Current status of prostate androgen receptors. Over the past eight years, many investigators have attempted to measure androgen receptors in human prostate cytosol and nuclear extracts. It is clear that the concentration of unoccupied cytosol receptors is quite low though measurable if appropriate techniques such as agar gel electrophoresis, sucrose density gradient centrifugation (SDGC) or protamine sulfate precipitation are employed. Attempts to correlate these values in untreated patients with their subsequent response to therapy have not been successful. Recent attempts to measure total cytosol receptors, whose concentration is much higher, have been encouraging and suggest that their determination should be further explored. Measurement of the concentration of nuclear receptors present in salt extracts of nuclei has shown that the level of these receptors is higher than in cytosol and that interference from ligand binding to sex steroid binding globulin or other interfering binding proteins is minimal. It seems likely that greater success in correlating androgen receptors with the clinical response will be achieved from measurements of these nuclear receptors.


Large needle biopsy diagnosis of thyroid carcinoma: problem areas. The major problem area in the diagnosis of thyroid cancer by large needle biopsy is in the identification of follicular carcinoma and the follicular variant of papillary carcinoma. Diagnosis of these tumors is facilitated by use of Bouin's solution as an initial fixative to duplicate the nuclear detail seen in the surgical specimen. As specimen size precludes demonstration of invasive characteristics, details of cellular and follicular architecture must be relied upon to indicate the need for surgical biopsy.


Palliation of esophageal carcinoma with intraluminal tubes: experience with 30 patients. Between 1968 and 1978, 26 patients with carcinomas of the thoracic esophagus and 4 with adenocarcinomas involving the esophagogastric junction were treated by the insertion of indwelling intraluminal (endoesophageal) tubes. Four different types of tube were inserted by the pull-through technique. Thirteen of the 30 patients died in the hospital within 30 days. However, among the 20 patients who did not have neoplasms of the upper third of the thoracic esophagus or who had not had a prior resection, only 5 died. The principal cause of death was aspiration pneumonia. Survival averaged 2.5 months. Four patients survived 5 to 7 months. Deglutition was adequate in most patients but was not as satisfactory as after esophagogastrectomy. Our best results were obtained in patients with carcinoma of the middle or lower third of the esophagus, with or without an esophagorespiratory fistula, who had not had a previous resection.


Isolation and immunological reactivity of soluble fractions from rat seminiferous tubule basement membrane. A preparation rich in basement membranes isolated from rat testes (STBM) was exposed to pepsin, collagenase, trypsin, and pronase to obtain soluble fractions. The immunological reactivity of these fractions was studied by gel immunodiffusion or by passive hemagglutination tests against an anti-STBM serum. All fractions reacted with the antiserum, but the highest titer was detected when the antiserum was reacted with a fraction that contained only traces of hydroxyproline (fraction 1), whereas low titers were obtained with collagen or collagen fragments isolated from STBM. Antibodies in the anti-STBM serum were mainly directed to the glycoproteins of STBM not related to collagen. Fraction 1, obtained by subsequent collagenase and trypsin digestion of STBM and purification by Sephadex G-200, was a high molecular weight glycoprotein that was free of half-cystine and methionine, had only traces of hydroxyproline, and contained 7.2% neutral sugars, 0.26% sialic acid, and 8.7 residues of glucosamine per 1000 residues of amino acids.


[Basic behavior and behavior after stimulation of inflammatory cellular response, leukocytary chemotaxis and phagocytosis in aged subjects with depression of cellular immunity]. A larger group of 70--75 years aged subjects was studied by means of various cutaneous tests, by E-rosette formation and by blastogenic response and 18 people showing a depressed cellular immunity were selected. These subjects and a similar control group with normal C.M.I. were further investigated in various condition of stimulation for cellular response in inflammation, chemotaxis, phagocytic activity and polysaccharide content in the granulocyte citoplasm. In general the former activity was not depressed, chemotaxis and phagocytic values were deficient, polysaccharide content appeared the same in the normal (control) and in the immune deficient group.


Plasma level of the prodrug midodrine and its active metabolite in comparison with the alpha-mimetic action in dogs. Midodrine, i.v. or orally administered, causes a prolonged elevation of blood pressure and a reduction in heart rate. These cardiovascular changes are not correlated to the plasma levels of the intact drug. On administration of either midodrine or its metabolite, ST-1059, formed by cleavage of the glycine residue, the elevation of blood pressure and the reduction in heart rate were significantly correlated to the plasma level of ST-1059. The results are in agreement with the assumption that the pressor activity of midodrine is mainly exerted by its metabolite ST-1059.


Viruses isolated from reptiles: identification of three new members of the family Rhabdoviridae. The growth of four viruses isolated from lizards in Brazil (Marco, Chaco, and Timbo viruses) and Australia (Almpiwar virus) was studied in a variety of continuous cell lines of mammalian, reptilian, amphibian, and piscine origin. Although replication was found in certain cell lines derived from the coldblooded species, cytopathic effect (CPE) was absent or minimal and growth was less than or equal to that in mammalian cells. Those observations appear to limit the value of poikilothermic cells for primary isolation of viruses from field-collected, cold-blooded vertebrates or arthropods that feed upon them. The four reptilian viruses were found to be naturally occurring temperature sensitive agents, with optima for growth of approximately 30 degrees C. Electron microscope studies showed three of the viruses (Marco, Chaco, and Timbo) to be new members of the family Rhabdoviridae. Marco virus particles were conically shaped and resembled bovine ephemeral fever virus, and two lyssaviruses (Kotonkan and Obodhiang). Chaco and Timbo viruses were cylindrical viruses resembling other rhabdoviruses with particle lengths longer than the prototype VSV. No serologic relationships were found in cross complement fixation tests between these viruses, Marco virus, and 34 other rhabdoviruses.


[Use of hematoxylin to demonstrate the microcirculatory bed in full-thickness objects and sections of the wall of the gastrointestinal tract]. Slight modifications of the widely applied hematoxylin method of staining make it possible to reveal completely microcirculatory bed in total film preparations and in histological sections of the gastrointestinal wall. Hematoxylin staining of all elements in the vascular wall and the surrounding tissue helps to differentiate definitely every component of the microcirculatory bed and to demonstrate vascular-tissue and vascular-neural interconnections in film preparations and histological sections.


Intracellular potential changes of Corti's organ with anoxia. Intracellular measurements of the resting cell membrane potentials of guinea pig Corti's organ were made in order to determine the sensitivity of this cell potential to anoxic hypoxia (a lowered oxygen state due to lack of respiratory oxygen) and to establish differences according to cell types or morphologic regions of the sensory epithelium. The negative cell potentials measured from successful electrode penetrations were found to be relatively more stable and resistant to change during a 120-s period of anoxia than was the positive endocochlear potential. The intracellular resting potentials were also much slower to recover after resumption of respiration. Data obtained from various cells in two different regions of Corti's organ is receiving oxygen from both the perilymph and the endolymph. An iontophoretic dye-marking technique was used to label some experimental cells for later histologic identification.


Relationship of centrifugal fibres to 'supporting' cells. We have described cholinergic fibres which cross the tunnel to three levels of the cells on the opposite side--from the level of the efferent network to the base of a plaque at the level of the nucleus of the first Deiters cell. These fibres are dependent upon the OCB being intact. The same is true for those fibres seen within the Hensen cell region. However, the cholinesterase-staining fine fibres travelling along the floor of the tunnel to the base of the plaque are independent of the integrity of the OCB.


The goitrogen 3-hydroxy-4(1H)-pyridone, a ruminal metabolite from Leucaena leucocephala: effects in mice and rats. Mice fed a diet containing 1% (w/w) 3-hydroxy-4(1H)-pyridone (DHP) developed goitre even with a diet high in iodine whereas mimosine (0.5% w/w) did not produce goitre even with a low-iodine diet. Thyroid enlargement was apparent (measured morphometrically) by the 7th week and was advanced by the 11th week. Histologically the goitre was hyperplastic in type. No marked histological changes were found in other organs of mice fed DHP or any organs of mice fed mimosine, except for some atrophy of hair follicles. A single intragastric dose of DHP inhibited the uptake of 125I by the thyroid in the rat but an equivalent dose of mimosine did not. Evidence is presented that the inhibition occurs at the iodine binding step, as with methyl thiouracil, rather than at the iodide trapping step, as with thiocyanate. Chronic treatment of mice with DHP, as with 6-methyl thiouracil, increased the avidity of the thyroid in taking up 125I. The major conjugated form of DHP in mammals, DHP-3-O-glucuronide, was almost as effective a goitrogen as the unconjugated compound when given by mouth but considerably less active than the free form in the blood stream. It was concluded that DHP is a potent antithyroid compound of the thiouracil type with low general toxicity, since mammals can tolerate a level of intake sufficient to produce goitre in spite of iodine supplementation.


Immunological similarities of mammalian xanthine oxidases. The degree of relatedness among mammalian xanthine oxidases (XO) was determined by microcomplement fixation. Rabbit anti bovine milk XO serum was tested against xanthine oxidase (homologous protein) and against the heterologous proteins of bovine liver, monkey liver, rat liver, lactating cow serum, nonlactating cow serum, and steer serum. The indices of dissimilarity for the heterologous proteins were expressed as units of immunological distance and the percent sequence differences among these proteins inferred from the y = 5x relationship where y is immunological distance and x is percent sequence difference. Rat liver XO differed by approximately 27% in amino acid sequence from bovine milk XO. In order of increasing immunological distance from bovine milk XO, the sources of XO ranked as follows: lactating cow serum less than nonlactating cow serum less than steer serum = beef liver less than monkey liver less rat liver. The monkey ranked much closer than the rat in order of phylogenetic kinship to the cow. Starch gel electrophoresis of liver, milk, and serum showed that the milk and the serum contained only cationic forms of xanthine oxidase while all the liver samples tested contained cationic as well as anionic forms of the enzyme. The electrophoretic mobility properties of xanthine oxidase confirmed the polymorphic nature of the enzyme as revealed by the immunological data.


Quantitative studies of the interaction of cholecalciferol ((vitamin D3) and its metabolites with different genetic variants of the serum binding protein for these sterols. Cholecalciferol (vitamin D3) and its 25-hydroxy metabolite are transported in plasma bound to a specific protein, the binding protein for cholecalciferol and its metabolites (DBP). DBP is identical with the group-specific component (Gc) proteins, which are known to display genetic polymorphism. Studies were conducted to explore whether or not major differences in the transport of cholecalciferol and its biological metabolites might exist among persons with different Gc phenotypes. Detailed quantitative studies were first carried out on the interaction of 25(OH)D3 with DBP in 21 different samples of serum, representing eight different Gc phenotypes. The studies used a filter disc assay method that provided highly reproducible quantitative results with cholecalciferol-related sterols. The Gc phenotypes studied included the three common types (Gc 1-1, 2-1, and 2-2) and several uncommon genetic variants (Gc Ab-Ab, Ab-1, Ab-2, Chip-1, and Chip-2). The binding affinities for 25(OH)D3 observed with these different sera were all fairly similar to each other. More extensive studies were then conducted to compare the binding of four cholecalciferol-related sterols to each of three genetic variants of DBP, by using sera from homozygous persons with the Gc 1-1, Gc 2-2 and Gc Ab-Ab phenotypes. The ligands tested included cholecalciferol, 25(OH)D3, 1,25(OH)2D3, and 24(R) 25(OH)2D3. The affinities of the three genetic types of DBP/Gc protein were found to be similar for each of the four cholecalciferol-related sterols. The apparent association constants for 25(OH)D3 and 24,25(OH)2D3 were similar (approx. 1--2 x 10(8) M-1); lesser affinities were observed for 1,25(OH)2D3 (kA approx. 1 x 10(7) M-1) and for cholecalciferol (kA approx. 3--4 x 10(5) M-1). Thus the common genetic variants of DBP/Gc protein, and the uncommon genetic variants studied here, all appear to have similar binding properties for cholecalciferol and its several metabolites.


Unaltered catabolism of desialylated low-density lipoprotein in the pig and in cultured rat hepatocytes. Removal of the terminal sialic acid residues from many serum glycoproteins results in exposure of their penultimate galactose residues and rapid clearance from circulation by the liver. Low-density lipoprotein is a glycoprotein containing 21 galactose and 9 sialic acid residues per particle. Studies in this laboratory and others have shown that both the liver and extrahepatic tissues contribute to the degradation of low-density lipoprotein. This study was undertaken to determine whether desialylation of pig low-density lipoprotein alters its removal from circulation. Low-density lipoprotein was incubated at 37 degrees C with an agarose-bound neuraminidase, proteinase-free, from Clostridium perfringens. After 18 h at pH 5.0, 70% of the sialic acid residues were removed. The desialylated 131I-labelled and native 125I-labelled low-density lipoproteins were simultaneously injected into a pig, and their disappearance from plasma was followed for 96 h. The turnovers of the two were identical. In contrast, neuraminidase-treated fetuin was cleared about 200-fold faster than native fetuin. Studies were also performed in cultured rat hepatocytes. Rates of degradation of native and neuraminidase-treated low-density lipoprotein were similar, whereas asialo-fetuin was degraded at six to ten times the rate of native fetuin. Thus desialylation does not appear to alter low-density-lipoprotein catabolism by hepatic or extrahepatic cells.


Synthetic peptides comprising sequences of the human immunoglobulin E heavy chain capable of releasing histamine. On the basis of previous studies on the structure-activity relationship of model polypeptide histamine liberators, a site within the Fc region of immunoglobulin E antibody molecules has been proposed as that responsible for the direct triggering of target mast cells after antigen challenge. Peptides comprising this region of the epsilon-chain have now been synthesized and shown to induce histamine release from normal rat peritoneal mast cells in a selective manner essentially similar to that mediated by anaphylactic antibody-antigen interaction.


Comparison of human alkaline phosphatase isoenzymes. Structural evidence for three protein classes. The structural relationships among human alkaline phosphatase isoenzymes from placenta, bone, kidney, liver and intestine were investigated by using three criteria. 1. Immunochemical characterization by using monospecific antisera prepared against either the placental isoenzyme or the liver isoenzyme distinguishes two antigenic groups: bone, kidney and liver isoenzymes cross-react with anti-(liver isoenzyme) serum, and the intestinal and placental isoenzymes cross-react with the anti-(placental isoenzyme) antiserum. 2. High-resolution two-dimensional electrophoresis of the 32P-labelled denatured subunits of each enzyme distinguishes three groups of alkaline phosphatase: (a) the liver, bone and kidney isoenzymes, each with a unique isoelectric point in the native form, can be converted into a single form by treatment with neuraminidase; (b) the placental isoenzyme, whose position also shifts after removal of sialic acid; and (c) the intestinal isoenzyme, which is distinct from all other phosphatases and is unaffected by neuraminidase digestion. 3. Finally, we compare the primary structure of each enzyme by partial proteolytic-peptide 'mapping' in dodecyl sulphate/polyacrylamide gels. These results confirm the primary structural identity of liver and kidney isoenzymes and the non-identity of the placental and intestinal forms. These data provide direct experimental support for the existence of at least three alkaline phosphatase genes.


Articular cartilage preservation and storage. I. Application of tissue culture techniques to the storage of viable articular cartilage. Articular cartilage slice explants were stored under various conditions, including freezing-thawing at various rates by using dimethyl sulfoxide (DMSO) as a cryoprotective agent, incubating in standard tissue culture medium (MEM Eagle:NCTC 135:15% fetal calf serum) in 5% CO2 and air at 4 degrees, 21 degrees, and 37 degrees C, and incubating in standard tissue culture medium containing 200 micrograms/ml alpha-tocopherol (vitamin E) at 37 degrees C after first ascertaining a dose-response curve of vitamin E. Results indicated that articular cartilage slice explants did not survive freezing or storage at 4 degrees and 21 degrees C as measured by 35S uptake. When stored at 37 degrees C in standard tissue culture in 5% CO2 and air, the slice explants remained viable for up to 60 days. The addition of alpha-tocopherol to the medium resulted in significantly less release of previously incorporated 35Sin stored cartilage slices and significantly less reduction of the amount of hexosamine present in the stored explants. alpha-Tocopherol in the medium also preserved safranin O staining. Thus, the application of tissue culture techniques to the storage of articular cartilage made it possible to preserve cartilage slice explants in a viable, biochemically "normal" state.


Anti-allergic properties of a new histamine antagonist, 4-(p-chlorobenzyl)-2- [N-methyl-perhydroazepinyl-(4)]-1-(2H)-phthalazinone hydrochloride (azelastine). Anti-allergic properties of 4-(p-chlorobenzyl)-2 [N-methyl-perhydroazepinyl-(4)]-1-(2H)-phthalazinone hydrochloride (azelastine, A-5610) were investigated focusing the most attention on its decongestive effect. Intravenous injection of azelastine into anesthetized dogs with doses more than 0.1 mg/kg prevented the changes in nasal impedance provoked by histamine sprayed into the nasal cavity. When azelastine was given orally, the minimum effective dose to abolish the impedance reduction due to histamine was 2 mg/kg, in the case of cleamastine the same dose was required. Histamine release from the rat mesentery pieces by the condensation product of N-methyl-homoanisylamine formaldehyde (compound 48/80) (0.005%) was inhibited almost completely by pretreatment with azelastine at the concentrations of 10(-4) to 10(-3) g/ml, and in those concentrations azelastine alone released histamine scarcely. When 5 mg/kg of azelastine was given i.v. to rabbits, the characteristic changes in EEG -- a high-voltage low-frequency pattern -- persisted more than 1 h, but not the least inhibition in arousal response was noted. With the dose of 0.5 mg/kg, diphenhydramine impaired arousal response and slow waves with high amplitude dominantly appeared in EEG.


4-(p-Chlorophenylthio)butanol (W-2719), a new non-antihistaminic drug for immediate hypersensitivity allergies. p-Chlorophenylthiobutanol (W-2719) has been found to effectively inhibit immunologic and non-immunologic histamine release and mast cell degranulation. It has been found to effectively suppress passive cutaneous anaphylaxis (PCA) reaction not be antihistaminic action, but by inhibiting the release of allergic mediators.


Rapid staining technique for Trichomonas vaginalis. Preliminary report. A simple, rapid, staining method for the identification of Trichomonas vaginalis has been tested on cultured trichomonads and specimens of vaginal discharge. Fifty-eight stained slides of vaginal discharge were examined and trichomonads were correctly identified in the 31 specimens from patients with confirmed vaginal trichomoniasis. No false-positive results were obtained. This staining procedure could prove a useful addition to wet-film and cultural methods.


[Counterimmunoelectrophoresis in the diagnosis of visceral leishmaniasis]. Technique of counter current immunoelectrophoresis (C.I.E.P.) was employed for the diagnosis of V.L. using an antigen grossly extracted (by means of repeated freezing and thawing) from culture of Leishmania. 6 lots of antigen were prepared - in various time - at the same way. Positive results are obtained in V.L. from 83.3 to 94% (according to various lots of antigens). Few false positivity (from 1.4 to 8.8%) are obtained in sera from patients with other diseases (especially) cirrhosis and blood disorders. No positivity in controls (blood donors). The reproducibility of results appear satisfactory. Our results suggested that C.I.E.P., rapid and less sophisticated test, can be applied for the diagnosis of V.L. But false positivity and false negativity limit the value of these test.


Identification of mouse bone marrow colony growth in agar-cultures by the nitroblue tetrazolium dye. Identification of mouse bone marrow colony growth in soft agar cultures has been realized by layering of the plates with nitroblue tetrazolium at the concentrations of 1 mg/ml. Granulocyte-macrophage colonies appear dark-blue coloured on the yellow surrounding medium and can be fixed, stored at 4 degrees C and counted few days later. This method allows the cultures to be assessed macroscopically and makes a better enumeration of small clusters even if spread and consisting of only 30-40 cells.


Location of the antigenic determinants of bovine pancreatic ribonuclease. Four antigenic regions of native bovine pancreatic ribonuclease have been located by using antibodies that react specifically with segments 1--13, 31--79, and 80--124. These antibodies were purified by affinity chromatography on columns to which these peptide segments were bound. Analysis of precipitin curves indicates that there are at least three antigenic determinants to which antibody molecules can bind simultaneously in the presence of excess antibodies. Analysis of binding data, however, for each purified specific antibody preparation, carried out by the method of Berzofsky et al. [Berzofsky, J. A., Curd, J. G., & Schechter, A. N. (1976) Biochemistry, 15, 2113], leads to an estimate of four for the number of antigenic determinants in ribonuclease; this estimate had also been made earlier by Stelos et al. [Stelos, P., Fothergill, J. E., & Singer, S. J. (1960) J. Am. Chem. Soc. 82, 6034]. We find that one determinant is associated with each of segments 1--13 and 80--124 and two with segment 31--79. No antigenic activity could be detected for segment 14--29 either in native ribonuclease or in the free fragment. These conclusions are based on (1) the use of specific peptides to isolate purified antibodies by affinity chromatography, (2) immunoprecipitation of an antigenic peptide from the peptic digest of ribonuclease, (3) competitive inhibition studies with various peptide and protein fragments [cyanogen bromide fragments 1--13, 31--79, and 80--124, the tryptic peptides 40--61 and 105--224, S-peptide, S-protein, and des(121--124)-RNase], and (4) comparison and evaluation of the published effects on antigenicity of chemical and enzymatic modifications and changes in sequence among homologous ribonucleases. These approaches provide evidence that the four antigenic determinants are localized around the alpha-helical portion of segment 1--10, somewhere in segment 40--61, at the beta bend in segment 63--75, and either at the beta bend or beta sheet in segment 87--104 of native ribonuclease.


Enzymic O-glycosylation of synthetic peptides from sequences in basic myelin protein. Nine synthetic peptides containing sequences in the region of a threonine residue at position 98 of bovine basic myelin protein were prepared by the Merrifield solid-phase method and tested for their ability to be glycosylated with [14C]uridinediphospho-N-acetylgalactosamine and a crude detergent-solubilized preparation of uridinediphospho-N-acetylgalactosamine:mucin polypeptide N-acetylgalactosaminyltransferase obtained from porcine submaxillary glands. The tetrapeptide Thr-Pro-Pro-Pro and all larger peptides containing this sequence were glycosylated. The glycosylation was greater for peptides containing residues N-terminal to the Thr-Pro-Pro-Pro. Under the conditions used, the peptide Val-Thr-Pro-Arg-Thr-Pro-Pro-Pro was glycoslyated twice as much as bovine basic myelin protein. Thr-Pro and Thr-Pro-Pro, as well as 10 other synthetic peptides which did not contain the Thr-Pro-Pro-Pro sequence, were not glycosylated. Treatment of the glycopeptide of Phe-Lys-Asn-Leu-Val-Thr-Pro-Arg-Thr-Pro-Pro-Pro-Ser with an alpha-N-acetylgalactosaminidase released N-acetylgalactosamine from the peptide, indicating that the hexosamine was covalently bonded to the peptide in an alpha linkage.


Characterization of an RNA-directed DNA-polymerase from a cell line derived from a radiation-induced lymphoma in mice. An RNA-directed DNA polymerase was purified from a cell line derived from a radiation-induced lymphoma in NIH Swiss mice which produced non-infectious type C virus particles. The enzyme was isolated from a high speed particulate fraction which bands at a density of 1.16--1.19 g/ml in a sucrose gradient, and purified by successive chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The purified DNA polymerase has a molecular weight of 68 000, a pH optimum of 7.5, a KCl optimum of 50 mM, and a Mn2+ optimum of 0.25 mM. It prefers (dT)15 . (A)n to (dT)15 . (dA)n as the primer template and transcribes the poly(C) strand of (dG)15 .(C)n and (dG)15 . (OMeC)n. It transcribes heteropolymeric regions of avian myeloblastosis virus 70 S RNA, and is inhibited by antiserum to Rauscher murine leukemia virus DNA polymerase. Comparison of the properties of DNA polymerase purified from radiation-induced lymphoma cells with the DNA polymerase purified from non-defective murine type C RNA tumor viruses shows that the mouse lymphoma enzyme is both biochemically and immunologically related to murine leukemia virus DNA polymerases.


Regulation of endogenous virus production by bromodeoxyuridine and dibutyryl cyclic AMP in Chinese hamster ovary cells. Either dibutyryl cyclic AMP or bromodeoxyuridine can enhance RNA type-C virus production in Chinese hamster ovary cells. The resultant effect of simultaneous treatment of cells with 1 mM cyclic AMP and 65 muM bromodeoxyuridine is greater than their additive effects when used separately. The increase in virus production does not seem to involve viral gene amplification, but correlates with an increase in viral RNA transcription. The virus is also shown immunologically to be unique to the Chinese hamster cells and does not appear to be a result of external infection by other common laboratory type-C viruses. The control of this endogenous viral genomic expression may therefore by proposed to be under the normal Chinese hamster gene regulatory mechanism.


Effect of alamethicin, gramicidin S and melittin upon the particulate guanylate cyclase from rat lung. The channel-forming antibiotic alamethicin activated rat lung particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2), and the activated enzyme was further stimulated by sodium nitroprusside when a thiol such as 2-mercaptoethanol was present. Similar effects were seen with the antibiotic gramicidin S and with melittin, a polypeptide purified from bee venom. All of these agents are amphiphilic polypeptides. Nitroprusside was not able to stimulate both particulate and soluble enzyme treated with the nonionic amphiphile, Lubrol PX, suggesting that the membrane-active polypeptides had a different mechanism of action. These polypeptides are known to alter the membrane matrix by binding to phospholipid, and we suggest that this alteration allowed greater access of substrate and of nitroprusside to the enzyme. Lubrol PX, however, may interact preferentially with the enzyme, and thus block nitroprusside activation. The most potent of these agents was melittin, which stimulated nitroprusside activation at a concentration which had little effect by itself (7 microns), and at which others have demonstrated lytic effects on cells.


Circular dichroic analysis of the secondary structure of myelin basic protein and derived peptides bound to detergents and to lipid vesicles. In aqueous solution bovine myelin basic protein exhibits no significant alpha-helical or beta-pleated sheet structure. However, in vivo this protein is associated largely with the myelin membrane: experiments have therefore been performed to determine the structure of the protein when bound to lipid bilayers. Circular dichroism spectra show that this protein undergoes a major conformational change on binding to lipid bilayer vesicles formed from diacylphosphatidylserine or diacylphosphatidic acid, and on binding to micelles of several detergents. Association with diacylphosphatidylcholine failed to induce a structural change: this observation is interpreted in terms of an earlier report that lysophosphatidylcholine does increase the alpha-helical content of basic protein. These circular dichroism measurements and studies of the binding to the bilayer-forming lipids appear to provide support for significant hydrophobic lipid-protein interactions. Similar studies using two peptides produced by cleavf basic protein indicate that a major structure-forming region in the middle of the protein has been disrupted by this scission.


Binding of bilirubin by bovine and human alpha-fetoprotein. alpha-Fetoprotein, a fetal protein associated with certain tumors, was found to bind bilirubin. Addition of human or bovine alpha-fetoprotein to bilirubin solutions enhanced the light absorbance of bilirubin and shifted its  maximum. Bovine alpha-fetoprotein caused a marked shift towards shorter wavelengths, while human alpha-fetoprotein gave a slight red shift. The spectral changes were used to study the characteristics of the binding of bilirubin by bovine alpha-fetoprotein. These studies indicated the presence of one binding site/molecule of alpha-fetoprotein with an association constant of about 1.1 . 10(6) M-1. A difference between the spectral changes brought about by alpha-fetoprotein and albumin allowed comparison of their relative affinities for bilirubin. The spectrum approximated the average between the spectra induced by the two proteins when the ratio of bovine alpha-fetoprotein to bovine albumin was 6.3 : 1, and of the human proteins 21 : 1, respectively. These results show that alpha-fetoprotein from two species binds bilirubin with an affinity somewhat lower than that of albumin. Binding of bilirubin by alpha-fetoprotein is in agreement with the recent demonstration of structural homology between alpha-fetoprotein and albumin. Whether alpha-fetoprotein plays a role in the metabolism of bilirubin or other degradation products of heme remains to be investigated.


Inhibition of lipolysis in hamster epididymal adipocytes by selective alpha-adrenergic agents. Evidence for cyclic AMP-dependent and independent mechanisms. In this study the role of cyclic AMP in the antilpolytic effect of the alpha-adrenergic agents methoxamine and phenylephrine in hamster epididymal adipocytes was studied. Both methozamine and phenylephrine lowered the very high levels of cyclic AMP that were produced by high concentrations of isoproterenol (10 muM) or ACTH (100 MU/ml), and partially inhibited lipolysis. When lower concentrations of isoproterenol were used, the antilipolytic effect of phenylephrine and methoxamine was still evident. Under these conditions methoxamine produced a slight suppression of cyclic AMP levels while phenylephrine increased accumulation of cyclic AMP. It follows, therefore, that the inhibition of lipolysis by the alpha agents is most likely unrelated to changes in cyclic AMP levels; in contrast, phenylephrine promoted lipolysis and increased cyclic AMP levels. When the stimulus for lipolysis was provided by methylxanthines a different picture emerged. Methoxamine antagonized lipolysis and lowered cyclic AMP levels. In the presence of propranolol, phenylephrine lowered cyclic AMP levels and suppressed methylxanthine-accelerated lipolysis. It is suggested that when methy xanthines provide the stimulus for lipolysis the antilipolytic effect of methoxamine and phenylephrine (in the presence of propranolol) may be mediated by the suppression in cyclic AMP levels.


Relations between fatty acids and oestrogen binding properties of pure rat alpha 1-foetoprotein. A delipidation procedure based on treatment with charcoal at pH 3 has been applied to highly purified rat alpha 1-foetoprotein preparations. The oestrogen binding properties of the delipidated proteins have been studied with an equilibrium dialysis technique, and compared with the properties of the untreated foetal protein, as well as those of preparations reconstituted from the defatted alpha 1-foetoprotein and the removed lipids. An important increase has been evidenced for the binding levels of oestrone, oestradiol-17 beta and diethylstilboestrol by the delipidated alpha 1-foetoprotein. A reversal of this effect has been obtained by incubating the delipidated protein either with the lipids extracted from the purified alpha 1-foetoprotein or with a potent competitor of the rat alpha 1-foetoprotein-oestrogen interaction, designated as 'L', previously demonstrated and isolated from whole rat sera, and tentatively characterized as a mixture of fatty acids. Scatchard analysis of the oestrone and oestradiol-17 beta binding parameters show that the enhanced fixation of the hormones after defatting is primarily due to a two-fold increase of the apparent number of binding sites/mol alpha 1-foetoprotein. The results are interpreted in terms of the probable, at least partial, identity between the lipids closely associated with the pure alpha 1-foetoprotein and the fatty acid mixture 'L' isolated from whole sera. The possible biological role of complex interplay between oestrophilic alpha 1-foetoproteins, phenolsteroids and fatty acids in the control of oestrogen levels during development is discussed briefly.


[Interaction between alpha-2 macroglobulin and trypsin with synthetic trypsin inhibitors]. The complex formed between trypsin (Tn) and alpha 2 Macroglobulin (alpha 2 M) retains the whole hydrolytic activity of the enzyme for synthetic substrates. Moreover synthetic inhibitors of low molecular weight stiel inhibit this activity. A comparative study of three inhibitors (Benzylamine, Butylamine, Benzamidine) has been carried out and shows that their behavior is similar. These inhibitors bind trypsin when it is bound to alpha 2 M and reciprocally alpha 2 M can bind Tn-inhibitor complex. Nevertheless the dissociation constant of the enzyme-inhibitor complex (Ki) is increased by alpha 2 M. In the case of Benzamidine the value of Ki is 2.22.10(-5) M for native enzyme and 13.4.10(-5) M for Tn-alpha 2 M and in the case of Butylamine this value increases from 0.5.10(-3) M to 2.95.10(-3) M. These variations of the Ki values are due to the modification of the accessibility of the inhibitor to the active site. Unpublished results show that the alpha 2 M molecule undergoes a deep structural modification in the course of the complex formation, which must lead to an increase of the value of Ki. This structural modification is probably irreversible so that the alpha 2 M complex has never been dissociated without altering the alpha 2 M molecule. The increase of the values of Ki cannot therefore result in an effective decrease of the association constant of the Tn-alpha 2 M complex.


Drug allergy: in vitro cross-allergenicity between amoxicillin and benzyl penicillin. The capacity of amoxicillin to elicit allergenic reactions in vitro was determined on benzyl-penicillin sensitive patients. RAST and histamine release were performed on blood of these patients using B pen linked to human serum albumin (BPO-HSA) as allergen. A relationship was noted between the results of the in vitro tests and the date of the allergic manifestations. When results of RAST and histamine release were significant, amoxicillin was tested for its capacity to elicit histamine release and to inhibit RAST performed with BPO-HSA. A cross-allergenicity was observed in six sera out of eight. The results suggested that amoxicillin may be capable to react with cell-bound or serum IgE to give hypersensitive manifestations in some penicillin sensitive patients.


Serum copper, ceruloplasmin, lactic-dehydrogenase and alpha 2-globulin in lung cancer. Twenty-six patients with primary lung cancer were studied in whose blood we found a significant increase in the levels of copper, ceruloplasmin, lactic-dehydrogenase and alpha 2-globulins. The role of copper in pulmonary cancerogenesis is discussed.


Laminar origin of projections from the superficial layers of the superior colliculus in the tree shrew, Tupaia glis. The laminar origin of the efferent projections from the superior colliculus to the pulvinar and to the dorsal and ventral lateral geniculate nuclei has been studied using the retrograde axonal transport of horseradish peroxidase. Following injections in either the dorsal or the ventral lateral geniculate nucleus, cells heavily labeled with the horseradish peroxidase reaction product are restricted primarily to the upper stratum griseum superficiale. These cells have small, fusiform somas with dendrites which extend dorsally and ventrally, perpendicular to the pial surface. In contrast, following injections in the pulvinar, cells labeled with reaction product are restricted primarily to the lower stratum griseum superficiale and to the most superficial part of stratum opticum. These cells typically have larger somas than cells in the upper stratum griseum superficiale, and often have dendrites which emerge horizontally from the cell body. When taken together with previous electrophysiological and anatomical studies, the present findings suggest that there is a laminar subdivision of the tree shrew stratum griseum superficiale, and that these subdivisions project selectively to different thalamic targets.


Cells of origin of the spinocerebellar tract in the rat, studied with the method of retrograde transport of horseradish peroxidase. Following injections of horseradish peroxidase into the cerebellum, the distribution of labeled neurons was studied in the whole length of the spinal cord of the rat. To find the ascending side of the axons, injections were made following hemisections at C1 or between C1 and C2. Labeled spinocerebellar tract neurons were classified into two groups according to the axonal course in the spinal cord; one is composed of neurons with uncrossed ascending axons and the other, neurons with crossed ascending axons. Neurons of origin of the uncrossed tracts were located in the medial part of lamina VI of C2 to C8, the central part of lamina VII of C4 to C8, lamina V of C7 to L3 and Clarke's column. Neurons of origin of the crossed tracts were found in the central cervical nucleus of C1 to C3, the intermediate zone and the ventral horn of the lower thoracic and the lumbar segments (T11 to L3), and in the dorsal horn, the medial part of lamina VII and the ventrolateral part of the ventral horn of the sacral and caudal spinal cord. In comparison with our previous results in the cat, it was suggested that the spinocerebellar system in the rat is organized in the same fashion as in the cat, in terms of the location and the intraspinal axonal course of the cells of origin.


Axonal transport in early experimental diabetes. In the present study fast axonal transport was examined in streptozotocin rats with 4 weeks duration in diabetes. Tritiated leucine and 14C-labelled glucosamine were injected into the fifth lumbar ganglion and TCA-soluble as well as insoluble activity were measured in segments of the sciatic nerve at various time intervals. (1) Time from injection until start of fast axonal transport was prolonged in diabetic rats whereas anterograde transport velocity was unchanged. (2) Incorporation of labelled leucine was reduced by 40%, whereas labelled glucosamine incorporation was unchanged. (3) Alterations observed in accumulations of labelled glycoconjugates proximal and distal to a collection crush might represent a decreased amount of retrograde transported material. The changes found in protein and glycoconjugate synthesis and transport could be related to the early reduction in axon calibre and conduction velocity in peripheral nerve of streptozotocin-diabetic rat.


Effects of anti-white matter serum on myelin and lipid synthesis in brain prisms. Tissue prisms prepared by choping whole mouse brain maintained respiratory capacity and ultrastructural integrity of 3 h in vitro. Normal rabbit serum (ca. 25%) caused no morphological change but inhibited the synthesis of galactolipids by the prisms. Heating the serum abolished the inhibition. Complement containing anti-white matter rabbit serum destroyed myelin and inhibited galactolipid synthesis to a greater degree than did normal serum. Structures other than myelin were unaffected by the antiserum. Incubation in the presence of heated anti-white matter serum eliminated the myelin destruction but resulted in specific morphological changes characterized by the doubling of the myelin lamellae at the intraperiod line. Immunoperoxidase studies suggest specific binding of immunoglobulin to components of myelin located at the intraperiod lone. These changes were similar to those found in organotypic cultures. Heated antiserum did not inhibit galactolipid synthesis but addition of complement (normal guinea pig serum) to the heated antiserum restored only that portion of the inhibition which exceeded that caused by normal serum. Heat labile factors in normal rabbit serum which inhibit myelin lipid synthesis in the prisms must be corrected for in studies in which the heating of serum is used to demonstrate that the effect is complement dependent. The prism system is simpler than that of organotypic cultures and may be useful in the study of myelinotoxic factors.


[Effect of organochlorine pesticides on the ocular electric response of the white mouse]. The injection of DDT or HCH resulted in a decrease of the amplitude of b-wave of electroretinograms without any modification of its latency, whereas a-wave remained unchanged. In conclusion, we may say that organochlorous pesticides do not act on photoreceptors or synapses, but they act only on bipolar and ganglionnar neurones.


[Neovascular glaucoma. Surgical technic]. We describe a surgical technique we have used in eight cases of neo-vascular glaucoma. Under the scleral flap of a trabeculectomy we insert a sheet of supramid. After a follow-up period of 8 to 24 months, half of the cases have a good functioning filtration bleb.


Comparative effects of lignocaine and lorcainide on conduction in the Langendorff-perfused guinea-pig heart. The effects of lignocaine and lorcainide on conduction through the AV node and the ventricular conduction system have been investigated in the Langendorff-perfused guinea-pig heart. Basal conduction times were estimated at 80 and 120/min; at each frequency extra-stimuli with variable coupling intervals were applied in order to determine the change in conduction velocity and estimate refractory periods. In most of the preparations paced at 80/min premature impulses were blocked at a site distal to the AV node (distal gate). In therapeutic concentrations (1 to 5 mg . litre-1) and at low frequency of stimulation lignocaine did not change basal conduction times but had a definite slowing effect on the conduction of extra-systoles. Slowing of basal conduction times became prominent at higher frequencies of stimulation or at concentrations above 5 mg . litre-1. In all conditions the effect on the ventricular conduction system was more pronounced than on the AV node. Compared with lignocaine, lorcainide was more effective and more selective. Important changes in basal conduction times and in conduction of premature impulses were observed at 0.31 and 0.63 mg . litre-1. The effect on conduction in the ventricular conduction system was more pronounced than on conduction in the AV node. The dose-effect relationship for different parameters was steeper with lorcainide than with lignocaine. At high concentrations of lorcainide (greater than 1.25 mg . litre-1) intermittent block, especially in the ventricular conduction system, was frequently observed.


Ultrastructural identification of non-adrenergic, non-cholinergic nerves in the rat anococcygeus muscle. The innervation of the rat anococcygeus muscle has been investigated ultrastructurally following fixation with a modified chromaffin reaction for the demonstration of biogenic amines (Tranzer and Richards, 1976). Three types of nerve profiles were revealed: (1) 60-70% of the profiles are adrenergic; (2) less than 5% of the profiles appear to be cholinergic; (3) up to 40% of the profiles are distinguished by the presence of a characteristically high proportion of electron-opaque, chromaffin-negative vesicles, 85-110 nm in diameter. This third type of profile was not affected by 6-OHDA, and is considered to represent the non-adrenergic, non-cholinergic inhibitory innervation of this tissue. Because of the morphological similarity of this nerve type, apart from the smaller vesicle size, to classical peptidergic nerve endings, they have been termed "small p-type" (sp-type). These results are discussed in relation to a previous report describing only two types of nerve profiles in this tissue (Gillespie and Lüllmann-Rauch, 1974).


Neural inputs to the prefrontal agranular insular cortex in the rat: horseradish peroxidase study. The prefrontal cortex, dorsal to the rhinal sulcus of the rat (hereinafter termed the agranular insular cortex) has been examined with the use of the retrograde transport of horseradish peroxidase. Labelled perikarya were seen in the region of the ipsilateral medial forebrain bundle, consistently rostral to the caudal edge of the posterior mammillary nucleus. These cells were interpreted as being the rostral members of the A10 dopaminergic cell group. Labelled cells were also seen in the dorsal raphe nucleus, the ipsilateral pyriform cortex, the contralateral agranular insular cortex, the ipsilateral basolateral amygdaloid nucleus, the ipsilateral locus coeruleus, and the medio-dorsal, ventromedial, and parafascicular thalamic nuclei. Detailed examination of the thalamic input confirmed a differential innervation of the dorsal and ventral regions of the agranular insular cortex. Possible functional correlates of these prefrontal afferents are discussed.


Neovascularity related to a mural thrombus. This is a case report on an unusual angiographic finding related to a left ventricular mural thrombus. An abnormal vascular blush of contrast was noted on performing coronary arteriography. This was attributed to neovascularity in a left ventricular mural thrombus. This abnormality must be distinguished from vascular fistulas, intracardiac communications, and tumors in making appropriate therapeutic decisions.


Cell-free synthesis and membrane insertion of mouse H-2Dd histocompatibility antigen and beta 2-microglobulin. Messenger RNA from SL2 lymphoma cells was translated in a cell-free system in the presence of microsomal membranes. Mouse H-2Dd histocompatibility antigen was correctly assembled in the microsomal membranes, and transmembrane insertion of the nascent chain was accompanied by glycosylation and cleavage of the signal sequence H-2Kd antigens, synthesized in vivo, comprised a transmembrane glycoprotein and an unglycosylated protein in the cytoplasm. The glycosylated forms of the H-2Dd and H-2Kd antigens were modified during intracellular transport from the endoplasmic reticulum to the cell surface. beta 2-Microglobulin was also synthesized in vitro, and transfer of this protein into microsomal vesicles was accompanied by cleavage of its signal sequence. In the endoplasmic reticulum, beta-microglobulin can bind to newly synthesized H-2d glycoproteins. The mRNAs coding for beta 2-microglobulin and H-2Dd antigen could be separated on aqueous sucrose gradients.


Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Three new mesenchymal phenotypes were expressed in cultures of Swiss 3T3 and C3H/10T1/2CL8 mouse cells treated with 5-azacytidine or 5-aza-2'-deoxycytidine. These phenotypes were characterized as contractile striated muscle cells, biochemically differentiated adipocytes and chondrocytes capable of the biosynthesis of cartilage-specific proteins. The number of muscle and fat cells which appeared in treated cultures was dependent upon the concentration of 5-azacytidine used, but the chondrocyte phenotype was not expressed frequently enough for quantitation. The differentiated cell types were only observed several days or weeks after treatment with the analog, implying that cell division was obligatory for the expression of the new phenotypes. Oncogenically transformed C3H/10T1/2CL8 cells also developed muscle cells after exposure to 5-azacytidine, but at a reduced rate when compared to the parent line. Five subclones of the 10T1/2 line which were the progeny of single cells all expressed both the muscle and fat phenotypes following 5-azacytidine treatment. The effects of the analog are therefore not due to the selection of preexisting myoblasts or adipocytes in the cell populations. Rather, it is possible that 5-azacytidine, after incorporation into DNA, causes a reversion to a more pluripotential state from which the new phenotypes subsequently differentiate.


[Ultrastructural identification of the nucleolus organizer by the silver staining technic]. The nucleolus organizer regions can be selectively stained in metaphase chromosome preparations by the Goodpasture and Bloom's technique which was adapted to electron microscopy analysis of cells during interphase. Using this technique, a selective accumulation of silver grains was observed over nucleolus light areas. This selective accumulation allows the identification of the interphase fibrillar centers as the nucleolus organizer regions. Ultrastructural relationships between fibrillar centers and dense fibrillar component are discussed.


[Histamine production in mixed culture between allograft donor and recipient lymphocytes]. An increased production of histamine has been demonstrated during mixed cultures between allograft donor and recipient lymphocytes. This phenomenon could result from the action of a non-dialysable factor released by recipient cells in the presence of donor cells. This factor is able to increase histamine production from normal spleen cells. Little or no increase in histamine production is found during primary and mixed lymphocyte cultures (without previous allograft).


The use of fluorescein conjugated antisera to identify proteins on stained and dried electrophoresis strips. Paraprotein bands discovered on routine electrophoresis may be identified immunologically without repeating the electrophoretic separation. It is only necessary to heat the stained and dried strip in a hot air oven to reactivate the antigenic sites and insolubilise the protein. The strip may then be stained using specific antisera conjugated with fluorescein. The absence of prozone phenomena greatly helps in typing of free light chains.


Evidence for heterogeneous or incomplete immunoglobulins in oligoclonal CSF studied by electroimmunofixation. 10 oligoclonal CSF have been studied by electroimmunofixation techniques on Cellogel RS. The gammaglobulin bands are either homogeneous or heterogeneous. The heterogeneous bands migrate preferentially in the cathodic part of the gamma zone. The light chain type of many homogeneous bands is lambda. Some oligoclonal bands may be composed of free homogeneous light chains. The free lambda chains have a more anodic migration than the free kappa chains. Furthermore, our results suggest the presence of free heavy chains in some instances.


125Iodine labeling of beta-hexosaminidase A without modifying its properties. Human placental beta-hexosaminidase A was labeled with 125iodine to high specific activity with the retention of conformational integrity as judged by the retention of enzymatic activity. The oligosaccharide structure also appeared to be intact since the labeled enzyme was cleared from the circulation of the rat with a half-life identical to that of the unlabeled enzyme and an excess of unlabeled enzyme effectively blocked the clearance of the labeled form. Furthermore, the pattern of inhibition of clearance of the native and labeled enzymes by asialofetuin and mannans was identical. This useful and mild procedure for labeling enzymes may be of general importance in the preparation of enzymes for metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.


Advanced head and neck cancer treated by combined radiotherapy and VBM cytotoxic regimen--four-year results. The 4-year results of treatment of advanced squamous cell cancer of the head and neck regions by radiotherapy combined with a cytotoxic regimen VBM (Vincristine, Bleomycin, Methotrexate) given synchronously are compared with those obtained with radiotherapy alone in similar patients. There was a statistically significant improvement in the results of the combined treatment at four years in terms of crude survival rate (56.1% v. 24.5%) and disease free survival rate (56.5% v. 21.8%). Both groups are examined for general prognostic features which might account for the difference. In the absence of such features it must be concluded that the addition of VBM to radiotherapy had produced a marked therapeutic improvement.


Discovery of an inherited bisatellited metacentric microchromosome in amniotic cell culture. The identification is reported of an extra bisatellited metacentric microchromosome in amniotic cell culture from the third pregnancy of an identical twin (amniocentesis being performed because of age), and its subsequent finding in the maternal parent as an inherited familial marker. The carriers of the microchromosome are all clinically normal and the parents opted for continuation of pregnancy. Only one other report was found in the literature of a similar microchromosome detected in amniotic fluid culture, but we believe ours to be the first bisatellited microchromosome to be clearly identified from an amniotic cell culture using silver staining.


Dual response of lymphocytes from multiple sclerosis patients to myelin basic protein. Peripheral blood lymphocytes from forty-nine patients with multiple sclerosis (MS), twelve patients with other neurological diseases and twenty-seven healthy individuals were examined by a two-stage procedure based on the Con A stimulation of the basic protein (BP) pre-treated lymphocytes. Enhanced BP-induced blastogenic stimulation (BS) (90--220% was observed in MS individual classified as active (A-MS), whereas suppressed BS (27--120%) was observed in MS patients classified as non-active (NA-MS). The difference was statistically significant (P less than 0.01). The results obtained with the MS patients' lymphocytes also differed significantly (P less than 0.05) from those of the two control groups. Either response obtained with MS lymphocytes could be elicited with a similar concentration of BP. The enhanced or suppressed response to BP did not correlate with the cellular response to common antigens such as candida and streptokinase-streptodornase. Most lymphocytes of individuals from the two control groups showed BS values of 100 +/- 20% in response to BP. However, some degree of stimulation or suppression was also obtained with lymphocytes from a few individuals of both control groups. The potential value and application of the two-stage procedure is discussed.


Synthesis and secretion of alpha 1-microglobulin by human lymphocytes. alpha 1-Microglobulin (alpha 1-m) was purified by column chromatography from a supernatant fluid of cultured T and B lymphocytes stimulated with mitogens. This protein had a molecular weight of 33,000 as determined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, migrated in the alpha 1-region on immunoelectrophoresis and proved immunologically identical to alpha 1-m which had been purified from the urine of patients with renal tubular disorders. Using indirect immunofluorescence, alpha 1-m was detected on the surface of both T and B lymphocytes, displaying the same intensity on each cell type. These findings indicate that alpha 1-m is actively produced and secreted by T and B lymphocytes.


Lymphocyte response to phytohaemagglutinin in the presence of serum from pregnant women: correlation with serum levels of alpha-foetoprotein. The effect of sera from women in different stages of pregnancy on cultures of human lymphocytes stimulated with phytohaemagglutinin (PHA) has been studied and correlated with alpha-foetoprotein (AFP) levels. Sera taken before the twentieth week of gestation showed low levels of AFP and failed to inhibit lymphocyte proliferation. Inhibition was obtained with 38.5% of sera taken from weeks 20 to 30 and with 51.7% of sera after the thirtieth week of pregnancy. Average serum levels of AFP also increased with advanced gestation, but a consistent correlation was not found between AFP concentration and inhibitory activity for every individual serum tested. Lymphocytes from pregnant women or from normal blood donors behaved in the same way in these tests. The mechanisms of inhibition and the role of AFP are discussed.


Antigenic properties of the Golgi derived scales of Pleurochrysis scherffelii. The cell wall of the marine alga Pleurochrysis scherffelii is composed of Golgi-derived scales with four distinct subcomponents: amorphous coating material, radial microfibrils, spiral coating substances and spiral microfibrils. The antigenic properties of the Pleurochrysis scales were studied. Antibodies were produced in rabbits to the amorphour to the amorphous coating material and to a heretofore undescribed peripheral subcompoent, known as the amorphoous rim modification substance. The antigenic subcomponents were detected by agglutination/precipitation, negative staining, and shadowing procedures. Scales of the Cricosphaera phase produced negative results when tested with Pleurochrysis scale antisera. A possible function of the amorphous rim modification substance in maintaining the integrity of spiral microfibrillar organization is introduced.


The differentiation and calcification of chondrocytes in primary cell cultures. The cartilage from a non-immobilized fracture undergoes a series of morphological and biochemical changes resembling the in vivo differentiation and calcification in the epiphyseal plate. The studies reported here demonstrate that a homogeneous population of chondrocytes isolated from fracture callus fibrocartilage undergoes the same changes in vitro. Chondrocyte primary cultures were grown for 28 days during which time the morphological, histological and histochemical properties of the cultures were studied. Demonstrated by various histological procedures, chondrocytes synthesized the characteristic cartilage matrix, and progressively calcified with increased culture age. This system can be used to elucidate the cellular and molecular mechanisms of calcification.


The effect of bleomycin on rat bone marrow cells. Experiments were carried out to determine the cytogenetic effects of the antibiotic bleomycin (BLM) in rats using the bone marrow system. A total of eighteen male and eighteen female Sprague-Dawley rats were injected with varying concentration of BLM, over varying periods of time. The results revealed that at low concentrations BLM showed no alteration in the ploidy or the morphology of rat chromosomes. This however, was not the case when the dose or administration period was increased.


Phleomycin and bleomycin: molecular model building studies. Molecular model building studies were conducted to simulate phleomycin-bleomycin (PB) antibiotics and thus assess a hypothetical polyphleomycin-DNA complex proposed earlier. While the latter model was not conclusively proved it was found to be quite consistent with the structure of PB compounds.


The intra-nucleolar localization of amplified rDNA in Xenopus laevis oocytes. A fluorescent Feulgen-stain was adapted in order to demonstrate DNA-containing structures inside the amplified nucleoli of Xenopus laevis. At all stages of oogenesis this method reveals granules or complex structures of DNA in each nucleolus. The micronucleoli which do not stain with this method and which do not reveal an internal structure in low molarity saline, unlike real nucleoli are considered as nucleolus-like bodies. The DNA-containing structures in the nucleoli can be composed of one or several granules, or they can be arranged in a linear, reticulated or circular form, independant of any correlation with the stage of oogenesis.


Observations on the synaptonemal complex in Armenian hamster spermatocytes by light microscopy. Using the silver staining technique, in somatic and meiotic chromosomes of the Armenian hamster (Cricetulus migratorius), it is possible to stain synaptonemal complexes (SCs) and the nucleolus organizer regions (NORs) in early spermatocytes. There are five pairs of autosomes (Nos. 2, 4, 6, 7, and 8) which have terminally located NORs. Synaptonemal complexes and accessory structures present in the sex chromosomes within the sex vesicle can be easily observed using light microscopy.


Chromosome core structure revealed by silver staining. Chromosomes were subjected to either prolonged hypotonic solution pretreatment or aging. Both conditions greatly loosened and dispersed the overlying epichromatin from the central chromosome core structure. This was followed by silver staining and examination with bright-field microscopy. The chromosome core selectively reduced the silver and stained black while the surrounding epichromatin stained yellow. A single core was seen extending the length of each chromatid. Nucleolus organizer regions appeared to be attached to the core, while kinetochores seemed to be specialized regions of the core itself. Cytochemical tests indicated that the core component(s) responsible for silver staining was non-histone protein(s).


Relation between the SCE points and the DNA replication bands. A method for obtaining a combination of differential sister chromatid staining and DNA replication banding is described. Using this method the SCE points can be precisely localized to particular bands of individual chromosomes. It was shown, that SCEs occur not only in the regions of early DNA replication (= euchromatic segments = negative G-bands), but also in the regions of late DNA replication bands (=heterochromatic segments=positive G-bands). SCEs occurred about three times more frequently in the euchromatic segments than in the heterochromatic segments. Furthermore, more SCEs were observed in the early replicating X-chromosome than in the late replicating X-chromosome.


Synaptonemal complexes of mouse and human pachytene chromosomes visualized by silver staining in air-dried preparations. A new method is described which allows light microscopy visualization of the synaptonemal complexes in primary spermatocytes. The conventional acetic acid-alcohol fixed and air-dried preparations can be used for the modified silver staining technique. Several hundred pachytene spermatocytes from a single mouse testis or human testicular biopsy can be examined. The prospective utilization of the present method is briefly discussed.


Immunofluorescent patterns of dissociated rat embryo cerebral cells during development in surface culture: distinctive reactions with neurite and perikaryon cell membranes. The presence of antigenic determinants on surface cultures of dissociated cells from rat embryo cerebral tissue has been investigated with the immunofluorescence technique. Antisera raised in rabbits against mature mouse brain reacted with the embryonic cells and enabled the identification of two distinct populations of immunofluorescent cells. One population clearly distinguishable by its ring fluorescence was involved in early cellular reaggregation and neurite formation. A second population which displayed dispersed fluorescence over the whole cell, did not establish intercellular contacts for prolonged periods of time in culture. Indirect evidence suggests that after several days in culture a great portion of the latter population was found in close proximity to the early aggregate, to form an immunofluorescent-heterotypic aggregate. A second antiserum, raised against embryonic rat cerebral cells, displayed immunofluorescent labeling patterns similar to those observed with the mature mouse brain antiserum, when reacted with freshly dissociated cerebral cells. As the cells differentiated in surface culture, distinctive patterns of immunofluorescent reactions developed. Large neuronal cells were labeled on their neuritic cell membranes by the antiserum to adult brain but not by the antiserum to embryonic cells. The results suggest that the anti-mature brain serum possess a class of antibodies which is able to react with antigenic determinants localized specifically on the neuritic plasma membrane.


Breach rhythm: the EEG related to skull defects. From 33 patients with skull defects 89 EEGs were recorded, most with X ray control of the electrode sites. In 10 patients records were made shortly before and about 10 days after bone replacement. The remaining cases had EEGs either with or without bone replacement. The amplitude of alpha and frontal fast rhythms might be increased over or near unilateral posterior and frontal defects respectively. This enhancement was by a factor of less than 3. Since the electrode involved might not be in the defect but on adjacent bone this does not seem to be an effect of greater proximity to the generators. In 21 cases with defects involving or near to electrodes C3(/) and T3(4) sharply focal mu-like rhythms at 6--11 c/sec, usually with faster components, were seen. They formed two groups, at C3(4), responsive to fist clenching and other stimuli (not to eye opening) and at T3(4), unresponsive to any stimulus. In both groups the waves often had spike-like negative phases, but true spikes and also random slow waves with the same restricted focus and responsiveness were sometimes seen. Because of this complexity we prefer the term breach rhythm to mu or mu-like. Bone (or acrylic) replacement abolished central breach rhythm in 3 cases, but not in others and it might or might not restore the symmetry of alpha rhythm or fast rhythm, but burr holes, saw cuts etc., always remain after craniotomy. It is argued that, with the possible exception of 2 patients, the breach rhythms described in this series do not represent enhanced normal mu rhythm. Breach rhythm, even when very spike-like, appears to have little relationship to epilepsy and is not an indicator of recurrence of a tumour.


Relation of frequency-analyzed EEG to monitoring behavior. Two experiments examined the relation of prestimulus electroencephalographic (EEG) frequency to choice RT. Based on previous studies, initial analysis was concerned with 3--7 c/sec activity. In well-rested subjects (Exp. 1), trial-by-trial analyses indicated large variations in prestimulus EEG activity which were unrelated to RT and large variations in RT which were unrelated to prestimulus EEG. In Exp. 2, subjects were deprived of sleep for 1 night and within-subject comparisons made between RT and EEG activity immediately preceding the 10 shortest and 10 longest RT trials, and 10 trials where the subject failed to respond. Significant univariate correlations were found, largely between RT and the frequencies in the 15--20 c/sec range of EEG activity and not in the 3--7 c/sec activity. A multiple regression analysis using up to 5 EEG frequencies indicated significant correlations of prestimulus EEG activity with RT; but with considerable subject-to-subject variability in the EEG frequencies contributing to the multiple R. The overall results suggest that there can be considerable variation in EEG activity which is unrelated to performance, except when the EEG fluctuations are secondary to changes in arousal which, in turn, effect performance.


Acute mouse and chronic dog toxicity studies of danthron, dioctyl sodium sulfosuccinate, poloxalkol and combinations. Because of an apparent typographic error in a US patent, there has been some confusion as to the acute oral toxicity of danthron and danthron in combination with dioctyl sodium sulfosuccinate (DSS). Acute oral toxicity studies in mice revealed LD50 values of greater than 7 gm/kg for danthron, 2.64 gm/kg for DSS and 3.42 gm/kg for danthron/DSS mixture (1:2 ratio). These results indicate that the lethality of these compounds is in the gm/kg range and not in the mg/kg range. A one year chronic toxicity study of danthron, dioctyl sodium sulfosuccinate, poloxalkol and combinations in dogs failed to reveal any toxic effects. In particular, there was no evidence of hepatotoxicity or of any changes in the myenteric plexuses in the chronically treated dogs.


Human slow auditory evoked potentials during natural and drug-induced sleep. The alterations of human slow auditory evoked potential (SAEP) were studied during the sleep-waking cycle. Simultaneous recording of the mean square and standard error curves proved helpful in obtaining more information on the EEG samples used for averaging. In both natural and drug-induced sleep the earlier SAEP components decreased or disappeared, while the later components increased. A significant lengthening of all SAEP peak latencies was found in all sleep stages during tone-burst stimulation. With click stimulation the peak latencies of most SAEP components remained constant. Due to the variability of individual SAEPs, the interpretation of averaged SAEPs was commonly more difficult in sleep as compared with wakefulness. The alterations of SAEPs during sleep are considered from the point of view of their exrralemniscal origin. The hypothesis is put forward that the activating (desynchronizing) and depressing (synchronizing) systems participate in the generation of the earlier and later SAEP components, respectively.


[Effect of cooling and electrical stimulation of nuclei of raphe system on states of alertness in cat]. In cats prepared in a 'semi-chronic' manner (spinal cord transected, brachial plexus sectioned bilaterally) all states of alertness are present; their quantitative evolution is characterized by an immediate postoperative period with continuous wakefulness and a secondary period of recovery of the states of sleep, which are then maintained. In such a preparation, localized moderate cooling (+10 degrees C) of the nucleus raphe dorsalis induces slow wave sleep and paradoxical sleep; the same type of cooling of the nucleus raphe magnus induces wakefulness; however, the electrical stimulation of these two nuclei always induces wakefulness. Cooling of the nuclei raphe centralis or pontis suppresses only the 'ponto-geniculo-occipital' (PGO) waves. Colling of the nuclei raphe obscurus and pallidus induces wakedfulness, but stage I of slow wave sleep may occur. Treatment with p-chlorophenylalanine (PCPA) before making the semi-chronic preparation produces the classical syndrome characterized by cortical activation and continuous discharge of PGO waves; in this case cooling of the nucleus raphe dorsalis no longer induces slow wave sleep but only paradoxical sleep; after injection of DL-5-HTP, slow wave sleep is obtained again through cooling; this effect diminishes progressively.


The effects of flurazepam hydrochloride on brain electrical activity during sleep. To further evaluate the effects of flurazepam on EEG during sleep, following 7 nights of placebo baseline, flurazepam (30 mg) was administered to 6 young adult poor sleepers for 10 additional nights while 6 other young adult poor sleepers continued to receive placebo capsules in a double-blind paradigm. Three placebo follow-up nights were recorded 2--3 weeks post-treatment. Twelve good sleepers received only placebo capsules for the first 7 nights. Delta waves, 0.5--2 c/sec, and sleep spindles were counted on-line by a phasic detector. Delta activity was also analyzed off-line by PDP-12 computer for only the first 4 h of sleep and involved a comparison over stages of sleep. Click-evoked K-complexes during NREM sleep were analyzed for 6 good sleepers and 11 poor sleepers. Repeated use of flurazepam caused a gradual decrease in delta amplitude and count, and a gradual increase in sleep spindle rate. The decrease in delta amplitude was seen in all sleep stages, but the decrease was significant only during SWS and stage 2. The decrease in delta amplitude was significant by the 3rd drug night, but the rate of amplitude decrease tended to slow with continued treatment. The decrease in delta count was less pronounced and more gradual over drug nights than the rate of decrease in amplitude. Flurazepam also significantly reduced evoked K-complex amplitude but did not affect latency. Sleep spindle rate was significantly increased by drug night 5. Results of this study indicate that the reduction of SWS with flurazepam during the initial drug nights is due primarily to the decrease in delta amplitude, but, with continued use, the decrease in delta count also contributes to the decrease in stage 4 sleep.


[Abolition by naloxone of the inhibitory effect of peripheral electrical stimulation on the blink reflex]. The effects of a low frequency (2 c/sec) peripheral stimulation (electro-acupuncture, EA) on the nociceptive (R2) response of the blink reflex elicited by supra-orbital nerve stimulation (0.1 msec, 1 shock/8 sec) were studied in 10 healthy subjects. EA stimulation produced a very significant inhibition of the reflex in 8 subjects. Double-blind injection of naloxone (0.8 mg) reversed this inhibition while no signiificant change was observed with placebo. These results suggest that EA stimulation induces the release of endogenous opiates.


Stimulus induced and seizure related changes in extracellular potassium concentration in cat thalamus (VPL). Extracellular potassium activity (ak) and field potentials (fp) were measured in the nucleus ventro-postero-lateralis (VPL) thalami in order to assess the extent of thalamic participation in cortical seizure activity. Small increases (up to 0.7 mmole/l) or decreases (up to 0.2 mmole/l) in ak were induced by electrical stimulation of the contralateral forepaw. These changes in ak were spatially more limited than the simultaneously recorded fp. Similar observations were made during weak electrical stimulation of the somatosensory cortex and during interictal spikes in a cortical penicillin focus. Large and widespread increases in ak to levels of 11.6 mmoles/l and slow negative fps of 8 mV accompanied seizure generation either in a cortical penicillin focus or during intense repetitive electrical stimulation of the cortical surface. Subsequent to such increases ak fell to subnormal levels. The amplitudes and durations of such undershoots were correlated with the amplitudes of the preceding increases in ak. Sometimes thalamic seizures ceases before cortical epileptic episodes. This resulted in a decrease of cortical EEG amplitudes. After ablation of the sensorimotor cortex seizures in forepaw-VPL could be induced by stimulation of the somatosensory cortex. These results further support the conclusion that specific thalamic nuclei participate in seizure generation and may serve as a subcortical route of seizure spread.


Relation between extracellular potassium concentration and neuronal activities in cat thalamus (VPL) during projection of cortical epileptiform discharge. Neuronal and potassium activities (ak) were measured in the nucleus ventro-posterolateralis thalami (VPL) during propagated epileptiform activity from the somatosensory cortex of cats. Seizures were induced by repetitive electrical stimulation of the cortical surface or by topical application of penicillin. The recruitment of VPL into a seizure resulted in large increases of ak to levels of up to 11.6 mmoles/l, accompanied by increased in neuronal discharge rate to 300/sec. Sometimes the rise in ak preceded active participation of a given thalamo-cortical relay (TCR) neuron in the seizure. After reaching a peak level, ak and neuronal discharge rate slowly declined during an ictal episode. After cessation of seizures all TCR neurons were inhibited, while ak fell to subnormal levels. The duration of these postictal depressions increased with the amplitude of preceding increases and subsequent undershoots in ak and could last up to 120 sec. During decay and undershoot in ak, relay capability of TCR neurons was reduced. Also the probability that action potentials elicited in intracortical endings of TCR cells would antidromically invade their cell bodies was decreased. The duration of these periods varied with the amplitude of undershoot in ak. Seizure threshold was increased during undershoots. These observations are consistent with a long-lasting postictal hyperpolarization of neuronal membranes. The hyperpolarization may be caused by the action of an electrogenic pump, which is probably involved in termination of seizure discharge.


Effects of diphenylhydantoin on the spontaneous activity of Purkinje, nucleus interpositus, red nucleus and motor cortex cells. (1) Extracellular multiunit recordings were made of the spontaneous activity in cerebellar Purkinje cells, nucleus interpositus, red nucleus and sensorimotor cortex in acute cat preparations. (2) Changes in this spontaneous neural activity produced by the administration of diphyenylhydantoin (DPH) were studied. DPH was infused i.v., generally at a concentration of 2.5 mg/ml and at a rate varying from 0.08 to 0.48 mg/kg/min. Two different patterns of infusion were used: fixed time, variable rate and variable time, fixed rate. Pulsed doses were also given at intervals of 5--10 min. (3) DPH at a level of 10--20 mg/kg produces a significative initial deceleration in all structures followed by a significative acceleration in the Purkinje cells, nucleus interpositus and red nucleus as a dose of 20--30 mg/kg is reached. Higher levels caused a profound depression of multiunit activity. (4) The activation produced by DPH is oscillatory (3--5/min) in character and is composed of 'trains' which occur at a rate of 20--30/sec with very rapid discharge frequencies (600--800 Hz). (5) A direct significant correlation was found between DPH serum levels and the intravenously administered dose. The activating DPH dose (20--30 mg/kg) corresponded to serum levels of 24--32 micrograms/ml. (6) The possibility is discussed whether the anticonvulsant action of DPH may be due in part to the production of rhythmic oscillatory activity in the cerebello-rubro-olivo-cerebellar ciruit and the depression of the cerebellothalamic-cortical pathway.


Sleep patterns in Kleine-Levin syndrome. Diurnal and nocturnal sleep records were obtained from a male and a female with Kleine-Levin syndrome, during excessive daytime sleep attacks and while they were asymptomatic. A common pattern of abnormal sleep was seen in both patients even during the asymptomatic period. The female, aflicted with a severe form of periodic hypersomnia, showed nocturnal and diurnal sleep onset REM periods. The different pattern of sleep abnormality in the female could be an expression of the severity of her symptoms or might indicate a variant of sleep abnormality present only in females with Kleine-Levin syndrome.


Single unit activity vs. amplitude of the epidural evoked potential in primary auditory cortex of awake cats. The study investigated, in primary auditory cortex (AI) of awake cats, the relationship over a range of stimuli between the amplitude and latency of the initial positive deflection (P1) of the primary evoked potential and the intensity of concurrent underlying evoked single unit activity. Epidural evoked potentials and extracellular responses of 155 single units to monaural 100 musec clicks ranging from 45 to 110 dB were recorded. At low stimulus levels, considerable unit response could occur with a very small P1. At middle stimulus levels, unit response was directly proportional to P1 amplitude. At higher stimulus levels, P1 amplitude continued to increase while unit response began to saturate.


Effect of stimulus intensity on short latency somatosensory evoked potentials. The peripheral and central potentials evoked by percutaneous electrical stimulation of the median nerve were investigated in a group of neurologically normal subjects. We found: (1) Motor threshold stimulation gave consistently submaximal responses and probably does not represent an optimal intensity for routine use. (2) The sum of motor plus sensory threshold gave potentials which were consistently at, or close to, maximal in amplitude. This intensity was comfortable for all subjects. (3) When stimulating at intensities above motor threshold, the increase in amplitude of peripheral potentials markedly exceeded that of the central potentials. There was evidence suggesting that amplitudes would decline at very high stimulus intensities. (4) The P13 peak latency and the P13--N9 interpeak latency declined and the N17--P13 interpeak latency increased with increasing intensities of stimulation. The N9 and N18 peak latencies remained stable.


A case of oesophageal cancer limited to the mucosa and submucosa. The authors report a case of an oesophageal cancer limited to the mucosa and the submucosa. This case is interesting because of its long history for more than 18 months, the difficulty of fiberoptic diagnosis and the diagnostic value of rigid instruments completed via vital staining by o-toluidine.


Methylene blue dye spraying method in patients with ulcerative proctitis: a comparative study with morphological findings and functional capacity of the rectal epithelium. Methylene Blue dye spraying method is currently used in the endoscopic assessment of the colonic mucosa of patients with chronic ulcerative proctocolitis. We have assessed the validity of this method in the evaluation of the functional capacity of the rectal mucosa in 17 patients with ulcerative proctitis and in 5 normal subjects. The ionic transmucosal fluxes of Na+ and K+ and the potential difference were used as parameters of the mucosal function. Our results indicate that methylene blue dye spraying method can be useful in the functional assessment of the rectal epithelium, especially for differentiating normal subjects and patients with mild ulcerative proctitis.


Increased urinary beta 2-microglobulin in cadmium exposure: dose-effect relationship and biological significance of beta 2-microglobulin. A study on the general population from both cadmium-polluted and control areas and on cadmium alloy workers indicates that beta 2-MG in urine is very closely correlated with aging, but it indicates an association with cadmium exposure. However, the age factor is stronger than cadmium exposre in both polluted and control areas among persons without clinical proteinuria. On the other hand, cadmium exposure is most likely correlated with beta 2-MG even in nonpolluted areas. Thus it seems that there is no noneffect level of cadmium dose in affecting the elevation of beta 2-MG in urine. beta 2-MG in serum indicated a very close correlation with cadmium in blood among cadmium alloy workers. This may suggest that an increase of beta 2-MG in both blood and urine in an early stage of cadmium exposure is caused by the increased level of beta 2-MG in blood, which may be a result of stimulation due to cadmium, but not necessarily by the clinical dysfunction of reabsorption of beta 2-MG in the renal.


Identification and characterisation of the major antiproteases in equine serum and an investigation of their role in the onset of chronic obstructive pulmonary disease (COPD). Three major antiprotease components in equine serum were identified and characterised. These were the acidic prealbumin Pr, the homologue of human alpha-1 antitrypsin and 2 protease binding proteins, the acidic prealbumin Xc and alpha-2 macroglobulin, both capable of inhibiting the proteolytic activity of trypsin, but with only limited inhibitory effect on its esterolytic activity. The possible role of these serum antiproteases in the onset of chronic obstructive pulmonary disease (COPD), analogous to the hereditary dysproteinaemia of alpha-1 antitrypsin in man, was investigated. There was no evidence of a genetically determined variation in the protease binding proteins but an increased frequency of the electrophoretically slower Pr antitrypsin alleles was present in horses affected with COPD. However, because of both the mixed breeding of the animals investigated and the lack of correlation with low serum trypsin inhibitory capacity, measured by inhibition of DL-BAPA hydrolysis, the significance of this observation could not be critically assessed.


Characterization of a thermosensitive sporulation mutant of Bacillus subtilis affected in the structural gene of an intracellular protease. A thermosensitive sporulation mutant (ts-15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42 degrees C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts-15R1 and ts-15R2) and were also transmitted together by transformation into the wild type. Immunological studies have shown that when ts-15 is grown at 42 degrees C it synthesizes a 'cryptic' protein with apparently the same antigenic properties as the wild type or as ts-15 mutant grown at the permissive temperature (30 degrees C). The intracellular proteases from the wild type and from ts-15 grown at 30 degrees C and 42 degrees C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts-15 grown at 30 degrees C was 64000--65000 in the absence of sodium dodecylsulfate and 31000--32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts-15 grown at 42 degrees C showed the same molecular weight of 32000--34000 in the presence or in the absence of sodium dodecylsulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts-15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form.


Biosynthesis of a blood-group-I determinant reacting with anti-I Ma serum (group 1). A blood-group-I determinant reacting specifically with anti-I Ma serum (group 1) has been produced biosynthetically by the action of a beta-galactosyl transferase isolated from human milk on a precursor glycoprotein produced by formic acid hydrolysis and beta-D-galactosidase action on blood-group H substance prepared from hog gastric mucosa.


Biological role of gramicidin S in spore functions. Studies on gramicidin-S-negative mutants of Bacillus brevis ATCC9999. Gramicidin-S-negative mutants of Bacillus brevis ATCC9999 have been isolated with a remarkly higher yield after ethidium bromide or acridine orange treatment, than after N-methyl-N'-nitro-N-nitrosoguanidine treatment. Four (MIV, Smr170, R5 and EB 16) of 38 isolated mutants were characterized with respect to the lesion in gramicidin-S-synthesizing activity. The mutants sporulate to the same extent as the parental strain except mutant Smr 170 which sporulates less. However, mutant spores were more heat-sensitive and possessed a reduced level of dipicolinic acid content. No significant difference was observed in the germination time of wild-type and mutant spores. All spores germinated after 80--110 min, but the outgrowth time was different: all gramicidin-S-negative mutants grew out immediately after germination whereas wild-type spores required a lag period of 9--10 h. When the mutants were allowed to sporulate in the presence of gramicidin S, the spores were found to be heat-resistant and their outgrowth postponed to the same period as the parent spores. The addition of gramicidin also eliminated the deficiency of dipicolinic acid. A new class of gramicidin-S-negative mutant, R5, which only activates L-valine and L-leucine, is described. A possible biological function of gramicidin S in the heat-resistance and in the timing of spore outgrowth is discussed.


Lysolecithin analogs as adjuvants in delayed-type hypersensitivity in mice. I. Characterization of the adjuvant effect. The adjuvant activity of 5 different lysolecithin analogs (LLA) has been studied in delayed-type hypersensitivity (DTH) using fowl gamma globulin, bovine serum albumin and the terpolymer L-glutamic acid 60-L-alanine30-L-tyrosine10(GAT), as antigens. Increased DTH responses, by factors of 1.8--2.0 in CBA and BALB/c mice, showed that LLA are immunopotentiators if given intraperitoneally (i.p.) or subcutaneously (s.c.) together with the antigen. The concentration range, within which LLA are active, is limited to 10--20 micrograms/mouse s.c. and 100--300 micrograms/mouse i.p. Adjuvanticity was tested as a function of the LLA structure. The most pronounced immunopotentiation was obtained with racemic 1-octadecyl-2-methylglycero-3-phosphorylcholine (ET18-O-CH3). The LLA became less active with decreasing numbers of C atoms in the alkyl chain.


In vitro action of combined salbutamol and theophylline on anaphylactic contractions, mediator release and cyclic 3',5'-adenosine monophosphate in lung parenchyma. The mechanisms involved in the anti-asthmatic action of combined bronchodilator therapy was studied by determining the effects of combined salbutamol and theophylline on anaphylactic contractions, histamine release, prostaglandin (PG)F2 ALha release, cyclic 3',5'-adenosine monophosphate (c-AMP) and smooth muscle tone in guinea-pig peripheral airways in vitro. Combined, salbutamol (3 X 10(=8) M) and theophylline (3 X 10(-4) M) markedly inhibited anaphylactic contractions (85.8%) in lung strips. The inhibition of anaphylactic contractions was significantly greater than inhibition of histamine and PGF2 alpha-induced contractions. Histamine release was reduced by 66.1% but PGF2 alpha was not significantly altered. Increased c-AMP was observed with combined salbutamol (3 X 10(-8) M) and theophylline (3 X 10(-4) M) in the absence of antigen. The combined effect of salbutamol and theophylline was always greater than the sum of their individual effects. The results demonstrate that in peripheral airways theophylline potentiates the action of salbutamol both directly via smooth muscle relaxation and indirectly via inhibition in mediator release.


Idiopathic sideroblastic anemia: presence of sideroblastic changes in the erythropoietic precursors cultured from peripheral blood. Peripheral blood mononuclear cells from five patients with idiopathic sideroblastic anemia were examined in clonal culture for circulating erythropoietic precursors. Three of these patients had relatively mild anemia and revealed significant burst formation. Two patients who had severe anemia and required regular blood transfusions showed severe diminution or absence of circulating precursors. These results could suggest that the number of circulating erythropoietic precursors reflects effective erythropoiesis in the marrow. Ultrastructural morphology and ultrastructural Prussian-blue staining of the bursts from a patient revealed the characteristic mitochondrial deposition of ferrugionous material in pathologic normoblasts. Peripheral blood clonal cell cultures would, therefore, appear to be a potentially useful tool for analysis of biochemical abnormalities in idiopathic sideroblastic anemia.


Hemopoietic stem cells bear sperm-associated antigens. A fluorescence-activated cell sorter has been used to sort out enriched populations of murine hemopoietic stem cells from mouse bone marrow, based on fluorescence of cells labeled with various antisera. Significant positive enrichment for stem cells was obtained with rabbit anti-human brain, human anti-human sperm and mouse anti-mouse F9 sera but not with mouse anti-mouse Ia serum. These results support the hypothesis that the hemopoietic stem cell exhibits 'unique' surface antigens cross-reactive with sperm, brain and embryonic antigens.


Recent evidence for the participation of vitamin A in glycoprotein synthesis. Recent evidence supports the concept that vitamin A plays some role in glycoprotein synthesis in a large-variety of tissues examined. Its involvement may be through participation of a retinol-linked sugar, mannosyl retinyl phosphate (MRP). Upon injection of [3H]retinol and [14C]mannose into rats, [14C, 3H]MRP could be isolated from liver and intestinal mucosa, and identified by chromatographic and hydrolytic experiments. The enzyme system that forms MRP from GDP-mannose and retinyl phosphate was located primarily in rough endoplasmic reticulum of fractionated liver cells, with some activity also in smooth membranes and Golgi apparatus. Vitamin A deficiency resulted in depressed synthesis of the rat serum glycoprotein alpha 1-macroglubin (alpha 1-MG), as shown by a decline in labeling. Analysis of the labeled alpha 1-MG from serum of normal and vitamin A-deficient rats showed this to be the result of a defect in glycosylation. The specific activity ratio (deficient:normal) of the alpha 1-MG of serum declined progressively with development of the deficiency, as a result of underglycosylation. Complete carbohydrate analysis of the alpha 1-MG of normal and deficient serum revealed a sugar loss in this glycoprotein as a result of vitamin A deficiency.


[Histological aspects of mycoses provoked by opportunistic fungi]. The histological study with Hotchkiss-Mc Manus staining and Grocott silver impregnation of tissues invaded by opportunistic fungi is a rapid and easy means of diagnosis. The inflammatory reactions around the fungus give the certitude that it is a pathogen and not a contaminant. The major opportunists among Canidida, Aspergillus, Mucor, Absidia and Cryptococcus species are presented in local and disseminated lesions, but all fungi, saprophytic in the normal host, can become pathogens in the immunodepressed patient.


Parathyroid hormone-induced calcium efflux from isolated renal cortical tubules: evidence for cyclic AMP mediation. Effects of parathyroid hormone (PTH) upon cyclic AMP and calcium efflux in isolated renal cortical tubules from hamsters were investigated. PTH caused a rapid rise in cyclic AMP levels, temporally preceding an increase in calcium efflux. Increases in both cyclic AMP levels and calcium efflux were noted over an identical PTH concentration range 0.007--0.7 U/ml). Other peptide hormones tested which had no effect upon cyclic AMP levels did not enhance efflux of calcium. The phosphodiesterase inhibitor methyl isobutylxanthine (MIX) was utilized in other studies to potentiate the cyclic AMP response, and produce a range of cyclic AMP concentrations in response to PTH. In these experiments a range of calcium efflux responses was noted which closely paralleled changes in cyclic AMP. Direct addition of cyclic AMP or dibutyryl cyclic AMP to isolated renal tubules caused increased efflux of calcium, while addition of 5'-AMP did not. These results indicate a role for cyclic AMP as a mediator of PTH-induced calcium efflux in this system and suggest that cyclic AMP may mediate the action of this hormone in enhancing renal conservation of calcium in vivo.


Studies on hemorrhagic tendency in cirrhosis of the liver. Recently it has been paid attention whether the liver diseases might induce the disseminated intravascular coagulation (DIC) or not, although it has well known that liver cirrhosis contribute to the hemorrhagic diathesis. In this paper, we studied on the hemorrhagic tendency and hemostatic tests in the patients with liver cirrhosis in order to clarify the coagulation and fibrinolytic activity. We can conclude that the hemorrhagic diathesis occurs due to the complex of decreases in platelet number, platelet functions, clotting factors, and increased fibrinolytic activity. The increased fibrinolytic activity may be primary and the frequency of clinically important DIC is quite low.