The relationship between concentration of a dual marker strain of Salmonella Typhimurium in bovine faeces and its probability of detection by immunomagnetic separation and culture

AIMS: To modify a strain of Salmonella serotype Typhimurium to express unique marker traits and then define how the concentration of the marker in bovine faeces affects the probability of its detection by culture preceded by immunomagnetic separation (IMS). METHODS AND RESULTS: DNA encoding for the production of green fluorescent protein (gfp) and resistance to kanamycin was inserted into the bacterial chromosome of Salm. Typhimurium. Transposon insertion was demonstrated by Southern blot hybridization. Varying amounts of one electroporant (gfpSal-1) were inoculated into suspensions of bovine faeces and attempts made to isolate gfpSal-1 using a protocol based on pre-enrichment incubation, IMS and enrichment in selective media. Isolates of gfpSal-1 were differentiated from wild strains of Salmonella using fluorescence under u.v. light and expression of kanamycin resistance. A logistic and Gompertz function each derived from the dose-response data partially explained the observations with the fit of the Gompertz function judged to be superior. The 10, 50 and 90% limits of detection from the Gompertz function were estimated to be 1.92, 2.03 and 2.27 CFU g(-1) respectively. CONCLUSIONS: Reliance on the traditional concept of 'limit of detection' could introduce unacceptable errors in the interpretation of test findings when the concentration of Salm. Typhimurium in bovine faeces (pooled or individual) is below ca 3 CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The dose-response curve can be used to aid the design of protocols for detecting Salmonella in individual and pooled faecal specimens. The experiments demonstrate that both reporter genes in tandem are useful for studying the performance of culture-based methods for detecting pathogens in faeces.     

Inactivation of a testis-specific Lis1 transcript in mice prevents spermatid differentiation and causes male infertility

Lis1 protein is the non-catalytic component of platelet-activating factor acetylhydrolase 1b (PAF-AH 1B) and associated with microtubular structures. Hemizygous mutations of the LIS1 gene cause type I lissencephaly, a brain abnormality with developmental defects of neuronal migration. Lis1 is also expressed in testis, but its function there has not been determined. We have generated a mouse mutant (LIS1GT/GT) by gene trap integration leading to selective disruption of a Lis1 splicing variant in testis. Homozygous mutant males are infertile with no other apparent phenotype. We demonstrate that Lis1 is predominantly expressed in spermatids, and spermiogenesis is blocked when Lis1 is absent. Mutant spermatids fail to form correct acrosomes and nuclei appear distorted in size and shape. The tissue architecture in mutant testis appears severely disturbed displaying collapsed seminiferous tubules, mislocated germ cells, and increased apoptosis. These results provide evidence for an essential and hitherto uncharacterized role of the Lis1 protein in spermatogenesis, particularly in the differentiation of spermatids into spermatozoa.     

Hoechst 33258 binds to G-quadruplex in the promoter region of human c-myc

In vitro binding of Hoechst 33258 to the promoter region of human c-myc, d(GG GGAGGG TGG GGA GGG TGG GGA AGG TGG GG) which forms G-quadruplex, both in vitro and in vivo in the presence of metal ions, was investigated by equilibrium absorption, fluorescence, and kinetic surface plasmon resonance methods. Hypochromic effect in UV absorption spectra and blue shift in fluorescence emission maxima of Hoechst in the presence of quadruplex revealed that Hoechst binds to the quadruplex. Analysis of UV and fluorescence titration data revealed that Hoechst binds to quadruplex with binding affinity of the order of 10(6). Anisotropy measurements and higher lifetime obtained from time-resolved decay experiments revealed that quadruplex-bound Hoechst is rotationally restricted in a less polar environment than the bulk buffer medium. From surface plasmon resonance studies, we obtained kinetic association (k(a)) and dissociation (k(d)) of 1.23+/-0.04 x 10(5)M(-1)s(-1) and 0.686+/-0.009 s(-1), respectively. As Hoechst is known to bind A-T-rich region of duplex DNA, here we propose the likelihood of Hoechst interacting with the AAGGT loop of the quadruplex.     

The anomalous pKa of Tyr-9 in glutathione S-transferase A1-1 catalyzes product release

The pKa of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to approximately 8.1 in the apoenzyme and approximately 9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pKa has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pKa values for the conformational transition were 7.9 +/- 0.1 and 9.3 +/- 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pKa for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pKa values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pKa of Tyr-9. In contrast, the Y9F/W21F/F222W mutant had no pH-dependent transition in KD or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of "open" and "closed" states of the C terminus are not determined solely by the ionization state of Tyr-9. However, the rates of transition between these states are faster for the ionized Tyr-9. The ionized Tyr-9 states provide a parallel pathway for product dissociation, which is kinetically and thermodynamically favored. In silico kinetic models further support the functional role for the parallel dissociation pathway provided by ionized Tyr-9.     

A nanosecond molecular dynamics study of antiparallel d(G)7 quadruplex structures: effect of the coordinated cations

Nanosecond scale molecular dynamics simulations have been performed on antiparallel Greek key type d(G7) quadruplex structures with different coordinated ions, namely Na+ and K+ ion, water and Na+ counter ions, using the AMBER force field and Particle Mesh Ewald technique for electrostatic interactions. Antiparallel structures are stable during the simulation, with root mean square deviation values of approximately 1.5 A from the initial structures. Hydrogen bonding patterns within the G-tetrads depend on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate different cations. However, alternating syn-anti arrangement of bases along a chain as well as in a quartet is maintained through out the MD simulation. Coordinated Na+ ions, within the quadruplex cavity are quite mobile within the central channel and can even enter or exit from the quadruplex core, whereas coordinated K+ ions are quite immobile. MD studies at 400K indicate that K+ ion cannot come out from the quadruplex core without breaking the terminal G-tetrads. Smaller grooves in antiparallel structures are better binding sites for hydrated counter ions, while a string of hydrogen bonded water molecules are observed within both the small and large grooves. The hydration free energy for the K+ ion coordinated structure is more favourable than that for the Na+ ion coordinated antiparallel quadruplex structure.     

Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves. In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17- and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb.     

Role of chromium supplementation in Indians with type 2 diabetes mellitus

Type 2 diabetes mellitus is a complex metabolic disorder with adverse cardiovascular risk. The role of micronutrients has not yet been well clarified in this condition, especially in India.THE OBJECTIVES OF THIS STUDY WERE TO: (1) evaluate chromium status in Indian subjects with type 2 diabetes mellitus, (2) assess the effect of chromium picolinate (200 &mgr;g trivalent chromium twice daily) administration on glycaemic control and lipid profile in these subjects and (3) comment on the possible mechanism of any beneficial effect noted above.Fifty subjects were studied in a double blind, placebo-controlled, crossover fashion, with each treatment arm (chromium/placebo) lasting 12 weeks and 4 weeks' wash-off period in between. 50 healthy age- and sex-matched volunteers served as controls. Serum chromium level appeared to be higher in the general population in our country compared to western countries (36.5-59.5 nmol/L as compared to 2.3-40.3 nmol/L) However, the local diabetics were found to have a lower serum chromium level than the healthy controls (32.3 nmol/L against 44.7 nmol/L; p < 0.0001) and a mean increase of 3.5 nmol/L was noted after 12 weeks of chromium supplementation that was, expectedly, not seen in the placebo phase (p < 0.0001).Significant improvement in glycaemic control was noted in the chromium-treated group (DeltaFasting serum glucose = 0.44 mmol/L, p < 0.001; DeltaPost-prandial serum glucose = 1.97 mmol/L, p < 0.001; Deltaglycated hemoglobin = 0.01; p = 0.04, in comparison to placebo) This was accompanied by a significant greater fall in fasting serum insulin in the chromium-treated group, p < 0.05.The change in lipid parameters (total serum cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol and triglycerides) did not show significant difference between the chromium and placebo groups.Clinically significant hematological, renal or hepatic toxicity were excluded by routine hemogram, serum urea, creatinine, alanine amino transferase (ALT) and alkaline phosphatase estimations.In conclusion, chromium supplementation seems to improve glycaemic control in type 2 diabetic patients, which appears to be due to an increase in insulin action rather than stimulation of insulin secretion.     

Beverage specific alcohol intake in a population-based study: Evidence for a positive association between pulmonary function and wine intake

Background  

Lung function is a strong predictor of cardiovascular and all-cause mortality. Previous studies suggest that alcohol exposure may be linked to impaired pulmonary function through oxidant-antioxidant mechanisms. Alcohol may be an important source of oxidants; however, wine contains several antioxidants. In this study we analyzed the relation of beverage specific alcohol intake with forced expiratory volume in one second (FEV₁) and forced vital capacity (FVC) in a random sample of 1555 residents of Western New York, USA.     

Synthesis and testing of 5-benzyl-2,4-diaminopyrimidines as potential inhibitors of leishmanial and trypanosomal dihydrofolate reductase

Dihydrofolate reductase is a drug target that has not been thoroughly investigated in leishmania and trypanosomes. Work has previously shown that 5-benzyl-2,4-diaminopyrimidines are selective inhibitors of the leishmanial and trypanosome enzymes. Modelling predicted that alkyl/aryl substitution on the 6-position of the pyrimidine ring should increase enzyme activity of 5-benzyl-2,4-diaminopyrimidines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Various compounds were prepared and evaluated against both the recombinant enzymes and the intact organisms. The presence of a substituent had a small or negative effect on activity against the enzyme or intact parasites compared to unsubstituted compounds.