Developmental Expression of HNK-1-Reactive Antigens in Rat Cerebral Cortex and Molecular Heterogeneity of Sulfoglucuronylneolactotetraosylceramide in CNS Versus PNS

Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex.     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic β-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in β-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba²⁺ was not inhibited by d-glucose. Ba²⁺ served as a substitute for Ca²⁺ both in maintaining the glucose metabolism after removal of extracellular Ca²⁺ and making it possible for glucose to stimulate insulin release. Furthermore, Ba²⁺ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the β-cell content of cyclic AMP. It is likely that the observed actions of Ba²⁺ are mediated by Ca²⁺, since Ca²⁺-dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba²⁺ specifically.     

Naltrexone partially inhibits the estrogen-induced increase in prolactin secretion and anterior pituitary weight

The effects of naltrexone, a specific opiate antagonist, on stimulation by estradiol benzoate (EB) of prolactin (PRL) release and anterior pituitary (AP) weight, were studied in gonadectomized female and male Sprague-Dawley rats. One week after castration, rats were injected for 10 days once daily with 2 μg EB alone, or together with twice daily injections of 2 mg naltrexone/kg body weight (BW). Blood was collected for radioimmunoassay of PRL by orbital sinus puncture on days 0 and 6, and by decapitation on day 11, at which time the AP was quickly removed, weighed and assayed for PRL.Serum PRL concentrations and AP weights were significantly increased by EB administration. These effects of EB were partially but significantly inhibited by naltrexone. These results suggest that endegenous opiates may be involved in the estrogen-induced rise in serum PRL and increase in pituatary weight.     

Long cytoplasmic processes in pancreatic polypeptide cells

Pancreatic polypeptide (PP) cells were studied in human endocrine pancreatic tumours and in normal human pancreata by immunohistochemical techniques and electron miscroscopy. The existence of long cytoplasmic processes was demonstrated both in tumours and normal tissue. These processes are in close contact with other endocrine cells or with acinar cells. This particular morphological aspect suggests that PP cells may control the function of other cells via paracrine secretion.     

Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

T-lymphocyte function and sensitivity to interferon in trisomy 21

Previous studies have shown that cells from subjects with trisomy 21 have enhanced sensitivity to the antiviral effects of interferon, presumably because of the location of the gene, IfRec, coding for the species-specific response to interferon on chromosome 21. Interferon is also known to have many other effects including the ability to inhibit the proliferation of many types of cells. To determine whether proliferating trisomic lymphocytes are more sensitive to the antiproliferative effect of interferon we have investigated, using healthy noninstitutionalized subjects with trisomy 21, the ability of interferon to inhibit the proliferation of lymphocytes stimulated with phytohemagglutinin P(PHA), concanavalin A (Con A), and tetanus toxoid. The trisomic subjects had normal numbers of peripheral blood leukocytes, and normal numbers and proportions of T and B lymphocytes. The production of interferon by PHA-stimulated trisomic T lymphocytes was normal. Trisomic lymphocytes also had normal proliferative responses to PHA and Con A. There were no differences between the inhibitory effects of interferon on the proliferation of PHA-stimulated trisomic and normal lymphocytes. However, trisomic lymphocytes stimulated with low doses of Con A did display significantly enhanced sensitivity to the antiproliferative effects of interferon. In contrast to normal lymphocytes, trisomic lymphocytes were not stimulated to proliferate by tetanus toxoid, and exposure to interferon resulted in enhancement, rather than inhibition, of DNA synthesis.     

Suppression of lymphokine production: I. Macrophage-mediated inhibition of MIF production

Macrophages have been found to suppress the in vitro production by stimulated T lymphocytes of a lymphokine, migration inhibitory factor. When macrophages isolated from primary MSV-induced tumors were added to antigen-stimulated MSV-immune spleen cells, a complete suppression of MIF production was observed. This suppression was nonspecific, since MIF production by antigen-stimulated alloimmune spleen cells and by PHA-stimulated normal spleen cells was also inhibited. Suppressor macrophages could also be induced by inoculation with Corynebacterium parvum, whereas light mineral oil-induced peritoneal macrophages had no detectable effect on MIF production. The failure to detect MIF in the supernatants of stimulated cultures containing activated macrophages appeared to be due to inhibition of lymphokine production rather than to absorption or inactivation of MIF or to interference with the assay for detection of MIF. Macrophages were able to suppress MIF production only when added during the first 4–5 hr of culture and they had no effect when added later. These data show that activated macrophages can nonspecifically suppress lymphokine production and that this appears to be due to inhibition of an early step in lymphocyte stimulation.     

Immunologic significance of nonspecific suppressor cells in spleens of tumor-bearing mice.

Spleen cells from mice bearing a progressively growing syngeneic tumor failed to respond to stimulation with mitogens in vitro. This lack of reactivity was due to the presence of nylon wool-adherent cells in the population that could inhibit the mitogen response of normal lymphocytes. Paradoxically, at times when strong suppressor cell activity could be detected in tumor-bearing mice, the animals responded normally to in vivo immunization with sheep erythrocytes and allogeneic tumors, and to in vitro sensitization with allogeneic tumor cells. Regression of a highly antigenic syngeneic tumor also was unaffected by the presence of these suppressor cells. Thus, the occurrence of nonspecific suppressor cells in the spleens of tumor-bearing mice did not influence the overall immunologic competence of these animals.     

Colorimetric determination of acetaldehyde in the presence of formaldehyde

A procedure is described that enables use of the p-phenylphenol color reaction to determine acetaldehyde in the presence of formaldehyde. The sample is first treated with an acidic 2,4-pentanedione reagent, which selectively removes formaldehyde. The method is applicable to blochemical reactions using tissue preparations.