Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglycemic (db/db) mice. Effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-3H]glucose, uptake of 3-O-[methyl-3H]glucose (methylglucose) and [2-14C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 +/- 18 and 180 +/- 9 microliter/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V- of methylglucose transport was decreased with no change in Km in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and 3H2O production from [5-3H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

HNF-4 increases activity of the rat Apo A1 gene.

Apolipoprotein A1 (Apo A1) is the major protein component of high density lipoprotein (HDL) particles. HDL particles mediate the removal of cholesterol from extra-hepatic tissues via a process known as reverse cholesterol transport. Augmented production of Apo A1 will likely be beneficial to those who suffer from the consequences of hypercholesterolemia. One approach to increase expression of the protein is to identify nuclear factor(s) that enhance Apo A1 promoter activity. Therefore, we have used transient transfection to study a limited portion (-474 to -7) of the gene and showed that a cis-regulatory element, site C had a permissive effect on the ability of an adjacent site B to increase promoter activity by 30-fold. The importance of element C prompted us to identify the factor(s) that interact with this site. Results showed that HNF-4, a new member of the thyroid/steroid hormone receptor superfamily interacts with site C to enhance activity of the promoter. Based on this observation and that of the known inhibitory effects of ARP-1 on site C, we postulate a model which may account for the tissue-specific expression of the rat Apo A1 gene.     

七种云南地衣植物的化学成分

本文报道分属于三个科的七种云南产地衣植物的化学成分。这些植物是亚洲树发(Alectoria asiatica DR.)、沟树发(Alectoria sulcata Nyl.)、长茎松萝(Usnea longissimaAch.)、胡子松萝(Usnea comosa(Ach.)Rohl.)、林石蕊(Cladonia arbuscula(Rabh.)Rabh.)、砖孢发(Oropogon loxensis(Fee.)Th.Fr.)和卷梢雪花衣(Anaptychiaboryi(Fee.)Mass.)。根据光谱数据测定,其化学成分为:松萝酸(Usnic acid)(1),维任西酸(Virensic acid)(2),赤星衣酸乙酯(Ethyl haematommate)(3),瑞藏酸(Rhizonic acid)(4),赤星衣酸(Haematommic acid)(5),扁枝衣酸乙酯(Ethyleverninate)(6),黑茶渍素(Atranorin)(7)和泽屋萜(Zeorin)(8)。上述资料,对开发利用这些植物资源提供了科学依据。     

PCR-based generation of shRNA libraries from cDNAs

Background  

The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries; one is to use chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries.     

Altered expression of C/EBP family members results in decreased adipogenesis with aging

Fat mass, adipocyte size and metabolic responsiveness, and preadipocyte differentiation decrease between middle and old age. We show that expression of CCAAT/enhancer binding protein (C/EBP)-alpha, a key regulator of adipogenesis and fat cell function, declined substantially with aging in differentiating preadipocytes cultured under identical conditions from rats of various ages. Overexpression of C/EBP alpha in preadipocytes cultured from old rats restored capacity to differentiate into fat cells, indicating that downstream differentiation-dependent genes maintain responsiveness to regulators of adipogenesis. C/EBP alpha-expression also decreased with age in fat tissue from three different depots and in isolated fat cells. The overall level of C/EBP beta, which modulates C/EBP alpha-expression, did not change with age, but the truncated, dominant-negative C/EBP beta-liver inhibitory protein (LIP) isoform increased in cultured preadipocytes and isolated fat cells. Overexpression of C/EBP beta-LIP in preadipocytes from young rats impaired adipogenesis. C/EBP delta, which acts with full-length C/EBP beta to enhance adipogenesis, decreased with age. Thus processes intrinsic to adipose cells involving changes in C/EBP family members contribute to impaired adipogenesis and altered fat tissue function with aging. These effects are potentially reversible.     

Broad spectrum identification of SUMO substrates in melanoma cells

Like phosphorylation, protein sumoylation likely represents a dynamic PTM to alter protein function in support of cell regulatory systems. The broad-spectrum impact of transient or chronic engagement of signal transduction cascades on protein sumoylation has not been explored. Here, we find that epidermal growth factor (EGF) stimulation evokes a rapid alteration in small ubiquitin modifier (SUMO) target selection, while oncogene expression alters steady-state SUMO-protein profiles. A proteomic SUMO target analysis in melanoma cells identified proteins involved in cellular signaling, growth control, and neural differentiation.     

Molecular isolation and sequence determination of the cDNA for the mouse sperm-specific lactate dehydrogenase-X gene

Several cDNA clones for the mouse lactate dehydrogenase-X (LDH-X), a sperm-specific glycolytic enzyme, were isolated from mouse testicular cDNA libraries constructed in the bacteriophage vectors, lambda gt11 and gt10. The largest cDNA clone contains an insert of 1135 base pairs in length and an open reading frame that encodes a 332 amino acid polypeptide with a molecular weight of 35.89 kD. The deduced amino acid sequence of this protein is in close agreement with the published sequence of mouse LDH-X obtained by direct protein sequencing. Northern analysis of RNA isolated from different tissues detected a single size mRNA of 1.5 kilobases in mouse testis but not in brain or liver. The Ldh-x structural gene was estimated to be about 12 kb in size as demonstrated by Southern hybridization analysis of mouse genomic DNA using the full-length cDNA as a probe.     

Dipsticks for rapid detection of Plasmodium in vectoring Anopheles mosquitoes

Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, transmitted by many species of Anopheles mosquitoes (Diptera: Culicidae). Monoclonal antibodies developed against specific circumsporozoite (CS) proteins of the main malaria parasites Plasmodium falciparum and P. vivax have been used previously for enzyme-linked immunosorbent assays (ELISA), widely employed for detection of malaria sporozoites in vector Anopheles for local risk assessment, epidemiological studies and targeting vector control. However, ELISA procedures are relatively slow and impractical for field use. To circumvent this, we developed rapid wicking assays that identify the presence or absence of specific peptide epitopes of CS protein of the most important P. falciparum and two strains (variants 210 and 247) of the more widespread P. vivax. The resulting assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. In laboratory assessment, dipsticks identified 1 ng/ mL of any of these three CS protein antigens, with sensitivity nearly equal to the CS standard ELISA. We have developed and are evaluating a combined panel assay that will be both qualitative and quantitative. This quick and easy dipstick test (VecTest Malaria) offers practical advantages for field workers needing to make rapid surveys of malaria vectors.