大鼠肝癌中α_2-巨球蛋白等几种分泌性蛋白基因表达的变化

用Northern blot方法对二乙基亚硝胺所诱发的大鼠肝癌中内源性蛋白酶抑制因子α_2-巨球蛋白(α_2-M)、非特异性免疫抑制剂α_1-酸性糖蛋白(α_1-AGP)及雄性激素正调控的α-2u球蛋白(α-2u)三种分泌性蛋白基因表达情况进行了分析。结果表明在大部分(14/16)肝癌样品中α_2-M RNA水平显著降低;而α_1-AGP RNA水平显著高于正常对照水平;α-2u RNA水平明显下降,但在某些雄性大鼠肝癌样品中该基因却有一定程度的表达。这些结果说明,一些肿瘤宿主血浆中α_2-M水平的显著下降及α_1-AGP水平的明显升高分别是由于基因表达活性的下降及升高所致。α-2u基因表达的异常提示,在癌变过程中机体的内分泌功能发生了某些变化。     

Proinflammatory cytokine profiles in prediabetic Saudi patients

Prediabetes is an increase-risk state for diabetes that is associated with an increase in blood glucose levels to more than normal, but not increased enough to be termed as type 2 diabetes mellitus (T2DM). A timely intervention and management of prediabetes can stop its further progression to the diabetic state. Many cytokines are involved in diseases including diabetes, however, their role in prediabetes is unknown. In this study, we attempted to analyze numerous proinflammatory cytokines in prediabetic patients. A total of 60 adult Saudi prediabetes patients and healthy control individuals were included in this study. To better understand the role of the proinflammatory cytokines in prediabetes patients and its potential link to the disease outcome, the variations in the levels of these cytokines were investigated using Multi-Analyte ELISA technique. The T helper cells (Th1 and Th2) immune response expression profiling of 84 genes was done using Real Time-quantitative PCR (RT-qPCR) technique. The present finding showed that serum Interleukin IL-2, IL-1β, and IL-1α levels of all prediabetes patients were increased when compared with healthy control cases (P < 0.05). Inductions of proinflammatory cytokines and upregulation of Th1 and Th2 immune genes might play a potential role during prediabetes status and may be linked to the disease outcome. Further studies are needed to investigate the underlying mechanism of these proinflammatory cytokines in diabetes development. A strong positive correlation was found between IL and 1α with glucose levels than with IL-1β and IL-2. In conclusion, cytokines, especially IL-1, may play a critical role in the development of diabetes.     

Effect of dimethylglycine and trimethylglycine (Betaine) on the response of Atlantic salmon (Salmo salar L.) smolts to experimental Vibrio anguillarum infection

Atlantic salmon (Salmo salar L.) were fed on a control diet or experimental diets containing betaine (15 mg g^-1) or dimethylglycine (DMG, I mg g^-1 or 5 mg g^-1). After 10 weeks of feeding, resistance to infection was assessed following inoculation with Vibrio anguillarum. Total blood and differential leucocyte counts were made, and plasma lysozyme and ceruloplasmin were assayed as non-specific humoral factors. The mortality during the bacterial exposure was of the same magnitude in all feeding groups. Betaine or DMG had no effect on the 'basal' levels of plasma total protein, lysozyme or ceruloplasmin, but 3 days postinjection the lysozyme and ceruloplasmin levels were higher in the control group compared with the experimental groups. In both DMG groups, the lymphocyte response took place 1-2 weeks earlier than in the control or betaine supplemented group indicating that DMG has an immunomodulating effect on salmon.     

Auto and transactivation of FGF expression: Potential mechanism for regulation of myogenic differentiation

Summary Fibroblast growth factors (FGFs) are potent inhibitors of myogenic differentiation. The recent observation that the endogenous expression of acidic and basic FGF by myogenic cells decreases coordinately with differentiation suggests a regulatory role for these growth factors in myogenesis. Inasmuch as other proteins known to influence myogenesis (e.g., MyoD1 and myogenin) activate their own expression as well as the expression of other members of their family, we hypothesized that the FGFs might be capable of similar autoregulation. We examined the effect of exogenously supplied FGF on the abundance of the mRNAs encoding acidic and basic FGF in Sol 8 myoblasts, and demonstrate that either acidic or basic FGF stimulate, through paracrine mechanisms, the accumulation of the mRNAs encoding both of these FGFs. Thus FGFs can auto- and transregulate their own expression in a manner analogous to that observed for the myogenic determination proteins. In addition, similar to that previously observed for MyoD1, both acidic and basic FGF suppress myogenin expression in myoblasts. These results suggest two mechanisms whereby endogenously produced FGFs participate in the maintenance of the undifferentiated state of myogenic cells. These data provide support for paracrine, and suggest potential autocrine, roles for FGFs in the regulation of myogenic differentiation.     

ABR frequency tuning curves in dolphins

Tone-tone masking was used to determine auditory brain-stem response tuning curves in dolphins (Tursiops truncatus) in a simultaneous-masking paradigm. The Q ₁₀ of the curves was as large as 16–19 in the frequency range 64–128 kHz. In the range 45–16 kHz, Q ₁₀ decreased proportionally to the frequency with the bandwidth of the curves being constant, about 3.5–4 kHz at the 10-dB level. Tuning curves below 45 kHz are supposed to reflect broad spectral bandwidth of the probe's effective part which is no longer than 0.5 ms, irrespective of actual probe duration. Tuning curves above 64 kHz are supposed to reflect the real frequency tuning of the dolphin's auditory system.Abbreviations ABR auditory brain stem response - AP action potential     

The mosquito dihydrofolate reductase gene functions as a dominant selectable marker in transfected cells

An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10–15 days in the presence of selective medium containing 1 μM methotrexate. The transformed clones contained an estimated 100–500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.     

Short communication: Variability of response to feed restriction in lactating Charolais cows

Involuntary temporary feed restriction on commercial cattle is likely to become more frequent with forage shortages in the context of climate change. If general consequences of feed restriction have been the subject of an abundant scientific literature, focus on the inter-individual variability of response is scarce. Here, we explore the response profile in terms of BW, body condition score, milk production, calf weight and cyclicity resumption of 293 lactations from 169 Charolais cows during a winter feed restriction in early lactation and its subsequent recovery at grazing using a principal component analysis followed by a hierarchical clustering on principal component. Results show a very continuous range of response profiles that was divided into three clusters: one with light animals having an intermediate response in terms of milk production and body maintenance, one with animals prioritising body maintenance and cyclicity resumption over milk production and calf weight, and the last one with animals prioritising milk production and calf weight over the rest. Among the animals performing more than one lactation, 57% remain in the same cluster on two successive lactations. This work highlights that an average group response to feed restriction may hide various resilience individual profiles. Further studies are required to determine the existence of a genetic component as well as the consequences of not taking this phenomenon into consideration with the regular use of feed restriction in commercial farms.     

The CD95 (Fas/APO-1) receptor is phosphorylatedin vitro andin vivo and constitutively associates with several cellular proteins

Different CD95 (Fas/APO-1) isoforms and phosphory lated CD95 species were identified in human T and B cell lines. We had shown previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts, was phosphorylatedin vivo. GST-CD95IC was phosphorylatedin vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291) and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also mediated interaction with a 35 kDa cellular protein. Immuno-precipitation of CD95 and chemical cross-linking revealed CD95-associated proteins of approximately 35, 45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex, and that a kinase activity is associated with CD95 not onlyin vitro but alsoin vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction. This work was in part supported by a grant from the Health Research Council of New Zealand (to JW).     

Tyrosine protein kinase assays

Protein kinases form a large family of enzymes that play a major role in a number of live processes. The study of their action is important for the understanding of the transformation mechanisms and of the normal and pathological growth events. The quality of an enzyme assay is often the key point of an enzymatic study. It must be flexible and compatible with various experimental conditions, such as those for the purification process, the screening of inhibitors and the substrate specificity studies. As will be shown in the present review, two categories of substrates, peptidic and proteic, should be distinguished. The use of peptide substrates facilitates the determination of the recognition requirements of the enzyme and of the kinetic effects of even minute variations in their sequence. These linear peptide structures are assumed to mimic a complex interaction between the enzyme and a proteic substrate in which distant amino acids in the sequence are vicinal in the folded substrate. Less amenable to a systematic study, but probably more adequate to investigate the natural substrate of a given kinase, are the proteic substrates. Obviously the tools to measure protein kinase activities are not the same in these two cases. The main difficulty in assaying protein kinases is the use of labelles γ-ATP, mostly at large excess concentration, since the final product of the reaction has to be separated from the non-reacted labelled ATP. In the case of peptide substrates, the difficulty is to separate them from ATP basing on differences of molecular mass. Despite the efforts of many investigators to rely upon differences in solubility, in charges or in “affinity”, this separation, which is crucial for the assay, is still an unsolved experimental problem. Chromatographic, as well as electrophoretic assays appeared relatively late in this domain, and more work in assessing new methodologies might bring new breakthroughs in the next few years. Specific, simple and reliable kinase assays are still a major challenge. Their improvement will help to conduct specificity studies, to elucidate complex growth mechanisms in which they are involved and to discover more selective potent inihibitors.