IMMUNOCHEMICAL PROPERTIES OF S-100 PROTEINS AND THEIR SUBUNITS

Abstract— The immunological activities of two populations of bovine S-100 proteins with anti-S-100 serum were studied by complement fixation and rocket immunoelectrophoresis. The reactivities of subunits of these two populations were studied by crossed immunoelectrophoresis and rocket immunoelectrophoresis. Although the two populations conformed in all respects to the properties of S-100 protein, the immunological reactivity of one, III-IVa-1, was significantly lower than that of the other, III-IVb-1. The difference was much larger when the S-100 protein fractions were isolated in the absence of aids (mercaptoethanol, EDTA, EGTA, protease inhibitors). With bovine S-100 fractions, the three subunits separated by differences in charge as well as the four subunits separated by differences in molecular weight all reacted with the same antibody molecules in the antiserum. The reactivities of the subunits showed large quantitative differences.
Two populations of S-100 proteins from rat brain also showed differences in reactivity with anti-S-100 serum. The two subunits in each of these fractions reacted with anti-S-100 serum but with quantitative differences, the larger having almost double the activity of the smaller. These results provide firm evidence for the heterogeneity of S-100 proteins based on immunological activity of their subunit components. Different molecular species of S-100 proteins probably differ considerably in their reactivity with antibodies to S-100 protein. Some of the more reactive molecular species also appear to be much more labile, since the reactivity of some S-100 protein fractions was considerably reduced when they were isolated in the absence of aids.     

Dynamics of neurospecific protein S-100 accumulation in the brain of AKR/J and DBA/2J strain mice in the process of postnatal development

The content of total S-100 protein and its water-soluble fraction was determined in the brain of DBA/2J and AKR/J mice during postnatal development. Reliable differences between the two strains were found in the content of total S-100 protein (from the 27th day after birth on) and its water-soluble fraction (from the 35th day).     

Appearance and accumulation of the brain specific S 100 protein in the developing nervous systems of rat, rabbit and guinea pig.

The appearance and accumulation of S 100 protein during postnatal development of rats and rabbit, and during pre- and postnatal development of guinea pig, was studied quantitatively by immunoelectrophoresis. High amounts of S 100 were found in newborn guinea pig brain. In rabbit and rat central nervous systems the content of S 100 rose linearily between the 1st and the 3rd and 4th weeks of postnatal life, respectively. From this later period to the adult animal there was a small increase in the amount of S 100 in rabbit and rat brain. The results of this study are compared with other studies on S 100 accumulation during postnatal development of rat, rabbit and guinea pig, using immunofluorescence and immunoelectron microscopic techniques.     

Proliferating cell nuclear antigen of the rat thyroid gland before and after birth]

We were studied the proliferative activity of the thyroid gland's cells of embryo and adult Wistar rats due to using the antiserum against the cell nuclear antigen (PCNA). The 100% of cells in thyroid's embryo was a positive on the 16th, 17th, 18th stages of the embryonic development (stages by Kornegy). The percent of PCNA-positive cells considerably increased to 67% on the 19th stage. This fact the 20th and 21th stages of prenatal development relatively the previous stage coordinate with starting of the thyroid hormones in fetal thyroid gland and the first follicles formation. The small increasing of number of PCNA-positive cells detected on the 20th and 21th stages of prenatal development relatively the previous stage. Considerable elevation of the proliferating cells to 75% immediately before the birth (22th stage). An infant rats had have the 39% of proliferating cells. The 51% cells divided on the 5th day of postnatal development. Considerable decreased of the cell's division was occurred until the postnatal day 60. Using of the PCNA antiserum allowed to study cell proliferation in thyroid gland during pre- and postnatal rat development.     

The brain-specific S-100 protein on neuronal cell membranes

The S-100 protein has been localized to the neuronal plasma membranes of isolated Deiters' neurons by fluorescence microscopy using fluorescein-conjugated antiserum to the protein and by immunoelectron microscopy using peroxidase-conjugated anti-S-100 antiserum. In the present study this is shown also by incubating neurons with Sepharose 4B or methylacrylate spherules to which were coupled anti-S-100 antibodies. The specificity of the antiserum is discussed in the text. The technique described can be used to study the topography of antigenic characteristics of nerve cells by using antisera insolubilized on spherules of suitable size.     

Immunocytochemical localization of S-100 beta beta protein in olfactory and supporting cells of lamb olfactory epithelium

By immunocytochemistry, we have identified two novel cell types, olfactory and supporting cells of lamb olfactory epithelium, expressing S-100 beta beta protein. S-100 immune reaction product was observed on ciliary and plasma membranes, on axonemes and in the cytoplasm adjacent to plasma membranes and to basal bodies of olfactory vesicles. A brief treatment of olfactory mucosae with Triton X-100 before fixation is necessary for detection of S-100 beta beta protein within olfactory vesicles. In the absence of such a treatment, the immune reaction product is restricted to ciliary and plasma membranes. On the other hand, irrespective of pre-treatment of olfactory mucosae, S-100 beta immune reaction product in supporting cells is restricted to microvillar and plasma membranes. The anti-S-100 beta antiserum used in these studies does not bind to basal cells of the olfactory epithelium or to cells of the olfactory glands, whereas it binds to Schwann cells of the olfactory nerve. An anti-S-100 alpha antiserum does not bind to cellular elements of the olfactory mucosa, Schwann cells, or axons of the olfactory nerve. The present data provide, for the first time, evidence for the presence of S-100 beta beta protein in mammalian neurons (olfactory cells).     

移植视网膜NOS阳性神经元的发育

目的 观察不同年龄组段大鼠正常视网膜及移植视网膜内NOS阳性神经元的发育情况及其定位分布。方法 实验分正常视网膜发育组和移植视网膜发育组,应用还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）组织化学方法显示。结果 1、NOS阳性神经元最早出现于生后第五天（P5）,P18时阳性神经元数目达到最高峰,2、移植视网膜具有正常视网膜的各层结构和相似的生长规律,NOS阳性神经元在生后第4天移植视网膜（TP4）中出现,TP12数量达到高峰值,TP22后降至正常成年鼠水平。结论 根据NOS阳性神经元的定位,分布,推测其为无长突细胞,移位无长突细胞及节细胞。     

Distribution of histamine in the rat kidney during pregnancy and development

Summary An antiserum against conjugated histamine and two oligonucleotide probes that detect the mRNA encoding L-histidine decarboxylase (HDC) involved in histamine synthesis were used to study the appearance of histamine and its location in the kidneys of fetal, newborn and young postnatal rats and in the kidneys of pregnant rats. On embryonic days 16 and 18 (E16 and E18), some HA-immunoreactive (HA-ir) cells were found within the largest S-shaped bodies. Histamine was found to appear rapidly between the 18th and 20th embryonic days in the convoluted tubules of the kidneys. On postnatal day 0 (P0), the distal convoluted tubules and collecting ducts exhibited bright fluorescence, the intensity of which decreased quickly so that it was faint on day P4 and absent at later stages. In kidneys of pregnant rats HA-ir was found in the epithelium of both the Bowman's capsule, collecting ducts and in a few cells within the tubules. Nonuniform HA-ir was also detected within glomeruli. No evidence for the presence of L-histidine decarboxylase mRNA in kidneys of fetuses or pregnant rats was seen. It is concluded that distinct structures in the developing rat kidney contain histamine during a period around birth from day E20 to day P4. In the pregnant rat, the epithelium that is in direct contact with the urine flow is immunoreactive for histamine from day 16 to 20 of pregnancy. The results suggest that histamine is not synthesized locally in the kidneys but rather originates from other tissues.     

Intracellular distribution of creatine kinase isoenzymes in the brains and hearts of rats at different stages of postnatal development]

The total activity and range of the creatine kinase (CK) isozymes have been studied in the homogenate and subcellular fractions (nuclei, mitochondria, cytoplasm) of the rat brain and heart during postnatal ontogenesis. The total activity of CK in the brain and heart of newborn rats was found to be 4 and 2 times less, resp., than in those of adults. The age patterns were established in the activity of cytoplasmic (CK-1, CK-2 and CK-3) and mitochondrial (CK-4) isozymes. During the whole postnatal development the rat brain contains only one cytoplasmic isozyme, CK-1. In the heart of newborn rats, as compared with adults, the content of CK-1 and CK-2 is much higher and that of CK-3 lower. On the 12-15th day of life the range of the CK isozymes approaches that characteristic of adult animals. The activity of CK-4 was found in the brain on the 5-7th day of life and in the heart on 12-15th day. In the range of the CK isozymes in the adult brain the content of mitochondrial CK amounts to 19.3% and in the heart to 16.5%. The data obtained complement the literary ones suggesting the low level of energy-forming processes in the brain and heart cells at the early stages of the rat postnatal development.     

Proliferative activity of cells of the intermediate lobe of the rat pituitary during the postnatal period.

Cellular proliferation was studied in the intermediate lobe (IL) of the pituitary gland of developing rats by labelling cells at the S-phase of the cell cycle with bromodeoxyuridine (BrdU). The number of BrdU-labelled cells in the IL decreased from birth until the 14th postnatal day and was low from that day until the end of the first month after birth. Throughout the postnatal period a large proportion of BrdU-labelled cells was found in the marginal layer (ML) of the IL, suggesting for the ML a role as a germinative layer of the IL during postnatal growth. Double immunostaining with anti-BrdU and anti-MSH showed that MSH cells actively proliferate as from the day of birth. Cells doubly immunostained with anti-BrdU and anti-S100 protein were first seen on the 14th postnatal day. From then onwards, most proliferating cells were labelled with either anti MSH or anti S-100 protein. This, together with the high proportion of proliferating cells found in the ML marks a clear difference with the pattern of cellular proliferation previously reported during a similar period in the anterior lobe of the rat pituitary.     

PHOSPHOCHOLINE DIGLYCERIDE TRANSFERASE ACTIVITY DURING DEVELOPMENT OF THE CHICKEN BRAIN

Abstract— The activity of chicken brain phosphocholine diglyceride transferase was followed during pre- and postnatal development. The specific activity of this enzyme increases from the 10th day of embryonic life, reaches a maximum at hatching and decreases thereafter. Total brain activity increases in parallel with the increase of brain lecithins. The apparent K _m of the enzyme for CDP choline is 1.5 × 10^-4 m before the 10th day of embryonic life, 2.5 × 10^-5 m between the 13th day of the embryo and the 10th day after hatching, and finally 1.3 × 10^-4 m after the 38th day of postnatal life. These data suggest the existence of isoenzymes, one of which appears at the beginning of myelination.     

Morphological studies on neuroglia

Immunohistochemical studies with the use of the peroxidase-antiperoxidase (PAP) method revealed that "amoeboid microglial cells", in the brains of neonatal rats and "brain macrophages" in lesioned brains of adult rats react positively to an antiserum raised against macrophages. In brains of neonatal rats, "amoeboid microglial cells" stained by means of the PAP-method were observed in the corpus callosum, internal capsule, dorso-lateral region of the thalamus, subventricular zone of the lateral ventricle, and the subependymal layer of the ventricular system. These cellular elements were not detected in brains of rats aged 21 days or older. Resting microglial cells displaying a typical ramified structure were not specifically stained. Cells reacting positively to the macrophage antiserum appeared (i) in the cerebral cortex of adult rats following placement of a stab wound, or (ii) in the hippocampal formation after kainic acid-induced lesions; in the damaged areas immunoreactive cells exhibited the typical features of "brain macrophages". "Brain macrophages" and "amoeboid microglial cells" are considered to belong to the class of exudate macrophages derived from blood monocytes. Thus, elements of hematogenous origin do exist in the intact brain parenchyma of neonatal rats and in lesioned brains of adult rats. The relationship between brain macrophages and resting microglial cells is discussed.     

HETEROGENEITY OF S-100 PROTEINS OF BRAIN: SUBUNIT COMPOSITIONS

Abstract— Two populations of S-100 proteins (III-IVa-1) and (III-IVb-1) were isolated from bovine brain by a simple three-step procedure involving precipitation with ammonium sulfate, ion exchange chromatography and gel electrophoresis. These two fractions reacted with anti-S-100 serum and showed all the properties of S-100 protein, but differed in the distribution of protein among the four subunits obtained in SDS-gels. These subunits had apparent molecular weights of 7800, 6800, 6100, and 5200. Separations based on charge differences revealed only three subunits. Similar fractions isolated from rat brain contained only two subunits with apparent molecular weights of 7800 and 6500. The two S-100 protein fractions also differed in the specific extinction at 280 nm and in the protein distribution among the separate bands which formed either in a 14% acrylamide-agarose gel-discontinuous buffer system or in 7.5% gels in the presence of calcium. The yield of S-100 protein isolated in the presence of aids (EDTA, EGTA, mercaptoethanol, protease inhibitors) was considerably higher than in their absence (80 mg vs 60 mg/kg tissue), the predominant loss occurring in fraction III-IVa-1. It is concluded that 'S-100 protein'contains a number of molecular species which have different subunit compositions and differences in stability.     

Effects of prenatal X-irradiation on the 14th-18th days of gestation on postnatal growth and development in the rat

Thirty-nine pregnant adult Wistar strain rats were randomly assigned to one of three exposure groups: 0, 0.75, or 1.50 Gy X-radiation total exposure. Animals were exposed from the 14th to the 18th days of gestation at 0, 0.15, or 0.30 Gy per day. At term, 15 rats were killed and morphologic analyses were completed. Twenty-four rats were allowed to deliver their offspring. On the first day of postnatal life, litters were reduced to a maximum of eight pups per litter, with equal numbers of male and female offspring wherever possible. A total of 187 pups were observed for the age of acquisition of five reflexes (air righting, surface righting, visual placing, negative geotaxis, auditory startle) and the appearance of four physiologic markers (pinna detachment, eye opening, vaginal opening, testes descent). There was significant dose-related weight reduction in term fetuses and offspring throughout the 86-day postnatal period. Postnatal growth rate (g gained/day) was unaffected. Adult offspring brain and gonadal weight and organ weight:body weight ratios were reduced. Using the PAC50 methodology, dose-related alterations occurred in the acquisition of several reflexes. All physiologic markers exhibited a dose-related delay in appearance. These results indicate that fractionated exposure to X-radiation during the fetal period in the rat results in dose-dependent alterations in postnatal growth and physiologic development. These studies are important for our understanding of the long-range effects of prenatal exposure to ionizing radiation late in gestation.     

Effect of anti-S-100 serum on36C1− ion permeability across the Deiters' neuron plasma membrane

1. A microtechnique allowing the study of single plasma membranes from the gamma-aminobutyric acid (GABA)-acceptive Deiters' neuron has been utilized in order to assess the effect of both S-100 protein and its antiserum on 36Cl- permeability through such membranes. 2. The results show that both S-100 (in the Ca2+ form) incorporation onto the external side of the membranes and their preincubation with anti-S-100 serum stimulate 36Cl- permeability. 3. These effects are not additive with that of GABA, indicating that both S-100 and anti-S-100 act via the GABAA receptor complexes on Deiters' membranes. 4. When the membranes were incubated first with S-100/Ca2+ and then with anti-S-100, the second treatment resulted in the disappearance of the S-100 effect. However, the anti-S-100 effect was fully displayed.     

Immunologic study of the cellular components of bacillus pyocyaneus. III. Immunochemical analysis, toxicity and protective properties of water-soluble antigenic complexes]

Aqueous extracts of two Ps. aeruginosa strains killed with acetone were subjected to fractionation by preparative ultracentrifugation and gel-chromatography. Toxic activity of the extract was found to be connected with the high-molecular, possibly glycoprotein components reacting with the corresponding antiserum in the immunoprecipitation test, and protecting 30--40% of rats against the generalized infection with Pseudomonas aeruginosa. This protective activity is apparently connected with the protein components (molecular weight--20000--60000 dalton), nontoxic for mice, not reacting with the corresponding antiserum in the immunoprecipitation test, and protecting 60 to 80% of rats against Ps. aeruginosa infecsion. Thus, as a result of preparative ultracentrifugation and gel-chromatography it was postible to divide the toxic and the nontoxic protective components of Ps. aeruginosa.     

Glial fibrillary acidic protein in the brain of prenatally stressed rats

The content of glial fibrillary acidic protein (GFAP) was studied in the brain structures of rats borne by intact females and females that underwent stress. In the offspring of stressed rats, the GFAP content in the brain gray and white matter on the 15th postnatal day noticeably dropped. On the 30th postnatal day, the GFAP content in the cortex and pons increased, while it somewhat decreased in the striatum and cerebellum. The results suggest that formation of the intermediate astrocyte filaments in the animals subjected to prenatal stress is markedly disturbed.     

Expression of Ext1, Ext2, and heparanase genes in brain of senescence-accelerated OXYS rats in early ontogenesis and during development of neurodegenerative changes

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) play a significant role in brain development, and their structural and quantitative changes are revealed during aging and in neurodegenerative disorders. The mechanism of these changes is not clear, but is likely to be associated with alteration in the expression and/or activity of enzymes responsible for HSPG biosynthesis and degradation. The contents of mRNAs of the genes Ext1 and Ext2 encoding polymerization enzymes and of gene Hpse of heparanase degrading HS were determined in the brain of prematurely aging OXYS rats during early postnatal development and during appearance of signs of brain accelerated aging (at age of 1, 7, 14, 30, 60, and 420 days). Wistar rats of the same age were used as controls. Expression levels of the genes Ext1, Ext2, and Hpse in the brain of rats of both strains were maximal during the two first weeks of life, and the contents of mRNAs of all genes in the brain of newborn and 7-day-old OXYS rats were significantly higher than in Wistar rats. By the 14th day of life the differences leveled, but at the age of 30 days on the background of a decrease in the contents of mRNAs of Ext1, Ext2, and Hpse in OXYS rats they became more pronounced (three-, four-, and twofold, respectively). Differences between the strains were absent at the age of 60 days and 14 months, and expression of all the genes was significantly lower than in the newborn animals. A strong positive correlation was found between contents of mRNAs of all the studied genes, and this suggested that heparanase should be involved in HSPG metabolism together with Ext1 and Ext2. Based on these and earlier findings, we conclude that development of the OXYS rat brain occurs on the background of significant alterations in HSPG metabolism that precede the development of neurodegenerative manifestations recently detected by magnetic resonance imaging.     

Formation of structural and ultrastructural organization of striatum rat postnatal ontogenesis upon changes in their embryonal development

By light microscopy (by Nissl and Golgi), electron microscopy, and immunohistochemistry methods, formation of structure of the brain striatum dorsolateral part from birth to the 3-month age was studied in rats submitted to acute hypoxia at the period of embryogenesis. It has been established that hypoxia at the 13.5th day (E13.5) leads to a delay of neuronogenesis for the first two weeks of postnatal development as compared with control animals, while the majority of large neurons at this period are degenerated by the type of chromatolysis with swelling cell body and processes and lysis of cytoplasmic organoids. By the end of the 3rd week, shrunk hyperchromic or picnomorphic neurons with the electron-dense cytoplasm and enlarged tubules of endoplasmic reticulum and Golgi complex were also observed. An increased number of swollen processes of glial cells was detected in neuropil around degenerating neurons. By the 30th day as well as in adult rats there was observed destruction of mitochondrial apparatus, an increase of the number of lysosomes, and the appearance of bladed nuclei - signs of apoptotic cell death, which was also confirmed by an increased expression of proapoptotic p53 protein and its colocalization with caspase-3 in a part of neurons. Morphometrical analysis has shown a decrease of density of striatum cell arrangement and a change of ratio of different cell types in the rats submitted to hypoxia as compared with control group. At early stages of postnatal ontogenesis there was the greatest decrease (42.3% at the 5th day, 14.2% at the 10th day, p < 0.01) of the number of large neurons with the area more than 80 microm2. After 3 weeks of postnatal development the number of middlesize neurons (30-95 microm2) decreased (by 11.8-19.2%) as compared with control. The obtained data show that a change of conditions of embryogenesis (hypoxia) at the period of the most intensive proliferation of the forebrain neuroblasts leads to disturbances of the process of formation of the striatum nervous tissue. This can be the cause of delay of development and disturbances of behavior and learning observed in rats submitted to prenatal hypoxia.     

Ontogenesis of corticotropes and lactotropes in situ in the pituitary gland of the hamster

Summary The development of corticotropes and lactotropes was investigated in the golden Syrian hamster using an anti-porcine ACTH antiserum and a homologous antihamster PRL antiserum. Oval corticotropes were first visible in the ventral region of the pars distalis at 13 days of gestation. By the end of gestation, corticotropes were found throughout the pars distalis and in the pars intermedia. Corticotropes in the pars distalis of postnatal hamsters were either round or irregularly-shaped, often appearing in clusters. Throughout development, corticotropes often appeared to be surrounding other cells. Scarce, very small lactotropes were first observed in the pars distalis of hamsters on the first postnatal day. The number of these cells, which were either round or polyhedral, increased dramatically between 4 and 20 days of postnatal life. These observations indicate that the sequence of appearance of corticotropes and lactotropes in the hamster is similar to that in other species and that lactotropes are confined to the pars distalis of postnatal hamsters.