Skin collagen can be accurately quantified through noninvasive optical method: Validation on a swine study.

BACKGROUND/PURPOSE: Diffuse reflectance spectroscopy (DRS) is a noninvasive optical technology characterized by relatively low system cost and high efficiency. In our previous study, we quantified the relative concentration of collagen for the individual keloid patient. However, no actual value of collagen concentration can prove the reliability of collagen detection by our DRS system. METHODS: Skin-mimicking phantoms were prepared using different collagen and coffee concentrations, and their chromophore concentrations were quantified using the DRS system to analyze the influence of collagen and other chromophores. Moreover, we used the animal study to compare the DRS system with the collagen evaluation of biopsy section by second-harmonic generation (SHG) microscopy at four different skin parts. RESULTS: In the phantom study, the result showed that coffee chromophore did not severely interfere with collagen concentration recovery. In the animal study, a positive correlation (r=.902) between the DRS system and collagen evaluation with SHG microscopy was found. CONCLUSIONS: We have demonstrated that the DRS system can quantify the actual values of collagen concentration and excluded the interference of other chromophores in skin-mimicking phantoms. Furthermore, a high positive correlation was found in the animal study with SHG microscopy. We consider that the DRS is a potential technique and can evaluate skin condition objectively.

Autophagy-dependent generation of Axin2+ cancer stem-like cells promotes hepatocarcinogenesis in liver cirrhosis.

Autophagy is a pathophysiological phenomenon in liver cirrhosis that can further progress into hepatocarcinoma. Liver cancer stem cells (CSCs) are believed to initiate hepatocarcinogenesis. To investigate the precise mechanism related to the origin of CSCs in liver cirrhosis and hepatocarcinogenesis, we labeled Axin2+ hepatic cells with EGFP in Axin2Cre;Rosa26EGFP transgenic rats, and then stratified clinical and rat liver cirrhosis samples by autophagy flux. Clinical follow-up and lineage tracing in transgenic rat liver cirrhosis revealed that while Axin2/EGFP+ hepatic cells were present in normal livers and cirrhotic livers without aberrant autophagy, hepatic Axin2/EGFP+CD90+ cells were generated exclusively in cirrhotic livers with aberrant autophagy and promoted hepatocarcinogenesis. Aberrant autophagy in liver cirrhosis resulted in hepatocyte growth factor (HGF) expression, leading to activation of Met/JNK and Met/STAT3 signaling in sorted hepatic Axin2/EGFP+ cells and their transition into Axin2/EGFP+CD90+ cells that possess CSC properties. In a transgenic rat liver cirrhosis model, induction or inhibition of autophagy in cirrhotic livers by systemic administration of rapamycin or chloroquine or transfection with Atg3- and Atg7-shRNAs significantly induced or suppressed HGF expression, which in turn increased or reduced generation of EGFP+CD90+ hepatic cells by activating or inactivating Met/JNK and Met/STAT3 signaling, thereby promoting or preventing hepatocarcinogenesis. Systemic treatment with HGF-shRNA, SP600125 or stattic also reduced generation of EGFP(Axin2)+ hepatic cell-originated CD90+ CSCs in aberrant autophagic cirrhotic livers by inactivating HGF/Met/JNK or HGF/Met/STAT3 signaling, further preventing hepatocarcinogenesis. These data suggest that activation of Met/JNK and Met/STAT3 signaling in Axin2+ hepatic cells via autophagy-dependent HGF expression and the resultant generation of Axin2+CD90+ CSCs is a major mechanism of hepatocarcinogenesis in cirrhotic livers.

[Related factors analysis for lymph node metastasis in papillary thyroid carcinoma: a series of 2 073 patients].

Objective: To investigate the related factors for lymph node metastasis (LNM), especially for high volume LNM (>5 metastatic lymph nodes) in papillary thyroid carcinoma (PTC). Methods: The medical records of 2 073 consecutive PTC patients who underwent lobectomy, near-total thyroidectomy or total thyroidectomy with ipsilateral or bilateral central lymph node dissection in Department of General Surgery, Peking Union Medical College Hospital from November 2013 to October 2014 were reviewed. Clinical and pathological features were collected. Univariate and multivariate analysis were performed to identify the related factors for LNM/high volume LNM. Results: In all 2 073 patients, LNM and high volume LNM were confirmed in 936 (45.15%) cases and 254 (12.25%) cases respectively. In univariate analysis, large tumor size, young patients (<40 years), male were associated with both LNM and high volume LNM. In multivariate analysis, tumor size >2.0 cm, young patients (<40 years), male were independent related factors of LNM (OR=5.262, 95% CI: 3.468 to 7.986; OR=2.447, 95% CI: 2.000 to 2.995; OR=1.988, 95% CI: 1.593 to 2.480, respectively, all P=0.000) and high volume LNM (OR=6.687, 95% CI: 4.477 to 9.986; OR=2.975, 95% CI: 2.224 to 3.980; OR=2.354, 95% CI: 1.737 to 3.191, respectively, all P=0.000). In 1 414 PTMC patients, a similar result was also demonstrated.Compared with young patients (<40 years), old patients (≥60 years) had lower incidence of LNM (25.47% vs. 52.24%, χ(2)=62.903, P=0.000) and high volume LNM (1.89% vs. 13.18%, χ(2)=37.341, P=0.000). Additionally, old patients also had lower risk of both LNM (OR=0.316, 95% CI: 0.194 to 0.517, P=0.000) and high volume LNM (OR=0.142, 95% CI: 0.034 to 0.599, P=0.000). Conclusions: The tumor size was the main related factor for both LNM and high volume LNM in PTC. The treatment should be more active in patients with tumor size >2 cm with consideration of higher incidence and risk for LNM and high volume LNM. Young patient was another important related factor for LNM and high volume LNM. In PTMC, old patients had lower incidence and risk for both LNM and high volume LNM. Dynamic observation or less surgical extent could be an option for these patients.

[Influences of DNA methylation upon neuroglobin sustained expression in oxygen- glucose deprivation model].

Objective: To investigate the influences of genomic DNA methylation upon neuroglobin sustained expression in oxygen- glucose deprivation model. Methods: With A549 cell strain as the research object, the control group were cultivated in the complete medium containing 10 µmol/L of 5-azacytidine for 4 days, and the control group was cultivated in the complete medium for 4 days.Then carried out oxygen glucose deprivation treatment for 4 h.Detecting neuroglobin expression, DNA methyltransferase expression, cell inhibition ratio and DNA methylation level at different time points. Results: DNA methylation level of the experimental group declined apparently[6 h : (1.0±0.0) vs (2.1±0.3); 12 h: ( 0.9±0.0) vs (1.4±0.0); 24 h: (0.9±0.0) vs (2.6±0.2); 36 h: (0.9±0.0) vs (2.9±0.1)], neuroglobin expression of the experimental group continued and was obviously higher than that of the control group at the same time point[NGB-PCR: 6 h: (3.3±1.1) vs (0.4±0.1); 12 h: (3.2±0.8) vs (0.1±0.1); 24 h: (4.6±0.6) vs (0.2±0.0); 36 h : (5.1±0.3) vs (0.1±0.1)], while the Cell inhibition ratio of the experimental group was obviously lower than that of the control group at the same time point[(6 h: (10.4±0.5) vs (14.1±0.7); 12 h: (22.0±1.3) vs (35.1±0.5); 24 h: (25.7±1.0) vs (40.6±1.3); 36 h: (30.0±0.8) vs (44.4±0.7)], differences had statistical significance (P<0.05).mRNA expression of three methyltransferases of the experimental group was higher than that of the control group at different time points, where, DNMT1 and DNMT3B showed great differences (P<0.05), while differences in DNMT3A of two groups had no statistical significance (P>0.05). Conclusions: In the OGD/R model of A549 cell strain, genomic DNA methylation resulted in unsustained expression of neuroglobin, but neuroglobin expression increased after demethylation inhibitor was used.

Triptolide inhibits the multidrug resistance in prostate cancer cells via the downregulation of MDR1 expression.

Triptolide (TPL) is a diterpenoid triepoxide derived from the Chinese herb Tripterygium wilfordii and possesses anti-tumor activity against a range of cancer cells. However, the effect of TPL on prostate cancer cells and its potential to overcome multidrug resistance (MDR) have not been explored. Therefore, in this study we used prostate cancer cell line DU145 as the experimental model and established DU145/ADM cell line resistant to adriamycin (ADM). Our results showed that TPL inhibited the proliferation and induced the cell cycle arrest and apoptosis of DU145 cells in a dose and time dependent manner. TPL decreased the levels of Cyclin D1 and anti-apoptotic protein Bcl-2, and increased the levels of pro-apoptotic proteins Fas and Bax. Furthermore, we found that TPL restored the sensitivity DU145/ADM cells to ADM in a dose dependent manner, and this was accompanied by the inhibition of MDR1 expression at both mRNA and protein levels. Taken together, these results provide strong evidence that TPL overcomes MDR in prostate cancer cells by downregulating MDR1 expression, and suggest that TPL is a promising agent for prostate cancer therapy, especially for chemoresistant prostate cancer.

Expression and characterization of a recombinant Cry1Ac crystal protein fused with an insect-specific neurotoxin ω-ACTX-Hv1a in Bacillus thuringiensis.

In order to assess possible enhancement of biopesticide activity, the fusion gene of crystal protein gene cry1Ac with the insect-specific neurotoxin ω-ACTX-Hv1a gene and egfp was expressed in Bacillus thuringiensis acrystalliferous strain Cry-B under the control of the native gene expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell and the GFP signal could be clearly observed. A 166 kDa full-length fusion protein was identified by immunoblot analysis. Virulence of the fusion inclusions was at least fivefold higher toward larvae of Spodoptera exigua. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in B. thuringiensis by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity.

Efficient constitutive expression of chitinase in the mother cell of Bacillus thuringiensis and its potential to enhance the toxicity of Cry1Ac protoxin.

Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine-Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry(-)B. The recombinant plasmid was stably maintained over 240 generations in Cry(-)B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l(-1) Na(2)CO(3)-0.2% beta-mercaptoethanol buffer at a wide range of alkaline pH values, and what's more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.

A Phytoplasma Associated with an Outbreak of an Unusual Disease of Chrysanthemum in China in 2008.

In August of 2008, a disease of chrysanthemum (Dendranthema morifolium (Ramat.) Tzvel) caused losses of 70 to 80% in one of the largest chrysanthemum gardens in Yangling, Shanxi Province, China. Chrysanthemum plants in nearby areas also were affected to various degrees. Symptoms included flattened stems, shortening of internodes, yellowing of leaf margins, root death, and dwarfing of plants. Affected plants eventually collapsed. On the basis of these symptoms, a phytoplasma was suspected. Total nucleic acids were extracted from 0.5 g of phloem tissue from stems of eight symptomatic and eight asymptomatic plants by the cetyltrimethylammoniumbromide (CTAB) method (1). To amplify phytoplasma DNA, primer pairs R16mF2/R16mR1, followed by R16F2n/R16R1 (2), were used in a nested PCR. A final amplicon product (1.2 kb) was obtained from all symptomatic plants but not from asymptomatic ones. Restriction fragment length polymorphism (RFLP) analyses of R16F2n/R16R1 amplicons with MseI, AluI, HhaI, HaeIII, KpnI, RsaI, and HpaII endonucleases indicated that all symptomatic plants, but none of the asymptomatic plants, contained a phytoplasma strain of group 16SrI, subgroup B (3). A search of rDNA sequences in GenBank revealed a similarity (>99%) to aster yellow phytoplasma, 16SrI group, thereby confirming strain identity based on RFLP analysis. These results indicate the disease of chrysanthemum is associated with a phytoplasma related to the aster yellow phytoplasma group. Sequences were deposited in GenBank (Accession No. FJ543467). A vector of this phytoplasma in chrysanthemum has not been identified. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 48:1153, 1998.

Vasopressin 1a receptor knockout mice have a subtle olfactory deficit but normal aggression.

Two receptors for vasopressin (Avp) are expressed in the brain, the Avp 1a receptor (Avpr1a) and the Avp 1b receptor (Avpr1b). To investigate the role of Avpr1a in behaviors in mice more extensively, we generated a line of mice lacking a functional Avpr1a (knockout, Avpr1a(-/-)). We first performed a baseline phenotypic screen of the Avpr1a knockouts followed by a more detailed analysis of their circadian rhythms and olfactory function. When free-running in constant darkness, the Avpr1a(-/-) mice have a longer circadian tau than the wild types. There are also subtle olfactory deficits in Avpr1a(-/-) mice as measured in an olfactory habituation/dishabituation test and in the discrimination of female urine from male urine using an operant testing paradigm. An extensive body of research has shown that manipulation of the Avpr1a alters behavior, including aggression and social recognition. Therefore, we expected profound behavioral deficits in mice lacking the Avpr1a gene. Contrary to our expectations, social aggression, anxiety-like behavior and social recognition are unaffected in this line of Avpr1a knockout mice. These data suggest either that the Avpr1a is not as critical as we thought for social behavior in mice or, more likely, that the neural circuitry underlying aggression and other social behaviors compensates for the life-long loss of the Avpr1a. However, the olfactory deficits observed in the Avpr1a(-/-) mice suggest that Avp and Avpr1a drugs may affect behavior, in part, by modulation of chemosensory systems.

Differential gene expression response to gonadal hormones by preoptic regulatory factors-1 and -2 in the female rat brain.

Steroids and neuropeptides interact in the central nervous system (CNS) to regulate reproductive function and behavior. The preoptic regulatory factors, PORF-1 and PORF-2, are unique neuropeptides for which roles in gender-related brain development and function have been proposed. PORF-1 and PORF-2 expression in rat brain are age, region and gender dependent, and castration or hypophysectomy alter the metabolism of the PORF-1 and PORF-2 mRNAs in male rat brain and testes. If these two peptides have a role in gender-dependent brain function, then gonadal steroids might well affect their expression. The present study was designed to investigate the response of the PORF-1 and PORF-2 mRNAs to sex steroids in the female rat brain and to compare this response to that of two peptides whose roles in the neuroendocrinology of reproduction are well established, gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY). Rats were ovariectomized and treated with placebo, estradiol (E2), progesterone (P4) or a combination of the two (E2/P4) and NPY, PORF-2, GnRH and PORF-1 mRNAs were quantified by nuclease protection assays. PORF-1, PORF-2 and GnRH mRNAs were also measured in intact rats during estrus and proestrus. Responses were compared in the preoptic anterior hypothalamus (POA), medial basal hypothalamus (MBH), cerebral cortex (CC) and hippocampus (HIPP). Expression of PORF-1 and PORF-2 was also confirmed in the female rat hypothalamus by in situ hybridization analysis. PORF-1 and PORF-2 mRNAs were detected in the adult female rat brain by both in situ hybridization and ribonuclease protection analyses. In situ hybridization analysis demonstrated that PORF-1 and PORF-2 mRNAs are expressed in hypothalamic neurons. RNase protection analysis showed that PORF-1, PORF-2 and NPY mRNAs were present in all four brain regions examined while GnRH expression was detected only in the MBH and POA. Estradiol alone upregulated expression of the PORF-1 and PORF-2 mRNAs in the ovariectomized rat in the POA and HIPP, and of NPY mRNA in the MBH and HIPP. Progesterone alone had a stimulatory effect on NPY mRNA in the MBH and HIPP. Treatment with a combination of E2/P4 downregulated PORF-2 mRNA in the POA as well as PORF-1, PORF-2 and NPY mRNAs in the CC. In contrast, E2/P4 upregulated the PORF-2 and NPY mRNAs in the HIPP and NPY mRNA in the MBH. In the cycling rat, PORF-1 mRNA levels were higher during proestrus than estrus in both the MBH and POA, while PORF-2 mRNA levels did not change. In contrast GnRH mRNA was lower in the POA and higher in the MBH during proestrus compared with estrus. Thus, intrinsic factors, most likely both ovarian and neuroendocrine, regulate PORF-1 and GnRH expression in the intact cycling rat CNS in a region-dependent manner. In the ovariectomized rat, PORF-1, PORF-2, NPY and GnRH mRNAs all respond in a region-specific manner to sex steroid treatment. These data support the role of PORF-1 and PORF-2 in gender-dependent brain function in the adult female rat.

A functional determinant in human luteinizing hormone and chorionic gonadotropin: differential effect of mutations about beta-GLN-54.

There is evidence that the conserved glutamine at residue 54 in the beta-subunit of human LH and and CG (hCG) is important for biological activity. Mutation to Arg in LH has been reported to impair receptor binding, leading to a documented case of hypogonadism, whereas in hCG the mutation has been shown to result in defective subunit association. Functional distinctions between LH and hCG have been described, but the significance of peptide-chain differences between the two has not been investigated systematically. We therefore compared the role of Gln-54 and its neighboring residues in both hormones, through replacement by amino acids with contrasting properties using site-directed mutagenesis. The mutant subunits were coexpressed with alpha-subunit in mammalian (Chinese hamster ovary) cells and the secreted hormones assayed for heterodimer formation, receptor binding, and steroidogenesis in murine Leydig cell tumor (MA-10) cells. Basic (Arg, Lys) substitution for Gln-54 in either hormone markedly impaired subunit association (<20% of wild-type) and the heterodimers that were formed were inactive (<5% of wild-type) in both assays. Arg-substituted hCG was also inactive in an adenylate cyclase assay using HEK-293 cells expressing rat LH/hCG receptor. After acidic (Glu) or neutral (Ala) substitution, heterodimer formation was less impaired (50-60% of wild-type), but effects on receptor interaction differed between the two hormones. The LH mutants still lacked binding activity, whereas the hCG products were fully active. The importance of residue 54 for receptor interaction appears to be sharply localized because mutation at adjacent positions (Pro-53 and Val-55) did not impair the activity of either hormone. Diminished heterodimer formation by Ile-53 mutation in LH (but not hCG), together with the similar effects of basic mutations at 54, imply long-distance effects as these residues are remote from alpha in the crystal structure. Our findings indicate that position 54 in LH and hCG is a determinant for both subunit association and receptor interaction. The differing responses between LH and hCG to certain mutations suggest that structural characteristics of the peptide chains may confer functional differences despite their close sequence homology.

Expression of the preoptic regulatory factor-1 and -2 genes in rat testis. Developmental and hormonal regulation.

Hormone-responsive peptides play a vital role in development and regulation of testicular function. The preoptic regulatory factors, porf-1 and porf-2, were originally discovered in the rat brain, but are also expressed in the rat and human testis. In the brain expression is age-related, hormone-responsive, region- specific, and gender-related, suggesting that porf-1 and porf-2 are involved in gender-specific brain development and function. Tissue-specific porf-1 and porf-2 mRNAs are also found in the testis and hypophysectomy may alter testicular porf-2 expression. It was thus of interest to further examine porf-1 and porf-2 expression in the testis to evaluate their potential as hormone-responsive peptides that regulate testicular development and function. Testicular expression of both porf-1 and -2 was analyzed as a function of maturational stage, aging and hypophysectomy by the solution hybridization/nuclease protection assay, and cellular location determined by in situ hybridization histochemistry. Expression was quantitatively compared in normal male rats at 15, 30 and 60 d (n = 4) and at 2, 6, 12, and 24 mo of age (n = 5). During development porf-1 is expressed at a constant level at 15, 30 and 60 d, then declines significantly with advancing age; levels at 24 mo are only 20% of those seen at 2 mo (p < 0.05). In contrast, porf-2 expression is highest at 15 d of age and steadily declines at 30 and 60 d, plateaus in the mature adult (6 and 12 mo), then exhibits an additional significant decline in the aged 24 mo animals (6 vs 24 mo, p < 0.05). Hypophysectomy of young adult rats at day 42 results in increased testicular expression 12 d later of both porf-1 (p < 0.05) and porf-2(p < 0.005) compared to intact 54-d-old rats (n = 5). In situ hybridization histochemistry confirms that both porf-1 and porf-2 are expressed in the mature testis at 60 d of age. Porf-2 mRNA is localized to immature germ cells including spermatogonia and primary spermatocytes. Porf-1 mRNA is associated with mature sperm and at low levels in the Sertoli cell cytoplasm surrounding spermatocytes. These data suggest that porf-2 is a pituitary hormone-responsive factor in the developing testis and that both porf-1 and porf-2 have cell-type specific functions in the germ cell compartment of the mature testis

Cross-replantation of lower extremities in multilimbed amputation: case report and literature review in China.

Presented is a case of a 20-year-old man on whom cross-replantation of the right leg onto the stump of the left was successfully conducted by means of unusual bone fixation. Follow-up showed that the result was satisfactory, with firm bone union and functional recovery of the replanted leg.

Sex-specific changes in preoptic regulatory factor-1 and preoptic regulatory factor-2 mRNA expression in the rat brain during development.

Preoptic regulatory factor-1 (Porf-1) and Preoptic regulatory factor-2 (Porf-2) are two novel peptide genes which are expressed in the central nervous system. Expression is modified by age and by hormones of the reproductive system. In this study nuclease protection assays were employed to investigate Porf-1 and Porf-2 mRNA expression in the cerebral cortex (CC), hippocampus (HIPP), preoptic area (POA) and medial basal hypothalamus (MBH) of male and female rats, aged 15, 30 and 60 days. Porf-1 and Porf-2 mRNA expression tended to decrease from 15 to 60 days, with two exceptions. Porf-2 in the hippocampus of female rats, and Porf-1 in the MBH of the male rats, were found instead to increase with age. There were distinctive sex differences inporf-1 andporf-2 gene expression. Consistently higher mRNA levels were measured for both genes in the POA of female rats at all ages examined, and this difference reached statistical significance for Porf-1 at the age of 60 days. In contrast, levels of Porf-2 mRNA were higher in MBH of male than female rat MBH at 15 days, and Porf-1 mRNA was substantially more abundant in male than in female rat MBH at 30 and 60 days of age. These results indicate that there is sexual dimorphism and regional specificity in the developmental expression of these genes.

Regional distinction in age-related changes of preoptic regulatory factor-1 and preoptic regulatory factor-2 mRNA expression in the rat brain.

Preoptic regulatory factor-1 (porf-1) and preoptic regulatory factor-2 (porf-2) are two novel neuropeptide genes expressed in the central nervous system and peripheral tissues. Other studies have shown that these genes may play a role in steroid-dependent brain development and functions. In this study, nuclease protection assays were employed to investigate Porf-1 and Porf-2 mRNA expression in male rat brains of different ages. The preoptic area (POA), cerebral cortex (CC), and hippocampus (HIPP) expressed both Porf-1 and Porf-2 mRNA, while only Porf-2 mRNA was detectable in the medial basal hypothalamus (MBH). Porf-1 mRNA in the POA was highest at the age of 2 months (young adult), decreased at the age of 6 months (mature adult), and remained low at the ages of 12 (middle aged) and 24 months (aged). Porf-1 mRNA in the CC was also the highest at the age of 2 months and decreased with age. However, there were no age-related changes for Porf-1 mRNA in the HIPP. Porf-2 mRNA in the HIPP was found to be low at the age of 2 months, increased at the ages of 6 and 12 months, and decreased at the age of 24 months. The effect of age on Porf-2 mRNA in the POA was similar to that seen for Porf-1, with the highest expression observed in the 2-month-old rats. There were no age-related Porf-2 mRNA changes in the MBH and CC. These results indicate differential regulation of expression of the porf-1 and porf-2 genes in the MBH, POA, HIPP, and CC. The possible roles of these two genes in maturation and aging of male rats are discussed.

Regulation of arginine vasopressin mRNA in rat fetal hypothalamic cell culture. Role of protein kinases and glucocorticoids.

Studies have been performed to investigate the regulation of arginine vasopressin (AVP) mRNA expression in fetal hypothalamic cultures. AVP mRNA-positive neurones were identified by in-situ hybridization histochemistry, and changes in mRNA expression were quantitated by nuclease protection assay. Both protein kinase C and protein kinase A activators increased the expression of AVP mRNA, in contrast to dexamethasone, which inhibited the responses to both protein kinase C and protein kinase A activation.

Mechanisms of norepinephrine mediated corticotropin-releasing factor-41 release from cultured fetal hypothalamic cells.

Catecholamines have been shown to activate hypothalamic corticotropin-releasing factor-41 (CRF) synthesis and release. In order to study the mechanisms involved, fetal hypothalamic cells were cultured and CRF release was measured by radioimmunoassay. Norepinephrine (NE) induced CRF release in a dose-dependent manner. Further studies were performed with a protein kinase C inhibitor, H-7(1-(5-isoquinolinesulfonyl)-2-methylpiperazine) and a protein kinase A inhibitor, IP-20. NE-stimulated CRF release was reduced by H-7 (5 and 50 microM) in a dose-dependent fashion, while 5 microM IP-20 resulted in a small but significant inhibition. Pretreatment of the cells for 15 h with 20 and 200 nM 12-O-tetradecanoylphorbol-13-acetate, which down-regulates protein kinase C activity, blocked the release of CRF in response to NE (1 microM), further supporting protein kinase C as a mediator for NE-activated CRF release. Pretreatment with 50 and 500 ng/ml pertussis toxin (15 h) resulted in a dose-dependent inhibition of NE-activated CRF release. Both dexamethasone and aldosterone at the concentrations of 1 microM reduced NE-induced CRF release. These results suggest that CRF can be released from hypothalamic neurons in response to NE through both protein kinase C- and protein kinase A-dependent mechanisms, and that pertussis toxin-sensitive G-proteins are also involved in this response. Furthermore, glucocorticoids and mineralocorticoids can reduce NE-activated CRF release from cultured hypothalamic cells.