Membrane-associated thiamin triphosphatase. II. Activation by divalent cations.

Activation of membrane-associated thiamin triphosphatase from rat brain requires a divalent cation (Mg2+, Ca2+, or Mn2+). The optimum concentration of Mg2+ necessary for maximal enzyme activity varies with substrate concentration; conversely, the maximal rate of hydrolysis attainbale by increasing thiamin triphosphate concentration is directly proportional to [Mg2+] for all levels of Mg2+ below that of the substrate. Under appropriate conditions, the Km of the thiamin triphosphatase for Mg2+ and for thiamin triphosphate are shown to be identical. Dissociation constants (Kd) for the binding of Mg2+ to thiamin triphosphate, thiamin diphosphate, and thiamin were determined; kinetic data re-expressed in terms of [Mg2+-thiamin triphosphate] conform to simple single substrate predictions, suggesting that the true enzyme substrate may be the Mg2+-thiamin triphosphate complex. Excess free Mg2+ inhibits thiamin triphosphatase activity competitively while excess free thiamin triphosphate in concentrations up to 10 times Km has no effect on the membrane-bound enzyme.


Reconstitution of Bacillus stearothermophilus 50 S ribosomal subunits from purified molecular components.

Bacillus stearothermophilus 50 S ribosomal subunits have been reconstituted from a mixture of purified RNA and protein components. The protein fraction of 50 S subunits was separated into 27 components by a combination of various methods including ion exchange and gel filtration chromatography. The individual proteins showed single bands in a variety of polyacrylamide gel electrophoresis systems, and nearly all showed single spots on two-dimensional polyacrylamide gels. The molecular weights of the proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An equimolar mixture of the purified proteins was combined with 23 S RNA and 5 S RNA to reconstitute active 50 S subunits by the procedure of Nomura and Erdmann (Nomura, M., and Erdmann, V. A. (1970) Nature 226, 1214-1218). Reconstituted 52 S subunits containing purified proteins were slightly more active than subunits reconstituted with an unfractionated total protein extract in poly(U)-dependent polyphenylalanine synthesis and showed comparable activity in various assays for ribosomal function. The reconstitution proceeded more rapidly with the mixture of purified proteins than with the total protein extract. Reconstituted 50 S subunits containing purified proteins co-sedimented with native 50 S subunits on sucrose gradients and had a similar protein compsoition. Initial experiments on the roles of the individual proteins in ribosomal structure and function were performed. B. stearothermophilus protein 13 was extracted from 50 S subunits under the same conditions as escherichia coli L7/L12, and the extraction had a similar effect on ribosomal function. When single proteins were omitted from reconstitution mixtures, in most cases the reconstituted 50 S subunits showed decreased activity in polypheylalanine synthesis.


Conformational changes required for pyruvate kinase activity as modulated by monovalent cations.

The interaction of a series of alkylamines with muscle pyruvate kinase was investigated by kinetic and physical studies in order to understand the mechanisms by which certain monovalent cations can activate the enzyme and to define several of the important conformational changes necessary for catalytic activity. Monomethylammonium ion interacts with pyruvate kinase to activate the enzyme. Dimethyland trimethylammonium ions do not activate, but are competitive inhibitors against activating cations. Tetramethylammonium ion neither activates nor inhibits pyruvate kinase activity. When the enzyme is in the presence of monomethylammonium ion or dimethylammonium ion, a conformational change is observed by ultraviolet difference spectroscopy. This conformational change is similar to that observed with other activating cations and appears to be a necessary but no sufficient conformational change in the formation of an active complex. The interaction of the substrate phosphoenolpyruvate with the pyruvate kinase-Mn2+ complex in the presence of these cations was studied by water proton relaxation rate measurements. The affinity of the enzyme-Mn2+ complex for phosphoenolpyruvate is decreased by a factor of 5 in the presence of any of the alkylamines compared to the affinity measured in the presence of K+ or NH4+. No change in the Km of phosphoenolpyruvate is observed however when it is measured in the presence of monomethylammonium ion, suggesting that the decrease in affinity for the substrate is not the reason for lack of enzymic activity. The conformation of the ternary enzyme-Mn2+-phosphoenolpyruvate complex about the bound Mn2+, as reflected by the enhancement values (epsilont) measured, differs depending upon the nature of the monovalent cation. The epsilon t values measured in the presence of the alkylamines are larger (epsilont - 5.7 +/- 0.2) than those measured in the presence of K+ or NH4+ (epsilont = 1.9 +/- 0.1).


Effects of serotonin, carbamylcholine, and ascorbic acid on leukocyte cyclic GMP and chemotaxis.

Serotonin, ascorbic acid, and carbamylcholine enhanced the chemotactic responsiveness of human monocytes to endotoxin-treated serum. These agents caused significant accumulation of cyclic GMP in monocytes. PMN leukocyte chemotaxis was also enhanced by these agents although significant increases in cyclic GMP were not demonstrated.


A simple method for freeze-fracture of monolayer cultures.

A simple method is described for the freeze-fracture in situ of monolayer cultures grown on gold carriers coated with a thin layer of silicon monoxide. Preliminary observations on 3T3 mouse embryo fibroblasts indicate that this technique exposes large areas of cell membrane, making it possible to determine how areas of membrane specialization are related to the cell as a whole and to regions of cellular interaction. 3T3 cells cultured on silicon monoxide show no modification of growth properties compared to cells growing on Falcon plastic, and other cell lines also appear to grow well on this substrate.


Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol.

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.


Autoradiographic studies of nicotinic acid utilization in human-mouse heterokaryons and inhibition of utilization in newly-formed hybrid cells.

Although most mammalian cell lines can utilize either nicotinic acid or nicotinamide for the biosynthesis of nicotinamide adenine dinucleotide (NAD), thymidine kinase-deficient, mouse 3T3-4F cells are unable to utilize nicotinic acid. When 3T3-4E cells were fused with human D98/AH2 cells, autoradiography showed that the resultant heterokaryons synthesized NAD from nicotinic acid at rates comparable to the human parental cell. The rate of nicotinic acid utilization in heterokaryons remained unchanged over the four-day period of study following cell fusion. In contrast to the results observed with heterokaryons, nicotinic acid utilization was markedly reduced in hybrid cells. Of 100 hybrid clones examined at four or five days following cell fusion, 60 utilized nicotinic acid at rates less than one tenth that of the parental human cell. Similar results were observed in hybrid clones at nine or ten days following fusion. Uniformly high rates of NAD biosynthesis were observed in hybrid clones with nicotinamide as the precursor. This excludes the possibility that the reduction in nicotinic acid utilization in hybrid cells is due to a general metabolic dysfunction. The biochemical mechanism by which nicotinic acid utilization is markedly reduced has not been determined with certainty, however, several observations suggest genetic suppression.


The role of adenosine 3',5'-monophosphate in the transformation of Cloudman mouse melanoma cells.

The role of adenosine 3',5'-monophosphate (cyclic AMP) in the regulation of mouse melanoma cell growth and differentiation was investigated. A variant melanoma (Cloudman S91-F) which displays a greater degree of transformation than the parental cell (Cloudman S91) was isolated. A correlation between cyclic AMP metabolism and transformation was made. Dibutyryl cyclic AMP depressed cell growth and increased pigmentation in both parental and variant cell lines. The parental cell line, however, was more responsive to melanocyte-stimulating hormone (MSH) which was found to affect cell growth and pigmentation by increasing cyclic AMP levels. The more transformed S91-F cell line contained lower levels of cyclic AMP than the parental cell line, and this fact correlated well with the higher degree of growth and lesser degree of pigmentation in the variant. Enzymatic analysis revealed that the hydrolysis of cyclic AMP in both cell lines was similar, while the adenylate cyclase activity of the variant cell line was lower than that of the parental cell line. Lineweaver-Burk plots demonstrated that the Km's for the enzymes in the two cell lines were the same but that the Vmax of the S91-F cell line was significantly less that that of the S91 cell line. Thus, the lesion in the S91-F cell which is responsible for its more transformed characteristics seems to be one which affects adenylate cyclase at the level of the cell membrane.


Pyridine nucleotide metabolism in mitotic cells.

The biosynthesis and turnover of nicotinamide adenine dinucleotide (NAD) have been examined in mitotic cells of the human culture line, D98/AH2. No significant difference in the incorporation of nicotinic acid or nicotinamide could be detected between mitotic and interphase cells. The distribution of newly-incorporated nicotinic acid among the various pyridine nucleotides was also identical in mitotic and interphase cells. Whereas previous results have shown that the nucleus is necessary for NAD biosynthesis, the present results show that an intact nucleus is not required. In contrast to the equivalent rates of biosynthesis in mitotic and interphase cells, the pyridine ring of NAD was lost twice as fast from mitotic as from interphase cells. Loss of the pyridine ring to the medium is not necessarily an accurate measure of turnover, and the difference between mitotic and interphase cells may reflect differential reutilization of the pyridine ring within the cell. However, it is clear that NAD turnover is substantial in mitotic cells and possibly greater in mitotic cells than interphase cells.


Regulation of growth and morphological modulation of HeLa65 cells in monolayer culture by dibutyryl cyclic AMP, butyrate and their analogs.

N6-O2'-Dibutyryl adenosine-3',5' monophosphate (DBcAMP) markedly altered the morphology of HeLa cells by increasing average cell size with an increase in total cell protein and RNA. Such effects were not caused by adenosine 3',5' monophosphate (cAMP) or related nucleosides and nucleotides. Butyrate, an enzyme catalyzed hydrolysis product of DBcAMP, induced a jagged spindle shape in HeLa cells within 8 hours and then caused them to enlarge and resemble those grown with DBcAMP. These effects were specific for butyrate (C4) and pentanoate (C5) and were not observed with isomers, substituted analogs, or other fatty acid derivatives. These morphological effects were prevented by blocking protein synthesis or by altering the cytoskeleton with Colcemide or cytochalasin B.


Effects of adenosine 5'-monophosphate and adenosine 3',5'-cyclic monophosphate on l cells.

The adenine nucleotides, 5'-AMP and 3',5'-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5'-AMP, and rates of uridine transport are increased after incubation with either 5'-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.


Calcium distribution in islets of Langerhans: a study of calcium concentrations and of calcium accumulation in B cell organelles.

Calcium concentrations of various pancreatic B cell organelles have been determined by X-ray microanalysis of areas of frozen sections of unfixed rat islets of Langerhans. Highest concentrations were detected in storage granules and in mitochondria, although calcium was also present in nuclei, in areas of endoplasmic reticulum and of cytoplasm. Accumulation of 45Ca by isolated organelles has been studied in homogenates and isolated subcellular fractions of rat islets of Langerhans. In the presence of a permeant anion (oxalate or phosphate), accumulation of 45Ca into mitochondria and microsomes was strongly stimulated by ATP. This net uptake was diminished during incubation of homogenates or of a mitochondria plus storage granule-rich fraction in the presence of cyclic AMP, dibutyryl cyclic GMP; 2:4-dinitrophenol or of ruthenium red. Investigations of the characteristics of 45Ca accumulation by homogenates prepared from storage granule-depleted islets showed no differences from those of normal islets, suggesting that the granules do not represent an important labile pool of calcium. With the exception of cyclic AMP and cyclic GMP none of the insulin secretagogues tested (glucose, leucine, arginine, adrenalin, noradrenalin, theophylline, glibenclamide) altered calcium accumulation by islet homogenates. On the basis of absolute calcium levels and of 45Ca uptake studies it is concluded that islet B cells contain a readily exchangeable mitochondrial calcium pool, and an endoplasmic reticulum pool containing a lower concentration of calcium which is also readily exchangeable. The storage granules, despite their high calcium content, do not appear to constitute a labile pool. It seems likely that the labile mitochondria and endoplasmic reticulum pools play a predominant role in the regulation of cytoplasmic free calcium levels, which may in turn be important in the regulation of rates of insulin secretion.


Silica gel thin-layer chromatography of acidic phospholipids. II. Chromatographic behaviour of phosphatidylserine and phosphatidic acid applied with different cation composition on adsorbents either free of metal ions or containing a surplus of divalent metal ions.

Different salt forms of phosphatidylserine and phosphatidic acid (two acidic phospholipids) have been subjected to thin-layer chromatography on two commonly used silica adsorbents, one of which (silica gel HR) is practically free of metal ions and the other (silica gel G) contains 13% of calcium sulphate as binder. The chromatographic behaviour was studied in an acidic, a neutral and a basic solvent. Both adsorbents provided usable systems for phosphatidylserine with each of the three solvents, except for silica gel G with the neutral solvent, in which system tailing was prominent. The inclusion of calcium sulphate in the silica gel tended to impair chromatography of phosphatidylserine in acidic and neutral solvents, but improved its chromatography in the basic solvent. In all the systems, the migration was independent of the cation composition of the applied phosphatidylserine samples. For the chromatography of phosphatidic acid, only three of the systems tested were usable, and in those three, the chromatographic behaviour was independent of the cation composition of the samples. The calcium sulphate in an adsorbent increased tailing of phosphatidic acid in acidic and neutral solvents, as it did for phosphatidylserine, whereas with the basic solvent, calcium sulphate in the adsorbent caused phosphatidic acid to remain at the origin. Two one-dimensional thin-layer chromatographic systems previously recommended for the chromatography of acidic phospholipids were unsuitable for the chromatography of phosphatidic acid under the conditions used here. For both phosphatidylserine and phosphatidic acid chromatographed in acidic systems, the solvent must contain water in addition to acetic acid if excessive tailing is to be avoided.


Solid phase radioimmunoassay for typing herpes simplex viral antibodies in human sera.

An indirect solid phase radioimmunoassay (RIA) was developed for typing antibody to herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2) in human sera. The test is based upon absorption of sera with uninfected, HSV-1-infected cells and testing for residual antibody. The high sensitivity of the RIA method for detecting HSV antibody permits examination of sera at high dilutions, and thus relatively small volumes of virus-infected cells are required for cross-absorption of antibodies. Results obtained in RIA typing of HSV antibodies showed good agreement with those obtained by microneutralization and inhibition of passive hemagglutination. The HSV antibody type(s) determined by RIA also showed good correlation with the virus type isolated from the individual, either from clinical specimens or sensory nerve ganglia. The technique was very sensitive for detection and typing of HSV antibdodies in cerebrospinal fluids. The RIA method was highly suitable for detecting two types of HSV antibody in the same serum specimen, and it was possible to show that a marked, type-specific antibody response to HSV-2 does occur in individuals with a primary HSV-2 infection who have experienced a prior infection with HSV-1.


Responses of isolator-derived Japanese quail and quail cell cultures to selected animal viruses.

Thirteen oncogenic and necrotizing animal viruses were assayed in LIFE Sciences, Inc. (LSI)-specific pathogen-free Japanese quail and LSI-specific pathogen-free chicken embryo cell cultures. Nine viruses produced similar titers in the quail and chicken cell systems, whereas four viruses showed significantly higher titers in chickens. Young Japanese quail and chickens were inoculated with five selected avain viruses and maintained in stainless-steel isolators. Comparable responses were noted in quail and chickens injected with Newcastle disease virus and avain leukosis virus, but quail were significantly more resistant than chickens to fowl pox virus, laryngotracheitis virus, and Marek's disease herpesvirus. Although no overt symptoms of disease were observed in Japanese quail inoculated with most avain viruses, neutralizing antibody or virus was detected, indicating presence of an inapparent infection. In one experiment, neutralizing antibody was detected in a comparable number of quail and chickens after inoculation with avian leukosis virus. Avian leukosis virus viremia was observed at 12 and 70 days postinoculation, with the COFAL (complement fixation for avian leukosis) titers similar for quail and chickens. Most quail infected with Marek's disease herpesvirus produced neutralizing antibody within 70 days but showed no classical symptoms of Marek's disease even when held for 5 months. In contrast, all chickens inoculated with Marek's disease herpesvirus died within 20 days. The utility of quail embryo cell cultured in the preparation of vaccines and biological reagents is discussed.


Longitudinal, serological study of cytomegalovirus infections in nurses and in personnel without patient contact.

Sera were obtained at intervals from 172 hospital employees for measurement of cytomegalovirus (CMV) complement fixation (CF) and indirect hemagglutination antibody. No fourfold rises or falls in titer were seen over a 19- to 27-month period among 71 employees with initially positive CMV CF titers. The concurrence rate between the CMV CF and the indirect hemagglutination antibody tests in identifying seronegative personnel was 96%. Five seroconversions were identified during an average follow-up period of 15 to 17 months per person among 65 pediatric nurses whose CMV CF titers had initially been less than 1:8. No seroconversions were seen during an average follow-up period of 29 months per person among 27 hospitad little patient contact. The rate of acquisition of CMV infections in seronegative pediatric nurses was 4.1 to 7.7% per year. Sera from 9 of the 172 employees studied (5.2%) gave inconsistent results at the lower limits of the CF test.


On the mechanisms responsible for the phosphaturia of bicarbonate administration.

Experiments were carried out in normal dogs to characterize the mechanisms by which sodium bicarbonate administration results in increased excretion of phosphate. Infusion of sodium bicarbonate alone increased fractional phosphate excretion from 0.8 to 29.3%. During bicarbonate administration, ionized calcium fell and mean parathyroid hormone values increased from 59.6 to 230.4 muleq/ml. In the same group of dogs, administration of sodium bicarbonate plus calcium prevented the fall in ionized calcium, and parathyroid hormone levels remained unchanged. In these dogs fractional phosphate excretion increased from 2.4 to only 4.9%. Similar results were obtained in thyroparathyroidectomized dogs receiving sodium bicarbonate. In these dogs fractional excretion of phosphate increased from 0.6 to 4.5%. Under all three experimental conditions no differences were observed in sodium or bicarbonate excretion or in urinary or plasma pH. Administration of hydrochloric acid, after phosphaturia had been induced by the infusion of bicarbonate, resulted in a decrease in plasma bicarbonate and an acid urine; however, the phosphaturia persisted even in the presence of an acid urine pH. In five thyroparathyroidectomized dogs infused with parathyroid hormone throughout, administration of identical amounts of sodium as either NaCl or NaHCO3 resulted in a similar degree of phosphaturia despite significant differences in urine pH. These experiments suggest that a rise in parathyroid hormone levels, resulting from a fall in ionized calcium, is the major mechanism by which bicarbonate administration produces phosphaturia. An increased natriuresis per nephron, as a consequence of extracellular fluid volume expansion, contributes to the phosphaturia. On the other hand, alkalinization of the urine does not play a significant role in the phosphaturia seen after bicarbonate administration.


Metabolism of apoprotein B of plasma very low density lipoproteins in the rat.

As an extension of metabolic studies of the cholesteryl ester component of rat very low density lipoproteins, we have studied the metabolism of the B apoprotein component labeled by intravenous injection of [3H]lysine. The B apoprotein separated from other apoproteins by delipidation and selective precipitation with tetramethylurea could not be distinguished from B apoprotein prepared by the conventional gel filtration technique. After injection of [3H]lysine, specific activity of B apoprotein was maximal in very low density and low density lipoproteins 1 and 11/2-h later, respectively, in a manner consistent with a precursor-product relationship. When protein-labeled very low density lipoproteins were injected into rats, the relationships of specific activity again indicated that B apoprotein of very low density lipoproteins may be the sole precursor of that of low density lipoproteins. However, less than 10% of the B apoprotein that disappeared from very low density lipoproteins appeared in density lipoproteins. To evaluate the sites of removal of B aproprotein of very low density lipoproteins from plasma, protein-labeled very low density lipoproteins were incubated with unlabeled high density lipoproteins to reduce radioactivity in non-B apoproteins selectively by molecular exchange. Most of the B apoprotein was rapidly removed by the liver. The extensive hepatic uptake of both the cholesteryl ester and B apoprotein components of rat very low density lipoproteins may explain the characteristically low concentrations of plasma low density lipoproteins in the rat.


Reduction in cholesterol and low density lipoprotein synthesis after portacaval shunt surgery in a patient with homozygous familial hypercholesterolemia.

The turnover of 125I-labeled low density lipoprotein (LDL) and the total body balance of cholestrol were studied in a 6-yr-old girl with the homozygous form of familial hypercholesterolemia (FH) before and after the surgical creation of an end-to-side portacaval shunt. The results were compared with those of similar studies simultaneously performed in untreated patients with the heterozygous form of FH and with the results of earlier studies performed on normolipidemic subjects. Before shunt surgery, the rate of synthesis of LDL in the FH homozygote (mg/kg per day) was fourfold higher than in normolipidemic subjects and twofold  higher than in her heterozygous mother. The fractional catabolic rate for LDL in the homozygote was decreased to 33% of normal control values. The rate of cholesterol synthesis, estimated by chemical sterol balance, was higher in the FH homozygote than in two FH heterozygotes of similar age studied simultaneously. When considered in relation to the markedly elevated level of plasma cholesterol, the observed rate of cholesterol synthesis in the FH homozygote was inappropriately elevated. Bile acid production was normal in all three children. 5 mo after shunt surgery, the rate of LDL synthesis in the homozygote had declined by 48% as compared with the preoperative value, and this caused a 39% drop in the plasma LDL cholesterol level despite a 17% reduction in the fractional catabolic rate of the lipoprotein. The rate of cholesterol synthesis fell by 62% as compared with the preoperative value. The findings of an inappropriately elevated rate of production of both cholesterol and LDL as well as a reduced fractional catabolic rate for the lipoprotein in the untreated FH homozygote are consistent with results of studies in cultured fibroblasts indicating that the primary genetic defect in FH involves a deficiency in a cell-surface receptor for LDL that regulates both cholesterol synthesis and LDL degradation. Although the mechanism for the decline in production of cholesterol and LDL after portacaval shunt surgery is unknown, it was observed that these changes were associated with marked increases in the plasma concentrations of bile acids and glucagon.


Extrusion of pyrophosphate into extracellular media by osteoarthritic cartilage incubates.

The distribution of calcium pyrophosphate mineral phase, almost exclusively confined to articular cartilage in chondrocalcinosis, and the high level of pyrophosphate (PPi) ion relative to serum in synovial fluid in patients with either chondrocalcinosis or advanced osteoarthritis led to an investigation of whether cartilage cells elaborate PPi ions. Incubates of articular cartilage from young rabbits but not from mature rabbits, as well as growth plates cartilage, released PPi into incubation media during a 4h period. Control rabbit ear cartilage and synovial membrane elaborated negligible amounts of PPi. The PPi was shown to be undialyzable but could be dissociated from the alkaline phosphatase by ultracentrifugation. In 16 patients with osteoarthritis, a substantial output of PPi by samples of articular cartilage from the knee was demonstrated. It is postulated that either rapid cell division and matrix synthesis found in the base of ulcerating osteoarthritic cartilage or remodeling calcified sites are the source of the PPi in such osteoarthritic cartilage. It is further hypothesized that this PPi output accounts at least in part for the elevated PPi levels found in synovial fluid of patients with osteoarthritis.


Conversion of very low density lipoprotein to low density lipoprotein. A metabolic study of apolipoprotein B kinetics in human subjects.

The interrelationship between apolipoprotein B in very low density lipoprotein (VLDL-B) and in low density lipoprotein (LDL-B) was studied in seven normal and hyperlipidemic men and women, with purified radioiodinated VLDL. The time-course of the appearance of radioactivity in LDL was followed. As the specific activity curves intersected at the masimal height of the LDL-B curve, it was inferred that all or most LDL-B peptide is derived from VLDL-B peptide. This transfer was further quantitated in seven normotriglyceridemic subjects by simultaneous i.v. injection of purified 131I-VLDL and 125I-LDL. By a deconvolution method, a quantitative description of the rate of entry of 131Ivldl-b into 131I-LDL-B was derived by analysis of 131I-LDL-B and 125I-ldl-b radioactivity in plasma. The results indicate that approximately 90% of VLDL-B mass is converted into LDL-B in subjects with normal serum triglyceride concentrations. The synthetic rates of VLDL-B and LDL-B peptide were simultaneously measured in six normal subjects, and two patients with heterozygous familial hypercholesterolemia (type IIa). The turnover rates for VLDL-B and LDL-B peptide were similar in these subjects. The findings in the three parts of this study were consistent with the view that most if not all VLDL-B is converted into LDL-B peptide, and most if not all LDL-B is derived from VLDL-B peptide in normotriglyceridemic subjects.


Apoprotein composition of very low density lipoproteins of human serum.

Methods for quantitation of the major apoproteins of human serum very low density lipoprotein have been developed employing tetramethylurea, which delipidates the lipoprotein and selectively precipitates apolipoprotein B. Six soluble apoproteins are separated by electrophoresis in polyacrylamide gel. One of these is a previously unrecognized species of R-alanine (R4-alanine), more anionic than the R3-alanine polypeptide. Conditions of staining have been found which yield reproducibly linear chromogenic response with native lipoprotein and with each purified apoprotein. Recovery of protein in the seven species measured accounts for over 97% of the total in the very low density lipoprotein of normolipidemic individuals and in most samples from individuals with endogenous hyperlipemia. The mean content of apolipoprotein B in 43 samples from normolipidemic subjects was 36.9(+/-1.2 SEM)% of total protein, The distribution of the major soluble apoproteins as mean (+/-SEM) percentage of the soluble fraction was : R-serine, 5.3+/-o.5; arginine-rich, 20.6+/-1.0; R-glutamic, 10.6+/-0.4; R2-alanine, 28.3+/-0.7; R3-alanine, 26.9+/-0.5; and R4-alanine, 8.0+/-0.5. Distribution of the apoproteins was a function of particle diameter of very low density lipoprotein in fractions separated by gel permeation chromatography and by density gradient ultracentrifugation. In fractions below 700-800 A, apolipoprotein B comprised an increasing percentage of the total protein with decreasing particle diameter. Among the soluble proteins the percentage of the arginine-rich and R-serine polypeptides increased and that of the R-glutamic polypeptide declined progressively with decreasing particle size. Apoprotein distribution was similar in fractions of similar particle size from normolipidemic and hyperlipemic subjects with the exception that all fractions from the hyperlipemic subjects contained more R-serine and some, more arginine rich polypeptide. Even in the absence of chylomicrons, the distribution of soluble apoproteins in particles of diameters greater than 700-800 A was usually similar to that of the smallest particles. This suggests that the largest particles may include products of the partial catabolism of chylomicrons.


The diagnosis of mucopolysaccharidoses by electron microscopy of skin biopsies.

An ultrastructural examination was carried out on the skin of six children suffering from Mucopolysaccharidosis I (MPSI or Hurler's disease) and MPS II (Hunter's disease). Both intracellular mucopolysaccharides and lipids were observed. The dermal cells, i.e. fibroblasts, macrophages, were loaded with multiple vacuolar inclusions thought to be of lysosomal origin. They appeared either content-free or filled with granular, fuzzy and/or pseudomyelinic structures. Identical abnormalities were observed within the Schwann cells, smooth muscle cells and keratinocytes. Mast cells showed peculiar "worm-like" inclusions apart from their normal granulations. Since ultrastructure of a skin sample may provide as much data as brain, liver or kidney, cutaneous electron microscopy can be recommended to confirm a diagnosis of MPS.


Plasma adrenocorticoid response to corticotropin in dairy cattle fed high silage diets.

Responses of plasma corticoids to adrenocorticotropic hormone (.32 U/kg body weight) administered during early lactation were measured in dairy cattle consuming large amounts of concentrate feeds and one of the following forage diets: alfalfa-timothy hay plus corn silage, alfalfa-timothy hay crop silage plus corn silage, or corn silage. Concentrations of plasma corticoids increased to maximum in all groups within 60 to 90 min. Concentrations remained at near maximum for 4 to 5 h and then declined to baseline. Maximum corticoid concentrations following adrenocorticotropic hormone injection differed among forage treatment groups. Responses of corn silage and hay crop silage groups (46.7 +/- 1.1 and 50.1 +/- 2.4 ng/ml) did not differ but were less than those in the hay group (65.5 +/- 2.9 ng/ml). Response measured as area under the curve of plasma corticoid response also differed among forage treatments: hay 443 +/- 17 h (ng/ml) is greater than hay crop silage 334 +/- 34 is greater than corn silage 245 +/- 36. Results are discussed in terms of stress susceptibility in dairy cattle consuming diets containing large amounts of silage.


How the human brain responds to aging.

The characteristic morphologic changes frequently observed in the brain of an old adult include a decrease in weight and volume, a change in the pattern of cerebral cortical convolutions, and an increase in ventricular size. Cell loss varies from region to region in the brain, and may be intensified in Alzheimer's disease and other disorders associated with senile dementia. Among the neuroglial cells, the microglia undergo the most significant changes with age. Although senile brain disease previously has been regarded as secondary to atherosclerosis, recent neuropathologic studies indicate that only 30 to 40 percent of senile brain disease arises from cerebrovascular pathologic lesions. The dilemma remains, however, of how much of the deterioration observed in the aged is related to disease and how much to senescence. The interaction between gene expression and environmental conditions in aging is another important question for the geriatrician. Progress in the control and treatment of disorders associated with old age depends upon further research into the mechanisms that underlie the process of aging in the brain.


Production of a nonwettable enamel surface.

A water-repellent enamel surface was achieved using a new material that is a combination of fumed silicon dioxide and a silane. The effects of this coating on enamel were studied. In vitro studies suggest that the material produces good water and saliva repellency. However, no reduction in bacterial colonization between treated and control enamel occurred. results of in vivo investigations of coated and control Styroflex strips showed a reduction in deposits on the former. Retention with or without previous acid etching of the enamel was poor.


[Radiography of breast cancers].

Malignant tumor in the breast create a stroma-reaction that gives the typical aspect of malignant picture with retractile fibrosis, oedema and microcalcifications (10 to 15%). The diagnosis is really uncertain in only 2% of the cases. Cytology after fine needle puncture permit the diagnosis.


[Viruses and mammary carcinogenesis].

Bittner virus has been extensively studied by recent electron microscopy and molecular biology techniques. The structure, the biochemical, physical and antigenic properties of the RNA tumor viruses - i.e. the mouse mammary tumor virus (MMTV) - are well known. Recent observations in human tissues of particles similar to animal viruses that are known to be oncogenic have raised the hypothesis of the role of viruses in human cancer. In mice, breast cancer can be caused by a virus - the Bittner virus or MMTV - that is usually transmitted from mother to offspring in the milk. The discovery of such virus particles in human milks and breast cancer tissues could provide data about a viral aetiology of human breast cancer.


[Hormonal receptors of malignant breast tumors].

Tissue samples from 166 primary and 136 metastatic breast cancers were analysed for the presence of estrogen receptors. It was found by measuring the affinity of the cytoplasmic fraction of these samples for 3H-estradiol-17 beta that receptors were present in 72 p. 100 and 54 p. 100 of primary and metastatic cancers respectively. Receptor concentration varied among sample in an apparently continuous distribution from zero to 2,080 femtomoles per mg tissue protein. This suggests that mammary tumors are different from one another more in a quantitative than in a qualitative way. Detectable amounts of receptors were found in samples from mammary dysplasia, fibroadenomas as well as from one papilloma; none was detected in samples from non-tumorous mammary gland, nipple areola or skin. At mastectomy, no correlation was found between presence or absence of receptors in the primary tumors, and presence or absence of metastatic axillary nodes. On the other hand both the primary and its axillary metastases almost always displayed the same characteristic as far as presence or absence of receptors was concerned. Analysis of clinical studies reported seems to indicate that women with advanced breast cancer respond in a fair proportion of cases to various endocrine treatments when tumor tissue biopsies contain estrogen receptors whereas the probability of a response is very low in their absence.


[Histological classification of malignant breast neoplasms. Recent concepts].

The recent histological classifications of breast malignant epithelial tumours place increased emphasis on several concepts: in situ carcinoma, difference of lobular carcinoma from other forms of breast cancer and histological factors of prognosis. The authors propose to discriminate: non infiltrating duct carcinoma (intraductal carcinoma); lobular carcinoma (in situ and infiltrating); infiltrating duct carcinoma in their usual form (80 p. 100 about of all breast carcinoma); among them, histological types with a less ominous prognosis, although relatively rare, are stressed (infiltrating papillary and comedo-carcinomas, tubular carcinoma, medullary carcinoma, colloid carcinoma, cylindroma, certain metaplastic variants, Paget's disease of the nipple); some features in unusual hosts are related. They mention the criteria of the Scarff and Bloom's "grading" and its importance from the point of view of prognosis, mainly for the usual infiltrating forms. Other malignant tumours of the breast (malignant cystosarcoma phyllodes, sarcomas, mammary metastases) are more scarcely seen (I p. 100 of the mammary neoplasms): their classification is succinctly recalled.


Enzyme cytochemistry of Candida albicans.

The application of a new preparation method for demonstrating the activities of hydrolytic and oxidative enzymes in Candida albicans is reported. The problem of inadequate penetration of fixatives into yeast cells has been solved by sectioning solidified pellets of the cells in the presence of glutaraldehyde, a procedure that yields a fairly well preserved ultrastructure and sufficient enzyme activities. The subcellular distribution of most specific and nonspecific phosphatases and of peroxidases is at variance with that found in mammalian cells. The activities toward beta-glycerophosphate, p-nitrophenylphosphate, adenosine triphosphate, adenosine monophosphate, thiamine pyrophosphate and glucose 6-phosphate are almost exclusively confined to the central vacuolar apparatus. Oxidative and peroxidative activities are demonstrated only in mitochondria. Specific marker enzymes for endoplasmic reticulum, plasmalemma, Golgi apparatus and peroxisomes in C. albicans are not found. The possible function of the various subcellular organelles in relation to their enzymatic content is discussed.


Ultrastructural visualization of virus-induced surface antigens. Comparative studies of three immunohistochemical techniques.

Three immunohistochemical techniques used for ultrastructural localization of cell surface antigens were compared with respect to ease of reagent preparation and qualitative aspects of cell surface staining. The techniques compared were: (a) ferritin-conjugated antibody, (b) "hybrid" antibody and (c) a modification of the "bridging" technique which employs soluble immune complexes. Used in conjuction with strain MC29 avian oncornavirus-infected chick embryo cells and chicken antiviral immune serum, the results of all three techniques were comparable. In terms of ease of preparation, the "bridging" technique employing soluble complexes was found to be the most satisfactory.


The distribution of collagenase in normal rat tissues.

A rabbit monospecific anti-rat uterus collagenase antibody has been used to study the distribution of collagenase in normal rat tissues by immunohistochemical methods. Indirect staining was performed with fluorescein-conjugated goat anti-rabbit immunoglobulin G antibody. The organs studied were brain, lung, myocardium, liver, spleen, kidney, adrenal, testes, uterus, xiphoid cartilage, tail tendon, skeletal (triceps) muscle and skin. Collagenase is widely present throughout the connective tissue structures in all organs examined. The enzyme is apparently bound to collagen fibers, reticulum fibers and basement membranes. The results suggest that control of collagenase activity depends on factors other than the presence of the enzyme in tissues.


Intracellular localization and size of glycogen particles in glycogen synthesized under histochemical conditions.

During the investigation of the histochemical synthesis of glycogen particles from glucose 1-phosphate by the phosphorylase-branching glycosyltransferase system in various tissue cells, it was observed that focal synthesis localized in a certain area of the cytoplasm occurred in some cells. This differed from the usual synthesis in which particles of similar size were synthesized within the cytoplasm. Otherwise, cytoplasmic particles of various size were also synthesized in other cells under the same histochemical condition. The possible significance of the presence of these patterns in glycogen synthesis is discussed.


Variation among strains of type A foot-and-mouth disease virus in the Eastern Mediterranean region 1964-1972.

Variants of type A FMD virus from the Eastern Mediterranean region over the years 1964-72 have been shown to belong to a group distinct from the Western European strains as represented by A5 Westerwald. This group appears to derive from the A22 strain first recognized in 1964 and indicates the possibility of new strains supplanting old in the field.


A solid phase micro-radioimmunoassay to detect minute amounts of Ig class specific anti-viral antibody in a mouse model system.

A simple and rapid micro-radioimmunoassay was developed to detect and quantitate class specific mouse anti-Sendai virus antibodies. Two different 125I-labelled indicator systems were studied. After incubation of test serum with antigen one system used 125I-rabbit anti-mouse IgG (RIA 1) and the second employed rabbit anti-mouse IgG, IgA or IgM followed by 125I-sheep anti-rabbit immunoglobulin reagent (RIA 2). The RIA 2 method was adopted for routine use as it was more sensitive, gave better discrimination between sample and background counts and eliminated the need for several labelled rabbit anti-mouse Ig class specific antisera. The technique was found to be about 100 times more sensitive than conventional HI tests, specific, reliable and economical of reagents and time.


An evaluation of adenosine 3',5'-cyclic monophospate-dependent protein kinase activity in atopic dermatitis.

Activity of adenosine 3',5'-cyclic monophosphate-dependent protein kinase has been measured in the skin of normal controls, patients with non-atopic skin disorders, and those with atopic dermatitis. All samples analyzed displayed the presence of this enzymatic activity. However, the enzyme from the atopic skin did not seem to be dependent on cyclic AMP for activity. Whether this is due to an artifact of isolation of protein kinase or is indeed the true in vivo nature of the enzyme remains to be established.


Human responses to two decavalent rhinovirus vaccines.

Two formalin-inactivated, decavalent rhinovirus vaccines were tested in humans for acceptability and antigenicity. Infectivity titers of the vaccine antigens were low and ranged from 10(1.5) to 10(5.5) 50% tissue culture infective doses/ml. There were minimal or no side effects to either vaccine. The first inoculation of one vaccine produced antigenic responses to 30% of the administered antigens. Limited testing for heterologous antibody responses to nonvaccine antigens showed scattered responses. These findings suggest that potent multivalent rhinovirus vaccines containing antigens selected for cross-reactivity should be tested in an attempt to develop practical control measures for rhinoviruses.


Humoral and cellular immune responses to an envelope-associated antigen of herpes simplex virus.

The humoral and cellular immune responses of rabbits and guinea pigs to the envelope-associated antigen of herpes simplex virus type I were studied. Neutralizing antibody (at high titer) and lymphocytes reactive to herpes simplex virus were detected in both guinea pigs and rabbits after immunization with the antigen. In a standard assay of cellular immunity to herpes simplex virus, the antigen stimulated blast transformation of herpes simplex virus-reactive splenic lymphocytes in vitro. Furthermore, immunization of rabbits with the envelope-associated antigen protected the animals from a lethal dose of live herpes simplex virus. Thus an antigen of herpes simplex virus can be prepared which contains neither infectious nor noninfectious viral particles and which stimulates immunity to the virus in laboratory animals.


Experimental horizontal transmission of Herpesvirus saimiri from squirrel monkeys to an owl monkey.

Herpesvirus saimiri was naturally transmitted from squirrel monkeys excreting the virus to one of two owl monkeys housed in the same cage. The owl monkey became infected approximately three months after contact was initiated. H. saimiri was consistently isolated from the peripheral lymphocytes until this animal died eight months later. During this period the owl monkey developed specific antibody to H. saimiri to a maximal neutralization index of 5.5 logs. The other monkey remained uninfected for an ovservation period of one year. The documentation of this horizontal transmission of H. saimiri infection from squirrel monkeys to an owl monkey suggests that owl monkeys developing spontaneous malignant lymphomas associated with H. saimiri infection may also have acquired the infection in this manner.


Adaptive enhancement of amino acid transport in human leukemia leukocytes: studies with alpha-aminoisobutyric acid.

Initial rates of accumulation (Vi) of [3-14C] alpha-aminoisobutyric acid (AIB) by human leukemic leukocytes increased markedly and progressively during 240 minute incubations in amino acid-deficient media. Absolute increments were greater in blast cells from patients with acute lymphoblastic leukemia and with acute myeloblastic leukemia than in lymphocytes from patients with chronic lymphocytic leukemia, but per cent increments did not differ. Time-related increases appear to be restricted to an active mechanism for AIB entry and could not be attributed to cell damage, concomitant alterations in the incubation media, or progressive reduction in transinhibition of AIB entry. Studies with two cell populations revealed these increases to be associated with an augmented Vmax and a reduction in apparent Km, suggesting enhanced capacity and affinity of the transport system for AIB. Time-related increases failed to develop in two chronic lymphocytic leukemia (CLL) lymphocyte populations and were partially reduced in two leukemic blast cell populations during continuous exposure to high extracellular AIB concentrations. Hence, this phenomenon may represent an adaptive response to environmental amino acid deprivation. Adaptation may involve de novo protein and RNA synthesis, since it was not seen in cells which were treated with cycloheximide or actinomycin D. However, unlike previous observations in nonmalignant cells, once triggered, the adaptive response was not completely suppressed in leukemic cells which underwent a large degree of adaptation.


Glomus jugulare tumours. (A series of 21 cases).

Twenty-one cases of glomus jugulare tumours were analysed in retrospect. Six had been treated surgically, ten by irradiation, and five by a combination of incomplete surgical excision followed by irradiation. Recurrences appeared in four cases, but no patient has died of the tumour during the follow-up period which is from a few months to eleven years. Metastases were not found in any case. The optimal time of irradiation in combined treatment is discussed.


Rhabdomyoma purum of the larynx.

A case of pure well-differentiated rhabdomyoma of the larynx occurring in a 50-year-old male is reported. The lesion was localized to the interarytenoid region. From an accurate revision of the world literature on the subject it appears that the cases so far described number only seven, including the one in the present report. Furthermore, the differential histological and histochemical criteria for distinguishing rhabdomyoma from granular cell Schwannoma are discussed and tabulated, as the two neoplasms may be easily confused. In our case, there has been no evidence of recurrence of the tumour for more than 1 year, which is in accordance with the previous cases reported in the literature.


Hyperbaric oxygen in anaerobic infection of the medistinum.

In the last decade hyperbaric oxygen has been recognized as an important therapeutic tool in a variety of instances in which either destruction of anaerobic bacteria is urgent or an improvement in the oxygenation level is mandatory. We have used it successfully in a case of mediastinal anaerobic infection (gas gangrene) after medical and surgical measures had failed to eradicate the disease. The causative organism of gas gangrene, Clostridium perfringens (Welchii), is widely distributed. It may be cultured from the soil, house dust, human skin and faeces, etc. For this reason infection with Clostridium is practically inevitable whenever suitable conditions arise. As an anaerobic bacterium Clostridium Welchii multiplies readily in damaged tissues without contact with the air and devoid of a normal blood supply. This occurs especially in road accidents and war wounds in which broken bones and crushed muscles provide a suitable medium for the infection (Altmeier, 1965). Sometimes this infection can occur too after abdominal or gynaecological operations (Hitchcock, 1965). We present here a case of mediastinal gas gangrene which was caused by perforation of the oesophagus by a swallowed foreign body.


Role of lamellar inclusions in surfactant production: studies on phospholipid composition and biosynthesis in rat and rabbit lung subcellular fractions.

Lamellar inclusion bodies in the type II alveolar epithelial cell are believed to be involved in pulmonary surfactant production. However, it is not clear whether their role is that of synthesis, storage, or secretion. We have examined the phospholipid composition and fatty acid content of rabbit lung wash, lamellar bodies, mitochondria, and microsomes. Phosphatidylcholine and phosphatidylglycerol, the surface-active components of pulmonary surfactant, accounted for over 80% of the total phospholipid in lung wash and lamellar bodies but for only about 50% in mitochondria and microsomes. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin accounted for over 40% of the total in mitochondria and microsomes but for only 6% in lung wash and 15% in lamellar bodies. The fatty acid composition of lamellar body phosphatidylcholine was similar to that of lung wash, but different from that of mitochondria and microsomes, in containing palmitic acid as a major component with little stearic acid and few fatty acids of chain length greater than 18 carbon atoms. The biosynthesis of phosphatidylcholine and phosphatidylglycerol was examined in the mitochondrial, microsomal, and lamellar body fractions from rat lung. Cholinephosphotransferase was largely microsomal. The activity in the lamellar body fraction could be attributed to microsomal contamination. The activity of glycerolphosphate phosphatidyltransferase, however, was high in the lamellar body fraction, although it was highest in the mitochondria and was also active in the microsomes. These data suggest that the lamellar bodies are involved both in the storage of the lipid components of surfactant and in the synthesis of at least one of those components, phosphatidylglycerol.


High-pressure liquid chromatography: separation of the metabolites of vitamins D2 and D3 on small-particle silica columns.

The high-pressure liquid chromatographic separation of all of the known metabolites of vitamin D(2) and vitamin D(3) found in biological fluids has been achieved. This technique has been successfully applied to the analysis of vitamin D mixtures, purification of vitamin D metabolites, and identification of radioactive peaks. Some theoretical bases for the observed resolutions are suggested.


Determination of B protein of low density lipoprotein directly in plasma.

Quantitation of the apoprotein constituents of lipoproteins has extended our knowledge of plasma lipid transport. Previously, B protein content of low density lipoprotein could be measured by radial immunodiffusion only after ultracentrifugation. However, if performed in 1.5% agarose gel with standards and measured at 18 hr rather than at equilibrium, low density lipoprotein B protein can be measured directly in plasma, eliminating the need to separate very low density lipoprotein.


Radioiodinated lipoproteins: absorption of 125I radioactivity by high density solutions.

Concentrations of potassium bromide commonly used for separation of lipoproteins were shown to cause absorption of 125I and thus reduce the counting efficiency of the labeled lipoproteins. Chloroform was shown to cause a 50% reduction in counting efficiency of lipid from 125I-labeled lipoprotein. No reduction of counting efficiency was observed in the presence of high density solutions when 131I was used as label.


Modification of myometrial activity in vivo by administration of cyclic nucleotides and theophylline to the pregnant rat.

Myometrial activity was recorded in vivo in unrestrained pregnant rats from day 19 of gestation using radiotelemetry. The effects of short-term infusions of theophylline, dibutyryl cyclic AMP and 8-bromo-cyclic GMP were investigated. All three compounds caused a decrease in oxytocin-induced, prostaglandin F2alpha-induced and spontaneous uterine activity. After cessation of the infusion of these compounds uterine activity returned to near pre-infusion levels within approximately 15 min in most animals. The possible roles of cyclic nucleotides in the control of myometrial contraction are discussed in the light of these observations.


Comparison of the characteristics and of the hormonal control of endometrial and myometrial progesterone receptors.

The characteristics of endometrial and myometrial progesterone receptor of guinea-pig were compared. Affinity for progesterone, hormone specificity, sedimentation properties (in oestrogen-primed animals), inhibition of binding by p-hydroxymercuribenzoate were found to be identical in both tissue. Differences were, however, observed in the hormonal control of the concentration of receptor. In all the situations studied, the concentration of receptors was higher in the endometrium than in the myometrium. In guinea-pigs ovariectomized at dioestrus the concentration was 3500 binding sites per diploid genome in the myometrium and 20300 in the endometrium; 1-3 days after oestradiol injection, this concentration was raised to 46000-38000 and 65000-83000 binding sites respectively. Thus the absolute rise was similar in both tissues but the relative increase was about 15-fold in the myometrium and only three- to fourfold in the endometrium. After injection of 2 mg progesterone, the concentration of receptor previously induced by oestrogen returned to very low values similar to those observed in non-hormonally treated controls. This difference between endometrial and myometrial receptors could be due either to a faster turnover of the latter or to the existence of a stable non-hormonally controlled population of receptors, present only in the endometrium.


Effect of magnesium deficiency and parathyroid hormone on cyclic AMP metabolism in rat renal cortex.

The influence of magnesium deficiency on cyclic AMP metabolism was investigated in rats on diets of normal and low calcium content. Magnesium deficiency itself did not significantly affect either the basal concentration or the parathyroid hormone-stimulated formation of cyclic AMP in the renal cortex. Magnesium-deficient rats with hypercalcaemia excreted more cyclic AMP in the urine, but similar rats that developed hypocalcaemia on low calcium intake excreted less than their respective controls. The former type of animals also tended to accumulate more cyclic AMP in the renal cortex in response to the injection of a standard dose of parathyroid hormone, whereas rats of the latter type accumulated less. The activity of parathyroid hormone-stimulated renal cortical adenylate cyclase in vitro was increased by magnesium and reduced by calcium under most conditions, but with low concentrations of magnesium small amounts of calcium had a stimulatory effect. These observations suggest that cyclic AMP metabolism is influenced by metabolic disorders developing secondary to magnesium deficiency.


Osmotic inhibition of the natriferic response of the toad urinary bladder to vasopressin.

The electrical potential difference and short-circuit current (scc, reflecting active transmural sodium transport) across the toad urinary bladder in vitro was unaffected by the presence of hypo-osmotic solutions bathing the mucosal (urinary) surface, providing that the transmural flow of water was small. Vasopressin increased the scc across the toad bladder (the natriferic response), but this stimulation was considerably reduced in the presence of a hypo-osmotic solution on the mucosal side, conditions under which water transfer across the membrane was also increased. This inhibition of the natriferic response did not depend on the direction of the water movement, for if the osmotic gradient was the opposite way to that which normally occurs, the response to vasopressin was still reduced. The natriferic response to cyclic AMP was also inhibited in the presence of an osmotic gradient. Aldosterone increased the scc and Na+ transport across the toad bladder but this response was not changed when an osmotic gradient was present. The physiological implications of these observations and the possible mechanisms involved are discussed.


Levels of corticotrophin analogues in the blood after infusion into rats.

An isolated rat adrenal cell bioassay was used to measure blood concentrations in rats after infusion of synthetic human ACTH, corticotrophin-(1-24)-tetracosapeptide or [D-Ser1, Lys17, Lys18]corticotrophin-(1-18)-octadecapeptide amide. Lower blood levels were found with the 1-24 peptide than with human ACTH and the highest levels were found with the 1-18 peptide. These results suggest that the 1-24 peptide which is almost equipotent with natural ACTH in vivo may be more potent at the receptor and corroborate findings to this effect obtained with isolated adrenal cells. The high potency and prolonged action of the 1-18 analogue in vivo are also explained by these results. Low arterial blood concentrations of the 1-24 peptide and human ACTH were found during infusion, suggesting that substantial inactivation must be occurring in a single passage through the lungs. The effects of renal ligature on blood concentrations indicated that the kidney is involved in handling the 1-18 peptide and that human ACTH is also cleared by this organ. After infusion the fall in blood concentrations was biphasic. It is suggested that the rapid phase is due to clearance of peptides in the circulation which results in a fall to lower blood concentrations which are sustained by slow release of peptide from binding sites which act as a depot.


[Neurulation and interkinetic nuclear migration in the chick embryo (author's transl)].

Neurulation and interkinetic nuclear migration was studied in cells of the forming neural tube of chick embryos submitted to a variety of treatments. Our results show that cytochalasin B (5 mug/ml) does not protect microtubules against disruption occurring after 3 h at 2 degrees C nor does it prevent their repolymerization once they are cold-disrupted. However, db-cAMP protects microtubules against such cold disruption. We indicate that the inhibitory effect of cytochalasin B on interkinetic nuclear migration cannot be ascribed to an effect on microtubules.


Effects of helium and other inert gases on Echinosphaerium nucleofilium (protozoa, heliozoa).

The effects of helium, nitrogen, argon and krypton on Echinosphaerium nucleofilum (Heliozoa) have been studied at partial pressures of 10-130 atm. Additional experiments have been carried out with hydrostatic pressure alone. Helium causes shortening of the axopods over the whole range of pressures, and damage to the cell body at pressures of 60-90 atm, both with a maximum at 80 atm. These effects cannot be explained in terms of hydrostatic pressure alone; a 'pressure reversal' effect may be operating, causing the peak at 80 atm. Nitrogen also causes both cell damage and axopod shortening, the severity increasing with increasing pressure. Argon and krypton cause cell damage but no shortening. The order of potency for cell damage is krypton greater than argon greater than nitrogen greater than helium. It is suggested that there may be tuo sites of action, possibly the microtubules (for axopod shortening) and the cell membrane (for cell damage). In appropriate mixtures of helium and argon, both the cell damage usually caused by argon, and the axopod shortening usually caused by helium, are prevented. Possible mechanisms include the effects of hydrostatic pressure on gas solubility coefficients, reversal of the effects of the gases by the increase in total pressure, and competition for sites of action.


Lymphocyte E rosette inhibitory factor: a regulatory serum lipoprotein.

Rosette inhibitory factor, RIF, previously described in serum from patients with hepatitis B virus infection, has been isolated and identified as a minor species of beta-lipoprotein of the low-density (LDL) class. It is unrelated to hepatitis B virus proteins or particles. Although discrete by reference to charge and density (1.050 +/- 0.004 g/cm3), RIF appears to be a complex macromolecular structure containing apolipoproteins A, B, and C. Greater than 400% recovery is achieved upon 300,000-fold purification from RIF+ sera suggesting activation of a precursor form that is not present in normal serum. RIF inhibits E rosette function of T lymphocytes in vitro with a lag period of approximately 4 h and maximal effect at 24 h consistent with a metabolically-induced event. RIF is functionally active at concentrations of 1 X 10(-12) M or greater, rapidly binds to lymphocytes, and has a functionally effective half-life of approximately 1.5 h. Approximately 2,900 receptors for RIF appear to be present per cell and a high mutual affinity is apparent (k approximately to 9 X 10(10) liters/mol). RIF has no detectable effect on mitogen (PHA) responsiveness of lymphocytes, but inhibits the capacity of lymphocytes to respond to histoincompatible cells in vitro at concentrations greater than 10(-8) M. Equivalent RIF- lipoprotein fractions from normal serum are equally inhibitory in the mixed lymphocyte reaction suggesting that this effect is not directly attributable to RIF activity. These data indicate that RIF is a unique and functionally specific species of LDL that represents either an association complex of lipoproteins or a hybrid molecule of unusual composition. The association of this factor with viral-induced hepatocellular injury underscores the need to elucidate its structure and function in greater detail.


Endogenous oncornaviral gene expression in adult and fetal mice: quantitative, histologic, and physiologic studies of the major viral glycorprotein, gp70.

Endogenous expression of the murine leukemia virus (MuLV) genome has been studied in a number of strains of mice. Expression of the major envelope glycoprotein, gp70, is restricted to certain anatomical sites and cell types, prominent among which are lymphoid and epithelial cells. On a quantitative basis, the major site of gp70 expression is the male genital tract. During development, gp70 first appears in the hematopoietic liver of 14-day-old embryos and by day 18, it is already expressed at anatomical sites similar to those of the adult. In toto, these results show that control of expression of the MuLV genome in adult and developing mice is linked to differentiation.


Relationship of infectious murine leukemia virus and virus-related antigens in genetic crosses between AKR and the Fv-1 compatible strain C57L.

In a further genetic study of murine leukemia virus (MuLV) and its components we examined the backcross C57L X (C57L X AKR). This population was selected because strains AKR and C57L are both Fv-1n, and the restriction which the Fu-1b allele imposes on the output of virus was thereby obviated. The segregants were scored for three characters: (a) infectious Gross-AKR-type MuLV (V), in the tail; (b) group-specific antigen indicative of p30 internal viral protein, in spleen; and (c) GIX antigen, now thought to be indicative of gp69/71 viral envelope glycoprotein, on thymocytes. Our conclusions are: (a) It is confirmed that the AKR mouse has two unlinked chromosomal genes, Akv-1 and Akv-2, each of which can independently give rise to the life-long high output of MuLV that is characteristic of AKR mice. (b) Of the eight phenotypes that could possibly be derived from segregation of the three pairs of independent alternative traits, seven were observed, but on progeny testing only three were shown to reflect stably heritable genotypes; these were V+p30+GIX+ and V-p30-GIX- (the parental types) and V-p30+GIX+. A third, newly identified AKR gene, designated Akvp, segregating independently of Akv-1 and Akv-2, also determines expression of p30 and GIX but in this case independently of XC-detectable MuLV. (c) The four remaining observed phenotypes, which did not breed true on progeny testing, involved mostly antigen-negative parents yielding antigen-positive progeny; it is likely that these discrepancies represented suppression of phenotype by a maternal resistance factor.


Comparative studies of purinergic nerves.

Purinergic nerves supply the gastrointestinal tract of vertebrates, including fish, amphibians, reptiles and birds, as well as mammals. Their cell bodies are located in Auerbach's plexus and their axons extend in an anal direction before innervating mainly the circular muscle coat. In the stomach they are controlled by preganglionic cholinergic fibres of parasympathetic origin. They are involved in "receptive relaxation" of the stomach, "descending inhibition" in peristalsis and reflex relaxation of oesophageal and internal anal sphincters. The terminal varicosities of purinergic nerves are characterised by a predominance of "large opaque vesicles," which can be distinguished from the "large granular vesicles" found in small numbers in both adrenergic and cholinergic nerves. Stimulation of purinergic nerves with single pulses produces hyperpolarisations of up to 25 mV (inhibitory junction potentials) in smooth muscle cells. These potentials are unaffected by atropine, adrenergic neuron blocking agents or sympathetic denervation, but are abolished by tetrodotoxin. The "rebound contraction" which characteristically follows cessation of purinergic nerve stimulation is probably due to prostaglandin. Evidence that ATP is the transmitter released from purinergic nerves includes: (1) synthesis and storage of ATP in nerves; (2) release of ATP from the nerves when they are stimulated; (3) exogenously applied ATP mimicking the action of nerve-released transmitter, both producing a specific increase in K+ conductance; (4) the presence of Mg-activated ATPase, 5'-nucleotidase and adenosine deaminase, enzymes which inactivate ATP; (5) drugs (including quinidine, some 2-substituted imidazolines, 2-2'pyridylisatogen and dipyridamole) which produce similar blocking or potentiating effects on the response to exogenously applied ATP and nerve stimulation. Speculations are made about the evolution and development of the nervous system, including the possibility that purinergic nerves are a primitive nerve type.


Comparative physiology and the challenge of design.

Analysis of the complexity of biological organization commonly takes us through an initial and necessary stage of over-simplification towards the ultimate goal of an appreciation of the subtle intricacy of nature. One example of this is that recent advances in studies of the phosphagens have failed to confirm earlier hopes that the distribution of these substances could be used to confirm phylogenetic relationships. It is now suggested that a previously unrecognized factor influencing this distribution may be competing demands for arginine in the development of the testes. Another illustration is the belief that the presence of iodine in ascidians may provide a clue to the origin of thyroidal biosynthesis. This belief is probably sound, but the presence of abundant iodine in the tunic may well be a false trail. More significant is the presence of much smaller amounts of bound iodine in the endostyle, where its presence is probably correlated with specialized protein metabolism associated with the ciliary feeding mechanism. A third illustration, discussed in more detail, is provided by recent recognition that the gut is a major component of the vertebrate endocrine system. This lays open attractive fields of research, ranging from problems of molecular and hormonal evolution to aspects of fish farming and the physiology of gut parasites. The need is thus plain for breadth of perspective in comparative physiological research, and for recognition that the ultimate concern of the biologist is with the life and relationships of the whole organism.


Failure of gonadotropins to induce in vitro maturation of mouse oocytes treated with dibutyryl cyclic AMP.

In sufficient concentration, dibutyryl cAMP (DBC) prevents the spontaneous in vitro maturation of mouse oocytes. The effects of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on this inhibition were tested in an oil-free chamber-slide culture system. Mouse oocytes devoid of cumulus cells were incubated in the presence of DBC and/or gonadotropins. Oocytes cultured with follicle cells were similarly treated. Whether follicle cells were present or absent, DBC (100 or 500 mug/ml) prevented germinal vesicle breakdown in more than 95% of the oocytes cultured. Neither LH nor FSH in a wide range of concentrations acted directly on the oocytes or indirectly through the follicle cells to initiate maturation in oocytes incubated with 100 mug DBC/ml. The combination of LH (5 mug/ml) and FSH (10 mug/ml) was also ineffective in overcoming the block induced by either 100 or 50 mug DBC/ml. Maturation of oocytes in each of the DBC-free LH or FSH treatments was comparable to that occurring in control medium which did not contain exogenous gonadotropins or DBC. It was concluded that cultured oocytes treated with DBC are not a satisfactory model for studying the steps by which gonadotropins trigger the resumption of meiosis in mammalian oocytes.


Mach bands in the lateral eye of Limulus.

Patterns of optic nerve activity were computed for stationary step patterns of illumination from theoretical models of lateral inhibiton based on revised Hartlin-Ratliff equations. The computed response patterns contain well-defined Mach bands which match closely in amplitude and shape those recorded from single optic nerve fibers of the Limulus lateral eye. Theory and experiment show that the amplitude of the Mach bands is reduced by in inhibitory nonlinearity, the width of the Mach bands is approximately equal to the lateral dimension of the inhibitory field, but the shapes of the Mach bands are poor indices of the precise configuration of the inhibitory field. Theorems are proved establishing the equivalence of Mach-band patterns for models of different dimensions and a uniqueness condition for solutions of the piecewise linear model.


Sodium chloride transport by rabbit gallbladder. Direct evidence for a coupled NaCl influx process.

The results of the present study that NaCl transport by in vitro rabbit gallbladder must be a consequence of a neutral coupled carrier-mediated mechanism that ultimately results in the active absorption of both ions; pure electrical coupling between the movements of Na and Cl can be excluded on the grounds of electrphysiologic considerations. Studies on the unidirectional influxes of Na and Cl have localized the site of this coupled mechanism to the mucosal membranes. Studies on the intracellular ion concentrations and the intracellular electrical potential are consistent with the notion that (a) the coupled NaCl influx process results in the movement of Cl from the mucosal solution into the cell against an apparent electrochemical potential difference; (b) the energy for the uphill movement of Cl is derived from the Na gradient across the mucosal membrane which is maintained by an active Na extrusion mechanism located at the basolateral membranes; and (c) Cl exit from the cell across the basolateral membranes is directed down an electrochemical potential gradient and may be diffusional. Finally, as for the case of rabbit ileum, the coupled NaCl influx process is inhibited by elevated intracellular levels of cyclic 3',5'-adenosine monophosphate. A working model for transcellular and paracellular NaCl transport by in vitro rabbit gallbladder is proposed.


Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP.

Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).


Receiver operating characteristic analysis. Application to the study of quantum fluctuation effects in optic nerve of Rana pipiens.

Receiver operating characteristic (ROC) analysis of nerve messages is described. The hypothesis that quantum fluctuations provide the only limit to the ability of frog ganglion cells to signal luminance change information is examined using ROC analysis. In the context of ROC analysis, the quantum fluctuation hypothesis predicts (a) the detectability of a luminance change signal should rise proportionally to the size of the change, (b) detectability should decrease as the square root of background, an implication of which is the deVries-Rose law, and (c) ROC curves should exhibit a shape particular to underlying Poisson distributions. Each of these predictions is confirmed for the responses of dimming ganglion cells to brief luminance decrements at scotopic levels, but none could have been tested using classical nerve message analysis procedures.


The respiratory system of Chromobacterium violaceum grown under conditions of high and low cyanide evolution.

The particulate fraction of disrupted Chromobacterium violaceum grown under cyanide-evolving conditions was unable to oxidize ascorbate plus N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD), but oxidized NADH and succinate by a linear respiratory pathway which was very resistant to inhibition by cyanide. When the bacteria were grown under conditions where little cyanide evolution occurred, particulate fractions developed the ability to oxidize ascorbate-TMPD by a pathway highly sensitive to cyanide inhibition; respiratory activity with NADH and succinate proceeded via both the cyanide-sensitive and-resistant pathways. Studies with respiratory inhibitors, and the cytochrome compositions of the fractions derived from cultures grown under both conditions, are presented. A soluble, carbon monoxide-binding cytochrome c was found, and this appears similar to those found recently in Beneckea natiegens, methylotrophic bacteria and the marine pseudomonad B16.


Genes involved in the uptake and catabolism of gluconate by Escherichia coli.

The isolation and properties of a mutant of Escherichia coli K12 that is totally unable to take up and utilize gluconate are described. Genetical analysis shows this phenotype to be associated with two lesions. One phenotype, designated GntM-, is the result of a mutation in a gene co-transducible with malA; the other, designated GNTS-, is the result of a mutation in a gene (GntS) co-transducible with fdp. The GntS--phenotype differs little from that of wild-type cells, but GntM- GntS+ organisms grow on gluconate only after a prolonged lag and form a gluconate uptake system that is strongly repressed by pyruvate. Moreover, such GntM- mutants readily give rise to further mutants that form a gluconate uptake system, gluconate kinase and 6-phosphogluconate dehydratase consititutively; in partial diploids, this constitutivity is recessive to the inducible character. It is postulated that the GntM- phenotype is due to malfunction of a negative control gene gntR, and that gntS+ specifies the activity of a gluconate uptake system.


The activity of polymyxins against dense populations of Escherichia coli.

The activities of polymyxin B sulphate, colistin (polymyxin E) sulphate and their sulphomethyl derivatives were compared by continuous turbidimetric monitoring of dense cultures of an Escherichia coli strain exposed to these agents. Judged by the concentration of antibiotic which caused a rapid fall in opacity of the culture, polymyxin B sulphate and colistin sulphate had similar activities, but the sulphomethyl compounds differed considerably: sulphomyxin sodium induced lysis of the culture at a concentration four times that of the parent compound, whereas colistin sulphomethate sodium induced a delayed fall in opacity consistent with recruitment of activity as the inactive sulphomethyl derivative was broken down to the parent compound. Durign overnight incubation, regrowth of cultures which had initially succumbed to polymyxin action occurred, apparently due to the selection of phenotypically resistant variants from within the population. In this way cultures could easily be adapted to growth in concentrations of antibiotic well above the conventionally-determined minimum inhibitory concentration. The comparative ease of adaptation was in the order: colistin sulphomethate greater than sulphomyxin greater than colistin sulphate greater than polymyxin B sulphate.


Loss of cytochrome oxidase in Saccharomyces cerevisiae during inhibition of mitochondrial protein synthesis by erythromycin and chloramphenicol.

There is a major reduction in respiratory competence, and inhibitionof growth, several hours after the addition of erythromycin or chloramphenicol to Saccharomyces cerevisiae growing in medium containing a non-fermentable carbon source. Spectrographic evidence is presented for a loss of cytochrome oxidase as a consequence of the antibiotic treatment. This loss is prevented by cyanide or oligomycin. When glucose is added, however, the loss occurs irrespective of the presence of the respiratory inhibitors. Cycloheximide does not affect respiratory competence or cause loss of cytochrome oxidase, and it prevents the loss elicited by erythromycin if both compounds are added together. However, if cycloheximide is added some time after the addition of erythromycin, it fails to block the response to the latter drug. The results cannot be accounted for on the basis of the segregation of a finite number of mitochondria into an increasing number of progeny cells but, rather, suggest that the mitochondria are modified during growth in chloramphenicol or erythromycin.


Alveolar hypoventilation and hyperosmnia in myotonic dystrophy.

A case of myotonic dystrophy accompanied by alveolar hypoventilation and hypersomnia is presented. Radiological studies and EMG examination of the intercostal muscles demonstrated that the respiratory muscles were affected by the disease, while polygraphic recordings showed that the alveolar hypoventilation and pulmonary hypertension worsened during sleep. The hypersomnia preceded the appearance of clinical signs of the muscular disease by many years and persisted even after treatment when the blood gas analysis values were greatly improved. During both diurnal and nocturnal sleep, the patient frequently fell asleep directly into a REM stage. The possibility is discussed that, concomitant with the respiratory musculature involvement, there is an alteration in the central nervous system in myotonic dystrophy which is at least partially responsible for both the alveolar hypoventilation and the hypersomnia.


Significance of muscle biopsies in neuronal ceroid-lipofuscinoses.

Muscle specimens obtained at necropsy from four cases of neuronal ceroid-lipofuscinosis (NCL), three of the juvenile and one of the late infantile type, and a muscle biopsy from a fifth patient with the juvenile type of NCL, all showed curvilinear bodies typical of NCL within the muscle fibres. The pigments were autofluorescent. It appears that skeletal muscle is a reliable tissue source for the diagnosis of these disorders by biopsy.


An ultramicroscopic study of the skin in the diagnosis of the  infantile and late infantile types of ceroid-lipofuscinosis.

A skin biopsy was carried out in two children suffering from early infantile (Santavuori) and late infantile (Jansky-Bielschowsky) types of ceroid-lipofuscinosis. In both cases cytoplasmic inclusions, identical with those found in neurones, were present in the skin. Skin biopsy thus appears as a simple technique for the diagnosis of the ceroid-lipofuscinoses.


Effects of collagenase upon the intervertebral disc in monkeys.

The authors studied 5 rhesus monkeys for the effects of annulotomy, with or without the addition of a bacterially-derived collagenase into the nuclear-evacuated centrum of lumbar intervertebral discs. The animals were sacrificed from 3 weeks to 21 months after the single or staged double procedures. The earliest radiographic changes were loss of height of the interspace at 7 days, erosion of the vertebral body margins at 3 weeks, malalignment at 6 weeks, osteophytic formation at 3 1/2 months, sclerosis of vertebral body surfaces at 9 1/2 months, and fusion of adjacent vertebral bodies at 13 1/2 months. Pathological changes included early loss of disc convexity, focal new home formation, osteophytic new growth, through-and-through bone formation, irregular cystification, and disc replacement by hyaline fibrous tissue. Changes were reminiscent of those seen in humans with naturally acquired disc disease or aging of the disc. Injuries were produced in a tissue that probably has a limited scope of variation in reaction to insult.


Measurement of acute myocardial infarcts in dogs with 99mTc-stannous pyrophosphate scintigrams.

Myocardial scintigrams using 99mTc-stannous pyrophosphate (99mTc-PYP) can be used to measure myocardial infarcts produced in dogs by proximal ligation of the left anterior descending coronary artery. Seven dogs had 99mTc-PYP myocardial scintigraphy performed 24--32 hr after ligation of the proximal left anterior artery. In each dog the scintigrams showed increased 99mTc-PYP uptake in the distribution of the artery. The scintigraphically visible areas of infarction, measured using interactive computer-aided techniques, were compared subsequently with independent histologic measurements of myocardial infarct size. Several methods for using the area measurements to estimate infarct size were tested. The most successful method (r = 0.92, p less than 0.01) assumed a linear relationship between the largest scintigraphic infarct area and the histologically determined infarct weight. The results suggest that 99mTc-PYP myocardial scintigrams provide a useful noninvasive method for measuring infarct size in dogs with proximal ligation of the left anterior descending coronary artery.


Effect of unilateral pulmonary hypovascularity on the bone scan: case report.

Two patients who had decreased pulmonary artery blood flow to the right lung showed increased clarity of rib detail on a bone scan over the affected hemithorax as contrasted with the uninvolved side. Decreased blood background is postulated to result in enhancement of rib detail on the affected side.


Ovarian carcinoma imaged by 99mTc-pyrophosphate: case report.

An ovarian carcinoma which avidly accumulated 99mTc-pyrophosphate was imaged during a bone scan. The primary tumor and its implants over the liver surface were both visualized. Although imaging of such an ovarian malignancy has not been previously reported, it offers a potential method for assessing neoplastic size and spread.


Enzyme activities in the epidermis of zinc-deficient rats.

Fifteen enzymes participating in epidermal energy metabolism in zinc-deficient and -supplemented rats were assayed utilizing fluorometric microchemical techniques. In the zinc-deficient group, the activities of six enzymes catalyzing glycolysis decreased by 30 to 50% of the control; the most dramatic decreases were found in phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. Zinc deficiency caused a 31% decrease in the activity of glucose-6-phosphate dehydrogenase, a 63% decrease in fumarate hydratase, a 46% decrease in glutamate dehydrogenase, and a 30 to 40% decrease in aminotransferases.


Effects of quantity and quality of dietary protein and variation in certain enzyme activities on glucose metabolism in the rat.

This study attempted to determine whether the quantity and the quality of protein intake could influence the activity of some enzymes involved in carbohydrate metabolism. Thus, adult rats were fed for 23 days a diet containing different levels (10 to 70%) and qualities (casein, wheat gluten, and egg yolk) of protein. Variations in liver enzyme activities of pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), glucose-6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK) were studied. Also the changes in enzyme activities were compared with changes in food intake and body weight gain. Increasing the protein level produced a progressive fall in the activities of ME and PK. The decrease in PK activity was greater when the biological value of the dietary proteins was higher (P less than 0.05). On the other hand, the activities of G6PDH and PEPCK increased as the protein level increased. The activity of G6Pase was unchanged. The relationship between the two opposing enzyme activities PK and PEPCK, in relation to protein intake, shows that for each protein studied, the equilibrium between glycolysis and gluconeogenesis was obtained at different protein intakes (1.5, 1.9, and 2.2 g of protein/day/100 g of body weight, respectively, for egg yolk, casein, and wheat gluten) regardless of daily consumption of energy as carbohydrate, which are similar (8 to 9 kcal/day/100 g of body weight). This equilibrium also corresponded to the maximum weight gain (5 g) of the experimental animals. In conclusion, the experimental method used permits a simultaneous assessment of the protein and carbohydrate requirements ensuring the best weight gain in young adult rats.


Synovial sarcoma of the neck: report of case.

Synovial sarcoma is an uncommon neoplasm that usually arises in the extremities and rarely in the head and neck. A case arising in the neck has been reported herein. The tumor is composed of two intimately associated cellular patterns; one resembles synovial structures and the other resembles fibrosarcoma. The tumor is fairly slow growing and is fairly well circumscribed but not actually encapsulated. There is a predilection for males; most occur in the third through fifth decades of life. Recurrence follows inadequate therapy. Although local excision was the therapy used in this case, the patient is alive and free of disease 30 months postoperatively.


The pathology of type II skeletal muscle glycogenosis. A light and electron-microscopic study.

Muscle biopsies were obtained from three infants under the age of 12 mth, each of whom was diagnosed as having Pompe's disease. The biopsies revealed a severe vacuolar myopathy with accumulation of large amounts of PAS positive material within the muscle fibres, changes similar to those in adult cases of the disease. In addition large amounts of metachromatic material were found within the muscle fibres in all three cases and in two of them scattered, rather sparse perivascular lymphocytic infiltrates were seen in the interstitial tissue. Review of material previously obtained from two adult cases showed no accumulation of metachromatic material in the older case and only moderate amounts in the younger. However, dense interstitial lymphocytic infiltrates were seen in the former, some concentrated around small vessels. These observations suggest that the pathogenesis of the muscle disorder in acid maltase deficiency may not depend on abnormal glycogen storage only.


Lethality of disrupted intestinal lamina propia cells for Trichinella spiralis in vitro.

The possible direct role of inflammatory cells in resistance to Trichinella spiralis was studied by observing the effects of lamina propria cells from the small intestine (LP cells) of immunized rats on various stages of the parasite. Effects produced by physically disrupted cells were compared to those produced by intact cells on worms exposed to phytohemagglutinin or immune serum. LP cells were isolated from the rat intestine by collagenase digestion of everted gut segments that were previously denuded of epithelium by treatment with hyaluronidase. Disrupted cells, but not intact ones, selectively killed T. spiralis juvenile and adult worms in vitro, whereas larvae were unaffected by similar treatment. Attempts to identify the lethal component of disrupted cells led to an evaluation of the enzyme, peroxidase. Mucosal peroxidase is localized in LP cells and its activity increases several-fold during intestinal trichinosis. It is presumed to be myeloperoxidase, a particulate-bound enzyme of myeloid-derived leukocytes that functions as part of a potent antimicrobial system in combination with H2O2 and a halide. Results indicated that the vermicidal component of LP cells was associated with the pellet fraction of disrupted centrifuged LP cells, but was not linked to a peroxidase-H2O2-halide system.


Acylation of phenol by cyclic and acyclic anhydrides in anhydrous acetic acid.

Acylation of phenol with succinic, glutaric, trans-1,2-cyclohexanedicarboxylic, maleic, phthalic, and cis-1,2-cyclohexanedicarboxylic anhydrides in anhydrous acetic acid generally resulted in phenyl acetate as the major product. The formation of phenyl acetate as the major reaction product could be rationalized as being due to the reactivity of the cyclic anhydrides with acetic acid to form acetic anhydride as well as the greater reactivity of phenol with formed acetic anhydride than with the cyclic anhydride.


Effects of choroid plexus peptide IIF on adenylate cyclase and 3',5'-cyclic adenosine monophosphate in adipose tissue.

Two hypophyseal lipolytic peptides, adrenocorticotropin (ACTH) and beta-melanocyte-stimulating hormone (beta-MSH), and the extrhypophyseal lipolytic peptide IIF, were compared with regard to their effects on free fatty acid production and 3',5'-cyclic adenosine monophosphate (cAMP) concentration in isolated rabbit and rat adipose tissue, and on adenylate cyclase activity in the tissue homogenates. ACTH at concentrations of 0.01 mug/ml or more increased lipolysis and cAMP levels in both tissues. beta-MSH at concentrations of 0.001 mug/ml or more increased lipolysis and cAMP in the rabbit tissue, but a concentration of 10 mug/ml did not stimulate lipolysis and did not alter nucleotide concentration in the rat tissue. Peptide IIF at 0.01 mug/ml or more stimulated lipolysis in rabbit adipose tissue and caused an accumulation of cAMP. A concentration of 100 mug/ml failed to stimulate free fatty acid production in the rat tissue and the cAMP level was also unaffected. In a medium containing 7.6 mEq/l of Mg++ and no Ca++, ACTH at 0.1 mug/ml or more stimulated adenylate cyclase activity in both rabbit and rat adipose homogenates by 6- to 12-fold. This effect was inhibited when Mg++ was replaced by Ca++, Na+ or K+. beta-MSH stimulated adenylate cyclase in rabbit, but not in rat, adipose homogenate in Mg++-containing incubation midium; again, the effect on rabbit adenylate cyclase was suppressed when Mg++ was replaced by Ca++, Na+ or K+. Peptide IIF failed to influence adenylate cyclase in the rabbit tissue homogenate in the Mg++-containing, Ca++-free medium; but when the medium contained 7.6 mEq/l of Ca++ in place of Mg++, 0.1 mug/ml or more of IIF caused a 4- to 15-fold increase in cyclase activity. IIF did not affect cyclase in the rat tissue homogenate in the presence or absence of Ca++. The data are consistent with the conclusion that extrahypophyseal lipolytic peptide IIF, as well as hypophyseal peptides ACTH and beta-MSH, accelerates lipolysis in susceptible adipocytes by stimulating adenylate cyclase to produce cAMP. The effect of IIF on cyclase requires the presence of exogenous Ca++; that of ACTH and beta-MSH requires exogenous Mg++.


Effect of albuterol and terbutaline, synthetic beta adrenergic stimulants, on the cyclic 3',5'-adenosine monophosphate system in smooth muscle.

The effect of albuterol and terbutaline on the cyclic 3',5'-adenosine monophosphate (cAMP) system was studied in rat uterus, aorta and myocardium and in dog bronchus, and was compared to that of isoproterenol in order to determine whether the tissue specificity observed in their functional effects is reflected in their effect on the cAMP system. Tissue specimens were either homogenized in Tris buffer for enzyme activity measurements or incubated in Krebs-Ringer-bicarbonate medium with the test drugs. Both albuterol and terbutaline produce an increase in cAMP content in the tissues due to a direct effect on adenylate cyclase. This effect can be potentiated by a phosphodiesterase inhibitor and antagonized by a beta adrenergic blocking compound. The cAMP response to each beta adrenergic agonist differs in the tissues examined: in uterus and aorta where the maximal effects are idenitcal, the ED50 values may reflect differences in affinity which may account for the different cAMP response to the compounds at the lower concentrations. In bronchus and myocardium, both the maximum effect and ED50 values of the compounds are different. Albuterol and terbutaline increases cAMP content in bronchus significantly and have only a small effect on cAMP cont in myocardium, whereas isoproterenol increases cAMP level significantly in both tissues. The results indicate that the tissue specificity of albuterol and terbutaline may have its origin at the level of the cAMP system.