Expression of Presenilin 1 mRNA in Rat Peripheral Organs and Brain

At least 50 different mutations in the presenilin 1 gene have been shown to cause early onset familial Alzheimer's disease. Although presenilin 1 has an obvious role in the pathogenesis of Alzheimer's disease, its function is still unknown. In the present study, the occurrence and distribution of presenilin 1 mRNA was examined in rat peripheral organs as well as in the brain by in situ hybridization histochemistry, using a radiolabelled oligonucleotide probe. In comparison to the brain, a high presenilin 1 mRNA expression was found in the testis, kidney, spleen, adrenal gland and thymus. It was also observed in skeletal muscle, liver, small intestine and lung, whereas no presenilin 1 could be detected in the heart, spinal cord and pancreas. Since presenilin 1 mRNA was found to be abundant in peripheral tissues which apparently are not affected in Alzheimer's disease, additional functions of presenilin 1 are suggested, unrelated to its role in the pathological processes of the disease.     

LIM结构域蛋白KyoT在成年小鼠睾丸的表达

报道了KyoT在成年小鼠体内的表达,以便进一步研究KyoT在体内的功能。为研究KyoT mRNA及蛋白质水平的表达,采用Northern印迹、RT－PCR、免疫组织化学SABC和原位杂交方法。睾丸中两种KyoT的mRNA水平均很高,睾丸间质细胞的免疫化学反应阳性,阳性物质分布于细胞质内,细胞核呈阴性反应。同样,KyoT的mRNA在睾丸间质细胞杂交信号呈阳性反应,阳性物亦分布于细胞质内,细胞核呈阴性反应。生精细胞及对照组均为阴性。上述结果提示睾丸中有KyoT的表达,且特异性分布于睾丸间质细胞。     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catabolic Rate of Body Protein in Adult Rat over an Entire Period of Protein Depletion

In order to clarify the magnitude of different labile body proteins and the over-all catabolizable body protein, the catabolic rate of total body nitrogen in adult rat was measured by nitrogen balance method up to the time of death due to protein depletion.

The more labile body protein having 83.3% of fractional catabolic rate per day and occupying 2.8% of the whole body protein mainly represented the so-called protein reserves at the beginning of protein depletion. The less labile one, the remainder, namely 97.2%, having 0.79% of fractional catabolic rate almost wholly represented the exponential decrease of body protein during the first 40 days of protein depletion. Urinary and fecal nitrogen in this period showed a similar exponential decrease. In the next 40 days of the depletion, body protein decreased almost linearly giving the constant excretions of urinary and fecal nitrogen. In the last 40 days, it decreased drastically accompanied by a remarkable increase in urinary nitrogen.

After 118 days, on the average, the animals died of protein depletion at 35.7% level of the initial body nitrogen. Contributions of various organs to the total nitrogen deficit up to the time of death, were considerably different in different organs, where muscle was the greatest in total amount but with less catabolizability than the viscera, such as liver, pancreas and spleen.     

Androgen Regulates Gene Expression of Cytoskeletal Proteins in Adult Rat Motoneurons

Expression of β-actin and β-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding chick β-actin and mouse β-tubulin, respectively. Both hybridizable β-actin and βtubulin mRNAs were localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both β-actin and β-tubulin mRNAs in the SNB motoneurons, whereas the changes were prevented by testosterone treatment. In contrast, castration or testosterone treatment induced little or no change in the expression levels of these mRNAs in the much less androgen-sensitive motoneurons of the retrodorsolateral nucleus (RDLN). These results suggest that androgen regulates the expression of β-actin and β-tubulin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.     

Expression of Trefoil Factor 1 in the Developing and Adult Rat Ventral Mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson’s disease.     

Localization of the Extracellular Matrix Protein SC1 to Synapses in the Adult Rat Brain

Extracellular matrix molecules play important roles in neural developmental processes such as axon guidance and synaptogenesis. When development is complete, many of these molecules are down-regulated, however the molecules that remain highly expressed are often involved in modulation of synaptic function. SC1 is an example of an extracellular matrix protein whose expression remains high in the adult rat brain. Confocal microscopy revealed that SC1 demonstrates a punctate pattern in synaptic enriched regions of the cerebral cortex and cerebellum. Higher resolution analysis using electron microscopy indicated that SC1 localizes to synapses, particularly the postsynaptic terminal. SC1 was also detected in perisynaptic glial processes that envelop synapses. This work was supported by the National Science and Engineering Research Council of Canada.     

Protein Synthesis in Hybrid Cells Derived from Fetal Rat × Mouse Chimeric Organs

Abstract. Malignant hybrid cells (As3) derived from fusion of rat hepatoma cells (Fu5AH) with mouse teratocarcinoma cells (OTT6050) were injected into genetically marked mouse blastocysts which were subsequently transferred into pseudopregnant surrogate mothers. From a total of 61 fetuses developed, four normally differentiated fetuses at day 18 of gestation showed hybrid cell contributions in their livers and a few other organs of endo-mesodermal origin. The chimeric tissues were briefly cultured in vitro and then further investigated for their protein synthesis using two-dimensional gel electrophoresis. After comparison of the protein patterns obtained from the corresponding normal rat and mouse organs, several rat-specific polypeptides were detected in the cultured chimeric tissues illustrating functional xenogeneic gene expression during in situ differentiation. In addition, some other rat proteins characteristic of the parental hybrid cell line disappeared. The tumorigenicity of the chimeric tissues was tested by subcutaneous transplantation into immunodeficient nude mice. Tumors originating from two of the four chimeric organs differed histologically from those formed by cells of the hybrid As3 line since they also contained muscle-like structures resembling rhabdomyosarcomas. The tumors were analyzed for their protein synthesis and compared with the three malignant cell lines of parental origin. The morphologic differences between the tumors derived from the chimeric organs and those developed from the As3 cell line were also reflected in characteristic differences of their protein synthesis patterns. Our results demonstrate that interspecific rat × mouse hybrid cells, when implanted into early mouse embryos, participate in fetal tissue differentiation and selectively repress certain rat gene products typical of the malignant parental cells as well as functionally reactivate other rat genes presumably required for normal development.     

n-3 Fatty Acid Deficiency Increases Brain Protein Synthesis in the Free-Moving Adult Rat

Abstract: The autoradiographic method with l -[³⁵S]methionine was used to determine the effects of an n-3 fatty acid deficiency on brain protein synthesis. Brain protein synthesis was significantly increased (from 50 to 150%) in 45 of the 52 brain structures studied in n-3 fatty acid-deficient rats as compared with control animals. Biochemical analysis confirmed the increase in overall rate of protein synthesis in brain as a whole.     

The Expression of Tenascin-X in Developing and Adult Rat and Human Eye

Tenascin-X has been studied in developing and adult rat eye and in foetal and adult human eyes, using immunohistochemistry and frozen sections. The data were compared with the distribution of tenascin-C. The immunoreactivity for tenascin-X was seen in a basement membrane-like feature in different structures of embryonic (E) day 16–17 rat eyes. Postnatal (P) day 2 and older rat eyes showed immunoreactivity for tenascin-X in different connective tissues. In the epithelial basement membrane zone of the cornea, immunostaining was positive in P5 eyes, negative in P10 and P15 eyes and again positive in P30 and adult eyes. In the 20-week-old human foetus, immunoreactivity for the tenascin was seen in the posterior parts of the conjunctival stroma adjacent to the sclera and in a basement membrane-like fashion in anterior conjunctiva. In the adult human eye, immunoreactivity for tenascin-X was seen in the anterior one-third stroma of cornea as thin fibrils, in the stroma of the limbus and conjunctiva, and in blood vessels. Immunostaining for tenascin-C was seen in the posterior aspect of the further cornea, and in mesenchyme adjacent to cornea in E16–17 rat eyes. Corneal keratocytes and Descemet's membrane showed immunoreactivity for tenascin-C in P2–P15 rat eyes. Sclera and the junction of the cornea, and sclera expressed tenascin-C in P2 and older rat eyes. In human foetal eyes, immunostaining for tenascin-C was seen in the anterior parts of the corneal stroma, in the basement membrane zone and Bowman's membrane of the corneal epithelium, in the posterior one-fifth of the corneal stroma and the sclera starting from the junction of the cornea and sclera. In normal human adult eyes, immunostaining for tenascin-X was seen in the anterior one-third stroma of cornea, in the stroma of limbus and conjunctiva, and in blood vessels. The association of tenascin-X and basement membranes in early development evokes a question of its potential function in the development of the basement membrane. The results also suggest the association of tenascin-X with connective tissue development as well as the association of tenascin-C with the migration of keratocytes during the development of the corneal stroma.     

大鼠CRIP2蛋白的表达和分离纯化

目的:获得大鼠crip2基因片段,并在大肠杆菌中表达、纯化大鼠CRIP2(cysteine-rich intestinal protein 2)蛋白。方法:从大鼠主动脉组织中提取总DNA,RT-PCR扩增出相应大小的crip2 DNA片段,与pGEM-T-easy载体连接后测序;将测序正确的crip2按照BamHⅠ和HindⅢ酶切位点克隆入原核表达载体pRSET A,将连接产物转化大肠杆菌BL21,挑出阳性克隆,IPTG诱导表达重组的6×His融合蛋白,通过镍柱进行纯化。结果:PCR获得的crip2序列与GenBank报道的一致(为707 bp);重组融合蛋白在大肠杆菌BL21中以可溶形式高效表达,经SDS-PAGE和Western印迹分析,在相对分子质量为27×103处有特异的蛋白条带,经镍柱纯化后,得到了高纯度的CRIP2融合蛋白。结论:克隆了大鼠crip2基因片段,并在大肠杆菌BL21中高效表达,亲和层析纯化后获得高纯度的CRIP2融合蛋白。     

Decreased Protein Kinase C Activity During Cerebral Ischemia and After Reperfusion in the Adult Rat

The possible activation of protein kinase C (PKC) during total cerebral ischemia was investigated in the rat. Translocation of PKC activity from the soluble to the particulate fraction was used as an index of PKC activation. There was a drop in the proportion of particulate PKC activity from 30% for controls to 20% by 30 min of ischemia (p less than 0.01). By 20 min of cardiac arrest, there was a 40% decline of the total cellular PKC activity (p less than 0.01). This was not accompanied by an increase in activator-independent activity, a finding indicating PKC was not being converted to protein kinase M. These data suggest that PKC was not activated during ischemia, but rather that ischemia causes a reduction in cellular PKC activity. Translocation of PKC activity to the particulate fraction was not observed in the cerebral cortex or hippocampus of reperfused brain for up to 6 h of recovery following 11-13 min of total cerebral ischemia. The level of total, soluble, and particulate PKC activity in the cerebral cortex was reduced (p less than 0.05), corresponding to the decrease observed by 15 min of ischemia without reflow. A similar decline in activity was also observed in the hippocampus. No increase in activator-independent activity was observed. These data suggest that PKC was inhibited during cerebral ischemia and that this reduced level of PKC activity was maintained throughout 6 h of recovery. We conclude that pathological activation of PKC was not responsible for the evolution of ischemic brain damage.     

Atomic Structures of Two Novel Immunoglobulin-like Domain Pairs in the Actin Cross-linking Protein Filamin

Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16–24. Similarly, as with the previously known compact domain pair of IgFLNa20–21, the two-domain fragments IgFLNa16–17 and IgFLNa18–19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NMR structures revealed that the domain packing in IgFLNa18–19 resembles the structure of IgFLNa20–21. In both domain pairs the integrin-binding site is masked, although the details of the domain-domain interaction are partly distinct. The structure of IgFLNa16–17 revealed a new domain packing mode where the adhesion receptor binding site of domain 17 is not masked. Sequence comparison suggests that similar packing of three tandem filamin domain pairs is present throughout the animal kingdom, and we propose that this packing is involved in the regulation of filamin interactions through a mechanosensor mechanism.Actin cytoskeleton is a dynamic network that is involved in many fundamental cellular processes such as cell differentiation, morphology, endocytosis, exocytosis, cytokinesis, and cell movement. These events are regulated by proteins that interact with monomeric and filamentous actin. Filamins are actin filament-binding and cross-linking proteins. Filamin A and filamin B are both ubiquitously expressed, and their mutations in human patients cause developmental abnormalities in brain, cartilage, bones, and epithelial tissues (1). Filamin C is muscle-specific, and mutations thereof cause myofibrillar myopathy (2). Mice with targeted deletion of any of the filamin genes die either during development or soon after birth (3–6). These phenotypes are thought to reflect the roles of filamins as scaffolds of signaling pathways required for cell differentiation, regulators of cell migration, and stabilizers of cytoskeleton and cell membranes (1, 7).Filamins bind to actin filaments mainly via their N-terminal actin-binding domains and interact with other proteins via the 24 filamin type immunoglobulin-like domains (IgFLN),³ also called filamin repeats (8). Especially the C-terminal IgFLNs 16–24 contain several protein-protein interaction sites (1). Our previous structural studies have revealed that many proteins interact with filamins by forming an additional β-strand next to strand C of an individual IgFLN. The platelet von Willebrand factor receptor, glycoprotein (GP) Ibα, interacts in this way with IgFLNa17 (9). The integrin family adhesion receptor β subunits interact with IgFLNa21 and to a lesser extent with IgFLNa19 (10, 11). Furthermore, some signaling proteins use a similar interaction mode: the adaptor protein migfilin interacts with IgFLNa21 (12), and the Rho family GTPase-activating protein FilGAP interacts with IgFLNa23 (13, 14).Although structural details are known from many filamin interactions, it is not completely clear how these interactions are regulated. In some cases the regulation involves competition between multiple binding partners (10, 11). Alternative splicing (15), proteolysis of filamin (16–18), and ligand phosphorylation (11) also contribute to the regulation. Recently, it has become apparent that conformational changes in filamins may also be involved. For instance, actomyosin contraction exposes hidden cysteine residues in filamins (19). This opens the possibility that forces transmitted through actin filament may open up binding sites, and filamin may thus be involved in mechanosensor signaling.We have recently found a structural mechanism by which mechanical forces could regulate interactions at the C-terminal part of filamin. Our recent crystal structure revealed that IgFLNa20 forms a compact pair with IgFLNa21, and in this pair the N-terminal part of IgFLNa20 masks the integrin-binding site on IgFLNa21 (15). It is possible that this masking could be released by mechanical forces. Four lines of evidence led us to hypothesize that in addition to the IgFLNa20–21 pair, other similar domain pairs could exist at the C terminus of filamin: (i) the overall structure of the C-terminal part (IgFLNs 16–24) of filamin is relatively more compact than the N-terminal part of the molecule (IgFLNs 1–15) (8); (ii) the N-terminal sequences of even-numbered domains 16, 18, and 20 differ from other IgFLNs (20) (sequence alignment is shown in supplemental Fig. S1); (iii) in single-domain solution NMR structures of IgFLNc16, IgFLNb16, 18, and 20, the N-terminal part is not folded with the rest of the domain; and (iv) according to biochemical experiments, IgFLNa18 masks integrin binding to IgFLNa19 (15). We report here small angle x-ray scattering (SAXS) analysis showing that IgFLNa16–17 and 18–19 have overall dimensions very similar to those of the previously known domain pair IgFLNa20–21. The IgFLNa22–23 construct was much more elongated, which is indicative for two independently folded noninteracting domains. Further, the atomic structures solved with NMR spectroscopy show that IgFLNa18–19 forms a pair similar to IgFLNa20–21, but the details of the interaction and orientation of the domains differ. On the other hand, IgFLNa16–17 forms an entirely novel type of domain pair. Sequence comparisons predict that these three interdependent domain pairs are conserved from nematodes to vertebrates, suggesting that the arrangement has special regulatory functions.     

大鼠热休克蛋白70的融合表达及纯化鉴定

目的：在大肠杆菌中高效表达并纯化大鼠热休克蛋白（HSP）70与麦芽糖结合蛋白（MBP）的融合蛋白,以进一步研究细胞外HSfr70的生物学功能。方法：用RT-PCR方法扩增目的基因,并将其克隆到原核表达载体pMAL-c2X中,酶切鉴定并进行DNA测序;将该重组表达载体转化大肠杆菌B121,用IPTG在不同温度及时间下进行诱导表达,建立最佳诱导表达条件;采用Amylose树脂预装柱对目的蛋白进行亲和纯化,并对不同表达条件下的产物进行SDS-PAGE及Westernblot分析。结果：克隆出目的基因,构建了融合表达载体pMAL-c2X／hsp70;诱导表达后经SDS-PAGE检测表明获得了目的条带,并纯化出纯度较高的融合蛋白;免疫印迹鉴定表明其具有抗原活性。结论：在大肠杆菌中高效表达并纯化了融合蛋白MBP-HSP70,为进一步研究细胞外HSP70的生物学效应提供了有用的材料。     

Selective Expression of Huntingtin-associated Protein 1 in ��-Cells of the Rat Pancreatic Islets

Huntingtin-associated protein-1 (HAP1) was initially identified as a binding partner of huntingtin, the Huntington''s disease protein. Based on its preferred distribution among neurons and endocrine cells, HAP1 has been suggested to play roles in vesicular transportation in neurons and hormonal secretion of endocrine cells. Given that HAP1 is selectively expressed in the islets of rat pancreas, in this study, we analyzed the expression pattern of HAP1 in the islets. In rats injected intraperitoneally with streptozotocin, which can selectively destroy β-cells of the pancreatic islets, the number of HAP1 immunoreactive cells was dramatically decreased and was accompanied by a parallel decrease in the number of insulin-immunoreactive cells. Immunofluorescent double staining of pancreas sections showed that, in rat islets, HAP1 is selectively expressed in the insulin-immunoreactive β-cells but not in the glucagon-immunoreactive α-cells and somatostatin immunoreactive δ-cells. In isolated rat pancreatic islets, ∼80% of cells expressed both HAP1 and insulin. Expression of HAP1 in the INS-1 rat insulinoma cell line was also demonstrated by immunofluorescent staining. Western blotting further revealed that HAP1 in both the isolated rat pancreatic islets and the INS-1 cells also has two isoforms, HAP1A and HAP1B, which are the same as those in the hypothalamus. These results demonstrated that HAP1 is selectively expressed in β-cells of rat pancreatic islets, suggesting the involvement of HAP1 in the regulation of cellular trafficking and secretion of insulin. (J Histochem Cytochem 58:255–263, 2010)     

Kainic Acid and Decorticating Lesions Stimulate the Synthesis of C1q Protein in Adult Rat Brain

Abstract: The first component of the classic complement cascade, C1q, was increased in whole rat brain after lesioning by intraperitoneally injected kainic acid (KA) (20-fold, 3 days after KA) and in the striatum ipsilateral to unilateral decortication (fivefold, 10 days after decortication). C1q was measured after purification by chromatography and electrophoresis. De novo biosynthesis of C1q 3 days after KA was increased >10-fold, as measured by the incorporation of [³⁵S]methionine into C1q after incubation of brain slices from KA-treated rats for 2 h. In parallel with these responses, KA induced fivefold increase of C1q bioactivity, as evaluated with C1q-dependent hemolysis. The contribution of C1q from entrapped cerebrovascular blood was evaluated by the effects of perfusion and was minor relative to the increases of C1q in response to KA lesioning. These findings support the hypothesis that the C1q protein detected by immunocytochemistry in senile plaques of Alzheimer brains and in the hippocampus after deafferenting lesions is synthesized by resident brain cells.