Identification of basic residues involved in activation and calmodulin binding of rabbit smooth muscle myosin light chain kinase.

It is postulated that basic residues in the regulatory region of myosin light chain kinase are important for conferring autoinhibition by binding to the catalytic core. To investigate this proposal, 10 basic amino acids within the regulatory region of rabbit smooth muscle myosin light chain kinase (Lys961-Lys979) were replaced either singularly or in combination with acidic or nonpolar residues by site-directed mutagenesis. All active mutant kinases were dependent on Ca2+/calmodulin for catalytic activity. None of the mutants was active in the absence of Ca2+/calmodulin, suggesting that the autoinhibitory region has not been defined completely. Charge reversal mutants at Arg974, Arg975, and Lys976 resulted in loss of high affinity binding of calmodulin and increased the concentration of calmodulin required for half-maximal activation (KCaM). The charge reversal mutant at Lys979 also increased KCaM but to a lesser extent. Charge reversal mutants at Lys965 and Arg967 resulted in an inactive myosin light chain kinase that could not be proteolytically activated. When these residues were mutated to Ala, the expressed kinase was dependent upon Ca2+/calmodulin for activity and exhibited a decrease in KCaM. Charge reversal mutants in Lys961 and Lys962 also had decreased KCaM values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.     

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H₂O₂ (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P = 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H₂O₂ (P < 0.01), unexpectedly, exogenous gliotoxin relieved H₂O₂-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ΔgliK²⁶⁹³³ deletion mutant. The A. fumigatus ΔgliK²⁶⁹³³ mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ΔgliK²⁶⁹³³ mutant than in those of the wild type (P = 0.0024 [fold difference, 24] and P = 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ΔgliK²⁶⁹³³ mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P = 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK⁴⁶⁶⁴⁵ mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus.     

Effect of recovery method on yield of bovine oocytes per ovary and their developmental competence after maturation, fertilization and culture in vitro

An important aim of an oocyte recovery method is to maximize the number of oocytes per ovary which can be employed for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). In this study, primary bovine oocytes were collected by 2 methods: aspiration of visible follicles (2 to 6mm in diameter) or surface dissection in which the ovary surface is finely dissected. The oocytes were classified on the basis of cumulus cover and cytoplasmic appearance. The total number of oocytes and the yield of good-quality oocytes recovered per ovary by surface dissection and aspiration were 44.2 and 13.9 and 13.5 and 4.6 (P<0.05), respectively. When a sample group of selected oocytes recovered by each method was measured, no significant difference was found in the mean diameter (144.11m vs 142.54m). A representative sample of good-quality oocytes recovered by each method was put through the IVM/IVF/IVC procedure: no significant difference in cleavage rate, cleavage index or blastocyst yield was found. However, when the blastocyst yield was compared on a per ovary basis, a significant difference was observed in favor of surface dissection (3.30+/-0.46 vs 0.96+/-0.16;P<0.05). When unselected oocytes recovered by surface dissection of the ovaries were put through the standard embryo production system, an average of 15.4 blastocysts per dam was obtained.     

Heparan sulfate 2-O-sulfotransferase (Hs2st) and mouse development

Heparan sulphate 2-O-sulphotransferase (Hs2st) acts at an intermediate stage in the pathway of biosynthesis of heparan sulphate (HS), catalysing the transfer of sulphate from 3-phosphoadenosine-5-phosphosulfate (PAPS) to the C2-position of selected hexuronic acid residues within the maturing HS chain. It is well established that 2-O-sulphation within HS, particularly of iduronate residues, is essential for HS to participate in a variety of high-affinity ligand-binding interactions. HS plays a central role in embryonic development and cellular function, modulating the activities of an extensive range of growth factors. Interestingly, in contrast to the early failure of embryos entirely lacking HS, Hs2st ^–/– mice survive until birth, but die perinatally due to a complete failure of kidney formation. The phenotype of Hs2st ^–/– mutant kidneys suggests that signalling between two tissues, ureteric bud and metanephric mesenchyme, is disrupted. We discuss candidate signalling molecules that may mediate this interaction. The HS generated by these mice lacks 2-O-sulphate groups but is extensively modified above wild type levels by O-sulphation at C-6 of glucosamine-N-sulfate (GlcNS) residues. We will discuss the potentially altered role of this atypical HS in growth factor signalling. Published in 2003.     

Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.     

Assessing the effectiveness of a community intervention for monkeypox prevention in the Congo basin

Background

In areas where health resources are limited, community participation in the recognition and reporting of disease hazards is critical for the identification of outbreaks. This is particularly true for zoonotic diseases such as monkeypox that principally affect people living in remote areas with few health services. Here we report the findings of an evaluation measuring the effectiveness of a film-based community outreach program designed to improve the understanding of monkeypox symptoms, transmission and prevention, by residents of the Republic of the Congo (ROC) who are at risk for disease acquisition.

Methodology/Principal Findings

During 90 days, monkeypox outreach was conducted for ∼23,860 people in northern ROC. Two hundred seventy-one attendees (selected via a structured sample) were interviewed before and after participating in a small-group outreach session. The proportion of interviewees demonstrating monkeypox-specific knowledge before and after was compared. Significant gains were measured in areas of disease recognition, transmission, and mitigation of risk. The ability to recognize at least one disease symptom and a willingness to take a family member with monkeypox to the hospital increased from 49 and 45% to 95 and 87%, respectively (p<0.001, both). Willingness to deter behaviors associated with zoonotic risk, such as eating the carcass of a primate found dead in the forest, remained fundamentally unchanged however, suggesting additional messaging may be needed.

Conclusions/Significance

These results suggest that our current program of film-based educational activities is effective in improving disease-specific knowledge and may encourage individuals to seek out the advice of health workers when monkeypox is suspected.     

Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia

Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X_{1–5, 7}) was detected in all sampled equine tissues, whereas P2X₆ receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X_{1–3, 7} antibodies, and these were used in immunohistochemistry studies. P2X_{1–3, 7} receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X₃ subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X₂ and P2X₃ receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X_1–3 receptor subunits, whereas glial cells were positive for P2X₇ and P2X_{1 and 2} receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.     

A Quantitative Diffuse Reflectance Imaging (QDRI) System for Comprehensive Surveillance of the Morphological Landscape in Breast Tumor Margins

In an ongoing effort to address the clear clinical unmet needs surrounding breast conserving surgery (BCS), our group has developed a next-generation multiplexed optical-fiber-based tool to assess breast tumor margin status during initial surgeries. Specifically detailed in this work is the performance and clinical validation of a research-grade intra-operative tool for margin assessment based on diffuse optical spectroscopy. Previous work published by our group has illustrated the proof-of-concept generations of this device; here we incorporate a highly optimized quantitative diffuse reflectance imaging (QDRI) system utilizing a wide-field (imaging area = 17cm²) 49-channel multiplexed fiber optic probe, a custom raster-scanning imaging platform, a custom dual-channel white LED source, and an astronomy grade imaging CCD and spectrograph. The system signal to noise ratio (SNR) was found to be greater than 40dB for all channels. Optical property estimation error was found to be less than 10%, on average, over a wide range of absorption (μ_a = 0–8.9cm^-1) and scattering (μ_s’ = 7.0–9.7cm^-1) coefficients. Very low inter-channel and CCD crosstalk was observed (2% max) when used on turbid media (including breast tissue). A raster-scanning mechanism was developed to achieve sub-pixel resolution and was found to be optimally performed at an upsample factor of 8, affording 0.75mm spatially resolved diffuse reflectance images (λ = 450–600nm) of an entire margin (area = 17cm²) in 13.8 minutes (1.23cm²/min). Moreover, controlled pressure application at the probe-tissue interface afforded by the imaging platform reduces repeated scan variability, providing <1% variation across repeated scans of clinical specimens. We demonstrate the clinical utility of this device through a pilot 20-patient study of high-resolution optical parameter maps of the ratio of the β-carotene concentration to the reduced scattering coefficient. An empirical cumulative distribution function (eCDF) analysis is used to reduce optical property maps to quantitative distributions representing the morphological landscape of breast tumor margins. The optimizations presented in this work provide an avenue to rapidly survey large tissue areas on intra-operative time scales with improved sensitivity to regions of focal disease that may otherwise be overlooked.