Substitution of Alanine at Position 184 with Glutamic Acid in <Emphasis Type="Italic">Escherichia coli</Emphasis> PBP5 Ω-Like Loop Introduces a Moderate Cephalosporinase Activity

Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic β-lactam resistance. Though PBP5 does not have β-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A β-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in β-lactamases. It is speculated that β-lactamases and DD-CPases might have undergone divergent evolution leading to distinct enzymes with different substrate specificities and functions indicating the versatility of the Ω-loops. Nonetheless, direct experimental evidence favoring the idea is insufficient. Here, aiming to investigate the effect of introducing a glutamic acid residue in the PBP5 Ω-like loop, we substituted A184 to E to create PBP5_A184E. Expression of PBP5_A184E in E. coli ?PBP5 mutant elevates the β-lactam resistance, especially for cephalosporins. However, like PBP5, PBP5_A184E has the ability to complement the aberrantly shaped E. coli septuple PBP mutant indicating an unaffected in vivo DD-CPase activity. Biochemical and bioinformatics analyses have substantiated the dual enzyme nature of the mutated enzyme possessing both DD-CPase and β-lactamase activities. Therefore, substitution of A184 to E of Ω-like loop alone can introduce the cephalosporinase activity in E. coli PBP5 supporting the phenomenon of a single amino acid polymorphism.     

Trace amounts of enhancing factor/phospholipase A2 in mouse peritoneal exudate cells

Enhancing factor (EF), a mouse phospholipase A₂ (PLA₂), has been purified from the small intestines, based on its ability to increase the binding of epidermal growth factor in a radioreceptor assay. EF/PLA₂ was found to be localized predominantly in the Paneth cells in the small intestines. Whether mouse intestinal EF/PLA₂ is identical/similar to mouse secretory PLA₂ was to be determined. Phospholipases are known to play a crucial role in the process of inflammation. This paper reports the presence of trace amounts of EF/PLA₂ in the peritoneal exudate cells. Western blot analysis of the acid extracts showed the presence of a 14 kDa immunologically cross-reactive protein. RT-PCR analysis using EF specific primers amplified a ∼700 bp product which was further confirmed to be EF-specific by nested PCR analysis and sequencing. Presence of EF in the peritoneal exudate cells could be a unique mode of transport of growth factor modulator to the site of injury to aid in regeneration/cell proliferation of damaged tissue.     

BDNF and trkB mRNA Expression in Neurons of the Neonatal Mouse Barrel Field Cortex: Normal Development and Plasticity after Cauterizing Facial Vibrissae

Development of the central somatosensory system is profoundly modulated by the sensory periphery. Cauterization of facial whiskers alters the segregation pattern of barrels in rodents only during a few days just after birth (critical period). Although a molecular basis of the segregation of barrel neurons and the critical period for the anatomical plasticity observed in layer IV barrel neuron is not clear yet, the accumulating evidence suggests that neurotrophins modulate synaptic connections including central nervous system. In this study, we showed by in situ hybridization that mouse barrel side neurons express brain-derived neurotrophic factor (BDNF) mRNA and both catalytic and non-catalytic forms of trkB mRNA. Cautery of row C vibrissae on the right side of the face within 24 h after birth (post natal day 0, PND0) reduced the expression of BDNF and trkB mRNA from the division region between the contralateral row C barrels at PND7. The vibrissae in row A, C, and E were cauterized at PND0 followed by quantitative RT-PCR for BDNF and trkB mRNA with total RNA isolated from the barrel region at PND7. The result showed that BDNF, but not trkB, mRNA was increased several-fold in the contralateral barrel region. These data suggest that the expression of BDNF mRNA is differentially regulated between injured barrels and actively innervated barrels. The differential expression of the mRNA encoding neurotrophins and their receptors may be important in regulating the injury-dependent re-segregation of barrels.     

Hemopexin increases the neurotoxicity of hemoglobin when haptoglobin is absent

Hemopexin (Hpx) binds heme with extraordinary affinity, and after haptoglobin may provide a second line of defense against the toxicity of extracellular hemoglobin (Hb). In this series of experiments, the hypothesis that Hpx protects neurons from Hb neurotoxicity was evaluated in murine primary cultures containing neurons and glial cells. Contrary to hypothesis, Hpx increased neuronal loss due to micromolar concentrations of Hb by 4‐ to 12‐fold, as measured by LDH release assay; conversely, the neurotoxicity of hemin was completely prevented. The endogenous fluorescence of Hpx was quenched by Hb, consistent with transfer of Hb‐bound heme to Hpx. This was associated with precipitation of globin chains, as detected by immunostaining and fluorescent Hb labeling. A portion of this precipitate attached firmly to cells and could not be removed by multiple washes. Concomitant treatment with haptoglobin (Hp) prevented globin precipitation and most of the increase in neuronal loss. Hpx weakly attenuated the increase in culture non‐heme iron produced by Hb treatment, quantified by ferrozine assay. However, Hb‐Hpx toxicity was iron‐dependent, and was blocked by deferoxamine and ferrostatin‐1. Up‐regulation of cell ferritin expression, a primary cell defense against Hb toxicity, was not observed on western blots of culture lysates that had been concomitantly treated with Hpx. These results suggest that Hpx destabilizes Hb in the absence of haptoglobin, leading to globin precipitation and exacerbation of iron‐dependent oxidative cell injury. Combined therapy with hemopexin plus haptoglobin may be preferable to hemopexin alone after CNS hemorrhage.

     

Patch depression in grazers: the roles of biomass distribution and residual stems

1. In this paper the results are presented of two experiments designed to examine the relationship between sward structure variables and the nature of patch depression experienced by a large grazing herbivore, Bos taurus, grazing from small patches of forage. In experiment 1 the spatial distribution of leaves within patches was varied. In experiment 2 the role of reproductive stems of different lengths as potential intake rate inhibitors was examined.
2. Both experiments were conducted by offering cattle hand-constructed swards of Orchardgrass ( Dactylis glomerata ) leaves attached to plywood boards. Each of three experimental animals was allowed to take 10, 20, 30, 45 or 60 bites from a patch and intake was estimated on the basis of pregrazing and postgrazing dry-matter. All trials were videotaped and the trial length in seconds was determined from playback of the tapes.
3. In both experiments patch depression (reduction in instantaneous intake rate) was due to decreases in bite mass concomitant with patch depletion. Handling time per bite was unaffected by depletion level. In experiment 1, different spatial configurations resulted in markedly different gain functions ranging from linear to sigmoid. In experiment 2, the presence in the sward of long (15 cm) reproductive stems slowed cumulative gains by restricting bite mass and by increasing the per-bite handling time relative to patches with shorter (5 cm) or no stems.
4. The study of patch depression mechanisms now needs to be generalized over a range of herbivore body sizes and feeding styles.     

Specific 50'CpG island methylation signatures of FHIT and p16 genes and their potential diagnostic relevance in Indian breast cancer patients

Even after tremendous molecular studies, early detection,more accurate and sensitive diagnosis, and prognosis of breast cancer appear to be a riddle so far. To stab the enigma, this study is designed to envisage DNA methylation signatures as cancer-specific and stage-specific biomarkers in Indian patients. Rigorous review of scattered scientific reports on aberrant DNA methylation helped us to select and analyze a potential tumor suppressor gene pair (FHIT and p16 genes) in breast cancer patients. Methylation signatures from 232 primary sporadic breast cancer patients were pinpointed by methylation-specific PCR (MSP). To increase the sensitivity, we combined both MSP and expression studies (RT-PCR and Northern blotting) in a reproducible manner. Statistical analysis illustrated that hypermethylation of FHIT gene ( p < 0.0001) and p16 gene ( p=0.04) may be used as a potential diagnostic marker to diagnose the early and locally advanced stages of breast cancer. Additionally, the study authenticates the dependency of methylation and expressional loss of p16 gene on FHIT gene silencing. This observation not only describes the severity of disease when both genes are silenced but also drives to speculate the molecular cross talk between two genes or genetic pathways dictated by them separately.     

Genetic and morphological characterisation of the Ankole Longhorn cattle in the African Great Lakes region

The study investigated the population structure, diversity and differentiation of almost all of the ecotypes representing the African Ankole Longhorn cattle breed on the basis of morphometric (shape and size), genotypic and spatial distance data. Twentyone morphometric measurements were used to describe the morphology of 439 individuals from 11 sub-populations located in five countries around the Great Lakes region of central and eastern Africa. Additionally, 472 individuals were genotyped using 15 DNA microsatellites. Femoral length, horn length, horn circumference, rump height, body length and fore-limb circumference showed the largest differences between regions. An overall F_ST index indicated that 2.7% of the total genetic variation was present among sub-populations. The least differentiation was observed between the two sub-populations of Mbarara south and Luwero in Uganda, while the highest level of differentiation was observed between the Mugamba in Burundi and Malagarasi in Tanzania. An estimated membership of four for the inferred clusters from a model-based Bayesian approach was obtained. Both analyses on distance-based and model-based methods consistently isolated the Mugamba sub-population in Burundi from the others.     

Expression of Dm-AMP1 in rice confers resistance to Magnaporthe oryzae and Rhizoctonia solani

Magnaporthe oryzae and Rhizoctonia solani, are among the most important pathogens of rice, severely limiting its productivity. Dm-AMP1, an antifungal plant defensin from Dahlia merckii, was expressed in rice (Oryza sativa L. sp. indica cv. Pusa basmati 1) using Agrobacterium tumefaciens-mediated transformation. Expression levels of Dm-AMP1 ranged from 0.43% to 0.57% of total soluble protein in transgenic plants. It was observed that constitutive expression of Dm-AMP1 suppresses the growth of M. oryzae and R. solani by 84% and 72%, respectively. Transgenic expression of Dm-AMP1 was not accompanied by an induction of pathogenesis-related (PR) gene expression, indicating that the expression of DmAMP1 directly inhibits the pathogen. The results of in vitro, in planta and microscopic analyses suggest that Dm-AMP1 expression has the potential to provide broad-spectrum disease resistance in rice.     

A role for the 30S subunit E site in maintenance of the translational reading frame

The exit (E) site has been implicated in several ribosomal activities, including translocation, decoding, and maintenance of the translational reading frame. Here, we target the 30S subunit E site by introducing a deletion in rpsG that truncates the β-hairpin of ribosomal protein S7. This mutation (S7ΔR77–Y84) increases both −1 and +1 frameshifting but does not increase miscoding, providing evidence that the 30S E site plays a specific role in frame maintenance. Mutation S7ΔR77–Y84 also stimulates +1 programmed frameshifting during prfB′-lacZ translation in many synthetic contexts. However, no effect is seen when the E codon of the frameshift site corresponds to those found in nature, suggesting that E-tRNA release does not normally limit the rate of prfB frameshifting. Ribosomes containing S7ΔR77–Y84 exhibit an elevated rate of spontaneous reverse translocation and an increased K_1/2 for E-tRNA. These effects are of similar magnitude, suggesting that both result from destabilization of E-tRNA. Finally, this mutation of the 30S E site does not inhibit EF-G-dependent translocation, consistent with a primary role for the 50S E site in the mechanism.