Characterization of α-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

α-mannosidase from Erythrina indica seeds is a Zn²⁺ dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 °C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol^− 1. N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3–8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of α-helical structure in the enzyme. α-Mannosidase from E indica exhibits immunological identity with α-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with α-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of α-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.     

Value of cytology in papillary condylomatous lesions of the cervix

OBJECTIVE: To determine the value of cytology in detecting mature and immature papillary condylomas of the uterine cervix. STUDY DESIGN: We evaluated 240 cases of plane cervical intraepithelial neoplasia, grade 1 (CIN 1), and 23 papillary condylomas by Pap smear and biopsy and classified histologic sections according to maturity and keratinization. We reevaluated corresponding cytologic smears and identified criteria of low grade squamous lesion (LSIL) and human papillomavirus (HPV) infection. RESULTS: Thirteen (56.5%) papillary lesions were histologically classified as mature, 6 (26%) as immature and 4 (17.3%) as mixed. Fifteen lesions (65.2%) were nonkeratinized, 2 (8.6%) keratinized and 6 (26%) partially keratinized. Corresponding smears were cytologically diagnosed as LSIL (6, 26%), atypical squamous cells of undetermined significance (ASCUS) (7, 30.4%) and negative (10, 43.4%). Careful cytologic review diagnosed only two of the 13 mature lesions; few cytological criteria of LSIL and HPV infection were observed. Koilocytes were seen in just 1 case. Sample limiting factors occurred in 4 cases: 2 cytologically diagnosed as LSIL, 1 asASCUS and 1 as negative for lesion. CONCLUSION: Cytology was not effective in the detection of cervical condyloma acuminatum, independent of limitations in sample adequacy and of the degree of maturity or keratinization of the lesions.     

A broad range of Fab stabilities within a host of therapeutic IgGs

Although the functional properties of IgGs are well known, little has been published concerning the stability of whole IgG molecules. Stability is, however, a requirement for the development of antibodies for therapeutic or diagnostic applications. The hypervariable antigen-binding region (Fv) is responsible for stability variations between IgGs of identical subclass. To determine the range of stabilities that may be expected for human(ized) antibodies, differential scanning calorimetry was performed on 17 human(ized) antibodies from various in-house programs. The antigen-binding fragments (Fabs) of these antibodies exhibited thermal unfolding transitions with midpoints (T(M)s) varying from 57 to 82 degrees C. Antibodies with very low Fab stabilities were found to aggregate and express poorly. Fab instability was often associated with high levels of uncommonly observed amino acids or CDR loop lengths particularly within the variable heavy chain domain. Overall, the study provides a thermostability range for IgGs and suggests possible stability guidelines for developing antibody diagnostics or therapeutics.     

Role of cardiovascular nitric oxide system in C-type natriuretic peptide effects

The aims were to evaluate the role of cardiovascular nitric oxide (NO)-system in C-type natriuretic peptide (CNP) actions and to investigate receptor types and signaling pathways involved in this interaction. Wistar rats were infused with saline or CNP. Mean arterial pressure (MAP) and nitrites and nitrates (NOx) excretion were determined. NO synthase (NOS) activity and NOS expression (Western blot) were analyzed in atria, ventricle and aorta. CNP decreased MAP and increased NOx excretion. CNP estimulated NOS activity, inducing no changes on cardiac and vascular endothelial NOS expression. NOS activity induced by CNP was abolished by suramin and calmidazoliumand but it is not modified by anantin. CNP would interact with NPR-C receptor coupled via G proteins leading to the activation Ca(2+)-calmodulin dependent endothelial NOS, increasing NO production which would induce the reduction in cardiac myocyte contractility and ANP synthesis and secretion in right atria and the relaxation of vascular smooth muscle.     

Impact of climatic variability on parameters used in typology and ecological quality assessment of surface waters—implications on the Water Framework Directive

In most cases the negative impacts of climate change to aquatic ecosystems cannot be mitigated by measures in the river basin management. Ignoring climate change by the Water Framework Directive may have strong implications for the typology and quality assessment systems used for water bodies. As a result of climate change, water bodies, especially those located near the type boundaries may change their type. Compared to typology characteristics, water quality parameters are even more labile and may be easily affected by climate change. The paper exemplifies that the anticipated deterioration of water quality within the time frame relevant for WFD implementation may be large enough to endanger the fulfillment of the set water quality objectives. The review of the river basin characterization every six years, as required by the WFD, might also include re-evaluation of reference conditions according to the changes observed at pristine reference sites. As a consequence, the restoration targets (i.e., the good ecological status) would also need to be evaluated periodically.     

Selective addressing of heteroditopic ligands by iron(II) and platinum(II)

The heteroditopic ligand 4′-(4,7,10-trioxadec-1-yn-10-yl)-2,2′:6′,2″-terpyridine, 2, contains an N,N′,N″-donor metal-binding domain that recognizes iron(II), and a terminal alkyne site that selectively couples to platinum(II). This selectivity has been used to investigate routes to the formation of heterometallic systems. The single crystal structures of ligand 2 and the complex [Fe(2)₂][PF₆]₂ are reported.     

Negative regulation of Bacillus anthracis sporulation by the Spo0E family of phosphatases

The initiation of sporulation in Bacillus species is controlled by the phosphorelay signal transduction system. Multiple regulatory elements act on the phosphorelay to modulate the level of protein phosphorylation in response to cellular, environmental, and metabolic signals. In Bacillus anthracis nine possible histidine sensor kinases can positively activate the system, while two response regulator aspartyl phosphate phosphatases of the Rap family negatively impact the pathway by dephosphorylating the Spo0F intermediate response regulator. In this study, we have characterized the B. anthracis members of the Spo0E family of phosphatases that specifically dephosphorylate the Spo0A response regulator of the phosphorelay and master regulator of sporulation. The products of four genes were able to promote the dephosphorylation of Spo0A approximately P in vitro. The overexpression of two of these B. anthracis Spo0E-like proteins from a multicopy vector consistently resulted in a sporulation-deficient phenotype. A third gene was found to be not transcribed in vivo. A fourth gene encoded a prematurely truncated protein due to a base pair deletion that nevertheless was subject to translational frameshift repair in an Escherichia coli protein expression system. A fifth Spo0E-like protein has been structurally and functionally characterized as a phosphatase of Spo0A approximately P by R. N. Grenha et al. (J. Biol. Chem. 281:37993-38003, 2006). We propose that these proteins may contribute to maintain B. anthracis in the transition phase of growth during an active infection and therefore contribute to the virulence of this organism.     

Functional and intracellular signaling differences associated with the Helicobacter pylori AlpAB adhesin from Western and East Asian strains

Following adhesion of Helicobacter pylori to gastric epithelial cells, intracellular signaling leads to cytokine production, which causes H. pylori-related gastric injury. Two adjacent homologous genes (alpA and alpB), which encode H. pylori outer membrane proteins, are thought to be associated with adhesion and cytokine induction. We co-cultured gastric epithelial cells with wild type H. pylori strains and their corresponding alpA/alpB-deleted mutants (DeltaalpAB). Results were confirmed by complementation. Flow cytometry confirmed that AlpAB was involved in cellular adhesion. Deletion of alpAB reduced interleukin (IL)-6 induction in gastric epithelial cells. Deletion of alpAB reduced IL-8 induction with East Asian but not with Western strains. All AlpAB-positive strains tested activated the extracellular signal-regulated kinase, c-Fos, and cAMP-responsive element-binding protein. Activation of the Jun-N-terminal kinase, c-Jun, and NF-kappaB was exclusive to AlpAB from East Asian strains. DeltaalpAB mutants poorly colonized the stomachs of C57BL/6 mice and were associated with lower mucosal levels of KC and IL-6. Our results suggest that AlpAB may induce gastric injury by mediating adherence to gastric epithelial cells and by modulating proinflammatory intracellular signaling cascades. Known geographical differences in H. pylori-related clinical outcomes may relate to differential effects of East Asian and Western types of AlpAB on NF-kappaB-related proinflammatory signaling pathways.