Primary structure of the nucleotide binding domain of the Ca,Mg-ATPase from cardiac sarcoplasmic reticulum

The nucleotide binding domain of the active site of the Ca,Mg-ATPase of cardiac sarcoplasmic reticulum (SR) has been isolated using fluorescein isothiocyanate (FITC) as an active site label and sequenced. After removal of non-specifically incorporated FITC with hydroxylamine, the amount of label incorporated was stoichiometric with residual ATPase activity, demonstrating that the label was incorporated uniquely at the active site. The SR was succinylated before digestion by trypsin in order to obtain a peptide of sufficient length to determine if the cardiac SR ATPase is a candidate for the unidentified cDNA clone recently sequenced by MacLennan et al. (Nature 316: 696-700, 1985). The sequence of the labeled SR peptide, obtained by affinity chromatography on a FITC antibody column, was T S M S K M F K G P E V I D R. This sequence was identical with that predicted by the unidentified clone and is significantly different from the sequence reported by Kirley et al. (Biochem. Biophys. Res. Commun. 130: 732-738, 1985) for a FITC labeled peptide isolated from cardiac SR.     

Disorders of the Menstrual Cycle in Elite Female Ice Hockey Players and Figure Skaters

The purpose of this study was to assess and compare the incidence of delayed menarche and menstrual dysfunction among elite ice hockey players and figure skaters. Forty-three ice hockey players (23.5 ± 4.8 years, 68.2 ± 1.2 kg, 1.68 ± 0.01 m) and 39 figure skaters (17.5 ± 3.4 years, 53.7 ± 5.8 kg, 1.64 ± 0.05 m) completed a self-administered questionnaire on their menstrual status and history, training regimens and lifestyle. Age at menarche did not differ significantly between ice hockey players (13.3 ± 1.3 years) and figure skaters (13.7 ± 1.4 years). Menarche was unrelated to nationality, vigorous training premenarche or age at which the athlete began her sport, but was correlated with the age at menarche of the athletes’ mothers (r = 0.39, p < 0.05). Hormonal contraceptives were used by 35% of ice hockey players and 15% of the figure skaters. Amenorrhea and oligomenorrhea were experienced by 7.1% and 38.7% of postmenarcheal, ice hockey players and figure skaters respectively not using hormonal contraceptives. Menstrual dysfunction was associated with both age and age at menarche in the ice hockey players only. Training factors, and psychological pressure were perceived by the athletes to contribute to menstrual dysfunction. A greater training volume, younger age at commencing sport, lower body mass, greater subjective body image pressure and younger biological and gynaecological age were reported among the figure skaters, and were proposed to explain the higher incidence of menstrual dysfunction among the figure skaters compared with the ice hockey players. Figure skaters appear at increased risk of amenorrhea and oligomenorrhea compared with ice hockey players, which may be linked to training and physical characteristics of the sports.     

Use of the EM algorithm to detect QTL affecting multiple-traits in an across half-sib family analysis

QTL detection experiments in livestock species commonly use the half-sib design. Each male is mated to a number of females, each female producing a limited number of progeny. Analysis consists of attempting to detect associations between phenotype and genotype measured on the progeny. When family sizes are limiting experimenters may wish to incorporate as much information as possible into a single analysis. However, combining information across sires is problematic because of incomplete linkage disequilibrium between the markers and the QTL in the population. This study describes formulæ for obtaining MLEs via the expectation maximization (EM) algorithm for use in a multiple-trait, multiple-family analysis. A model specifying a QTL with only two alleles, and a common within sire error variance is assumed. Compared to single-family analyses, power can be improved up to fourfold with multi-family analyses. The accuracy and precision of QTL location estimates are also substantially improved. With small family sizes, the multi-family, multi-trait analyses reduce substantially, but not totally remove, biases in QTL effect estimates. In situations where multiple QTL alleles are segregating the multi-family analysis will average out the effects of the different QTL alleles.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Endemic infection reduces transmission potential of an epidemic parasite during co-infection

Endemic, low-virulence parasitic infections are common in nature. Such infections may deplete host resources, which in turn could affect the reproduction of other parasites during co-infection. We aimed to determine whether the reproduction, and therefore transmission potential, of an epidemic parasite was limited by energy costs imposed on the host by an endemic infection. Total lipids, triacylglycerols (TAG) and polar lipids were measured in cockroaches (Blattella germanica) that were fed ad libitum, starved or infected with an endemic parasite, Gregarina blattarum. Reproductive output of an epidemic parasite, Steinernema carpocapsae, was then assessed by counting the number of infective stages emerging from these three host groups. We found both starvation and gregarine infection reduced cockroach lipids, mainly through depletion of TAG. Further, both starvation and G. blattarum infection resulted in reduced emergence of nematode transmission stages. This is, to our knowledge, the first study to demonstrate directly that host resource depletion caused by endemic infection could affect epidemic disease transmission. In view of the ubiquity of endemic infections in nature, future studies of epidemic transmission should take greater account of endemic co-infections.     

Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.