Crystallization and a preliminary X-ray diffraction study of isozyme 3-3 of glutathione S-transferase from rat liver

Crystals of the homodimeric isozyme 3-3 of glutathione S-transferase from rat liver have been obtained with the hanging drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of the enzyme required the presence of both the enzyme inhibitor (9R, 10R)-9, 10-dihydro-9-(S-glutathionyl)-10-hydroxyphenanthrene and the detergent beta-octylglucopyranoside. The crystals belong to the monoclinic space group C2, with cell dimensions of a = 88.24(8) A, b = 69.44(4) A, c = 81.28(5) A, beta = 106.01(6) degrees, and contain four dimeric enzyme molecules per unit cell. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination at high resolution.     

Effect of melatonin on postpartum anestrus in beef cows

The effect of melatonin treatment on intervals from calving to first postpartum estrus and ovulation was determined in Shorthorn cows which calved May 8 to June 14. Melatonin (500 mg in beef tallow) was injected subcutaneously (s.c.) into 20 cows on June 15 (4 to 38 d postpartum). Ovulation was determined from progesterone concentrations in jugular venous blood collected weekly from June to August. Mean intervals to first estrus and first ovulation were significantly longer in primiparous than in multiparous cows (85 +/- 4 vs 55 +/- 3 d and 83 +/- 4 vs 52 +/-3 d). Melatonin treatment caused a significant increase in the intervals to first postpartum estrus (68 +/- 4 vs 58 +/- 5d) and ovulation (68 +/- 4 vs 55 +/- 5 d). Mean plasma melatonin concentrations during the daytime were significantly higher in treated than in control cows one and two weeks after melatonin injection and were within the lower range of nighttime values reported previously for cows. Thus melatonin treatment raised daytime plasma concentrations of melatonin and delayed the onset of estrus and ovulation. These results support the possibility of a role of photoperiod through melatonin secretion in the onset of postpartum ovarian activity in cattle.     

Changes and availability of P fractions following 65 years of P application to a calcareous soil in a Mediterranean climate

The fate and availability of P derived from granular fertilisers in an alkaline Calcarosol soil were examined in a 65-year field trial in a semi-arid environment (annual rainfall 325 mm). Sequential P fractionation was conducted in the soils collected from the trial plots receiving 0–12 kg P ha⁻¹crop⁻¹, and the rhizosphere soil after growing wheat (Triticum aestivum L. cv. Yitpi) and chickpea (Cicer arietinum L. cv. Genesis 836) for one or two 60-day cycles in the glasshouse. Increasing long-term P application rate over 65 years significantly increased all inorganic P (Pi) fractions except HCl–Pi. By contrast, P application did not affect or tended to decrease organic P (Po) fractions. Increasing P application also increased Olsen-P and resin-P but decreased the P buffer capacity and sorption maxima. Residual P, Pi and Po fractions accounted for an average of 32, 16 and 52% of total P, respectively. All soil P fractions including residual P in the rhizosphere soil declined following 60-day growth of either wheat or chickpea. The decreases were greater in soils with a history of high P application than low P. An exception was water-extractable Po, which increased following plant growth. Changes in various P fractions in the rhizosphere followed the same pattern for both plant species. Biomass production and P uptake of the plants grown in the glasshouse correlated positively with the residual P and inorganic fractions (except HCl–Pi) but negatively with Po in the H₂O-, NaOH- and H₂SO₄-fractions of the original soils. The results suggest that the long-term application of fertiliser P to the calcareous sandy soil built up residual P and non-labile Pi fractions, but these P fractions are potentially available to crops.     

Time to treatment with thrombolytic therapy: determinants and effect on short-term nonfatal outcomes of acute myocardial infarction

OBJECTIVES: To characterize the extent of delay in administration of thrombolytic therapy to patients with acute myocardial infarction (AMI) in Canada, to examine patient-specific predictors of such delay and to measure the effect of delay on short-term nonfatal cardiac outcomes. DESIGN: Secondary cohort analysis of data from the first international Global Utilization of Streptokinase and tPA for Occluded Coronary Arteries (GUSTO-I) trial. SETTING: Sixty-three acute care hospitals across Canada. SUBJECTS: All 2898 Canadian patients with an AMI enrolled in GUSTO-I. MAIN OUTCOMES: Time before arrival at a hospital ("symptom-to-door" time) and time from arrival to administration of therapy ("door-to-needle" time) for patients who had an AMI outside of a hospital, in clinically relevant categories; proportions of patients with nonfatal, serious cardiac events, including shock, sustained ventricular tachycardia, ventricular fibrillation and asystole. RESULTS: Of the total number of patients enrolled, records were complete for 2708; 2542 of these patients (93.9%) had an AMI outside of a hospital. These 2542 patients presented a median 81 (interquartile range 50 to 130) minutes after the onset of symptoms, and the median time to treatment in hospital was 85 (interquartile range 61 to 115) minutes. Whereas a greater proportion of Canadian patients than of patients enrolled in GUSTO-I in other countries reached hospital within 2 hours of symptom onset (71.5% v. 61.2%, p < 0.001), a greater proportion of Canadian patients experienced in-hospital treatment delays of more than 1 hour (75.3% v. 57.1%, p < 0.001). In an analysis of all 2708 patients with complete records, both the unadjusted and adjusted odds of nonfatal cardiac events for those treated 4 to 6 hours after symptom onset were significantly higher than for those treated within 2 hours (odds ratio 1.60, 95% confidence interval 1.09 to 2.37). CONCLUSION: After arrival at a hospital, Canadian patients enrolled in GUSTO-I received thrombolytic therapy more slowly than trial enrollees in other countries. Such delays are already known to decrease the rate of short-term survival after AMI. The findings further show that long time to treatment also increases the odds of nonfatal, serious cardiac events. Hospitals and physicians caring for patients with AMI should routinely assess whether and how they can improve door-to-needle times.     

Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.     

The effect of restricted feeding on the wheel-running activity rhythms of the predatory marsupial Dasyurus viverrinus

Summary The effects of restricted feeding schedules on the circadian rhythms of wheel-running of Dasyurus viverrinus were examined under a light/dark cycle and in constant darkness (experiment 1) and in constant light (experiment 2). The results of the 2 experiments showed that: (1) in contrast to the light/dark cycle, restricted feeding is only a weak zeitgeber for the wheel-running activity rhythms of D. viverrinus; (2) restricted feeding elicits meal anticipatory activity in D. viverrinus comparable to that elicited by restricted feeding in the rat; (3) transient cycles of the anticipatory activity free-run with a period different to that of the main component of activity for several cycles after the termination of restricted feeding; and (4) activity suggestive of beating between 2 oscillators occurs during restricted feeding and after the termination of restricted feeding. Taken together the latter 3 observations suggest that the activity rhythms of D. viverrinus are controlled by at least 2 separate circadian oscillators.     

N-delta-acetylornithine and S-methylcysteine in blood plasma

N-delta-Acetylornithine and S-methylcysteine have been identified as minor components of deproteinized blood plasma of human and bovine blood. Human blood plasma contains a variable amount of acetylornithine, averaging 1.1 +/- 0.4 mumol/l (range 0.8--0.2 mumol/l). Urine contains a very small amount of acetylornithine, approximately 1 nmol/mg creatinine (1 mumol/day). Human blood plasma contains 3.9 +/- 1.9 mumol/l (range 1.4--6.5 mumol/l) of S-methylcysteine. Urine contains approximately 5 nmol/mg creatinine; after acid hydrolysis the amount is increased to 20 nmol/mg creatinine.