Structural Bases for Stability-Function Tradeoffs in Antibiotic Resistance

Preorganization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein; consequently, enzymes can be dramatically stabilized by substitutions that attenuate the size and preorganization “strain” of the active site. How this stability-activity tradeoff constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities or how these new activities manifest structurally. These questions are both germane and easy to study in β-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of β-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C β-lactamases, which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100-fold and 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability (from 1.7 kcal mol^− 1 to 4.1 kcal mol^− 1), consistent with the stability-function hypothesis. Intriguingly, several of the substitutions were 10-20 Å from the catalytic serine; the question of how they conferred extended-spectrum activity arose. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by X-ray crystallography. Distinct mechanisms of action, including changes in the flexibility and ground-state structures of the enzyme, are revealed for each mutant. These results explain the structural bases for the antibiotic resistance conferred by these substitutions and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.     

Evaluation of Hypoglycemic Activity of Inorganic Constituents in Nelumbo nucifera Seeds on Streptozotocin-Induced Diabetes in Rats

The seeds of Nelumbo nucifera (Lotus) have been used in the traditional system of medicine for various ailments including diabetes. The present study was aimed at analyzing the levels of biologically important trace elements in the lotus seeds by atomic absorption spectroscopy and evaluating the hypoglycemic properties of seed ash on streptozotocin-induced diabetes in rats. Diabetic rats treated with lotus seed ash at a concentration of 200 mg/kg body weight orally for 30 days exhibited significant hypoglycemic activity. The presence of trace elements in appreciable amounts in the seeds may play a direct or indirect role on insulin secretion or its action in a synergetic manner. The hypoglycemic activity of the ash was comparable with glyclazide. The role of trace elements in disorders related to diabetes is also discussed briefly.     

Activities of enzymes of fermentation pathways in the leaves and roots of contrasting cultivars of sorghum (Sorghum Bicolor L.) during flooding

Flooding evoked a differential response in the activities of enzymes of fermentation pathway in leaves and roots of flood sensitive (S-308) and flood-tolerant (SSG-59-3) cultivars of sorghum. Activities of alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH) and alanine aminotransferase (AlaAT) enhanced in roots of SSG-59-3 during 72 h of flooding. In contrast, a transient increase in the activities was discerned in roots of S-308 up to 24 h flooding followed by a decline in activities of these enzymes. In leaves of SSG-59-3, the activities of ADH and LDH increased to about three fold during flooding stress as compared to that in the non-flooded control plants. Though elevation in activities of these enzymes was observed in leaves of S-308 up to 48 h of flooding, the magnitude of enhancement was much lower than that in SSG-59-3. Alanine aminotranferase activity depressed in leaves of both the cultivars but the level of decline was more pronounced in sensitive cultivar S-308 as compare to tolerant SSG-59-3. The amount of alcohol, lactic acid and alanine were higher in both roots and leaves of SSG-59-3 than that in S-308 during flooding stress. It is thus apparent that roots and leaves of flood tolerant variety tends to attain greater capacity to perform reactions of various fermentation pathways to sustain production of ATP under flooded conditions.     

PTP1B Targets the Endosomal Sorting Machinery: DEPHOSPHORYLATION OF REGULATORY SITES ON THE ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT COMPONENT STAM2*

Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated STAM2 specifically suppressed Akt activation, and a phosphorylation-deficient STAM2 mutant displayed prolonged localization on endosomes following EGF stimulation. These results reveal a novel link between the dephosphorylation and endocytic machinery and suggest that PTP1B can affect RTK signaling in a previously unrecognized manner.     

Polymorphisms in the pituitary growth hormone gene and its receptor associated with coronary artery disease in a predisposed cohort from India

We investigated the promoter polymorphisms of the pituitary growth hormone gene (GH1) and exon 3 deletion polymorphism (GHRd3) in its receptor gene (GHR) in 299 angiographically proven patients with coronary artery disease (CAD) and 231 asymptomatic controls enrolled in the ongoing Indian Atherosclerosis Research Study. Real time PCR based analysis of the GHR variant showed significant association of the GHRd3 deletion allele with CAD (OR 0.48, 95% CI: 0.30–0.76, P = 0.0014) and a dominant model of inheritance (Akaike information criterion = 482). The deletion allele showed significant association with high plasma HDL-c levels (P = 0.001). Sequencing of the proximal promoter region of GH1 revealed 12 novel polymorphisms and a TAGA haplotype constituted by the functional SNPs rs2005171, rs11568828, rs2005172 and rs6171, that showed significant association with CAD alone (adjusted OR of 3.31 (95% CI = 1.33–8.29, P = 0.011) and in CAD patients with diabetes (P = 0.019). Mean standardized height was associated with three of the four haplotype-tagging SNPs in the cohort (P ≤ 0.03). Eleven of the 12 polymorphic promoter SNPs contributed to 14.7% of variation in height in females in the whole dataset (P = 0.029). CAD patients with history of stroke exhibited marginally significantly lower mean height as compared to rest of the cohort (P < 0.006). In conclusion, genetic polymorphisms in the GHR gene and its ligand, GH1, may modulate the risk of CAD in the Asian Indian population.     

The effect of chain length on protein solubilization in polymer-based vesicles (polymersomes)

Using a mean-field analysis we derive a consistent model for the perturbation of a symmetric polymeric bilayer due to the incorporation of transmembrane proteins, as a function of the polymer molecular weight and the protein dimensions. We find that the mechanism for the inhibition of protein incorporation in polymeric bilayers differs from that of their inclusion in polymer-carrying lipid vesicles; in polymersomes, the equilibrium concentration of transmembrane proteins decreases as a function of the thickness mismatch between the protein and the bilayer core, whereas in liposomes the presence of polymer chains affects the protein adsorption kinetics. Despite the increased stiffness of polymer bilayers (when compared to lipid ones), their perturbation decay length and range of protein-protein interaction is found to be relatively long. The energetic penalty due to protein adsorption increases relatively slowly as a function of the polymer chain length due to the self-assembled nature of the polymer bilayer. As a result, we predict that transmembrane proteins may be incorporated in significant numbers even in bilayers where the thickness mismatch is large.     

Stimulation of perivascular nitric oxide synthesis by oxygen

We hypothesized that elevated partial pressures of O(2) would increase perivascular nitric oxide (*NO) synthesis. Rodents with O(2)- and.NO-specific microelectrodes implanted adjacent to the abdominal aorta were exposed to O(2) at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA). Exposures to 2.0 and 2.8 ATA O(2) stimulated neuronal (type I) NO synthase (nNOS) and significantly increased steady-state.NO concentration, but the mechanism for enzyme activation differed at each partial pressure. At both pressures, elevations in.NO concentration were inhibited by the nNOS inhibitor 7-nitroindazole and the calcium channel blocker nimodipine. Enzyme activation at 2.0 ATA O(2) appeared to be due to an altered cellular redox state. Exposure to 2.8 ATA O(2), but not 2.0 ATA O(2), increased nNOS activity by enhancing nNOS association with calmodulin, and an inhibitory effect of geldanamycin indicated that the association was facilitated by heat shock protein 90. Infusion of superoxide dismutase inhibited.NO elevation at 2.8 but not 2.0 ATA O(2). Hyperoxia increased the concentration of.NO associated with hemoglobin. These findings highlight the complexity of oxidative stress responses and may help explain some of the dose responses associated with therapeutic applications of hyperbaric oxygen.     

IgE binding synthetic peptides of Alt a 1, a major allergen of Alternaria alternata

Alternaria alternata protein, Alt a 1 is a major allergen associated with allergy in atopic patients. Although the molecule binds strongly to IgE antibody from patients, the epitopes involved have not been identified or defined. In the present study, we synthesized overlapping peptides spanning the whole sequence and evaluated their IgE binding with sera from patients with Alternaria-induced allergy. The results identified four IgE binding linear regions. Two of these regions K41-P50 and Y54-K63 showed consistent reactivity with all four patients studied. The specific epitopes involved in the immune response may be of value in the immunodiagnosis and probably also in specific immunotherapy.     

Immunolocalization of detergent-susceptible nucleoplasmic lamin A/C foci by a novel monoclonal antibody

The A-type lamins are localized in the interior of the nucleus as well as on the nuclear periphery. In this study, we have characterized a monoclonal antibody LA-2F9 produced against recombinant rat lamin A which stains a subpopulation of various cell types in a pattern of small nucleoplasmic foci that are unusually susceptible to mild detergent/salt extraction. The specific reactivity of mAb LA-2F9 towards lamins was confirmed by immunoblotting of HeLa and C3H10T(1/2) whole cell lysates and nuclear lysates. The epitope for LA-2F9 was narrowed down to amino acid residues 268-278 (SAKLDNARQSA). To check whether the appearance of lamin foci was cell-cycle-dependent, C3H10T(1/2) cells were serum-starved and then refed to trigger cells to enter the G(1) phase of the cell-cycle. The intensity of staining increased 3.5-fold within 6 h of refeeding, when the maximum number of cells were labeled with LA-2F9. We also checked whether the LA-2F9 foci colocalized with nuclear proteins known to be distributed in small foci such as hnRNPs, snRNPs, SC-35, and p80 coilin, but did not find evidence of colocalization. Our studies suggest that LA-2F9 has a novel and specific reactivity towards detergent-susceptible lower order lamin structures that are likely to be assembly intermediates.