Metabolism of benzo(a)pyrene by guinea pig pancreatic microsomes.

The in vitro microsomal metabolism of the strain 13 guinea pig pancreas was investigated by determining the benzo(a)pyrene (BP) hydroxylase activity in the 9000 x g supernatant and microsomal pellet. BP hydroxylase activity in both 9000 x g supernatant and microsomal pellet of the pancreas was less than 1% of the activity in the respective liver fractions, However, pretreatment of animals with methylcholanthrene or BP at 20 mg/kg, for either 1 day or 3 consecutive days, markedly enhanced the BP hydroxylase activity of pancreatic microsomes over that of controls; the induction in the liver microsomes was less than 2-fold over that of controls. The hydroxylation of BP by pancreatic microsomes was linear with time over a 30-min period, with the rate of hydroxylation dependent on both the enzyme and substrate concentrations.


A time factor in the success or failure of immune rejection of transplanted tumors.

Changes in susceptibility to immune rejection have been studied and compared during the initial stages of s.c. and pulmonary establishment of a transplanted syngeneic C3H mouse mammary carcinoma. The time of immunological attack on the implanted tumor cells was varied by two experimental procedures: In one experiment, the test mice that were immunologically suppressed by the presence of a large s.c. tumor implant were surgically cured before s.c. challenge. However, the immune recovery, which normally follows directly after tumor removal, was delayed for increasing lengths of time after challenge by injections of irradiated cells of the same tumor. In another experiment, the test mice were immunologically impaired by sublethal whole-body irradiation before s.c. and i.v. challenge. Immune rejection reactivity was then introduced, by passive transfers of lymph node cells from immunized mice, at increasing delays after the challenge implantations of tumor cells. In both of the two experiments, an increase in the number of tumor "takes" was observed if tumor immunity was reduced or absent for at least 3 days after challenge. If tumor immunity was restored or provided by the third day after challenge, there was an abrupt decrease in the number of observed tumors. The reduction in the effectiveness of immunosupportive treatments about the third day after tumor implantation may indicate a reduction in the vulnerability to immune rejection that coincides with vascularization of the implants.


The inhibition of thymidine kinase in glial tumor cells by an amino sugar, D-glucosamine.

Thymidine kinase activity in rat C6 glioma cells is inhibited by 50 to 70% after 4 hr incubation with 20 mM D-glucosamine. The inhibition is uncompetitive with respect to thymidine, reducing both the apparent Km and Vmax of the enzyme. The inhibition does not appear to be caused by the reversible combination of the enzyme with a cytoplasmic inhibitor, including D-glucosamine and its metabolites. The addition of D-glucosamine or its metabolites to cell-free thymidine kinase produced an inhibition which differed quantitatively and qualitatively from that which resulted from treatment of intact cells with D-glucosamine. The presence of a reversible cytoplasmic inhibitor of the enzyme was also excluded by mixing experiments. D-Glucosamine inhibited the incorporation of labeled uridine and amino acids into acid-precipitable material. The magnitude of inhibition of thymidine kinase activity and amino acid incorporation by D-glucosamine was comparable to that produced by cycloheximide, suggesting that the inhibition might arise from interference with enzyme synthesis. However, whereas the kinetics of recovery of amino acid incorporation from inhibition was rapid, thymidine kinase activity was depressed for at least 6 hr after drug washout. The results presented are best explained by assuming either that two forms of thymidine kinase are present in rat C6 cells and are differently affected by D-glucosamine or that D-glucosamine acts by two separate mechanisms to inhibit a single form of the enzyme.


Synchronization of hepatocellular DNA synthesis in regenerating rat liver by continuous infusion of hydroxyurea.

Hydroxyurea (HU) inhibited DNA synthesis in the livers of partially hepatectomized rats. After an i.v. infusion of HU begun in the late G1 phase and continued for periods of up to 30 hr, all hepatocytes scheduled to embark on DNA synthesis in a characteristic intralobular sequence during this time interval accumulated at the G1-S boundary. The effective dose was 1.25 mmoles/kg/hr, preceded by a single injection of 1.69 mmoles/kg. Serum levels of HU rose slightly during continuous infusion and decreased after termination, with a half-life of about 80 min. Liver weight increased during HU infusion. The G1-S blockade was rapidly reversed in the liver after the end of HU infusion. The specific activity of DNA increased to a maximum between 3 and 5 hr. [3H]Thymidine labeling indices reached about 80%. Intralobular distribution of labeled hepatocytes was congruent to the pattern seen in partially hepatectomized rats after a continuous [3H]thymidine infusion of equal duration. The beginning of DNA synthesis in nonparenchymal cells was delayed, as compared with hepatocytes. Vincristine infusion for 12 hr after release from the HU block arrested about 40% of the hepatocytes in mitosis, indicating that a large fraction of cells progressed through the cycle after the prolonged HU block. Partially resected rat liver appeared to be rather resistant to the unfavorable consequences of "unbalanced growth" during the protracted inhibition of DNA synthesis, providing a useful model for synchronization of DNA synthesis in a differentiated resting organ triggered into active growth.


Corynebacterium parvum and cyclophosphamide as combination treatment for a murine mammary adenocarcinoma.

Weekly i.p. injections of killed Corynebacterium parvum and of cyclophosphamide (given on different days) strongly inhibited growth of a transplantable murine mammary adenocarcinoma. A significant portion (40 to 80%) of animals could be made tumor free by means of combined therapy. No tumor-free survivors were obtained with C. parvum alone, and tumor-free mice were obtained with cyclophosphamide alone only at the expense of a high incidence of deaths due to drug toxicity. No evidence of tumor rejection immunity was detected in the tumor-free survivors from the combined treatment protocols, suggesting that this therapeutic regimen is not associated with tumor rejection immunity.


The inhibition of thymidine metabolism in tumor cells treated with D-glucosamine.

The amino sugar, D-glucosamine, inhibits the preformed route of thymidine metabolism in rat C6 glioma cells. This inhibition results from a concatenation of several distinct effects, including the inhibition of thymidine uptake, the reduction of thymidine phosphorylation, and an increased leakage of thymidine to the extracellular space. Each of these effects, while ostensibly small in magnitude, is significant and contributes to the observed inhibition of acid-precipitable thymidine incorporation by D-glucosamine. These effects of D-glucosamine, together with its known ability to reduce uridine nucleotide pools, may contribute to its toxicity toward certain experimental animal tumors.


Quantitation of fibrinolytic activity of Syrian hamster fibroblasts using 3H-labeled fibrinogen prepared by reductive alkylation.

Tritium-labeled fibrinogen with a specific activity of 2.0 X 10(7) cpm/mg was prepared by the method of reductive alkylation. The use of the 3H-fibrinogen as a substrate for detection of both intracellular and extracellular fibrinolytic activity derived from cultures of benzo(a)pyrene-transformed Syrian hamster cell lines was examined in cell-free assays, 3H-fibrinogen enabled reliable quantitation of the fibrinolytic activity associated with neoplastic cells. The elevated extracellular fibrinolytic activity in the transformed cell lines as compared to normal hamster embryo cultures was demonstrated with this substrate. The ease with which large quantities of 3H-fibrinogen of high specific activity and prolonged half-life can be prepared makes the use of this substrate an attractive alternative to 125I-labeled fibrinogen.


Radioimmune assay and characteristics of antibodies to macromomycin (NSC 170105).

A radioimmune assay for the antitumor agent, macromomycin, using purified, radioiodine-labeled macromomycin and antisera raised in rabbits against a carbodiimide-catalyzed macromycin-Limulus polyphemus hemocyanin complex has been developed. Radiolabeled macromomycin was prepared by direct iodination of the polypeptide antibiotic with the use of iodine monochloride or solid-state lactoperoxidase. Antibody-bound drug was isolated from free macromomycin with dextran-coated, activated charcoal. The standard curve of the sequential saturation assay was linear on a logit-log plot and indicated a lower limit of sensitivity of approximately 100 pg macromomycin. The radioimmune assay was suitable for measuring macromomycin in the presence of other antitumor drugs, and detection of macromomycin was quantitative when it was added to normal human serum or urine. Drug binding to melanoma and mammary carcinoma cell surfaces could be inhibited by preincubating macromomycin with affinity-purified antimacromomycin antibodies. However, once the drug was bound to cell surfaces, addition of antimacromomycin antibodies did not result in removal of the drug from cell surfaces or in reversal of macromomycin-induced inhibition of thymidine incorporation into cellular DNA. Antimacromovide useful tools for developing pharmacokinetic and toxicity studies of macromomycin, as well as for analyzing the mechanism(s) of action of the drug.


Metabolic activation of benzo(a)pyrene and binding to DNA in cultured human bronchus.

Human bronchus is one target site for the carcinogenic action of tobacco smoke, which contains chemical carcinogens, including benzo(a)pyrene. Human bronchi were obtained from surgery or "immediate" autopsy and then cultured in a chemically defined medium. The cultured bronchi were exposed to either benzo(a)pyrene or its metabolites, and their levels of binding to DNA were measured. One of the benzo(a)pyrene metabolites. (-)-trans-7,8-diol, is more active in binding to DNA than benzo(a)pyrene and several of its metabolites, including (-)-trans-4,5-diol, (-)-trans-9,10-diol, and phenols. The predominant metabolite formed by human bronchus from the (-)-trans-7,8-diol is found by high-pressure liquid chromatographic analysis to be the diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene. The results suggest that this diol-epoxide is the major benzo(a)pyrene metabolite bound to DNA in human bronchus.


Nucleic acid content and nuclear chromatin structure of human bladder cell culture lines as studied by flow cytofluorometry.

Two human bladder cell lines, T-24 and HCV-29, are studied with flow cytofluorometry and acridine orange staining to determine relative DNA and RNA content per cell and to measure resistance to thermal denaturation of DNA in situ. The RNA/DNA ratio for HCV-29 is over twofold higher than that for T-24, a difference that is consistent with the differences in cytological morphology and staining characteristics of these two cell lines and is sufficient to distinguish them completely, although measurements of DNA or RNA alone may not. In addition, the two cell lines show differences in DNA "melting" curves that indicate structural or conformational differences in nuclear chromatin. It is evident that the features are related to nuclear and cellular morphology, and they may be of value as additional parameters for characterizing tissue culture cell lines.


Prevention of renal failure in rats receiving cis-diamminedichloroplatinum(II) by administration of furosemide.

The clinicopathological signs of renal failure induced in rats by weekly i.v. administration of cis-diamminedichloroplatinum(II) were prevented by pretreatment with furosemide. Weight loss, anemia, and generalized toxic effects of the drug were not effected by furosemide.


Cytotoxicity of 5,6-dihydroxytryptamine in dimethylhydrazine-induced carcinomas of rat colon.

Male Sprague-Dawley rats were given weekly s.c. injections of 1,2-dimethylhydrazine (21 mg/kg) for 20 weeks. The injections were then discontinued, and, after an interval of 2 to 8 weeks, experimental animals were given i.p. injections of 5,6-dihydroxytryptamine (5,6DHT) at a dose of 40 mg/kg and sacrificed at intervals of 1, 2, 6, 16, and 48 hr later. Specimens of descending colon and carcinomas of the descending or transverse colon from sacrificed animals were examined using light microscopy and transmission electron microscopy. The results show that 5,6DHT at a dose of 40 mg/kg is cytotoxic to malignant colonic epithelial cells but not cytotoxic to adjacent nonmalignant colonic epithelial cells. In malignant colonic epithelial cells, ultrastructural changes in cytoplasmic membranes and mitochondria were evident at 1 hr after 5,6DHT treatment. At 6 hr after 5,6DHT treatment, light microscopy of sections of tumor showed areas of cell necrosis and disrupted tumor morphology. Sections of specimens taken 16 hr after treatment showed widespread destruction of malignant cells.


Epidermal hyperplasia after topical application of benzo (a) pyrene, benzo (a) pyrene diol epoxides, and other metabolites.

The effects of benzo(a)pyrene (BP) and 22 derivatives upon the number of nuclei per unit length of epidermis, the number of cell layers of epidermis, and the thickness of the epidermal layer were studied. Several derivatives of BP induced changes in epidermal morphology that are typical of those produced by various agents that promote skin tumorigenesis after application of an initiator. The most potent compounds tested were the BP diol epoxides, (+/-)-7beta,8alpha-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydrobenzo-(a)pyrene (diol epoxide 1) and (+/-)-7beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol epoxide 2). These derivatives were followed in activity by 9-hydroxybenzo(a)pyrene, 2-hydroxybenzo(a)pyrene, and by 9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. The possible implications of these results with regard to the carcinogenicity of BP on mouse skin are discussed.


Oxidation of a branched-chain alditol by acetobacter suboxydans: a stereospecific synthesis of L-dendroketose.

A synthesis of L-dendroketose (5) has been achieved by microbiological oxidation by Acetobacter suboxydans of the branched-chain alditol 2-C-(hydroxy-methyl)-D-erythro-pentitol (4). Treatment of the oxidation product with acetone, copper(II) sulfate, and sulfuric acid afforded the two di-O-isopropylidene-L-dendro-ketose derivatives 6 and 7. Assignment of configuration at the branching carbon atom (C-4) and at the anomeric center in 6 and 7 was made on the basis of the carbon-13 magnetic resonance spectra of these derivatives.


Facile synthesis of 2-methyl-[4,6-di-O-acetyl-1,2-dideoxy-3-O-(2,3,4,6-tetra-O-acetyl-D-glycopyranosyl)-alpha-D-glucopyrano-[2',1':4,5]-2-oxazolines, key intermediates for the synthesis of oligosaccharides.

A simple synthesis of disaccharide oxazolines has been developed. Condensation of methyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-glucopyranoside with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide, followed by removal of the 4,6-O-benzylidene group from the resulting disaccharide derivative, gave crystalline methyl 2-acetamido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-alpha-D-glucpyranoside which, on acetolysis with acetic anhydride-acetic acid-sulfuric acid, provided 2-methyl-[4,6-di-O-acetyl-1,2-dideoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-alpha-D-glucopyrano]-[2',1':4,5]-2-oxazoline (7). Synthesis of the related alpha-D-mannopyranosyl compound was similarly accomplished. The glycosylating capability of 7 was employed for the synthesis of 6-(benzyloxycarbonylamino)hexyl-2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside (18). An alternative synthesis of compound 18 is also described.


The synthesis of allyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside and of chitobiose derivatives by the oxazoline procedure.

Controlled, partial benzylation of allyl 2-acetamido-3-O-benzyl-2-deoxy-alpha-D-glucopyranoside gave a mixture of the 3,4-di-, 3,6-di- (15), and 3,4,6-tri-O-benzyl derivatives, the major product being 15. Condensation of 15 with 2-methyl-(3,4,6-tri-O-acetul-1,2-dideoxy-alpha-D-glucopyrano)-[2,1-d]-2-oxazoline gave a disaccharide which, after purification, removal of the allyl group, and hydrogenolysis of the benzyl substituents, gave 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-i-deoxy-beta-D-glucopyranosyl)-2-deoxy-alpha-D-glucopyranose. This compound was further converted into di-N-acetyl-hexa-O-acetylchitobiose by acetylation, or into 2-methyl-[4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-3,6-di-O-acetyl-1,2-di-deoxy-alpha-D-glucopyrano]-[2,1-d]-2-oxazoline, a starting material for the preparation of di-N-acetyl-alpha-chitobiosyl phsophate.


The morphology of the oval nuclei of neonatal Torpedo marmorata.

The morphology of the oval nucleus of neonatal Torpedo marmorata is described at the light and electron microscopic level of examination. The nucleus is unique relative to other central electromotor centers of electric fish so far described being bilaterally symmetrical, composed of two nerve cell types, and possessing no gap junctions between neurons and their processes. This particular structural plan presents difficulties in accounting for presumed synchronous discharge since it has been strongly argued that electrotonic coupling by means of gap junctions is the primary process by which synchronization is accomplished. Close membrane apposition and dendritic bundling, common features within the nucleus, are discussed as possible alternative structural correlates.


Electron microscopy of human melanosomes in unstained, fresh air-dried hair bulbs and their examination by electron probe microanalysis.

Electron microscopy of unstained, fresh air-dried spreads of plucked human hair bulbs revealed relatively well preserved melanocytes and dense storage granules of melanin. Energy dispersive X-ray microanalysis of melanosomes and premelanosomes from black facial hair bulbs disclosed high peaks for potassium and sulfur, intermediate peaks for magnesium and calcium, and low but distinct ones for phosphorus and chlorine. No peaks for magnesium were found in the central portion of mature melanosomes, but at their edges and in premelanosomes conspicuous peaks for this element were obtained.


Fine structure of seminiferous tubules in antarctic seals.

The fine structure of seminiferous tubules from 5 crabeater, 2 leopard and 2 Ross seals showed that during the nonbreeding season the tubules were essentially similar in possessing spermatogenic and Sertoli cells. However, the tubules of leopard and Ross seals had more primary and secondary spermatocytes and spermatids than the crabeater seals. In general, the tubules were devoid of spermatozoa. The spermatids showed stages of maturation such as Golgi phase of acrosome formation, acrosomal cap formation and condensation of nuclei. Some spermatids degenerated in tubules. Both maturing and degenerating spermatids were closely associated with Sertoli cells. Junctional complexes with plaques of filaments were observed between Sertoli cells and the spermatogenic cells. Sertoli cells, irregular and polygonal, contained highly convoluted nuclei, strands of rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi complexes, small mitochondria, variable amounts of lipid droplets, lysosomes, lipofuscin granules and highly plicated plasma membranes. In brief, the spermatogenic activity had practically ceased in the testes and the animals probably secreted low levels of testosterone during the nonbreeding season.


Neursecretory fibres in the median eyes of the scorpion, Androctonus australis L.

The retina of the median eyes of the North African scorpion, Androctonus australis L., is supplied with numerous neurosecretory nerve fibres which establish synaptoid contacts on the retinula cells. The number of fibres or profiles of varicosities of fibre terminals associated with a retinular unit (five retinula cells with a fused rhabdom) varies between 10 and 20. Electron-opaque vesicles with a diameter of 80-100 nm are abundant within the axonal profiles. The synaptoid junctions are characterized by postsynaptic electron-dense material on the inner leaflet of the retinula cell membrane and, frequently, presynaptic submembranous dense material. Because of these ultrastructural features, the junctions observed here resemble typical interneuronal synaptic contacts. Hence this kind of neurosecretory junction appears to be unique among arthropods. It is suggested that the neurosecretory fibres within the retina represent the efferent pathways for the control of the circadian pigment movements within the retinula cells.


Scanning electron microscopy of rat maturation ameloblasts.

Post-secretory, maturation-phase ameloblasts were studied by scanning electron microscopy of freeze-fractured or dry-dissected rat incisors. These cells are in contact with the enamel which they secreted at an earlier time and which undergoes a process of continuing mineralization. The lateral intercellular compartment between maturation ameloblasts is sometimes continuous with the intercellular space of the papillary layer of the enamel organ, but often closed by basal ring contacts which correspond to terminal bars seen in transmission electron microscopy. The distal poles of the cells sometimes possess striated borders. Lateral cell surfaces may show longitudinal gutter-like depressions between ridges from which numerous intercellular connections arise; or a maze of lateral folds and ridges; or they may have mostly microvillous surface projections bordering a minimal intercellular space compartment. Preliminary correlations of groupings of basal, lateral and distal cell features indicate that "basal-closed plus distal striated border" cells may show every type of lateral surface. Cell without a striated border, whether open or closed basally, have ridge or maze lateral surfaces bordering a wide intercellular compartment. "Basal-open plus striated border" cells have microvillous or maze-like surfaces. These combinations of features are encountered a few times along the length of the maturation zone of individual incisors and suggest tha existence of cyclical changes in the type of activity of maturation ameloblasts.


Ultrastructure of lymphatic capillaries in the human dental pulp.

Occlusal intradentinal cavities, prepared in normal human premolars and third molars to be extracted for orthodontic reasons, were filled for 7 to 11 days with gutta percha. A superficial pulpitis with localized small abscesses developed in the pulp chamber. Under local anesthesia, 0.2 to 0.3 cc of sterile colloidal carbon was injected in the pulp horn and the teeth were extracted 1 to 3 h later. Lymphatic capillaries could thus be identified in the pulpal tissues. They were characterized by a thin endothelium with occasional large intercellular clefts, absence or incompleteness of basement membrane, absence of pericytes, absence of luminal red blood cells, and presence of a filamentous material between the endothelium and the surrounding collagen fibrils. Moreover, some structural variations were observed.


Cytophotometric studies on the reaction kinetics of acid phosphatase in motoneurons marked by injection of Evans blue into the soleus muscle of the mouse.

Evans blue was injected into the soleus muscle of albino mice in order to mark retrogradely the corresponding motoneurons of the spinal cord. Subsequently, reaction kinetics of acid phosphatase were studied in the marked nerve cells. 41-51 motoneurons per animal were counted. They are located in the dorsolateral portion of the Rexed zone IX where they form motor cell columns approximately 1.5 mm in length. After identification of the motoneurons, the enzyme reaction for acid phosphatase was performed by covering the section with a gel film containing naphthol-AS-BI-phosphate-hexazonium-pararosanilin. The formation of the azo dye was measured cytophotometrically at 520 nm. During the first 10 min of registration, a linear decrease in transmission of 0.4 per cent/min was shown. Because of the unimodal distribution of changes in transmission, the motoneurons of the soleus muscle could not be characterized as fast or slow types on the basis of the reaction kinetics of acid phosphatase.


The influence of phenylthiourea (PTU) on the pigment epithelium and the photoreceptor cells in the developing retina of Haplochromis burtoni (Cichlidae, Teleostei).

The effect of phenylthiourea (PTU) on the pigment epithelium and the photoreceptor cells in the developing retina of Haplochromis burtoni was studied by electron microscopy. In the retinal pigment epithelium of 6-day old embryos, both types of melanin granule (spindle-shaped and rod-shaped) are already found. PTU inhibits the biosynthesis of melanin but does not influence the formation of premelanosomes so that in PTU-treated embryos there are no melanosomes, but an abundance of premelanosomes. The structure of the premelanosomes is described. It differs completely from that of all other vertebrates. Other changes: an increase in polysomes, retarded development of the inner segment of the photoreceptor cells and enlargement of the intercellular space between the inner and outer leaflet of the retina, may be due to a toxic effect of PTU.


Light and electron microscopic study on the pineal complex of the coelacanth, Latimeria chalumnae Smith.

The pineal complex of the coelacanth, Latimeria chalumnae was studied light and electron microscopically. It consists of two vesicles representing parapineal and pineal organs. Both occur intracranially and openly communicate with each other and the brain ventricle. The entire complex shows a striking photoreceptor morphology with sensory, ependymal and nerve cells. The last cell type is more abundant in the parapineal vesicle than in the pineal organ. The following ultrastructural details of the parapineal are noteworthy: 1. The sensory cells possess large inner and outer segments protruding freely in the vesicular cavity. The outer segments measure 8-10 mum in length and consist of as many as 275 lamellae. The basal processes of these cells terminate in neuropil-like regions. Occasionally, dense granules (500-1000 A) of uncertain identity occur in the perinuclear and inner segment cytoplasm of the cells. 2. The supporting cells are of the ependymal type. Their cytoplasm contains a filamentous feltwork and pinocytotic vesicles, but lacks secretory granules. Cytosomes are particularly abundant in cell processes in the neuropil-like zones. The basal end-feet of these cells isolate the receptor and nerve cells from the perivascular space. 3. In the neuropil-like regions, terminals of sensory cells make synaptic contacts with neuronal dendrites. Synaptic ribbon-like profiles in the terminals characterize the contact zones. Only unmyelinated nerve fibers could be observed in the small area of the tissue examined. The results are discussed with regard to photoreceptive and secretory functions of the pineal complex and its evolution in lower vertebrates.


The use of ionic lanthanum as a diffusion tracer and as a marker of calcium binding sites.

The distribution of ionic lanthanum (La3+) was studied after perfusion of the isolated rat heart with a buffered salt solution containing lanthanum chloride. We have demonstrated that the localization of lanthanum differed from that shown in previous studies in which hearts were exposed to ionic or colloidal lanthanum during or after fixation. La3+ passed rapidly through the intercellular cleft between endothelial cells. There was relatively little transport by micropinocytosis and no evidence of transendothelial passage through the cytoplasm. Perfusion with a solution containing 1 to 15 mM LaCl3 resulted in localization of La3+ on the entire cytoplasmic membrane system of muscle cells, i.e.: the non-specialized parts of the muscle cell membranes were stained by La3+ as well as the membranes of micropinocytotic vesicles, of the T-system and of the intercalated disc. The cytoplasmic leaflets of gap junctions were especially stained. However, the cell membranes of non-muscle cells were free of lanthanum precipitates. Freeze-fracturing of muscle cells showed concomitant alteration in the distribution of membrane-associated particles. The gap junction membranes also showed changes in particle aggregation. Intracellular binding of La3+ are restricted to muscle cells. Lanthanum was found in the cell nucleus associated with heterochromatin, in the nucleolus, and along the nuclear membrane. The myofibrils were stained mainly over the I-band.


The effects of different methods of fixation on central nervous system synaptic pinocytotic vesicles.

Synaptic pinocytotic vesicles (invaginating from the surface membrane) and coated vesicles inside rat mossy fiber endings were counted after the use of different kinds of fixatives. Significantly greater numbers of pinocytotic vesicles and coated pinocytotic vesicles per unit length of membrane were found when osmium was used as the first fixative. A high positive correlation was found between these values and the number of coated vesicles per unit area of mossy fiber ending profiles. The results emphasize the need for caution when considering the theory that in vivo synaptic vesicle recycling involves a coated vesicle invagination of the surface membrane followed by internalisation and loss of coat of the vesicle.


The uptake of horseradish peroxidase by cortical synapses in rat brain. An in vivo study.

Horseradish peroxidase (HRP) was introduced directly into the cerebral cortex of adult rats, which were allowed to survive for 60 min before perfusion fixation. After the tissue had been incubated to demonstrate HRP at the LM and EM levels, blocks of cortical tissue were taken at varying distances from the injection site. These eight blocks of tissue constituted a time sequence for HRP diffusion. Qualitative examination of the presynaptic terminals showed that the most commonly encountered profiles are the plain synaptic vesicles, many of which accumulate tracer. In some terminals labelled vesicles are "lined-up" in tubular fashion. Other profiles commonly labelled are coated vesicles, tubular and vacuolar cisternae, and plain and coated pinocytotic vesicles. Quantitative analyses based on the number of terminals containing labelled profiles demonstrate an early rise in the rate of labelling of both plain synaptic vesicles and coated vesicles, after which synaptic vesicle labelling rises slowly towards a plateau. By contrast, there is a late parallel increase in the rate of labelling of coated vesicles and cisternae. A more detailed analysis, based on the actual numbers of labelled and total profiles within each presynaptic terminal, highlight early and late periods of rapid labelling for plain synaptic vesicles, coated vesicles and cisternae. A further aspect of HRP incorporation studied, concerns its uptake into four delineated regions of the presynaptic terminal. Our data indicate that membrane uptake into the presynaptic terminal is accomplished mainly via coated vesicles, although plain synaptic vesicles may also be involved. Coated vesicles, in turn, appear to give rise directly to plain synaptic vesicles, with some coalescing to produce vacuolar cisternae. The latter are involved in a two-way interchange of membrane with tubular cisternae, plain synaptic vesicles and coated vesicles. An additional source of plain synaptic vesicles are the tubular cisternae. Exocytosis of plain synaptic vesicles constitutes the mechanism by which transmitter is released from the presynaptic terminal.


Surface morphology of taste buds in catfish barbels.

External taste buds abound on barbels of the adult catfish Corydoras arcuatus. When examined by scanning electron microscopy, they are visualized as a series of punctate, conical elevations projecting from the general surface epithelium. All taste buds were found to be of one type. Both their external and internal surface features could be clearly elucidated on intact barbels and in barbels fractured transversely at various positions along their length. An extensive nerve terminal network penetrates the base of each taste bud. Two populations of elongated cells bearing prominent microvilli project through the central pore at the tip of each bud. One set of microvilli is thicker, longer and more club-shaped than its counterpart. While both are randomly distributed within each central pore, the small, short microvilli appear to outnumber the larger ones. A third population of cells, devoid of any apical microvilli, was also seen in some of the taste buds examined internally. These cells do not project to the external surface and are interpreted as "basal" cells described in previous light and transmission electron microscope studies of taste buds in other vertebrate species. The functional significance of some of these morphological findings is discussed.


Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey.

Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface. The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45 degrees. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain "intraluminal vesicles" and are continuous with apical ends of cytoplasmic tubules.


Fine structural studies on the thyroid gland of the normal domestic fowl.

The ultrastructure of the thyroid gland of the domestic fowl has been investigated and found to be similar to that of mammals. The differences were found at subcellular level in the distribution of the "dark bodies" which were mainly apical and in the sizes of primary lysosomes. These were found to range from 100 to 500 nm in diameter. All organelles described in mammals as being concerned with the production of thyroglobulin and the two hormones thyroxine and triiodothyronine were found to be present.


A possible role for placental lysosomes in the formation of villous syncytiotrophoblast.

An ultrastructural and ultrahistochemical study of first trimester human placentae confirms previous reports that the cytotrophoblastic cells show a spectrum of differentiation, that dissolution of the limiting membrane of the cytotrophoblastic cells occurs and that fragments of free membrane can be found in the syncytiotrophoblast. There is an aggregation of primary lysosomes in the region of approximation of the cytotrophoblast to the syncytiotrophoblast, free lysosomal enzymes are found in the space between the two trophoblastic components, secondary lysosomes have been noted in the vicinity of fragmenting cytotrophoblastic cell membrane and the incorporation of a segment of free membrane into a vesicular structure has been noted. It is suggested that placental lysosomes mediate the dissolution of the cytotrophoblastic cell membranes that is a necessary prerequisite for their full differentiation into syncytiotrophoblast and it is further suggested that one of the principal roles of placental lysosomes is in the structural refashioning of the organ that occurs during the first trimester.


Kinetics of biosynthesis of acetylcholine receptor and subsequent incorporation into plasma membrane of cultured chick skeletal muscle.

20% of the acetylcholine receptors in cultured chick skeletal muscle remain unbound following long-term growth of muscle in medium containing a potent, essentially irreversible receptor-blocking agent, alpha-bungarotoxin. About half the receptors which are unavailable for interaction with extracellular alpha-bungarotoxin are newly synthesized molecules which presumably are being processed and transported to the plasma membrane. When the muscle cultures are switched to a medium containing 2H, 13C, 15N-amino acids, these receptors are rapidly labeled, the fraction of labeled molecules beginning to plateau at 3 hr. Few labeled receptors appear in the plasma membrane during the first 3 hr of labeling with 2H, 13C, 15N-amino acids. After 3.5 hr of labeling, virtually all the receptors being incorporated into the plasma membrane are labeled receptors. The kinetics of labeling of the "pool" and "surface" receptors with 2H, 13C, 15N-amino acids confirm the "precursor-product" type relationship of pool and surface acetylcholine receptors. In this study, receptors synthesized in medium containing 2H, 13C, 15N-amino acids were resolved from 1H, 12C, 14N-receptors by velocity sedimentation in sucrose-deuterium oxide and sucrose-H2O gradients, and their densities were estimated from sedimentation rates in shallow gradients of various average density. Estimated densities were 1.32 g/cm3 for 1H, 12C, 14N-receptors and 1.41 g/cm3 for 2H, 13C, 15N-receptors. This density difference corresponds to 80% substitution of normal aminoacyl residues by 2H, 13C, 15N-residues in the denser receptor.


Regression of mouse mammary gland anlagen in recombinants of Tfm and wild-type tissues: testosterone acts via the mesenchyme.

In the male mouse, regression of the mammary gland anlagen is induced by testosterone during embryonic life. In the androgen-insensitive Tfm mouse, the gland anlagen are resistant to the testosterone action. To analyze cellular interactions in this process, we isolated the mammary gland anlagen from Tfm- and wild-type embryos. The epithelial buds were separated from the mesencyme by trypsin-pacreatin treatment. From the epithelial and mesenchymal components, reciprocal recombinations were prepared and cultivated on millipore filter in the presence of testosterone. In combination with androgen-insensitive Tfm- mesenchyme, the wild-type buds survived the action of testosterone. On the other hand, in combination with wild-type mesenchyme, the androgen-insensitive Tfm epithelial buds were destroyed. The results show that testosterone induces detachment and degeneration of the buds via the mesenchyme.


Transcription of specific genes in isolated nuclei by exogenous RNA polymerases.

Mouse plasmacytoma (MOPC) 460 cells contain two chromatographic forms of RNA polymerase III (IIIA and IIIB) in addition to the major class I and II RNA polymerases. Nuclei isolated from these cells actively synthesize RNA. Among the discrete transcription products observed are the 5S and 4.5S RNAs and additional low molecular weight RNA species (approximately 5.8S, 6.3S, and 6.6S in size). The 4.5S RNAs appear to be tRNA precursors since they can be converted in vitro to 4S RNAs. Studies with alpha-amanitin have shown that the synthesis of these discrete RNA species, and other uncharacterized transcripts somewhat larger in size, is mediated by an endogenous RNA polymerase III activity(ies). Nuclear RNA synthesis is stimulated by exogenous purified RNA polymerases. Exogenous MOPC class III RNA polymerases stimulate the synthesis of each of the distinct low molecular weight species (including 5S and 4.5S RNAs) about 3-6 fold. The hybridization of nuclear transcripts to purified 5S genes (5S DNA) confirms that exogenous class III RNA polymerases stimulate (approximately 4 fold) the synthesis of ribosomal 5S RNA. The 5S RNA genes in nuclei are transcribed asymmetrically by both the endogenous and the exogenous class III enzymes. Exogenous RNA polymerase III from Xenopus laevis ovaries stimulates 4.5S and 5S RNA synthesis in MOPC nuclei as effectively as do the MOPC class III RNA polymerases. However, exogenous MOPC class I and II RNA polymerases do not stimulate 4.5S and 5S RNA synthesis, suggesting that this effect is specific for the structurally similar class III RNA polymerases.


Synthesis and turnover of polysomal mRNAs in sea urchin embryos.

The synthesis and turnover kinetics of polysomal mRNA have been measured in sea urchin embryos. Polysomes were isolated from stages ranging between mesenchyme blastula and late gastrula Strongylocentrotus purpuratus embryos which had been exposed to exogenous 3H-guanosine. The amount of radioactivity incorporated into messenger and ribosomal RNAs was determined separately as a function of time, and the precursor pool specific activity was measured in the same embryos. Synthesis and decay rate constants were extracted from the data by a least-squares procedure. Per embryo, the rate of mRNA synthesis was calculated to be about 0.13 pg min-1, while the rate of rRNA synthesis is about 0.022 pg min-1. The newly synthesized mRNA turns over with a half-time of 5.7 hr. The data support only a single decay rate for the mRNA, but small fractions of mRNA decaying at different rates cannot be excluded. Previous studies have shown that a minor fraction of the mRNA includes the least abundant, most highly diverse set of messages ("complex class" mRNAs). To determine whether mRNAs of the complex class are synthesized and degraded at similar rates, labeled mRNA was measured in hybrids formed in mRNA excess reactions with single copy DNA. These experiments showed that complex class mRNAs represent an approximately proportional amount of the new mRNA symthesis, and turn over at the same average rate as does the bulk of the mRNA. Most of the mRNAs in the embryo polysomes are newly synthesized, rather than maternal. This statement refers both to complex class mRNAs and to prevalent mRNAs. Considering the sequence homology between embryo and oocyte mRNAs shown earlier, these results indicate that many of the same structural genes active during oogenesis are being transcribed in embryos at these stages.


The distribution of poly(A)+ and poly(A)- protamine messenger RNA sequences in the developing trout testis.

Protamine messenger RNA was isolated in a very pure form from trout testes and used as a template for the synthesis of labeled complementary DNA (cDNA) of high specifiv activity. The cDNA was found to be a full-length transcript of protamine messenger RNA and was used as a probe for hybridization reactions with RNA preparations isolated from three subcellular compartments of differentiating trout testis cells. The RNA populations from the nuclei, polysomes, and postribosomal supernatant of these cells were fractionated into poly(A)-containing [poly(A)+] and poly(A)-free [poly(A)-] RNA to determine the distribution of these two forms of protamine mRNA in these cell compartments. At the early protamine stage of testis development, polysomal and postribosomal supernatant fractions contain almost equal quantities of poly(A)+ protamine mRNA, but poly(A)- protamine mRNA was found almost entirely in the polysomes.


A heteroduplex study of the sequence relationships between the RNAs of M-MSV and M-MLV.

The regions of sequence homology between the RNA genomes of a murine sarcoma virus (clone 124 Moloney-MSV) and its parental helper virus (clone 3 Moloney-murine leukemia virus (M-MLV)) have been mapped. Long complementary DNA transcripts of the M-MLV RNA were hybridized to M-MSV RNA, and the structures of the hybrids were observed in the electron microscope. Beginning at the 5' end, the two RNAs are homologous for a region of length 2.25 kb (kilobases). In the next region, of length approximately 4.2 kb on the MLV genome, there are several homology segments between MLV and MSV, but there are also several short sequences present on MLV and deleted in MSV. There is then a major substitution loop; with a sequence (beta L) of length 2.9 kb present on MLV and missing on MSV, and a sequence (beta S) of length 1.5 kb present on MSV and missing on MLV. At the 3' end, there is a homology sequence of length 0.8 kb. On the basis of these results, other data on genes expressed in M-MSV-transformed cells, and by analogy with the avian gene map, we suggest that the gag genes (internal structural proteins) lie in the 2.25 kb region of homology near the 5' ends of M-MSV and M-MLV RNAs, and that the beta S segment contains a sarcoma (src) gene. Some of the heteroduplexes and some of the MLV cDNA/MLV RNA homoduplexes are circular, thus showing that cDNA transcription is initiated at an internal position in the RNA, proceeds to the 5' end, and them "jumps" to the 3' end.


Phosphorylation of murine type C viral p12 proteins regulates their extent of binding to the homologous viral RNA.

The purified p12 phosphoprotein of Rauscher murine leukemia virus was fractionated by ion exchange chromatography into subpopulations of molecules containing different amounts of covalently linked phosphate. Of the various phosphorylated forms of p12 protein purified from virions, only a species containing relatively little phosphate can bind in vitro to purified homologous 70S viral RNA. Using ultraviolet irradiation to stabilize ribonucleoprotein complexes in intact virions, the same molecular species of p12 phosphoprotein can be isolated in close association with the 70S viral genome. The results show that phosphorylation of type C viral p12 proteins influences the extent, but not the specificity, of their interaction with homologous viral RNA.


Hemodynamic effects of rapid atrial stimulation in adult and young dogs.

We evaluated the use of rapid atrial stimulation (RAS) to produce 2:1 atrioventricular (AV) block as a method for treating supraventricular tachycardias. We studied the cardiovascular hemodynamic changes before and after block in eight adult dogs and nine young dogs (3-4 1/2 weeks old). In each dog an electrocardiogram; cardiac index; aortic, pulmonary, and left ventricular (LV) systolic and end-diastolic pressures; and LV (dp/dt) p were recorded simultaneously and pulmonary ans systemic resistances were calculated. Measurements were obtained during (1) the control state, (2) RAS with maximal 1:1 AV conduction, (3) RAS with 2:1 AV block, and (4) RAS with 1:1 AV conduction at 50% of the rate which resulted in 2:1 AV block. Comparison of the hemodynamic effect of RAS with maximal 1:1 AV conduction and hemodynamics in the control state showed that there was a significant decrease in cardiac index and aortic mean pressure of 39% and 15%, respectively. When hemodynamic changes during RAS with 2:1 AV block were compared with those during RAS with maximal 1:1 AV conduction, cardiac index, LV systolic pressure, and aortic mean pressure increased by 52%, 17%, and 22%, respectively. LV (dp/dt) p increased by 20% and was significantly higher during RAS with 2:1 AV block than at higher ventricular rates obtained with RAS and maximal 1:1 AV conduction. We found that in this acute study the hemodynamic findings for the young dogs were similar to those for adult dogs. The data suggest that (1) significant hemodynamic improvement is obtained by RAS causing 2:1 AV block when compared to higher ventricular rates associated with 1:1 AV conduction; (2) in addition to increasing the diastolic filling period, reducing the very high ventricular rates improves the cardiac contractile state, and (3) RAS and 2:1 AV block may provide a useful technique for the management of some patients with intractable supraventricular tachycardia.


Effect of physical training on calcium transport by rat cardiac sarcoplasmic reticulum.

Cardiac microsomes enriched in fragmented sarcoplasmic reticulum (SR) were isolated from hearts of physically trained rats and were compared to those from sedentary rats. Preparations from conditioned rats were found to transport calcium to a greater extent than those from sedentary rats both in the absence and in the presence of 1 mM oxalate. Higher oxalate concentrations abolished the differences in calcium accumulation by cardiac microsomes from conditioned and sedentary rats and indicated a qualitative change in SR from conditioned hearts. We conclude that the increased transport by SR from hearts of conditioned rats may provide one mechanism for enhanced contractile reserve in these hearts.


Steady state aldosterone dose-response relationships.

The steady state effects of different infusion rates of aldosterone on plasma concentrations of sodium and potassium, plasma renin activity, sodium space, and mean arterial pressure were determined. Measured amounts of aldosterone were infused continuously into adrenalectomized dogs for 13 weeks. Four rates of aldosterone administration were used: 16 +/- 1 mug/day, 48 +/- 3 mug/day (approximately the normal secretory rate for 22-kg dogs on a daily sodium intake of 27 mEq, potassium intake of 27 mEq), 91 +/- 4 mug/day, and 219 +/- 10 mug/day. Each rate of infusion was continued until the dogs were in sodium and potassium balance and measured variables were steady. Decreasing the rate of aldosterone administration below normal led to sharp decreases in plasma sodium concentration and sodium space, while raising the rate above normal had little effect. Plasma potassium concentration varied inversely and significantly with changes in aldosterone administration over the entire range of rates. Plasma renin activity rose extremely rapidly as the level of aldosterone fell below normal and went to zero at aldosterone infusion rates slightly above normal. Arterial pressure increased as aldosterone rose above normal but did not fall below normal at subnormal aldosterone levels, probably because of the pressor effects of simultaneously generated angiotensin II.


Chronic one-kidney hypertension in rabbits. III. Renopressin, a new hypertensive substance.

The blood pressure of rabbits with chronic one-kidney hypertension can be lowered to normal by immunization with hog kidney cortex preparations that do not contain renin, thus providing evidence for a new factor essential for the maintenance of an elevated blood pressure. A search for the new factor has led to the discovery of a hypertensive substance which we have named renopressin. Subcutaneous injection of the new substance into normal rabbits produces a delayed, slow increase in blood pressure, and after a few days the development of a moderate hypertension which persists indefinitely. The response of the blood pressure to renopressin is totally unlike that to renin. While the pressor action of renin can be blocked by an angiotensin II antagonist, the hypertension caused by renopressin cannot. Renin can increase the blood pressure of hypertensive rabbits; renopressin has no effect. The blood pressure of hypertensive rabbits and of normal rabbits made hypertensive by injection of renopressin can be lowered to normal by passive immunization with the same antibody preparation. The evidence is consistent with the possibility that renopressin and the agent causing the elevation of blood pressure in hypertensive rabbits are similar or identical.


Change in liver blood flow and blood content in dogs during direct and reflex alteration of hepatic sympathetic nerve activity.

A mean decrease of 60% in liver blood volume was recorded by a plethysmographic technique during electrical stimulation of the hepatic nerves in anesthetized, vagotomized dogs. A decrease in pressure in the vascularly isolated carotid sinus to 40 mm Hg, from a mean control of 144 mm Hg, decreased liver blood volume by a mean of 16%; arterial blood pressure increased by a mean of 77 mm Hg. Carotid sinus hypotension was accompanied by respective mean increases of 16% and 1.4% in hepatic arterial and portal venous blood flows, and of 45% and 22% in arterial and portal resistances. Increase in sinus pressure to 240 mm Hg increased liver blood volume by a mean of 20%; arterial blood pressure decreased by 90 mm Hg. Sinus hypertension was accompanied by respective mean decreased of 10% and 1.5% in hepatic arterial and portal venous blood flows, and of 44% and 18% in arterial and portal resistances. Interruption of afferent vagal traffic from cardiopulmonary receptors was maximally effective in decreasing liver blood volume at a carotid sinus pressure of 40 mm Hg and was ineffective at carotid sinus pressures greater than 160 mm Hg. Combined withdrawal of  carotid and cardiopulmonary vasomotor inhibition decreased liver blood volume by 42%; of this 37% was due to the cardiopulmonary and 63% to the carotid baroreflex. The study showed the canine liver to function as a blood reservoir by active mobilization of a portion of its blood volume.


Regional myocardial function and dimensions early and late after myocardial infarction in the unanesthetized dog.

Pairs of ultrasonic dimension gauges and a micromanometer implanted in the subendocardium of the left ventricles of unanesthetized dogs were used to analyze serial changes in hemodynamic status and segmental function for up to 4 weeks after permanent circumflex coronary artery occlusion. Regional function was studied in control segments and in segments identified as marginal (hypokinetic) and ischemic. In three dogs, after transient regional dysfunction, no myocardial infarction developed, whereas in five dogs regional dysfunction at 3 hours after occlusion was followed by the development of persistent dysfunction and infarction. Left ventricular end-diastolic segment length (EDL) changes over time; EDL of the control segments increased progressively, but in marginal segments EDL was 12% below control and in the ischemic segments 30% below control by 4 weeks. Progressive increases in percent active shortening occurred in control segments; but holosystolic bulging was replaced by akinesia in ischemic segments, and persistent reduction in shortening was present in marginal segments at 4 weeks. Correlations were found between percent scar and reductions in percent shortening, EDL, and the ratio of change in diastolic length to change in diastolic pressure. These methods have detected hyperfunction in normal regions and variable segmental loss of contractile function, together with reduction of subendocardial dimensions and changes that may reflect decreased diastolic compliance in ischemic regions. We conclude that this model for the conscious animals may be useful for studying the influence of therapy on the extent of myocardial damage after experimental coronary occlusion.


Cardiac output and renal blood flow in glycerol-induced acute renal failure in the rat.

Cardiac output (CO) and renal blood flow (RBF) were simultaneously evaluated by the microsphere method in water-drinking and chronic saline-drinking rats at 3, 12 and 24 hours after induction of acute renal failure by glycerol injection. Threee hours after glycerol injection CO and RBF decreased to 36% and 20% of the respective controls in water-drinking rats and to 41% and 24% of the controls in saline-drinking rats. Renal vascular resistance (RVR) increased significantly in both groups at this time. Isoncotic plasma expansion (3% of body weight) restored the RBF and RVR to normal in water-drinking rats 3 hours post-glycerol injection, althought CO increased to only 70% of the control. Twelve hours after glycerol injection, CO and RBF returned to normal in saline-drinking rats, whereas they remained lower than controls in water-drinking rats. Twenty-four hours post-glycerol injection, when acute renal failure was evident as indicated by blood urea nitrogen (BUN) values of 116.9 and 63.8 mg/100 ml in water- and saline-drinking rats, respectively, CO and RBF returned to normal, except that the CO of of water-drinking rats was slightly higher than control. Thus, we conclude that decreased CO is an important determinant of the early decrease in renal perfusion in glycerol-induced acute renal failure. Furthermore, the observed earlier return of CO and RBF to normal in saline-drinking rats may  be partly responsible for reproducing the severity of acute renal failure.


The distribution of labeled albumin across the rabbit thoracic aorta in vivo.

125I-albumin was injected intravenously into normal conscious rabbits. The rabbits were killed after 10 minutes to 24 hours, and the descending thoracic aorta was excises immediately, opened longitudinally, rinsed, and frozen. Samples of frozen aorta were sectioned paralled to the intimal surface and washed with trichloroacetic acid (TCA) prior to counting. TCA-soluble tissue radioactivity slowly increased with time, suggesting that 125I was cleaved gradually from the labeled albumin within the aortic wall. At up to 4 hours, transmural concentration profiles of TCA-precipitable radioactivity had steep gradients near the intimal surface, moderate gradients near the medial-advential border, and were relatively falt in the middle of the media. After 24 hours, the steep intimal gradient had disappeared. Concentrations were otherwise comparable to those at 4 hours. The rate of accumulation of TCA-precipitable radioactivity was rapid initially (measurable concentrations were found throughout the media after only 10 minutes) and decreased with time. The results are consistent with entry of 125I-albumin into the media from both the luminal and adventitial sides. Approximate calculations indicate that the albumin mass transfer resistance associated with the intimal endothelium is about 1 order of magnitude greater than that associated with the media.


Effects of angiotensin II on steroid metabolism and hepatic blood flow in man.

Metabolic clearance rates (MCR) of aldosterone, cortisol, 11-deoxycorticosterone (DOC), corticosterone, and progesterone were simultaneously measured by constant infusion in eight control subjects before and during angiotensin II infusion in subpressor (3 ng/min per kg) and pressor (22 ng/min per kg) doses. Plasma levels of aldosterone and cortisol, the heat-labile protein-bound fraction of aldosterone, and hepatic blood flow (HBF) (as estimated by the fractional clearance of indocyanine green) were determined concomitantly. Angiotensin II in a subpressor dose produced a significant decrease of the MCR of aldosterone (by 23%), cortisol (by 16%), DOC (by 26%), corticosterone (by 14%) and progesterone (by 33%). The pressor dose further decreased the respective MCR by 37%, 21%, 40%, 28%, and 42% of the baseline value. Plasma aldosterone levels rose by 317% with subpressor and by 434% with pressor doses. HBF decreased by 18% with subpressor and by 33% with pressor doses of angiotensin II. Furthermore, there were significant negative correlations between the MCR of each steroid and the respective values of the fractional clearance of indocyanine green. We conclude that angiotensin II, by its vasoconstrictive action on the splanchnic vascular bed, decreases the MCR of aldosterone, cortisol, DOC, corticosterone, and progesterone. This decrease has to be taken into account when considering the stimulatory effect of angiotensin II on various plasma steroid concentrations.


Distribution of coronary artery flow to the canine right atrium and sinoatrial node.

We assessed segmental distribution of blood flow to the right atrium and the region of the sinoatrial node using micropheres (7-10 mum) in 20 anesthetized dogs. Mean right atrial flow averaged 83+/-7 (SE) ml/min X 100 g, which was 56% of the left ventricular blood flow. The distribution of right atrial flow was not homogeneous. For example, flow to anterior right atriaater (105+/-8 ml/min X 100 g) than mean right atrial flow. Following ligation of the sinus node artery, perfusion of the segment containing the sinus node decreased by only 36%. Relative preservation of perfusion to the sinus node following sinus node artery ligation may explain why ligation of the sinus node artery does not alter heart rate. Furthermore, we also found that cannulation and pump perfusion of the sinus node artery at pressures 10 and 50 mm Hg greater than systolic pressure did not alter the distribution of right atrial flow. Thus, because cannulation and perfusion of the sinus node artery do not artifactually distort regional right atrial blood flow, we conclude that this should be a useful method for evaluating responsiveness of the sinus node to various interventions.


Volume changes in sarcoplasmic reticulum of rat hearts perfused with hypertonic solutions.

To explore whether morphometry of intracellular membrane-limited subcompartments can be used to follow physiological volume changes in such subcompartments in hearts rapidly fixed by perfusion fixation, we have measured osmotically induced volume changes in electron micrographs of longitudinally oriented sarcoplasmic reticulum (LSR) and terminal cisterns (TC) of rat left ventricular myocardial cells. Vascular perfusion with solutions whose osmolality varied from 0.67 to 1.88 isomolal showed that in the hyperosmolal range LSR volume decreased linearly. Approximately 79% of LSR luminal volume participated in the osmotic rey unresponsive. By contrast, we found that the TC responded by dilation when hearts were perfused with hypersomolal NaCl, NaI, LiCl, or sucrose. Furthermore, with hyperosmolal NaCl the dilation developed within 1 minute; its rate and extent of development were concentration-dependent; it manifested an obligate association with prior or concomitant T-tubular dilation and was not readily reversible. We conclude that (1) the technique sensitively measures in situ changes of LSR volume; (2) most of LSR luminal water is osmotically responsive, but a significant fraction may not be; (3) exposure to hyperosomolal solutions may bring about (perhaps irreversible) structural changes in the diadic membrane complex, leading to changes in its solute permeability.


Length-dependent calcium inotropism in cat papillary muscle.

We studied changes in the relationship of isometric developed force to muscle length when extracellular calcium concentration was altered in isolated cat papillary muscles. In two series of experiments at temperatures of 30 degrees C and 32 degrees C, and rates of 12 and 30 beats/min, 21 muscles were exposed to calcium concentrations of 1.125, 2.25 and 4.5 mM. Muscle lengths were varied between 80% and 100% of the length at which maximum developed force occurred (Lmax). Peak developed force and the time from stimulus to peak were measured. The data indicate that force is not altered proportionately at all lengths when calcium concentration is changed. Rather, we found that a substantially greater modification of force occurs at short lengths than at long lengths. Similarly, the time to peak force increases with length at a rate which is more than 4 times greater at the low calcium concentration than it is at the high concentration. Small but consistent shifts of Lmax also are seen. We observed that Lmax is longer when inotropic changes reduce force and shorter when force is increased. These results indicate that the inotropic effect of extracellular calcium concentration changes is dependent on muscle length.


Balloon-induced right ventricular outflow obstruction: a new approach to control of acute interventricular shunting after myocardial infarction in canines and swine.

Current management of ventricular septal defect (VSD) after myocardial infarction (MI) is aimed at improving left ventricular (LV) performance by afterload reduction as a means of hemodynamic stabilization or shunt control. The current investigation was undertaken to determine whether primary manipulation of right venticular (RV) performance by afterload enhancement was an effective means of reducing MI-VSD shunting. In five open-chest dogs an external LV-RV shunt was created with pulmonary-systemic flow ratios (Qp/Qs) averaging 2.26:1. Inflation of a balloon-tipped catheter in the main pulmonary artery (PA) reduced average QP/Qs to 1.28:1 and shunt flow from 783 to 343 ml/min. However, this increase in RV afterload caused further significant increases in RV systolic and end-diastolic pressure and suggested that deterioration of RV function might be limiting the usefulness of this technique. To investigate whether inotropic support for the RV would overcome this limitation, a similar shunt was created in 11 open-chest swine. We then investigated the effects of dopamine, infused at 24, 60, and 120 mug/min, on QP/QS and other hemodynamic variables both with and without PA balloon inflation. Optimal shunt control was obtained when effects of dopamine were added to those of PA balloon inflation. Shunt flow that had been 1,633 ml/min was reduced to 892 ml/min with the PA balloon and reduced further to 757 ml/min with dopamine, which also lowered RV and LV end-diastolic pressure and reduced total systemic vascular resistance. In four other swine with left anterior descending ligations, PA balloon inflation and dopamine infusion again favorably affected hemodynamics. Epicardial mapping in these swine showed an increase in S-T segment displacement, suggesting that the cumulative effect of these interventions allowed hemodynamic improvement at the expense of enhanced ischemic injury. These data indicate that acute RV outflow obstruction with a balloon catheter is an effective means of temporarily reducing acute ventricular shunting and that dopamine may be a useful pharmacological agent to use with RV afterload manipulation to stabilize a patient prior to transfer to a medical center for more definitive therapy.


Renal cortical blood flow distribution in obstructive nephropathy in rats.

To examine the role of intrarenal hemodynamics in in obstructive nephropathy, we determined cortical blood flow distribution (CBFD) in rats with bilateral ureteral occlusion (BUO) and unilateral ureteral occlusion (UUO) during and after release of obstruction. Prior to release of obstruction of 24 hours' duration, we found that outer cortical perfusion decreased by 20+/-5% in both BUO and UUO rats. Furthermore, one hour after release of BUO, there was rapid normalization of CBFD associated with a modest return of glomerular filtration rate (GFR), an almost complete return of renal blood flow (RBF), and a marked postobstructive diuresis. In contrast, after release of UUO, we observed that outer cortical perfusion remained decreased by 21+/-31%, both GFR and RBF remained markedly depressed, and no diuresis occurred. These data demonstrate (1) marked ischemia of the outer cortex in both BUO and UUO during obstruction, (2) a rapid return of CBFD to a normal pattern after release of BUO, but (3) persistent outer cortical ischemia following release of UUO.


Directional coronary collateral growth with chronic circumflex occlusion in the dog.

The object of this study was to determine whether coronary collateral resistances were dependent on the direction of perfusion and to investigate whether a pattern of collateral growth with gradual circumflex occlusion could be discerned. In 12 dogs an Ameroid occluder was placed on the circumflex for 1 month, and six dogs for 3 months; 12 dogs served as controls. The circumflex, left anterior descending, and right coronary arteries were separately but simultaneously perfused in an isolated heart preparation in which the vasculature was maximally dilated with dipyridamole. Collateral flows were determined by measuring retrograde flows for two vessels simultaneously. The results showed that collateral flows from the right to the left coronaries in control dogs were 3.5-fold larger than when these collateral beds were perfused in the opposite direction. This difference in the 1- and 3-month Ameroid groups was approximately 20-fold. Relative to the control group, the collateral resistances from right to left coronary vessels were an average of 10-fold less in the 1- and 3-month groups, but there was no significant differences in resistance in the collaterals perfused from the left to the right. The results strongly suggest that collateral proliferation occurs in response to hypoxia rather than to a pressure gradient, and that collateral development is toward the hypoxid area.


On the mechanism underlying the action of D-600 on slow inward current and tension in mammalian myocardium.

D-600 the methoxy derivative of verapamil, is said to affect the force of cardiac contraction and the slow inward current (LSi) specifically by reducing the membrane conductance for Ca2+ (gsi). However, it is apparent that many effects of D-600 cannot be adequately explained solely by an effect on gsi. We studied the effects of D-600 on membrane current and tension of cat papillary muscle, using a conventional single sucrose gap voltage clamp technique. The results indicate that D-600 not only reduces the maximal Ca conductance but also, depending on concentration and duration of exposure, alters both the kinetics of the Ca-carrying system and the amplitude of the steady state outward current. No changes in the steady state activation and inactivation variables or in the rate of Isi inactivation were found. However, a substantial increase in the time to peak Lsi, as much as 7 times normal, was observed after exposure to D-600 (0.5 X 10(-6) to 2.0 X 10(-6) M) for at least 20 minutes. Because approximately only 75% of the reduction in Lsi induced by D-600 could be attributed to change in the maximum value of gsi (gsi), we conclude that the change in time to peak and about 25% of the reduction in Isi must be due to a change in the activation kinetics of the Ca-carrying system, Calculations suggest that the time to 70% activation of gsi can be prolonged to as much as 10 times normal by prolonged exposure to negatively inotropic concentrations of D-600.


Characteristics of left ventricular receptors with nonmedullated vagal afferents in cats.

Activity from left ventricular receptors with non-medullated afferents was recorded in the right cervical vagus in anesthetized, thoracotomized cats. Probing of the open heart demonstrated that the receptors were distributed throughout the free wall and the interventricular septum. Fibers from posterior receptors pass along the posterior descending coronary artery and the lateral surface of the right atrium to join the right main cardiac nerve; those from the anteriolateral region pass behind the aorta and the pulmonary trunk. The control receptor discharge was 1.4 (range, 0-6) impulses/sec at a mean left ventricular end-diastolic pressure (LVEDP) of 4.5 (range, 2-10) mm Hg. It was observed that receptor  discharge increased with progressive increase in LVEDP produced either by transfusion or by aortic occlusion. At a mean LVEDP of 8 mm Hg, the mean discharge rate was 3.2 impulses/sec, and at 16 mm Hg it was 7.0 impulses/sec. It was also found that propranolol reduced and isoproterenol increased the discharge frequency at any given LVEDP. Measurement of total conduction times indicated that during the increase in LVEDP the receptors were activated principally in the systolic portion of the cardiac cycle. Despite this there was no obvious relationship between the discharge frequency and left ventricular systolic pressure.


Beneficial effect of arachidonic acid during hemorrhagic shock in the dog.

Arachidonic acid (AA), precursor of the bisenoic prostaglandins was infused at a rate of 120 mug/kg per min into the vena cava of dogs subjected to hemorrhagic shock to assess the effects of stimulation of the prostaglandin (PG) synthetase system on the shock state. Hemorrhagic shock was induced by bleeding to a mean arterial blood pressure (MABP) of 40 mm Hg for 150 minutes followed by reinfusion of all remaining shed blood. In sham shock dogs receiving AA vehicle (0.1 M Na2C03), there were no significant changes in MABP, superior mesenteric artery flow (SMAF), renal artery flow (RAF), PGF2 or PGF2alpha concentrations, or in cathepsin D or myocardial depressant factor (MDF) activities during a 260-minute experimental period. During oligemia, untreated hemorrhagic shock dogs exhibited dramatic reductions in MABP, SMAF, and RAF which were transiently restored following reinfusion, but markedly decreased 100 minutes after reinfusion. Cathepsin D, MDF, PGE2, and PGF2alpha values increased significantly in these dogs. AA given during oligemia did not prevent changes in SMAF or RAF, but maintained MABP at near-normal values after reinfusion. AA also significantly protected against the plasma accumulation of both cathepsin D an MDF is hemorrhagic shock dogs. Circulating PGF2alpha and PGE2 values increased rapidly in AA-treated dogs and plateaued at 3.6 and 4.8 times control values, respectively, during oligemia. Hemorrhagic shock dogs receiving AA plus Na meclofenamate, a PG synthetase inhibitor, were not significantly different from shock dogs receiving vehicle except that the circulating PG concentrations did not increase. Thus, products of the PG synthetase system appear to prevent the plasma accumulation of lysosomal hydrolases nand of MDF, and may significantly preserve MABP after hemorrhagic shock in the dog.


Diagnostic effectiveness of biochemical liver-function tests, as evaluated by discriminant function analysis.

I evaluated the diagnostic value of routinely ordered liver-function tests in 175 biopsy-proven cases of hepatic disease by use of stepwise discriminant analysis. The tests studied-total and "direct" bilirubin, alkaline phosphatase, lactate dehydrogenase, and aspartate aminotransferase-correctly classified 45-73% of cases, depending on the homogeneity of the diagnostic groups. Aspartate aminotransferase and alkaline phosphatase were the best discriminators. When all tests were used in the most homogeneous groups (tumors, cirrhosis, and hepatitis), there was a stepwise improvement in diagnostic accuracy from 51 to 73%.


Acid phosphatase isoenzymes in Gaucher's disease.

Acid phosphatase (EC 3.1.3.2) isoenzyme profiles of extracts of splenic tissue and serum from patients with Gaucher's disease were measured by a mini-column ion-exchange chromatographic method [Clin. Chem., 23, 000 (1977)]. Diagnosis of Gaucher's disease in the five patients studied was confirmed by demonstrating decreased (2.3 to 4.1% of normal) glucocerebrosidase activity in the spleen. With p-nitrophenyl phosphate as substrate, increased acid phosphatase activity (three-to eight-fold normal) was demonstrated in spleen tissue from Gaucher;s disease patients; isoenzyme profiles by the ion-exchange column technique showed acid phosphatase isoenzyme 5 to be the predominant isoenzyme. Comparison of acid phosphatase isoenzyme profiles from patients with Gaucher's disease and prostatic carcinoma revealed distinct differences in the activities  of isoenzymes 2 and 5. The isoenzyme-5 measurement thus appears to provide a diagnostic test for Gaucher's disease that can be done reapidly and easily in the routine clinical chemistry laboratory.


Measurement of procainamide and N-acetylprocainamide in serum by high-performance liquid chromatography.

We measured procainamide (I) and its metabolite, N-acetylprocainamide (II), in human serum samples by solvent extraction, high-performance liquid chromatography on a reverse phase column, and detection at 280 nm, with use of external standards. The method requires 0.2 ml of serum and is sensitive to 0.3 mg of I and 0.6 mg of II per liter of serum, with intra-assay standard deviations of 0.22 and 0.24 mg/liter, respectively, at 5 mg/liter (N=10) and inter-assay standard deviations of 0.63 and 0.81 mg/liter, respectively, at 7.5 mg/liter (CV 8.4 and 10.5%, respectively, n = 20). Concentrations measured by high-performance liquid chromatography and by an established fluorescence technique correlated well (r = 0.98 for I and 0.97 for II). No interfering substances were found in 20 randomly selected sera from patients receiving a large number of other drugs. Of the pure drug substances tested only sulfathiazole interfered with the assay of II. The method is therefore suitable for routinely monitoring these compounds in serum in a clinical laboratory. The high concentrations of the metabolite in a significant number of patients demonstrate the need to consider it as well as the parent drug as guides in optimizing dosage regiments for I.


Photometry of inulin and polyfructosan by use of a cysteine/tryptophan reaction.

Described are a macromethod for measuring 2 to 30 mug of polyfructoside in sample volumes of 0.5 ml and a micromethod for measuring 40 to 400 ng in 10-mul samples. The absorptivity of the cysteine/tryptophan color product is 2.5-to 10-fold those found with use of five other colorimetric methods for inulin. The method is highly specific for alkali-stable polyfructoside. Color, measured at 515 nm, was maximal on development at 56 degrees C for 25 min in dilute (57-58% by vol) sulfuric acid, after the addition of the combined cysteine hydrochloride/tryptophan reagent. Analytical recoveries of inulin and polyfructosan from plasma and urine were complete. With initial alkaline heating, the endogenous blank inulinoid values averaged 0.6 +/- 0.47 (SD) mg/liter for plasma and 0.05 +1- 0.024 mg/min for urine. The coefficient of variation of the macromethod was less then 3%; that for the micromethod ranged from 1.3 to 6.3%. The micromethod appears adaptable to the determination of nanogram quantities of polyfructoside in nanoliter volumes of biological fluids.


Creatine kinase: stability, inactivation, reactivation.

I determined the in vitro biological half-lives or decay constants for creatine kinase isoenzymes at various temperatures. Values at 37 degrees C are consistent with values reported by others in vivo, which suggests that in vivo irreversible inactivation is primarily thermal. Reversible inactivation appears to be an oxidation-reduction phenomenon. Proteins and some inactivators (urate, catecholamines) retard irreversible inactivation and preserve isoenzyme integrity. Dilution and thiols promote reversal of inactivity. Mercaptoethanol is the preferred thiol, particularly for storage and reactivation of isoenzyme MB. MB is sensitive to light and to freeze-thawing. I recommend that specimens be cooled promptly after drawing, that mercaptoethanol (10 mmol/liter) be added, and that they be stored refrigerated. Avoid prolonged exposure to light and freezing. A model of inactivation is proposed, which is based on the assumed existence of four monomer types: active, denatured, oxidized, and insulated. The model is consistent with dilution and thiol reactivation, lag phase variations, and subtype heterogeneity.


Separation of tissue and serum acid phosphatase isoenzymes by ion-exchange column chromatography.

I describe a simple, rapid ion-exchange column-chromatographic technique for separating the acid phosphatase (EC 3.1.3.2) isoenzymes in human serum and tissue. Extracts of platelets, spleen, liver, erythrocytes, and prostate were used to determine optimum conditions for separating these isoenzymes. Samples layered on mini-colunms of DEAE-Sephadex A-50 were eluted stepwise with sodium chloride (100, 200, and 300 mmol/liter, buffered with tris (hydroxymethyl)aminomethane). Activity in column effluents was measured with p-nitrophenol phosphate as substrate, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Comparision of activity patterns so derived for various tissues revealed prostatic tissue to be a rich source of acid phosphatase isoenzyme 2 activity. Evaluation of sera from six patients with prostatic cancer revealed isoenzyme patterns with prominent amount of isoenzyme 2 (3.8 to 27.6 U/liter). sera from 10 healthy laboratory technicians contained isoenzyme 2 in the range of 0.3-0.5 U/liter. Samples from two patients with abnormally high activity owing to nonprostatic conditions (Gaucher's disease and carcinoma of lung) exhibited less than 2 U of isoenzyme 2 per liter and acid phosphatase isoenzymes 3-5 that were 50- to 100-fold the normal range. Quantification of isoenzyme 2 by DEAE-Sephadex column chromatography as described appears to provide a more sensitive and specific approach to diagnosis of prostatic cancer.


The immediate reaction between bromcresol green and serum as a measure of albumin content.

I investigated the immediate ( less than 30s) reaction between bromcresol green and serum, as a measure of albumin content. Fifty sera were analyzed in duplicate for albumin by electrophoresis on cellulose acetate by a procedure previously shown to give results in good agreement with two immunoprecipitation techniques, and by a bromcresol green method with the absorbance being read as soon as possible after mixing. Results by the dye method were 3.0 g/liter higher than those by electrophoresis; this difference was independent of the albumin content of the serum. Slight turbidity, jaundice, or added hemoglobin did not significantly interfere with albumin determination. Aparently the immediate reaction between serum and the dye reagent provides a simple, reliable measure of albumin content when 3.0 g/liter is subtracted from the result obtained.


Determination of nonsuppressible insulin-like activity in human serum by a sensitive protein-binding assay.

We describe a sensitive protein-binding assay for non-suppressible insulin-like activity in human serum. It can detect as little as 0.2 microunits (corresponding to 0.5 ng) of the activity in 0.4 ml of the assay mixture. It is measured in a low-molecular-weight fraction (termed "biological material") obtained by chromatography of serum on Sephadex G-50 in 1 mol/liter acetic acid. This fraction has been shown earlier to contain nearly all this billogically active material that is present in serum. A partially purified carrier protein from human serum is used as the binding protein; different concentrations of a partially purified preparation of material with the activity serve as standards, which compete with 125l-labeled tracer for binding. Biological material dilutes more or less in parallel with the standard over a 10-fold concentration range. In the chromatographed serum fractions, displacing activity appears between 50 and 80% bed volume, with the peak at 60%, and coincides with the distribution and the peak of radioactivity obtained by chromatography of tracer. A good correlation (r = 0.88) is observed between the values determined for this activity in the rat fat-pad assay and the protein-binding assay, although the latter yields about two fold higher results (190 +/- 37 milliunits/liter vs. 345+- 65 milliunits/liter, mean values for 18 normal sera). Values determined in the protein-binding assay are decreased in hypopituitary patients (183 +/- 27 milliunits/liter) and increased in acromegalics (486 +/- 88 milliunits/liter), in accord with the results of the bioassay (68 +/- 21 milliunits/liter for hypopituitary patients, 293 +/- 53 for acromegalics).


Ultrafiltration for improved assay of urinary enzymes.

Ultrafiltration of urine and rapidly separates urinary enzymes from inhibitors, activators, and heat-stable, spurious activities present in urine. Using eight enzymes of clinical interest as examples, we verified the reliability of results obtained after ultrafiltration by comparsion with results obtained after gel filtration.


Analysis for procainamide and N-acetyl procainamide in plasma or serum by high-performance liquid chromatography.

A high-performance liquid chromatography method is presented for simultaneous analysis for procainamide and N-acetyl procainamide in plasma or serum. The procedure involves internal-standard addition, organic extraction, and separation on a reverse-phase column. The detection limit for procainamide is 0.1 mg/liter and the calibration plot is linear to at least 30 mg/liter. Comparison with a colorimetric assay for procainamide gave a correlation coefficient of 0.989. We checked for interference by a large series of appropriate drugs, and found none, nor did icteric or lipemic sera present problems.


The gallium melting-point standard: its role in our temperature measurement system.

The latest internationally-adopted temperature scale, the International Practical Temperature Scale of 1968 (amended edition of 1975), is discussed in some detail and a brief description is given of its evolution. The melting point of high-purity gallium (stated to be at least 99.99999% pure) as a secondary temperature reference point is evaluated. I believe that this melting-point temperature of gallium should be adopted by the various medical professional societies and voluntary standards groups as the reaction temperature for enzyme reference methods in clinical enzymology. Gallium melting-point cells are available at the National Bureau of Standards as Standard Reference Material No. 1968.


The gallium melting-point standard: a determination of the liquid-solid equilibrium temperature of pure gallium on the International Practical Temperature Scale of 1968.

The sharpness and reproducibility of the gallium melting point were studied and the melting temperature of gallium in terms of IPTS-68 was determined. Small melting-point cells designed for use with thermistors are described. Nine gallium cells including three levels of purity were used in 68 separate determinations fo the melting point. The melting point of 99.99999% pure gallium in terms of IPTS-68 is found to be 29.771(4) +/- 0.001(4) degree C; the melting range is less than 0.0005 degree C and is reproducible to +/- 0.0004 degree C.


The gallium melting-point standard: its role in manufacture and quality control of electronic thermometers for the clinical laboratory.

I discuss the traceability of calibration of electronic thermometers to thermometric constants of nature or to the National Bureau of Standards, form a manufacturer's basic standards through the manufacturing process to the user's laboratory. Useful electrical temperature sensors, their advantages, and means for resolving their disadvantages are described. I summarize our development of a cell for realizing the melting phase equilibrium of pure gallium (at 29.770 degrees C) as a thermometer calibration fixed point, and enumerate its advantages in the routine calibration verification of electrical thermometers in the clinical chemistry laboratory.


The gallium melting-point standard: its application and evaluation for temperature measurements in the clinical laboratory.

We are impressed with the ease and certainty of calibration electronic thermometers with thermistor probes to +/- 0.01 degree C at the gallium melting point, 29.771(4) degrees C. The IFCC reference method for measuring aspartate aminotransferase activity in serum was run at the reaction temperature of 29.771(4) degrees C. By constantly referencing to gallium as an integral part of the assay procedure, we determined the absolute reaction temperature to IPTS-68 (International Practical Temperature Scale of 1968) to +/- 0.02 degrees C. This unique temperature calibration standard near the center of the range of temperatures commonly used in the clinical laboratory is a valuable addition and can be expected to improve the accuracy of measurements, especially in clinical enzymology.


Plasma fibrinogen, cholinesterase activity, and anemia: utility of fibrinogen in multiphasic screening and in assessing the activity of diseases.

I present the general pattern relationship between (a) plasma fibrinogen and cholinesterase activity and (b) plasma fibrinogen and hemoglobin, in 250 and 310 hospitalized adults, respectively. Although responses of cholinesterase were often unrelated to fibrinogen, when its activity in plasma was depressed, above-normal fibrinogen concentrations were present in about 90% of the subjects. Further, despite the observed independence of hemoglobin and fibrinogen concentrations, whenever frank anemia was present (hemoglobin less than 110 to 120 g/liter), hyperfibrinogenemia was also present in about 75-85% of such anemic patients. Thus, these studies affirm that fibrinogen determination would be an appropriate component of tests on hospital admission and also that hyperfibrinogenemia is a very sensitive "acute phase" reactant, and an important reaction for gauging the course of numerous disorders.


Acetaminophen in serum and plasma estimated by high-pressure liquid chromatography: a micro-scale method.

We describe a capillary-sampling method for serum or plasma acetaminophen by cation-exchange chromatography. As little as 1.5 mul of plasma or serum and an equal volume of the internal standard (N-butyryl-p-aminophenol) were run, with a precision of +/- 5% between duplicates. Acetaminophen and the internal standard chromatographed in 32 and 50 min, respectively, distinct from intrinsic plasma peaks and peaks caused by other medications.


Use of tetramethylbenzidine in plasma hemoglobin assay.

Plasma hemoglobin can be assayed by substituting 3,3',5,5'-tetramethylbenzidine for benzidine (a carcinogen) as the chromogenic reducing substance. Sensitivity, precision, and accuracy compare well with methods in which benzidine is used. Plasma inhibition can be accommodated by preincubation with hydrogen peroxide.


Spectrophotofluorometric assay for isoniazid and acetyl isoniazid in plasma adapted to pediatric studies.

We modified the micro-scale spectrophotofluorometric method of Miceli et al. [Biochem. Med. 12, 348 (1975)] for the assay of "apparent" isoniazid (isoniazid acid-lable hydrazones) to improve its clinical application. We also adapted the method for determination of acetyl isoniazid. Data are presented showing how long plasma containing isoniazid may validly be stored. The applicability of the method was demonstrated in studies on children and small animals.


Photometric determination of total protein in lipemic sera.

We compared various procedures and biuret reagents suggested for determination of protein in lipemic sera. A modified procedure, consisting of the precipitation of serum protein with acetone and dissolution of the precipitate in a biuret reagent, has been developed. The sample (0.02 ml) is mixed with 0.1 ml of distilled water and 2 ml of acetone. The mixture is shaken, centrifuged, and the precipitate dissolved in 1.5 ml of ethylenediaminete-traacetate-chelated biuret reagent. This method is suitable for both clear and lipemic sera. Values obtained with the procedure proposed are in good agreement with values by the Kjeldahl method.


Interference with the enzymic measurement of cholesterol in serum by use of five reagent kits.

We describe the effects of uric acid, hemolysis, drugs, ascorbic acid, lipemia, and bilirubin on the enzymic measurement of cholesterol in serum by use of reagent kits from Abbott, Beckman, Boehringer Mannheim, Calbiochem, and Worthington. In all of these, the chromogen formed from the reaction of hydrogen peroxide with phenol and 4-aminoantipyrene is measured. The absorbance was measured at 500 nm vs. a serum blank for each kit--except Abbott's with which the recorded absorbances were the differences between readings at 500 and 600 nm. With all reagents kits, there was no interference from uric acid up to 200 mg/liter, hemoglobin up to 1.0 g/liter, or drugs (clofibrate, phenobarbital, Ketochol, Ovral-28), but negative interferences from ascorbic acid. Except for the Abbott kit, the cholesterol values obtained for lipemic samples were lower than found with the comparison method [Abell et al., Stand. Methods Clin. Chem. 2, 26 (1958)]. With Abbott's reagents, for most lipemic samples, the values were the same. Bilirubin at concentrations of 200 mg/liter significantly decreased the cholesterol values with Beckman, Calbiochem, and Worthington reagent kits. With Boehringer Mannheim reagent a small negative interference was observed and with Abbott reagent a small positive interference was observed when the bilirubin concentrations were 200 mg/liter.


Xanthine oxidase and guanase activities in normal and psoriatic epidermis.

Xanthine oxidase and guanase activities in normal and psoriatic epidermis were demonstrated and accurately assayed by the new micro-assay methods which rely on the isolation of 14C-labelled end-products, xanthine and uric acid, from the substrates, hypoxanthine and guanine, by electrophoresis on cellulose acetate membrane using 0.1 M borate buffer, pH 9.0. The average specific activities of xanthine oxidase and guanase in normal epidermis were 10.2 and 55.0 pmol/min/mg protein, respectively, and in psoriatic epidermis, 52.1 and 980.6 pmol/min/mg protein, respectively. Increased activities of these enzymes in psoriatic epidermis suggested that urate formation might be enhanced in the psoriatic lesions resulting from an increased turn-over of nucleic acids.


The clearance of lipoprotein X in vitro, and effect of phospholipase A on serum lipoprotein X.

The in vitro effect of post-heparin and post-heparin plus post-protamine normal serum on serum lipoprotein X (LP-X) is described. Serum LP-X is cleared after incubation with post-heparin normal serum, and serum LP-X remain unmodified when it is incubated with post-heparin plus post-protamine normal serum. The action of phospholipase A from snake venom on serum LP-X is also studied. A very small quantity of phospholipase is necessary to degrade LP-X and to transform lecithin into lysolecithin. It is concluded that phospholipase seems to be the enzyme that most likely induces the LP-X changes after heparin administration.


Urinary oxalate estimation.

A method of estimating urinary oxalate is described. Impurities which might otherwise interfere with the estimation are removed by means of an ion-exchange resin. The oxalate is then reduced to glycolic acid, which forms a coloured compound with chromotropic acid. It can then be estimated colorimetrically. No special equipment is required, oxalate recovery is good, and the method is reproducible and precise.


Activities of acid hydrolases in fibroblasts from normal and cystinotic children.

A study of the lysosomal hydrolases bete-galactosidase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and arylsulphatases A and B has been carried out on fibroblasts cultured from seven patients with cystinosis and eight control subjects. beta-Galactosidase activity was found to be consistently lower in cells derived from cystinotics, while the other enzymes studied showed no significant differences between normals and cystinotics.


Blood glucosylceramide levels in Gaucher's disease and its distribution amongst lipoprotein fractions.

Plasma glucosylceramide levels in eight patients with Gaucher's disease showed a 2 to 7-fold elevation over normal levels whereas erythrocyte glucosylceramide levels were within the normal range. The majority of the glucosylceramide elevation was associated with the low density lipoprotein fraction.


Neutral arylamidase in urine of healthy and nephrotic children.

The neutral arylamidase activity in urine of healthy and nephrotic children was determined using L-leucyl-beta-napthylamide as substrate. The neutral arylamidase activity in urine samples from 13 nephrotic children (22.4+/-3.0 (S.E.) mumol/min/g of creatinine) was significantly higher (p less than 0.001) than that from 27 normal children (8.5+/-0.4). Two forms of neutral arylamidase were demonstrated by polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration in urine samples from normal children. One was a high molecular weight arylamidase (more than 1 000 000), the other had a molecular weitht of 240 000 as estimated by Sephadex G-200 gel filtration. Two additional electrophoretically distinct froms of neurtral arylamidase were demonstrated on polyacrylamide gel in urine samples from nephrotic children. Two neutral arylamidases from normal children, one additional arylamidase from normal children, one additional arylamidase from nephrotic children and kidney membrane-bound neurtral arylamidase had identical KM values, effect of inhibition by L-methionine and heat stability. One of four urinary arylamidases from nephrotic children was suggested to be derived from plasma. It is concluded that an increase in urinary neutral arylamidase activity in nephrotic children is not only due to increased released from kidney membranes, but also due to leakage of plasma neutral arylamidase in the urine.


[On GLC of bile acids in serum and bile (author's transl)].

Some modifications to previous methods are reported in this paper to make this chromatographic assay more specific and more specific and more convenient in practice. 1. Bile acids were deconjugated via an enzymatic route with choloylglycine hydrolase (EC 3.5.1.24). 2. By acetylation of methyl derivatives, chromatography on alumina which led to a loss of lithocholic acid, can be supressed. Moreover, the compounds obtained are thermically more stable than those obtained by trifluoroacetylation. 3. The use of nor-cholic acid as internal standard led to a better quantitation of bile acids than others because its relative retention time is located between cholic and chenodesoxycholic acid. 4. Because of the hight sensitivity of this method, the assay of duodenal samples can be made with minimal volumes (0.1 ml) allowing a direct extraction by organic solvents.


[Inaccuracy of serum albumin measurements in renal failure patients using a dye-binding method (author's transl)].

A survey of serum albumin determinations in a group of patients with renal failure revealed that albumin reasurements using the 2-(4'-hydroxyphenylazo)-benzoic acid (HABA) dye-binding method were understimated when compared to results obtained with the biuret method. Equilibrium dialysis of the HABA dye binding to albumin are reported. Scatchard plot analysis showed that an average of 5.7 binding sits per molecule of albumin were unavailable in renal failure patients. The binding interference encountered in these patients indicates that alubumin measurements using dye binding techniques should not be used for these patients.