Transient co-localization of calretinin,parvalbumin, and calbindin-D28k in developing visual cortex of monkey

Brain Cell Biology - This paper reports a double-labelling immunocytochemical study of the three calcium-binding proteins calretinin, parvalbumin, and calbindin-D28k in developing and adultMacaca...     

Developmental sex differences in calbindin-D(28K) and calretinin immunoreactivity in the neonatal rat hypothalamus

The proteins calbindin-D(28K) and calretinin buffer intracellular calcium and are speculated to be involved in the integration of neuronal signaling. Using Western blot analysis, we compared the levels of calbindin-D(28K) and calretinin in the developing male and female rat hypothalamus on postnatal days (PN) 0, PN2, PN4, PN6, PN8, and PN10. Analysis of variance (ANOVA) of mean calbindin levels indicated a significant effect of sex (p 相似文献   

Adenylyl cyclase and the control of cell differentiation in Dictyostelium dicoideum.

Adenylyl cyclase is part of a biochemical network that controls cell differentiation in Dictyostelium discoideum. At a certain stage of development the enzyme is rhythmically activated, with periods of about 8 min. These oscillations are superimposed upon an increase of the basal activity extending over a period of hours. The basal activity remains low in a mutant blocked at an early stage of development. In strain Ax-2 two periods of strongly increasing basal activity have been found: the first from 2 to 4 h after the end of the growth phase, the other beginning at about 8 h. Based on the periodic regulation of adenylyl cyclase, cyclic AMP is released into the extracellular space in the form of pulses. Application of cyclic-AMP pulses, but not its continuous influx, stimulates the increase of basal adenylyl cyclase activity. Two other constituents of the cyclic-AMP signal system cyclic-AMP receptors and cell-surface phosphodiesterase, are similarly controlled. The principal importance of positive feedback loops in a network controlling cell differentiation is discussed.     

The effect of different maternal deprivation paradigms on the expression of hippocampal glucocorticoid receptors, calretinin and calbindin-D28k in male and female adolescent rats

Maternal deprivation (MD) is a well-established protocol used to investigate neurobiological changes that are associated with the etiology of and vulnerability to stress-related diseases in animal models. The resulting psychophysiological effects, the timing and duration of these adverse stimuli, and the method by which they exert their effects on the animals remain unclear. This study characterized differences in the hippocampal expression of glucocorticoid receptors (GRs) and the calcium-binding proteins calretinin (CALR) and calbindin-D28k (CALB) in male and female rats that underwent different MD paradigms during the stress hyporesponsive period (SHRP). Both GRs and the two calcium-binding proteins were much more abundant in females than in males. MD paradigms had a significant effect on CALR and CALB expression in both males and females but affected GR levels only in males. Additionally, expression of the two calcium-binding proteins in the hippocampus responded differently to MD-induced stress, especially in females. Taken together, these results indicate that females are able to modulate their response to stress better than males.     

Ontogeny of calbindin-D28K and calbindin-D9K in the mouse kidney, duodenum, cerebellum and placenta.

The appearance of the calcium-binding proteins (CaBP-D28K and CaBP-D9K) in embryonic mice tissues was determined using a sensitive immunohistochemical assay. CaBP-D28K first appears in myenteric nerve plexuses of the duodenum on day E15, in duodenal villus cells on day E16, in Purkinje cells of the cerebellum on day E19, in cells of the mesonephric duct on day E11 and in the metanephric duct on day E12. CaBP-D9K first appears in enterocytes of the duodenum on day E18, in trophoblastic giant cells (TGC) of the placenta on day E10, and in the metanephric duct on day E15. A differential time of appearance and colocalization of the two CaBPs is demonstrated in the embryonic mouse kidney, suggesting either that vitamin D does not control both CaBPs in the foetus or that the vitamin D control is unequal. The early appearance and location of CaBP-D9K in TGCs may suggest that these cells play an important role in transplacental transfer of calcium.     

Adenylate cyclase in membrane fractions of RIN-A2-cells: studies with forskolin, NaF, GppNHp and NEM

In crude membrane fractions of rat pancreatic islets and of RIN-A2-cells, forskolin and NaF stimulated adenylate cyclase activity. Basal and stimulated enzyme activity was approximately 3 to 6 fold higher in membranes of RIN-A2-cells than in membranes of islet cells. In RIN-A2-cells GppNHp and NEM inhibited forskolin-stimulated enzyme activity. The inhibitory effect of GppNHp could be reduced by NEM. It is suggested that the adenylate cyclase system of RIN-A2-cells contains inhibitory and stimulatory N-proteins and that there are critical thiols related to Ni, Ns and/or the catalytic unit. Thus, membrane fractions of RIN-A2-cells may be an appropriate model for studies on the adenylate cyclase system of insulin-producing cells.     

Activation of renal adenylate cyclase by forskolin: assessment of enzymatic activity in animal models of the secondary hyperparathyroid state

The effects of forskolin on kidney slice cyclic AMP content and membrane adenylate cyclase activity were studied in order to determine whether or not activation of the enzyme by forskolin was affected in experimental animal models of the secondary hyperparathyroid state. Forskolin was found to be a potent activator of renal adenylate cyclase in rats and chicks, and the diterpene produced a marked potentiation of the cyclic AMP response to parathyroid hormone (PTH). The diterpene had no effect on the binding of PTH to renal receptors. Activity of adenylate cyclase in the presence of forskolin was similar in renal membranes from either vitamin D-deficient rats or chicks compared to control. Forskolin did not restore full responsiveness to PTH in renal slices from chicks raised on diets that were deficient in either vitamin D or calcium although the diterpene was capable of potentiating the cyclic AMP response to PTH in these tissues. Forskolin also augmented the activation of membrane adenylate cyclase by PTH although this effect of the diterpene was much less prominent in membrane preparations than that observed in renal slices. This study provided additional evidence that the downregulation of renal PTH-dependent adenylate cyclase in experimental models of secondary hyperparathyroidism is due to a specific reduction in receptor-mediated regulation of cyclic AMP formation. Adenylate cyclase activity as assessed by forskolin-stimulated enzyme activity was fully maintained in kidney membranes from these animal models. Thus, forskolin appears to be a useful drug for measuring total enzymatic activity in situations where altered responsiveness of adenylate cyclase to hormones has been demonstrated to be mediated by changes in hormone receptors.     

Calmodulin-dependent adenylate cyclase activity in rat cerebral cortex: effects of divalent cations, forskolin and isoprenaline

The role of calcium-calmodulin (Ca2+-CaM) in the modulation of beta-adrenergic adenylate cyclase activity in rat cerebral cortex has been studied. In addition, the effects of manganese (Mn2+) and forskolin on CaM-dependent enzyme activity were investigated. At 2 mM magnesium (Mg2+) low concentrations of Ca2+ stimulated the enzyme activity (Ka 0.25 +/- 0.08 microM), whereas higher Ca2+ levels (greater than 2 microM) inhibited the activity. No activating effect of Ca2+ was observed in CaM-depleted membranes, but the inhibitory effect persisted and the stimulatory action of Ca2+ could be restored by addition of exogenous CaM. The ability of Ca2+ to activate the enzyme was reduced by increasing concentrations of Mg2+. At 10 mM Mg2+ the apparent Ka of Ca2+ was 0.55 +/- 0.16 microM and half-maximal inhibition was observed at 80-120 microM Ca2+. A synergistic effect was observed between Ca2+ and isoprenaline on the adenylate cyclase activity. Calcium did not alter the apparent Ka of isoprenaline (0.9 +/- 0.27 microM) and isoprenaline did not change the apparent Ka of Ca2+. However, isoprenaline decreased the apparent Ka of CaM; 0.11 +/- 0.07 micrograms vs. 0.32 +/- 0.1 micrograms (0.5 ml assay mixture)-1, with and without isoprenaline, respectively. A synergistic effect was also observed between Ca2+ and forskolin, but no change in their apparent Ka values was found. Furthermore, Mn2+ was found to activate the enzyme through CaM. These data demonstrate that Ca2+ -CaM potentiates beta-adrenergic adenylate cyclase activity and thus is able to modulate neurotransmitter stimulation in cortex. Furthermore, both forskolin and Mn2+ affect CaM-dependent enzyme activity. Forskolin potentiates Ca2+-CaM stimulation, while Mn2+ increases the activity by activating the enzyme through CaM.     

IL-28A,IL-28B,and IL-29: Promising cytokines with type I interferon-like properties

IL-28A, IL-28B and IL-29 (also designated type III interferons) constitute a new subfamily within the IL-10–interferon family. They are produced by virtually any nucleated cell type, particularly dendritic cells, following viral infection or activation with bacterial components, and mediate their effects via the IL-28R1/IL-10R2 receptor complex. Although IL-28/IL-29 are closer to the IL-10-related cytokines in terms of gene structure, protein structure, and receptor usage, they display type I interferon-like anti-viral and cytostatic activities. Unlike type I interferons, the target cell populations of IL-28/IL-29 are restricted and mainly include epithelial cells and hepatocytes. These properties suggest that IL-28/IL-29 are potential therapeutic alternatives to type I interferons in terms of viral infections and tumors. This review describes the current knowledge about these cytokines.     

Calbindin-D28k and calretinin in the rat posterior pituitary; Light and electron microscopic localization and upregulation with dehydration

Ca²⁺ binding proteins (CaBPs), calbindin-D_28k (calbindin) and calretinin, are thought to contribute to the regulation of intracellular Ca²⁺ in many neuronal populations and perhaps more importantly, signal functional modulation in neuronal activity. In the present experiments, light microscopic immunohistochemistry revealed that the immunoreactivity of calbindin and calretinin was contained in varicose axons in the posterior pituitary. The dual labeling study with confocal microscopy demonstrated that calbindin immunoreactivity was present in the terminals of both oxytocin (OXT) and arginine-vasopressin (AVP) neurons. However, calretinin immunoreactivity was exclusively seen in the OXT terminals. Moreover, the dual labeling study showed that most calretinin-positive terminals contained calbindin immunoreactivity, demonstrating the colocalization of calbindin and calretinin in the same OXT nerve terminals. By electron microscopy, calbindin and calretinin immunoreactivities were seen in the neurosecretory axons and nerve terminals. These immunoreactive nerve terminals were seen to contain more clear microvesicles than dense-core neurosecretory granules. This immunoelectron microscopic observation suggests that both calbindin and calretinin localize preferentially in the active zone of the nerve terminals, which usually face the perivascular space around fenestrated capillaries. In spite of similar localization of calbindin and calretinin within the posterior pituitary, Western blot analysis showed some differences between the two CaBPs. Calbindin was present mostly in the soluble fraction with little in the insoluble fraction, but a substantial portion of calretinin was present in both the insoluble and soluble fractions. Moreover, dehydration induced by drinking 2% NaCl solution and deprivation of drinking water increased calretinin levels in the posterior pituitary as compared with control, but the calbindin level was not changed. The present findings demonstrate that calbindin and calretinin colocalize in the active zones of OXT nerve terminals, but only calretinin is upregulated with dehydration, suggesting different physiological role of calbindin and calretinin in the nerve terminals.     

Muscle activity in steady swimming scup, Stenotomus chrysops, varies with fiber type and body position

The red and pink aerobic muscle fibers are used to power steady swimming in fishes. We examined red and pink muscle recruitment and function during swimming in scup, Stenotomus chrysops, through electromyography and high-speed ciné. Computer analysis of electromyograms (EMGs) allowed determination of initial speed of muscle recruitment and duty cycle and phase of muscle electromyographic activity for both fiber types. This analysis was carried out for three longitudinal positions over a range of swimming speeds. Fiber type and longitudinal position both affected swimming speed of initial recruitment. Posterior muscle is recruited at the lowest swimming speed, whereas more anterior muscle is not initially recruited until higher speeds. At more anterior positions, the initial recruitment of pink muscle occurs at a higher swimming speed than the recruitment of red muscle. The duty cycle of pink muscle EMG activity is significantly shorter than that of red muscle, reflecting a difference in the onset time of activation during each cycle of length change: pink muscle onset time follows that of red. The different patterns of usage of red and pink muscle reflect differences in their contraction kinetics. Because pink muscle generates force more rapidly than red muscle, it can be activated later in each tailbeat cycle. Pink muscle is used to augment red muscle power production at higher swimming speeds, allowing a higher aerobically based steady swimming speed than that possible by red muscle alone.     

Morphogenesis of the saccus vasculosus of turbot Scophthalmus maximus: assessment of cell proliferation and distribution of parvalbumin and calretinin during ontogeny

The ontogenesis of the saccus vasculosus (SV) of turbot Scophthalmus maximus is described using histological and immunohistochemical methods to assess the general morphology, as well as the distribution of proliferative cells and several calcium‐binding proteins (CaBP). The results reveal that the SV begins to differentiate on hatching, when immature coronet cells are morphologically distinguishable. Further morphogenesis involves the formation of a tubular avascular SV, which remains until premetamorphic larval stages. Folding and vascularization of the SV occurs mostly during metamorphosis, when S. maximus settle down on the bottom. Proliferative cells were placed within the SV itself and in the neighbouring infundibular hypothalamus. Their putative relationship with the growth of the SV is discussed. The CaBPs analysed are expressed in coronet cells. Parvalbumin is expressed in these cells from the beginning of their differentiation, while calretinin expression arises in the tubular SV and becomes more widespread over time. These data emphasize the importance of calcium buffering in the function of coronet cells.     

Calbindin D28k, parvalbumin, and calretinin immunoreactivity in the main and accessory olfactory bulbs of the gray short-tailed opossum, Monodelphis domestica

The vertebrate main and accessory olfactory bulbs (MOB and AOB) are the first synaptic sites in the olfactory pathways. The MOB is a cortical structure phylogenetically well conserved in its laminar structure and overall synaptic organization, while the AOB has significant species variation in size. In order to better understand signal processing in the two olfactory systems and the species differences, immunocytochemical staining and analysis were done of the neuronal expression patterns of the calcium-binding proteins calbindin D28k (CB), parvalbumin (PV), and calretinin (CR) in the MOB and AOB in a marsupial species, the gray short-tailed opossum, Monodelphis domestica. In the MOB, antibody to CB labeled periglomerular cells, superficial short axon cells / Van Gehuchten cells; antibody to PV labeled Van Gehuchten cells; and antibody to CR immunostained periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. In the AOB, CB immunoreactivity was detected in periglomerular cells and a subpopulation of granule cells; antibody to PV labeled the superficial short axon cells / Van Gehuchten cells and granule cells; and antibody to CR labeled a small number of periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. These results showed that the patterns of CB, PV, and CR expression differ in the opossum main and accessory olfactory bulbs and differ from that in other animal species. These varying patterns of neuronal immunostaining may be related to the different functions of the main and accessory olfactory bulbs and to the differing signal processing features.     

Simultaneous purification of merozoites and schizonts of Eimeria tenella (Apicomplexa) by Percoll flotation and assessment of cell viability with a double fluorescent dye assay.

The asynchronous development of Eimeria tenella in orally infected chickens makes it possible to purify second generation merozoites (meros) and shizonts from a single mucosal homogenate. After centrifugation in 30% Percoll in phosphate-buffered saline (Percoll-PBS), debris, villi, and schizonts float, whereas meros and erythrocytes are pelleted. Erythrocytes are lysed by a mild hypotonic shock; meros are filtered through a cotton wool plug and collected by centrifugation. The 30% Percoll-PBS supernatant fraction is diluted to 25% Percoll-PBS and centrifuged to sediment mature schizonts. By repeated slow-speed centrifugation, schizonts are separated from nuclei and small-sized debris. In less than 3 hr, 8.8 +/- 2.3 x 10(8) meros and 7.2 +/- 3.9 x 10(6) schizonts are collected from 10 infected chickens. Contamination with host material is 2% for meros but variable for schizonts. For the assessment of cell viability, ethidium bromide (EB) and acridine orange (AO) have been used as markers for dead and living cells, respectively, in a single step method. More than 95% of the schizonts and meros accumulate AO and no EB, whereas lysed erythrocytes and all cells hosting a schizont are permeable to EB. After incubation of meros and schizonts in synthetic media with [5,6- 3H]uracil, label accumulates in the perchloric acid-soluble and -insoluble fractions, indicating transport, salvage, and incorporation of the pyrimidine precursor in nucleic acids. If stored on ice, meros and schizonts retain metabolic activity for at least 5 hr, but metabolism declines rapidly during incubation at 41 C.     

Distribution of P2Y6 and P2Y12 receptor: their colocalization with calbindin, calretinin and nitric oxide synthase in the guinea pig enteric nervous system

The distribution of P2Y₆ and P2Y₁₂ receptor-immunoreactive (ir) neurons and fibers and their coexistence with calbindin, calretinin and nitric oxide synthase (NOS) has been investigated with single and double labeling immunostaining methods. The results showed that 30–36% of the ganglion cells in the myenteric plexus are strongly P2Y₆ receptor-ir neurons; they are distributed widely in the myenteric plexus of stomach, jejunum, ileum and colon, but not in the submucosal plexus, with a typical morphology of multipolar neurons with a long axon-like process. About 42–46% of ganglion cells in both the myenteric and submucosal plexuses show P2Y₁₂ receptor-ir. About 28–35% of P2Y₆ receptor-ir neurons were found to coexist with NOS and 41–47% of them coexist with calretinin, but there was no coexistence of P2Y₆ receptor-ir with calbindin. In contrast, all P2Y₁₂ receptor-ir neurons were immunopositive for calbindin, although occasionally P2Y₁₂ receptor-ir neurons without calbindin immunoreactivity were found, while none of the P2Y₁₂ receptor-ir neurons were found to coexist with calretinin or NOS in the gastrointestinal system of guinea pig. The P2Y₁₂ receptor-ir neurons coexpressing calbindin-ir in the small intestine are Dogiel type II/AH, intrinsic primary afferent neurons.     

Alteration with dietary state of the activity and zonal distribution of adenylate cyclase stimulated by glucagon, fluoride and forskolin in microdissected rat liver tissue

Adenylate cyclase activated by glucagon, fluoride and forskolin was measured in liver homogenates and microdissected periportal and perivenous tissue of fed and fasted rats. A radiochemical microtest, more sensitive by 2-3 orders of magnitude as compared with the usual assay, was established for the determination of the activity in liver samples corresponding to 200-600 ng dry weight. In liver homogenates from fasted as compared to fed animals the glucagon-stimulated and fluoride-stimulated activity was increased by 1.65-fold, while the basal and the forskolin-stimulated activity remained the same. In microdissected tissue of both fed and fasted animals the activity was stimulated in about 60% of the samples by glucagon, fluoride and forskolin (responsive samples). However, in about 40% of the microdissected tissue samples the activity could not be stimulated by any of the above activators (non-responsive samples). In responsive microdissected tissue of fasted as compared to fed animals, the glucagon-stimulated and fluoride stimulated activity but not the basal and the forskolin-activated activity was increased by 2-3-fold. In responsive microdissected samples of fed animals neither the basal nor the stimulated activities showed a significant periportal to perivenous gradient. In samples of fasted animals, however, a zonal gradient was observed for the glucagon-stimulated activity exhibiting a 1.5-fold higher rate in the perivenous zone.     

Germ-line origins of mutation in families with hemophilia B: the sex ratio varies with the type of mutation.

Previous epidemiological and biochemical studies have generated conflicting estimates of the sex ratio of mutation. Direct genomic sequencing in combination with haplotype analysis extends previous analyses by allowing the precise mutation to be determined in a given family. From analysis of the factor IX gene of 260 consecutive families with hemophilia B, we report the germ-line origin of mutation in 25 families. When combined with 14 origins of mutation reported by others and with 4 origins previously reported by us, a total of 25 occur in the female germ line, and 18 occur in the male germ line. The excess of germ-line origins in females does not imply an overall excess mutation rate per base pair in the female germ line. Bayesian analysis of the data indicates that the sex ratio varies with the type of mutation. The aggregate of single-base substitutions shows a male predominance of germ-line mutations (P < .002). The maximum-likelihood estimate of the male predominance is 3.5-fold. Of the single-base substitutions, transitions at the dinucleotide CpG show the largest male predominance (11-fold). In contrast to single-base substitutions, deletions display a sex ratio of unity. Analysis of the parental age at transmission of a new mutation suggests that germ-line mutations are associated with a small increase in parental age in females but little, if any, increase in males. Although direct genomic sequencing offers a general method for defining the origin of mutation in specific families, accurate estimates of the sex ratios of different mutational classes require large sample sizes and careful correction for multiple biases of ascertainment. The biases in the present data result in an underestimate of the enhancement of mutation in males.     

Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester

This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.