounds with specific flavor notes, in the detection and measurement of specific substances such as nitrosamines or pesticides, or in following the progress of a biochemical reaction. Analytical speed and the sensitivity of the method may also be important, as well as the ability of reactive compounds to survive the analysis. Toward all of these goals, glass capillary gas chromatography offers distinct advantages.


The determination of oxalic acid in urine by high performance liquid chromatography with electrochemical detection. A system for the determination of oxalic acid in human urine using ion exchange-ion pair, high performance liquid chromatography with electrochemical detection (HPLCEC) is described. Urine is acidified with HCl, and excess CaCl2 is added to precipitate oxalate ion. The precipitate is isolated, redissolved in dilute sulfuric acid, and separated on a strong cation exchange column using an acetic acid-solium acetate-tetrabutylammonium tetrafluoroborate mobile phase adjusted to pH 2.8. Using an electrochemical detector at 1.25 volts vs. the saturated calomel electrode (SCE), oxalic acid exhibits a linear dynamic range from 1 to 1000 mg/liter with a detection limit of 0.1 mg/liter. Quantitative data are obtained by the method of standard addition in the clinically significant range from 5 to 40 mg/liter. Percentage recovery for spiked urine samples was 97.8% with a relative standard deviation of 2.5%.


Yersinia intermedia: temperature-dependent bacteriocin production. Yersinia enterocolitica and related Yersinia species share many temperature-dependent biological attributes. The present report documents temperature-dependent production of a bacteriocin-like substance only among Yersinia strains which ferment L-rhamnose, raffinose, and melibiose and which have been tentatively designed Y. intermedia. When tested by the "lawn-spotting" technique at 25 and 37 degrees C, 7 of 15 Y. intermedia strains produced a bacteriocin only at 25 degrees C with a uniform spectrum of activity against 2 strains of Y. enterocolitica, 1 rhamnose-positive, raffinose-negative, melibiose-negative strain tentatively designated Y. frederiksenii, 8 Y. intermedia, and 3 sucrose- and acetylmethylcarbinol-negative yersinial isolates tested. Bacteriocin-like activity was not detected among the Y. enterocolitica, Y. frederiksenii, or sucrose-negative yersinial strains tested. The exclusive activity of the Y. intermedia antibacterial substance only against yersiniae and not against other representative Enterobacteriaceae tested supports the placement of these microorganisms within the genus Yersinia and further establishes the singularity of Y. intermedia.


Differentiation of Peptococcus and Peptostreptococcus by gas-liquid chromatography of cellular fatty acids and metabolic products. Gas-liquid chromatographic (GLC) profiles of cellular fatty acids and metabolic products were useful in identifying strains of Peptococcus saccharolyticus, Peptococcus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus micros, and Streptococcus intermedius. The GLC results supported the recent taxonomic decision to transfer aerotolerant Peptostreptococcus species to the genus Streptococcus. Because inconsistencies in the results prevented our differentiating Peptococcus prevotii. Peptococcus magnus, and Peptococcus variabilis by GLC, additional strains will have to been examined. These GLC techniques are amenable to routine use; however, for interlaboratory results to be meaningful, the classification and nomenclature of the anaerobic gram-positive cocci should be standardized.


Comparison of direct and standard antimicrobial disk susceptibility testing for bacteria isolated from blood. To determine the reliability of early antimicrobial susceptibility testing, we compared the results of direct and standard single-disk diffusion methods for 581 positive blood cultures processed routinely by the clinical microbiology laboratory. The direct procedure differed from the standard one only in that the 0.5 McFarland inoculum was prepared from 1 ml of turbid broth rather than five isolated colonies from a subculture plate. A major discrepancy in results was defined as a change from susceptible to resistant or vice versa according to interpretive standards for zone diameters, whereas a minor discrepancy was defined as a shift to or from the intermediate category when paired direct and standard tests were compared. The overall agreement between the two methods was 94.6% of 2,308 comparisons. There were 119 minor (5.2%) and 6 major (0.3%) discrepancies. The major discrepancies were seen with three strains of Staphylococcus epidermidis and one strain each of S. aureus, Escherichia coli, and Enterobacter sp. Direct susceptibility testing of positive blood cultures that were pure by gram-stained smear provided reliable results 24 to 36 h earlier than conventional procedures; therefore, we recommended this procedure to guide early antimicrobial therapy in patients with bacterial sepsis.


Longitudinal survey of the distribution of various serotypes of Streptococcus mutans in infants. The establishment of various serotypes of Streptococcus mutans was studied serologically in plaque samples collected from label surfaces of upper primary incisors of 22 infants (starting age, 5 to 13 months) over a period fo 30 months. Clinical examinations were also performed. No clear-cut association between the initiation of dental caries and previous detection of S. mutans was noted. However, all 12 of the infants with caries had S. mutans isolated at some time during the course of this study. The most common serotype isolated at the initial establishment of S. mutans on the tooth surfaces was serotype c, whereas types d, e, and g became established in a few cases. During the test period, changes in the distribution of serotypes of S. mutans were observed in some cases. The initiation of carious lesions could be found in a few cases even when S. mutans comprised about 1% or less of the total streptococcal count of the specimen from the tooth surfaces. Serotype d/g strains tended to develop carious lesions on smooth surfaces, although serotype c was isolated from almost all individuals who developed caries.


Rapid differentiation of Streptomyces from Nocardia by liquid chromatography. A liquid chromatographic method is described for analysis of aerobic actinomycetes for isomers of diaminopimelic acid. One or two colonies of organism were hydrolyzed with 6.0 mol of HCl per liter at 121 degrees C for 15 min. The hydrolysate was neutralized and buffered with an NaOH solution (3 mol/liter) containing 0.15 mol of sodium borate per liter. Precolumn derivatization with dansyl chloride was used to form a fluorescent product for detection. Analysis was performed by reversed-phase, ion-pair chromatography. The L-diaminopimelic acid isomer was detected in all 10 strains of Streptomyces tested, and the meso-diaminopimelic acid isomer was detected in all 10 strains of Nocardia tested. Liquid chromatography was compared simultaneously with thin-layer chromatography in the analysis of three strains of aerobic actinomycetes. Liquid chromatography required less growth of the organisms, and analysis was completed within 1 h, compared with the 3 to 5 days required by thin-layer chromatography.


Immunoglobulin A antibodies reactive with Streptococcus mutans in saliva of adults, children, and predentate infants. Immunoglobulin A (IgA) antibodies reactive with Streptococcus mutans MT3 cells (serotype c) were sought, using an indirect enzyme-linked immunosorbent assay, in the saliva of humans who either harbored or did not harbor detectable levels of this organism. Samples of unstimulated whole saliva from three adults and one child who were infected with S. mutans contained IgA which bound to MT3 cells. Saliva samples of two adults studied also contained IgA which reacted with S. mutans strains of serotypes e, g, a, and b, the latter two of which are rarely isolated from humans. The saliva of three children who did not harbor detectable levels of S. mutans and of three of five predentate infants also contained IgA reactive with MT3 cells. The latter observation is of special interest since S. mutans does not colonize the mouth before eruption of teeth. Thus, the presence of salivary IgA reactive with S. mutans cells is not necessarily related to present or past infection by this organism. Absorption with MT3 cells markedly reduced the reactivity of adult saliva without greatly altering the total concentration of IgA present; this suggests that the IgA was not binding to S. mutans MT3 cells via Fc receptors. The possibility that the antibodies which reacted with S. mutans MT3 may have been induced to other bacteria with cross-reactive antigens was supported by the finding that absorption of saliva with mixed bacterial growth derived from common dairy products significantly reduced its reactivity. Absorption experiments further suggested that a significant portion of the salivary IgA antibodies was binding to glucans on the cell surface.


Synonymy of strains of Center for Disease Control group DF-1 with species of Capnocytophaga. Of eight strains of Center for Disease Control group DF-1 examined, seven had 62 to 87% deoxyribonucleic acid homology with the neotype strain of Capnocytophaga ochracea and one had 72% deoxyribonucleic acid homology with the type strain of C. gingivalis. Deoxyribonucleic acid homology of four strains of Bacteroides ochraceus with the neotype strain of C. ochrecea was 76 to 86%.


Detection, identification, and comparison of Capnocytophaga, Bacteroides ochraceus, and DF-1. Working independently, three laboratories had recognized considerable similarity among certain strains of dysgonic, fermentative, capnophilic, surface translocating, gram-negative bacilli referred to as Capnocytophaga, Bacteroides ochraceus, and Center for Disease Control biogroup DF-1. To determine the relationship among these groups, 21 strains were exchanged and independently characterized by the three laboratories. Additionally, a fourth laboratory examined the deoxyribonucleic acid homologies of the same strains. Using methods common to dental microbiology, eight of the strains had been isolated from the gingival sulcus and periodontal lesions and identified as Capnocytophaga. Three strains isolated from blood and transtracheal aspirate had been characterized by conventional anaerobic methods and recorded as B. ochraceus. Ten strains isolated from sputum, blood, throat, spinal fluid, and tracheal aspirate had been identified as DF-1 with the methods of E. O. King and a buffered single-substrate technique. All strains were similar in respect to colonial and microscopic morphology, surface translocation, biochemical features, gas-liquid chromatograms of metabolic end products, and deoxyribonucleic acid composition. We conclude that these biogroups should be termed Capnocytophaga species.


Plate assay for detection of Leptospira interrogans serovar pomona hemolysin. A plate assay which utilizes the addition of a blood agar overlay for detection of hemolysin by colonies of Leptospira interrogans serovar pomona is described.


Survival of Ureaplasma urealyticum on different kinds of swabs. The survival of Ureaplasma urealyticum on five different types of swabs routinely used for obtaining clinical specimens is limited to 1 h. Wooden applicator sticks inhibit the alkaline color change in broth and result in a significant loss of isolates if broth alone is used.


Sulfones and sulfonamides in dermatology today. Although dapsone and sulfapyridine have been used for years in dermatology, there has not always been a clear understanding of how these agents work. Recent investigation, however, has shed new light on thes agents which now allows a more rational approach to their use. This review is an attempt to familiarize the clinician with how these agents work, in what disease states they are effective, how to administer them, what adverse effects may occur, and how to monitor the patient to detect these adverse effects.


Heck's disease (focal epithelial hyperplasia). A patient and his mother noted the insidious appearance of a distinctive benign papular eruption confined to the mucosa of the lips. The clinical pattern and histologic findings fulfill the criteria of a recently described entity, focal epithelial hyperplasia. An electron microscopic search for viral particles was negative. Arguments for both a viral and a hereditary pathogenesis have been put forth, but the etiology of the condition remains controversial. This disorder may be more frequently reported as dermatologists become increasingly aware of its existence.


Eruptive nevocytic nevi following erythema multiforme. A patient with erythema multiforme developed many nevocytic nevi, including a large cluster of such lesions at the sites of healed blisters. The development of eruptive nevocytic nevi in this pattern suggests that cutaneous injury plays a role in their pathogenesis.


Bowen's disease and 5-fluorouracil.

Bowen's disease and 5-fluorouracil. Sixty-six confirmed cases of Bowen's disease (intraepidermal squamous cell carcinoma) were seen from 1965 through 1976: twenty-four cases were treated surgically-four by excision (two recurrences) and twenty by desiccation and curettage (four recurrences); one was treated with x-ray radiation; and forty-one were treated with topical 5-fluorouracil (5-FU) (three recurrences). Of the six surgical recurrences (25% recurrence), one was retreated by desiccation and curettage and five with 5-FU. Of the three recurrences after 5-FU (8% recurrence), all three were successfully retreated with 5-FU. In view of the generally high recurrence rate after all treatment modalities in Bowen's disease, related to both the ill-defined lateral margins and the frequent follicular involvement with the Bowen's process, prolonged treatment time is required for success with 5-FU. Treatment times employed were 4 to 12 weeks, with a median of 9 weeks. 5-FU concentration ranged from 1% to 3 % in propylene glycol. A 1% concentration of 5-FU was generally adequate on the face, except for occasional recalcitrant areas, such as the nose and eyebrow. A concentration of 2.5% to 3.0% was usually necessary for treatment of Bowen's disease on the other body areas.


Localized acquired cutaneous pseudoxanthoma elasticum. Localized acquired cutaneous pseudoxanthoma elasticum is a new designation proposed for a nonheritable form of pseudoxanthoma elasticum (PXE), lacking the retinal and vascular stigmata associated with the inherited form of the disorder, but having skin lesions that are clinically, histologically, and ultrastructurally similar to those seen in the inherited type. A patient is presented with skin lesions confined to the chest and abdomen. Similar cases from the literature, some apparently idiopathic in multiparous black females and others with histories of cutaneous exposures to calcium salts, are grouped together as examples of the acquired form of PXE.


Acanthosis nigricans presenting as hyperkeratosis of the palms and soles. A patient with an unusual form of palmoplantar hyperkeratosis is described. On thorough physical examination, widespread acanthosis nigricans was discovered. We believe this patient's distinctive keratoderma represents acanthosis nigricans.


Malignant neoplasms associated with nevus sebaceus of Jadassohn. Nine patients with nevus sebaceus of Jadassohn (NSJ) developed associated morphologically aggressive neoplasms. Four of the tumors were apocrine carcinomas, two of which metastasized to regional lymph nodes, and in one of the two generalized metastases developed. Three tumors were adnexal carcinomas with probable pilar differentiation, but none showed progression of the disease after wide surgical excision. One patient has squamous cell carcinoma and died with generalized metastasis, and one patient had a complex adnexal and squamous cell carcinoma with a large area of anaplastic carcinoma that recurred and invaded the skull.


Influence of bromocriptine on plasma levels of prolactin and steroid hormones in the 20th week of pregnancy. The influence of bromocriptine, a prolactin antagonist, on maternal plasma and amniotic fluid prolactin (PRL) was investigated in two pregnancies at the 20th week with medical indication for abortion. Voluntary consensus of the patients was obtained. Blood sample determinations demonstrated that bromocriptine inhibits the secretion of PRL both in plasma and amniotic fluid. Since no changes were observed in peripheral maternal steroid concentrations of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, delta 4-androstene-3, 17-dione, testosterone, estradiol, estriol, and cortisol, it is concluded that PRL does not seem to affect maternal and fetal adrenal cortex as supposed in amenorrhoic patients with hyperprolactinemia.


Correlation of platelet taurine levels with thyroid function. Changes in neurotransmitter or neuromodular activity and levels have been thought to be responsible for some of the behavioral characteristics observed in patients with thyroid disease. To investigate these relationships, taurine, a proposed inhibitory neurotransmitter or modulator, was measured in platelets, which may serve as biochemical model for synaptosomes. Blood of euthyroid, hypothyroid and hyperthyroid patients was collected. Thyroxine was determined by RIA. Platelets were separated using differential centrifugation techniques. The taurine in platelets was separated by ion exchange chromatography and was assayed spectrophotometrically as a product produced by coupling with dinitrofluorobenzene. In hypothyroid, untreated hyperthyroid and euthyroid individuals, taurine/platelet and number of platelets/ml of blood were inversely related. In propranolol-treated hyperthyroid patients, such a relation was not found. Following initial treatment with either antithyroid drugs (i.e. methimazole or propylthiouracil) and/or propranolol, taurine/platelet returned to euthyroid levels while plasma thyroxine levels remained elevated. These data implicate a relationship between taurine in platelets and the thyroid state in man.


Elevated blood pressure after pinealectomy in the rat. Previous observations have suggested a role for the pineal gland in regulation of blood pressure. To investigate this possibility, pinealectomy was performed in prepubertal (43-day-old) and postpubertal (55-day-old) male rats. Systolic blood pressure (BP) was measured by the tail cuff method in conscious pinealectomized (Px) and sham pinealectomized (ShPx) rats at various times. In the 3 weeks following surgery, the BP increase after Px was twice that after ShPx (p less than 0.05). During administration of 1% saline as the sole fluid source from day 96-123, hypertension (BP greater than 150 mmHg) was observed in those animals Px at day 43 (p less than 0.05 vs ShPx) but not in those Px on day 55. Moreover, saline consumption was increased in the hypertensive group compared to ShPx controls. There was no significant difference in mean body weight or heart rate between Px or ShPx groups. These data suggest that pinealectomy in the prepubertal period may result in a tendency to hypertension, measurable in conscious rats, which is unmasked by saline administration.


Study of bioassayable somatomedin on renal artery and vein. Somatomedin was measured by rat bioassay in samples obtained from renal artery and vein, in order to study the role of this organ in the production of somatomedin activity. The results showed a significant difference, with an increase of the activity in the venous samples. According to the data reported in the literature with bioassay (low values) and radioreceptor assay (normal or high values) in nephropathic subjects, our results seem to suggest that the kidney mainly inactivates a specific somatomedin inhibitor.


Detection of candida antigenemia by counterimmunoelectrophoresis in patients with invasive candidiasis. Because of the difficulty in diagnosis of invasive candidiasis, an assay that makes use of counterimmunoelectrophoresis (CIE) was developed to detect candida antigen. A cell-wall polysaccharide of Candida was extracted with hot formamide, ethanol precipitation, and gel filtration. Rabbit antiserum was tested by CIE against sera from 20 healthy individuals, 15 patients with mucosal candidiasis, and 48 compromised patients with cultures positive for Candida. Antigen was detected in sera from 13 patients by CIE; eight of these patients were eventually proven to have invasive disease. None of the antigen-negative patients for whom autopsy was performed had evidence of invasive candidiasis. Healthy individuals and patients with superficial mucosal candidiasis were also antigen-negative. The detection of candida antigenemia by CIE is specific for invasive disease and serves as a guide in initiation and follow through of antifungal therapy.


An acute rise in serum phosphorus after single-dose oral metyrapone. Metyrapone, an 11 beta-hydroxylase inhibitor, is widely used as a pharmacologic test for ACTH reserve. During studies on the pharmacodynamics of metyrapone and the response of plasma 11-deoxycortisol and cortisol, we fortuitously noted a consistent change in serum phosphate but no other routine laboratory chemistry determination. For these studies 12 control subjects were given oral methyrapone (30 mg/kg) at midnight. Serum phosphate (P) concentration was measured in fasting subjects at 08:00 on the morning before and after the administration of metyrapone. In 11 of 12 subjects, serum P increased from a mean of 3.6 +/- 0.7 (SD) mg/100 ml to a mean of 4.2 +/- 0.07 (SD) mg/100 ml on the morning following the drug. No increase in P was noted following the same dose of metyrapone in four patients on acute or chronic steroid therapy or in patients with primary or secondary hypoadrenocorticism. In addition, two addisonian patients were given either a saline or a hydrocortisone infusion for four hours. While the hydrocortisone infusion resulted in a drop in serum P, there was no decrease during saline infusions. These findings suggest that an acute rise in serum P after a single dose of metyrapone might be a rapid indicator of adrenocortical blockade leading to increased ACTH secretion, and cortisol and/or ACTH might play a role in P homeostasis.


Gene duplication at an isocitrate dehydrogenase locus in Scaphiopus. Spadefoot toads of the subgenus Scaphiopus have two isocitrate dehydrogenase loci, with no intergenic interaction between them. Toads of the subgenus Spea have three Idh loci, with intergenic enzymes formed between two of them, providing strong evidence for their homology and the origin of one through a duplication process. The Idh phenotype of interspecific hybrids is consistent with the theory of a gene duplication.


Evaluation in humans of a new, inactivated vaccine for Venezuelan equine encephalitis virus (C-84). A new, formalin-inactivated vaccine for Venezuelan equine encephalitis (VEE) virus (C-84), prepared from an attenuated vaccine strain of virus (TC-83), was tested in humans. Only occasional, mild, local and systemic reactions were noted in 28 volunteers; no meaningful changes in clinical laboratory values occurred. The vaccine augmented preexisting titers of serum neutralizing antibody to VEE virus in seropositive recipients of TC-83 vaccine, and it induced high titers of neutralizing antibody in nonimmune subjects after one primary and two booster vaccinations. Circulating antibody persisted for at least 14 months in these persons. The neutralizing antibody produced after one dose of C-84 vaccine in immune subjects and after booster doses in nonimmune subjects had broad cross-reactivity within the VEE virus complex. The C-84 vaccine induced a VEE virus-specific lymphocyte transformation response. The vaccine was safe, and immunologic results showed it to be highly antigenic in healthy immune and nomimmune adults.


Protective efficacy of immunization with capsular antigen against experimental infection with Bacteroides fragilis. The protective efficacy afforded by immunization with the capsular antigen of Bacteroides fragilis against abscess formation and bacteremia due to this organism was studied in an experimental rat model of intraabdominal sepsis. Of unimmunized animals, animals immunized with methylated bovine serum albumin and complete Freund's adjuvant, and animals immunized with lipopolysaccharide of Bacteroides thetaiotaomicron, greater than 90% developed abscesses when challenged intraperitoneally with strains of B. fragilis or Bacteroides distasonis (given with an enterococcus) or with the cecal contents of meat-fed rats. In contrast, animals immunized with B. fragilis capsular polysaccharide, given with or without methylated bovine serum albumin and complete Freund's adjuvant, and animals immunized with the outer membrane of B. fragilis strain 23745 were protected to a significant degree from abscesses caused by challenge with B. fragilis or B. distasonis. Such immunization had no overall effect on the development of abscesses in animals challenged with the entire cecal contents of meat-fed rats; however, B. fragilis was eliminated from the abscesses of these animals. Animals immunized with the capsular polysaccharide were protected from early B. fragilis bacteremia.


Adverse effects of nosocomial infection. The effects of noscomial infection on duration of hospital stay and outcome of hospitalization were measured by matching two control patients to each of 85 patients found to have qctive nosocomial infection during a prevalence survey at Boston City Hospital in 1973. The control patients were selected from all patients discharged from this hospital during the same time period; they were matched by exact primary discharge diagnosis, similar operative procedure, and age. Patients with a single infection remained in hospital on average 13.0 days longer than their matched controls, and those with two such infections stayed on average 35.4 days longer. This effect of extra stay associated with nosocomial infection was consistent when data were stratified by primary discharge diagnosis, hospital service, site of infection, or outcome of hospitalization. The outcome of hospitalization for these infected patients was slightly, but not significantly, worse than for their matched controls.


Development of a statewide program for surveillance and reporting of hospital-acquired infections. In 1974, a statewide program was begun to improve surveillance of nosocomial infection in Virginia hospitals. Infection control practitioners were trained at the University of Virginia Hospital, Charlottesville, and were encouraged to submit monthly surveillance reports for analysis. In the first three years of the project, 141 students from 65 hospitals within the state attended a two-week basic course, with eight to 10 students per class. Of the 98 Virginia hospitals that sent students, 75 (73%) submitted monthly reports. The consistency of reporting (number of monthly reports received divided by the number of possible reporting months) was 83%. The sensitivity of reported data was estimated in comparative daily prospective surveys to be 69% for participating hospitals, and the specificity was 99%. The crude infection rate for the first 1.1 million patients at risk was 3.3%.


Lack of specific effect of adenine arabinoside, human interferon, and ribavirin on in vitro production of hepatitis B surface antigen. PLC/PRF/5 hepatoma cells continue to produce hepatitis B surface antigen (HBsAg) after greater than 80 passages in vitro, but they do not express other markers of heaptitis B virus (HBV) replication. In this respect, they resemble most liver cells that are persistently infected with HBV. PLC/PRF/5 cells were cultured in the presence of adenine arabinoside, human fibroblast interferon, and ribavirin to determine whether production of HBsAg was sensitive to these antiviral agents. HBsAg released into culture media was detected by radioimmunoassay, and cellular protein synthesis was assessed by [3H]amino acid incorporation studies. A dose-related inhibition of HBsAg occurred with each antiviral agent that was tested, but in each case, this inhibition was matched by a reduction of cellular protein synthesis to a similar degree. Thus, no specific effect on the production of HBsAg was found with any of the antiviral agents tested.