An extracellular fibrillar matrix in gastrulating sea urchin embryos

Fine structural studies of fractured developing sea urchin embryos revealed the existence of a voluminous, fibrillar, extracellular matrix composed of fine filaments, twisting fibers and granules lining the blastocoel of midgastrula embryos. Glycine disaggregated embryos also exhibited this material. The fibrillar matrix is closely associated with the basal lamina of the ectodermal cells of the embryo and histochemical studies suggest it is composed mostly of sulfated glycosaminoglycans. The position of the matrix within the blastocoel as well as its organized association with embryonic cell surfaces is consistent with the hypothesis that it plays a major role in guiding the invaginating archenteron during gastrulation.     

Biosynthesis of alpha-galactosidase A in cultured Chang liver cells

An investigation of the structure and biosynthesis of alpha-galactosidase A (alpha-D-galactoside glycohydrolase, EC 3.2.1.22) and its N-linked oligosaccharide chains was undertaken by metabolic labeling of Chang liver cells with [2-3H]mannose, immunoprecipitation of the activity, and examination of the resulting immunoprecipitates. From cells pulse labeled for 3 h, two radioactive bands with Mr = 58,000 and 49,000 were detected by SDS-gel electrophoresis; following a 20-h chase, only the Mr = 49,000 band was observed. Examination of the oligosaccharide fraction derived from pulse-labeled enzyme revealed that 18% of the asparagine-linked oligosaccharides were complex and 82% were high-mannose type. After a 20-h chase, 48% of the oligosaccharides were complex and 52% were high mannose. The high-mannose oligosaccharides of alpha-galactosidase A immunoprecipitated from both pulsed and pulse-chased cells had the same mobilities as Man8-9GlcNAc on thin-layer chromatography and Bio-Gel P-4. Two fractions of complex glycopeptides derived from the alpha-galactosidase A of pulsed and pulse-chased cells had the same migration on Bio-Gel P-4 as glucose oligomers containing 14 and 19-39 glucose units. Based on their apparent size and their behavior on concanavalin A-Sepharose, the complex oligosaccharides are believed to be composed of tri- and/or tetraantennary structures.     

Culture-generated suppressor cells: evidence for an adherent cell component

Cells which suppress mixed lymphocyte reaction (MLR) and cytotoxic T-lymphocyte (CTL) generation can be demonstrated after culture for 3 to 5 days in the absence of added antigen. Such precultured suppressors are resistant to the cytotoxic effects of commercial rabbit anti-mouse brain serum, congenic anti-Thy-1.2 serum, and monoclonal anti-Thy-1.2 reagent. In addition, these cells, which adhere to both nylon wool and Sephadex G-10, ingest carbonyl iron. These properties suggest that precultured suppressors may not be thymus-processed. Precultured suppressor cells, when irradiated, are able to suppress only the MLR, and not the generation of allo-CTL. This finding, taken with our previously published work on the differential ontogeny of cells which suppress the MLR or CTL generation in culture, suggests that at least two kinds of suppressors are generated. One suppressor acts on CTL generation, is radiosensitive, and develops late in ontogeny; the other suppressor acts on proliferative cells in the MLR, is radioresistant, and develops early in ontogeny. Both kinds of suppressors, however, are adherent to nylon wool and Sephadex G-10, ingest carbonyl iron, and are resistant to anti-T-cell sera.     

A convenient procedure for purification of thymidylate synthase from L1210 cells

Several laboratories have described procedures for purification of thymidylate synthase (TMP synthase) that utilize folates or folate analogs covalently attached to a matrix. The principle of separation is the formation of a ternary complex between dUMP, TMP synthase, and the bound ligand and the subsequent elution of the enzyme with buffers that do not contain dUMP. We have successfully used 10-formylfolic acid as the bound ligand for the purification of TMP synthase. As compared to other ligands that have been used, 10-formylfolic acid has the advantages that it can be easily synthesized, it is stable, and the enzyme is eluted as a sharp peak. Application of this procedure to L1210 leukemia cells gave 1765-fold purification of TMP synthase with a recovery of 39%. The native enzyme had a molecular weight of 78,000, which is about the same as that reported.     

Human teratoma cells share antigens with mouse teratoma cells.

Cytochalasin B inhibits the penetration of sperm nuclei into Urechis eggs without inhibiting sperm-induced egg activation. The acrosome reaction appears normal, and plasma membranes of the acrosomal tubule and egg become closely apposed. It is uncertain whether or not the drug blocks fusion of these membranes; however, sperm penetration cone formation is inhibited.     

Reversal of insulin effects in fat cells may require energy for deactivation of glucose transport, but not deactivation of phosphodiesterase

The reversal of insulin effects on sugar transport and phosphodiesterase in fat cells was studied after arresting further actions of insulin with KCN, NaN₃, 2,4-dinitrophenol, or dicumarol. These agents rapidly lower the ATP concentration and concomitantly block the actions of insulin added later. Contrary to our expectation, the above inhibitors failed to initiate deactivation of the hormone-stimulated transport system. Instead, in the presence of the agents the transport system remained activated even after cells had been washed with an insulin-free buffer. This effect of the inhibitors was reversed when cells were washed with an inhibitor-free buffer containing glucose or pyruvate. The above inhibitors also blocked the deactivation of sugar transport stimulated by mechanical agitation. The effects of the inhibitors could not be explained by their possible effects on the basal transport activity, the intracellular urea space, or the cell count. The insulin-stimulated phosphodiesterase activity was rapidly lowered when cells were exposed to the above inhibitors. Apparently, these agents did not denature phosphodiesterase itself since the latter could be reactivated by insulin when inhibitor-treated cells were washed with a glucose-containing buffer. None of the above agents, except dicumarol, significantly inhibited phosphodiesterase activity in a cell-free system. It is suggested that the effects of insulin on sugar transport and phosphodiesterase are reversed by different mechanisms. ATP or metabolic energy may be involved in the deactivation of sugar transport, but not in that of phosphodiesterase.     

Nuclear matrix as acceptor for cAMP-binding proteins

Using [³²P]-8-N₃-cAMP, a photoaffinity analog of cAMP, we have established that nuclear binding of cAMP is preferentially localized in the “nuclear matrix”. Two major radioactive bands corresponded to proteins of M_r 40 K and 50 K, and three minor bands to proteins of M_r 55, 150 and 200 K. Even though the molecular weight of the major nuclear binding proteins in the matrix are similar to those of the cytosolic cAMP binding proteins, the characteristics of the binding reaction in the nucleus were markedly different from those in the cytosol.     

Independent responses of nucleoside triphosphatase and protein kinase activities in nuclear envelope following thioacetamide treatment

We have monitored the binding of heparin to thrombin as well as antithrombin by fluorescence techniques. The interaction of mucopolysaccharide with thrombin is characterized by a stoichiometry of 2:1 with K_DISS^H1T = K_DISS^H2T = 8 × 10^?7 M. The interaction of heparin with antithrombin is typified by a stoichiometry of 1:1 with K_DISS^H-A = 5.7 × 10^?8 M. A plot of the initial velocity of the thrombin-antithrombin reaction versus mucopolysaccharide concentration exhibits an ascending limb, descending limb and final plateau. The ascending limb of the kinetic profile corresponds to the level of heparin-antithrombin complex. The descending limb of the initial velocity plot coincides with the formation of tertiary complexes between two molecules of heparin and one molecule of thrombin. Thus, our data demonstrates that the generation of heparin-antithrombin complex is responsible for the mucopolysaccharide dependent acceleration of enzyme neutralization. Furthermore, our results also suggest that interactions between heparin bound to inhibitor and free enzyme can account for only a small fraction of the total kinetic effect of the complex carbohydrate. Therefore, we conclude that the direct binding of heparin to antithrombin is probably responsible for the anticoagulant effect of the mucopolysaccharide.     

Prolongation of cardiac allograft survival by pretreatment of recipient mice with donor blood or spleen cells plus cyclophosphamide.

Using six mouse strain combinations, we attempted to prolong cardiac allograft survival by pretreatment of recipients with a single iv injection of donor-specific whole blood or spleen cells plus a single ip injection of cyclophosphamide (Cy). Significant prolongation of cardiac allograft survival occurred in a small proportion of pretreated mice of some strain combinations, with some grafts surviving for periods longer than 6–9 months. Cy injected alone did not influence the normal cardiac allograft rejection time of between 1 and 2 weeks. Depending upon the strain combination, accelerated rejection of all or some of the grafts occurred in mice pretreated with blood or spleen cells or myocardial cells alone.     

Resistance and susceptibility to infection in inbred murine strains. I. Variations in the response to thymic hormones in mice infected with Candida albicans

Nine inbred murine strains were either highly resistant or highly susceptible to intravenous challenge with 4 X 10(4) to 1 X 10(5) cells of Candida albicans. The resistant strains had the capacity to develop delayed footpad reactions on appropriate sensitization and challenge; the susceptible strains did not have this innate capacity. Administration of thymosin fraction 5 beginning on the day of infection greatly increased the resistance of the susceptible strains to infection, but decreased the resistance of the resistant strains. In contrast, thymosin fraction 5 enhanced the delayed footpad responses of resistant-sensitized mice to specific antigen, but did not have a detectable effect on the delayed footpad reactions of the susceptible strains. Reinfection of the two types of strains had different effects, in that, depending on the strain, resistance could be increased, decreased, or not influenced at all.     

NMR study of the lability of the thiazolium proton in thiamin and its polyphosphoric esters

The purpose of the present paper is to study the exchange rate of the hydrogen at the C-2 position of the thiazolium ring in thiamin and its polyphosphoric esters, by NMR spectroscopy. This rate is determined by following the corresponding signal intensity of the NMR spectrum in ²H₂O.It has been found that the rate of exchange increases with pH, and that this increase is greater as the polyphosphoric chain becomes longer.Data show us that the half-life time of this exchange for thiamin at a pH value of about 9 is the same as that for diphosphothiamin at a lower pH range.     

Differential maintenance of penile responses and copulatory behavior by gonadal hormones in castrated male rats

Sexually experienced male rats were castrated and immediately received implants of Silastic tubing containing either testosterone (T), dihydrotestosterone (DHT), estradiol (E), or nothing (blank). The ability of these hormone treatments to maintain precastration levels of copulatory behavior and ex copula penile responses was assessed for 40 days after castration. Throughout the study T- and E-treated males, but not males with DHT or blank implants, maintained normal copulatory behavior. In contrast males treated with T and DHT, but not E or blanks, maintained penile responses ex copula. In blank-treated males, penile-response latencies increased more rapidly than did intromission latencies. These results, together with those of previous studies, appear to rule out a role for estradiol and reinforce the role of androgens in the activation of rats' penile-response potential ex copula. Similarly, the results support the conclusion that in castrated male rats estradiol treatment is sufficient for the activation of masculine copulatory behavior, and that the penile actions necessary for intromission are not dependent on androgen. Thus, the evocability of penile actions and their relative androgen dependence are context sensitive.     

Activation of calcium and phospholipid-dependent protein kinase by epidermal growth factor (EGF) in A431 cells: attenuation by 12-0-tetradecanoylphorbol-13-acetate (TPA)

A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity.     

Contribution of cholinesterase to developmental decreases in the time course of synaptic potentials at an amphibian neuromuscular junction

Urodele limbs are able to regulate for intercalary deletions created when distal regeneration blastemas are grafted to a more proximal level. Using several morphological markers, pigmentation differences between white and dark axolotls, and the difference in nucleolar number between diploid and triploid animals, we show that the entire intercalary regenerate is derived from the stump when wrist or tarsus blastemas are grafted to the midstylopodium of the fore- or hindlimb. The transplanted prospective autopodium forms no more than would be expected in situ. Thus, the rule of distal transformation is not violated during intercalary regeneration in salamanders. The advantages and disadvantages of the several marking techniques are discussed.     

Changes in the insulin and glucagon receptors in the regenerating liver following intoxication with carbon tetrachloride.

The response of rat liver plasma membrane adenylate cyclase was studied from one to 14 days after a single dose of carbon tetrachloride (CCl₄). The response to glucagon was diminished to a greater extent than that of fluoride and was due to a deficiency in hormone binding. In contrast, insulin binding increased 300% over control; the change was due to increased number of binding sites. The “affinities” of receptors for either hormone were not altered. The tissue levels of adenosine 3′:5′ -monophosphate increased following CCl₄ poisoning reaching a peak precisely when the adenylate cyclase response to glucagon was at its lowest level. These studies present evidence that receptors for pancreatic hormones change differently when liver is damaged and during its regeneration following CCl₄ intoxication. The change in pattern is remarkably similar to changes reported previously in fetal liver development or following partial hepatectomy of adult rat.     

Lipopolysaccharide (LPS) modulation of human monocyte accessory cell function in promoting T-cell colonies: Inability of LPS and IL-1 to abrogate the need for monocytes with high HLA-DR expression

The abilities of human monocytes differentially expressing HLA-DR and of lipopolysaccharide (LPS) to influence T-cell colony responses were investigated. Optimal T-cell colony responses stimulated by soluble Staph protein A were crucially dependent on monocytes. Also, monocyte facilitation of colony responses was markedly inhibited by 10 μg/ml LPS and the addition of indomethacin reversed this inhibition. In contrast the inhibition of T-cell colony responses with 100 μg/ml LPS was not reversed with indomethacin and preincubation experiments with high concentrations of LPS showed the inhibition could be mediated through T cells by mechanisms other than prostaglandins. The treatment of monocytes with a monoclonal anti-HLA-DR reagent + C reduced the frequencies of monocytes expressing high levels of HLA-DR ~ fivefold and the resulting monocytes which expressed low levels of HLA-DR also poorly functioned in the promotion of colony responses compared to controls. LPS in the presence of indomethacin improved the ability of monocytes expressing low levels of HLA-DR to promote colony responses. However, these monocytes consistently failed to augment colony responses to those levels observed with untreated monocytes and their failure was not secondary to deficient interleukin 1 release. These results indicate that although LPS can somewhat potentiate the accessory cell function of certain human monocytes, it cannot abrogate an additional requirement for those monocytes expressing high levels of HLA-DR.     

Murine cell surface glycoproteins: immunochemical analysis of a major differentiation alloantigen of phagocytic cells

Four independent monoclonal antibodies derived from spleen cells of rats immunized with mouse NIH/3T3 cells were found to precipitate an 80,000-dalton plasma membrane glycoprotein, identified as a polymorphic differentiation antigen of murine mesenchymal cells. The homology of the four immunoprecipitated polypeptides was proven by analysis of partial proteolytic cleavage products. The genetic polymorphism detected by the four antibodies was shown to reside in a single antigenic site by several criteria: (i) The expression of the antigenic determinant among different strains of mice; (ii) cross-inhibition of antibody binding; (iii) precipitation of partial proteolytic cleavage fragments of the 80,000-dalton glycoprotein; (iv) the kinetics of heat inactivation. These antibodies thus define a single polymorphic site of a major phagocytic cell surface glycoprotein and provide the basis for genetic and functional characterization of this glycoprotein.     

A comparative analysis of extracellular fluid volume of several tissues as determined by six different markers

The uptake and distribution of 6 different extracellular markers were analyzed in ten tissues of the rat. The saccharides, ³H-mannitol, ³H-raffinose, ³H-inulin, and ¹⁴C-inulin, reached a steady-state distribution in all tissues within ≈15 min after intraperitoneal injection; ²²Na and ³⁶Cl followed similar kinetics in all tissues except the choroid plexuses and the thyroid, which required > 1 hr to obtain a steady-state plateau. In most tissues, the steady-state spaces of ³H-raffinose, ³H-inulin, and ¹⁴C-inulin (60 min) were not significantly different; however, the ³H-mannitol, ²²Na and ³⁶Cl spaces were on average 45, 54, and 79%, respectively, greater than the ³H-inulin space.     

Nisoxetine prevents the 6-hydroxydopamine induced decrease in post-decapitation convulsions

Intraventricular injection of 6-hydroxydopamine (60HDA) to rats caused a marked reduction in post-decapitation convulsions (PDC), which was also observed in rats given 60HDA systemically at birth. The reduction in PDC and norepinephrine (NE) content in brain and spinal cord was completely prevented by pretreatment with the selective norepinephrine uptake inhibitor, nisoxetine, but not by fluoxetine, a specific serotonin uptake inhibitor. Presumably nisoxetine prevented the reduction in PDC and NE levels by blocking the entry of 60HDA into the neuron via the membrane uptake pump, and thus preventing subsequent NE depletion and neuron degeneration. These data imply that NE neurons are involved in the neurological mechanism of PDC, although this does not exclude a role for other neurotransmitters such as serotonin (5HT) and dopamine (DA).     

Characterization of a stabilizing factor of rat liver and of the nature of its interaction with ATP-citrate lyase

A stabilizing factor, previously reported to protect phosphofructokinase (EC2.7.1.11) from thermal or lysosomal inactivation, has been shown to stabilize ATP-citrate lyase (EC 4.1.3.8) from thermal inactivation (B. Osterlund and W. A. Bridger, 1977, Biochem. Biophys. Res. Commun., 76, 1–8). We now report that this factor protects ATP-citrate lyase from inactivation by proteases extracted from lysosomes. While it has been suggested that the stabilizing factor may play a role in the turnover of other lipogenic enzymes, we have found that the factor has no stabilizing or other effects on NADP⁺-malic enzyme (EC 1.1.1.40). In order to assess the properties and mode of action of the stabilizing factor with regard to interaction with its target enzyme(s), the factor has been extensively purified from rat liver and characterized as to its composition. Although glutathione appears to copurify with the factor, and glutathione exerts some stabilizing effects on ATP-citrate lyase, the factor is clearly distinguishable from glutathione on the basis of its mode of action and its concentration dependence. Several other biological compounds have been tested in attempts to identify the chemical nature of the stabilizing factor. Thus, biotin, pyridoxal phosphate, glucose tolerance factor, substrates for ATP-citrate lyase, and oxidized glutathione have been eliminated as possible identities for the stabilizing factor. In contrast to results reported by other workers (who investigated stabilization of phosphofructokinase) we find this factor to be insensitive to treatment by proteases or sulfhydryl reagents when tested by its ability to protect ATP-citrate lyase from inactivation.