The regulatory G protein signaling complex, Gß5-R7, promotes glucose- and extracellular signal-stimulated insulin secretion.

G protein-coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gß5-R7, a protein complex consisting of the atypical G protein ß subunit Gß5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit ß 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M-stimulated glucose-stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5-/- islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K+-evoked membrane depolarization. These results indicate that Gß5-R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gß5-R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release.

Association of maternal anti-HLA class II antibodies with protection from allergy in offspring.

BACKGROUND: Recent studies have suggested that the birth order effect in allergy may be established during the prenatal period and that the protective effect may originate in the mother. HLA class II disparity between mother and foetus has been associated with significantly increased Th1 production. In this study, we investigated whether production of HLA antibodies 4 years after pregnancy with index child is associated with allergic outcomes in offspring at 8 years. METHODS: Anti-HLA class I and II antibodies were measured in maternal serum (n = 284) and levels correlated to numbers of pregnancies and birth order, and allergic outcomes in offspring at 8 years of age. RESULTS: Maternal anti-HLA class I and II antibodies were significantly higher when birth order, and the number of pregnancies were larger. Anti-HLA class II, but not class I antibodies were associated with significantly less atopy and seasonal rhinitis in the offspring at age 8 years. Mothers with nonatopic (but not atopic) offspring had a significant increase in anti-HLA class I and II antibodies with birth order. CONCLUSION: This study suggests that the 'birth order' effect in children may be due to parity-related changes in the maternal immune response to foetal antigens. We have observed for the first time an association between maternal anti-HLA class II antibodies and protection from allergy in the offspring. Further work is required to determine immunologically how HLA disparity between mother and father can protect against allergy.

Acute alcohol intoxication suppresses natural killer cell activity and promotes tumor metastasis.

Alcohol consumption is associated with increased morbidity and mortality related to infectious diseases and malignancy (1-5), although immune mediation of these relationships is controversial. Specifically, the activity of natural killer (NK) cells, which are involved in the resistance to infections and metastasis, can be suppressed in the presence of ethanol in vitro. However, acute consumption or infusion of ethanol in vivo exerts no effects on NK activity assessed in vitro thereafter. Therefore, we have developed and used a method to study the effects of ethanol on NK activity in living rats by using an NK-sensitive metastatic process and selective depletion of NK cells in vivo. Acute ethanol intoxication caused a marked suppression of NK activity in vivo and a tenfold increase in the number of MADB106 tumor metastases. Ethanol had no effect in rats selectively depleted of NK cells or when an NK-insensitive tumor (C4047) was used. These findings suggest that even acute ethanol intoxication markedly suppresses NK activity in the living organism. This suppression may underlie some aspects of the association between alcoholism, infectious disease and malignancies.

Vitamin d3-induced calcium-binding protein in chick intestinal mucosa.

The administration of vita-min D(3) to rachitic chicks induces in intestinal mucosal tissue the formation or elaboration of a calcium-binding factor which is found in the supernatant of the mucosal homogenate. The enhanced binding of Ca by the "vitanmin D" supernatant (in contrast to "rachitic" supernatant) was indicated by a slower rate of diffusion of Ca(45) across a cellophane dialyzing membrane and by a lesser amount of Ca(45) being bound to an ion-exchange resin (Chelex-100) in the presence of vitamiiin D(3) supernatant. The binding activity was only associated with the protein fraction from a Sephadex G-25 column and was destroyed by trypsin digestion. This and other evidence suggest that the soluble factor is a protein. The vitamin D(3)-enhanced duodenal absorption of Ca(47) in rachitic chicks occurred almost simultaneously with the appearance of the vitamin D(3)-induced factor, and there was good correlation between the concentration of binding factor and the rate of absorption of Ca(47).

Morphine and enkephalin: analgesic and epileptic properties.

Systemic and intracerebroventricular administration of analgesic doses of morphine resulted in large increments of spontaneous multiple unit activity in the periaqueductal gray matter of the awake rat. Intracerebroventricular injection of methionine enkephalin gave analgesia in only 8 of 19 rats, but in all 8, and in no others, increased periaqueductal multiple unit firing was also seen. These findings support the view that the periaqueductal gray matter is actively involved in endogenous mechanisms of analgesia. A striking observation was that enkephalin caused electrographic and behavioral epileptic phenomena in most animals. This observation together with other recent findings suggests that endogenous enkephalin may play some role in epileptogenesis.

Resting-state global functional connectivity as a biomarker of cognitive reserve in mild cognitive impairment.

Cognitive reserve (CR) shows protective effects in Alzheimer's disease (AD) and reduces the risk of dementia. Despite the clinical significance of CR, a clinically useful diagnostic biomarker of brain changes underlying CR in AD is not available yet. Our aim was to develop a fully-automated approach applied to fMRI to produce a biomarker associated with CR in subjects at increased risk of AD. We computed resting-state global functional connectivity (GFC), i.e. the average connectivity strength, for each voxel within the cognitive control network, which may sustain CR due to its central role in higher cognitive function. In a training sample including 43 mild cognitive impairment (MCI) subjects and 24 healthy controls (HC), we found that MCI subjects with high CR (> median of years of education, CR+) showed increased frequency of high GFC values compared to MCI-CR- and HC. A summary index capturing such a surplus frequency of high GFC was computed (called GFC reserve (GFC-R) index). GFC-R discriminated MCI-CR+ vs. MCI-CR-, with the area under the ROC = 0.84. Cross-validation in an independently recruited test sample of 23 MCI subjects showed that higher levels of the GFC-R index predicted higher years of education and an alternative questionnaire-based proxy of CR, controlled for memory performance, gray matter of the cognitive control network, white matter hyperintensities, age, and gender. In conclusion, the GFC-R index that captures GFC changes within the cognitive control network provides a biomarker candidate of functional brain changes of CR in patients at increased risk of AD.

Binding proteins from animals with possible transport function.

Several proteins from various animal tissues with possible transport function have been briefly described, with emphasis given to a vitamin D-induced calcium-binding protein (CaBP) implicated in calcium translocation across epithelial membranes. The latter protein was shown to be present in the small intestine, colon, kidney, and the uterus (shell gland) of the chicken. CaBP was also found in the small intestine of the rat, dog, bovine, and monkey. This protein has been isolated in high purity from chick intestinal mucosa and some of its properties determined. Its molecular weight is about 28,000, its formation constant, about 2.6 x 10(5) M(-1), and its binding capacity, 1 calcium atom per protein molecule. Correlative studies have shown that CaBP concentration in intestinal mucosa varies with the calcium absorptive capacity of the gut, thereby suggesting that CaBP is intimately involved in the process of calcium absorption. CaBP has been localized in the brush border region of the intestinal absorptive cell and within goblet cells. Among other proteins mentioned were the intrinsic factor required for vitamin B(12) absorption and the protein(s) associated with iron translocation.

Persistent alteration of pituitary-adrenal function in the rat by prepuberal corticosterone treatment.

The possible existence of periods of sensitivity to elevated levels of corticosterone in the postnatal development of the pituitary-adrenal axis was investigated. Long-Evans rats were implanted ip with corticosterone (25 mg/100 g body wt) mixed with an equal amount of cholesterol on postnatal days 3, 6, 12 or 18. Eight blood samples were obtained from each animal at various ages by cardiac puncture. Steroid treatment during the first postnatal week resulted in lowered adult basal levels of plasma corticosterone without affecting the diurnal rhythm. Males treated on day 3 had lowered peak (PM) and trough (AM) levels of the steroid as adults, while males treated on day 6 showed only a lowered PM level. A similar reduction in basal levels of plasma corticosterone was observed in immature females, but did not persist into adulthood. Steroid treatment at day 12 had no effect on basal or diurnal pituitary-adrenal activity. Treatment at day 18 reduced the amplitude of the adult diurnal rhythm in both sexes and delayed the onset of the rhythm in males. No differences in stress responsiveness or adrenal weights were detected. Thus, two distinct periods exist in the postnatal development of the rat during which basal and diurnal pituitary-adrenal activity can be differentially and persistently altered by treatment with the naturally occurring glucocorticoid.

Effects of ethanol on CRF release in vitro.

Acute ethanol exposure produces activation of the brain-pituitary-adrenal (BPA) axis, resulting in the release of ACTH, beta-endorphin, and glucocorticoids. While elevated levels of plasma glucocorticoids are also found after chronic ethanol administration, plasma ACTH and beta-endorphin are normal or reduced. It is also unclear whether chronic ethanol exposure results in tolerance to the stimulatory effect of ethanol on BPA activity. To determine the site and mechanism of ethanol action on the BPA axis we studied the CRF secretory profile in a superfused rat hypothalamic preparation after chronic ethanol administration in vivo and the CRF responses after acute ethanol exposure in vitro. Superfused hypothalami from normal and pair-fed control rats released CRF-like immunoreactive material (CRF-LI) in a pulsatile manner, with a mean (+/- SE) frequency of 5.1 +/- 0.7 pulses/h. In contrast, the pulse frequency of CRF-LI release from hypothalami of rats receiving chronic ethanol treatment (fed an alcohol-containing liquid diet for 2 weeks) increased dramatically; the basal mean CRF level, pulse amplitude, and pulse duration remained unchanged. Hypothalamic CRF content was decreased. This chronic ethanol exposure also altered the dose-response characteristics of CRF release when ethanol was introduced acutely, as a pulse, into the in vitro preparation. Acute exposure to 20 mg/100 ml ethanol produced greater release of CRF-LI from control hypothalami than from chronic ethanol-exposed hypothalami. A further elevation above basal levels was produced by 200 mg/100 ml ethanol in control, but not ethanol-exposed, hypothalami. Secretion of CRF from ethanol-exposed hypothalami in response to depolarizing concentrations of potassium chloride was suppressed. Chronic ethanol treatment had no effect on CRF-LI and CRF bioactivity responses to stimulation with acetylcholine. These findings suggest the presence of a high frequency pulse-generating mechanism for CRF release in the hypothalamus. This pulsatile secretory mechanism is altered by chronic ethanol exposure of the animals in vivo. Chronic intoxication resulted in tolerance to the stimulatory effect of ethanol on CRF release in vitro.

Immunodiffusion using tissue sections.

A simple radial immunodiffusion technique is described for determining relative antigen content of frozen tissue sections. Its use is illustrated with frozen sections of chick intestine containing the vitamin D-induced calcium-binding protein (CaBP). Diffusion of the intestinal CaBP from the sections placed on agar containing specific anti-CaBP anti-serum produced a precipitin ring, whose diameter was proportional to the CaBP content of the intestine. The technique allowed the determination of the relative antigen content in sections of tissue adjacent to others in which immunocytochemical localization or other histochemical studies were to be performed.

Fetal alcohol exposure attenuates interleukin-1beta-induced fever: neuroimmune mechanisms.

Central mechanisms for the attenuating effects of fetal alcohol exposure (FAE) on interleukin-1beta (IL-1beta)-induced fever were studied in adult male offspring of dams fed a liquid diet supplemented with ethanol (E), in pair-fed (P) control and in normal (N) offspring. Hypothalamic levels of IL-1 were significantly lower in E than in N rats at 2 h, but not at 4 and 6 h, after intraperitoneal administration of lipopolysaccharide. Fever induced by intracerebroventricular (i.c.v.) IL-1 was significantly lower in E than in N and P rats. In contrast, E rats showed a normal febrile response to i.c.v. prostaglandin-E2. Thus, whereas FAE does not affect central thermoregulatory mechanisms, per se, FAE alters the kinetics of hypothalamic IL-1 production/appearance and decreases the responsiveness of central mechanisms which mediate the febrile response to IL-1.

Intracerebroventricular interleukin-1beta impairs clearance of tumor cells from the lungs: role of brain prostaglandins.

We have previously demonstrated that central administration of interleukin (IL)-1beta suppresses natural killer (NK) cell activity, impairs NK-mediated lung clearance of tumor cells, and enhances tumor colonization. The central pathways activated by IL-1beta are as yet unknown. Using an in vivo model of tumor colonization, this study examined the role of central noradrenergic, opioid and prostaglandin mechanisms in mediating the effect of IL-1beta on lung clearance of tumor cells. We demonstrate that central noradrenergic and opioid systems are not critically involved in this effect. Neither depletion of central noradrenergic pathways, or administration of the opioid antagonist, naltrexone (50 ug), blocked the impaired lung clearance of MADB106 tumor cells induced by central administration of IL-1beta (20 ng). Central prostaglandins (PGs) do, however, appear to play a critical role. Central administration of the prostaglandin antagonist, diclofenac (250 ug), but not ibuprofen, completely blocked the effect of IL-1beta on lung clearance of tumor cells. Antagonism of the effects of IL-1beta was shown to be due to the effects of centrally and not of peripherally acting prostaglandins.

The development of sexual dimorphism in natural killer cell activity and resistance to tumor metastasis in the Fischer 344 rat.

The development of sexual dimorphism in the number and activity level of natural killer (NK) cells was studied in the inbred Fischer 344 rat from prepubescence to maturity. Additionally, in view of the biological significance of NK cells in controlling cancer, especially the metastatic process, we used a syngeneic mammary tumor (MADB106) to assess the host anti-metastatic activity. This tumor model was used because NK cells control the lung clearance of i.v.-injected MADB106 tumor cells, a process that critically affects the metastatic colonization of these tumor cells in the lungs. The results indicated that although prepubescent (36 days of age) males and females exhibited greater NK cytotoxicity (assessed in vitro) and higher anti-metastatic activity, evidenced by fewer tumor cells retained in the lungs. On the other hand, the mature males (140-170 days of age) displayed greater LGL/NK number and activity per ml blood, retained fewer tumor cells, and developed fewer lung tumor colonies compared to the females. During early postpubescence (63 days of age), a transitional stage between prepubescence and maturity, females and males exhibited equivalent numbers of circulating LGL/NK cells, and females displayed slightly greater NK cytotoxicity per ml blood yet retained somewhat greater numbers of tumor cells compared to the males. Overall, whereas the males exhibited increasing levels of NK number and activity throughout the age span tested, the females, despite displaying greater NK function compared to the males at prepubescence and slight improvement at postpubescence, fell behind the males in these indices of NK function at maturity.

The glossopharyngeal nerve as a novel pathway in immune-to-brain communication: relevance to neuroimmune surveillance of the oral cavity.

Glossopharyngeal afferents may be the neural channel by which immune challenge of the posterior oral cavity conveys information to the brain. If this is the case, then bilateral transection of the glossopharyngeal nerves (GLOx) should disrupt this communication. Injection of lipopolysaccharide (LPS) or interleukin (IL)-1beta into the soft palate (ISP) of sham-operated rats induced a dose-related febrile response. GLOx significantly attenuated the febrile response induced by ISP injection of both LPS and IL-1beta. In contrast, GLOx did not affect the febrile response when LPS or IL-1beta were injected intraperitoneally, indicating that the effect of GLOx is not systemic. These results provide experimental evidence for a novel neural pathway for immune-to-brain communication.

Intestinal vitamin D-induced calcium-binding protein: time-course of immunocytological localization following 1,25-dihydroxyvitamin D3.

The vitamin D-induced calcium-binding protein (CaBP) was localized in histological sections of chick duodenum using the peroxidase-antiperoxidase immunocytochemical technique. The time-course of appearance of CaBP in rachitic chicks was investigated from 0 to 120 hr after stimulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). CaBP was not routinely detected at 0 hr after 1,25(OH)2D3 administration. CaBP was first noted in some, but not all, of the samples taken 2 hr following 1,25(OH)2D3 and was detected in all 2 1/2 hr samples. The number of CaBP-containing absorptive cells and the apparent CaBP concentration both increased to a maximum at about 16-24 hr. At later times, as CaBP free cells migrated up the villi, the CaBP-containing cells decreased in number, but even at 120 hr post 1,25(OH)2D3 dose there were significant numbers of CaBP-containing cells present. The relationships between time-course of CaBP location on intestinal villi, enterocyte migration rates, and the time-course of 1,25(OH)2D3 stimulated intestinal calcium transport are discussed.

Immunocytochemical localization of the vitamin D-induced calcium-binding protein: relocation of antigen during frozen section processing.

The vitamin D-induced calcium-binding protein (CaBP) was localized in chick duodenum by the indirect fluorescent antibody technique after tissue was prepared by three different rapid-freezing methods: freeze-thaw, freeze-drying, and freeze-substitution. Sections prepared by freeze-thawing demonstrated CaBP-specific fluorescence over goblet cells and at the absorptive surface of villi, but not in absorptive cells. Sections from the same or adjacent segments prepared by freeze-drying or freeze-substitution produced virtually the opposite pattern of CaBP distribution. CaBP-specific fluorescence was associated with absorptive cell cytoplasm but not with goblet cells. From the results of experiments in which 6 micrometer freeze-dried sections were rehydrated, it was concluded that in the presence of an aqueous environment CaBP migrated from absorptive cells to discrete localization sites bound to goblet cell mucus, possibly to the calcium present in the mucus. By analogy, it was concluded that CaBP in association with goblet cells and the absorptive surface in sections prepared by the freeze-thaw method represented an artifactual localization. The true in situ localization of CaBP in chick duodenum was that which was present in absorptive cell cytoplasm.

Tooth formation and the 28,000-dalton vitamin D-dependent calcium-binding protein: an immunocytochemical study.

Antiserum to the 28-kilodalton vitamin D-dependent calcium-binding protein (CaBP) was used to localize CaBP in histologic sections of the continuously erupting incisor in mandibles obtained from normal rats. With the peroxidase--anti-peroxidase technique, no CaBP was detected in undifferentiated ameloblasts or in those which had become columnar and were facing pulp. Calcium-binding protein was first noted in the cytoplasm of random ameloblasts facing dentin in the presecretion zone. As the ameloblasts became more mature in the zone of enamel secretion, CaBP was uniformly present in their cytoplasm. Ameloblasts with Tome's processes clearly contained CaBP in these processes as well as in the cell-body cytoplasm. Near the later developmental stages of the zone of enamel secretion, some of the adjacent underlying cells of the stratum intermedium also contained CaBP in their cytoplasm. In some stratum intermedium cells and papillary cells, CaBP extended into the zone of enamel maturation, but not to the end of that zone. Cytoplasmic CaBP continued to be present in ameloblasts as they progressed through the zone of enamel maturation to the final, shortened cells at the gingival margin of the erupting incisor. No CaBP was detected in odontoblasts, pulpal cells, the stellate reticulum, or the outer dental epithelium.

1,25-Dihydroxyvitamin D3-induced calcium-binding protein: localization in organ-cultured embryonic chick duodenum.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces de novo biosynthesis of a specific calcium-binding protein (CaBP) in embryonic chick duodenum in organ culture. Using a highly sensitive and specific, peroxidase-antiperoxidase immunocytochemical procedure, 1,25(OH)2D3-induced CaBP in the organ-cultured duodenum was found only in the cytoplasm of absorptive cells, corresponding to its localization in rachitic chick duodenal cells after a single injection of 1,25(OH)2D3 in vivo. This observation, along with evidence correlating CaBP with calcium transport, strongly supports the use of the embryonic chick duodenal organ culture system as a physiologically relevant model of the vitamin D-dependent calcium absorptive mechanism.

Calbindin-D immunolocalization in developing chick thyroid: a light and electron microscopic study.

Antiserum to calbindin-D, a 28 KD vitamin D-dependent calcium binding protein, was used to localize the protein immunocytochemically in developing chick thyroid by both light and electron microscopy. The protein first appeared in future follicular cells of developing thyroid tissue from 8-day-old embryos. The number of calbindin-D-containing cells increased rapidly to a near-plateau level at day 10; this concentration was sustained until day 15, and then declined to an undetectable level just before hatching. The protein was distributed throughout organelle-free areas of the follicular cell cytoplasm and extended into the nucleus; it was not present in the follicular colloid. Comparison of the time course of changes in calbindin-D content with known differentiative changes taking place in follicular cells suggests that the protein may function in some yet to be determined mechanism related to normal development of the thyroid.

Immunocytochemical localization of rat intestinal vitamin D-dependent calcium-binding protein.

Vitamin D-dependent calcium-binding protein (CaBP) was localized in intestinal tissue sections obtained from rats raised under three different nutritional conditions: a normal vitamin D-replete diet, a vitamin D-free diet followed by supplementation with vitamin D3, or a vitamin D-free diet without additional supplementation. An indirect immunoperoxidase technique, with immunocontrols, was used to visualize the specific sites of CaBP. CaBP was visualized only in the cytoplasm of absorptive cells. In the duodenum of animals raised on a normal diet, CaBP was present in absorptive cells from the upper crypt region to the villus tips. In the jejunum, many fewer absorptive cells contained CaBP, while in the ileum only random absorptive cells near the villus tips contained CaBP. In rats raised on a vitamin D-deficient diet then supplemented with vitamin D3, CaBP was present in cells at the full depth of the crypts and in absorptive cells along the total villus length in the duodenum. Rats raised on the same deficient diet but without supplementation with additional vitamin D exhibited no CaBP in crypt cells nor in absorptive cells more than half way up the villi. Absorptive cells higher on the villi contained immunoreactive CaBP but the intensity of immunostaining and number of CaBP-containing cells was markedly reduced compared to the vitamin D-supplemented group.