Plasma and acrosomal membrane lipid content of saltwater crocodile spermatozoa.

This study describes the chemical lipid composition of the sperm plasma and acrosomal membranes of the saltwater crocodile Crocodylus porosus with the aim of providing new insights into sperm physiology, particularly that associated with their preservation ex vivo. The specific fatty acid composition of the sperm plasma and acrosomal membranes is documented. The mean (±s.d.) ratio of unsaturated to saturated membrane fatty acids within the plasma membrane was 2.57±0.50, and was determined to be higher than a similar analysis of the lipids found in the acrosomal membrane (0.70±0.10). The saltwater crocodile sperm plasma membrane also contained remarkably high levels of cholesterol (mean (±s.d.) 40.7±4.5 nmol per 106 sperm cells) compared with the spermatozoa of other amniote species that have so far been documented. We suggest that this high cholesterol content could be conferring stability to the crocodile sperm membrane, allowing it to tolerate extreme osmotic fluxes and rapid changes in temperature. Our descriptive analysis now provides those interested in reptile and comparative sperm physiology an improved baseline database for interpreting biochemical changes associated with preservation pathology (e.g. cold shock and cryoinjury), epididymal sperm maturation and capacitation/acrosome reaction.

Multiple-strain colonization in nasal carriers of Staphylococcus aureus.

Staphylococcus aureus is a commensal that can also cause invasive infection. Reports suggest that nasal cocolonization occurs rarely, but the resources required to sequence multiple colonies have precluded its large-scale investigation. A staged protocol was developed to maximize detection of mixed-spa-type colonization while minimizing laboratory resources using 3,197 S. aureus-positive samples from a longitudinal study of healthy individuals in Oxfordshire, United Kingdom. Initial typing of pooled material from each sample identified a single unambiguous strain in 89.6% of samples. Twelve single-colony isolates were typed from samples producing ambiguous initial results. All samples could be resolved into one or more spa types using the protocol. Cocolonization point prevalence was 3.4 to 5.8% over 24 months of follow-up in 360 recruitment-positives. However, 18% were cocolonized at least once, most only transiently. Cocolonizing spa types were completely unrelated in 56% of samples. Of 272 recruitment-positives returning ≥12 swabs, 166 (61%) carried S. aureus continuously but only 106 (39%) carried the same single spa type without any cocolonization; 31 (11%) switched spa type and 29 (11%) had transient cocarriage. S. aureus colonization is dynamic even in long-term carriers. New unrelated cocolonizing strains could increase invasive disease risk, and ongoing within-host evolution could increase invasive potential, possibilities that future studies should explore.

USA300 methicillin-resistant S. aureus (USA300 MRSA) colonization and the risk of MRSA infection in residents of extended-care facilities.

To examine the pathogenesis of USA300 MRSA infection in long-term care residents, we performed a retrospective cohort study of 1691 adult residents of two extended-care facilities from 2003 to 2007 to assess whether the risk of subsequent MRSA infection is higher in USA300 MRSA-colonized residents compared to non-colonized residents or non-USA300 MRSA colonized residents. Six per cent of residents were colonized with USA300 MRSA; 12% of residents were colonized with non-USA300 MRSA; and 101 residents developed MRSA infection. The risk of infection was twofold higher in residents colonized with USA300 MRSA compared to residents not colonized with MRSA [adjusted hazard ratio 2·3, 95% confidence interval (CI) 1·1-4·5]. The risk of infection in USA300 MRSA-colonized residents was similar to USA300 MRSA non-colonized residents (relative risk 1·1, 95% CI 0·5-2·3). Our findings show that colonization with USA300 MRSA increases the risk of MRSA infection suggesting a similar pathogenesis.

Flying-fox (Pteropus spp.) sperm membrane fatty acid composition, its relationship to cold shock injury and implications for cryopreservation success.

The very large acrosome of Pteropus species spermatozoa is prone to damage during cooling procedures. Cryogenic succuss has been linked to membrane composition, therefore the lipid composition of five Pteropus species sperm acrosomal and plasma membranes were investigated to provide insight into reasons for cold shock susceptibility. Rapid chilling and re-warming of spermatozoa from three Pteropus species resulted in a decrease (P<0.05) in acrosomal integrity. Biochemical analysis of lipids revealed that stearic acid (18:0) was the predominant saturated fatty acid and oleic acid (18:1, n-9) the predominant unsaturated fatty acid in both acrosomal and plasma membranes. Linolenic acid (18:3, n-3) was only detected in plasma membranes of Pteropus hypomelanus and was detected in acrosomal membranes of all Pteropus spp. studied (except Pteropus giganteus). Although detected in both plasma and acrosomal membranes of Pteropus vampyrus, docosahexaenoic acid (22:6) was not detected at all in Pteropus poliocephalus, only in trace levels in the acrosomal and plasma membranes of P. giganteus and P. hypomelanus and not in acrosomal membranes of Pteropus rodricensis. No difference was seen in the levels of polyunsaturated fatty acids (PUFAs) within plasma membranes, however PUFAs were lower (P<0.05) in acrosomal membranes of P. giganteus compared with P. vampyrus. Pteropus spp. spermatozoa have a very low ratio of unsaturated/saturated membrane fatty acids (<0.5). Membranes containing more PUFAs are more fluid, so the use of cryogenic media which improves membrane fluidity should improve Pteropus spp. spermatozoal viability post-thaw.

The hormonal profile of hip fracture female patients differs from community-dwelling peers over a 1-year follow-up period.

UNLABELLED: Hormone levels were compared over a 1-year period between elderly women who had sustained a hip fracture and women of similar age and functional ability. Our study suggests progressive hormonal changes that may contribute to severe bone loss during the year following hip fracture. INTRODUCTION: Alterations in hormones affecting the musculoskeletal system may increase risk of hip fracture or poor post-fracture recovery in postmenopausal women. Most studies lack appropriate reference groups, and thus cannot assess the extent to which these alterations are attributable to hip fracture. METHODS: Women aged ≥65 years hospitalized for an acute hip fracture (Baltimore Hip Studies, BHS-3; n = 162) were age-matched to 324 women enrolled in the Women's Health and Aging Study I, a Baltimore-based cohort with similar functional status to the pre-fracture status of BHS-3 women. Both studies enrolled participants from 1992 to 1995. Insulin-like growth hormone-1 (IGF-1), parathyroid hormone (PTH), 1,25 dihydroxyvitamin D [1,25(OH)2D], and osteocalcin were evaluated at baseline and 2, 6, and 12 months post-fracture, and at baseline and 12 months in the comparison group. Between-group differences in trajectories of each hormone were examined. RESULTS: Baseline mean IGF-1 levels were significantly lower in hip fracture patients than the comparison group (75.0 vs. 110.5 µg/dL; p < 0.001). Levels increased by 2 months post-fracture, but remained significantly lower than those in the comparison group throughout the 12-month follow-up (p < 0.01). Levels of PTH and osteocalcin were similar between groups at baseline, but rose during the year post-fracture to significantly differ from the comparison women (p < 0.001). 1,25(OH)2D levels did not differ between the hip fracture and comparison women at any time. CONCLUSIONS: Older women who have sustained a hip fracture have progressive changes in hormonal milieu that exceed those of women of similar health status during the year following fracture.

Retrieval failure induced by electroconvulsive shock: reversal with dissimilar training and recovery agents.

Amnesia was obtained following electroconvulsive shock in rats trained at one-trial passive avoidance of immersion in ice water. Avoidance behavior was restored when noncontingent foot shock was administered outside the training apparatus. The qualitative differences between ice water and foot shock demonstrate that the agent inducing recovery of memory need not be physically similar to the reinforcer used during training. These findings are interpreted as supporting a retrieval failure view of experimental amnesia.

Retrograde amnesia produced by electroconvulsive shock after reactivation of a consolidated memory trace.

Rats had a memory loss of a fear response when they received an electroconvulsive shock 24 hours after the fear-conditioning trial and preceded by a brief presentation of the conditioned stimulus. No such loss occurred when the conditioned stimulus was not presented. The memory loss in animals given electroconvulsive shock 24 hours after conditioning was, furthermore, as great as that displayed in animals given electroconvulsive shock immediately after conditioning. This result throws doubt on the assertion that electroconvulsive shock exerts a selective amnesic effect on recently acquired memories and thus that electroconvulsive shock produces amnesia solely through interference with memory trace consolidation.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: a case study with melanocortin-4 receptor agonists.

1-[(2R)-2-([[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl]amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a lambda(max) at approximately 450 nm. The species responsible for this absorbance required the presence of beta-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the k(inact) and K(I) parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution alpha to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

Examining Differences in Recovery Outcomes between Male and Female Hip Fracture Patients: Design and Baseline Results of a Prospective Cohort Study from the Baltimore Hip Studies.

BACKGROUND: Incidence of hip fractures in men is expected to increase, yet little is known about consequences of hip fracture in men compared to women. It is important to investigate differences at time of fracture using the newest technologies and methodology regarding metabolic, physiologic, neuromuscular, functional, and clinical outcomes, with attention to design issues for recruiting frail older adults across numerous settings. OBJECTIVES: To determine whether at least moderately-sized sex differences exist across several key outcomes after a hip fracture. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study (Baltimore Hip Studies 7th cohort [BHS-7]) was designed to include equal numbers of male and female hip fracture patients to assess sex differences across various outcomes post-hip fracture. Participants were recruited from eight hospitals in the Baltimore metropolitan area within 15 days of admission and were assessed at baseline, 2, 6 and 12 months post-admission. MEASUREMENTS: Assessments included questionnaire, functional performance evaluation, cognitive testing, measures of body composition, and phlebotomy. RESULTS: Of 1709 hip fracture patients screened from May 2006 through June 2011, 917 (54%) were eligible and 39% (n=362) provided informed consent. The final analytic sample was 339 (168 men and 171 women). At time of fracture, men were sicker (mean Charlson score= 2.4 vs. 1.6; p<0.001) and had worse cognition (3MS score= 82.3 vs. 86.2; p<0.05), and prior to fracture were less likely to be on bisphosphonates (8% vs. 39%; p<0.001) and less physically active (2426 kilocalories/week vs. 3625; p<0.001). CONCLUSIONS: This paper provides the study design and methodology for recruiting and assessing hip fracture patients and evidence of baseline and pre-injury sex differences which may affect eventual recovery one year later.

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys.

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.

Multi-site and nasal swabbing for carriage of Staphylococcus aureus: what does a single nose swab predict?

BACKGROUND: Carriage of Staphylococcus aureus is a risk for infections. Targeted decolonization reduces postoperative infections but depends on accurate screening. AIM: To compare detection of S. aureus carriage in healthy individuals between anatomical sites and nurse- versus self-swabbing; also to determine whether a single nasal swab predicted carriage over four weeks. METHODS: Healthy individuals were recruited via general practices. After consent, nurses performed multi-site swabbing (nose, throat, and axilla). Participants performed nasal swabbing twice-weekly for four weeks. Swabs were returned by mail and cultured for S. aureus. All S. aureus isolates underwent spa typing. Persistent carriage in individuals returning more than three self-swabs was defined as culture of S. aureus from all or all but one self-swabs. FINDINGS: In all, 102 individuals underwent multi-site swabbing; S. aureus carriage was detected from at least one site from 40 individuals (39%). There was no difference between nose (29/102, 28%) and throat (28/102, 27%) isolation rates: the combination increased total detection rate by 10%. Ninety-nine patients returned any self-swab, and 96 returned more than three. Nasal carriage detection was not significantly different on nurse or self-swab [28/99 (74%) vs 26/99 (72%); χ2: P=0.75]. Twenty-two out of 25 participants with first self-swab positive were persistent carriers and 69/71 with first self-swab negative were not, giving high positive predictive value (88%), and very high negative predictive value (97%). CONCLUSION: Nasal swabs detected the majority of carriage; throat swabs increased detection by 10%. Self-taken nasal swabs were equivalent to nurse-taken swabs and predicted persistent nasal carriage over four weeks.

Thrombospondin in ligament, meniscus and intervertebral disc.

The presence of thrombospondin in extracts of ligament, meniscus and human nucleus pulposus was demonstrated by Western blot analysis using an anti-human thrombospondin antibody. Metabolic labeling of ligament fibroblast and meniscal fibrochondrocyte cultures followed by immunoprecipitation with anti-human thrombospondin revealed a single band corresponding to the molecular weight of thrombospondin when analyzed by SDS-PAGE and fluorography under reduced and non-reduced conditions. Thrombospondin, therefore, is a matrix constituent of ligament, knee joint meniscus and the nucleus pulposus and is synthesized by ligament fibroblasts and meniscal fibrochondrocytes in vitro.

Analysis of ambulatory electrocardiograms in 15 patients during spontaneous ventricular fibrillation with special reference to preceding arrhythmic events.

Fifteen patients sustained ventricular fibrillation during ambulatory electrocardiographic recording in a period of 3.5 years over which time 16,500 ambulatory electrocardiograms were analyzed (prevalence = 0.09% or 1/1,100). Eight patients died, and seven survived cardiopulmonary resuscitation. Quantitative analysis of hourly ventricular arrhythmias prior to ventricular fibrillation revealed an increased frequency of premature ventricular beats and ventricular tachycardia, especially in the 2 hours immediately before ventricular fibrillation. Ventricular fibrillation was initiated by ventricular tachycardia in all 15 cases. These runs of ventricular tachycardia were characterized by their unusual length (mean = 560 +/- 536 beats) and their rapid rate (241 +/- 45 beats/min). Although an R on T premature ventricular beat initiated ventricular tachycardia and ventricular fibrillation occasionally, the mean prematurity index of the initiating premature ventricular beat was not early (mean = 1.27 +/- 0.28). QT prolongation was present in only 3 of the 15 patients (mean QTc interval = 0.42 +/- 0.06). Left ventricular dysfunction (mean left ventricular ejection fraction = 34.9 +/- 9.9%) and coronary artery disease were nearly always present. The cardiac medications most frequently associated with these patients at the time of ventricular fibrillation were digitalis and quinidine.

Stem-loop SL4 of the HIV-1 psi RNA packaging signal exhibits weak affinity for the nucleocapsid protein. structural studies and implications for genome recognition.

Encapsidation of the genome of the human immunodeficiency virus type-1 (HIV-1) during retrovirus assembly is mediated by interactions between the nucleocapsid (NC) domains of assembling Gag polyproteins and a approximately 110 nucleotide segment of the genome known as the Psi-site. The HIV-1 Psi-site contains four stem-loops (SL1 through SL4), all of which are important for genome packaging. Recent isothermal titration calorimetry (ITC) studies have demonstrated that SL2 and SL3 are capable of binding NC with high affinity (K(d) approximately 140 nM), consistent with proposals for protein-interactive functions during packaging. To determine if SL4 may have a similar function, NC-interactive studies were conducted by NMR and gel-shift methods. In contrast to previous reports, we find that SL4 binds weakly to NC (K(d)=(+/-14 microM), suggesting an alternative function. NMR studies indicate that the GAGA tetraloop of SL4 adopts a classical GNRA-type fold (R=purine, N=G, C, A or U), a motif that stabilizes RNA tertiary structures in other systems. In combination with previously reported gel mobility studies of Psi-site deletion mutants, these findings suggest that SL4 functions in genome recognition not by binding to Gag, but by stabilizing the structure of the Psi-site. Differences in the affinities of NC for SL2, SL3 and SL4 stem-loops can now be rationalized in terms of the different structural properties of stem loops that contain GGNG (SL2 and SL3) and GNRA (SL4) sequences.

Quantification of hyperaemia bordering ischaemic myocardium in experimental myocardial infarction.

To quantify and determine the time course of hyperaemia adjacent to acutely ischaemic myocardium, 19 dogs underwent open-chest coronary-artery occlusion and 4 had sham-occlusion. At 15 min (19 animals) and 45 min (9 animals) post-occlusion regional myocardial blood flow (RMBF) was measured by the radionuclide-labelled microsphere technique. The dogs were sacrificed after 24 hours, the hearts were excised, sectioned into four segments from apex to base, and stained with triphenyl tetrazolium chloride (TTC). Transmural samples were taken from necrotic areas, normal areas, and the zone bordering the infarct. Each sample was divided into an endocardial and epicardial layer. At 15 min post-occlusion RMBF in the border zone was 16% greater than in the normal myocardium (116+/-16 vs 100+/-12 cm3xmin(-1)x100g(-1);P<0.05). In addition, the ratio of endocardial to epicardial flow was lower in the border zone (0.87+/-0.05) compared with normal myocardium (1.01+/-0.02; P<0.05) indicating that the hyperaemia observed was predominantly epicardial. From 15 to 45 min hyperaemic endocardial flow tended to decline: 126 to 108 cm3xmin(-1)x100g(-1) (P<0.05). In four sham-occlusion dogs there were no differences (P>0.05) in RMBF between sites equivalent to those sampled in occluded animals. These studies document a hyperaemic border zone persisting at least 45 min post-occlusion. Hyperaemia appears to be more prominent and of longer duration in the epicardium than endocardium.

Antibiotic-associated colitis.

Among 26,294 hospitalized patients monitored by the Boston Collaborative Drug Surveillance Program (BCDSP), 8,948 (34%) received at least one antibiotic, and none were diagnosed as having drug-induced colitis to in-hospital antibiotic exposure. Seven patients who had taken antibiotics as outpatients, however, were admitted with antibiotic-associated colitis. Six of these patients had taken lincomycin prior to the onset of symptoms; one had taken ampicillin. Six of the patients were hospitalized at a New Zealand hospital and one at a hospital in Canada. The five patients with lincomycin-associated colitis at the New Zealand hospital were admitted over an 11-month period. Severe colitis due to antibiotics has been a rare event in the BCDSP experience, especially in the United States.

Comparative effects of tolamolol and propranolol on cardiac and peripheral circulatory function in patients with coronary artery disease.

In a clinical study comparing the cardiocirculatroy effects of intravenous tolamolol to those of propranolol, tolamolol, 16 mg, induced similar reduction in resting heart rate as 8 mg propranolol in 16 coronary patients. Tolamolol did not disturb cardiac pump performance and exerted less negative inotropic action than propranolol as assessed by mechanical contractility indices. Myocardial beta-one chronotropic and inotropic stimulation by exogenous epinephrine was blocked equally by tolamolol and propranolol. Tolamolol exerted less systemic vascular beta-two blockade than propranolol as assessed by the peripheral resistance and vasopressor responses to epinephrine infusion. Tolamolction than propranolol and is thereby suitable for careful extension of beta blockade therapy to certain patients with pulmonary and ventricular dysfunction.

Effects of prazosin on forearm resistance and capacitance vessels.

The effects of oral prazosin on the peripheral circulation were evaluated in 10 subjects, including 7 patients with chronic congestive failure due to coronary heart disease. To achieve this purpose the actions of 30 to 50 micrograms/kg body weight prazosin were assessed on both the forearm arteriolar and venous beds simultaneously with the use of the limb plethysmographic technique. Prazosin produced marked decline of forearm venous tone (FVT) from 44.5 +/- 12.3 to 14.3 +/- 3.1 mm Hg/ml (p less than 0.01) concomitant with marked decrease in forearm vascular resistance (FVR) from 70.2 +/- 11.4 to 48.4 +/- 4.9 mm Hg/ml/100 gm/min (p less than 0.05) which persisted for at least 60 mins without change in heart rate. These findings, demonstrating that prazosin induces systemic venodilation and peripheral arteriolodilation, indicate potential benefits of the oral drug for relief of pulmonary congestion and elevations of cardiac output in patients with chronic congestive heart failure.

Efficacy of beta adrenergic blockade in coronary heart disease: propranolol in angina pectoris.

Despite widespread clinical application of propranolol (P) in angina pectoris, convincing evidence of its efficacy has been incomplete, thereby resulting in continued controversy. Its antianginal effectivess was investigated in 20 patients with documented coronary heart disease in a 44-wk study incorporating a prolonged 12-wk lead-in period, individualization of P dosage in a 6-wk dose-finding period, and a 24-wk doule-blind crossover phase. On double-blind placebo, patients had 10.5 +/- 2.1 anginal attacks and consumed 12.8 +/- 3.0 nitroglycerin tablets (NTG) each week compared to 6.6 +/- 1.5 anginal episodes (- 37%, p less than 0.001) and 8.0 +/- 1.7 NTG (-38%, p less than 0.001) when on P. No patient experienced more angina with P than with placebo. In addition, time to onset of chest pain during treadmill exercise was prolonged by P from 190 +/- 16 to 248 +/- 22 sec (+31%, p less than 0.02) and ST depression was reduced from 1.7 +/- 0.21 to 0.99 +/- 0.18 mm (-42%, p less than 0.05). There was correlation (r = 0.64 p less than 0.01) between per cent declines in anginal frequency and resting double product with P. Thus, propranolol favorably altered several indices of myocardial ischemia in severe coronary heart disease. This investigation clearly documents the clinical efficacy of optimal beta adrenergic blockade in coronary disease and provides objective justification for the judicious application of propranolol in treatment of angina pectoris.