Gender specific LRP5 influences on trabecular bone structure and strength

A mutation in LRP5 (low-density lipoprotein receptor-related protein 5) has been shown to increase bone mass and density in humans and animals. Transgenic mice expressing the LRP5 mutation (G171V) demonstrate an increase in bone mass as compared to non-transgenic (NTG) littermates. This study evaluated LRP5 gene and gender-related influences on the structural and biomechanical strength properties of trabecular and cortical bone in femurs and vertebrae (L5) of 17-week-old mice. Micro-computed tomography was used to evaluate the trabecular bone structure of distal femurs and vertebrae ex vivo. Mechanical testing of the trabecular bone in the distal femur was done to determine biomechanical strength. Differences due to genotype and gender were tested using two-way ANOVA at a significance level of p<0.05. Trabecular bone structural parameters (BV/TV, trabecular thickness, number, etc.) at the distal femur, femoral neck, and vertebral body sites were greater in the transgenic as compared to the NTG mice. In addition, vertebral cortical thickness and trabecular strength parameters (ultimate and yield loads, stiffness, ultimate and yield stresses) in the distal femur were greater in the transgenic mice as compared to NTG. The increasing trends of cortical thickness were also noted in the transgenic mice as compared to NTG. Within LRP5 (G171V) mutant mice, there were significant gender-related differences in some of the trabecular bone structural parameters at all the sites (distal femur, femoral neck, and vertebral body). However, unlike trabecular structural parameters, the gender-specific differences were not found in the trabecular strength of LRP5 transgenic mice. In summary, these findings suggest that the LRP5 (G171V) mutation results in greater trabecular bone structure and strength at both the distal femurs and vertebral bodies as compared to NTG. In addition, only the trabecular structure parameters were affected by gender within the LRP5 (G171V) mutation.     

Structural insights into the recognition of substrates and activators by the OSR1 kinase

The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signalling cascade regulating the activity of Na(+)/K(+)/2Cl(-) co-transporters (NKCCs) in response to osmotic stress. Both kinases have a conserved carboxy-terminal (CCT) domain, which recognizes a unique peptide (Arg-Phe-Xaa-Val) motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as its substrates (NKCC1 and NKCC2). Here, we describe the structural basis of this recognition event as shown by the crystal structure of the CCT domain of OSR1 in complex with a peptide containing this motif, derived from WNK4. The CCT domain forms a novel protein fold that interacts with the Arg-Phe-Xaa-Val motif through a surface-exposed groove. An intricate web of interactions is observed between the CCT domain and an Arg-Phe-Xaa-Val motif-containing peptide derived from WNK4. Mutational analysis shows that these interactions are required for the CCT domain to bind to WNK1 and NKCC1. The CCT domain structure also shows how phosphorylation of a Ser/Thr residue preceding the Arg-Phe-Xaa-Val motif results in a steric clash, promoting its dissociation from the CCT domain. These results provide the first molecular insight into the mechanism by which the SPAK and OSR1 kinases specifically recognize their upstream activators and downstream substrates.     

Screening methods used to determine the anti-microbial properties of Aloe vera inner gel

The emergence of antibiotic resistant bacterial strains is a growing problem and is an important concern for patients, physicians, healthcare managers, and policymakers as it results in poorer health and economic outcomes. This has led to an urgent global call for new antimicrobial drugs, particularly from natural resources. We have been studying the antimicrobial properties of the inner leaf gel component of Aloe barbadensis Miller and have used a number of different, simple in vitro assays to establish a scientific basis for the potential use of Aloe vera on a range of clinically relevant bacteria. The bacteria used include Shigella flexneri, Methicillin-Resistant Staphylococcus aureus (MRSA), Enterobacter cloacae and Enterococcus bovis. In this paper, we compare standard methods recommended by the Clinical and Laboratory Standards Institute (CLSI) with a microtitre assay using a metabolic colour indicator Alamar blue. All the techniques described have shown that Aloe vera has an antimicrobial effect, however, the microtitre assay enables high throughput screening, under similar conditions and is less wasteful of plant material.     

Dexamethasone attenuates acute macromolecular efflux increase evoked by smokeless tobacco extract.

The purpose of this study was to determine whether dexamethasone attenuates the acute increase in macromolecular efflux from the oral mucosa elicited by an aqueous extract of smokeless tobacco (STE) in vivo, and, if so, whether this response is specific. Using intravital microscopy, we found that 20-min suffusion of STE elicited significant, concentration-related leaky site formation and an increase in clearance of fluorescein isothiocyanate-labeled dextran (FITC-dextran; mol mass 70 kDa) from the in situ hamster cheek pouch (P < 0.05). This response was significantly attenuated by dexamethasone (10 mg/kg iv). Dexamethasone also attenuated the bradykinin-induced leaky site formation and the increase in clearance of FITC-dextran from the cheek pouch. However, it had no significant effects on adenosine-induced responses. Dexamethasone had no significant effects on baseline arteriolar diameter and on bradykinin-induced vasodilation in the cheek pouch. Collectively, these data indicate that dexamethasone attenuates, in a specific fashion, the acute increase in macromolecular efflux from the in situ oral mucosa evoked by short-term suffusion of STE. We suggest that corticosteroids mitigate acute oral mucosa inflammation elicited by smokeless tobacco.     

Time course for bone formation with long-term external mechanical loading.

Increased mechanical loading of bone with the rat tibia four-point bending device stimulates bone formation on periosteal and endocortical surfaces. With long-term loading cell activity diminishes, and it has been reported that early gains in bone size may reverse. This study examined the time course for bone cellular and structural response after 6, 12, and 18 wk of loading at 1,200-1, 700 microstrain (muepsilon). Bone formation rates, measured by histomorphometry, were compared within groups, between loaded and contralateral nonloaded tibiae, and between weeks. Formation surface, mineral apposition rate, and bone formation rate on periosteal and endocortical surfaces were elevated after 6 wk of loading. By 12 wk of loading, periosteal and endocortical formation surface and endocortical mineral apposition rates were elevated. By 18 wk of loading, periosteal adaptation appeared complete, whereas endocortical mineral apposition rate remained elevated. No periosteal resorption was observed. Average thickness of new bone formed, from baseline to collection, was greater in loaded than nonloaded tibiae by week 6 and was maintained through week 18. Early increases in bone formation result in periosteal apposition of new bone that persists after formation ceases.     

Tyrosine kinase inhibitors modulate agonist-induced vasodilation in the hamster cheek pouch.

The purpose of this study was to determine whether inhibitors of tyrosine kinase attenuate vasodilation elicited by endogenously elaborated and exogenously applied nitric oxide in the in situ peripheral microcirculation. Using intravital microscopy, we found that pretreatment with genistein (1.0 microM) and tyrphostin 25 (10.0 microM), two structurally unrelated tyrosine kinase inhibitors, significantly attenuated acetylcholine-, bradykinin- and nitroglycerin-induced dilation of second-order arterioles (51 +/- 1 microm) in the in situ hamster cheek pouch (P < 0.05). Both inhibitors nearly abrogated acetylcholine-induced responses but only partially blocked bradykinin- and nitroglycerin-induced vasodilation. Genistein and tyrphostin 25 alone had no significant effects on resting arteriolar diameter and on adenosine-induced vasodilation in the cheek pouch. On balance, these data indicate that tyrosine kinase inhibitors attenuate endogenously elaborated and exogenously applied nitric oxide-induced vasodilation in the in situ hamster cheek pouch. However, the extent of tyrosine kinase inhibitor-sensitive pathway involvement in this response appears to be agonist dependent.     

Evidence for peroxynitrite formation during S-nitrosoglutathione photolysis in air saturated solutions

Flash photolysis of S-nitrosoglutathione (GSNO) in aerated solutions at pH 10 gave rise to an absorption with a maximum around 310-320 nm. This peak is spectrally similar to that displayed by ONOO-. The decay kinetics of this absorption was compared to that of authentic ONOO-, generated independently. An excellent correlation was obtained. Further proof of ONOO- generation was provided by HPLC studies showing the production of 3-nitrotyrosine on irradiation of GSNO in the presence of tyrosine at pH 7.4. In addition, the nitration yield was increased approximately 5-fold in the presence of bicarbonate and totally eliminated with DMPO, indicating the requirement of a radical intermediate for peroxynitrite production during S-nitrosothiol photolysis.     

Use of Internal Transcribed Spacer Primers and Fungicide Treatments to Study the Anther-Smut Disease, Microbotryum violaceum (=Ustilago violacea), of White Campion Silene alba (=Silene latifolia)

We report the construction and use of polymerase chain reaction (PCR) primers for detection of Microbotryum violaceum in Silene alba. Such primers show that the pathogen is present in the aboveground bud meristems, flower pedicels, and flower-stem internodes but not in the flowering-stem leaves and roots of flowering plants. Use of primers also showed that the pathogen was absent from plants that had been inoculated with the fungus but remained healthy. The fungicides Folicur and Bayleton cured plants of the disease, and the absence of the fungus from such cured plants was confirmed using the PCR primers.     

Drought Tolerance in Mung Bean is Associated with the Genotypic Divergence,Regulation of Proline,Photosynthetic Pigment and Water Relation

Drought is one of the critical conditions for the growth and productivity of many crops including mung bean (Vigna radiata L. Wilczek). Screening of genotypes for variations is one of the suitable strategies for evaluating crop adaptability and global food security. In this context, the study investigated the physiological and biochemical responses of four drought tolerant (BARI Mung-8, BMX-08010-2, BMX-010015, BMX-08009-7), and four drought sensitive (BARI Mung-1, BARI Mung-3, BU Mung-4, BMX-05001) mung bean genotypes under wellwatered (WW) and water deficit (WD) conditions. The WW treatment maintained sufficient soil moisture (22% ± 0.5%, i.e., 30% deficit of available water) by regularly supplying water. Whereas, the WD treatment was maintained throughout the growing period, and water was applied when the wilting symptom appeared. The drought tolerant (DT) genotypes BARI Mung-8, BMX-08010-2, BMX-010015, BMX-08009-7 showed a high level of proline accumulation (2.52–5.99 mg g⁻¹ FW), photosynthetic pigment (total chlorophyll 2.96–3.27 mg g⁻¹ FW at flowering stage, and 1.62–2.38 mg g⁻¹ FW at pod developing stage), plant water relation attributes including relative water content (RWC) (82%–84%), water retention capacity (WRC) (12–14) as well as lower water saturation deficit (WSD) (19%–23%), and water uptake capacity (WUC) (2.58–2.89) under WD condition, which provided consequently higher relative seed yield. These indicate that the tolerant genotypes gained better physiobiochemical attributes and adaptability in response to drought conditions. Furthermore, the genotype BMX- 08010-2 showed superiority in terms of those physio-biochemical traits, susceptibility index (SSI) and stress tolerance index (STI) to other genotypes. Based on the physiological and biochemical responses, the BMX-08010-2 was found to be a suitable genotype for sustaining yield under drought stress, and subsequently, it could be recommended for crop improvement through hybridization programs. In addition, the identified traits can be used as markers to identify tolerant genotypes for drought-prone areas.