Procurement of spore-free Bacillus anthracis for molecular typing outside of BSL3 environment.

AIMS: To (i) develop a protocol that would eliminate or greatly reduce sporulation within Bacillus anthracis vegetative cells, and (ii) harvest an adequate number of cells and sufficient DNA suitable for molecular methods including Riboprint analysis and pulse field gel electrophoresis (PFGE). METHODS AND RESULTS: Seven strains of B. anthracis (Ames, French B2, Heluky, Kruger, Pasteur, Sterne, and Vollum) were grown at 37, 42 and 45 degrees C under normal air, enhanced CO(2), microaerophilic, and anaerobic conditions on solid media and subcultured in two broths with and without supplements. The bacterial cells were centrifuged and washed. Slides made from the cell pellets were stained with Malachite Green and observed for the presence of spores. Cell preparations were subjected to 80 degrees C for 30 min and processed for and analysed by either Riboprinte or PFGE. Multiple pellets of each strain were processed, stained, placed onto solid culture media, incubated for 7 days and observed for growth. The cell preparations yielded clear and reproducible results with both molecular methods. None of the cell preparations yielded growth on the culture media. CONCLUSIONS: This method eliminated viable spores in cell preparations of B. anthracis, yet still allowed the growth of vegetative cells to provide sufficient DNA suitable for analysis by Riboprinter and PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This method will provide safe cell preparations, prevent instrument contamination, and may be useful for other aerobic and anaerobic spore-formers.

Insertion of a coiled-coil peptide from influenza virus hemagglutinin into membranes.

The trimeric protein hemagglutinin (HA) of the influenza viral envelope is essential for cell entry. To investigate the interaction of HA with membranes, two 40-residue, cysteine-substituted peptides comprising the loop region and the first part of the coiled-coil stem were synthesized and modified with a nitroxide spin label. Electron paramagnetic resonance analysis revealed that the peptide inserts reversibly into phospholipid vesicles under endosomal pH conditions. This result suggests that some or all of the long coiled-coil trimer of HA may insert into membranes, which could bring the viral and cell membranes closer together and facilitate fusion.

Structure and function of a human TAFII250 double bromodomain module.

TFIID is a large multiprotein complex that initiates assembly of the transcription machinery. It is unclear how TFIID recognizes promoters in vivo when templates are nucleosome-bound. Here, it is shown that TAFII250, the largest subunit of TFIID, contains two tandem bromodomain modules that bind selectively to multiply acetylated histone H4 peptides. The 2.1 angstrom crystal structure of the double bromodomain reveals two side-by-side, four-helix bundles with a highly polarized surface charge distribution. Each bundle contains an Nepsilon-acetyllysine binding pocket at its center, which results in a structure ideally suited for recognition of diacetylated histone H4 tails. Thus, TFIID may be targeted to specific chromatin-bound promoters and may play a role in chromatin recognition.

Sequence-based design of a peptide probe for the APC tumor suppressor protein.

BACKGROUND: Proteins form specific associations, but predictive rules for protein pairing are generally unknown. Here, we describe amino-acid sequence patterns capable of mediating specific pairing of a widespread protein motif: the parallel, dimeric, alpha-helical coiled coil. The pairing rules were tested by designing a 54-residue peptide (anti-APCp1) that is predicted to dimerize preferentially with a coiled-coil sequence from the adenomatous polyposis coli (APC) tumor suppressor protein. RESULTS: As judged by circular dichroism, ultracentrifugation and native gel electrophoresis, anti-APCp1 formed a specific, helical, dimeric complex with the target APC coiled coil. On western blots of APC fragments expressed in Escherichia coli, the designed peptide detected a pattern of bands identical to the pattern detected by an antibody directed against the APC coiled coil. Peptide-mediated precipitation experiments showed that anti-APCp1 bound and sequestered wild-type and mutant APC proteins in extracts of human colon cancer cell lines. In addition, binding of the designed peptide preserved native APC-beta-catenin complexes. CONCLUSIONS: These biochemical experiments demonstrate that the anti-APC peptide preferentially forms a heterodimeric coiled coil with mutant and full-length APC proteins. The specificity of the designed peptide is sufficient to support several applications that commonly use antibodies. The observed specificity of anti-APCp1 validates the pairing rules used as the basis for the probe design, and it suggests that residues in the core positions of coiled coils help impart pairing selectivity.

Insulin resistance in aging is related to abdominal obesity.

Studies have shown that insulin resistance increases with age, independent of changes in total adiposity. However, there is growing evidence that the development of insulin resistance may be more closely related to abdominal adiposity. To evaluate the independent effects of aging and regional and total adiposity on insulin resistance, we performed hyperinsulinemic euglycemic clamps on 17 young (21-33 yr) and 67 older (60-72 yr) men and women. We assessed FFM and total and regional adiposity by hydrodensitometry and anthropometry. Insulin-stimulated GDRs at a plasma insulin concentration of approximately 450 pM averaged 45.6 +/- 3.3 mumol.kg FFM-1 x min-1 (mean +/- SE) in the young subjects, 45.6 +/- 10.0 mumol.kg FFM-1 x min-1 in 24 older subjects who were insulin sensitive, and 23.9 +/- 11.7 mumol.kg FFM-1 x min-1 in 43 older subjects who were insulin resistant. Few significant differences were apparent in skin-fold and circumference measurements between young and insulin-sensitive older subjects, but measurements at most central body sites were significantly larger in the insulin-resistant older subjects. Waist girth accounted for > 40% of the variance in insulin action, whereas age explained only 10-20% of the total variance and < 2% of the variance when the effects of waist circumference were statistically controlled. These results suggest that insulin resistance is more closely associated with abdominal adiposity than with age.

Multi-site phosphorylation of Pho4 by the cyclin-CDK Pho80-Pho85 is semi-processive with site preference.

As part of a nutrient-responsive signaling pathway, the budding yeast cyclin-CDK complex Pho80-Pho85 phosphorylates the transcription factor Pho4 on five sites and inactivates it. Here, we describe the kinetic reaction between Pho80-Pho85 and Pho4. Through experimentation and computer modeling we have determined that Pho80-Pho85 phosphorylates Pho4 in a semi-processive fashion that results from a balance between kcat and k(off). In addition, we show that Pho80-Pho85 phosphorylates certain sites preferentially. Phosphorylation of the site with the highest preference inhibits the transcriptional activity of Pho4 when it is in the nucleus, while phosphorylation of the lowest-preference sites is required for export of Pho4 from the nucleus. This method of phosphorylation may allow Pho80-Pho85 to quickly inactivate Pho4 in the nucleus and efficiently phosphorylate Pho4 to completion.

Improvement in glucose tolerance after 1 wk of exercise in patients with mild NIDDM.

We investigated the effects of 1 wk of intense exercise on glucose tolerance in 10 men with abnormal glucose tolerance [7 had mild non-insulin-dependent diabetes mellitus (NIDDM), and 3 had impaired glucose tolerance]. The 7 days of exercise did not result in significant changes in body weight or maximal oxygen uptake. Plasma glucose concentration at 120 min averaged 227 +/- 23 mg/dl in an oral glucose tolerance test (OGTT) before and 170 +/- 18 mg/dl after the 7 days of exercise (P less than .001). There was a 36% reduction in the area under the glucose tolerance curve. Plasma insulin concentration at 120 min of the OGTT averaged 172 +/- 27 microU/ml before and 106 +/- 13 microU/ml after 7 days of exercise (P less than .001); the area under the insulin curve was decreased by 32%. In contrast to the response to 7 days of exercise, one bout of exercise did not result in an improvement in glucose tolerance. These results provide evidence that regularly performed, vigorous exercise can be effective in decreasing insulin resistance and improving glucose tolerance within 7 days in some patients with mild NIDDM.

Random circular permutation leading to chain disruption within and near alpha helices in the catalytic chains of aspartate transcarbamoylase: effects on assembly, stability, and function.

A collection of circularly permuted catalytic chains of aspartate transcarbamoylase (ATCase) has been generated by random circular permutation of the pyrB gene. From the library of ATCases containing permuted polypeptide chains, we have chosen for further investigation nine ATCase variants whose catalytic chains have termini located within or close to an alpha helix. All of the variants fold and assemble into dodecameric holoenzymes with similar sedimentation coefficients and slightly reduced thermal stabilities. Those variants disrupted within three different helical regions in the wild-type structure show no detectable enzyme activity and no apparent binding of the bisubstrate analog N:-phosphonacetyl-L-aspartate. In contrast, two variants whose termini are just within or adjacent to other alpha helices are catalytically active and allosteric. As expected, helical disruptions are more destabilizing than loop disruptions. Nonetheless, some catalytic chains lacking continuity within helical regions can assemble into stable holoenzymes comprising six catalytic and six regulatory chains. For seven of the variants, continuity within the helices in the catalytic chains is important for enzyme activity but not necessary for proper folding, assembly, and stability of the holoenzyme.

Mass spectrometric analysis of the fragments produced by cleavage and reduction of rat prolactin: evidence that the cleaving enzyme is cathepsin D.

The site(s) at which mammary tissue enzymatically cleaves rat (r) PRL, and the possibility that the cleaving activity is cathepsin D, were investigated using mass spectrometry and enzyme inhibitors. Cleavage of intact rPRL [22,566 atomic mass units (amu)] by either mammary gland-conditioned medium or cathepsin D (both at pH 3) reduced the mass of the molecule by 397 amu. Subsequent reduction of the large rPRL fragment cleaved by either method generated two fragments of 16,364 and 5808 amu. The mass of the smaller fragment is consistent with the report of Vick et al. (Biochim Biophys Acta 931: 196-204, 1987) that its amino-terminal residue is Ser149. These results indicate that both enzyme preparations cleave rPRL by excision of the tripeptide Leu-Val-Trp (mass = 397 amu) between residues 145 and 149. The ability of both enzyme preparations to cleave rPRL at pH3 was inhibited by pepstatin A but not by phenylmethane sulfonyl fluoride, and both preparations were essentially inactive at pH7. Accordingly, the PRL-cleaving activity of rat mammary tissue is probably cathepsin D.

Endocrine responses to chronic androstenedione intake in 30- to 56-year-old men.

In young men, chronic ingestion of 100 mg androstenedione (ASD), three times per day, does not increase serum total testosterone but does increase serum estrogen and ASD concentrations. We investigated the effects of ASD ingestion in healthy 30- to 56-yr-old men. In a double-blind, randomly assigned manner, subjects consumed 100 mg ASD three times daily (n = 28), or placebo (n = 27) for 28 days. Serum ASD, dihydrotestosterone (DHT), free and total testosterone, estradiol, prostate-specific antigen (PSA), and lipid concentrations were measured at week 0 and each week throughout the supplementation period. Serum total testosterone and PSA concentrations did not change with supplementation. Elevated serum concentrations of ASD (300%), free testosterone (45%), DHT (83%), and estradiol (68%) were observed during weeks 1-4 in ASD (P < 0.05). There was no relationship between age and changes in serum ASD (r2 = 0.024), free testosterone (r2 = 0.00), or estradiol (r2 = 0.029) concentrations with ASD, whereas the serum DHT response to ASD ingestion was related to age (r2 = 0.244; P < 0.05). Serum concentrations of high-density lipoprotein cholesterol were decreased by 10% during the supplementation period (P < 0.05). These results suggest that the ingestion of 100 mg ASD, three times per day, does not increase serum total testosterone or PSA concentrations but does elicit increases in ASD, free testosterone, estradiol, and DHT and decreases serum high-density lipoprotein cholesterol concentrations.

Can allergic exposure provoke psychological symptoms? A double-blind test.

Clinical ecologists report that exposure to allergens can induce cognitive and emotional symptoms as well as somatic symptoms in susceptible individuals, but controlled tests are meager. In a test of the hypothesis that sublingual exposure to allergens would produce cognitive-emotional symptoms in allergy patients, double-blind provocative testing was conducted at an allergy clinic; 30 allergy patients complaining of at least one psychological symptom were selected. Self-report, heart-rate, and several mood and psychological performance measures were obtained. MMPI scores indicated a pathological sample. Reported cognitive-emotional symptoms were greater for allergens than for placebos (p = 0.001), while placebo symptoms were equal to base rate. Greater variability of heart rate change was found for allergens than for placebos (p = 0.008). Severe reactions occurred more frequently to allergens (p = 0.008). Severe reactions occurred more frequently to allergens (p = 0.02). Other dependent measures were not affected by the allergens or the placebos. It is concluded that allergens may contribute to psychopathology in some individuals.

Role of modified glutamic acid in the helical structure of conantokin-T.

Circular dichroism (CD) and 2-dimensional NMR were used to study the solution conformation of conantokin-T (Con-T), a small peptide toxin found in the venom of fish-hunting cone snails, and its Glu-substituted analog. Con-T lacks disulfide bonds but contains many gamma-carboxyglutamic acids (Gla), a post-translationally modified residue. Our results show that Con-T adopts an alpha-helical conformation in aqueous solution even in the absence of calcium. Glu replacements diminish both helicity and function of Con-T. The helical content of Con-T is higher than most natural helical peptides of this length in aqueous solution. The sequence of this small toxin incorporates several known elements that stabilize alpha-helical structure in peptides. Gla residues form several salt bridges that stabilize helical conformation of Con-T.

Biological actions of synthetic locust ion transport peptide (ITP).

Locust Ion Transport Peptide (ITP) a member of the arthropod neuropeptide family which includes hyperglycemic, vitellogenesis-inhibiting, and moult-inhibiting hormones (CHH, VIH, MIH, respectively) was synthesized as proposed by Meredith et al. (1996) with terminal amidation of amino acid residue 72 and with 3 disulphide bridges. This is the first member of this family to be synthesized. Biological activities of synthetic ITP (synITP) were very similar to those previously reported for ITP purified from Schistocerca corpora cardiaca (ScgITP) and partially sequenced by Audsley et al. (1992a, b). Dose-response curves for both synITP and ScgITP on ileal transport of Cl- (measured as increased short-circuit current, delta Isc), were similar with a EC50 of 1-2 nM. The Isc time course and maximum delta Isc across ileal epithelia at different dosages of synITP and ScgITP had similar patterns as did changes in transepithelial open-circuit potential (Vt) and resistance (Rt), reflecting changes in salt transport which drives fluid absorption. Disulphide bridges were shown to be required for biological activity of synITP, which caused the same 4-fold increase in ileal fluid transport rate (Jv) as previously reported for ScgITP. Both synITP and ScgITP caused only partial stimulation of rectal Isc and had no significant effect on rectal Jv. These results indicate that the structure of ITP predicted earlier from cDNA is correct.

Muscle metabolism during exercise in the heat in unacclimatized and acclimatized humans.

The effect of heat acclimatization on aerobic exercise tolerance in the heat and on subsequent sprint exercise performance was investigated. Before (UN) and after (ACC) 8 days of heat acclimatization, 10 male subjects performed a heat-exercise test (HET) consisting of 6 h of intermittent submaximal [50% of the maximal O2 uptake] exercise in the heat (39.7 degrees C dB, 31.0% relative humidity). A 45-s maximal cycle ride was performed before (sprint 1) and after (sprint 2) each HET. Mean muscle glycogen use during the HET was lower following acclimatization [ACC = 28.6 +/- 6.4 (SE) and UN = 57.4 +/- 5.1 mmol/kg; P less than 0.05]. No differences were noted between the UN and ACC trials with respect to blood glucose, lactate (LA), or respiratory exchange ratio. During the UN trial only, total work output during sprint 2 was reduced compared with sprint 1 (24.01 +/- 0.80 vs. 21.56 +/- 1.18 kJ; P less than 0.05). This reduction in sprint performance was associated with an attenuated fall in muscle pH following sprint 2 (6.86 vs. 6.67, P less than 0.05) and a reduced accumulation of LA in the blood. These data indicate that heat acclimatization produced a shift in fuel selection during submaximal exercise in the heat. The observed sparing of muscle glycogen may be associated with the enhanced ability to perform highly intense exercise following prolonged exertion in the heat.

Effects of eccentric exercise on insulin secretion and action in humans.

The effects of an exhaustive bout of eccentric exercise on insulin secretion and action were determined using the hyperglycemic clamp technique. Clamps were performed on eight healthy men after 7 days of inactivity and approximately 36 h after a bout of eccentric exercise. Eccentric exercise consisted of 10 sets of 10 repetitions of combined knee extensions and flexions for each leg at a mean torque 84 +/- 5% of peak concentric torque. During the hyperglycemic clamp procedure, plasma glucose concentration was acutely raised to 10 mmol/l and was maintained near this level for 120 min. Arterialized blood samples were obtained from a heated hand vein to determine plasma glucose and insulin concentrations. Eccentric exercise appeared to produce marked muscle damage, as indicated by a 50-fold increase in plasma creatine phosphokinase (100 +/- 17 vs. 5,209 +/- 3,811 U/l, P < 0.001) and subjective reports of muscle soreness. Peak insulin response during the early phase (0-10 min) of the hyperglycemic clamp was higher after eccentric exercise (183 +/- 38 microU/ml) than after the control clamp (100 +/- 23 microU/ml, P < 0.005). Late-phase (10- to 120-min) insulin response was not altered after eccentric exercise. Peak plasma C-peptide concentrations were higher during the early phase (5.0 +/- 0.7 vs. 4.3 +/- 0.8 ng/ml, P < 0.05) and the late phase (7.5 +/- 0.9 vs. 5.4 +/- 0.6 ng/ml, P < 0.05). Prior eccentric exercise had no significant effect on whole body glucose disposal or glucose disposal rate adjusted for prevailing plasma insulin concentration. These data provide evidence that a single bout of eccentric exercise causes an increase in pancreatic beta-cell insulin secretion in response to hyperglycemia.

Effect of 10 days of physical inactivity on glucose tolerance in master athletes.

Master athletes who exercise regularly appear to avoid the development of insulin resistance and deterioration of glucose tolerance (GT) commonly seen with aging. To evaluate the possibility that exercise prevents rather than masks the aging-related changes responsible for development of insulin resistance, we investigated the effects of 10 days of physical inactivity in 14 master athletes aged 61 +/- 2 (SE) yr. The response of 10 of these men to inactivity was similar to that of young athletes, with an unchanged plasma glucose response and a significantly greater insulin response to an oral glucose tolerance test (OGTT) after 10 days of inactivity. These 10 athletes appeared to have been protected against the aging-related changes in GT because their plasma glucose and insulin levels during the OGTT after 10 days of inactivity were not significantly different from those of young lean sedentary men. In contrast, a deterioration in GT occurred in four of the master athletes during 10 days of inactivity; this was sufficiently marked in two of them to be classified as impaired GT. We conclude that regular exercise may 1) protect against the development of insulin resistance and decline in GT with aging in individuals with normal GT and 2) normalize GT by means of short-term effects of exercise in some individuals with abnormal GT.

Effects of exercise and lack of exercise on insulin sensitivity and responsiveness.

Insulin action is enhanced in people who exercise regularly and vigorously. In the present study, the hyperinsulinemic, euglycemic clamp procedure was used to determine whether this enhanced insulin action is due to an increased sensitivity and/or an increased responsiveness to insulin. To avoid the variability that exists between individuals and complicates cross-sectional studies, the same subjects were studied in the trained exercising state and again after 10 days of physical inactivity. When the plasma insulin concentration was maintained at approximately 78 microU.ml-1 (a submaximal level), glucose disposal rate averaged 8.7 +/- 0.5 mg.kg-1.min-1 before and 6.7 +/- 0.6 mg.kg-1.min-1 after 10 days of activity (P less than 0.001). When the plasma insulin concentration was maintained at approximately 2,000 microU.ml-1 (a maximally effective concentration), the rate of glucose disposal was not significantly different before (15.3 +/- 0.5 mg.kg-1.min-1) compared with after (14.5 +/- 0.4 mg.kg-1.min-1) 10 days without exercise. These results provide evidence that the reversal of enhanced insulin action that occurs within a few days when exercise-trained individuals stop exercising is due to a decrease in sensitivity to insulin, not to a decrease in insulin responsiveness.

Exercise and recovery ventilatory and VO2 responses of patients with McArdle's disease.

This study was designed to determine whether patients with McArdle's disease, who do not increase their blood lactate levels during and after maximal exercise, have a slow "lactacid" component to their recovery O2 consumption (VO2) response after high-intensity exercise. VO2 was measured breath by breath during 6 min of rest before exercise, a progressive maximal cycle ergometer test, and 15 min of recovery in five McArdle's patients, six age-matched control subjects, and six maximal O2 consumption- (VO2 max) matched control subjects. The McArdle's patients' ventilatory threshold occurred at the same relative exercise intensity [71 +/- 7% (SD) VO2max] as in the control groups (60 +/- 13 and 70 +/- 10% VO2max) despite no increase and a 20% decrease in the McArdle's patients' arterialized blood lactate and H+ levels, respectively. The recovery VO2 responses of all three groups were better fit by a two-, than a one-, component exponential model, and the parameters of the slow component of the recovery VO2 response were the same in the three groups. The presence of the same slow component of the recovery VO2 response in the McArdle's patients and the control subjects, despite the lack of an increase in blood lactate or H+ levels during maximal exercise and recovery in the patients, provides evidence that this portion of the recovery VO2 response is not the result of a lactacid mechanism. In addition, it appears that the hyperventilation that accompanies high-intensity exercise may be the result of some mechanism other than acidosis or lung CO2 flux.