Up-regulation of cystatin C expression in the murine hippocampus following perforant path transections.

Cystatins are endogenous cysteine protease inhibitors that modulate the turnover of intracellular and extracellular proteins. These inhibitors are strongly implicated in a variety of pathological processes such as tumor metastasis and many degenerating CNS disorders. Here we report the expression of cystatin C, a major cysteine protease inhibitor of mammalian animals, in the murine hippocampus at 3, 7, 15 and 30 days following perforant path transections. Northern blot analysis showed that cystatin C transcripts were up-regulated in a transient manner with a significant increase at 7 and 15 days post-lesion (219% and 185% of control, respectively) in the rat hippocampus after entorhinal deafferentation. In situ hybridization and immunohistochemistry confirmed the time-dependent up-regulation of both cystatin C mRNA and protein expressions in a mouse model which initiated at 3 days post-lesion, reached maximal levels 7-15 days post-lesion, and remained slightly elevated by day 30 post-lesion. The modulation of cystatin C expression was observed to occur specifically in the entorhinally denervated zones: the stratum lacunosum-moleculare of the hippocampus and the outer molecular layer of the dentate gyrus. Double labeling by either a combination of in situ hybridization for cystatin C with immunohistochemistry for glial fibrillary acidic protein or double immunofluorescence staining for both proteins in mouse hippocampus at 7 and 15 days post-lesion revealed that most cystatin C-expressing cells are astrocytes. From these results we suggest that the spatiotemporal up-regulation of cystatin C in the hippocampus is induced by entorhinal deafferentation and that cystatin C may be involved in the astroglia-mediated neural plasticity events in the hippocampus following perforant path transections.

Maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice.

Effects of maternal dietary zinc deficiency on prenatal and postnatal brain development were investigated in ICR strain mice. From d 1 of pregnancy (E0) until postnatal d 20 (P20), maternal mice were fed experimental diets that contained 1 mg Zn/kg/day (severe zinc deficient, SZD), 5 mg Zn/kg/day (marginal zinc deficient, MZD), 30 mg Zn/kg/day (zinc adequately supplied, ZA) or 100 mg Zn/kg/day (zinc supplemented, ZS and pair-fed, PF). Brains of offspring from these dietary groups were examined at various developmental stages for expression of nestin, an intermediate filament protein found in neural stem cells and young neurons. Immunocytochemistry showed nestin expression in neural tube 10.5 d post citrus (dpc) as well as in the cerebral cortex and neural tube from 10.5 dpc to postnatal d 10 (P10). Nestin immunoreactivities in both brain and neural tube of those zinc-supplemented control groups (ZA, ZS, PF) were stronger than those in zinc-deficient groups (SZD and MZD). Western blot analysis confirmed that nestin levels in pooled brain extracts from each of the zinc-supplemented groups (ZA, ZS, PF) were much higher than those from the zinc-deficient groups (SZD and MZD) from 10.5 dpc to P10. Immunostaining and Western blots showed no detectable nestin in any of the experimental and control group brains after P20. These observations of an association between maternal zinc deficiency and decreased nestin protein levels in brains of offspring suggest that zinc deficiency suppresses development of neural stem cells, an effect which may lead to neuroanatomical and behavioral abnormalities in adults.

[An exploration of animal behavior screen platform for novel gene function in central nervous system].

For the purpose of large-scale screening of novel gene functions in mammalian nervous system, we have developed an animal behavior-monitoring platform employing antisense-oligo technology. Twenty genes of different categories were chosen from a low abundant gene (c)DNA sub-library of rat brain. Antisense oligo-nucleotides of these genes were designed and synthesized according to the homologues of the genes in mouse for mouse behavior tests. These antisense oligos were injected into the lateral ventricles of mouse brain using a Hamilton micro-syringe, with saline and oligos of scramble sequences as controls. These mice were tested with the following behavior model paradigms: metabolism, open field behavior, tail flick latency, and step-down test. Out of the 20 genes tested, 14 genes showed significant behavioral differences from the control groups at the level of P value less than 0.05 or 0.001 in different behavior animal models.

[Effect of dietary zinc on microtubule-associated protein 2 expression in the brain of mice].

Zinc deficiency could result in reduction of microtubule polymerization, which may cause impairment of brain development and function. The relationship between zinc deficiency and microtubules polymerization is still unclear. In this paper, microtubule-associated protein 2 (MAP2) expression in the brain was examined in order to explore the mechanism of zinc regulated microtubule polymerization. 80 pregnant ICR mice, randomized into 5 groups, were fed with experimental diets of different zinc levels (from 1 to 100 mg/kg) during pregnancy and lactation. The MAP2 expression in the brain of offsprings was examined by Western blot assays. The results showed that MAP2, including MAP2a, MAP2b and MAP2c, were expressed in brain from embryonic day 15, but not found on embryonic day 10. The high molecular weight of MAP2a and MAP2b expressed continuously from embryonic day 15 to postnatal day 70 (adult). While the low molecular weight of MAP2c was down-regulated from embryonic day 15 to non-existing on postnatal day 70. The expression of MAP2 in cerebrum and cerebellum kept closely at the positive dependence with dietary zinc level. The order of the levels of expression of MAP2 of the various groups administrated with different amounts of zinc is as follows: 1 mg/kg < 5 mg/kg < 30 mg/kg < 100 mg/kg. The above results suggest that zinc deficiency may inhibit the MAP2 expression, while zinc supplement exerts much improvement. The lowered level of MAP2 expression is one of important mechanisms underlying impairments of microtubule polymerization, as a result of zinc deficiency.

[Nestin expression during P19 neuron differentiation].

Mouse nestin, an intermediate filament gene, is transiently expressed during the development of the central nervous system. In order to find the clue of its function during neural development, we tried to find out the gene expression pattern during the neuronal differentiation of P19 EC cells induced RA. RT-PCR showed that nestin was transiently expressed during P19 neuron differentiation, with a peak at day 4 of this process. However, BMP4, a neural precursor cell marker, was transiently expressed with its highest level at day 6, while NF160 kD a terminal differentiated neuronal marker, was increasingly expressed during the whole process. These results implied that nestin might play some roles during the process of neural progenitor cells differentiating into neural precursor cells. Moreover, immunostaining showed that nestin was located in the neurite and the growth cone of the P19 neuron, suggesting that nestin might be also involved in the process of the establishment of neural connection.

Evidence for a mouse brain-specific variant of alpha-tubulin.

While studying the neural precursor cell intermediate filament protein known as nestin in the developing mouse brain, we observed a strong cross-reaction of our nestin antibody with a 50 kDa protein that appeared on embryonic day 10 and continued to accumulate until postnatal day 1. Here we report evidence that this protein is a brain-specific variant form of alpha-tubulin and discuss its implications.

[The separation and detection of the bioactive proteins in nerve regeneration conditioned fluids].

OBJECTIVE: To understand the biological activities of the nerve regeneration conditioned fluid (NRCF). METHODS: Nerve regeneration chamber was made by using silicone tube bridging distal and proximal ends of severed SD rat's sciatic nerve. The biological activities of the proteins in NRCF, which were separated by natural polyacrylamide gel electrophoresis (PAGE), were analysed by being cocultured with excised neonatal dorsal root ganglia (DRG). RESULTS: Eight separated protein bands of NRCF were observed between 67-669 ku in molecular weight, and the protein bands between 232-440 ku showed strong neurotrophic and chemotactic function. CONCLUSION: NRCF has the promoting effects on nerve regeneration.

Identification of a serine protease with nerve growth promoting activity from snake venom.

A new protein with nerve growth promoting activity was purified from the crude venom of the Agkistrodon halys Pallas, a Chinese snake. Its amino-terminal sequence unexpectedly showed high homology with serine proteases, suggesting that it is a new member of the serine protease family. It also cross-reacted with antibodies against thrombin-like enzyme and possessed weak arginine esterase activity, amounting to about 3% of the activity of trypsin. However, its nerve growth promoting activity was comparable to that of nerve growth factor (NGF). It was named NGF-like protease (NLP). Northern blot analysis further demonstrated different patterns of induction of c-myc, vgf and trkA mRNA transcription in PC12 pheochromocytoma cells treated with NGF and NLP, respectively. These data suggested that NLP represents a novel potent neurotrophic factor.

[Neural differentiation of Wnt-1 overexpression P19 cells].

Wnt-1 gene plays important roles during the development of the mouse central neural system. and shown a transient expression during neuronal differentiation of P19 EC cells. We transfected Wnt-1 gene into P19 cells and found the Wnt-1 transfected cells could differentiate into neurons without the induction of RA. However, MASH-1, a crucial gene for the early neural differentiation, was expressed later than P19 cells induced by RA.

[The sub-cellular distribution of beta-catenin in the neural differentiation of RA induced P19 EC cells].

Wnt signals have been shown to play an important role in the development of the central nervous system (CNS) of mouse. In our previous work, it was demonstrated that Wnt signal could initiate differentiation of P19 EC cells. In the present investigation, it was examined with RT-PCR whether expression of beta-catenin, a downstream gene of Wnt in its signal transduction pathway, is regulated. It was found that the level of protein or transcript beta-catenin during P19 neuronal differentiation was not changed. However, immunostaining data showed that beta-catenin was translocalized into nuclei after retinoic acid induced P19 cell aggregates were trypsinized and cultured in serum free N2 medium for 2 and 4 d. In this period, transcription of En-2, a downstream target gene of Wnt signal, increased evidently. The above data suggest that Wnt signals are involved in the early stage of neuronal differentiation process of P19 cell. Meanwhile, the distribution of beta-catenin on the neurites indicates that this protein may also be involved in neuritis outgrowth process.

[An antibody recognizing neuron specific tubulin].

A 20-residue peptide corresponding to the C-terminal amino acid sequence of rat nestin was synthesized by the solid phase method. The anti-peptide antibody (designated Anti-Nes-2) against nestin was prepared. Western blots showed that Anti-Nes-2 recognized not only mouse nestin with a MW of 240 kD but also a band with a MW of 50 kD. N-terminal amino acid sequence showed that this 50 kD protein is alpha-tubulin. Western blots with Anti-Nes-2 and with monoclonal antibodies against alpha- and beta-tubulin revealed that this 50 kD band could only be detected in different stages of mouse brain and in the primary culture of neural precursor cells (NPCs), with higher expression during the development of mouse brain and the maturation of NPCs; whereas alpha- and beta-tubulin were expressed in different cell lines and tissues of adult mouse. Taken together, these results indicate that 50 kD protein recognized by Anti-Nes-2 is a neuron-specific alpha-tubulin and could be a neuron-specific posttranslational modification isotype of alpha-tubulin.

[Nestin mRNA expression during the development of mouse central nervous system].

Intermediate filament Nestin is a marker of neural precursor cells. Its expression pattern during mouse brain development was studied in the present investigation. A pair of primers based on the sequence of mouse Nestin were found best to be suit for the reverse transcriptional PCR (RT-PCR) with this pair of Nestin Primer. Using RT-PCR, Nestin transcripts were detected during the development of the central nervous system (CNS) of mouse, from embryonic day 10 to adult brain. We found that the Nestin showed a transient expression in the development of cerebrum. At the embryonic day 14, the expression of Nestin mRNA reached the highest peak, and was then down-regulated. In the postnatal cerebellum, Nestin gene was also expressed in a transient pattern. The highest peak of Nestin mRNA expression emerged at postnatal day 5, and was then down-regulated. Nestin mRNA expression in 10 kinds of adult mouse tissues did not yield any positive results.

Stearylamine liposome as a new efficient reagent for DNA transfection of eukaryotic cells.

The efficiency of stearylamine (SA) liposome in transfecting DNA into eukarytic cells was evaluated and compared with that of the Lipofectin reagent and the calcium phosphate transfection method. Results with the 21 cell lines tested varied, depending on the cell type. The overall indication was that SA liposomes had certain advantages over the Lipofectin reagent and both of them were superior to the calcium phosphate method, showing the greatest effect in 8 different cell lines, while the calcium phosphate method was the best in only 4 cell lines. There were 2 cell lines which could only be transfected by SA liposomes, another 2 only by the calcium phosphate method, but none depended solely on the Lipofectin reagent. The number of cell lines failed to be transfected was 2 for SA liposomes, 3 for calcium phosphate method, and 4 for the Lipofectin reagent. The advantage of the SA liposome method is discussed.

[The induction and differentiation of a human teratocarcinoma cell line (PA-1) in vitro].

PA-1 cell line is derived from human ovary teratocarcinoma. When it grows in 10% fetal calf serum, it can be induced to differentiate by 10(-5) mol/L retinoic acid (RA). Some morphological changes can be observed after RA induction. By immunostaining of cultured cells, we found that the expression and distribution pattern of desmin and some extracellular matrix molecules, such as fibronectin, laminin and tenascin, had been changed after induction, and these changes were associated with the morphological changes. Cell growth study showed that RA treatment had no effects on growth and autocrine activities of PA-1 cells. These results suggest that some PA-1 cells were induced to differentiate along muscle cells pathway by RA.

Secretion of polypeptides related to epidermal growth factor and insulinlike growth factor I by a human teratocarcinoma cell line.

To identify polypeptide growth factors for human teratocarcinoma cells, we studied the malignant ovarian teratoma-derived cell line, PA-1, that grew autonomously in serum-free medium. Medium conditioned by undifferentiated PA-1 cells strongly stimulated proliferation of the mouse mammary tumor cell line, GR 2H6, which is responsive to epidermal growth factor (EGF) and insulinlike growth factor-I (IGF-I). After ammonium sulfate precipitation, PA-1 conditioned medium was analyzed by anion exchange chromatography and bioassay of elution fractions on GR 2H6 cells that were grown in medium deficient in either EGF or insulin. The results demonstrated that PA-1 CM contained factors that can substitute for EGF and IGF-I in stimulating growth of GR 2H6 cells. Western blots of peak mitogenic fractions revealed low molecular weight polypeptides that were immunoreactive with either anti-EGF or anti-IGF-I antibodies. Indirect immunofluorescence staining of PA-1 cells with monoclonal antibodies localized receptors for each growth factor, and binding of human EGF and IGF-I to these cells was quantified by radioreceptor assays. Secretion of factors closely related to EGF and IGF-I by PA-1 cells under serum-free conditions may provide a novel model system to study molecular mechanisms of autocrine growth stimulation in teratocarcinomas.