It's all in the timing: coital frequency and fertility awareness-based methods of family planning

Fertility awareness-based methods of family planning help women to identify the days of the cycle they should avoid unprotected intercourse to prevent pregnancy. Therefore using fertility awareness-based methods influences the timing of sexual activity, which may affect the nature of the sexual relationship. Data are used from the clinical trials of two fertility awareness-based methods--the Standard Days Method and the TwoDay Method--to determine the frequency and timing of intercourse during the cycle, and the determinants of coital frequency. The mean coital frequency of study participants was similar to that reported by users of other methods. Results suggest that coital frequency increases with consecutive cycles of method use. At the same time the frequency of intercourse during the identified fertile days and during menses decreases. This evidence implies a behavioural change as couples get more experience using their method and communicating about the fertile days. Coital frequency was also influenced by the method used and by the study sites. Potential differences between the methods and sites that may contribute to this effect are discussed.     

Involvement of Rho kinase pathway in the mechanism of renal vasoconstriction and cardiac hypertrophy in rats with experimental heart failure

Rho-dependent kinases serve as downstream effectors of several vasoconstrictor systems, the activities of which are upregulated in congestive heart failure (CHF). We evaluated renal and cardiac effects of Y-27632, a highly selective Rho kinase inhibitor, in an experimental model of volume-overload CHF. Effects of acute administration of Y-27632 (0.3 mg/kg) on renal hemodynamic and clearance parameters and effects of chronic treatment (10.0 mg.kg(-1).day(-1) for 7 days via osmotic minipumps) on cardiac hypertrophy and cumulative Na+ excretion were studied in male Wistar rats with aortocaval fistula and control rats. The Y-27632-induced decrease in renal vascular resistance (from 40.4 +/- 4.6 to 26.0 +/- 3.1 resistance units, P < 0.01) in CHF rats was associated with a significant increase in total renal blood flow (+34%) and cortical and medullary blood flow (approx +37 and +27%, respectively). These values were significantly higher than those in control rats and occurred despite a decrease in mean arterial pressure (-15 mmHg). Despite the marked renal vasodilatory effect, Y-27632 did not alter glomerular filtration rate and renal Na+ excretion. Chronic administration of Y-27632 did not alter daily or cumulative renal Na+ excretion in CHF rats but was associated with a significant decrease in heart-to-body weight ratio, an index of cardiac hypertrophy: 0.32 +/- 0.007, 0.46 +/- 0.017, and 0.37 +/- 0.006% in control, CHF, and CHF + Y-27632 rats, respectively. The findings suggest that Rho kinase-dependent pathways are involved in the mechanisms of renal vasoconstriction and cardiac hypertrophy in rats with volume-overload heart failure. Selective blockade of these signaling pathways may be considered an additional tool to improve renal perfusion and attenuate cardiac hypertrophy in heart failure.     

Binding of the universal minicircle sequence binding protein at the kinetoplast DNA replication origin

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation.     

Calpain 11 is unique to mouse spermatogenic cells

The calpains are a family of calcium-dependent thiol proteases involved in intracellular processing of proteins. They occur as heterodimers containing one of various large subunits and a common small subunit. Some of the large subunits are expressed ubiquitously and others are expressed in a restricted set of tissues. We have cloned the cDNA for mouse calpain 11 and demonstrated that it is expressed specifically in the mouse testis. The mRNA begins to accumulate in the testis between days 14 and 16 after birth, corresponding to the period of pachytene spermatocyte development. The protein is detected by day 18 after birth, during mid to late pachytene spermatocyte development, and is present in the acrosomal region of spermatozoa from the cauda epididymis. The expression of calpain 11 during spermatogenesis and its localization in spermatozoa suggest that it is involved in regulating calcium-dependent signal transduction events during meiosis and sperm functional processes.     

Evaluation of antioxidants: Scope, limitations and relevance of assays

Peroxidation of lipids, particularly polyunsaturated fatty acid residues (PUFA) of phospholipids and cholesterol esters, is a process of marked implications: it shortens the shelf-life of food and drugs, it causes fragmentation of DNA, it damages cellular membranes and it promotes the genesis of many human diseases. Much effort is therefore devoted to a search for "potent antioxidants", both synthetic and from natural sources, mostly plants. This, in turn, requires a reliable, simple, preferably high throughput assay of the activity of alleged antioxidants. The most commonly used assays are based on measurements of the total antioxidant capacity (TAC) of a solution, as evaluated either by determining the rate of oxidation of the antioxidant or by measuring the protection of an easily determined indicator against oxidation by the antioxidants. The commonly used assays utilized for ranking antioxidants share three common problems: (i) They usually evaluate the effects of those antioxidants that quench free radicals, which constitute only a part of the body's antioxidative network, in which enzymes play the central role. (ii) Both the capacity and potency of antioxidants, as obtained by various methods, do not necessarily correlate with each other. (iii) Most estimates are based on methods conducted in solution and are therefore not necessarily relevant to processes that occur at the lipid-water interfaces in both membranes and micro emulsions (e.g. lipoproteins). Given this "state of art", many researchers, including us, try to develop a method based on the formation of hydroperoxides (LOOH) upon peroxidation of PUFA in lipoproteins or in model membranes, such as liposomes. In these systems, as well as in lipoproteins, the most apparent effect of antioxidants is prolongation of the lag time preceding the propagation of a free radical chain reaction. In fact, under certain conditions both water soluble antioxidants (e.g. vitamin C and urate) and the lipid soluble antioxidant tocopherol (vitamin E), promote or even induce peroxidation. Based on the published data, including our results, we conclude that terms such as 'antioxidative capacity' or 'antioxidative potency' are context-dependent. Furthermore, criteria of the efficacy of antioxidants based on oxidation in solution are not necessarily relevant to the effects of antioxidants on peroxidation in biological systems or model lipid assemblies, because the latter processes occur at water/lipid interfaces. We think that evaluation of antioxidants requires kinetic studies of the biomarker used and that the most relevant characteristic of 'oxidative stress' in the biological context is the kinetics of ex vivo peroxidation of lipids. We therefore propose studying the kinetics of lipid-peroxidation in the absence of the studied antioxidant and in its presence at different antioxidant concentrations. These protocols mean that antioxidants are assayed by methods commonly used to evaluate oxidative stress. The advantage of such evaluation is that it enables quantization of the antioxidants' efficacy in a model of relevance to biological systems. In view of the sensitivity of the lag time preceding peroxidation, we propose studying how much antioxidant is required to double the lag observed prior to rapid peroxidation. The latter quantity (C(2lag)) can be used to express the strength of antioxidants in the relevant system (e.g. LDL, serum or liposomes).     

ParSOC1, a MADS-box gene closely related to Arabidopsis AGL20/SOC1, is expressed in apricot leaves in a diurnal manner and is linked with chilling requirements for dormancy break

Dormancy break is a physiological phenomenon associated with the ability of plants to cope with changing environmental conditions and adjust their growth habits accordingly. In order to understand the potential role of genes in the control of dormancy break ParSOC1, a distinct apricot MADS-box gene which is most closely related to the AGL20/SOC1 MADS-box family was studied in several apricot cultivars that differ in their chilling requirements. The ParSOC1 gene is expressed in a diurnal manner and is highly polymorphic among apricot cultivars in the transcribed region upstream to the putative ATG translation initiation site. Genotyping of 48 different apricot cultivars revealed 13 different ParSOC1 alleles. By associating the chilling requirements of the apricot cultivars with their ParSOC1 genotype, it was possible to demonstrate a significant correlation between the presence of specific ParSOC1 alleles and chilling requirements. The data provided suggest that ParSOC1 or a gene in its close proximity could be involved in the regulation of dormancy break of vegetative shoots in apricot.     

Interleukin-1β Regulates Fat-Liver Crosstalk in Obesity by Auto-Paracrine Modulation of Adipose Tissue Inflammation and Expandability

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity.     

Blood pressure modulation following activation of mast cells by cationic cell penetrating peptides

Short cell penetrating peptides (CPP) are widely used in vitro to transduce agents into cells. But their systemic effect has not been yet studied in detail. We studied the systemic effect of the cell penetrating peptides, penetratin, transportan and pro-rich, on rat hemodynamic functions. Intra-arterial monitoring of blood pressure showed that injection of the positively charged penetratin and transportan in a wide range of concentrations (2.5-320 μg/kg) caused highly significant transient decrease in the systolic and diastolic blood pressure in a dose dependent manner (p<0.01). Pretreatment with histamine receptors blockers or with cromolyn, a mast cell stabilizing agent, significantly attenuated this effect. Furthermore, in vitro incubation of these both peptides with mast cells line, LAD2, caused a massive mast cell degranulation. In vitro studies showed that these CPP in a wide range of concentrations were not cytotoxic without any effect on the survival of LAD2 mast cell line. In contrast, the less positively charged and proline-rich CPP, pro-rich, had no systemic effects with no effect on mast cell degranulation. Our results indicate that intravenously administrated positively charged CPP may have deleterious consequences due to their induced BP drop, mediated by mast cell activation. Therefore, the major effect of mast cell activation on BP should be considered in developing possible future drug therapies based on the injection of membrane-permeable and positively charged CPP. Nevertheless, lower levels of such CPP may be considered as a treatment of systemic high BP through moderate systemic mast cell activation.     

Achieving broad molecular insights into dynamic protein interactions by integrated structural-kinetic approaches

A network of dynamic protein interactions with their protein partners, substrates, and ligands is known to be crucial for biological function. Revealing molecular and structural-based mechanisms at atomic resolution and in real-time is fundamental for achieving a basic understanding of cellular processes. These technically challenging goals may be achieved by combining time-resolved spectroscopic and structural-kinetic tools, thus providing broad insights into specific molecular events over a wide range of timescales. Here we review representative studies utilizing such an integrated real-time structural approach designed to reveal molecular mechanisms underlying protein interactions at atomic resolution.