Developmental appearance of the Ca2+-binding proteins parvalbumin,calbindin D-28K,S-100 proteins and calmodulin during testicular development in the rat

Summary Calcium and intracellular Ca²⁺-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.     

Ultrastructural localization of the calcium-binding protein parvalbumin in neurons of the song system of the zebra finch,Poephila guttata

Summary The distribution of parvalbumin (PV) within neurons of the vocal motor nucleus hyperstriatum ventralepars caudalis (HVc) was investigated in the forebrain of adult male zebra finches by means of light and electron microscopy using the indirect immunoperoxidase technique. Parvalbumin-reaction product was located in the amorphous material of perikarya, dendrites and nuclei, and associated to microtubuli, postsynaptic densities and intracellular membranes; it was found in some axons and Gray type-2 boutons, but rarely in type-1 boutons and never in the Golgi apparatus. These observations suggest that parvalbumin may regulate calcium-dependent processes at the postsynaptic membrane and in the cytosol. Furthermore, the partial association of parvalbumin to microtubuli points to an involvement in calcium-dependent tubular functions. Calcium currents and microtubular assembly or transport may be relevant for the known functions of HVc in song learning.     

CBP-18, a Ca2+-Binding Protein in Rat Brain: Tissue Distribution and Localization

Abstract: The distribution of a novel calcium-binding protein with a molecular mass of 18 kDa (CBP-18) in the rat brain was studied by means of biochemical methods and immunohistochemistry on cryostat-sectioned tissue and compared with staining patterns of parvalbumin on adjacent sections. The biochemical analysis revealed high levels of CPB-18 in cortex and cerebellum, low levels in the lungs, and undetectable levels in all other tissues tested. Immunohistochemically, the polyclonal rabbit-derived antibody for CPB-18 showed selective affinity with periglomerular cells and dendrites in the olfactory bulb. Distinct immunostaining of scattered cells and their proximal dendrites was found in the anterior olfactory nuclei and in the perirhinal and entorhinal cortex. Strong staining of neuropil with recognizable but diffusely outlined cells was observed in the retrosplenial cortex, central amygdala, hippocampal rudiment, septum, area preoptica, hypothalamus, colliculus superior, and parabrachial nuclei. The cerebellum showed strong neuropil staining of both the molecular and the granule cell layer. Less intense neuropil staining and a few scattered cells were found in the neocortex, the remaining basal forebrain, and in the entire brainstem. Immunoreactivity was barely detectable or missing in the striatum, the hippocampus, the thalamus, and in the colliculus inferior. Thus, CPB-18 shows a unique staining pattern in the CNS, different from all other Ca²⁺-binding proteins studied so far.     

S100 protein-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

S100-immunoreactivity (ir) was examined in tooth pulp primary neurons of the rat. An immunofluorescence method demonstrated that the molar tooth pulp contained S100-immunoreactive (ir) nerve fibers. In the root pulp, pulp horn and roof of the pulp chamber, S100-ir smooth and varicose fibers ramified and formed subodontoblastic nerve plexuses. All the fibers became varicose at the base of the odontoblastic layer and extended to the odontoblastic layer. Some varicose endings could be traced into the dentin. The trigeminal neurons retrogradely labeled with fluorogold (FG) from the first and second maxillary molar tooth pulps exhibited S100- and parvalbumin-ir. Approximately 60% and 24% of the labeled cells were ir for S100 and parvalbumin, respectively. Virtually all parvalbumin-ir FG-labeled cells showed S100-ir, while 40% of S100-ir ones coexpressed parvalbumin-ir. An immunoelectron microscopic method revealed that all myelinated axons and half of the unmyelinated axons in the root pulp contained S100-ir. In the odontoblastic layer, predentin and dentin, S100-ir neurites lost the Schwann cell ensheathment and made close contact with cell bodies and processes of odontoblasts. The odontoblastic layer also contained parvalbumin-ir neurites. These neurites were devoid of the Schwann cell ensheathment and in close apposition to cell bodies and processes of odontoblasts. S100-ir pulpal axons seemed to be insensitive to repeated neonatal capsaicin treatment. This study suggests that S100-ir tooth pulp primary neurons are mostly myelinated and that S100-ir unmyelinated axons in the root pulp are preterminal segments of myelinated stem axons.     

慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin的表达变化

目的:观察慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin(PV)的表达变化,为其功能的研究提供形态学依据。方法:将30只健康雄性SD大鼠随机分为吗啡依赖组和生理盐水对照组。吗啡依赖组大鼠腹膜腔注射吗啡,2次/d,起始剂量为5 mg/kg,逐日递增5mg,至第10d为50mg/kg;对照组注射同体积的生理盐水。于末次注射后动物分别存活3h、3 d和14d。用免疫组化方法和相对平均灰度值检测杏仁核内PV的表达。结果:在生理盐水处理组各存活时间点,杏仁核内PV的表达相同。和生理盐水对照组相比,3h时杏仁核内PV的表达明显增加(P<0.05)。第3d时,杏仁核内PV的表达减少,明显低于第3 h组(P<0.05)。至第14d时,PV的表达又开始增加,明显高于第3 d组(P<0.05)。结论:本结果提示慢性吗啡处理及戒断后杏仁核PV的表达具有时相特异性;这种变化在戒断早期可能主要与躯体依赖相关,而戒断晚期主要与精神依赖相关。     

Identification of commercial hake and grenadier species by proteomic analysis of the parvalbumin fraction

Analysis of parvalbumin fractions through proteomic methodologies allowed the differential classification of ten commercial, closely related species of the family Merlucciidae. Muscle extracts from nine hake species of the genus Merluccius including two subspecies of Merluccius australis (australis and polylepsis) and one grenadier species Macruronus novaezelandiae with two populations (novaezelandiae and magellanicus) were evaluated by 2-DE and MALDI-TOF MS. 2-DE demonstrated that the species tested displayed a low intra-specific degree of polymorphism and the isoform patterns were noticeably species-specific. MALDI-TOF mass fingerprints showed clear differences in the pattern of peptides produced by tryptic digestion between the Merluccius and the Macruronus, making the genus differentiation possible. In addition, a selective peptide mass present in the spectra from certain hakes allowed its classification in two groups: Euro-African and American hakes. Besides, some specific masses allowed a clearly individual identification for M. bilinearis, M. australis polylepsis, M. australis australis, M. productus, M. paradoxus and M. polli, while the rest of the hake species can be grouped in two clusters, comprising M. hubbsi and M. gayi in one and M. merluccius and M. capensis in the other.     

大鼠坐骨神经结扎后脊髓钙结合蛋白PV的表达变化

目的:研究大鼠坐骨神经结扎模型钙结合蛋白Parvalbumin(PV)在脊髓的时空变化规律,为探讨其在神经再生中的作用与机制提供实验依据。方法:SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1,3,7,14或21d,采用免疫组化结合图像分析技术观察PV在脊髓的表达变化。结果:在对照组,PV免疫阳性神经元主要分布于腰髓背角Ⅱ层,Ⅲ～Ⅵ层只观察到少量散在分布的PV样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见少量多极的大型阳性神经元。术后各时间点PV样阳性神经元表达下降,14d下降最显著,21d表达有所上升,但还是低于7d组。脊髓后角PV免疫阳性产物灰度值测定结果显示:术后14d后角PV表达最低,与对侧和对照组以及1、3d组相比有统计学意义(P<0.05)。结论:坐骨神经结扎后PV表达变化呈现一定的时空模式,为进一步揭示PV在神经系统疾病中的作用提供实验依据。     

5-HT<Subscript>1A</Subscript> receptor expression in pyramidal neurons of cortical and limbic brain regions

We studied expression of the 5-HT_1A receptor in cortical and limbic areas of the brain of the tree shrew. In situ hybridization with a receptor-specific probe and immunocytochemistry with various antibodies was used to identify distinct neurons expressing the receptor. In vitro receptor autoradiography with ³H-8-OH-DPAT (³H-8-hydroxy-2-[di-n-propylamino]tetralin) was performed to visualize receptor-binding sites. In the prefrontal, insular, and occipital cortex, 5-HT_1A receptor mRNA was expressed in pyramidal neurons of layer 2, whereas ³H-8-OH-DPAT labeled layers 1 and 2 generating a columnar-like pattern in the prefrontal and occipital cortex. In the striate and ventral occipital cortex, receptor mRNA was present within layers 5 and 6 in pyramidal neurons and Meynert cells. Pyramid-like neurons in the claustrum and anterior olfactory nucleus also expressed the receptor. Principal neurons in hippocampal region CA1 expressed 5-HT_1A receptor mRNA, and ³H-8-OH-DPAT labeled both the stratum oriens and stratum radiatum. CA3 pyramidal neurons displayed low 5-HT_1A receptor expression, whereas granule neurons in the dentate gyrus revealed moderate expression of this receptor. In the amygdala, large pyramid-like neurons in the basal magnocellular nucleus strongly expressed the receptor. Immunocytochemistry with antibodies against parvalbumin, calbindin, and gamma aminobutyric acid (GABA) provided no evidence for 5-HT_1A receptor expression in GABAergic neurons in cortical and limbic brain areas. Our data agree with previous findings showing that the 5-HT_1A receptor mediates the modulation of glutamatergic neurons. Expression in the limbic and cortical areas suggested an involvement of 5-HT_1A receptors in emotional and cognitive processes.This work was supported by the German Science Foundation (SFB 406; C4 to G.F.).     

Fluorescence quenching of the buried tryptophan residue of cod parvalbumin

Cod parvalbumin (isotype III) is a single tryptophan-containing protein. The fluorescence characteristics of this tryptophan residue (lambda em approximately 315 nm) suggest that it is buried from solvent and that it is located in an apolar core of the protein. Solute quenching studies of the tryptophan fluorescence of parvalbumin reveal dynamic quenching rate constants, kq, of 1.1 X 10(8) and 2.3 X 10(9) M-1 s-1 (at 25 degrees C) with acrylamide and oxygen, respectively, as quenchers. From temperature dependence studies, activation energies of 6.5 +/- 1.5 and 6.0 +/- 0.5 kcal/mol are found for acrylamide and oxygen quenching. The kq for acrylamide quenching is found to be relatively unchanged (+/- 10%) by an 8-fold increase in the bulk viscosity (glycerol/water mixture). These temperature and viscosity studies argue that the acrylamide quenching process involves a dynamic penetration of the quencher, facilitated by fluctuations in the protein's structure.