Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration

Human procathepsin L has been expressed in E. coli in the form of inclusion bodies. The recombinant protein was isolated, refolded and processed at pH 5.5 by the addition of dextran sulfate which increased the overall yield of cathepsin L almost 10-fold. After the auto-activation of the 38 kDa procathepsin L at least three processing sites were determined by N-terminal amino acid sequencing. After replacing the Ala205 residue by glutamic acid, cathepsin B-like specificity was introduced into cathepsin L. This mutation resulted in a 15-fold increased activity toward the substrate Z-Arg-Arg-AMC and in a 29-fold decreased activity toward Z-Phe-Arg-AMC. Residue 205 is thereby confirmed experimentally to be critical for the specificity of cathepsins B and L.     

In vitro regulation of an inducible-type NO synthase in the rat seminiferous tubule cells

S-Adenosyl-L-methionine:L-methionine S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-L-methionine (SMM) from L-methionine and S-adenosyl-L-methionine. SMM content increases during barley (Hordeum vulgare L.) germination. Elucidating the role of this compound is important from both a fundamental and a technological standpoint, because SMM is the precursor of dimethylsulfide, a biogenic source of atmospheric S and an undesired component in beer. We present a simple purification scheme for the MMT from barley consisting of 10% to 25% polyethylene glycol fractionation, anion-exchange chromatography on diethylaminoethyl-Sepharose, and affinity chromatography on adenosine-agarose. A final activity yield of 23% and a 2765-fold purification factor were obtained. After digestion of the protein with protease, the amino acid sequence of a major peptide was determined and used to produce a synthetic peptide. A polyclonal antibody was raised against this synthetic peptide conjugated to activated keyhole limpet hemocyanin. The antibody recognized the 115-kD denatured MMT protein and native MMT. During barley germination, both the specific activity and the amount of MMT protein increased. MMT-specific activity was found to be higher in the root and shoot than in the endosperm. MMT could be localized by an immunohistochemical approach in the shoot, scutellum, and aleurone cells but not in the root or endosperm (including aleurone).     

Epidemiologic and genetic approaches in the study of gene-environment interaction: an overview of available methods

The purpose of this study was to assess what impacts on organized mosquito control the implementation of an Endangered Species Protection Program for the Houston toad might have in Chambers and Harris counties, Texas. The study was also intended to demonstrate the value of using geographic information system (GIS) techniques and methodologies in making such assessments to those in mosquito control who are unfamiliar with GIS and its applications. Using the GIS, Geographical Analysis Support System (GRASS), databases were developed on the habitats and patterns of mosquito control insecticide usage occurring in Chambers and Harris counties. These databases were then employed by means of various utilities associated with GRASS and computer-supported, rule-based reasoning processes to create maps depicting the amount and locations of toad habitat and the areas treated annually with insecticides by districts in Chambers and Harris counties. This map information was then used via other GRASS utilities to identify and depict zones of overlap or coincidence between toad habitat and areas treated with insecticides for mosquito control in the 2 counties. As compared to existing maps for toad habitat, our resulting GIS-generated maps gave more precise, easy-to-use information that could be used to make decisions as to how to protect the toad in the zones of coincidence in each county without causing undue disruption to mosquito control activities in these zones.     

Apolipoprotein(a) binds via its C-terminal domain to the protein core of the proteoglycan decorin. Implications for the retention of lipoprotein(a) in atherosclerotic lesions

Although it is known that lipoprotein(a) (Lp(a)) binds to proteoglycans, the mechanism for this binding has not been fully elucidated. In order to shed light on this subject, we examined the interactions of decorin, a proteoglycan with a well defined protein core and a single glycosaminoglycan (GAG) chain, with Lp(a) and derivatives, namely Lp(a) deprived of apo(a), or Lp(a-), free apo(a), and the two main proteolytic fragments, F1 and F2. By circular dichroism criteria, the decorin preparations used had the same secondary structure as that previously reported for native decorin. Authentic low density lipoprotein from the same human donor was used as a control. In a solid phase system, Lp(a-)and low density lipoprotein bound to decorin in a comparable manner. This binding required Ca2+/Mg2+ ions, was lysine-mediated, and was markedly decreased in the presence of GAG-depleted decorin, suggesting the ionic nature of the interaction likely involving apoB100 and the GAG component of decorin. Free apo(a) also bound to decorin; however, the binding was neither cation-dependent nor lysine-mediated, unaffected by sialic acid depletion of apo(a), and markedly decreased when either reduced and alkylated apo(a) or reduced and alkylated decorin was used in the assay. Of note, the binding of apo(a) was unaffected when it was incubated with a spectrally native decorin that had been renatured from either 4 M guanidine hydrochloride by extensive dialysis or cooled from 65 to 25 degrees C. On the other hand, the binding significantly increased when decorin was depleted of GAGs, which by themselves had no affinity for apo(a). The binding of apo(a) to the decorin protein core was also elicited by the C-terminal domain of apo(a), and it was favored by high NaCl concentrations, 1 to 2 M. No binding was exhibited by the N-terminal domain accounting for the lack of effect of apo(a) size polymorphism on the binding. In the case of whole Lp(a), the binding to immobilized decorin was mostly GAG-dependent and ionic in nature. A minor contribution by apo(a) was detected when GAG-depleted decorin was used in the assay. Our results indicate that the binding of Lp(a) to decorin involves interactions both electrostatic (apoB100-GAG) and hydrophobic (apo(a)-decorin protein core), and that the binding of apo(a) requires decorin protein core to be in its native state.     

Abciximab therapy and unplanned coronary stent deployment: favorable effects on stent use, clinical outcomes, and bleeding complications. EPILOG Trial Investigators

BACKGROUND: Recent studies indicate that eradication of Helicobacter pylori might prevent peptic ulcer formation in patients treated with non-steroidal anti-inflammatory drugs (NSAIDs). On the other hand, gastric adaptation after repeated exposures to aspirin (ASA) is well documented but the influence of H. pylori on this process remains to be elucidated. AIM: To compare gastric damage and adaptation following repeated exposures to ASA in a group of patients with H. pylori infection, before and after eradication of the bacterium, and in H. pylori-negative controls. METHODS: Eight healthy volunteers without H. pylori infection and eight patients with duodenal ulcer (DU) history and H. pylori infection before and after H. pylori eradication were given ASA 2 g/day for a period of 14 days. Mucosal damage was evaluated by endoscopy and histology of biopsy samples. Gastric microbleeding, DNA synthesis in the gastric mucosa and mucosal expression, as well as luminal content of transforming growth factor-alpha (TGFalpha) were determined on days 0, 3, 7 and 14 of the ASA course. RESULTS: In all patients aspirin-induced gastric damage reached a maximum on day 3. In H. pylori-positive patients, this damage was maintained at a similar level up to day 14, whereas in H. pylori-negative controls and H. pylori-eradicated patients this damage significantly lessened on day 14 and was accompanied by elevated DNA synthesis as well as increased mucosal expression and luminal release of TGFalpha.     

Involvement of ovarian steroids in the opioid-mediated reduction of insulin secretion in hyperinsulinemic patients with polycystic ovary syndrome

To evaluate the possible involvement of ovarian steroids on the opioid-mediated disorders of insulin in patients affected by polycystic ovary syndrome (PCOS), we studied 40 PCOS women. All patients underwent an oral glucose tolerance test (OGTT; 75 g) and basal hormone assay; based on the insulin response to OGTT, 26 women were classified as hyperinsulinemic and continued the study protocol. Patients were randomly divided into three groups characterized by different treatments: group A (nine patients) was treated with GnRH analog (one ampule every 28 days for 2 months), group B (eight patients) was treated with naltrexone (an oral opioid antagonist, 50 mg/day, orally) for 8 weeks, and group C (nine patients) was treated with GnRH analog plus naltrexone for 2 months. After continuation of treatment, all patients repeated the basal study in a second hospitalization. Naltrexone treatment significantly reduced the insulin response to OGTT, whereas GnRH analogue administration did not significantly change the insulin secretion after the glucose load. The GnRH analog/ naltrexone cotreatment was not able to influence the insulin secretory pattern; in fact, the insulin area under the curve was superimposable before and after therapy. These data could lead to the hypothesis that the opioidergic regulation of insulin secretion requires a normal steroidogenic pattern, thus suggesting that ovarian steroids modulate opioid activity also at peripheric districts.     

The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly

The piggyBac (IFP2) short inverted terminal repeat transposable element from the cabbage looper Trichoplusia ni was tested for gene transfer vector function as part of a bipartite vector-helper system in the Mediterranean fruit fly Ceratitis capitata. A piggyBac vector marked with the medfly white gene was tested with a normally regulated piggyBac transposase helper at two different concentrations in a white eye host strain. Both experiments yielded transformants at an approximate frequency of 3-5%, with a total of six lines isolated having pigmented eyes with various levels of coloration. G1 transformant siblings from each line shared at least one common integration, with several sublines having an additional second integration. For the first transformant line isolated, two integrations were determined to be stable for 15 generations. For five of the lines, a piggyBac-mediated transposition was verified by sequencing the insertion site junctions isolated by inverse PCR that identified a characteristic piggyBac TTAA target site duplication. The efficient and stable transformation of the medfly with a lepidopteran vector represents transposon function over a relatively large evolutionary distance and suggests that the piggyBac system will be functional in a broad range of insects.     

Subjective symptoms and sleep apnea among persons referred to a sleep center. A questionnaire survey

The aim of this study was to evaluate the relation between symptoms and clinical characteristics and the frequency of increased apnoea and hypopnoea activity. The study population consisted of 135 persons referred to a sleep centre. One hundred and fifteen patients (85%) completed a questionnaire and were subsequently monitored during one night's sleep. Fifty-two persons (45%) had an Apnoea Hypopnoea Index (AHI = the mean number of apnoea and hypopnoea episodes per hour of sleep) > or = 10. Forty-one (36%) had an Apnoea Index (AI = the mean number of apnoea per hour of sleep) > or = 10. Symptoms and clinical characteristics had no significant correlation to increased apnoea activity; however, there was a significant relationship between self-reported frequent breathing pauses during sleep and increased AHI and AI. Symptoms and clinical characteristics are not capable of identifying persons with increased apnoea activity.     

Potent and selective bicyclic lactam inhibitors of thrombin: Part 2: P1 modifications

The synthesis and antithrombotic activity of a series of nonpeptide bicyclic thrombin inhibitors is described. We have explored the SAR with modifications to the P1 site. The introduction of arginine mimetics at the P1 site led to potent and selective thrombin inhibitors.