TEM-E1: a novel β-lactamase conferring resistance to ceftazidime

A novel beta-lactamase, conferring resistance to ceftazidime, has been identified to be encoded by a 31 kb plasmid (pUK720) in a clinical E. coli strain isolated in Belgium. The beta-lactamase, new designated TEM-E1, has a pI of approximately 5.4 and lies in between the iso-electric focused bands of the beta-lactamases TEM-1 and TEM-7. The TEM-E1 beta-lactamase has a similar molecular weight of 22,000 to the TEM-1 and it is also inhibited by clavulanic acid. However, the TEM-E1 enzyme differs from TEM-1 by its low rates and efficiency of hydrolysis for ceftazidime and cefotaxime, TEM-E1 has similar efficiency of hydrolysis values for ceftazidime and cefotaxime, but only confers resistance to ceftazidime.     

The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O⁶-carboxymethylguanine (O⁶-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O⁶-CMG is not a substrate for the human version of the DNA damage reversal protein O⁶-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O⁶-alkylguanine lesions by removing alkyl groups from the O⁶-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O⁶-methylguanine (O⁶-MeG) or O⁶-CMG effectively inactivate MGMT in vitro (IC₅₀ 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O⁶-alkyl group and its transfer to the active-site cysteine residue of MGMT. O⁶-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O⁶-CMG is a potential causative agent.     

The effects of manufacturing tolerances and assembly force on the volumetric wear at the taper junction in modular total hip arthroplasty

Abstract

Fretting and corrosion at the taper-head interface in total hip arthroplasty has been reported as a potential cause of early failure of the implant system. The finite element (FE) method can be used to study the mechanics at the taper junction that are difficult to assess experimentally. Taper mismatch is one of the factors that can influence the performance of the taper junction. In this study we have assessed the effect of taper mismatch, in combination with assembly force on the volumetric wear. The study showed that higher assembly forces and smaller mismatches result in the least volumetric wear.     

Inhibition of neutrophil apoptosis by ATP is mediated by the P2Y11 receptor

Neutrophils undergo rapid constitutive apoptosis that is delayed by a range of pathogen- and host-derived inflammatory mediators. We have investigated the ability of the nucleotide ATP, to which neutrophils are exposed both in the circulation and at sites of inflammation, to modulate the lifespan of human neutrophils. We found that physiologically relevant concentrations of ATP cause a concentration-dependent delay of neutrophil apoptosis (assessed by morphology, annexin V/To-Pro3 staining, and mitochondrial membrane permeabilization). We found that even brief exposure to ATP (10 min) was sufficient to cause a long-lasting delay of apoptosis and showed that the effects were not mediated by ATP breakdown to adenosine. The P2 receptor mediating the antiapoptotic actions of ATP was identified using a combination of more selective ATP analogs, receptor expression studies, and study of downstream signaling pathways. Neutrophils were shown to express the P2Y11 receptor and inhibition of P2Y11 signaling using the antagonist NF157 abrogated the ATP-mediated delay of neutrophil apoptosis, as did inhibition of type I cAMP-dependent protein kinases activated downstream of P2Y11, without effects on constitutive apoptosis. Specific targeting of P2Y11 could retain key immune functions of neutrophils but reduce the injurious effects of increased neutrophil longevity during inflammation.     

Immunoexpression of aquaporins 1, 2, 3 and 4 in kidney of yak (Bos grunniens) on the Qinghai-Tibetan Plateau

Aquaporins (AQP) 1, 2, 3 and 4 belong to the aquaporin water channel family and play an important role in urine concentration by reabsorption of water from renal tubule fluid. Renal AQPs have not been reported in the yak (Bos grunniens), which resides in the Qinghai Tibetan Plateau. We investigated AQPs 1?4 expressions in the kidneys of Yak using immunohistochemical staining. AQP1 was expressed mainly in the basolateral and apical membranes of the proximal tubules and descending thin limb of the loop of Henle. AQP2 was detected in the apical plasma membranes of collecting ducts and distal convoluted tubules. AQP3 was located in the proximal tubule, distal tubule and collecting ducts. AQP4 was located in the collecting ducts, distal straight tubule, glomerular capillaries and peritubular capillaries. The expression pattern of AQPs 1?4 in kidney of yak was different from other species, which possibly is related to kidney function in a high altitude environment.     

Local photorelease of caged thymosin beta4 in locomoting keratocytes causes cell turning

The broad aim of this work was to explore the feasibility of using light-directed perturbation techniques to study cell locomotion. Specifically, a caged form of thymosin beta4 (Tbeta4) was photoactivated in a defined local region of locomoting fish scale keratocytes and the resulting perturbation of locomotion was studied. Purified Tbeta4 was produced in an inactive form by "caging" with ([n-nitroveratryl]oxy)chlorocarbamate. In vitro spectrophotofluorometric assays indicated that caged Tbeta4 did not change the normal actin polymerization kinetics, whereas photoactivated Tbeta4 significantly inhibited actin polymerization. With an a priori knowledge of the cytoplasmic diffusion coefficient of Tbeta4 as measured by fluorescence recovery after photobleaching experiments, the rapid sequestration of actin monomers by uncaged Tbeta4 and the consequent reduction in the diffusional spread of the Tbeta4-actin complex were predicted using Virtual Cell software (developed at the Center for Biomedical Imaging Technology, University of Connecticut Health Center). These simulations demonstrated that locally photoactivating Tbeta4 in keratocytes could potentially elicit a regional locomotory response. Indeed, when caged Tbeta4 was locally photoactivated at the wings of locomoting keratocytes, specific turning about the irradiated region was observed, whereas various controls were negative. Additionally, loading of exogenous Tbeta4 into both keratocytes and fibroblasts caused very rapid disassembly of actin filaments and reduction of cellular contractility. Based on these results, a mechanical model is proposed for the turning behavior of keratocytes in response to photoreleased Tbeta4.     

Detection of Dientamoeba fragilis in fresh stool specimens using PCR

Dientamoeba fragilis is a trichomonad parasite that causes human gastrointestinal disease. Currently microscopy is considered to be the gold standard for diagnosis of D. fragilis infection. However, this method is time-consuming and relatively insensitive. A PCR assay based on the small-subunit ribosomal RNA gene of D. fragilis for the specific detection of D. fragilis DNA in fresh unpreserved stool samples was developed. The D. fragilis PCR was positive in 29/31 samples with positive microscopy and did not cross-react with other protozoan parasites. The PCR protocol showed a specificity of 100% and a sensitivity of 93.5% and the entire procedure can be performed in one day.