Competition radioimmunoassay for mason-pfizer monkey virus: comparison with recent isolates. The major core protein of Mason-Pfizer monkey virus was purified by DEAE ion exchange column chromatography and shown to be 27,000 daltons (p27). Following the characterization of monospecific antisera prepared against p27, a radioimmunoassay was developed with these reagents and competition experiments were done with come of the recent M-PMY-like isolates as well as with other oncornaviruses. Results suggest that three of the viruses tested, AO, X-381 and FTP-1, are similar to M-PMV while J-96 virus is related, but not identical, to M-PMV. It is also shown that competition RIA can be used successfully to detect the presence of viral proteins in tissue homogenates and cell extracts.


Demonstration of acid mucopolysaccharides in the trabecular meshwork of the Rhesus monkey. The ability to demonstrate AMPS in the trabecular region in the normal eye of the Rhesus monkey was shown to be critically dependent upon technical variation. Staining the fixed specimen prior to dehydration and embedding permits the uniform demonstration of AMPS in the trabecular region of the Rhesus monkey and shows it to be hyaluronidase-sensitive. Electron microscopy using the modified technique shows the reaction products to be present within the trabecular band, the intertrabecular spaces, and the canal of Schlemm. More impressive distribution was seen in the basement membrane of trabecular endothelium intimately related to the cell wall and in the ground substance and basement membrane of the endothelium of the  inner wall of the canal Schlemm. The technique is also successful in the human eye and suggests a greater abundance of trabecular AMPS in open-angle glaucoma.


Early cytologic changes of Fraser cataract. An electron microscopic study. The cataractous lens of the Fraser strain mouse was studied electron microscopically in its early developmental stage. Cytologic abnormalities were found in the cells of the invaginating ectodermal cell of the early optic cup. Morphogenetically degenerating cells were more numerous in this stage. In general, cells of the posterior layer of the developing lens elongated normally. However, they contained great numbers of dilated rough endoplasmic reticulum, vacuoles, and mitochondria, but less amounts of crystalline substance and polysomes. During the further differentiating stage (thirteenth and fourteenth fetal day), apical portions of these cells began to swell and the lens became opaque. The swollen lens cells degenerated after this stage.


Immunologic evaluation of human renal cell carcinoma. In vitro studies. Attempt were made to initiate cell lines from 11 specimens obtained from nine patients with renal cell carcinoma. Primary cultures were obtained in seven instances with only five long term cells lines. Two of these cell lines were obtained from metastatic tumors in two patients. Using microcytotoxicity assay, both autochthonous and allogeneic lymphocytotoxicity, specific to renal cell tumor, was demonstrated. This would suggest a common cross-reacting tumor-associated antigen. No lymphocytotoxicity could be demonstrated using autochthonous lymphocytes aganist two metastatic tumor target cell lines. This would suggest some antigenic differences between primary tumor and its metastases. In seven instances significant complement-dependent cytotoxicity was demonstrated using six different renal cell carcinoma target cell lines. Serums from three patients with renal cell carcinoma, one without any recurrent tumor and two with metastases, appear to significantly block the autochthonous and allogeneic lymphocyte cytotoxicity.


The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hydroxytryptamine in whole mounts. The reactions of glyoxylic acid with peripheral stores of noradrenaline and 5-hydroxytryptamine to provide a fluorescence histochemical method for their localization have been investigated. Incubation in glyoxylic acid, followed by drying and heating of whole mount preparations gives an intense and well localized reaction. For incubation, a concentration of 2% glyoxylic acid, buffered to pH 7 at room temperature for 30 minutes gives ideal results. The method is equally good if the pH is varied in the range 6 to 9 or if the tissue is stored in the incubation mixture for up to 6 hours. Ideal development of the fluorophore requires an initial excess of moisture in the tissue, that this moisture is driven off during development, and that the tissue is protected from further moistening. A suitable method of achieving these ends is to heat partially dried tissue at 100 degrees C for 4 minutes and then cover it with paraffin oil. 5-hydroxytryptamine can be readily distinguished from noradrenaline because it forms a fluorophore after reaction at pH 3.5, whereas noradrenaline does not. Both amines can be visualized after incubation at neutral pH. Comparison with the formaldehyde vapour technique reveals three main advantages (and no disadvantages) of the glyoxylic acid method: (1) it gives a finer localization with higher fluorescence yield, (2) the glyoxylic acid method is less susceptible to variations in procedure and, (3) it is both simpler and quicker to apply.


The histochemistry and structure of tentacle cartilage tissues in the marine polychaete, Sabella melanostigma. The cartilages (or "chondroid" tissue) in tentacles of the polychaete annelid, Sabella melanostigma, have been examined by electron microscopy and a series of histochemical techniques for the demonstration of mucopolysaccharides and protein end-groups. The ultrastructural studies indicated that the cartilages possess an investing layer of dense connective tissue which differs significanly from the matrix material secreted between the chondrocytes. The cartilage matrix was positive for acidic mucins with levels of sulfation above those of mammalian chondroitins A and C. This matrix as well as the investing connective tissue were intensely PAS-positive. Sabella cartilage was also stained intensely by methods for demonstrating tryptophan, tyrosine, side-chain carboxyl groups, disulfide groups, and amino groups. It was not stained by the procedure for sulfhydryl groups. Some evolutionary aspects of cartilage and chondroid tissues were discussed.


A study of DNA depolymerisation during Feulgen acid hydrolysis. The binding of Schiff dye molecules after acid hydrolysis (1 M HCl) for varying lengths of time was studied in ascites tumour cells. The amount of dye bound to the tumour cells closely followed the number of aldehyde groups, calculated from the extraction of radioactive nucleotides. This constant dye to aldehyde ratio did not change when the hydrolysis was performed at a lower acid concentration (0.3 M HCl). The conclusion drawn is that Feulgen dye measurements represent, in a constant way, the number of aldehydes on DNA at any given time during hydrolysis. The alteration of the hydrolysis pattern of chromatin fixed in formalin was found to be due to a slower extraction of DNA depolymerisation products, the purine liberation being unaffected. A similar explanation is offered for the extreme pattern obtained from hydrolysis of bull spermatozoa chromatin.


[Critical study of extra-lysosomal acid phosphatase localizations in thyroid follicular cells by the Gomori reaction (author's transl)]. With the Gömöri technique, lead precipitates have been found in thyroid follicle cells in unusual localizations such as apical hyaloplasm and microvilli; it has been established that they were actually significant for acid phosphatase activity: constant results in spite of repeated controls and several variations from the original cytochemical technique, allow to think that lead precipitates were not merely artefactual, but actually significant of enzymatic activity. However it is pointed to the fact that the origin of the enzyme has to be questioned; it is assumed that most likely acid phosphatase has diffused from its original lysosomal site. Such diffusion implies variations of the selective permeability of lysosomal membranes; inappropriate relation between the quantity of enzyme present in these organelles and the quantity of substrate used might also be considered, though changes in the amount (resp. concentration) of substrate remained ineffective and induced no modification in the localization of observed enzymatic activity. In addition, one point of interest is an obvious relation between the observed enzyme diffusion and the state of activity resp. rest of the cell; in the present state of investigations, this remains unexplained and likely related to factors escaping control during processing; moreover, no explanation can be provided for the fact that it revealed impossible to avoid such diffusion even by means of variations of the numerous parameters involved in the Gömöri technique. So that it finally appears necessary to remain on a critical position regarding the results at the ultrastructural level of this standardized technique, and there is no doubt it would reveal useful that several assumptions in the literature about extra lysosomal acid phosphatase activity should be reinvestigated with a similar critical purpose.


Cytochemistry and distribution of polysaccharides in an electroreceptor: the tuberous organ of Gnathonemus petersii (Mormyrids). The polysaccharides were studied in an electroreceptor organ, the tuberous organ of Gnathonemus petersii (Mormyridae). Histochemical methods (P.A.S., alcian blue, toluidine blue and iron colloidal reactions) allowed us to demonstrate the existence of glycogen in the sensory cytoplasm, and P.A.S. positive polysaccharides in the sensory cavity. The polysaccharides were shown to be amylase proof; they display an acidity due to the existence of sulphated radicals. The histochemical study was completed by a cytochemical analysis: a treatment with thiocarbohydrazide (TCH) according to the Thiery's method. This method allowed us to estimate the glycogen concentration, its localization, and relationship with cellular organites within the sensory cytoplasm, as well as to differentiate the highly glycogenous type II cells of the platform from the other accessory cells (Derbin and Szabo, 1968). After a treatment for 20 hours with TCH, silver stained grains were visible on the polysaccharide filaments of the sensory chamber, between the microvilli and the vacuoles of the epidermal cells lining to the sensory cavity. Silver grains coated the outer surface of the microvilli. Such polysaccharides were not identical to the filamentous polysaccharides of the cavity. In order to determine the cytochemical localization of the polysaccharide acid groups, sections were stained with iron salts. The colloidal iron constitutes a deposit opaque to electrons and located both on the filamentous polysaccharides of the sensory cavity and in the vacuoles of the epidermal cells, indicating that only these filamentous polysaccharides display acid radicals.


Fluorescent demonstration of basement membranes with hydroxy-naphthoic acid hydrazide. The hydroxy-naphthoic acid hydrazide (HNAH) procedure can be employed not only for selective fluorochroming of side chain carboxyl groups of proteins, but also for demonstration of certain exoplasmic structures---particularly basement membranes. The demonstration of basement membranes by the HNAH procedure is likely related to the relatively high levels of available side chain carboxyl groups in the noncollagen glycoproteins of basement membranes.


Myoid fibrils in epithelial cells: studies of intestine, biliary and pancreatic pathways, trachea, bronchi, and testis. Cytoplasmic filaments have been studied extensively by electron microscopy, but the histochemical nature of such fibrils in non-keratinizing epithelia has not been systematically investigated. During studies of early arterial lesions we observed structures with the staining properties of myosins in epithelial cells of various organs. The configurational staining, polarization and fluorescence microscopic properties of these myoid structures were compared with those of myofibrils in smooth muscle and classical myoepithelial cells. The following structures showed the characteristics of myofibrils: the terminal web in columnar epithelial cells of intestine, trachea, bronchi, bile ducts, pancreatic ducts and ductus epididymidis, the pericanalicular layer of bile and pancreatic canaliculi, fibers in the caudal tube of spermatids and the flagella of spermatozoa. Cilia, e.g. of respiratory epithelium, tonofibrils in squamous epithelium and nerve axons did not react. These studies indicate significant histochemical differences between cytoplasmic filaments. Different types of intracellular fibrils can be found in the same cell, e.g. in respiratory epithelium.


The use of glycol methacrylate embedding for studies of chromatin distribution and fine structure in salmon spermatid nuclei. Glycol methacrylate has been used as an embedding medium for studies of spermiogenesis in the salmon. DNA and basic proteins are shown to stain with the same specificity in thick (0.5-1.0 mu) sections of GMA-embedded salmon testes as in sections of comparably fixed, paraffin-embedded testes. Stain can be localized far more precisely in GMA sections than in paraffin sections due to the thinness of the sections and to the excellent structural preservation of nuclei. In addition, ultra-thin sections of GMA-embedded salmon testes can be observed with the electron microscope, and this permits exact correlation between nuclear fine structure and chemical composition in consecutive sections of the same nuclei.


Purification of mouse alpha1-fetoprotein and preparation of specific peroxidase conjugates for its cellular localization. Mouse alpha1-fetoprotein (AFP) was isolated from amniotic fluid by immunoadsorbent columns and preparative electrophoresis. Specific antibodies were isolated from monospecific hyperimmunsera by use of immunoadsorbents, and subsequently coupled with horseradish peroxidase. At the light microscopical level, purified antibody-peroxidase conjugates were used for the cellular localization of AFP in fetal liver by direct and indirect staining methods. Fixatives containing ethanol or aldehydes were tried for antigen staining. Prior to immunocytological reactions, endogenous peroxidases were inhibited by hydrogen peroxide.


Cytochemistry and morphology of synaptic structures in the cerebral cortex of rat. Studies have been carried out on the synapses in the cerebral cortex of rat by using impregnation with ethanolic solution of phosphotungstic acid, contrast staining with ruthenium red and impregnation with bismuth iodide, with or without subsequent uranyl acetate and lead citrate staining. It has been established that dense projections are adequately visualized with methods demonstrating basic chemical groups (phosphotungstic acid and bismuth iodide), whereas the synaptic vesicles are stained by techniques demonstrating acid chemical groups (ruthenium red and uranyl acetate and lead citrate). On the basis of these observations a hypothesis is forwarded concerning the mechanisms of migration of synaptic vesicles towards the presynaptic membrane. Measurements of the parameters of the dense projections suggest that the configuration of the presynaptic vesicular grid is not uniform along the presynaptic areas.


Benzene hexachloride poisoning in cattle. Of 174 cattle dipped in an emulsified preparation of benzene hexachloride labeled for plant use, 18 were fatally poisoned. The preparation contained 0.14% active ingredient, gamma isomer of benzene hexachloride (BHC), a normally safe concentration for cattle. Analyses revealed 0.08% gamma BHC in the used dip and 0.5, 7.9, and 198 ppm in liver, kidney, and hairskin specimens, respectively, from a fatally affected cow. Microscopic examination of the freshly prepared dip demonstrated emulsion droplets ranging from 5 to 60 mu in diameter.


A study of the distribution of chromaffin-positive (CH+) and small intensely fluorescent (SIF) cells in sympathetic ganglia of the rat at various ages. Small intensely fluorescent (SIF) and chromaffin-positive (CH+) cells were independently investigated by formol-induced fluorescence and by chromaffin techniques in the superior cervical, thoracic and coeliac-mesenteric ganglia of neonatal (2--10 days), adolescent (2--4 months) and adult (6--15 months) rats. Identification of CH+ cells was facilitated by glutaraldehyde fixation prior to chromatin. Intraganglionic blood vessels were displaced by antemortem injection of either India ink or the fluorescent dye Thioflavine-S. SIF and CH+ cells were randomly distributed through the ganglia, either singly or in pairs related to principal neurons, or in variably-sized, highly vascularized groups. In chromaffin preparations these groups either consisted entirely of CH+ cells or else they contained a mixture of CH+ and CH- cells. CH+ cells were present in some adolescent and adult ganglia of all types, and in the neonatal coeliac-mesenteric ganglion at 10 days. In neonatal material generally, SIF cells were mostly green fluorescent, occurring separately or in homogeneous or mixed groups, but both yellow and green cells occurred in coeliac-mesenteric ganglia at 10 days. All ganglia in adolescent and older animals contained both yellow and green cells. There were more green than yellow cells, and more SIF than CH+ cells in all ganglia studied.


Collaborative study of a semiautomated method for the analysis of acenocoumarol, phenprocoumon, and potassium warfarin tablets. This collaborative study was undertaken to determine if the anticoagulants acenocoumarol, phenprocoumon, and potassium warfarin could be analyzed by the automated analysis system described in the collaborative study for the analysis of sodium warfarin and dicumarol. Collaborators were supplied with a composited tablet sample of each anticoagulant. Results agreed well with the National Formulary methods for phenprocoumon and potassium warfarin, and an unpublished method for acenocoumarol. For acenocoumarol, coefficients of variation on individual sets of data ranged from 0.30 to 1.94% For phenprocoumon, coefficients of variation ranged from 0.52 to 1.20%. For potassium warfarin, coefficients of variation ranged from 0.54 to 1.79%. The results of this study show that acenocoumarol, phenprocoumon, and potassium warfarin can be analyzed by the official AOAC method for the analysis of sodium warfarin and dicumarol tablets.


Symptom formation as an expression of disordered information processing in schizophrenic children. The possible mechanisms that underlie symptom formation in childhood schizophrenia are discussed. A body of research evidence has been reviewed in which dissociation in relation to information processing was examined for its possible consequence in the formation and expression of symptomatology. Schizophrenic children have been found to exhibit dissociation of integrative processes among the sense systems at a level which is several years below normal expectation, and they usually fail to improve as age increases. The clinical manifestations of schizophrenia are considered to be the consequence of the conflict, distortion, and deprivation that derive from failure in information processing. These consequences can best be understood within a developmental framework which encompasses the different age-stages of function. This approach to the understanding of symptom formation is discussed in relationship to other evidence which suggests that primary neurological abnormality is present in schizophrenic children. Thus the identification of abnormality of intersensory integrative function may increase our understanding of etiology as well as of the mechanisms of symptom formation in schizophrenic children.


Active transport of manganese in isolated membrane vesicles of Bacillus subtilis. Membrane vesicles isolated from cells of bacillus subtilis W23 accumulate manganese in the presence of an energy source. The artificial electron donor system ascorbate and phenazine methosulfate or reduced nicotinamide adenine dinucleotide and phenazine methosulfate can supply the energy for the uptake. D-Lactate in the presence or absence of phenazine methosulfate would not support manganese accumulation. Anaerobiosis, cyanide, m-chlorophenyl carbonylcyanide hydrozone, valinomycin, gramicidin, and p-hydroxy-mercuribenzoate inhibit the uptake. The inhibition by p-hydroxymercuribenzoate is prevented by excess dithiothreitol. Potassium fluoride or sodium arsenate has no effect on the uptake. The manganese transport system in the B. subtilis vesicles exhibits Michaelis-Menten kinetics with a Km of 13 muM and a Vmax of 1.7 nmol/min per mg (dry weight) of membranes. The uptake of manganese is specific and is not inhibited by 0.1 mM CaCL2 or Mgcl2.


Radiocopper in L-tryptophan 2,3-dioxygenase isolated from Pseudomonas acidovorans grown in the presence of 64Cu (II). L-Tryptophan 2,3-dioxygenase (EC 1.13.11.11), isolated from L-tryptophan-induced Pseudomonas acidovorans, ATCC 11299b, which has been grown in a medium containing 64Cu(NO3)2, has been shown to contain radiocopper. At several stages of purification of the enzyme samples were taken, and these were subjected to disc acrylamide gel electrophoresis in the presence of 10 mM L-tryptophan. After electrophoresis the position of the yellow heme band, corresponding to tryptophan oxygenase, was visually located, and the gels were sliced and counted. A large peak of radioactivity was seen to occur at the location on the gel of tryptophan oxygenase no matter what the stage of purification. Treatment of each sample before electrophoresis for 30 min at 37 degrees with gamma-globulins prepared from rabbits sensitized to homogeneous pseudomonad tryptophan oxygenase greatly reduced this peak of radioactivity, whereas treatment of each sample with rabbit preimmune gamma-globulin did not. This direct demonstration of the presence of coper in pseudomonad tryptophan oxygenase, using 64-Cu, avoided the problems and artifacts inherent in the usual techniques of copper analysis and unequivocally refutes the recent contention of Ishimura and Hayaishi ((1973) J. Biol.Chem. 248, 8610-8612) "that copper is not an essential component of L-tryptophan 2,3-dioxygenase of Pseudomonas." The presence of copper in pseudomonad and rat liver tryptophan oxygenases, previously reported by us (Brady, F. O., Monaco, M. E., Forman, H. J., Schutz, G., and Feigelson, P. (P. (1972) J. Biol. Chem. 247, 7915-7922), is reaffirmed by the experiments reported herein.


Postillumination adenosine triphosphate synthesis in Rhodospirillum rubrum chromatophores. II. Stimulation by a K+ diffusion potential. Addition of valinomycin, nonactin, or monactin plus KCl in the dark to preilluminated chromatophores induced the synthesis of a large amount of ATP. This stimulation of postillumination ATP synthesis by a dark-imposed K+ diffusion potential was different from the stimulation caused by addition of permeant anions or cations in the light, since it increases when the pH of the light stage decreased from 8.0 to 6.0. It was thus most pronounced when the chromatophores were preloaded with protons but the light-induced proton concentration gradient (deltapH) was low. Imposition of a Kplus diffusion potential resulted however in stimulation of ATP synthesis even when the light-induced deltapH was already above the threshold value required to initiate postillumination ATP synthesis. This situation was realized when valinomycin plus KCl were added in the dark to chromatophores preilluminated above pH 6.7 with thiocyanate as the permeant anion, and the amount of ATP formed was the sum of the yields obtained with each of these affectors by itself. On the other hand addition of thiocyanate together with valinomycin plus KCl in the dark led to inhibition of ATP synthesis. In this case the permeant anion could not affect the light-induced deltapH but it did eliminate the diffusion potential by decreasing the difference between the permeabilities of Kplus and the anion present in the reaction mixture.


A twenty-one-year review of a case of congenital indifference to pain. A boy aged three with indifference to pain was followed up until his death from amyloid disease some twenty-one years later. A full necropsy was done and the neuropathology suggested a sensory neuropathy.


Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. A rapid method for the flow microfluorometric determination of the DNA content per cell is described. Incubation of cells in a hypotonic solution of propidium iodide results in disruption of the cell membrane and rapid staining of nuclear chromatin. DNA distribution histograms generated from cells stained by this method are identical to those generated after fixation and RNase digestion. In contrast to some earlier described methods, the present technique is rapid (5 min of processing), requires a minimal amount of material, and avoids formation of cell clumps.


The binding of cationized ferritin at the surfaces of ehrlich ascites tumor cells: the effect of pH and glutaraldehyde fixation. The densities of cationized ferritin (CF) particles binding to the surfaces of cultured Ehrlich ascites tumor cells were determined at pH 7.4, where the ferritin stain was applied either prior to or following glutaraldehyde fixation. The densities were also determined with CF adjusted to pH 1.9 and applied  after fixation. For all fixed samples there was a higher density of particles bound to microvilli than to the spaces between them. Treatment with neuraminidase removed more particles from microvilli than from the inter-microvillus spaces, but did not reduce the levels of binding to the same value. When cationized ferritin is applied prior to fixation, an aggregation of the CF particles at the cell surface was observed, with the internalization of some clusters. This effect was independent of neuraminidase treatment.


The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons. This study of the slow component of axonal transport was aimed at two problems: the specific identification of polypeptides transported into the axon from the cell body, and the identification of structural polypeptides of the axoplasm. The axonal transport paradigm was used to obtain radioactively labeled axonal polypeptides in the rat ventral motor neuron and the cat spinal ganglion sensory neuron. Comparison of the slow component polypeptides from these two sources using sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis revealed that they are identical. In both cases five polypeptides account for more than 75% of the total radioactivity present in the slow component. Two of these polypeptides have been tentatively identified as tubulin, the microtubule protein, on the basis of their molecular weights. The three remaining polypeptides with molecular weights of 212,000, 160,000, and 68,000 daltons are constitutive, and as such appear to be associated with a single structure which has been tentatively identified as the 10-nm neurofilament. The 212,000-dalton polypeptide was found to comigrate in SDS gels with the heavy chain of chick muscle myosin. The demonstration on SDS gels that the slow component is composed of a small number of polypeptides which have identical molecular weights in neurons from different mammalian species suggests that these polypeptides comprise fundamental structures of vertebrate neurons.


Differentiation of erythroleukemic cells in vitro: irreversible induction by dimethyl sulfoxide (DMSO). The inclusion of DMSO in the media of suspension cultures of Friend erythroleukemia cells results in the erythroid differentiation of these cells. The studies reported here were directed towards answering two questions. (1) How long an exposure to DMSO is necessary to induce the differentiation of these cells; and (2) What is the fate of the differentiating cells when DMSO is removed from the medium. Exposure to DMSO for less than 24 hours failed to produce any detectable evidence of erythroid differentiation. On the other hand, culture in the presence of DMSO for 24 hours followed by culture in DMSO-free medium for four additional days produced a small but detectable increment in the proportion of benzidine positive cells in the culture. Once the differentiation of an individual cell was initiated, the process continued after removal of DMSO from the medium. The cell became progressively more differentiated as evidenced by increases in the intensity of benzidine staining as well as the rate of heme synthesis and heme content. However, when cells which had been induced to differentiate by DMSO were cultured in DMSO-free medium for more than 3--4 days, they became vacuolated and apparently died. This latter phenomenon, as well as the more rapid proliferation of the undifferentiated cells in the culture, accounts for the observation that when new cultures are established from cultures which have been grown in the presence of DMSO for several days, the culture which results ultimately contains only differentiated cells.


The pattern of microtubules in the axonemes of Gymnosphaera albida sassaki: evidence for 13 protofilaments. The axonemes of Gymnosphaera albida radiate from a central axoplast. Their proximal ends form a shell around the axoplast. Within the shell each axoneme is enveloped by a fibrillar sheath and the microtubules are interconnected by electron-dense linkages, which sometimes appear to be double. In nearly transverse sections the microtubules and their linkages form hexagons of 2 irregular types arranged in alternating rows. The shapes of the hexagons vary from one axoneme to the next. The variation is caused largely by the inclination of the axonemes to the line of sight, but also by distortion occurring during the preparation, observation and photography of the sections. Calculations show that, of a number of likely basic patterns (as would be seen in strictly transverse section), only one is compatible with measurements made on 9 of the axonemes. This involves only one type of hexagon oriented in 2 directions to form a 'parquet-floor' pattern. The hexagon is bilaterally symmetrical and its 6 microtubules all have the same set of angles between their linkages, namely an unpaired angle of 138 degrees 28' and paired angles of 110 degrees 46'. Because these angles are in the ratio of 5:4:4, it is deduced that the microtubules have 13 protofilaments forming their walls. Morphogenetically the lateral growth of the pattern is governed by 2 rules: (1) there must be one, and only one, direction of 2-step zig-zagging of the linkages, and (2) linkages forming opposite sides of a hexagon must be in parallel.


The Giemsa-staining centromeres of Nigella damascena. The centromere regions of each chromosome in the complement of Nigella damascena (2n equals 2x equals 12) stain differentially with Giemsa at interphase and throughout all the principal stages of mitosis and meiosis. Each centromere is seen to consist of a pair of sister half-centromeres which appear as 2 differentially stained dots. The appearance and behaviour of these dots indicates that they are kinetochores. The technique used does not stain centromeres in other plant species investigated, a fact which shows that the centromeres of Nigella are in some way different. The implications of this observation in relation to centromere polymorphism are discussed.


Analysis of insecticide synergists.

Analysis of insecticide synergists. The utility of a variety of chromatographic procedures (GLC, TLC, and GLC/MS) has been described for the determination of a variety of methylene dioxyphenyl insecticide synergists. Particular focus was placed on chromatographic aspects of the principal synergist, piperonyl butoxide, in relation to its trace impurities, determination in formulations, metabolism, residues, and stability.


Preparation of 125-I-labeled human thyroxine-binding alpha globulin and its turnover in normal and hypothyroid subjects. A protein with the electrophoretic, immunologic, and hormone-binding properties of thyroxine-binding globulin (TBG) has been prepared from human plasma and labeled with radioiodine (125-I) by an enzymatic method of iodination. The [125-I]TBG retained the electrophoretic and immunologic characteristics of unlabeled TBG but exhibited a partial loss of thyroxine-binding activity, as assessed by affinity chromatography. The in vivo behavior of [125I]TBG was studied in six euthyroid subjects (controls) with normal serum levels of TBG as measured both by radioimmunoassay and by determination of maximal T4-binding capacity and in four male patients with untreated primary hyperthyroidism, three of whom had elevated serum TBG. The half-time of the final slope of the plasma disappearance curve averaged 5.0 days plus or minus 1.2 (SD) in the controls and ranged from 3.9 to 109 days in the hypothyroid patients. The distribution volume was similar in the two groups, 6.7 plus or minus 1.3 vs. 7.1 plus or minus 2.1 liters. The catabolic clearance rate averaged 0.99 plus or minus 0.33 liters plasma/24 h in the controls and 0.92 plus or minus 0.46 in the hypothyroids. The absolute turnover rate of TBG, calculated from the catabolic clearance rate multiplied by the serum concentration of radioimmunoassayable TBG, averaged 17.8 plus or minus 2.1 mg/day in the controls and ranged from 14.8 to 33.2 mg/day in the hypothyroids. Among the entire group of subjects there was no correlation between the serum TBG concentration and the absolute turnover rate of TBG.


Distribution of labeled antibiotics in different components of milk following intramammary and intramuscular administrations. In crossover trials, four lactating goats were given intramammary infusions and intramuscular injections of radioactivelabeled benzylpenicillin, spiramycin, chloramphenicol, dihydrostreptomycin, and tetracycline. Milk was collected after each treatment and the antibiotic contents in whole milk, skim milk, and whey were determined microbiologically and radiochemically and in cream and casein by radiochemical assay methods. Uptake of antibiotics by cream and casein was highly dependent on drug concentration, increasing with the decrease in antibiotic content in whole milk. Lipophilic chloramphenicol and tetracycline were concentrated in cream to a higher degree than the less lipophilic benzylpenicillin and dihydrostreptomycin. Antibiotic uptake by cream separated from whole milk after intramuscular injection was higher than after intramammary infusion. Antibiotic uptake by casein was independent of the route of administration and was highest for dihydrostreptomycin and tetracycline and lowest for benzylpenicillin.


Herpes simplex virus type 1 replication in a gingival cell line. Propagation of herpes simplex virus type 1 (HSV-1) in gingival cells is advantageous since these cells are more definitive than HeLa cells in demonstrating viral-induced cytopathic effects and are easily and economically maintained in the laboratory. They also provide a potential medium for studying oral tissue reactions to HSV-1 infection. HSV-1-induced cytopathic effects are clearly characterized following inoculation with virus from standard laboratory cultures and human infections.


Staining reaction of dental plaque after various extraction procedures. After water and dilute alkali extraction, a sample of dental plaque that was stained by the PA-CrA-silver technique showed staining intracellularly on the cell wall and extracellularly. When more prolonged and stronger alkali extraction was used, reacting material was retained on the cell wall and intracellualarly. There was a noticeable loss of extracellular reacting material.


Effect of infusing selected chemical compounds on dentinal fluid movement in the rat. Intra-arterial infusion of carbamyl phosphate or of carbamyl-DL-aspartic acid into rats on a cariogenic diet greatly stimulated the movement of fluid through the odontoblastic processes. The infusion of sodium cyanate also stimulated fluid movement. Guanidine HCL and L-asparagine were active at higher concentrations. Purifying the urea on a mixed-bed ion exchange resin virtually removed its stimulatory effect on dentinal fluid movement. The action of urea is apparently attributable to contamination with sodium cyanate.


Changes with age in protein-bound iodine (PBI) and body temperature in the mouse. Protein-bound iodine (PBI), body temperature, and the response of the thyroid gland to different doses of thyrotropin (TSH) were examined in males of two inbred strains of mice at different ages. Results indicate persistent decline in PBI with advancing age that is significantly greater in DBA/2J than C57BL/6J mice. The magnitude of the PBI response to TSH diminishes in both strains with advancing age but is significantly greater in strain DBA/2J than C57BL/6J. Body temperature was found to decline with advancing age and was highly correlated with thyroid function. Although additional research is needed, a tentative conclusion is that advancing age imparts a diminished response of the thyroid gland to endogenous thyrotropin. This is the first report of age-related changes and particularly correlation between PBI and body temperature in genetically controlled mouse strains. The indications are that such thyroid-temperature relationships are genotype dependent.


The specificity of a solid phase radioimmunoassay for human immunoglobulins. The specificity of a solid phase radioimmunoassay for immunoglobulin antigens, human Fab-mu in the case illustrated, has been assessed. The anti-Fab-mu serum available reacted well on gel diffusion with all Fab-mu- and light chain-containing proteins. In contrast the radioimmunoassay, in which unlabelled test antigen competes with radiolabelled Fab-mu for anti-Fab-mu coupled to Sephadex beads, was sensitive only to proteins containing entire Fab-mu regions. A double antibody radioimmunoassay showed comparable or greater sensitivity for Fab-mu-containing proteins, but was much less specific. By using Fab-gamma as the labeled antigen, with only light chain determinants reacting with the anti-Fab-mu, the solid phase assay was rendered polyspecific in being sensitive to all proteins containing light chain. Reasons for the specificity of the solid phase assay are discussed: steric factors can be expected in some cases to permit strong binding of antigen by multiple bonds, in others to restrict the number of molecular determinants simultaneously available to antibody.


Divergent results in radial immunodiffusion with antisera differing in precipitation properties with respect to individual immunoglobulin subclass. II. The antiserum factor. Differences in the antibody composition of antisera influence results obtained by radial immunodiffusion. If more than one subclass of the same immunoglobulin class is present in an antigen mixture, the occurrence of one or more subclass specific antibody species in antisera used for radial immunodiffusion leads to the production of precipitation rings smaller than rings produced by comparable antisera without subclass activity. The presence of antibodies against common immunoglobulin determinants only leads to production of larger precipitation areas. If one or more subclass specific antibody species are present in the antiserum this may further lead to production of double precipitation rings in radial immunodiffusion, depending on the subclass ratio in the antigen mixture. The production of antisera suitable for radial immunodiffusion is discussed.


Serodiagnostic application of immunohistoperoxidase reactions on antigen-coupled agarose beads. Agarose beads to which antigens were covalently bound were subjected to indirect immunohistoperoxidase procedures. For detection and titration of serum antibodies against bovine gamma globulin (BGG) this method appeared to be specific and sensitive. One advantage is that no special instruments are needed. As an example of diagnostic applicability the system was successfully used for demonstration of antibodies in human serum containing antibodies against the trematode Schistosoma mansoni. At the microscopical level the technique is suitable for study of basic problems. Microspectrophotometric absorbance scanning of the beads revealed that the method can provide quantitative information, and is probably capable of quantifying stoichiometric relations in immunological reactions.


A radioimmunoassay of cellular surface antigens on living cells using iodinated soluble protein A from Staphylococcus aureus. Soluble protein A from Staphylococcus aureus does carry great promise as a marker for cellular surface determinants, due to its specific reaction with, and high affinity for, most subclasses of mammalian IgG. In this article we present the different parameters involved in a radioimmunoassay using 125-I-labelled protein A. Using such an approach the actual technical procedures involved are reported in detail together with tests for mammalian alloantigens, including HL-A in the human, H-2 in the mouse and AgB antigenic sites in the rat, as this presents an unique opportunity to compare them with already widely used assays for transplantation antigens. The different parameters of the assay are analysed in view of measuring with precision quantities of cell-surface IgG molecules, thereby allowing possible determinations of antigenic site numbers in a new and simplified manner.


Cell-mediated immunity in systemic lupus erythematosus: alterations with advancing age. Cell-mediated immunity (CMI) was tested in young patients with systemic lupus erythematosus (SLE) (mean age 30.9 years), in elderly patients with SLE (mean age 70.8 years), and in young and elderly control subjects. Pre-existing CMI as evaluated by skin test response to PPD and mumps antigens and the response of peripheral blood lymphocytes in culture to the mitogen phytohemagglutinin was not significantly different in thefour groups. However, the mean area of induration to Candida antigen was significantly less in both groups of elderly patients, suggesting a diminution of CMI with advancing age. Elderly control subjects and elderly patients with SLE had a significant decrease in incidence and severity of skin-test response when sensitized with 2-4-dinitrochlorobenzene (DNCB), suggesting that the capacity to respond to a new antigen is impaired in the elderly, with and without SLE. Young SLE patients were easily sensitized to DNCB and developed strong skin-test responses to Candida antigen suggesting normal CMI in the young SLE group. SLE is a milder disease in the elderly. If, as seems likely, cell-mediated immune injury is of importance in the pathogenesis of SLE, then these data are consistent with the possibility that an altered state of CMI in the elderly modifies the clinical expression of SLE.


Morphological responses of benign human prostatic hypertrophic tissue to chemotherapeutic agents in an in vitro culture system. The in vitro maintenance of hypertrophic tissue with fluid containing cytotoxic drugs showed more degenerative changes in prostatic epithelium and stroma of the tissue compared with concurrently cultured aliquots of control tissue. It is suggested that theses morphological changes can be related with functional activity of the tissue. The mechanism of such an action is at present unsettled, but an interference on known enzymatic systems regulating prostatic growth seems likely. This system provides an added technique for the evaluation of drugs considered for possible therapeutic testing in human prostatic cancer states.


Induction of T-lymphocyte responses to a small molecular weight antigen. III. T-T cell interactions to determinants linked together: suppression vs. enhancement. The experiments presented in this paper demonstrate the existence of T-T cell interactions in responses to azobenzenearsonate (ABA)-protein conjugates, and also make the point that the spectrum of T-cell regulation from facilitation (i.e., help) at one end to suppression at the other, which has been well documented in T-B cell interactions, is also followed in T-cell regulation of other T lymphocytes. The data extend the activity of ABA-specific suppressor cells, which were shown to specifically suppress the development of delayed hypersensitivity to ABA-T, to T cells responsible for delayed hypersensitivity to protein antigens provided immunization is carried out with ABA conjugates of these antigens. Thus, suppressor T cells acting on the development of delayed hypersensitivity are not limited in their effects to T cells bearing the same specificity but can effectively suppress responses on immunologically unrelated T cells if they are specific for carrier antigens covalently linked to the ABA-T determinant. Moreover, these studies demonstrate that, as is true of T-B cell interactions, the most efficient T-T cell interactions occur to determinants linked together on the same molecule thus supporting the concept that development of effector T-cell function may involve participation of at least two distinct precursor cells, each of which may convey independent determinant specificities and/or genetic control.


Molecular relationship between private and public H-2 antigens as determined by antigen redistribution method. Molecular relationship of public H-2 antigens 1, 5, 6, 8, 11, 13, 25, and 28 to private antigens controlled by K and D regions was studied using the technique of antibody-induced resistance to complement-mediated cytotoxicity.  The results indicate physical association in the cell membrane between H-2 antigens 1 and 23 of H-2-a, 8 and 31 of H-2-d, 11 and 17 of H-2-q, 13 and 30 of H-2-q, 25 and 23 of H-2-k, and 28 and 31 of H-2-g.  These results are in agreement with genetic mapping placing the determinants of antigens H-2.8, 11 and 25 in the K region , the determinant of antigen H-2.13 in the D region, and the determinants of antigens H-2.1 and 28 in either the K or the D region.  In contrast to genetic mapping placing the determinant for antigen H-2.6 in the D region, we found that in the H-2-b haplotype the antigen is associated with K region antigen H-2.33 and H-2.32, and interpreted this result as evidence for two homologous H-2.5 sites controlled by opposite ends of the H-2 complex.  Although the data do not prove that public antigens are carried by the same molecules as private ones, they demonstrate a close physical association in the membrane between the two groups of loci, K and D, coding for the first 33 classical H-2 antigens (with the exception of antigen H-2.7), and thus support the two-locus model.  The data also support the duplication model of H-2 by demonstrating two homologous H-2.5 sites associated with K and D molecules.


Anti-immunoglobulin-induced histamine secretion by rat peritoneal mast cells studied by immunoferritin electron microscopy. We have used ferritin-conjugated divalent and monovalent anti-Ig antibodies to study simultaneously, histamine secretion and the ultrastructural distribution and redistribution of Ig receptors on rat peritoneal mast cells.  We conclude that (a) divalent anti-Ig is required for both receptor redistribution and for calcium-dependent degranulation and histamine release, (b) divalent anti-Ig induces patching and pinocytosis but not capping of Ig molecules, (c) neither capping nor pinocytosis are required for triggering and if clustering is necessary, then less than 10 Ig molecules are required per cluster, and (d) degranulation (and histamine release) is not an all or none response of the mast cell.


Immunological analysis of plasminogen activators from normal and transformed hamster cells.  Evidence that the plasminogen activators produced by SV40 virus-transformed hamster embryo cells and normal hamster lung cells are antigenically identical. Rabbits were immunized against the plasminogen activator released by SV4- virus-transformed hamster embryo cells.  The resulting antiplasminogen activator immunoglobulin (APA-IgG) inhibited the enzymatic activity of the plasminogen activator produced by SV40-transformed hamster cells, and the plasmin-catalyzed release of these cells from the tissue culture dish.  APA-IgG was not cytotoxic for these cells even in the presence of complement and did not inhibit their release of plasminogen activator.  APA-IgG formed a single precipitin line in immunodiffusion plates using highly purified plasminogen activator as antigen.  APA-IgG inhibited the plasminogen activator produced by newborn hamster lung cells and by an established diploid line (DON) of hamster lung cells, but did not inhibit plasminogen activators produced by normal or transformed hamster kidney cells or by cells of other species (mouse and human).  We derive three major conclusions from these data: (a) There are several immunologically distinguishable forms (isozymes) of plasminogen activators in normal hamster tissues. (b) The plasminogen activators produced by normal hamster lung cells and by SV40 virus-transformed hamster embryo cells share antigenic determinants and are presumably the same isozyme. (c) The plasminogen activators produced by different hamster tumor cells do not share antigenic determinants and are presumably different isozymes.


Binding properties of immunoglobulin combining sites specific for terminal or nonterminal antigenic determinants in dextran. Binding constants of the dextran-reactive BALB/c mouse IgA myeloma proteins W3129 and QUPC 52 have been determined for each member of the isomaltose series of oligosaccharides and for methyl alphaDglucoside. Protein W3129 has maximum complementarity for isomaltopentaose (IM5) deltaf degrees = 7,180 cal/mol) with 55-60% of the total binding energy directed against methylalphaDglucoside. Protein QUPC 52 gives maximum binding with isomaltohexaose (IM6) (deltaF degrees = -5,340 cal/mol) and has about 70% of its total binding energy for isomaltotriose (IM3), but at most only 5% for isomaltose (IM2) or methyl alphaDglucoside. Protein W3129 precipitates with branched dextrans high in alpha (1 yields 6) linkages and reacts with but does not precipitate a synthetic alpha (1 yields 6)-linked linear dextran. Protein QUPC 52 precipitates both branched and linear dextrans. Thus, the immunodominant group for protein W3129 is mimicked by methyl alphaDglucoside and this protein reacts exclusively at the terminal nonreducing ends of alpha (1 yields 6)-linked dextran chains. Protein QUPC 52 has an immunodominant group which is expressed by IM3 but not smaller oligosaccharides and this protein can react at nonterminal locations along alpha (1 yields 6)-linked dextran chains. Precipitation of linear dextran seems to be a valid although not quantitative assay for antidextrans with nonterminal specificity. Quantitative precipitin reactions with branched and linear dextrans suggest that alpha (1 yields 6)-specific human antidextrans are mixtures of molecules having terminal and nonterminal specificities and that the fraction of each type can vary among individuals. Rabbit antisera against IM3 or IM6 coupled to bovine serum albumin also appear to contain antibodies with nonterminal specificity for dextran chains although a large fraction has terminal specificity. Low molecular weight clinical dextran N-150N (congruent to 60,000) reacted more like linear dextran than like its parent native-branched dextran B512. This is thought to result from an abundance of nonterminal determinants in clinical dextran N-150N but a very small number of functional terminal determinants per molecule. An appreciation of terminal and nonterminal specificities and of the different immunodominant structures in isomaltosyl chains has proven to be of a great value in understanding the immunochemical reactions of dextrans. Moreover, certain previous findings with fructosan-reactive mouse myeloma proteins and human antilevans (55, 84) also suggest terminal and nonterminal specificities for levan chains.


RNA-instructed DNA polymerase activity in a cytoplasmic particulate fraction in brains from Guamanian patients. Nervous system tissues from a number of patients with idiopathic neurological disorders were examined for biochemical evidence of RNA tumor virus infection.  RNase-sensitive DNA polymerase activity was found in a cytoplasmic particulate fraction from two patients with Guamanian amyotrophic lateral sclerosis (ALS) but not in brains from two normal U.S. individuals.  The buoyant density of the enzyme-containing fraction was 1.16-1.18 g/ml and could be converted to a denser region of the gradient (1.24 g/ml) by treatment with the nonionic surfactant, Sterox.  The cation and detergent requirements for the endogenous RNase-sensitive DNA polymerase reaction were determined.  The early (5 min) endogenous reverse transcriptase product was analyzed by cesium sulfate gradient centrifugation.  RNase- and heat-sensitive RNA-DNA hybrids were detected in the product analysis of two ALS, one Parkinsonism-dementia (PD) brain, and two brains from asymptomatic Chamorros but not in brains from normal U.S. individuals and a number of patients with neuro-psychiatric disorders.  The DNA product was a 4.5S heteropolymer that hybridized more extensively to RNA extracted from the enzyme-containing pellet from PD brain as compared to a similar fraction from normal U.S. brain.  The DNA product appeared to be unrelated to Rausvher or visna virus 70S RNA as determined by RNA-[-3H]DNA hybridization.


Taxonomic distribution of the antigen eliciting bactericidal antibody for Bordetella pertussis. Strains of Bordetella pertussis varied in their ability to elicit (in mice) an antibody bactericidal for an antiserum-sensitive strain of B. pertussis, although antibody was usually detectable after only one injection. High titres were produced by a course of seven injections with all strains of B. pertussis tested (six of phase I and three of phase IV) but not with three strains of other Bordetella species nor with two unrelated organisms, a finding of possible taxonomic value. Preliminary investigations have not revealed whether strain vaiations are due to quantitative or qualitative differences in either the bacterial lipopolysaccharide or the carrier protein necessary for antibody production, or whether they may be due to differences in heat lability of 'bactericidal antigen'.


Attenuation and immunogenicity of ts mutants of Eastern encephalitis virus for mice. Preliminary experiments were intiated to test the attenuation for mice of chemically induced temperature-sensitive (Ets) mutants of a virulents strain of Eastern encephalitis (E) virus, and the potential of such mutants as live virus vaccines for mice. The reversion frequencies of eight mutants to temperature insensitivity were measured and the defects in their biosynthesis at the non-permissive temperature were studied. All eight mutants were less virulent for mice, but the extent of avirulence varied with the mutant and route of injection. The mutants selected as potential vaccines protected mice against subsequent challenge at 10 to 21 days by the virulent virus of strain E. Neutralizing antibody activity was detected in almost all the mutant-infected mice after 10 days, and was found in all infected mice at 21 days. Immunization by two doses of virus induced a very high protection against intracerebral challenge by virus strain E.


Poliovirus temperature-sensitivie mutants defective in cytopathic effects are also defective in synthesis of double-stranded RNA. The proportion of cells absorbing trypan blue (tb-+ character) can be used to measure the late c.p.e. of wild-type poliovirus (ts-+. tb-+), which was the same at restrictive (39-2 to 39-6 degrees C) or permissive (37 degrees C) temperatures. Of twenty ts mutants, seven showed normal c.p.e. at 37 degrees C but were defective in C.P.E. (TB) AT 39-5 degrees C; all seven tb mutants have previously been shown (Cooper et al. 1971) to give evidence of a primary defect in replicase 1 activity (to make the complementary or minus strand of virus RNA). The remainder (tb-+) have all previously been shown to give evidence of a primary defect either in replicase II activity (to make progeny plus strands) or in structural protein. Thus, the late c.p.e. is dependent on a product of the replicase I gene, of which the in vivo effector is probably double-stranded RNA. Late c.p.e. is not caused by prevention of host protein, RNA or DNA synthesis and is not necessarily correlated with lysosomal enzyme release. The tb mutants were also defective in inducing early changes in chromatin (chr) and in prevention of thymidine incorporation (pti), but the tb and pti/chr characters are probably independent expressions of replicase I activity. Virus growth does not depend on repression of DNA synthesis. Poliovirus represses the activities of host DNA-dependent RNA polymerase I and II to an equal extent. There is no evidence that repression of DNA or RNA synthesis results from direct interaction of virus protein with the DNA.


Host components in hepatitis B antigen.

Host components in hepatitis B antigen. Purified [125I]-labelled 20 to 25 nm hepatitis B antigen particles were found to give low affinity immunoprecipitation reactions with antisera to several normal serum components, which were immunologically distinct from the reaction due to the classical hepatitis B antigen surface determinant. These additional antigenic determinants were acid-stable and tightly bound to the particles; they could not be released by treatment with Tween 80 or ether, but were removed by protease digestion with the preservation of particle integrity. It was not possible to distinguish whether they were due to the presence of trace amounts of partly denatured serum components, or to a weak cross-reaction with antigens present in normal serum. The implications of this finding for hepatitis B antigen and antibody detection in sensititive assays are discussed. No evidence was found for native antigenic material, present in normal serum or normal liver cells, being integral to the structure of these particles.


Type C virus production by a continuous line of pig oviduct cells (PFT). Late passages of the PFT porcine oviduct cell line spontaneously release a typical type C virus antigenically related to the type C virus released from the PK(15) porcine kidney cell line. The PFT virus was non-infective for a large variety of cell lines. Type C virus can also be induced by BrdU treatment from earlier passages of the PFT cell line.


No evidence for particles encapsulating RNA-instructed DNA polymerase and high molecular weight virus-related RNA in herpesvirus induced tumours of non-human primates. The simultaneous detection test gave no evidence for the presence of RNA tumour viruses in herpesvirus induced malignant lymphomas of non-human primates. The 12 tumours tested were obtained from three different monkey species inoculated with Herpesvirus saimiri or herpesvirus ateles. Particles encapsulating RNA-instructed DNA polymerase and high mol. wt. virus-related RNA were easily demonstrated in tumours of the mouse induced by type-C or type-B oncornaviruses and in human lymphoid cells infected with simian sarcoma virus type I which were examined in parallel. Attempts to demonstrate partial expression of an oncornavirus genome in the herpesvirus induced tumours and attempts to detect an interspecies antigen related to monkey oncornaviruses were negative and strengthened the observations made with the simultaneous detection test.


Protein patterns of cerebrospinal fluid in hereditary ataxias and hereditary spastic paraplegia. The CSF findings in hereditary ataxias and allief disorders have hitherto mostly been reported as normal if one excludes Refsum's syndrome. The CSF-protein patterns found on isoelectric focusing and quantitative paper electrophoresis were studied in 12 patients with hereditary ataxias and hereditary spastic paraplegia. Using a recently-developed technique of isoelectric focusing of CSF-proteins in flat beds of polyacrylamide gel, the authors could show abnormal CSF-protein patterns in all but 1 of the present cases. The aberrant CSF-protein patterns found showed differences between the syndromes studied. Two unique patterns with conspicuous fractions in the acid range were observed in patients with Marie-Sanger-Brown's ataxia (mother and daughter) and Holmes' ataxia. A third CSF-protein pattern was found in a sibship with Friedreich's ataxia including a double fraction in the acid region (pI 5.9-6.1) in all 4 subjects and a highly alkaline fraction (HAF) with pI about 9.3, in 3 of them. Similar acid fractions (pI 5.9-6.1) were also detected in 3 of 4 patients with hereditary spastic paraplegia, a brother and sister showing a very similar CSF-protein pattern. Double fractions with pI 5.9-6.1 and/or HAF may also occur in other neurological diseases, mostly, however, associated with other distinctive features of their CSF-protein patterns. A possibility in the future of distinguishing hereditary CNS-diseases by examination of the CSF-protein pattern is suggested.


The fine structure of hyaline inclusions (pseudopsammoma bodies) in meningiomas. "Hyaline inclusions" of a meningothelial meningioma were examined under the electron microscope. These eosinophilic, PAS-positive proteinaceous structures, which under the light microscope are seen in both extra- and intracellular location, were found by electron microscopy to consist of granular masses surrounded by cell membranes with microvilli. The "intracellular" bodies were also surrounded by similar microvilli in a space within the cells. Such spaces, variously known as intracellular ductules and "neolumen formation", have been previously described in mammary cancer, bronchiolar carcinoma and pleural mesotheliomas, among others, and have been, in the latter instances, regarded as signs of secretory differentiation. Thus, hyaline inclusions of meningiomas are different from truly intracellular hyaline bodies of neoplastic astrocytes (found by Rubinstein and Herman to be bodies within autophagic vacuoles) and may be regarded as a possible factor in ultrastructural differential diagnosis between meningiomas and gliomas.


Early sterile autopsy in etiological studies on multiple sclerosis. The clinical findings and course in the first 2 cases of multiple sclerosis are described, in whom it was possible to isolate a virus from brain tissue by early sterile autopsy and fusion technique. It is noteworthy that in one of these cases multiple sclerosis probably occurred in female members of the family through three consecutive generations. A report is made on an additional case, in which no virus could be isolated, but in which electron microscopic studies showed two types of virus particles: nucleocapsid-like structures and particles identical in form and size with papova virus. Electron microscopic findings and attempts to cultivate a virus in multiple sclerosis published by other authors are discussed. It is pointed out, that the results described provide as yet no proof for the viral etiology of multiple sclerosis. They support the hypothesis, however, that a virus may play an essential role in the etiology and/or pathogenesis of this disease. Problems in relation to brain autopsy in multiple sclerosis are discussed. Early sterile autopsy is considered the most practicable possibility for obtaining tissue material for culturing and for ultrastructural studies in multiple sclerosis.


Multiple sclerosis and parainfluenza 1 virus. History of the isolation of the virus and expression of phenotypic differences between the isolated virus and Sendai virus. 54 cultures were established from brain tissue obtained 2-3 hrs after death from 1 case of multiple sclerosis and 30 cultures from another case. Following fusion with indicator cells in the presence of lysolecithin, a parainfluenza type 1 virus (6/94 virus) was isolated from cultures representing one plaque area in the first case and one plaque area in the second case. A cell line chronically infected with the 6/94 virus has been maintained for more than 100 passages in vitro. A close relationship to the Sendai Hemagglutinating Virus of Japan (HVJ) is indicated from RNA-RNA hybridization and the patterns of electrophoretic mobilities of viral polypeptides. Conversely, differences in optima for growth-requirement temperatures, hemolytic activity and the capability to fuse mammalian cells, distinguishes 6/94 virus and HVJ as distinct phenotypic entities of a closely related genotype.


Movement-associated cortical potentials with unilateral and bilateral simultaneous hand movement. Cortical potentials associated with unilateral and bilateral simultaneous thumb opposition were studied in 9 healthy subjects. The P1 component, "pre-motion positivity", was recognized in 5 out of 9 subjects on unilateral thumb movement. In all subjects in whom P1 was recognized, P1 was dominant over the cerebral hemisphere ipsilateral to the contracting muscles. On bilateral simultaneous thumb movement, however, P1 was not discerned in any subject. This is compatible with the assumption that P1 is related to an inhibition of imitative movement of the opposite hand (mirror movement). The N2 component had its onset after the start of muscle contraction in most cases, suggesting that N2 might not reflect activation of the corticospinal pathways.


[Diagnostic value of siderophages in the cytogram of cerebrospinal fluid]. The demonstration of siderophages in the cerebrospinal fluid is a very reliable diagnostic tool for identifying hemorrhage in the subarachnoid space and ventricular system. Siderophages are found 4 days after spontaneous or traumatic subarachnoid hemorrhage and persist in some cases up to 120 days, which makes them a reliable indicator of a previous hemorrhage. These findings are the result of an investigation in which 110 samples of cerebrospinal fluid, obtained from 105 patients, were examined for siderophages by the membrane filtration method.


[Anticonvulsant drugs and liver damage (Author's transl)]. 540 autopsies of institutionalized epileptics who had been treated with anticonvulsant drugs were evaluated as to type and frequency of pathological changes in the liver and compared with a control series of 270 nonepileptics. A specific liver-toxicity of antiepileptic drugs, or combination of drugs, could not be established. Thus, only in 3 cases, one of which was fatal, was a relationship found between anticonvulsant drug therapy and liver damage. Therefore, there seems to be no reason to discontinue anticonvulsant therapy or to reduce the usual dose for fear of causing liver damage. On the other hand, it was found that tuberculosis may have adverse effects upon the liver of patients undergoing anticonvulsant drug therapy. From 41 cases of active tuberculosis we found 13 with severe liver damage, 3 of which were fatal. 10 patients had, and 3 had not, received antituberculosis chemotherapy.


[The advantages and limitations of an automatic ultrasound determination of the cerebral midline--a study of 1889 cases]. The examination of the brain with ultrasound has become an indispensible diagnostic tool in all cases of intracranial pathology. Valuable information is obtained both in emergencies and in follow-up studies. Since long practice is necessary before the results of echoencephalograms can be interpreted accurately, a need was felt for a standardization of the examination which would eliminate the subjective influence of the investigator. The automatic midline computer, called Midliner, which has recently become available, makes fewer demands on the experience of the investigator although the diagnostic possibilities are not as broad. The position of the midline echo is of primary interest, although the width of the third ventricle can be successfully determined in occasional cases. this study, which presents the results of the Midliner examinations in three clinics, is grouped according to diagnosis. Through a comparison with conventional A-scan echoencephalograms, as well as with neuroradiological and operative findings in 1889 cases, the reliability of this method is clearly demonstrated. The advantages and disadvantages of the examination are discussed and the field of application indicated.


[Development of an apparatus for measuring attention in patients with cerebral damage and functional disorders]. 58 patients with various underlying neurological diseases, who had an impairment of attention, were examined. 12 patients without clinically evident disorders of attention were examined as a control group. The aim of the study was the development of a standardized procedure for the assessment of impaired attention. An additive, 4 step scale of the "susceptibility to stimulation" (Guttman scale) was constructed out of several, heterogeneous stimuli. In addition, an additive 4 step scale of "reactivity" was constructed out of a catalogue of defined reactions. The two scales permitted the assessment of reactivity and susceptibility to stimulation, the degree of which could be demonstrated as being proportional to one another. The transformation of susceptibility to stimulation into reactivity is thought to be performed by an internal system which is attributed to attention.


Mechanisms of contractile response of cerebral artery to externally-applied fresh blood. The authors report and analyze the in vitro contractions of fresh bovine cerebral artery on exposure to blood. The vessel does not contract significantly until blood clots on its surfaces; the contraction begins at the moment of clotting and is maintained as long as extracellular calcium is available. Comparative studies with vasoconstrictor amines suggest that serotonin, liberated from platelets by the clotting process, is responsible for this contraction and that the adherent clot itself concentrates the serotonin of the surface of the vessel. This contraction persists throughout the"viability" of the in vitro preparation, approximately 10 hours. Serotonin is capable of producing this sustained contraction by increasing smooth muscle cell membrane permeability to extracellular calcium, which in turn activates native actinomycin.


Depressed immune response in the magnesium-deficient rat. The effects of dietary magnesium on growth, food efficiency, organ development, splenic nucleic acids, and serum antibody were studied in two experiments with male Wistar rats. Diets containing 30% protein from casein were fed ad libitum. Rats were immunized intravenously with sheep red blood cells. Blood was obtained 5 and 9 days after immunization. In experiment 1, a group of weanling rats was fed 10 ppm Mg for 8 days, followed by 142 ppm for 37 days. Group 2 (controls) was fed 480 ppm Mg for 45 days. Group 1 weighed less but  had larger spleens, kidneys, and testes relative to body size than did group 2. Nucleic acids per gram spleen were similar in both groups as were serum gamma-globulin and its 19S and 7S components. Antibody log titers for group 1 were 45 and 65% of control agglutinin levels and 44 and 80% of control hemolysin values on days 5 and 9, respectively. In experiment 2,200-g rats were fed 10 (group 3) or 480 ppm (group 4) Mg for 38 days. Most effects of the 10 ppm Mg diet were similar to those seen in magnesium deficiency in experiment 1. Antibody titers for group 3 were 30 and 25% of control agglutinin and 43 and 53% of control hemolysin values on days 5 and 9, respectively. Total serum gamma-globulin and its 19S fraction were similar in both groups, while the 7S fraction of group 3 was only 64% of the control value.


Marginal adaptation of composite resins. The historical development and important properties of composite resins are briefly reviewed. Experimental work has been undertaken to examine the marginal adaptation of three commerically available composite resins in 100 Class II and Class V restorations; dye penetration tests were used and measurement of the gap at the tooth/restoration interface as recorded in photographs obtained by scanning electron microscopy of replicas. In Class V restorations showing marginal leakage by dye penetration the gap at the tooth/restoration interface was in all cases greater cervically than occlusally. The results underline the uncertainly of obtaining a good marginal seal with composite resins both peripherally and along internal line angles.


Liver tumors in children in the particular reference to hepatoblastoma and hepatocellular carcinoma: American Academy of Pediatrics Surgical Section Survey--1974. A review of 227 cases of hepatoblastoma, hepatic cell carcinoma in children seen in the United States over a 10-yr period is presented. Both tumors were seen most commonly in infancy, but the hepatocellular carcinoma shows a second peak of incidence around puberty. Males predominated in both diseases more so in hepatoblastoma. Presenting symptoms in both diseases were very similar, most commonly an upper abdominal mass or abdominal enlargement associated with anorexia and weight loss. In the preoperative evaluation the presence of alpha-feto protein was one of the most helpful diagnostic tests. Disturbances of liver function were usually mild but were more marked in those children with hepatocellular carcinoma. Preoperative x-rays were abnormal in a large percentage of cases with the hepatic arteriogram and vena cavagram being the most useful diagnostic x-rays for liver tumors. Liver scans were positive for liver tumor in 95% of the children when this test was carried out. The follow-up for these patients ranged from 2 to 10 yr. The size of the primary tumor did not appear to correlate with survival but bilateral location of the tumor, 33% in hepatoblastoma and 45% in hepatocellular carcinoma, made many of these tumors inoperable. Multicentric tumors were also found in a large number of patients, being more common in hepatocellular carcinoma. There was a high rate of local recurrence or local extension after operation in both diseases, and metastatic spread was similar being most common to the lungs and abdomen. A wide variety of surgical procedures were carried out in these patients from biopsy only to extended hepatic lobectomy. When incomplete excision or biopsy only was carried out no patient survived in either group. Among the hepatoblastoma patients, 45 of 78 patients who had complete excision are surviving. In the hepatocellular carcinoma patients where the operability rate was much lower 12 of 33 patients are surviving when tumor was completely excised. Complications were frequent, the most common being excessive blood loss at operation. There were eight operative deaths and 17 postoperative deaths in the combined group. There was no evidence that radiation therapy or chemotherapy controlled disease which could not be completely excised surgically. The only direct evidence of a favorable effect of radiation and chemotherapy were three cases of hepatoblastoma in which the tumor changed from inoperable to operable by a combination of radiation therapy and multiple drug chemotherapy. Both tumors are highly malignant, and 90% of the children who died of hepatoblastoma died within 12 mo of diagnosis. In the hepatocellular carcinoma 80% of the deaths occurred within 1 yr of diagnosis. At this time it seems that operative excision offers the only chance of cure in children with these tumors and cure rates of 60% can be expected with hepatoblastoma and 33% in hepatocellular carcinoma if the tumor can be completely excised.


An analysis of the specificity in pharmacological inhibition of the passive cutaneous anaphylaxis reaction in mice and rats. An antiserum obtained from mice, immunized to produce an antiovalbumin antibody of the IgE type, was employed in a 48-hour passive cutaneous anaphylaxis (PCA) reaction in both mice and rats. The antiserum contained an antibody which, "fixed" to skin for at least 6 days, was heat labile and eluted from diethylaminoethyl cellulose in the reagin peak. In both rats and mice, the PCA reaction was mediated by a combination of histamine and serotonin and was inhibited by specific antagonists. Various drugs were tested for inhibition of the PCA reaction in recipients also injected with compound 48/80 and histamine. Drugs which have been reported to cause an increase in intracellular cyclic adenosine monophosphate levels [prostaglandins (PG) E1 and E2 and theophylline] all selectively inhibited the PCA reaction at low doses. By varying the length of time of drug administration prior to antigen challenge, the pharmacological half-life of PGE1 was determined to be approximately 9 minutes. At high doses, theophylline also inhibited the 48/80 reaction, and PGE1 inhibited all three reactions, whereas PGE2 only inhibited PCA. Disodium cromoglycate, when given to rats, inhibited only the PCA reaction without effect on the 48/80 or histamine wheal. It was totally ineffective on any parameter measured in the mouse. It is suggested that the PCA reaction in the rodent is induced by an IgE-like antibody and mediator release is, to some extent, sensitive to intracellular levels of cyclic adenosine monophosphate. Analysis of the specificity of drug activity depends upon dose-response studies, species differences and consideration of nonspecific systemic effects.


Elicidation by a H-2-receptor antagonist of the significance of mucosal histamine mobilization in exciting acid secretion. 1. The consequence of H-2-receptor blockade for the secretory responses of the gastric mucosa to hormonal or cholinergic stimulation was studied in conscious rats with Heindenhain pouches or Pavlov pouches with the antrum retained or resected. 2. Metiamide almost completely abolished acid secretion induced by pentagastrin without altering significantly the amount of histamine excreted in the urine. Histamine mobilization on pentagastrin infusion determined in vitro, seemed to be larger during H-2-receptor blockade than with pentagastrin alone. 3. CCK-PZ mobilized mucosal histamine to a considerable extent; the secretory response to this hormone was completely abolished by H-2-receptor blockade. 4. Acid secretion in response to 2-deoxy-D-glucose was inhibited by H-2-receptor blockade in the presence or absence of the antrum; however the inhibition was less complete than with hormone-induced secretion. 5. The acid secretory response to 100 mg/kg of 2-deoxy-D-glucose appeared to be less susceptible to H-2-receptor blockade than that of 50-mg/kg of 2-deoxy-D-glucose. 6. Feeding induced a secretory response in the Pavlov pouch which initially was more effectively inhibited by H-2-receptor blockade than the response to 2-deoxy-D-glucose. In the absence of antral gastrin secretion by either stimulus was equally inhibited. 7. Methacholine-induced acid secretion was inhibited by infusion of the H-2-receptor antagonist, an inhibition that was absent when pentagastrin was concomitantly infused. 8. Although acid secretion induced by cholinergic stimuli was readily inhibited by the H-2-receptor antagonist, slight or nor inhibition was noted on pepsin secretion. 9. The role of histamine as a physiological stimulus for the parietal cell is discussed in view of the fact that the secretory effect of natural stimuli, known or demonstrated to mobilize mucosal histamine, is restrained by H-2-receptor blockade.


Membrane potentials in pinched-off presynaptic nerve ternimals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials. 1. Some physiological properties of tissue fractions from rat brain homogenates have been examined. Of the three fractions studied (presynaptic nerve terminals, mitochondria and fragmented membranes), only the nerve terminals (synaptosomes) have the ability to accumulate 42K from physiological salt solutions. 2. The ability to accumulate and retain K is lost if synaptosomes are exposed to very hypotonic solutions. The K uptake and total K content is reduced by ouabain and by inhibitors of glycolysis and oxidative phosphorylation. 3. These results suggest that synaptosomes in physiological saline accumulate K against a concentration gradient, and may have K diffusion potentials across their surface membranes. The voltage-sensitive fluorescent probe, 3,3'-dipentyl 2,2'-oxacarbocyanine (CC5), was used to test this possibility. 4. In the squid axon, the fluorescent emission of CC5 is directly proportional to membrane potential; depolarization causes an increase in fluorescence. 5. The fluorescence of synaptosomes ('synaptosome fluorescence') treated with CC5 is increased when [K]o is increased or [K]o is reduced; replacement of external Na by Li or choline has little effect on the synaptosome fluorescence. In quantitative terms, synaptosome fluorescence is proportional to log ([K]o plus 0-05[Na]o). Rb is about as effective as K in enhancing synaptosome fluorescence; Cs is about 1/4 as effective. The effect of increased [K]o is reversible. 6. The fluorescence data provide corroborative evidence that there is normally a large K gradient ([K]o smaller than [I]i) across the synaptosome surface membrane. The data suggest the [K]i may be in excess of 100 mM. 7. Replacement of Cl- by methylsulphate did not significantly affect the relationship between synaptosome fluorescence and [K]o, nor did removal of external Ca. 8. The fluorescence of CC5-treated mitochondria, membrane fragmnets, or lysed synaptosomes is unaffected by changes in the K concentration of the medium. 9. Veratridine and gramicidin D, both of which enhance Na permeability (PNa) in some intact tissues, increase synaptosome fluorescence when added to the standard medium. The increment is greatly reduced or abolished when external Na is replaced by choline. 10. If synaptosomes are first Na-loaded (by pre-treatment with cyanide + iodoacetate), and then placed in a choline medium, addition of gramicidin D significantly decreases fluorescence. This effect could be explained if, with [Na]o smaller than [Na]i, the increase in PNa causes the synaptosomes to hyperpolarize. 11. The veratridine-induced increase in synaptosome fluorescence was prevented by 3 times 10- minus 7M tetrodotoxin, which also blocks the depolarizing effect of veratridine in intact neurones. 12. The main conclusion is that synaptosomes may retain resting membrane potentials and the ability to increase Na permeability.


Amniotic fluid macrophages and the antenatal diagnosis of anencephaly and spina bifida. The macrophage content of amniotic fluid has been measured and the upper limit of normal on an arbitrary scale is 41. Amongst 65 amniotic fluids collected for antenatal diagnostic studies before 22weeks' gestation there were eight which had macrophage counts ranging from 82 to 6226,three of these were shown to have anencephaly and two spina bifida. The reasons for three apparently false positives are as yet undetermined. Rhesus iso-immunized amniotic fluids were found to have macrophage counts of up to 276 and a possible explanation for this is considered. It is argued that an elevated aminotic fluid macrophage count may indicate a CNS defect or possibly other fetal abnormality.


Alpha-feto-protein during development and in disease. An alpha-feto-protein (AFP) is present in many mammals, in birds, and in sharks during development. The AFP present in different species have similar physicochemical properties and often have common antigenic determinants. Their study, both in health and disease, has provided a useful model for the understanding of other phase-specific antigens and the activation of the genes which control their synthesis. In the human fetus, the level of AFP falls with increasing maturity. The more sensitive methods of detection have disclosed that this fetal protein persists in trace amounts throughout life and its level increases in maternal blood during pregnancy. The principal sites of synthesis are the fetal liver and in some mammals, the yolk sac splanchnopleur. In humans as well as in mice and cows, it is notable that the synthesis of AFP is increased in liver cancer cells and that high levels of this protein are present in serum. Elevated values of AFP have also been detected in human subjects with undifferentiated tumours of the testis and ovary. A fall to normal levels has been noted in cases of complete remission after surgery and a return to high levels in patients who develop metastases. In some patients with hepatitis a temporary rise in the level of AFP has also been observed. In recent years, the detection of high levels of AFP in amniotic fluid has proved to be of great value for the prenatal diagnosis of neural-tube defects. Abnormal levels have also been found in the amniotic fluid or in maternal serum in cases of spontaneous abortion. Such measurements are now being assessed as a methodof monitoring abnormal pregnancy.


Trisomy 22. Two new cases and delineation of the phenotype. Two unrelated children, not affected with Down's syndrome, with strikingly similar phenotypes and an extra G-like chromosome are presented. Quinacrine and trypsin-Giemsa banding identified the extra chromosome as No. 22. The phenotype of these patients and the review of 15 additional similar cases from the literature permit a definition of the cardinal features of trisomy 22; mental and growth retardation, microcephaly and craniofacial asymmetry, strabismus, beaked and prominent nose, long philtrum, cleft palate, micrognathia, large low set ears with preauricular tags and/or pits, long slender fingers, congenital heart disease, inguinal hernia, and hip dislocation.


A patient with congenital anomalies and a deletion of the long arm of the long arm of chromosome 4 [46,XY,del(4)(q31)]. This paper describes the clinical symptoms and cytogenetic findings in a patient previously described in a doctoral thesis (van Kempen, 1969). The patient is a boy with multiple congenital anomalies and a deletion of the long arm of chromosome 4. A recent Giemsa banding study showed absence of the terminal G-band, as was found in the patient described by Golbus  et al (1973). The symptoms and other data on the three patients known to have a deletion of the long arm of chromosome 4 are presented to facillitate comparison of these cases. However, the number of cases so far on record is too small to warrant conclusions on the basis of this comparison.


Dicentric Y chromosome in mixed gonadal dysgenesis. A 15-year-old girl was investigated because of ambiguous genitalia. Her chromosome studies showed a 45, X/45, Xdic(Yq) mosaicism. The identity of the dicentric Y chromosome was demonstrated by its typical fluorescent banding patterns. Histological evidence of mixed gonadal dysgenesis with intragonadal tumour was observed, confirming the occurrence of gonadoblastoma associated with mosaicism in which at least one cell bears a Y chromosome.


T-mycoplasmas: a study of the morphology, ultrastructure and mode of division of some human strains. The morphology of 10 strains of T-mycoplasma was studied in wet preparations of viable cells by darkfield, phase-contrast and interference microscopy, and in fixed preparations by various techniques of electron microscopy. Mycoplasma-like artefacts in the horse-serum component of the medium were eliminated by filtration. All 10 strains were similar. Individual cells were spherical, 0-25-1-0 mum in size, with a bounding trilaminar membrane, 10 nm thick and containing 7-5-12-5-nm particles, and a layer of pilus-like projections, 5-8 nm long, on the outer surface. A possible capsular matrix was observed only by the pseudoreplica technique. The cells contained 12-15-nm ribosomes, nuclear fibroids 7-5-9 nm wide, and vacuoles. During replication, the cell elongated slightly and the ribsomes migrated to the ends of the cell leaving a ribosome-free area into which the bounding membrane invaginated to form a bud. The bud eventually separated by completion of the process of invagination; a cross-septum did not form. Usually only a single bud developed but sometimes two appeared simultaneously.


The permeability of liposomes to nonelectrolytes. II. The effect of nystatin and gramicidin A. The process of selective permeation of nonelectrolytes across liposomes of different lipid composition and amount of cholesterol has been studied. The extent of the selectivity for diffusion within the membranes has been found to be related to the physical state of the hydrocarbon chains. It has been also found that incorporation of cholesterol into egg-lecithin membranes decreases the overall permeability by affecting the dehydration step more than the subsequent diffusion of the solute. The incorpporation into liposomes of the antibiotics nystatin and gramicidin A produces changes in the selective permeation of nonelectrolytes that are consistent with the formation by these molecules of aqueous pores of fixed dimensions. Finally, comparisons are made between the process of permeation in biological membranes and in liposomes with and without antibiotics.


Handling and staining epoxy resin sections for light microscopy. Epoxy resin sections 0 1-1.0 mum thick of specimens embedded for electron microscopy, were collected from the ultramicrotome using strips of cover slip, and, after drying on a hot plate, were bulk stained in specially made troughs. After treatment with periodic acid, the sections were stained first in basic fuchsin at 70 degrees C and then in alkaline methylene blue at room temperature. The handling technique allows accurate collection of serial sections without interrupting the sectioning process, and the stain combination is suitable for different specimens in different epoxy resins and is unaffected by storage for more than 1 year.


Identification of nerve fibres in the scanning electron microscope. The use of silver as a "stain" for nerve fibres in scanning electron microscopy (SEM) preparations has been investigated. Samples of cardiac tissues were treated according to the Bielschovsky silver impregnation method. Following embedding in paraffin, successive sections were selected for light microscopy and for SEM studies, respectively. The silver impregnated fibres, when examined in SEM preparations, appeared nearly white against a greyish background of cardiac tissue. They were therefore easy to localize even at a low magnification. These nerve fibres were identified in the same tissue, but different blocks by means of transmission electron microscopy (TEM) studies and by fluorescence microscopy.


Preparation of nematodes for scanning electron microscopy. Nematodes from the orders Tlyenchida and Rhabditida were fixed and processed in several different ways for examination with the scanning electron microscope (SEM). Four processes produced good preparations of fixed nematodes. Drying from acetone was the simplest of these techniques and most useful for regions of the tylenchid nematodes supported by skeletal tissue. Critical point drying, a more complicated procedure, gave good preparations, but they required special care in processing. Nematodes infiltrated with glycerol and a conducting agent were the most life-like but were difficult to examine. Specimens infiltrated with an epoxy resin looked natural and this was the most promising process tried.


Antibody-induced capping of measles virus antigens on plasma membrane studied by electron microscopy. Antibodies specific for measles virus could redistribute ("cap") virus antigens on infected HeLa cells as shown by transmission and scanning electron microscopy. Using an indirect immunoperoxidase technique, infected cells showed diffuse, circumferential distribution of virus antigens over the cell surface when mixed with antibody at 4 C. At 37 C, virus-coated microvilli concentrated on one pole of the cell, leaving the remainder of the plasma membrane devoid of both viral antigens and microvillus projections. Whereas extreme polar displacement of virus-antibody complexes frequently occurred, endocytosis was rarely seen. The findings indicate that antiviral antibodies can move and cluster virus on plasma membranes and suggest that virus-antibody complexes are stripped and shed from the cell surface.


Biological and physical modifications of a murine oncornavirus by 2-deoxy-D-glucose. 2-Deoxy-D-glucose (2-DG) inhibited the release of transforming Kirsten murine sarcoma-leukemia virus [KiMSV(KiMuLV)] from transformed rat kidney (NRK-K) cells. At a concentration of 30 mM 2-DG, RNA synthesis in NRK-K cells was inhibited by approximately 30 percent and protein synthesis was inhibited by as much as 80 percent of control levels. RNA synthesis was not inhibited in nontransformed normal rat kidney (NRK) cells, although protein synthesis was equally suppressed in NRK and NRK-K cells. After treatment with 2-DG, the release of physical particles of KiMSV(KiMuLV) from NRK-K cels was not reduced as determined by equilibrium density gradient centrifugation and assays for RNA-dependent DNA polymerase of culture fluids. The ability to detect virion-associated radioactivity in equilibrium density gradients was dependent on the conditions of labeling. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of KiMSV(KiMulLV) proteins revealed marked structural alterations after propagation of the virus in 30 mM 2-DG. These alterations may account for the observed loss of transforming ability of KiMSV(KiMuLV).


Structural proteins of mammalian RNA tumor viruses: relatedness of the interspecies antigenic determinants of the major internal protein. The relatedness of antigenic determinants of purified major core proteins of the murine, feline, RD 114/baboon, and woolly monkey/gibbon ape groups of RNA tumor viruses was examined by competition radioimmunoassay. In assay systems of a homologous antigen and antiserum, high affinity competition for binding to all of the antibodies was observed only with the homologous unlabeled protein; the core proteins of other groups of viruses showed only low affinity binding of a small fraction of antibodies, presumably those reactive with the interspecies determinants, at concentrations of competing protein 10- to 100-fold greater than that of the labeled antigen. The cross-reactive (interspecies) antigens of every two viruses were selectively examined by precipitating the purified 125-I-labeled protein with antiserum against each of the other proteins. The extent to which these shared determinants were common to the other viruses was then tested by the effectiveness of the proteins of each virus to compete for antibody binding. Several classes of interspecies determinants were distinguished: those common to two of the groups of viruses, others to three, and some to all four. Moreover, an even greater variety of interspecies determinants was indicated by differences in the affinity of the individual proteins for antibody binding, supporting the hypothesis that there are at least several, if not many, different interspecies determinants with a broad spectrum of antigenic cross-reactivity. These studies suggest that the murine and feline viruses are closely related as they contain cross-reactive antigenic determinants not shared with the other viruses, that the feline virus is more closely related to the woolly monkey virus than to RD 114, and that the RD 114 and woolly monkey viruses retain interspecies determinants shared relatively equally with each of the other viruses.


Inhibition of viral transcriptase by immunoglobulin directed against the nucleocapsid NS protein of vesicular stomatitis virus. In search of an anti-transcriptase, antibody was raised in rabbits to partially purified, soluble NS protein present in cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis (VSInd) virus. This antiserum gave specific reactions of identity by agar immunodiffusion with both cytoplasmic and virion NS protein. NS antiserum also preferentially precipitated NS 3-H-labeled protein from infected cytoplasmic extracts, whereas anti-whole VSInd virion serum also precipitated N 3-H-labeled protein from extracts both of infected cytoplasm and virion nucleocapsids. Transcriptase activity of VSInd cytoplasmic or virion-derived nucleocapsids was effectively inhibited by ribonuclease-free immunoglubulin prepared from homologous NSInd antiserum or from anti-whole vesicular stomatitis virus serum. Transcriptase activity of heterologous New Jersey serotype (VSNJ) nucleocapsids and virions was not appreciably affected by anti-NSInd or by anti-whole VSInd virion gamma globulin. Anti-NS gamma glubulin immediately switched off RNA synthesis by actively transcribing VSInd nucleocapsids, a finding which suggests that NS antibody inhibits RNA chain elongation.


Serological studies with low-molecular-weight polypeptides from the Moloney strain of murine leukemia virus. Major virion low-molecular-weight polypeptides were isolated from the Moloney strain of murine leukemia virus (type C) by agarose chromatography in 6M guanidine hydrochloride and were shown to have molecular weights of 15,000 (p15), 12,000 (p12), and 10,000 (p10) by their elution volumes and by their relative mobilities in sodium dodecyl sulfate-polyacrylamide gels. Each polypeptide could be iodinated and employed in double antibody radioimmunoassay procedures. All three polypeptides demonstrated a high degree of type-specificity in serologic immunoprecipitation analysis and in corresponding competition immunoassays. The p15 was immunologically distinct from other viron polypeptides including p12 and p10; the p12 and p10 were highly related to each other but not to other virion polypeptides and were even more type-specific than the p15 in serologic tests. Competition immunoassays with p15 and p10 indicate that the Moloney strain of MuLV is only a distant relative of the Friend-Rauscher group. The combined use of the Kirsten and Moloney low-molecular-weight polypeptide immunoassays suggest that xenotropic viruses constitute yet another group(s) of murine leukemia virus with distinct type-specific antigens, further expanding an already heterogeneous group of mouse type C viruses.