Large-scale production of mouse mammary tumor virus in the absence of endogenous murine leukemia virus. A system for the large-scale production and purification of mouse mammary tumor virus in the absence of detectable endogenous murine leukemia virus is described. By utilizing the Mm5mt/c1 cell line established from an adenocarcinoma of a C3H mouse, the continuous production of over 25,000 liters of mouse mammary tumor virus-containing tissue culture fluids has been achieved. By the strict adherence to well-defined tissue culture conditions, mammary tumor virus production was accomplished without the expression of murine leukemia virus. Various biochemical and immunological systems were established for the rapid and precise detection of the endogenous leukemia virus, the expression of which could be enhanced under conditions of culture stress.


Human prostatic acid phosphatases: I. Isolation. A new purification procedure is described for the human prostatic acid phosphatase. The procedure included carboxy-methyl-Sephadex and Concanavalin A affinity column chromatography. The purified enzyme has a high specific enzyme activity and is free from any extraneous proteins judging from immunochemical criteria and biochemical criteria such as SDS-polyacrylamide gel electrophoresis. The purified enyzme produced a monospecific anti-PAP antisera in animals and this anti-PAP antibody did not cross-react with other human acid phosphatases.


Human prostatic acid phosphatases: II. A double--antibody radioimmunoassay. A double-antibody radioimmunoassay method for prostate-specific acid phosphatase (PAP) is presented. Experimental details are outlined to assess the reproducibility and reliability of the method under assay conditions. The upper limit of the serum PAP levels in the present assay was set at 2.4 ng/100 microliter by 162 determinations of normal serum samples. The serum PAP levels of patients with nonprostatic malignant tumors fell in the normal range, whereas the levels higher than 4.0 ng/100 microliter were found in patients with prostatic carcinoma.


Cytochemical identification of T and B cells in situ in mouse lymphoid tissue and lymph nodes from the rat, gerbil and cat. The use of enzymes as markers of T or B cells in tissue sections has been studied in mouse lymphoid tissue and lymph nodes from the gerbil, rat and cat. Lymphocytes in the T-cell areas of murine lymph nodes and spleen contained discrete dots of non-specific esterase and N-acetyl-beta-D-glucosaminidase (beta-glucosaminidase) activity, with weak acid phosphatase activity. Lymphocytes in the B-cell areas lacked this discrete staining. Cortical thymocytes contained slight esterase activity while medullary thymocytes were strongly positive for both esterase and beta-glucosaminidase. Lymphocytes with a T-cell staining pattern were only occasionally seen in lymph nodes from Nude (nu/nu) mice. ATPase staining was restricted to lymphocytes in the B-cell areas; weak 5'-nucleotidase staining was only present in a frew lymphocytes in both T- and B-cell areas. Blast cells stimulated by in vivo injection of ConA or PHA in the mouse showed strong discrete enzyme activity for non-specific esterase and beta-glucosaminidase. Lipopolysaccharide-stimulated blast cells and cells within germinal centres lacked this discrete staining. Comparison of lymph nodes from the gerbil, rat and cat suggested at least on enzymes as a T-cell marker in each species although considerable variation in staining profiles was seen in the different species.


Human prostatic acid phosphatases: III. Counterimmunoelectrophoresis for rapid identification. When serial dilution of standard prostatic acid phosphatases (PAP) was reacted with constant amounts of anti-PAP serum by counterimmunoelectrophoresis (CIEP), the detection end point of enzyme concentration was 0.25 ng in a 10 microliter sample volume. The PAP concentrations in unknowns can be quantitated by comparing the dilution end points of reference PAP with the testing samples. Serum PAP levels were determined by a radioimmunoassay (RIA) and CIEP using normal male and female sera and serum samples from patients with prostatic cancer and nonprostatic tumors. An excellent correlation was observed between the two assay results. According to RIA data, the concentration of PAP higher than 0.4 ng per 10 microliter (or 4.0 ng per 100 microliter) signify the elevation of serum or bone marrow PAP level beyond normal range (normal value 1.6 +/- 0.8 ng/100 microliter). Thus, the CIEP assay will be a simple and reliable screening method for the serum PAP levels in the clinical diagnosis of prostatic cancer.


Effect of glucose on 22Na+ efflux in pancreatic islets. Glucose provokes an immediate and possibly sustained increase in the fractional outflow rate of 22Na+ in isolated islets of Langerhans.


Mechanism of T-cell suppression in tolerance to HGG: arguments in favor of a T-cell-T-cell interaction. The specific soluble factor from suppressor T cells of CBA mice rendered tolerant to human gammaglobulin was further investigated. Immunochemical analysis proved the low zone tolerance (LZT) factor to be similar as the one described in high zone (HZT), e. g. absorbed by anti-Iak or IGG but not anti-HGG or anti-IgG. Molecular weight was ascribed as 45--55,000 by Sephadex chromatography and "Amicon" ultrafiltration. The HZT factor acts only if administered early after challenge, and on T cell as judged by double transfer experiments, suggesting impairment of early T-cell function (helper?) by suppressor cells.


Rat-liver cell compartition as revealed by correlations between redox-quotient changes following alloxan treatment, starvation, carbohydrate- and fat-diet. As reported elsewhere (FERAUDI, 1976a & b), we have studied the mathematical relations between metabolite concentrations in the rat liver at various redox states and expressed them algebraically. In the present work we have measured the liver-cell concentrations of lactate, pyruvate, glycerol 3-phosphate, dihydroxyacetone phosphate, malate, oxaloacetate, beta-hydroxybutyrate, acetoacetate, 2-oxoglutarate, ribulose 5-phosphate as pentose phosphates, gluconate 6-phosphate, isocitrate, aspartate in untreated and treated rats (alloxan-diabetic, insulin-treated alloxan-diabetic or starved rats as well as rats fed on carbohydrate- or fat diet). Through analysis of the algebraic correlation between metabolite concentrations, we arrived at the following statements: 1. Under certain physiological conditions the concentration of some metabolites in one compartment determines their total quantity in the cell; 2. NADP and NADPH are comparted within the cytosol; 3. Reduced cosubstrate/oxidized cosubstrate ratios of some enzymic reactions are under certain physiological conditions in mutual equilibrium; 4. Such relationships are first verified after treatment and therefore characterize the metabolite status.


Dynamic dialysis utilizing a hollow fibre unit as a rapid method for studying protein binding. A useful approach for determining the binding ability which occurres in a protein-small molecule system was studied by a method of dynamic dialysis utilizing a hollow fibre unit. A dependency on experimental variables in the absence of protein was examined in order to characterize the nature of the dialytic process. It was shown that the rate of small molecule escaped across the dialytic membrane was dependent on stirring or flowing rate, temperature and pH, but independent on buffer strength or viscosity. The protein binding ability was examined by means of comparing amounts of small molecules in the absence and presence of protein. Kinetic parameters for the binding behaviour analysed from the Scatchard plot in the present experiment were in agreement with values previously reported.


The relations between intestinal alkaline phosphatase and carbohydrates with regard to calcium absorption. The effect of carbohydrates on calcium absorption were studied in situ following the injection of a solution containing CaCl2 (+45Ca) into the ileal loop. The increase in Ca absorption was proportional to the concentration of carbohydrates injected and could be attributed to a progressive increase in the duration of absorption. In the ileal loop, sorbitol was much more effective than L-arabinose at equal concentrations in activating absorption. Such differences in the action of these carbohydrates were also observed in vitro with alkaline phosphatase extracted from the ileum. The transphosphorylating effect of the enzyme was much more pronounced in the case of sorbitol. Since the carbohydrate is a phosphate acceptor, it might influence the duration of absorption by reducing the inhibition exerted by phosphate upon a transfer mechanism which involves phosphatase, another possibility is that carbohydrate could postpone calcium insolubility through the formation of a phosphocarbohydrate complex.


Tissue specific glucose-trehalose variations during spinning in the eri-silkworm, (Philosamia ricini). The beginning of spinning process in Eri-silkworm is characterized by sharp fall in the levels of glucose and trehalose in silkgland which gets completely decomposed after 6 days of spinning. Fatbody actively synthesizes trehalose during early days of spinning which results in enhanced level of this disaccharide in haemolymph. On the other hand, decreased glucose concentration of body fluid during early days of spinning is apparently because of its absorption by silkgland for its energetic needs.


Diurnal rhythmicity in folic acid metabolism of rat liver and kidney. In rat liver and kidney, tetrahydrofolate formylase activities show diurnal rhythms with maxima in the middle of the dark phase. The rhythms are not generated by de novo synthesis of enzyme protein. Dihydrofolate reductase activity is arrhythmic in both liver and kidney.


About some common biochemical and pharmacological properties of bile salts and non steroidal anti-inflammatory drugs. Glyco- and tauro-deoxycholate, -chenodeoxycholate, -dehydrocholate and -cholate uncouple the mitochondrial oxidative phosphorylation in this order of potency. They also induce pseudo-energized mitochondrial swelling and lymphocyte swelling in the same order of potency. Similar swelling properties were observed for glyco- and tauro-lithocholate. Glyco- and tauro-deoxycholate induce alkalinization of the intra-mitochondrial space as determined on bromothymol blue pre-loaded mitochondria. These two bile salts conjugates also increase the amount of -SH groups available on lymphocyte membranes for sulfhydryl-disulfide interchange reactions. All these biochemical properties have been described for non-steroidal anti-inflammatory drugs. They could thus be implicated in the jaundice remission observed in various inflammatory states.


[Role of duodenal serotonin in the pharmacological effects of DOPA and dopamine in the isolated rat duodenum]. We have studied the responsibility of tissue serotonin reserves in the excito-motor effects induced by DOPA and dopamine on the isolated rat duodenum in vitro in certain experimental conditions. Two groups of experiments have been performed: first the determination of serotonin endogenous stores after administration of repeated high doses of DOPA and dopamine in the organ bath, secondly the evaluation of motor effects of DOPA and dopamine on rat duodenums experimentally depleted of their endogenous serotonin stores. Serotonin levels were lowered after DOPA and the excito-motor effect of this compound was suppressed in serotonin-depleted duodenums. After dopamine, serotonin tissue levels were not significantly lowered, and the excito-motor effect was observed whatever the serotonin stores may be, depleted or not. Our results are consistent with a relationship between the excito-motor effects of DOPA and serotonin release from endogenous stores; but, concerning dopamine, experimental proofs supporting this hypothesis have not been obtained.


Effect of massive proximal small bowel resection on intestinal brush border membrane proteins in the dog. Dog enterocyte brush border proteins have been studied after a 75% proximal resection of the small bowel. This study was carried on microvillar membrane preparations purified from ileal mucosa sampled before and after regeneration on neighbouring intestinal segments, each animal acting as its own control. After six weeks of regeneration a statistically significant decrease of the following enzyme specific activities was observed: lactase, cellobiase, maltase, sucrase, palatinase, dextranase, trehalase, alkaline phosphatase, aminopeptidase and gamma-glutamyl transferase. Analysis of brush border proteins by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate have shown after regeneration a decreased rate for the proteins with a molecular weight higher than 100,000 daltons. Modifications of electrophoretic patterns seem to be related to the specific activity decreases observed for brush border enzymes after regeneration, since the molecular weight of these enzymes were found between 116,000 and 285,000 daltons, after gel filtration.


Interaction of phenolsulphonphthalein dyes with rabbit plasma and rabbit serum albumin. Interaction of various substituted phenolsulphonphthalein dyes to rabbit plasma and rabbit serum albumin has been studied by ultrafiltration, equilibrium dialysis and spectrophotometry. The results obtained by ultrafiltration and equilibrium dialysis showed that the degree of binding of these dyes to protein increases in the following order: Phenol red less than bromophenol blue less than bromocresol green less than bromothymol blue. Analysis of binding results revealed that five molecules of bromothymol blue bound very strongly to a molecule of rabbit albumin, whereas only two and three molecules of bromophenol blue and bromocresol green strongly interact with the protein, respectively. It is suggested that strong binding of these substances to protein may be related to the hydrophobicity of these compounds. Finally, an attempt has been made to evaluate the possibility, whether the spectral changes occurred during interaction of dyes to albumin can be utilized for the determination of binding of these ligands to proteins.


Effects of denervation on the rate of entry of inorganic phosphate into rat slow and fast muscles: selective inhibition of denervation changes by actinomycin D. Actinomycin D abolishes the post denervation increase in inorganic phosphate flow observed in the fast gastrocnemius muscle. In the slow soleus muscle, the initial decrease in phosphate flow is unaffected but the secondary rise is suppressed in the same manner as in the fast muscle. These observations put the post denervation increase in inorganic phosphate flow on a par with the development of extrajunctional cholinergic receptors in being the result of the synthesis of new proteins. It has the added advantage of being suitable to quantitative assessment at the whole muscle level.


[Effects of diets rich in triglycerides and phospholipids on lipase and phospholipase A activities in rat pancreatic juice and pancreas]. Lipase and phospholipase A activities were increased in the pancreas and pancreatic juice of rats ingesting during two months 20% phospholipids- or triglycerides-rich diets. The activity of lipase is higher with the triglycerides-rich diet and the phospholipase A activity is higher with the phospholipids-rich diet. After seven days of the experiment, these two enzymes activities were less increased with diet containing 40% lipids.


[Comparison between the potential maps, first measured in a Rijlant's vectocardiographic network and secondly calculated for a homogenous sphere when, in both cases, the source and the sink are opposite on the external boundary]. In order to predict quantitatively how a Rijlant's vectocardiographic network operates, we did calculate the electrical potential map generated by two opposite poles situated at the ends of a diameter in a conducting sphere isolated on its external boundary. We also measured the potentials similarly arising at each point of the network. The relation between the corresponding values may be usefully approximated. These relations are the best ones but by no means the only ones, and thus any generalization is not yet possible.


[Evaluation of sino-atrial function using the method of extrasystole induced by constant-relative premature impulses. 1. Method and normal results]. Atrial pacemaking under conditions of relative constancy (40 or 50% of the preceding cycle) enables us to calculate the immediate sino-atrial conduction time (retrograde and antegrade) (SACT). 17 patients were chosen for their normal sino-atrial function under spontaneous changes of the sinus cycle (SC). In each case, a significant inverse linear relationship was found between SACT and the corresponding SC. The mean correlation slope was -0.36 in 10 patients with no post-pacing depression (PPD). The slope was greater in 7 patients with a PPD (-0.89); if this depression is taken into account when the SACT is calculated, the slope decreases. In 5 patients, atropine (1 mg I.V. reduced the mean value of SC, and shortened (constant relative value) the SACT. The mechanisms for the spontaneous and induced variations in the sinus output are discussed; it may be that there are substitute pacemakers within the cells of the sino-artrial node, which are affected by variations in sympathetic or parasympathetic activity or by pacing. In clinical practice, automatism and conduction with the sinus node should be interpreted as inter-related functions, both under normal conditions and after vagal block.


[Evaluation of sino-atrial function using the method of extrasystole induced by constant-relative premature impulses. 2. Application in 50 normal and abnormal studies]. The total immediate sino-atrial conduction time (SACT) as calculated by constant relative pacing, 40 or 50% of the immediate sinus cycle (SC), normally varies inversely with the SC. 50 patients were investigated by this method. In 38 patients without sino-atrial block (SAB) on the surface ECG, it was found that the mean slope of correlation between SACT and SC varied with the shape of the curve of Strauss and with the presence or absence of a post-pacing depression. The smallest slope was found in the group in which the curve of Strauss was horizontal in zone II, and in which there was no depression. In the groups with a rising zone II, comparative use of pacing with a fixed relationship in milliseconds suggested a phenomenon of decreasing retrograde conduction. In cases with sinus arrhythmia and a Strauss curve with scattered coordinates, the relationship between SACT and SC was maintained. 1st degree right-sided SAB should therefore be defined as a function of the immediate SC. In 12 other patients with SAB on the surface ECG, the immediate SACT was greatly lengthened so as to be immeasurable, and bore no relationship to the SC.


[Possibilities and various results of the use of contact microscopy in pathological practice]. A method for tissue staining for contact microscopy is described. Preliminary results of using a contact microscope in histological examinations of operation, biopsy and autopsy materials are presented. The proposed method permits a rapid microscopic diagnostic examination of diverse pathological material in any dissecting room of wide profile.


[Use of semithin sections from epoxy blocks in the cytological analysis of the retina]. The cytological features of the cell structure of various layers of the retina are revealed better and with greater detail when semi-thin sections from epoxy blocks are stained with basophilic stains (toluidine blue, methylene blue, thionine) than when conventional histological sections are stained with the same stains. Semi-thin sections are suitable for light microscope cytological analysis of relatively large segments of the retina (3 X 8 mm). In future, this material will probably be used for electron microscopic examinations. The currently available methods for staining of semi-thin sections with basophilic stains are relatively simple and produce stable results.


Routine antenatal tests.

Routine antenatal tests. Numerous tests have been proposed in recent years to screen the antenatal population for various diseases which might contribute to perinatal and even maternal morbidity and mortality. Screening tests of any kind are currently the centre of controversy as measures are being strenuously taken to contain health costs. This paper critically examines twelve such screening tests.


Carcinoma of the large bowel in patients aged 70 years and older. In a prospective series of 443 cases of large-bowel cancer there were 192 cases occurring in patients aged 70 years and over. The resectability rate in this group was 84.9% and was almost identical with that of the whole series (84.6%). There was an overall operative mortality rate of 7.3% in the older age group, compared with 6.4% in the whole series. However, when octogenarians were considered, the resectability rate fell to 74.6% and the overall operative mortality rate increased to 19%. In the treatment of large-bowel cancer it would seem that special consideration should be given to octogenarians.


Immunogenetic study on the polymorphism of serum alpha2-lipoproteins in mink. II. Identification of allotypes Lpm-7 and Lpm-8 and genetic control of seven markers of the Lpm system. By means of alloimmunization of mink, two new antigens, Lpm-7 and Lpm-8, were detected in their sera. Lpm-7 and Lpm-8 allospecificities were referred to a very high density alpha2-lipoprotein (Lpm) by the following criteria: histochemical tests, immunoelectrophoresis, preparative ultracentrifugation, and coalescence of alloprecipitates with heteroprecipitates in double diffusion tests. Genetic analysis indicated that Lpm-7 and Lpm-8, together with the earlier described Lpm-1, Lpm-2, Lpm-3, Lpm-4, and Lpm-5, share a common immunogenetic system. Polymorphism for the seven markers is conditioned by the genetic units Lpm8, Lpm4, Lpm4,8, Lpm4,7, Lpm3,4,8, Lpm1,8, Lpm1,2,7, and Lpm2,4,5,7, which behave as alleles. Of these units, the latter six are probably haploid sets of closely linked genes.


Effects of zinc ion on the conformation of antigenic determinants on insulin. Comparison of c.d. spectra of Zn-insulin with Zn2+-free insulin demonstrated significant differences. It has been proposed that these differences are due to either changes in the structure of insulin monomers within aggregated insulins or the results of insulin aggregation. The effect of Zn2+ on the immunological activity of insulin indicated that the antigenic determinants of insulin were also altered. The apparent loss of immunological activity of monoiodotyrosylinsulin was demonstrated to be due to the loss of Zn2+ rather than the substitution of iodine. The immunological activity of Zn-insulin and Zn2+-free insulin was compared in both the radioimmune and immune haemolysis-inhibition assays by using an identical population of antibodies and concentrations of inhibitor. Relative to Zn-insulin, Zn2+-free insulin had a markedly attenuated immunological activity in the immune haemolysis-inhibition assay, whereas in the radioimmune assay slightly greater immunological activity was observed with the Zn2+-free insulin. These observations are submitted as evidence that the removal of Zn2+ perturbs the conformation of determinants that react with antibodies operative in the immune haemolysis-inhibition assay (immune haemolysis determinants) and has a minimal effect on the conformation of determinants that react with antibodies operative in the radioimmune assay (radioimmune determinants).


Rat alpha-foetoprotein. Purification, physicochemical characterization, oestrogen-binding properties and chemical modification of the thiol group. 1. Rat alpha-foetoprotein, an oestrogen-binding foetal globulin, was isolated in large quantities from amniotic fluid and serum by preparative electrophoresis on polyacrylamide slab gels or by chromatography on an immunoadsorbent column. Subsequently the two electrophoretic forms of this protein were separated by electrophoresis on the same medium. 2. Both forms were found to show identical binding with oestradiol. From the extrinsic fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonic acid it was shown that the polarity of the binding site is practically identical for both forms. One residue of tryptophan was determined for both forms. The two electrophoretic variants display the same amount of secondary structure as demonstrated by circular dichroism. 3. The affinity of total alpha-foetoprotein for oestradiol as a function of pH was studied by using a Sephadex G-25 gel-equilibration method. Maximal binding occurred at pH8.5. Only a fractional number of binding sites per molecule could be measured at pH7.4, whereas at higher pH the number of sites was very close to unity. There was no significant effect of pH on the value of the association constant (K(a)=4.3x10(7)+/-1.2x10(7)m(-1)). 4. Displacement experiments of bound labelled oestradiol with various steroids have permitted investigation of the specificity of alpha-foetoprotein. This foetal globulin binds rather strongly compounds that display the rigid structure of the oestratriene skeleton (oestradiol, oestrone). Diminished binding for diethylstilboestrol and a diethylstilboestrol affinity label was observed. No binding was measured with a more flexible structure such as hexoestrol [4,4'-(1,2-diethylethane-1,2-diyl)bisphenol]. 5. Chemical modification of cysteine residues of alpha-foetoprotein with two alkylating reagents [iodoacetic acid and 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid] has very little effect on the oestrogen binding. It is suggested that the oestrogen-binding site does not contain a cysteine residue. From the kinetics of alkylation and from the fluorescence properties of the chemically bound thiol reagent 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid], it was demonstrated that the very-slow-reacting thiol group is probably located in a non-polar region of the molecule.


A comparison of maternal serum levels of alpha-fetoprotein in normal and pre-eclamptic pregnancies. Alpha-fetoprotein (AFP) concentrations were measured by radioimmunoassay in serum from women with normal and pre-eclamptic pregnancies. An analysis of the results obtained in normal pregnancy was made using arithmetic and semi-logarithmic scales, and a statistical conversion of the AFP values in relation to gestational age was introduced to allow an easier interpretation of results. In pre-eclampsia, significantly lower mean AFP values were obtained, with the majority of individual values being lower than the mean for normal pregnancy. These low levels were not associated with fetal death, but appeared to be related to the severity of disease. The significance of these findings remains to be evaluated.


Hematologic and serologic studies in 6-sulfanilamidoindazole arthritis. 6-sulfanilamidoindazole (6-SAI), an antibacterial sulfonamide, is known to cause an acute, self-limited inflammatory reaction in the hind paws of older rats. Administration of 6-SAI results in hyperfibrinogenemia which precedes the onset of clinical arthritis. The hyperfibrinogenemia is associated with the prolongation of dilute blood clot lysis times, appearance of heparin-precipitable fibrinogen, elevations in fibrin degradation products, and complement titers and serum electrophoretic pattern changes that consist of decreased albumin concentrations and increased beta globulin concentrations. Alterations in 6-SAI medicated young rats are less marked and require higher doses.


Inhibition of replicative DNA synthesis and induction of unscheduled DNA synthesis in permeable sarcoma  cell by bleomycin. Unscheduled DNA synthesis was induced by bleomycin in isolated rat liver nuclei and in permeable mouse ascites sarcoma cells. ATP significantly enhanced the bleomycin effect of inducing unscheduled DNA synthesis. Replicative DNA synthesis in permeable mouse ascites sarcoma cells was inhibited by bleomycin. The apparent inhibition or stimulation by bleomycin of in vitro DNA synthesis was thought to be determined by a balance between inhibited DNA replicase activity and induced unscheduled DNA synthesis.


Aminoacylation of undermethylated mammalian transfer RNA. To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.


Informosomal and polysomal messenger RNA. Differential kinetics of polyadenylation and nucleocytoplasmic transport in Chinese hamster ovary cells. The relative kinetics of cytoplasmic appearance and polyadenylation were determined for informosomal (ribosome-free) and polysomal (ribosome-associated) mRNAs of cultured Chinese hamster cells. Label appeared in polysomal mRNA 17--20 min and into informosomal mRNA 2--5 min after addition of radio-labelled uridine. Adenosine appeared in polysomal mRNA 4--5 min before uridine. In contrast, adenosine label preceded uridine into informosomal mRNA by less than 1 min. About one-third of newly formed informosomal and two-thirds of newly formed polysomal mRNA are poly(A+). The data indicate that newly formed informosomal mRNA cannot be simple precursor to polysomal mRNA. Further, the pronounced difference in time required for polyadenylation and cytoplasmic  appearances of these messenger ribonucleoproteins suggests that there may be fundamental differences in their mode of processing.


Light-induced pH changes in sub-bacterial particles of Halobacterium halobium. Effects of ionophores. The kinetics of light-induced acidification and of the subsequent dark-induced alkalization in suspensions of sub-bacterial particles of Halobacterium halobium may be expressed as the sum of two exponentials, indicating two processes (Eisenbach, M., Bakker, E.P., Korenstein, R. and Caplan, S.R. (1976) FEBS Lett. 71, 228--232). We studied the effects of carbonyl cyanide p-trifluoromethyoxy phenyl-hydrazone, nigericin, gramicidin D, valinomycin, and monactin on the extents and the rate constants of the two processes. The various ionophores affected the two processes differently and in general the slower process was more sensitive to their presence. Valinomycin and monactin had relatively minor effects, apparently due to the high ionic strength of the suspension. When an artificial membrane potential was created in the dark, the light-induced acidification was preceded by a transient alkalization as is usually observed in intact cells. These results are discussed in the light of a suggested model accounting for the two processes (Caplan, S.R., Eisenbach, M., Cooper, S., Garty, H., Klemperer, G. and Bakker, E.P. (1977) in Bioenergetics of Membranes (Packer, L., Papageorgiou, G.C. and Trebst, A., eds.), pp. 101--114, Elsevier/North-Holland Biomedical Press, Amsterdam), taking into account the different selectivities of the ionophores applied.


Microheterogeneity of rat, mouse and human alpha1-fetoprotein as revealed by polyacrylamide gel electrophoresis and by crossed immuno-affino-electrophoresis with different lectins. Polyacrylamide gel electrophoresis and crossed immuno-affino-electrophoresis with several free lectins have been used to characterize and to compare the molecular heterogeneity of rat, mouse and human alpha1-fetoproteins. Each alpha1-fetoprotein contains a variable number of electrophoretic variants depending on the gel porosity. In SDS electrophoresis, two molecular size populations are present in rat alpha1-fetoprotein (Mr = 74 000 and 72 000) and in mouse alpha1-fetoprotein (Mr = 73 000 and 72 000) but only one is observed in human alpha1-fetoprotein (Mr = 70 000). The crossed immuno-affino-electrophoresis patterns square with affinity chromatography results and reveal a marked and characteristic heterogeneity for the three alpha1-fetoprotein species with Concanavalin A, Ricinus communis and Lens culinaris lectins. No lectin-alpha-fetoprotein interaction is apparent with Ulex, Lotus and wheat germ lectins. Since similar patterns are obtained whether with purified alpha1-fetoprotein or with unfractionated fresh fetal sera, it is likely that this heterogeneity is not a consequence of artefactual molecular modifications arising during the purification procedure.


Cyclic AMP synthesis in Xenopus laevis oocytes: inhibition by progesterone. [alpha-32P]ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.


[Biosynthesis of beta-phenylethanol in rose petals]. Biosynthesis of beta-phenylethanol is studied in a cell-free rose petals extract. This compound found to be synthesized via two intermediates: either trans-cinnamic acid, or phenylacetaldehyde. Subsequently beta-phenylethanol is glucosodized into its transport form, beta-phenylethanol- beta-D-glucoside. A scheme of beta-phenylethanol and its beta-D-glucoside is given.


Synthesis of alpha-fetoprotein, albumin and total serum protein in neonatal pigs. Studies on the incorporation of 3H-leucine established that synthesis of alpha-fetoprotein (AFP) does occur in the neonatal pig. This synthesis probably accounts for the increase in AFP concentration from 0.46 mg/ml on the day after birth to the maximum value of 1.02 mg/ml found 5 days after birth. After the 5th day, the concentration decreases successively and AFP cannot be detected by electroimmunoassay in the sera of 8-week-old pigs. During the first 3 weeks of extrauterine life, the albumin concentration in serum increases from 3 mg/ml to approximately 30 mg/ml due mainly to increased synthesis of albumin. The concentration of AFP in the fetal pig serum decreases from 3 mg/ml at 6.5 cm crown-to-rump (CR) length to 0.8 mg/ml at 28 cm CR length, whereas the albumin concentration increases from 0.2 mg/ml at 6.5 cm CR length to 1.0 mg/ml at 28 cm CR length. The total serum protein content in the fetus is approximately 20 mg/ml irrespective of gestational age. During the first day after birth there is a marked increase in both the synthesis and the concentration of total serum proteins. The concentration rises to 70 mg/ml during the first day and then slowly declines to 65 mg/ml during the 3 weeks following birth.


Relationship of incorporation of radioprecursors into protein and phospholipids of the plasmalemma of Guinea pig (neonate) intestinal epithelium and the cessation of uptake of macromolecules (closure). The incorporation of radioprecursors into intestinal epithelial cell proteins and phospholipids was studied to assess the role of protein and phospholipid synthesis and/or turnover in the cessation of the absorption of macromolecules (closure). Radiophosphorus incorporation into cellular phospholipids was enhanced when pinocytosis was stimulated. The specific activity of cellular and brush border phospholipids and specific phosphatides increased during the period of active endocytosis. No alteration in specific phosphatide percent composition was observed. Radioamino acid incorporation into cellular protein was not influenced by age. These data are consistent with the idea that the period of active macromolecule absorption by the guinea pig intestinal epithelium is defined by the exhaustion of membrane available for endocytosis.


Small angle x-ray diffraction studies of mucopolysaccharides in collagen. Small angle X-ray diffraction (SAXD) was used to locate mucopolysaccharides (MPS) at regular intervals along the collagen axis under physiological conditions. Ruthenium red was used to stain the MPS specifically. The difference in electron density between ruthenium red-stained and unstained moist native rat tail tendon should correspond to the position of the MPS. This difference was calculated from the SAXD intensity data by using difference Fourier transform calculations. Phases calculated independently from the amino acid sequence of collagen by two laboratories were used in this calculation, and the results were compared. At least four to seven bands of MPS per 660 A were found at regular intervals along the collagen axis. Some of these bands match in position to the cross-striations observed by freeze-etching. Electron micrographs of ruthenium red-stained native fibrils also showed bands close in position to the ones calculated.


Specificity of interproton nuclear Overhauser effects in gramicidin-S dissolved in deuterated ethylene glycol. The 250-MHz high-resolution proton magnetic resonance spectra of gramicidin-S in solution in deuterated methanol, deuterated ethylene glycol, and binary mixtures of these solvents have been recorded. Starting from previously published partial assignments for deuterated methanol solution, the solvent transition yields partial assignments in deuterated ethylene glycol solution. In the latter the rotational correlation time for the peptide backbone, tauc, is calculated to be 14 ns at 25 degrees C. The long tauc leads to proton spin relaxation behavior that mimics that of moderate-sized proteins in water, and yields negative nuclear Overhauser effects, which have been measured for the protons of the phenylalanine ring. The results suggest that there is rapid and efficient spin-diffusion within closely-connected "islands" of protons, and less efficient spin-diffusion between islands. The results are compatible with the accepted solution conformation of gramicidin-S.


The quantitation of nuclear Overhauser effect methods for total conformational analysis of peptides in solution. Application to gramicidin S. The [1H:1H] nuclear Overhauser effects (NOE's) and spin-lattice relaxation times (T1's) are reported for the backbone protons of the decapeptide gramicidin S. Several methods for calculating interproton distances from these measurements are presented. Ratios of interproton distances were obtained from [1H:1H] NOE's and from the combination of [1H:1H]NOE'S and T1 values. Actual proton-proton distances were calculated from these ratios either by using the known distance between two geminal protons or distances derived from scalar coupling constants. The interproton distances calculated for gramicidin S are consistent with a II' beta-turn/antiparallel beta-sheet conformation.


Orientation of nuclei as indicators of smooth muscle cell alignment in the cerebral artery. Geometric measurements were made of the nuclei of the muscle cells of the tunica media of human intracranial arteries. The nuclei measured 37 +/- (SD) 6.4 micron in length, and 2.0 /+- (SD) 0.83 micron average width and have a number density of 10(5) nucei/mm(3) (number per unit volume of tunica media). From the work of others it was known that the nuclei are centrally located within the cytoplasm of the muscle cell; because of this we have used the nuclei as indicators of alignment of the muscle cells themselves, making measurements from light micrographs of arterial sections stained with haematoxylin and eosin. On average, the nuclei were oriented at an angle that was not significantly different from a zero-degree pitch for the five sections analyzed in detail.


Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187. The role of endogenous adenosine triphosphate (ATP) in histamine release from rat mast cells induced by the ionophore A23187 in vitro has been studied. 2 The amount of histamine released by calcium from rat mast cells primed with the ionophore A23187 was dependent on the ATP content of the mast cells. 3 In aerobic experiments a drastic reduction in mast cell ATP content was found during the time when histamine release induced by A23187 takes place. 4 Anaerobic experiments were performed with metabolic inhibitors (antimycin A, oligomycin, and carbonyl cyanide p-trifluorometroxyphenylnydrazone), which are known to block the energy-dependent calcium uptake by isolated mitochondria. The mast cell ATP content was reduced during A23187-induced histamine release under anaerobic conditions in the presence of glucose. This indicates an increased utilization of ATP during the release process. 5 The observations are consistent with the view that energy requiring processes are involved in ionophore-induced histamine release from rat mast cells although part of the ATP reduction in the aerobic experiments may be due to an uncoupling effect of calcium on the oxidative phosphorylation.


Cryosurgery in the management of rectal tumours. Ten patients with rectal tumours were treated by cryosurgery. Good palliation without local complications was achieved in the cases of rectal carcinoma and a complete cure followed treatment of benign rectal tumours. Low rectal carcinomas with clinical evidence of dissemination are ideally suited for cryosurgery and complications are minimal. Cryosurgery is a simple, safe procedure which can be performed without a general anaesthetic, and this study indicates that it has a place in the management of rectal tumours.


The cells of origin of the commissural afferents to the area dentata in the mouse. The hippocampal commissural projection to the area dentata of the mouse was studied using the retrograde horseradish peroxidase (HRP) technique. Small volumes of HRP injected into the molecular layer of the fascia dentata or various subareas of regio inferior of the hippocampus (fields CA3a-c) resulted inlabeled perikarya in the contralateral hippocampus and area dentata. The commissural projection to the fascia dentata was observed to originate exclusively from cells within the hilus fasciae dentatae (CA4) of the contralateral area dentata. There was evidence of a considerable spread of commissural innervation along the septotemporal axis preferentially in the septal direction, confirming earlier observations. In contrast to the septotemporal spread, a sharp homotopic spatial organization was found in the mediolateral direction. For example, injections into the lateral portion of field CA3 (CA3a) resulted in HRP-positive cell bodies only in the contralateral field CA3a. When injections were made which apparently labeled all of the commissural fibers, the HRP reaction product was found in neurons both in the entire regio inferior and as far as the innermost point of the hilus fasciae dentatae; the majority of labeled cells were located in hippocampal subfield CA3c. No labeled cells were observed beyond the tip of the mossy fibers in regio superior.


Radioautographic characterization of a serotonin-accumulating nerve cell group in adult rat hypothalamus. Intensely labeled nerve cell bodies were identified by radioautography within the pars ventralis of nucleus dorsomedialis hypothalami (hdv), following intraventricular perfusion with 10(-5) or 10(-4) M tritiated serotonin [3H]5-HT in adult rats pretreated with a monoamine oxidase inhibitor. This selective reaction, which involved approximately 1000 neurons on each side of the third ventricle, was unaltered by concomitant administration of 10(-3) M non-radioactive norepinephrine, and was absent after intraventricular injection of 10(-5) or 10(-4) M tritiated norepinephrine. The 3H-labeled 5-HT nerve cell bodies were loosely grouped within the inner and caudal half of the hdv, and appeared morphologically similar to the unreactive neurons among which they were interspersed. Within the same region, numerous labeled axonal varicosities were also detected, which were never found in synaptic contact with the reactive cells. If the 3H-labeled 5-HT neurons contain endogenous 5-HT, they might constitute an intrinsic source of 5-HT innervation in the adult rat hypothalamus.


Retinofugal pathways in fetal and adult spiny dogfish, Squalus acanthias. Retinofugal pathways in fetal and adult spiny dogfish were determined by intraocular injection of [3H]proline for autoradiography. Distribution and termination of the primary retinal efferents were identical in pups and adults. The retinal fibers decussate completely, except for a sparse ipsilateral projection to the caudal preoptic area. The decussating optic fibers terminate ventrally in the preoptic area and in two rostral thalamic areas, a lateral neuropil area of the dorsal thalamus and more ventrally in the lateral half of the ventral thalamus. At this same rostral thalamic level, a second optic pathway, the medial optic tract, splits from the lateral marginal optic tract and courses dorsomedially to terminate in the rostral tectum and the central and periventricular pretectal nuclei. The marginal optic tract continues caudally to terminate in a superficial pretectal nucleus and also innervates the superficial zone of the optic tectum. A basal optic tract arises from the ventral edge of the marginal optic tract and courses medially into the central pretectal nucleus, as well as continuing more caudally to terminate in a dorsal neuropil adjacent to nucleus interstitialis and in in a more ventrally and medially located basal optic nucleus. Comparison of the retinofugal projections of Squalus with those of other sharks reveals two grades of neural organization with respect to primary visual projections. Squalomorph sharks possess a rostral dorsal thalamic nucleus whose visual input is primarily, if not soley, axodendritic, and an optic tectum in which the majority of the cell bodies are located deep to the visual terminal zone. In contrast, galeomorph sharks are characterized by an enlarged and migrated rostrodorsal thalamic visual nucleus, and an optic tectum in which the majority of the cell bodies are located within the visual terminal zone. These data suggest that evolution of primary visual pathways in sharks occurs by migration and an increase in neuronal number, rather than by the occurrence of new visual pathways.


Management of carcinoma of the extrahepatic bile ducts. The records of 80 cases of carcinoma of the extrahepatic bile ducts were studied. The median duration of survival, in months, of patients who were treated by curative surgery, palliative surgery and radiation, palliative surgery alone and biopsy alone was 21.7, 9.3, 5.5 and 1, respectively. The best results were obtained by excision of the tumour, but only 10% of tumours were considered resectable. A more aggressive surgical approach to tumours still confined to the bile ducts might be expected to increase the resectability rate and improve survival. The use of a U tube is recommended because it ensures greater comfort even though survival is no longer than when a T tube is used. The roles of adjuvant radiotherapy and chemotherapy need further study. Because of some encouraging responses with the use of radiotherapy and the infusion of 5-fluorouracil the authors are currently evaluating these two methods of treatment.


Biology and virology of the human malignant lymphomas: 1st Milford D. Schulz Lecture. Permanent cell lines have been established from twelve diffuse histiocytic lymphomas (SU-DHL-1 to -12), three American Burkitt's lymphomas (SU-AmB-1 to -3), two acute lymphoblastic leukemias (SU-ALL-1 and -2), and three diffuse undifferentiated lymphomas (SU-DUL-1, -2, and -3). The cultured cells displayed neoplastic characteristics, as manifested by heterotransplantability in congenitally athymic nude mice and by the presence of cytogenetic abnormalities in early passage generations. Functional and marker studies revealed that the three American Burkitt's lymphomas, as well as several of the diffuse histiocytic and undifferentiated lymphomas, were of B-lymphocytic origin, whereas the two acute lymphoblastic leukemias were both of T-lymphocytic origin. Two of the cell lines, SU-DHL-1 and -2, appeared to be of true histiocytic origin; two others exhibited no markers and were designated as "null" cells. All ten of the DHL cell lines studied to date, as well as SU-DUL-1, have been devoid of Epstein-Barr virus (EBV) genomes by the EBNA test, whereas two of the three American Burkitt's lymphoma cell lines were positive. Spontaneous production of a C-type RNA virus was first detected in post-mitochondrial cytoplasmic fractions and culture fluids of the SU-DHL-1 cell line. Screening assays for the detection of reverse transcriptase-positive particles in the culture fluids of the other cell lines indicate that eight of the fifteen cell lines tested to date have spontaneously initiated C-type RNA virus production. After partial purification by ion-exchange and affinity chromatography, the reverse transcriptases of the virus isolated from SU-DHL-1 cells is partially inhibited by antibodies to the reverse transcriptases of C-type viruses of subhuman primate and endogenous feline, but not of murine, origin. Conversely, antibody prepared against the purified SU-DHL-1 viral reverse transcriptase, at concentrations which maximally inhibit the homologous enzyme, partially inhibits the reverse transcriptases of subhuman primate C-type viruses, but has little or no inhibitory activity against the reverse transcriptases of feline or murine leukemia viruses. The viruses produced by the SU-DHL-1 and SU-AmB-3 cell lines have been shown to be infectious for normal human peripheral blood mononuclear cells, normal human bone marrow cells, and certain human lymphoblastoid cell lines. After infection by these viruses, normal human peripheral blood mononuclear cells and human bone marrow cells have exhibited striking changes in growth behavior and morphology which, though not permanently sustained, have many of the features of abortive transformation.


Sequential hemi-body radiotherapy in advanced multiple myeloma. Eleven patients with advanced multiple myeloma refractory to standard chemotherapy were treated with a regimen of sequential hemi-body radiotherapy consisting of 800 rad midplane in a single dose to each half. 9/10 patients experienced significant relief of skeletal pain and there were 5/11 objective tumor responses with one complete remission. Treatment-related morbidity was significant and consisted primarily of nausea and emesis, bone marrow suppression, and pneumonitis. This therapy is helpful in the management of advanced myeloma, and should be studied earlier in the course of the disease.


Aortico-pulmonary paraganglioma: report of a case with ultrastructural study and review of the literature. The light microscopic and ultrastructural features of an aortico-pulmonary paraganglioma (A-PP) are presented. The tumor was characterized by organoid clustering of neoplastic chief cells to form Zellballen. Argyrophilic granules were demonstrated within chief cell cytoplasm using a modified Grimelus technique. Ultrastructurally, three distinct cell types were present within the tumor: endothelial cells, pericytes and neoplastic chief cells. Membrane-bound neurosecretory granules were present and measured 100 to 2000 nm in diameter. "Light" and "dark" chief cells were less distinct than previously reported in other head and neck paragangliomas. Analysis of the 36 documented A-PP reported in the English literature reveals that the tumor has been either incompletely excised or has been considered unresectable in one-third of the cases. The reported surgical mortality is 9%, or approximately equal to the incidence of malignant behavior. The treatment of choice is surgical resection but when this is not possible, radiation may be a useful adjunct in therapy.


Arterial occlusion of pelvic bone tumors. Arterial occlusion of the internal iliac artery was successful in the relief of pain due to primary and secondary neoplasms of the bony pelvis in 8 of 9 patients. These included 3 giant cell tumors, 1 aneurysmal bone cyst, 1 recurrent chondrosarcoma, 3 metastatic renal cell carcinoma and 1 metastatic clear cell sarcoma. Calcification of the margin of the lesion occurred in 3 of 4 primary neoplasms after infarction. The transcatheter arterial occlusion was accomplished utilizing Gelfoam and stainless steel coils. Although most patients experienced pain and fever for several days following the procedure, no permanent sequelae or complications were encountered.


Serum acute-phase proteins and immunoglobulins in patients with gliomas. Cellular immune competence was found to be impaired in previous studies of patients with malignant brain tumors. In patients with nonneural tumors, we recently found that serum levels of acute-phase proteins were related to immune status as well as to tumor extent. To determine whether the serum proteins in patients with central nervous system tumors show similar changes, levels of acute-phase proteins (alpha1-acid glycoprotein, alpha1-antitrypsin, haptoglobin, C-reactive protein) and immunoglobulins (immunoglobulins G, M, and A) were assayed in patients with gliomas prior to treatment. Compared to normals, significant increases (p less than 0.001) in the acute-phase proteins were found, and the levels were similar to those in patients with nonneural solid neoplasms. Serum immunoglobulins were not significantly increased in patients with gliomas.


Antigenic specificity and cellular mechanisms in leukocyte adherence inhibition analysis of immunity to simple proteins and hapten-protein conjugates. The hemacytometer leukocyte adherence inhibition (LAI) assay was investigated with respect to immunological relevance, specificity, and cellular mechanisms. Humans were immunized to keyhole limpet hemocyanin, and rats were immunized to dinitrophenyl-bovine gamma-globulin. LAI analysis disclosed classic patterns of immune response kinetics. The LAI response was dose dependent in vitro with no inhibition at relatively high antigen doses. In vitro specificity in rats was restricted to the immunizing conjugate. Cells forming spontaneous E-rosettes were required for LAI reactions. Lymphokine production required the presence of E-rosette-forming cells. E-rosette-forming cells from normal donors lost adherence in the presence of lymphokine. The requirement for T-lymphocytes was confirmed in a human osteosarcoma system using independent criteria. Thus, the hemacytometer LAI depends upon T-lymphocyte collaboration via a lymphokine. It should be distinguished from the tube and microplate variants of LAI analysis because these appear to depend upon different mechanisms.


Association of melanoma tumor antigen activity with beta2-microglobulin. The majority of melanoma tumor antigen activity present in melanoma extracts derived from fresh tumor tissue binds to a Sepharose-anti-beta2-microglobulin adsorbent. Removal of HLA antigens from the extracts of melanoma tissue by using a KBr flotation technique did not reduce either the tumor antigen activity of the extracts or the binding of melanoma tumor antigen (MTA) activity to the Sepharose-anti-beta2-microglobulin adsorbent. The complete blocking of MTA activity by pretreating the anti-beta2-microglobulin adsorbent with beta2-microglobulin and the lack of detectable MTA binding to a Sepharose anti-normal human serum adsorbent demonstrated the specificity of the binding of MTA to the anti-beta2-microglobulin adsorbent.


Hemocytometer leukocyte adherence inhibition technique. The hemocytometer leukocyte adherence inhibition technique was employed in a criss-cross experimental design with two cancer patients (melanoma and colon carcinoma) and the corresponding tumor extracts. These extracts had been repeatedly tested for specific reactivity and lyophilized before transport to the workshop. The patients' leukocytes were mixed singly with each extract in the presence of normal serum, and the adherences of the cells were determined in hemocytometer chambers. Actual cell counts (total cells before washing and adherent cells after washing) are given in detail for the first time. Blood samples and reaction mixtures were coded by an independent observer. Determination of mean % adherence (+/- S.E.) showed that the melanoma patient's leukocytes (original adherence 70.8 +/- 2.8) reached with the melanoma extract (40.7 +/- 2.9; p less than 0.001) but not significantly with the colon carcinoma extract (61.5 +/- 4.1; p greater than 0.05). Similarly, the colon carcinoma patient's leukocytes (original adherence 68.6 +/- 2.7) reacted with the colon carcinoma extract (43.2 +/- 2.3; p less than 0.001) but adherence was not inhibited by the melanoma extract (76.9 +/- 2.6). The cancer patients were thus correctly identified with regard to tumor type in a simple blind trial.


Demonstration of tube leukocyte adherence inhibition assay with coded samples of blood. Heparinized samples of blood from three different patients were coded by impartial observers. The buffy coat leukocytes from the coded samples of blood were isolated and incubated separately with extracts of colon and pancreatic cancer in the tube leukocyte adherence inhibition assay. At the completion of the assay, the leukocytes from Patient 1 were equally nonadherent to both cancer extracts with a nonadherence index value of 8. By contrast, leukocytes from Patient 2 exhibited increased nonadherence to the extract of colon cancer (p = 0.02) with a nonadherence index value to colon cancer antigen of 89. Leukocytes from Patient 3 displayed increased nonadherence to the extract of pancreatic cancer (p less than 0.05) with a nonadherence index value to pancreatic cancer antigen of 39. When the code was broken, patients 1, 2, and 3 had diagnoses of malignant melanoma, colon cancer, and pancreatic cancer, respectively. Hence, this was a classical criss-cross experiment; the patient with malignant melanoma reacted to neither of the antigens, whereas the patients with colon and pancreatic cancer reacted to the sensitizing cancers which had unique organ-type specific neoantigens.


The need to establish whether the leukocyte adherence inhibition test is a reliable assay of tumor immunity in humans. Lymphocytes from human cancer patients can react to tissue-type-specific antigens shared by many tumors of the same histological type and different for tumors of different types. Such reactions have been detected, for example, by using microcytotoxicity assays and leukocyte migration inhibition techniques. Work presented at this workshop indicates that the leukocyte adherence inhibition test is an excellent assay for detecting reactivity to antigens shared by neoplasms of the same histological type. However, very little is known of the nature of these antigens; for example, we do not know whether they are tumor specific or just normal tissue antigens. The usefulness of the leukocyte adherence inhibition test for patient monitoring and for diagnostic purposes also needs to be studied more.


[Demonstration of amyloid using histological methods]. The authors present their personal experience with common histological methods for amyloid demonstration. For practical use, they recommend staining with Congo Red followed by differentiation in alkaline alcohol and another method using staining with a 2% solution of Saturn Red in 1% NaCl. The latter method permits to differentiate between amyloid and hyalin without employing polarized light.


Increased in vitro histamine release by radiographic contrast media in patients with history of incompatibility. This study was designed to compare in vitro leucocyte histamine release in patients with a history of previous radiographic contrast media (RCM) reactions and normal controls. Peripheral leucocytes of ten patients with a positive history of RCM imcompatibility and nineteen normal volunteers were stimulated in vitro with different RCM in different concentrations and the amount of histamine released was measured in the supernatant. There was a significant increase in histamine release induced by RCM in low doses (0.02-0.1 M) in the patients as compared to the normals. At the high doses (0.2-0.3 M), no significant differences were found. Leucocytes from four of the patients were stimulated preferentially by the dye responsible for the incompatibility. Six patients showed no such preference. The increased "releasability" of the patients' leucocytes could not be transferred by serum. Normal leucocytes, when incubated with serum from "high releasing" patients did not show increased histamine release after stimulation with the respective dye. It is suggested that an excessive non-immunological response of basophil leucocytes to RCM stimulation might, in part, account for the adverse clinical reactions observed. Furthermore, leucocyte histamine release might be a useful diagnostic tool for detecting patients with a high risk of developing contrast media reactions.


Detection of sulphated carbohydrates in primitive yolk granules of the hen's oocyte. Application of several staining methods specific for sulphated carbohydrates have indicated the presence of one or more of these macromolecular species in primitive yolk granules of the hen oocyte. The rationale for the various methods used are detailed in the Materials and methods section. Discussion centres on whether the substances taking up stain may be chondroitin sulphates or other sulphated glycosaminoglycans and the possible roles of such molecules in early development.


Effects of distamycin A on human leukocytes in vitro. Distamycin A, an oligopeptide antibiotic, supplied at various concentrations for 24 h to human leukocytes in culture, has induced the appearance on some chromosomes of specific areas lacking spiralization. In particular, the centromeric regions of chromosomes 1, 3 and one C-group chromosome and the distal part of the long arm of the Y chromosome were despiralized. The possible nature of these regions is discussed.


Identification of the facultative heterochromatic X chromosome in females of 25 rodent species. Treatment of the chromosomes of 25 rodent species with a 50 degrees C hypotonic solution and Giemsa staining permitted identification of the heterochromatic X chromosome in 24 species. With this technique, the facultative of the heterochromatic X chromosome or the facultative portion of large, composite-type X chromosoms is stained darker than the other chromosomes, allowing it to be distinguished from the homologous euchromatic X chromosome in female metaphase cells. Intense staining of the single X chromosome was not observed in male metaphase cells. It is suggested that this differential staining of one of the two X chromosomes might be due to qualitative differences in chromosomal proteins rather than to differences in the degree of chromosomal condensation or in DNA base sequence.


Induction of high frequencies of endoreduplication in mammalian cell cultures with 33258 Hoechst and rubidazone. Cells of the mouse L strain and the Chinese hamster CHO line were treated with 33258 Hoechst, rubidazone, and a combination of these. Recovering cell populations following the drug removal exhibited a high frequency (20--50%) of metaphases with diplochromosomes (endoreduplication), especially in the combination treatment series (up to more than 70% in the L strain). Such a procedure should be useful in probing the mechanisms of the endoreduplication process.


Comparison of the karyotypes of four Cercopithecoidae: Papio papio, P. anubis, Macaca mulatta, and M. fascicularis. The karyotypes of two species of baboons, Papio papio and P. anubis, and of two species of Macaca, M. mulatta and M. fascicularis, are compared after the use of numerous banding techniques. No difference was detected between the karyotype of the two Papio species. However, a minor change in the T-staining of a short segment, probably heterochromatic, could be detected between the Papio species and M. mulatta. A paracentric inversion exists between these three and M. fascicularis. These karyotypes are briefly compared with those of the Pongidae and man. The value of the karyotypic criteria and of the methods used for taxonomy is discussed.


Reassessment of presumed Y/22 and Y/15 translocations in man using a new technique. A new chromosome banding technique, distamycin A plus DAPI, has been used to reexamine cases of presumed Y/autosome translocations. In contrast with the results obtained with quinacrine fluorescence (Q-banding), the satellites of acrocentric chromosomes do not fluoresce brightly with this new (DA-DAPI) method, making it more specific for the long arm of the Y chromosome. Previous cases with intensely Q-fluorescent and abnormally long short arms on a chromosome 22 were considered as presumptive 22/Y translocations: The new technique clearly shows that, in these cases, the additional material on 22p is not derived from Yq. In contrast, in other cases the Yq nature of additional material on 15p, in conjunction with the presence of an extra Y-body in interphase nuclei and the presence of a male-specific DNA, supports the previous diagnosis of a presumptive 15/Y translocation.


Isolabeling of the long arm of the human Y chromosome demonstrated by the FPG technique. Isolabeling segments were found in the distal region of the long arm of Y chromosomes derived from human leukocytes grown through two replication cycles in medium containing BrdU and stained by the FPG technique. Three main types of Y chromosome staining patterns were demonstrated: I-Y chromosome with typical SCD, II-Y chromosome with weakly stained distal regions of long arms (isolabeling segments), III-Y chromosome with both terminal regions displaying SCD interrupted by one isolabeled segment. The existence of different types of Y chromosome staining patterns was explained on the basis of the previously described hypothesis of unequal distribution of thymine residues between two DNA polynucleotide chains in the distal part of the long arms of human Y chromosomes.


Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia, Urodela). The mitotic chromosomes of six specimens from Triturus vulgaris meridionalis have been examined by both in situ hybridization with 3H 18S + 28S rRNA and AS-SAT staining method. The results of these two sets of experiments can be summarized as follows: 1) in each specimen the NORs and the additional ribosomal sites, which react positively to in situ hybridization with 3H 18S + 28S rRNA, are also stained by silver; 2) other chromosomal regions, which do not hybridize in situ with 3H 18S + 28S rRNA, are on the other hand stained by the AS-SAT method. These latter AG-positive sites show a species-specific pattern of chromosomal distribution.


[Development of educational technology in dentistry in Brazil]. This article reviews the experiments with educational technology conducted in the School of Dentistry of the Federal University in Rio Grande do Sul, Brazil. An account is given of the production in the Audiovisual Center of teaching-learning materials for dentistry. A summary description is given of programmed instruction techniques for use with an electronic programmer, modules, and sequences of slides with accompanying tape recordings. Some comments are offered on the improvements made with this equipment and techniques in the institution itself and in other universities of Brazil.


Environmental pollutants and the epidemiology of cancer. Cancer etiology involves the interplay of genetic and environmental factors. Striking geographic differences and changes in cancer incidence over time have led epidemiologists to infer that probably the major etiologic component is environmental. Recent experiences with vinyl chloride, kepone, and polybrominated biphenyl illustrate the problems involved in epidemiologic studies of proven or suspected environmental carcinogens. While epidemiologic studies will continue to be an essential means for monitoring potential human risks, the long latent periods involved in human carcinogenesis severely limit the usefulness of such approaches for disease prevention. While in vitro and animal test systems can never fully supplant human studies, they represent our only means for detecting potential carcinogenicity before human exposure has become widespread or long established.


Models for mRNA translation: theory versus experiment. Three models for mRNA translation are discussed in the light of available experimental data. It is concluded that the elongation rates vary along a messenger, possibly as a result of a coupling between ribosome movement and mRNA secondary structure. Some promising areas of further experimentation are indicated.


Amino acids of the Krebs-Henseleit cycle of median and lateral lobes in human benign prostatic hypertrophy. The levels of citrulline, ornithine and arginine were determined in median and lateral lobes of benign prostatic hypertrophy. No statistically significant differences could be stated. In connnection with the concentration of total protein and urea, some hypotheses were formulated for the very high glutamate level of human prostatic tissue.


Enhancement of dicoumarol bioavailability by concomitant food intake. The influence of food intake on the bioavailability of dicoumarol from a non-micronized formulation was examined in ten healthy volunteers, by examination of its single-dose kinetics after ingestion of dicoumarol 250 mg with a standardized breakfast and on an empty stomach. Blood samples were collected at regular intervals from 0 to 72 h, and the serum concentration of unmetabolized dicoumarol was determinded by spectrophotometry. Postprandial AUC (area under the curve) values were significantly (p less than 0.01) greater than the preprandial figures, the mean increase being 85 per cent. The results suggest that dicoumarol should always be taken with food.


Effect of TRH and dopamine on cyclic AMP levels in enriched mammotroph and thyrotroph cells. Populations of normal anterior pituitary cells enriched in thyrotrophs or mammotrophs prepared by velocity sedimentation were used to investigate the effect of modulators of TSH and prolactin secretion on cyclic AMP accumulation. In both thyrotroph-enriched and mammotroph-enriched fractions, IBMX increased cyclic AMP accumulation. In the presence of IBMX, TRH invoked an increase in cyclic AMP suggesting that TRH modulates cyclic AMP accumulation in both of these cell types from normal pituitary glands. In the mammotroph-rich fraction, dopamine inhibited the increase in cyclic AMP induced by TRH. In contrast however, in the thyrotroph-enriched fraction dopamine lowered neither cyclic AMP concentration nor TSH secretion. Thus the inhibiting effect of dopamine on cyclic AMP appears to be specific for prolactin-secreting cells.


[The influence of pregnancy specific serum factors on the mitogen induced culture of lymphocytes (author's transl)]. The addition of 10% serum of pregnant patients to a culture of lymphocytes from healthy non-pregnant control persons inhibits the stimulation of these lymphocytes by the mitogens phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanvalin A (Con A). The inhibitory effect increases with increasing duration of pregnancy. Sera from pregnant patients which already inhibit during the first trimester of pregnancy show an increase of this effect with increasing length of gestation. The influence of several hormones and proteins was investigated in order to detect a factor in the pregnant serum responsible for the inhibition of the lymphocyte culture. Compounds which are known to increase during gestation were added as pure compounds in various concentrations to the lymphocyte culture. An inhibitory effect on the lymphocyte culture in vitro was shown dependent on the amount, by human placental lactogen, alpha-fetoprotein, human chorionic gonadotropin, cortisol, progesterone and SPI glycoprotein and alpha-2-macroglobulin. The estrogens estriol and estradiol in physiologic dosages did not show an inhibitory effect and showed a stimulatory effect in higher dosages.


Possibility of immunodiagnosis in ovarian cancer. An antigenic material possibly associated with ovarian cancer has been demonstrated by the Ouchterlony technique in the sera from 42(66.7%) of 63 patients with primary ovarian malignancy of various histological types. It was more frequently found in the sera of preoperative patients in the later stages of the disease and those in relapse. Positive reactions could also be elicited from control sera of a very few persons without ovarian cancer. in none of the sera examined could the corresponding antibodies by demonstrated. The antigenic material was found to be an alpha2- or beta-glycoprotein on preliminary identification. There is an antigenic relationship between this material and fetal or normal adult ovaries. The possibility of immunodiagnosis of ovarian cancer is briefly discussed.


Searching for euthanatos: the hospice alternative. American culture, with its insistence on happiness and its obsession with technology, discourages a realistic approach to death. Within the concept of Euthanatos, a "good death," however, patients get the chance in the comfortable surroundings of the hospice to integrate dying into their total life experience. Here, their emotional as well as physical needs receive medical attention.


Hillhaven Hospice: a free-standing, family-centered program. Hillhaven Hospice, Tucson, exemplifies characteristics of the hospice concept: patient-and-family-centered care, emphasis on symptom control, provision of both inpatient and home care, a team approach to serving patients, and service to the family through their mourning period.


Requirements for beta1H globulin and C3b inactivator in the control of the alternative complement pathway in human serum. Using beta1H-depleted and C3b inactivator (C3b1NA)-deficient sera we have investigated the regulatory roles of beta1H and C3bINA in the turnover of the alternative pathway. Spontaneous turnover of C3 and factor B occurred in both beta1H-depleted and C3bINA-deficient sera. In neither case was C3d generated. Prevention of activation could be achieved by the addition of the missing protein, but not by increasing the concentration of the remaining protein. Thus both beta1H and C3bINA must be present simultaneously to prevent spontaneous activation of the alternative pathway.


Serum concentrations of alpha-macrofeto-protein (acute-phase alpha2-macroglobulin), a proteinase inhibitor, in pregnant and neonatal rats and in rats with acute inflammation. Alpha macrofetoprotein (AMF) or acute-phase alpha2 macroglobulin serum concentrations are elevated in pregnant, fetal, and newborn rats and in rats with experimentally induced acute inflammation when quantitated using a double-antibody radioimmunoassay. Concentrations of AMF are 17 +/- 2 microgram/ml and 32 +/- 6 microgram/ml in normal adult rat sera. Both maternal (1.2 mg/ml) and neonatal (9.3 mg/ml) concentrations are maximal at term. AMF serum concentrations of rats injected with croton oil into the hind footpads are unchanged for the first 4 h after injection, then increase to a maximum of 10.5 mg/ml 36 h after injection. Serum AMF concentrations correlate directly with the dose of croton oil and the increase in size (swelling) of the injured foot, and remain elevated during the course of inflammation for at least two weeks. The possible function of AMF as an antiproteinase limiting the extent of inflammation after acute tissue injury is discussed.


Serological identification of neoantigens on mouse fibroblasts which have undergone "spontaneous" malignant alteration in vitro. ST-L1 is a cell line established from lung explants from a normal ST/a mouse. The ST-L1 cells have undergone spontaneous malignant alteration in vitro. The cells were rejected after inoculation into syngeneic immunocompetent hosts, and a syngeneic humoral immuneresponse against the ST-L1 cells has been detected. The specificity of this humoral response was investigated. The syngeneic response to ST-L1 was characterized by indirect immunofluorescence tests and by immunoprecipitation of radiolabelled cells and of a C-type virus produced by the cell lines. The specific anti-ST-L1 reactivities were found to be directed against the envelope glycoprotein of an endogenous C-type virus expressed by the antigenic cell line.


A quantitative assay for tumor antigen based on inhibition of cytotoxicity of sensitized lymphocytes. Specific inhibition of cell-mediated cytotoxicity can be used as a quantitative measure of soluble tumor antigen if highly cytolytic cells are obtained. In vitro secondary stimulation of spleen cells sensitized in vivo to the syngeneic 13762A mammary adenocarcinoma results in a lymphocyte population consistently more cytolytic than lymphocytes after primary stimulation. Maximal cytolysis requires removal of dead lymphocytes from the effector population. Soluble tumor antigen (STA) is detected only in supernatants of 13762A mammary tumor cultures grown in the  presence of the proteolytic enzyme inhibitor, epsilon-amino caproic acid. Soluble MTM antigen preparations block lymphocytes immune to the mammary tumor but not lymphocytes immune to a second mammary adenocarcinoma (R3230) or to allogeneic spleen cells. Soluble antigen preparations from other tumors do not inhibit lymphocytes specifically cytolytic to the 13762A tumor. Additional evidence that the STA preparation contains tumor antigen is its ability to induce specific cytolytic lymphocytes and partial protection from challenge with live MTA tumor cells.


Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. IV. Antigenic differences between a metastasizing variant and the parental tumor line revealed by cytotoxic T lymphocytes. The syngeneic cytotoxic T-cell response against a metastasizing murine lymphoma variant was investigated and compared with the response against the non-metastasizing parental tumor line Eb. Anti-tumor cytotoxicity was not detectable  in a 4-h 51Cr release assay in spleens taken directly from tumor-bearing animals (primary CMC). After restimulation in vitro (secondary CMC) however, high anti-tumor cytotoxic activity was detected. This activity was mediated by immune T lymphocytes as shown by its sensitivity to treatment with anti-Thy 1.2 serum and complement. Ten cells of the metastasizing tumor ESb, inoculated subcutaneously, were sufficient to raise a local tumor and metastases and to induce cytotoxic T memory cells in the spleens. In contrast, about 104 cells were required to raise a local tumor and to induce splenic cytotoxic T memory cells, when the parental tumor Eb was tested. The specificity studies of the anti-tumor cytotoxic activity demonstrated that cytotoxic T cells could distinguish unrelated, chemically induced syngeneic tumors and also recognize antigenic differences between the parental tumor Eb and its variant ESb. Eb and ESb tumor cells were recognized as carrying distinct antigens at the responder cell level, the stimulator cell level and the target cell level. The in vivo significance of these findings is discussed.


Cell mediated immunity to human myelin basic protein in Vogt-Koyahagi-Harada syndrome. An immunological basis for neurological involvement in Vogt-Koyanagi-Harada syndrome was sought by means of the migration-inhibition factor technique with human myelin basic protein. The test was carried out in four patients who had recent manifestations of the syndrome, two of whom were evaluated both before and after initiation of steroid treatment; in one patient 4 years after recovery from the syndrome; in three patients having uveitis of other causes, and in 12 healthy controls. The results were positive in all four patients who had recent manifestations, whereas they were negative in all the others. This finding may constitute evidence of a cell-mediated immunity toward components of the nervous system in this disease entity.


A histochemical study in Huntington's disease and control cases. 1. Extracellular deposits of cerebrosides and free fatty acids were found in the formaldehyde fixed frozen sections of the frontal lobe in 8 cases of Huntington's disease, in one case of the infantile form of Gaucher's disease, 2 cases of Krabbe's globoid cell leucodystrophy, 2 cases of metachromatic leucodystrophy, one case with multiple sclerosis, 2 cases with cerebral contusion and one case with bacterial meningitis. 2. The cerebroside deposits were present in the white matter as well as in the grey matter. 3. The significance of these findings in relation to their etiology is discussed.


[Histochemical demonstration of glycogen phosphorylase (E.C. 2.4.11) through the use of semipermeable membranes (author's transl)]. A method is described for the histochemical demonstration of phosphorylase in unfixed cryostat sections of rat liver using semipermeable membranes and a gel medium. In comparison to the conventional methods this procedure has the following advantages: 1. Staining of sections through a semipermeable membrane prevents diffusion of cellular glycogen and guarantees optimal localisation of phosphorylase activity. 2. Since diffusion is effectively prevented by the membrane the total activity of this highly soluble enzyme can be demonstrated. 3. Tissue and cell structures are well preserved.


Some communicative properties of the bipersonal field. This paper delineates three major types of communicative bipersonal fields: Type A, the symbolic field of illusion and play; Type B, the field of action in discharge and projective identification; and Type C, the static field characterized by impenetrable barriers, falsifications, destruction of meaning, and a rupture of the link between patient and therapist or analyst. The development of this conceptualization is traced through a review of the author's prior contributions and the relevant literature. Several illustrative clinical vignettes are presented. A number of major implications of the basic formulation are discussed, including their relevance to individual communicative styles, the nature of contents and defenses in each field, the distinctive interventions required, and the interaction between patient and therapist in each of the three communicative modes.


Detection of aldehydefuchsin-positive neurosecretory granules in the cells of the suprachiasmatic nucleus of the rat. Application of dark field microscopy to sections fixed with picric acid--formalin and stained with crotonaldehydefuchsin allows the demonstration of neurosecretory granules in the neurones of the suprachiasmatic nuclei of normal rats.


Histochemical staining of lymphatic anchoring filaments. Lymphatic anchoring filaments are stained by means of histochemical methods that demonstrate disulfide-groups. Thiosulfation of sections either followed by aldehyde-fuchsin staining or by Alcian Blue +0.8 M MgCl2 staining has been used. Lymphatic anchoring filaments display striking fine structural similarities to "elastic fiber microfibrils" and both kinds of fibers are characterized by disulfide content.


[Age-dependency of toluidine-blue dichroism and birefringence of fibers within the costo-chondral junction of human ribs (author's transl)]. The chondral fibers running parallel to the chondrocyte columns within die costo-chondral junction of human ribs were investigated quantitatively following standardized Toluidine blue staining by using the Leitz-Mikrospektrograph for observing the absorption spectrum, and by employing both a Berek- and a Brace-Köhler compensator for polarization measurements. Autopsy material was obtained from the ribs of 35 children and adults at the age of 0 to 22 years. Refractivity was measured in stained and unstained paraffin sections, whereas dichroism and metachromatic index (MI) of the fibers were determined in stained specimens. All three parameters showed characteristic, quantitative age-dependent variations indicating that the highest degree of molecular and structural adjustment and alignment of the fibers is already being reached at the age of 8 years. The continuously higher refractivity of the fibers with increasing age and bone maturity may be explained by a progressive loss of water by the fibers.


[Investigation of the anisotropy of glycocalyx stained with 1.9-dimethyl methylene blue and N,N'-diethylpseudoisocyanine chloride (author's transl)]. In the present study the anisotropic staining of the erythrocyte membrane with 1.9-dimethyl methylene blue and N,N'-diethylpseudoisocyanine chloride was studied and simultaneously compared with the toluidine blue topo-optical staining. The difference between anisotropic toluidine blue and 1.9-dimethyl methylene blue staining, except after KMnO4-oxidation, was only of quantitative nature. On the contrary, striking differences were observed between N,N'-diethylpseudoisocyanine chloride staining, and toluidine blue or 1.9-dimethyl methylene blue staining. Enzymatic and chemical degradation resulted the disappearance of N,N'-diethylpseudoisocyanine chloride staining. Following these treatment membrane birefringence could be restored by aldehyde bisulfate and/or KMnO4-oxidation, while the N,N'-diethylpseudoisocyanine chloride staining was restored only after KMnO4-oxidation. After methylation or acetylation the membrane birefringence disappears, while after KMnO4-oxidation both topo-optical reactions return. The digitonin reaction brought about a rearrangement of the glycocyalyx components. The results draw attention to the spatial orientation of the glycoprotein of the erythrocyte membrane. The role of glycocalyx in the three topo-optical reactions was thus clearly demonstrated.


[The suitability of further thiazine and quinoline dyes for topo-optical reactions on the plasmalemma (author's transl)]. The present studies prove that the thiazin dyes, azure B, azure C and thionin, and the quinolin dyes pinacyanol and its hydrochloride, are suitable for topo-optical staining of the plasmalemma. On the membrane surface the orientated bound dye molecules become stabilized, and with subsequent precipitation the anisotropic effect is reinforced. On optical analysis, the thiazin dye molecules (azure B, AZURE C and Thionin) are bound radially on the membrane. The molecules of the previously studied quinolin dye, N,N'-diethylpseudoisocyanide chloride are bound parallel to the membrane, while pinacyanol and its hydrochloride, like the thiazin dyes, are bound in the radial position.


Light and electron histochemistry of phosphoamidase with p-chloranilidophosphonic acid and with cyclophosphamide (endoxan). The original metal-salt technique of Gomori (1948a) employing p-chloranilidophosphonic acid as a substrate for the demonstration of the activity of phosphoamidase has been used with varying success by a number of investigators for light microscopy. Cyclophosphamide (endoxan) which is a cytotoxic drug known to activate phosphoamidase and other lysosomal enzymes in neoplasm (Grillo, 1971) is proposed as another substrate for the enzyme for both light and electron microscopy.


Combination of metallic impregnation and autoradiography of brain sections. A method for differentiation of proliferating glial cells in the brain of adult rats and mice. After labeling with 14C-thymidine, frozen sections or paraffin sections of the brain of adult mice or rats were first stained by metallic impregnation and then coated with chrome alum gelatine and with an emulsion layer of about 10 micron. On the autoradiographs 14C-tracks are readily recognized above labelled astrocytes or oligodendrocytes, and these can be well discriminated, if the sections are processed by the silver carbonate method of Rio-Hortega. In contrast, no labelling is obtained, if the gold chloride sublimate method of Cajal is applied.


Polarization and fluorescent microscopy--simple methods for demonstrating pulmonary surfactant lipids. Birefringence and fluorochrome lipid staining with benzpyrene are demonstrated as simple morphological methods to reveal the presence or absence of surfactant lipids in human newborn lung tissue. Lack of lipid birefringence proves to be an associated finding in the lungs of premature infants with hyaline membrane disease, indicating the possible pathogenetical importance of surfactant deficiency.


A synthesis of dihydrostreptomycin.

A synthesis of dihydrostreptomycin. A key protected streptidine derivative (3) useful for the synthesis of antibiotics of streptomycin series was prepared by hydrolysis of an acylated dihydrostreptomycin (DHSM) derivative (2), and it was condensed with a protected dihydrostreptobiosaminyl chloride (5) to give two condensation products (6 and 7). By deblocking, 6 was led to DHSM and 7 to a biologically inactive isomer (8) of DHSM. From the PMR spectrum of 4-O-mesyl derivative (4) of 3, the benzyloxycarbonyl and acetyl groups were concluded to be attached to the end nitrogens of the guanidine groups.


Biological studies on the degradation products of 3-[(S)-1'-phenylethylamino]propylaminobleomycin: a novel analog (pepleomycin). Pepleomycin (PEP), 3-[(S)-1'-phenylethylamino]propylaminobleomycin has potent activity and is less pulmonary toxic than bleomycin (BLM). Biological activity and toxicity of the following degradation products of PEP have been studied in detail: the product of carbamoyl migration (ISO), the product of decarbamylation (DC), the product of ring closure of the side chain on the pyrimidine moiety (RC), the depyruvamide product (DP) and the product of an enzymatic inactivation (DA). These degradation products showed much lower activity than PEP in vitro: antimicrobial and anti-HeLa activities, inhibition of DNA synthesis in AH66 cells and the DNA strand cleavage. Acute toxicity and pulmonary toxicity were tested in mice. Results indicated much lower acute toxicity corresponding to the decreased in vitro activity when compared to PEP. DP and RC did not cause lung fibrosis in mice, while ISO and DC showed 1/2.6 and 1/5.7 degree of pulmonary toxicity, respectively, in comparison with PEP.


The effect of bleomycin on prolyl hydroxylase and DNA chain breakage: structure-activity relationship. The activity of purified prolyl hydroxylase (proline, 2-oxoglutarate dioxygenase, EC 1.14.11.2) was enhanced about 3-fold by addition of bleomycin in the assay mixture. Various members of the bleomycin family, their derivatives and degradation products were investigated for activities against prolyl hydroxylase together with their activities of DNA chain breakage to determine relationships between the structure of bleomycin and its various actions. All the bleomycins with various terminal amine parts and desamide bleomycin stimulated the enzymatic activity but did not exhibit an effect on DNA chain breakage. The stimulatory activity of bleomycin was not decreased by hydrolysis with 0.3 N H2SO4 at 80 degrees C for 6 hours, conditions which liberates the sugar moiety, but was eliminated by hydrolysis with 6 N HCl at 105 degrees C for 24 hours. In contrast both treatments decreased the DNA chain breakage activity of bleomycin. Optical spectral studies revealed that all the bleomycins and their hydrolysates which stimulated the prolyl hydroxylase activity made complexes with ferrous ion, one of the cofactors of this enyzme.


Electron microscopic observations of polysaccharide components in polymyxin B treated outer membranes from Serratia marcescens. Treatment of outer membranes of Serratia marcescens with polymyxin B results in the formation of blebs. This effect is thought to be due to the action of the antibiotic on the lipopolysaccharides, proteins, phospholipids or a combination thereof. It is unclear whether this effect is dissociative, degradative or due to an inhibition of the assembly of outer membrane components. Prior studies showed that lipopolysaccharides and polymyxin B form complexes, but direct visualization of the in situ action of polymyxin B had not been accomplished. Isolated outer membranes normally exhibit a periodicity of the polysaccharide molecules when stained by the thiosemicarbazide-silver technique. Polymyxin B treated outer membranes display a change in their basic morphology. This effect is very drastic in the sensitive strain as demonstrated by the large gaps in the deposition of the granules in the modified outer membrane structure. Thus it appears that the polysaccharide molecules (probably the lipopolysaccharide) either alone or in association with protein or phospholipids are the primary targets of the antibiotic.


Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa. Membrane vesicles were prepared by osmotic lysis of spheroplasts of Pseudomonas aeruginosa strain P14, and the active transport of amino acids was studied. D-Glucose, gluconate, and L-malate supported active transport of various L-amino acids. The respiration-dependent leucine transport was markedly stimulated by Na+. Moreover, without any respiratory substrate, leucine was also transported transiently by the addition of Na+ alone. This transient uptake of leucine was not inhibited either by carbonyl cyanide p-trifluoromethyoxyphenylhydrazone or by valinomycin, but was completely abolished by gramicidin D. Increase in the concentration of Na+ of the medium resulted in a decrease of the Km for L-leucine transport, whereas the Vmax was not significnatly affected. Active transport of leucine was inhibited competitively by isoleucine or by valine, whose transport was also stimulated by Na+. On the other hand, Na+ was not required for the uptake of other L-amino acids tested, but rather was inhibitory for some of them. These results show (i) that a common transport system for branched-chain amino acids exists in membrane vesicles, (ii) that the system requires Na+ for its activity, and (iii) that an Na+ gradient can drive the system.


Structure of the complex oligosaccharides of fetuin. The complete structure of the complex oligosaccharides of fetuin has been established. The three fractions of complex oligosaccharide which were isolated by ion exchange chromatography following pronase digestion (F-I, F-II, and F-III) had identical molar ratios of sialic acid (Sia), galactose, mannose, and N-acetylglucosamine of 3:3:3:5. A combination of methylation analyses, Smith periodate degradations, and endoglycosidase and exoglycosidase digestions were utilized to establish the structure which is proposed to be: (see article of journal). Features of this structure not previously established include the presence of 2 residues of alpha2,3- and 1 residue of alpha2,6-linked sialic acid and their location relative to the mannose branch points. Also unusual is the presence of an alpha-linked branch mannose with substituents at positions 2 and 4 which is in turn linked to position 6 of the beta-linked, branch mannose. These features result in unexpected resistance to specific exoglycosidases.


A requirement for ATP for beta-galactoside transport by Bacillus alcalophilus. Lactose-grown cells of Bacillus alcalophilus actively transported methylthio-beta, D-galactoside (TMG) in a range of pH values from 7.5 to 10.5 with a pH optimum at 8.5. The TMG was accumulated in a chemically unmodified form, and cell extracts failed to catalyze either ATP or P-enolpyruvate-dependent phosphorylation of TMG. At pH 8.5, the lactose-grown cells exhibited a transmembrane proton gradient (deltapH) of 1.38 units, interior acid, and a transmembrane electrical potential (delta psi) of -132 mV. Accordingly, the total protonmotive force at this pH was very low, -51mV. Several lines of evidence indicate that the protonmotive force or delta psi did not directly energize TMG transport but, rather, that ATP was directly required: (a) in cells treated with arsenate so that the delta psi was unaffected and cellular ATP levels were markedly lowered, TMG transport was inhibited in proportion to the reduction of cellular ATP, while electrogenic alpha-aminoisobutyric acid transport was not; (b) when a valinomycin-induced potassium diffusion potential was established in starved cells, alpha-aminoisobutyric acid transport, but not TMG transport, was stimulated; and (c) in a series of experiments in which the delta psi was rapidly abolished by treatment with gramicidin, ATP levels declined slowly and the rate of TMG transport correlated directly with ATP levels rather than with the delta psi. Consumption of cellular ATP concomitant with TMG transport could be demonstrated.


Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus. Highly purified preparations of RNA-directed DNA polymerase from avian myeloblastosis virus (AMV) contain a Mn2+-activated endonuclease activity capable of nicking supercoiled DNA. This endonuclease activity co-sediments in glycerol gradients with the alphabeta form of AMV DNA polymerase, and co-chromatographs with DNA polymerase activity on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. It is also present in AMV alphabeta-DNA polymerase purified by electrophoresis through nondenaturing polyacrylamide gels and subsequently chromatographed on poly(C)-agarose. alphabeta-associated endonuclease is co-immunoprecipitated with DNA polymerase activity by antiserum directed against alphabeta holoenzyme. The alpha form of AMV DNA polymerase lacks this activity. In its enzymatic properties, alphabeta-associated endonuclease resembles the endodeoxyribonuclease activity associated with the AMV p32 protein, which has been shown to be structurally related to the beta (but not the alpha) subunit of AMV DNA polymerase.