High fat diet-induced hyperglycemia: prevention by low level expression of a glucose transporter (GLUT4) minigene in transgenic mice.

High-fat intake leading to obesity contributes to the development of non-insulin-dependent diabetes mellitus (NIDDM, type 2). Similarly, mice fed a high-fat (safflower oil) diet develop defective glycemic control, hyperglycemia, and obesity. To assess the effect of a modest increase in the expression of GLUT4 (the insulin-responsive glucose transporter) on impaired glycemic control caused by fat feeding, transgenic mice harboring a GLUT4 minigene were fed a high-fat diet. Low-level tissue-specific (heart, skeletal muscle, and adipose tissue) expression of the GLUT4 minigene in transgenic mice prevented the impairment of glycemic control and accompanying hyperglycemia, but not obesity, caused by fat feeding. Thus, a small increase (< or = 2-fold) in the tissue level of GLUT4 prevents a primary symptom of the diabetic state in a mouse model, suggesting a possible target for intervention in the treatment of NIDDM.

A maternal diet high in n − 6 polyunsaturated fats alters mammary gland development, puberty onset, and breast cancer risk among female rat offspring

We hypothesized that feeding pregnant rats with a high-fat diet would increase both circulating 17β-estradiol (E2) levels in the dams and the risk of developing carcinogen-induced mammary tumors among their female offspring. Pregnant rats were fed isocaloric diets containing 12% or 16% (low fat) or 43% or 46% (high fat) of calories from corn oil, which primarily contains the n − 6 polyunsaturated fatty acid (PUFA) linoleic acid, throughout pregnancy. The plasma concentrations of E2 were significantly higher in pregnant females fed a high n − 6 PUFA diet. The female offspring of these rats were fed with a laboratory chow from birth onward, and when exposed to 7,12-dimethylbenz(a)anthracene had a significantly higher mammary tumor incidence (60% vs. 30%) and shorter latency for tumor appearance (11.4 ± 0.5 weeks vs. 14.2 ± 0.6 weeks) than the offspring of the low-fat mothers. The high-fat offspring also had puberty onset at a younger age, and their mammary glands contained significantly higher numbers of the epithelial structures that are the targets for malignant transformation. Comparable changes in puberty onset, mammary gland morphology, and tumor incidence were observed in the offspring of rats treated daily with 20 ng of E2 during pregnancy. These data, if extrapolated to humans, may explain the link among diet, early puberty onset, mammary parenchymal patterns, and breast cancer risk, and indicate that an in utero exposure to a diet high in n − 6 PUFA and/or estrogenic stimuli may be critical for affecting breast cancer risk.

Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity

Perilipin coats the lipid droplets of adipocytes and is thought to have a role in regulating triacylglycerol hydrolysis. To study the role of perilipin in vivo, we have created a perilipin knockout mouse. Perilipin null (peri−/−) and wild-type (peri+/+) mice consume equal amounts of food, but the adipose tissue mass in the null animals is reduced to ≈30% of that in wild-type animals. Isolated adipocytes of perilipin null mice exhibit elevated basal lipolysis because of the loss of the protective function of perilipin. They also exhibit dramatically attenuated stimulated lipolytic activity, indicating that perilipin is required for maximal lipolytic activity. Plasma leptin concentrations in null animals were greater than expected for the reduced adipose mass. The peri−/− animals have a greater lean body mass and increased metabolic rate but they also show an increased tendency to develop glucose intolerance and peripheral insulin resistance. When fed a high-fat diet, the perilipin null animals are resistant to diet-induced obesity but not to glucose intolerance. The data reveal a major role for perilipin in adipose lipid metabolism and suggest perilipin as a potential target for attacking problems associated with obesity.

Expression of Batis maritima methyl chloride transferase in Escherichia coli

Methyl chloride transferase, a novel enzyme found in several fungi, marine algae, and halophytic plants, is a biological catalyst responsible for the production of atmospheric methyl chloride. A previous paper reports the purification of this methylase from Batis maritima and the isolation of a cDNA clone of the gene for this enzyme. In this paper, we describe the isolation of a genomic clone of the methylase gene and the expression of recombinant methyl chloride transferase in Escherichia coli and compare the kinetic behavior of the wild-type and recombinant enzyme. The recombinant enzyme is active and promotes the production of methyl chloride by E. coli under in vivo conditions. The kinetic data indicate that the recombinant and wild-type enzymes have similar halide (Cl−, Br−, and I−)-binding capacities. Both the recombinant and wild-type enzymes were found to function well in high NaCl concentrations. This high salt tolerance resembles the activity of halobacterial enzymes rather than halophytic plant enzymes. These findings support the hypothesis that this enzyme functions in the control and regulation of the internal concentration of chloride ions in halophytic plant cells.

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

p21WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.

Chrysanthemum chlorotic mottle viroid: Unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes

The causal agent of chrysanthemum chlorotic mottle (CChM) disease has been identified, cloned, and sequenced. It is a viroid RNA (CChMVd) of 398–399 nucleotides. In vitro transcripts with the complete CChMVd sequence were infectious and induced the typical symptoms of the CChM disease. CChMVd can form hammerhead structures in both polarity strands. Plus and minus monomeric CChMVd RNAs self-cleaved during in vitro transcription and after purification as predicted by these structures, which are stable and most probably act as single hammerhead structures as in peach latent mosaic viroid (PLMVd), but not in avocado sunblotch viroid (ASBVd). Moreover, the plus CChMVd hammerhead structure also appears to be active in vivo, because the 5′ terminus of the linear plus CChMVd RNA isolated from infected tissue is that predicted by the corresponding hammerhead ribozyme. Both hammerhead structures of CChMVd display some peculiarities: the plus self-cleaving domain has an unpaired A after the conserved A9 residue, and the minus one has an unusually long helix II. The most stable secondary structure predicted for CChMVd is a branched conformation that does not fulfill the rod-like or quasi-rod-like model proposed for the in vitro structure of most viroids with the exception of PLMVd, whose proposed secondary structure of lowest free energy also is branched. The unusual conformation of CChMVd and PLMVd is supported by their insolubility in 2 M LiCl, in contrast to ASBVd and a series of representative non-self-cleaving viroids that are soluble under the same high salt conditions. These results support the classification of self-cleaving viroids into two subgroups, one formed by ASBVd and the other one by PLMVd and CChMVd.

Glucose intolerance caused by a defect in the entero-insular axis: A study in gastric inhibitory polypeptide receptor knockout mice

Mice with a targeted mutation of the gastric inhibitory polypeptide (GIP) receptor gene (GIPR) were generated to determine the role of GIP as a mediator of signals from the gut to pancreatic β cells. GIPR−/− mice have higher blood glucose levels with impaired initial insulin response after oral glucose load. Although blood glucose levels after meal ingestion are not increased by high-fat diet in GIPR+/+ mice because of compensatory higher insulin secretion, they are significantly increased in GIPR−/− mice because of the lack of such enhancement. Accordingly, early insulin secretion mediated by GIP determines glucose tolerance after oral glucose load in vivo, and because GIP plays an important role in the compensatory enhancement of insulin secretion produced by a high insulin demand, a defect in this entero-insular axis may contribute to the pathogenesis of diabetes.

The Tail of a Yeast Class V Myosin, Myo2p, Functions as a Localization Domain

Myo2p is a yeast class V myosin that functions in membrane trafficking. To investigate the function of the carboxyl-terminal-tail domain of Myo2p, we have overexpressed this domain behind the regulatable GAL1 promoter (MYO2DN). Overexpression of the tail domain of Myo2p results in a dominant–negative phenotype that is phenotypically similar to a temperature-sensitive allele of myo2, myo2–66. The tail domain of Myo2p is sufficient for localization at low- expression levels and causes mislocalization of the endogenous Myo2p from sites of polarized cell growth. Subcellular fractionation of polarized, mechanically lysed yeast cells reveals that Myo2p is present predominantly in a 100,000 × g pellet. The Myo2p in this pellet is not solubilized by Mg++-ATP or Triton X-100, but is solubilized by high salt. Tail overexpression does not disrupt this fractionation pattern, nor do mutations in sec4, sec3, sec9, cdc42, or myo2. These results show that overexpression of the tail domain of Myo2p does not compete with the endogenous Myo2p for assembly into a pelletable structure, but does compete with the endogenous Myo2p for a factor that is necessary for localization to the bud tip.

Nascent Polypeptide–associated Complex Stimulates Protein Import into Yeast Mitochondria

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide–associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.

Degradation of Hepatic Stearyl CoA Δ9-Desaturase

Δ9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt–washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.

Identification of a Second Myosin-II in Schizosaccharomyces pombe:: Myp2p Is Conditionally Required for Cytokinesis

As in many eukaryotic cells, fission yeast cytokinesis depends on the assembly of an actin ring. We cloned myp2+, a myosin-II in Schizosaccharomyces pombe, conditionally required for cytokinesis. myp2+, the second myosin-II identified in S. pombe, does not completely overlap in function with myo2+. The catalytic domain of Myp2p is highly homologous to known myosin-IIs, and phylogenetic analysis places Myp2p in the myosin-II family. The Myp2p sequence contains well-conserved ATP- and actin-binding motifs, as well as two IQ motifs. However, the tail sequence is unusual, since it is predicted to form two long coiled-coils separated by a stretch of sequence containing 19 prolines. Disruption of myp2+ is not lethal but under nutrient limiting conditions cells lacking myp2+ function are multiseptated, elongated, and branched, indicative of a defect in cytokinesis. The presence of salt enhances these morphological defects. Additionally, Δmyp2 cells are cold sensitive in high salt, failing to form colonies at 17°C. Thus, myp2+ is required under conditions of stress, possibly linking extracellular growth conditions to efficient cytokinesis and cell growth. GFP-Myp2p localizes to a ring in the middle of late mitotic cells, consistent with a role in cytokinesis. Additionally, we constructed double mutants of Δmyp2 with temperature-sensitive mutant strains defective in cytokinesis. We observed synthetic lethal interactions between Δmyp2 and three alleles of cdc11ts, as well as more modest synthetic interactions with cdc14ts and cdc16ts, implicating myp2+ function for efficient cytokinesis under normal conditions.

The barrier-to-autointegration protein is a host factor for HIV type 1 integration

In vivo, retroviral integration is mediated by a large nucleoprotein complex, termed the preintegration complex (PIC). PICs isolated from infected cells display in vitro integration activity. Here, we analyze the roles of different host cell factors in the structure and function of HIV type 1 (HIV-1) PICs. PICs purified by size exclusion after treatment with high salt lost their integration activity, and adding back an extract from uninfected cells restored this activity. In parallel, the native protein–DNA intasome structure detected at the ends of HIV-1 by Mu-mediated PCR footprinting was abolished by high salt and restored by the crude cell extract. Various purified proteins previously implicated in retroviral PIC function then were analyzed for their effects on the structure and function of salt-treated HIV-1 PICs. Whereas relatively low amounts (5–20 nM) of human barrier-to-autointegration factor (BAF) protein restored integration activity, substantially more (5–10 μM) human host factor HMG I(Y) was required. Similarly high levels (3–8 μM) of bovine RNase A, a DNA-binding protein used as a nonspecific control, also restored activity. Mu-mediated PCR footprinting revealed that of these three purified proteins, only BAF restored the native structure of the HIV-1 protein–DNA intasome. We suggest that BAF is a natural host cofactor for HIV-1 integration.

Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases δ and ɛ, is involved in DNA replication as well as in diverse DNA repair pathways. In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases δ and ɛ. In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques. Exposure of normal cells to γ-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40–45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling. The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h. The PCNA foci formed after γ-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion. Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after γ-irradiation. We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks. The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells. Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks. Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks.

The structure of the human βII-tryptase tetramer: Fo(u)r better or worse

Tryptases, the predominant serine proteinases of human mast cells, have recently been implicated as mediators in the pathogenesis of allergic and inflammatory conditions, most notably asthma. Their distinguishing features, their activity as a heparin-stabilized tetramer and resistance to most proteinaceous inhibitors, are perfectly explained by the 3-Å crystal structure of human βII-tryptase in complex with 4-amidinophenylpyruvic acid. The tetramer consists of four quasiequivalent monomers arranged in a flat frame-like structure. The active centers are directed toward a central pore whose narrow openings of approximately 40 Å × 15 Å govern the interaction with macromolecular substrates and inhibitors. The tryptase monomer exhibits the overall fold of trypsin-like serine proteinases but differs considerably in the conformation of six surface loops arranged around the active site. These loops border and shape the active site cleft to a large extent and form all contacts with neighboring monomers via two distinct interfaces. The smaller of these interfaces, which is exclusively hydrophobic, can be stabilized by the binding of heparin chains to elongated patches of positively charged residues on adjacent monomers or, alternatively, by high salt concentrations in vitro. On tetramer dissociation, the monomers are likely to undergo transformation into a zymogen-like conformation that is favored and stabilized by intramonomer interactions. The structure thus provides an improved understanding of the unique properties of the biologically active tryptase tetramer in solution and will be an incentive for the rational design of mono- and multifunctional tryptase inhibitors.