Factors affecting cryopreservation-associated damages in sperm motility of cockerels (Gallus gallus domesticus).

1. Sperm are exposed to severe osmotic stress during cryopreservation, which results in impairment of fertilisation ability, including motility and viability, in poultry. Sperm osmotolerance is regulated by many extracellular factors and varies widely in birds, leading to uncertainty in the nature of the osmotic injury.2. Tail bending is a primary response resulting from cell swelling from excessive osmotic stress. However, the underlying mechanism responsible for tail bending is largely unknown. This study examined the relationship between osmotic stress and post-thaw motility, with a particular focus on the role of Na+/K+ ATPase (NKA) in the tail bending response.3. Cryopreserved sperm exhibited rapidly reduced motility when maintained at 37°C. The combination of temperature change and osmotic stress was a primary factor responsible for tail bending. This work tested a hypothesis known to be associated with post-thaw tail abnormality in other species and found that cold shock, that is not accompanied by an apoptotic response, may occur. Ouabain inhibition of Na+/K+ ATPase activity alleviated the tail bending response in fresh and post-thaw sperm.4. These results demonstrated that the combination of temperature change and osmotic stress has a primary impact on the reduction of post-thaw motility, with a particular role in NKA activity, in the tail bending response of chicken sperm. These results provide a foundation for establishing cryopreservation methodology to ensure the optimal fertilisation potential of cryopreserved chicken sperm.

Demographics, health-related behaviors, eating habits and knowledge associated with vegetable intake in Japanese adults.

OBJECTIVES: To analyze demographic, health-related behaviors, eating habit and knowledge associated with vegetable intake. METHODS: Secondary analyses using the dataset from the National Health and Nutrition Survey 2003. Food intake data measured by the food-weighing method in one-day and a questionnaire assessed the dietary intake and health-related behaviors, eating habit and knowledge. This study was made in Japan. The data of 1742 men and 2519 nonpregnant/nonlactating women, aged 20-69 years, energy intake between percentiles 1 and 99 were included. Vegetable intake was analyzed according to the Japanese vegetable recommendation (>or=350 g/day) after age adjustment. RESULTS: Average of VI was 307 g/day in men and 297 g/day in women. Only 35% of men and 31% of women met the recommended amount of vegetable intake. Japanese from city areas, aged 60-69 years, had the highest vegetable intake and subjects from metropolitan areas had the lowest vegetable intake. Depending on the age groups, risks for low vegetable intake in Japanese were found in subjects with skipping meals, alcohol intake and history of smoking. CONCLUSIONS: To increase vegetable intake, it is necessary to provide more nutritional education and lifestyle-related diseases education.

Observer performance in diagnosing osteoporosis by dental panoramic radiographs: results from the osteoporosis screening project in dentistry (OSPD).

Mandibular cortical erosion detected on dental panoramic radiographs (DPRs) may be useful for identifying women with osteoporosis, but little is known about the variation in diagnostic efficacy of observers worldwide. The purpose of this study was to measure the accuracy in identifying women at risk for osteoporosis in a worldwide group of observers using DPRs. We constructed a website that included background information about osteoporosis screening and instructions regarding the interpretation of mandibular cortical erosion. DPRs of 100 Japanese postmenopausal women aged 50 years or older who had completed skeletal bone mineral measurements by dual energy X-ray absorptiometry were digitized at 300 dpi. These were displayed on the website and used for the evaluation of diagnostic efficacy. Sixty observers aged 25 to 66 years recruited from 16 countries participated in this study. These observers classified cortical erosion into one of three groups (none, mild to moderate, and severe) on the website via the Internet, twice with an approximately 2-week interval. The diagnostic efficacy of the Osteoporosis Self-Assessment Tool (OST), a simple clinical decision rule based on age and weight, was also calculated and compared with that of cortical erosion. The overall mean sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the 60 observers in identifying women with osteoporosis by cortical erosion on DPRs were 82.5, 46.2, 46.7, and 84.0%, respectively. Those same values by the OST index were 82.9, 43.1, 43.9, and 82.4%, respectively. The intra-observer agreement in classifying cortical erosion on DPRs was sufficient (weighted kappa values>0.6) in 36 (60%) observers. This was significantly increased in observers who specialized in oral radiology (P<0.05). In the 36 observers with sufficient intra-observer agreement, the overall mean sensitivity, specificity, PPV, and NPV in identifying women with osteoporosis by any cortical erosion were 83.5, 48.7, 48.3, and 85.7%, respectively. The mean PPV and NPV were significantly higher in the 36 observers with sufficient intra-observer agreement than in the 24 observers with insufficient intra-observer agreement. Our results reconfirm the efficacy of cortical erosion findings in identifying postmenopausal women at risk for osteoporosis, among observers with sufficient intra-observer agreement. Information gathered from radiographic examination is at least as useful as that gathered from the OST index.

Structure and property of self-assemble valinyl bolaform amides having different chirality.

Bolaform amides were designed from N,N'-bis(carboethoxy-L-valinyl)-diaminoethane (1) by linking t-butyloxycarbonyl-valine through ethylenediamine (EDA) to enable spectroscopic and X-ray diffraction analyses. N,N'-Bis(Boc-L-valinyl)-diaminoethane (2) and N,N'-bis(Boc-D-valinyl)-diaminoethane (3) were composed of L-Val and D-Val, respectively. N-(Boc-L-valinyl)-N'-(Boc-D-valinyl)-diaminoethane (4) was composed of both L-Val and D-Val, and was achiral (meso-peptide). Peptide 5 was a 1:1 mixture of 2 and 3, and was also achiral (racemate). These peptides mediated gelation of corn oil at a concentration of approximately 1%. Within crystals, the peptides formed beta-sheet ribbons, but differences were observed in hydrogen-bonding patterns and side-chain arrangements. These differences were also deduced from temperature dependence of amide protons. Force-field calculations based on the crystal structures indicated that association of beta-sheet ribbons had energy benefits, and it was assumed that molecular aggregation progressed spontaneously. These structural studies indicated the chirality of amino acids affected for the properties of bolaform amides.

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.

Outcomes of radiotherapy for inoperable locally advanced (T4) esophageal cancer-retrospective analysis-.

To assess the treatment outcome after radiotherapy with or without chemotherapy for inoperable locally advanced (T4) esophageal cancer (EC), a retrospective analysis was performed. We enrolled 37 patients with T4 EC and analyzed 35 patients. A total of 28 patients were treated with radiotherapy and chemotherapy (median dose, 60 Gy) and seven patients were treated with radiation alone (median dose, 60 Gy). Many mainly received high-dose cisplatin (CDDP) and 5-fluorouracil (FU) or continuous infusion of low-dose CDDP and 5-FU. Among the 35 patients, there were eight survivors and 27 deaths. The median follow-up period was 15.5 months (3.5 to 74 months). The one-year, 3-year, and 5-year survival rates were 37.5%, 10.0%, and 10.0%, respectively, with a median survival time (MST) of 7.3 months. When the patients were divided into two groups, a complete response (CR) group and a non-CR group, MST was 16.5 and 6.2 months, respectively, showing significant differences between the two groups (p=0.0317). CR patients showed more satisfactory long-term outcomes than non-CR patients.

Male hybrid sterility of mice with the genomic region of the KitW mutation and the KitS allele from Mus spretus.

Two congenic strains, C57BL-KitW and C57BL-KitS, were generated. The KitW allele originated from strain WB-KitW and the KitS allele from Mus spretus. The KitW/KitS males showed hybrid sterility with small testes, but the females were fertile. The development of the seminiferous tubules of KitW/KitS males stopped before the spermatocyte stage and they were almost free of sperm. The Kit gene is located at position 42 on chromosome 5. We investigated in the C57BL-KitS congenic strain which part of the chromosomal region adjacent to the KitS allele is introduced from SPR into a C57BL background. The region between positions 42 and 44 was derived from SPR. Eleven amino acid substitutions of the KitS cDNA were detected by comparison with the sequence data of the +Kit cDNA from C57BL; seven were in the extracellular domain, one in the transmembrane domain, two in the kinase I domain, and one in the carboxy-terminal tail. The Kit mRNA derived from both KitW and KitS alleles was expressed in the sterile testes of KitW/KitS males.

Isoform-specific regulation of vascular endothelial growth factor (VEGF) family mRNA expression in cultured mouse brown adipocytes.

We have shown that brown adipose tissue (BAT), a thermogenic organ in mammals, expresses high levels of vascular endothelial growth factor (VEGF) mRNA in response to exposure to cold, which may contribute to angiogenesis associated with cold-induced hyperplasia of this tissue. In the present study, we examined mRNA expression of not only VEGF, but also VEGF-B and VEGF-C, recently cloned VEGF isoforms, in vitro using immortal brown adipocytes (HB2) isolated from mouse BAT. HB2 preadipocytes expressed detectable levels of VEGF, VEGF-B and VEGF-C mRNA, but a low level of VEGF. After HB2 cells differentiated into adipocytes, the VEGF mRNA level increased without a noticeable change in the VEGF-B and VEGF-C mRNA levels. When HB2 cells were stimulated by norepinephrine, the VEGF mRNA level increased without a change in that of VEGF-B, while the VEGF-C mRNA level decreased. A marked reduction of VEGF-C mRNA expression was also found when HB2 cells were treated with agonists of peroxisome proliferator-activated receptor gamma (PPARgamma, troglitazone), retinoic acid receptor (RAR, all-trans retinoic acid) and retinoid X receptor (RXR, 9-cis retinoic acid). These results suggest a specific adrenergic mechanism for up-regulation of VEGF expression different from those for other VEGF isoforms, and thereby the major contribution of VEGF to the cold-induced angiogenesis in BAT. In addition, the agonists of PPARgamma, RAR and RXR are suggested to be inhibitory to angiogenesis through the reduction of VEGF-C production.

Patterns of failure in carcinoma of the uterine cervix treated with definitive radiotherapy alone.

The purpose of this retrospective study was to evaluate the patterns of failure for the patients treated with definitive radiotherapy, and to discuss future treatment strategies for the uterine cervical cancer. From 1986 to 1995, 177 patients with stages I-III cervical cancer treated with a combination of two-axial conformal radiotherapy and radium brachytherapy were analyzed. The first treatment failures were pelvic failure in 11%, and distant metastases (DM) in 16% of the 177 patients. Paraaortic lymph nodes (PAN) were the most frequently metastatic regions (71%). In the pelvic control group, DM were in 6% of patients for stages I-II, and in 32% of patients for stage III. In the pelvic failure group, DM were in 75% of patients for stages I-II, and in 19% of patients for stage III. In stages I-II, the DM rate was significantly correlated with pelvic tumor control. However, there was no correlation in stage III. To improve survival, it is important to increase the pelvic tumor control rate for patients with stage I-II, and to increase the pelvic tumor and metastatic control rate in stage III. Additional treatments such as chemotherapy and/or PAN irradiation using conformal radiotherapy are required in stages I-II with locally bulky tumor and in stage III.

Treatment results of chemoradiation for T1 esophageal cancer with lymph node metastases.

PURPOSE: We clinically evaluated 12 patients with advanced superficial esophageal cancer who had undergone chemoradiotherapy. PATIENTS AND METHODS: A retrospective analysis was performed between October 1992 and December 1998 on 12 patients with M1 metastasis in lymph nodes or direct invasion of enlarged lymph nodes (A3) to adjacent structures. Combined treatment with radiotherapy and chemotherapy was applied to all patients. High-dose cisplatin (CDDP) and 5-fluorouracil (5-FU) were administered to five patients, low-dose CDDP and 5-FU to five patients, and others to two patients. Ten patients were treated by external irradiation alone, and two patients were treated by external and intracavitary irradiation. RESULTS: Of the 12 patients, the response of the primary tumor to this therapy resulted in 11 CRs (91.6%) and one PR, while the response of the largest metastatic lesion in lymph nodes in each patient resulted in three CRs (25%), four PRs (33.3%), and five NCs (41.6%). The 2-year survival rate of all patients was 51.9%, and MST was 28.9 months. The 2-year survival rates of the M1 and A3 patients were 60.0% and 41.7%, respectively. CONCLUSION: This study revealed that the present chemotherapy regimen is ineffective in treating metastatic lymph node lesions. Therefore, it will be necessary to evaluate the effectiveness of chemoradiotherapeutic agents from the perspective of their effects on metastatic lymph nodes.

Mutated human beta3-adrenergic receptor (Trp64Arg) lowers the response to beta3-adrenergic agonists in transfected 3T3-L1 preadipocytes.

Wild-type or mutated human beta3-adrenergic receptor (Trp64Arg) cDNAs were stably expressed in mouse 3T3-L1 cells. Saturation binding study using a beta-adrenergic ligand revealed that there was no significant difference in the receptor density and the equilibrium dissociation constant between the two cell lines. However, the ability of the mutant beta3-adrenergic receptor to accumulate cyclic AMP (cAMP) in response to isoproterenol was much reduced and Kact for cAMP accumulation was lowered as compared to the wild type receptor. The amount of alpha subunit of stimulatory GTP-binding protein (GSalpha) and adenylyl cyclase activity in response to forskolin were not different in the two cell lines. The responses of the mutant receptor to epinephrine, norepinephrine and L-755,507, a highly specific agonist for human beta3-adrenergic receptor, were also reduced, but the reduction of Kact for L-755,507 was more evident than other agonists tested. The cAMP accumulation in response to some conventional beta3 agonists was less than 10% of that to isoproterenol even in the cells expressing the wild type receptor. These results suggest that the Trp64Arg mutant beta3-adrenergic receptor has less ability to stimulate adenylyl cyclase, and that lipolytic activity through the beta3-adrenergic receptor by catecholamines in subjects carrying this mutation might be suppressed.

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro.

Both fertilization promoting peptide (FPP) and adenosine stimulate capacitation and inhibit spontaneous acrosome loss in epididymal mouse spermatozoa; these responses involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. However, it was unclear whether these responses were restricted to the mouse or possibly common to many mammalian species. To address this question, the response of boar spermatozoa to FPP and/or adenosine was evaluated. FPP is found in nanomolar concentrations in seminal plasma of several mammals, but not the pig. When cultured in caffeine-containing Medium 199 for 2 hr, chlortetracycline fluorescence evaluation indicated that neither FPP nor adenosine stimulated boar sperm capacitation per se but did inhibit spontaneous acrosome loss. However, in caffeine-free medium, FPP and adenosine both stimulated capacitation and inhibited spontaneous acrosome loss, suggesting that boar spermatozoa have receptors for both FPP and adenosine. Gln-FPP, a competitive inhibitor of FPP in mouse spermatozoa, has recently been shown to inhibit mouse sperm responses to adenosine as well, suggesting that FPP receptors and adenosine receptors interact in some way. Used with boar spermatozoa, Gln-FPP also significantly inhibited responses to both FPP and adenosine. These responses suggest that mechanisms whereby FPP and adenosine can regulate sperm function, via AC/cAMP, are of considerable physiological significance. Mouse, human, and now boar spermatozoa have been shown to respond to FPP, suggesting that these mechanisms may be common to many mammalian species. We also suggest that the effects of FPP and adenosine could also be exploited to maximize monospermic fertilization in porcine in vitro fertilization.

[Concurrent chemoradiotherapy using protracted infusion of low-dose CDDP and 5-FU and radiotherapy for esophageal cancer].

PURPOSE: We evaluated the effects and safety of concurrent chemoradiotherapy for patients with esophageal cancer. MATERIALS AND METHODS: Between March 1994 and April 1998, concurrent chemoradiotherapy using protracted infusion of low-dose cisplatin (CDDP: 3-6 mg/m2/24h), 5-fluorouracil (5-FU: 200 mg/m2/24h) and radiotherapy was given to 26 patients. The median age was 70 yr, with a range from 58 to 86 yr. With regard to TNM classification (1987), six patients were stage II, five stage III, and 15 stage IV. Radiotherapy was performed by external irradiation alone in 23 patients and external irradiation plus brachytherapy in three patients. One patient underwent surgery after a dose of 40 Gy owing to the possibility of idiopathic bleeding from the stomach. RESULTS: Locally, primary effects resulted in complete response in 11 patients (42.3%) and partial response in 15 (57.7%). Acute toxicity was primarily hematologic. Leukopenia and thrombocytopenia of grade 3 or 4 occurred in eight (30.7%) and six (23.0%) of 26 patients, respectively. In patients administered CDDP at more than 5 mg/m2/day, hemotoxicity was severe because in five of the 10 patients administered 5 mg/m2 CDDP and one of the two patients administered 6 mg/m2 CDDP, thrombocytopenia of grade 3 or 4 occurred. CONCLUSION: Protracted infusion of low-dose CDDP and 5-FU with concomitant radiation therapy is effective, but from the point of acute toxicity, the optimal dose of CDDP and 5-FU needs further investigation.

Cold-induced mRNA expression of angiogenic factors in rat brown adipose tissue.

Brown adipose tissue (BAT) is the major site of non-shivering thermogenesis in rodents. Rapid angiogenesis is induced in association with adaptive hyperplasia of this tissue when the animal is exposed to cold. We demonstrated previously adrenergic activation of mRNA expression of vascular endothelial growth factor (VEGF) in rat BAT and its possible contribution to the cold-induced angiogenesis in this tissue. In the present study, we examined the effect of cold exposure on mRNA expression of other two angiogenic factors, VEGF-B and basic fibroblast growth factor (bFGF), in rats. Conventional Northern blot analysis revealed abundant mRNA expression of VEGF-B as well as VEGF, but not bFGF, in BAT. When rats were exposed to cold at 4 degrees C, the VEGF mRNA level was increased by 2.7-fold in 1-4 hr and returned to the basal level within 24 hr. In contrast, the VEGF-B mRNA level did not change throughout the course of cold exposure. A significant expression of bFGF mRNA was detected in BAT by reverse transcription-polymerase chain reaction (RT-PCR). To evaluate the tissue bFGF mRNA level quantitatively, a competitive RT-PCR method was developed using a shorter RNA fragment as a competitor. The bFGF mRNA level in BAT was found to increase by 2.3-fold in 4 hr and decreased to the basal level within 24 hr after cold exposure. These results suggest that cold exposure leads to induce VEGF and bFGF rapidly and transiently in BAT, which in turn stimulate the proliferation of vascular endothelial cells in this tissue.

Immortal brown adipocytes from p53-knockout mice: differentiation and expression of uncoupling proteins.

Brown adipose tissue (BAT) is the specific site for metabolic heat production in mammals. To establish a novel immortal brown adipocyte cell line, the stromal-vascular fraction containing preadipocytes was obtained from interscapular BAT of mice deficient of a tumor-suppressor gene p53. The p53-deficient cells, tentatively named as HB2 cells, could be cultured in vitro after repeated passages and differentiated into adipocytes in the presence of insulin, T3 and/or troglitazone, expressing some adipocyte-specific genes and accumulating intracellular lipid droplets. The mRNA level of uncoupling protein 1 (UCP1), a mitochondrial protein specifically present in brown adipocytes, was undetectable in HB2 preadipocytes, but increased after adipose differentiation. In HB2 adipocytes, UCP1 mRNA expression was markedly activated after stimulation of the beta-adrenergic receptor pathway. The mRNA of UCP2 and UCP3, recently cloned isoforms of UCP1, were also detected in HB2 adipocytes, but their levels were not influenced by adrenergic stimulation. Thus HB2 cells seem useful for in vitro studies of BAT and UCP functions.

Inhibition of mitochondrial protein synthesis impaired C2C12 myoblast differentiation.

Various factors are required for the regulation of muscle cell differentiation. In an attempt to elucidate the mechanism underlying myogenesis, we examined the possible contribution of mitochondria to terminal differentiation of murine myoblast cell line, C2C12, using a specific inhibitor for mitochondrial protein synthesis, tetracycline. Tetracycline impaired myotube formation and induction of muscle creatine kinase activity which was specifically observed in differentiated myocytes. Transcript levels of muscle-specific proteins, creatine kinase and troponin-I were also significantly suppressed in a dose-dependent manner. However, those proteins with myogenic regulatory factors, MyoD and myogenin, and common proteins including glycolytic enzymes were not affected. Cellular viability, mitochondrial transcription, and mitochondrial proliferation were confirmed not to be impaired by tetracycline treatment. These results suggest that mitochondrial stress may affect regulation of differentiation-specific gene expression. This system may contribute to an understanding of mechanisms for differentiation inhibition caused by inhibitors of mitochondrial protein synthesis that have also been observed in other kinds of cells.

Association of erythrodermic cutaneous T-cell lymphoma, superantigen-positive Staphylococcus aureus, and oligoclonal T-cell receptor V beta gene expansion.

Forty-two patients with cutaneous T-cell lymphoma, including 31 with exfoliative erythroderma or Sezary syndrome and 11 with mycosis fungoides, were studied for the occurrence of staphylococcal infection. Thirty-two of 42 (76%) had a positive staphylococcal culture from skin or blood. One half of the patients with positive cultures grew Staphylococcus aureus. This group included 11 with Sezary syndrome and 5 with rapidly enlarging mycosis fungoides plaques or tumors. All of the S aureus carried enterotoxin genes. Surprisingly, 6 of 16 strains were the same toxic shock toxin-1 (TSST-1)-positive clone, designated electrophoretic type (ET)-41. Analysis of the T-cell receptor V beta repertoire in 14 CTCL patients found that only 4 had the expected monoclonal expansion of a specific V beta gene, whereas 10 had oligoclonal or polyclonal expansion of several V beta families. All patients with TSST-1+ S aureus had overexpansion of V beta Z in blood and/or skin lesions. These studies show that S aureus containing superantigen enterotoxins are commonly found in patients with CTCL especially individuals with erythroderma where they could exacerbate and/or perpetuate stimulate chronic T-cell expansion and cutaneous inflammation. Attention to toxigenic S aureus in CTCL patients would be expected to improve the quality of care and outcome of this patient population.

Trans-regulation of myogenin promoter/enhancer activity by c-ski during skeletal-muscle differentiation: the C-terminus of the c-Ski protein is essential for transcriptional regulatory activity in myotubes.

c-ski gene product is a nuclear protein with myogenesis-promoting and transforming activities. We have analysed the effects of c-ski transfection on the promoter/enhancer activity of the upstream region of the myogenin gene during in vitro myogenesis using CAT reporter assay. When co-transfected with c-ski into myogenic C2C12 cells, promoter/enhancer activity was efficiently suppressed in proliferating cells, but the myogenesis-induced increase in activity was potentiated approximately ten times more (150-fold in the ski-transfected cells) than the ordinary increase (12-fold in the mock) 48 h after induction of differentiation. In non-myogenic 10T1/2 cells, c-ski transfection caused persistent suppression of promoter/enhancer activity in both proliferating and growth-arrested (i.e. myogenesis-inducing) conditions. Thus the ski-dependent potentiation of myogenin gene transcriptional activity appears to be specific for myogenesis. The C-terminal region (amino acids 595-663) of the c-Ski protein was essential for the potentiating activity in myotubes. Other members of the ski-gene family, snoN and snoA, were ineffective in transactivation, possibly because of the defect in the corresponding C-terminal region. c-Ski protein underwent a mobility shift on SDS/PAGE after in vitro myogenesis which may explain the conversion of the activity from suppressive in myoblasts to potentiating in myotubes. Deletion analysis of the upstream region of the myogenin gene revealed that a responsive element to c-ski in myotubes is located at a distinct site upstream of the basal promoter/enhancer region.

Adrenergic activation of vascular endothelial growth factor mRNA expression in rat brown adipose tissue: implication in cold-induced angiogenesis.

Cold exposure produces adaptive hyperplasia and growth of brown adipose tissue (BAT), the major site of non-shivering thermogenesis in rodents, associated with increased angiogenesis in this tissue. Vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors, was found to be expressed abundantly in BAT of the rat. When rats were exposed to cold at 4 degrees C, the VEGF mRNA level in BAT was increased by 2-3-fold in 1-4 h, but returned to the basal level within 24 h. VEGF expression in other tissues such as heart, kidney and lung did not change after cold exposure. The cold-induced increase in VEGF mRNA was abolished by surgical sympathetic denervation, but mimicked by administration of noradrenaline or a beta3-adrenoceptor agonist CL316,243, indicating the critical role of the beta-adrenergic pathway in VEGF expression in BAT. Among three isoforms of VEGF, the mRNA of a short form (VEGF120) lacking heparin-binding activity was preferentially increased after cold exposure and treatment with the adrenergic agonists. These results suggest that cold exposure activates the sympathetic nerves and leads to a rapid increase in synthesis of VEGF in BAT, which in turn stimulates the proliferation of surrounding vascular endothelial cells.

Negative regulation of two hyperproliferative keratinocyte differentiation markers by a retinoic acid receptor-specific retinoid: insight into the mechanism of retinoid action in psoriasis.

Retinoids down-regulate the expression of metalloproteinases, cytokines, and other genes involved in cell proliferation and inflammation. Tazarotene (AGN 190168), a retinoic acid receptor (RAR)-specific retinoid, is effective in the treatment of psoriasis, a hyperproliferative and inflammatory skin disease. Because negative regulation of genes appears to be important in the antiproliferative and antiinflammatory action of retinoids, we studied the down-regulation of genes in skin raft cultures by this antipsoriatic retinoid. By subtraction hybridization, we found that migration inhibitory factor-related protein (MRP-8) and skin-derived anti-leukoproteinase (SKALP) are down-regulated by AGN 190168. MRP-8 and SKALP are overexpressed in psoriatic lesions as compared to the normal epidermis, and they are markers of hyperproliferative keratinocyte differentiation. We also show that MRP-8 expression is retinoid inhibitable in cultured keratinocytes induced to differentiate with 10% serum or IFN-gamma, and that MRP-8 is inhibited by RAR but not by retinoid X receptor-specific retinoids in a dose-dependent manner. Finally, MRP-8, SKALP, and the previously characterized differentiation marker, transglutaminase I, are all down-regulated in vivo in psoriatic lesions after treatment with AGN 190168 in comparison to placebo. Taken together, these data suggest that these markers may be down-regulated by tazarotene in psoriasis through direct action on keratinocyte gene expression rather than by an overall tazarotene effect on lesional therapeutic status.