Structural homologies among type I restriction-modification systems.

Structural homologies among different restriction systems of Escherichia coli and several Salmonella species have been investigated by immunological methods using antibodies prepared against two subunits of the E. coli K12 restriction enzyme, and by DNA hybridization experiments using different fragments of the E. coli K12 hsd genes as probes. The results with both techniques show a strong homology between the E. coli K12 and B restriction-modification systems, weaker but nevertheless marked homology between E. coli K12 and the Salmonella systems SB, SP, and SQ and, surprisingly, no homology between the E. coli K12 and A systems.     

Crystal structure of a complex formed between proteolytically-generated lactoferrin fragment and proteinase K

Lactoferrin is an iron binding glycoprotein with a molecular weight of 80 kDa. The molecule is divided into two lobes representing the N-terminal and C-terminal halves of the polypeptide chain, each containing an iron binding site. The serine proteinases such as trypsin, chymotrypsin, and pepsin hydrolyze lactoferrin into two unequal halves while proteinase K divides this protein into two equal halves. In the first step of hydrolysis by proteinase K, the C- and N-lobes, each having a molecular weight of approximately 40 kDa, are generated. In the next step, the lobes are further hydrolyzed into small molecular weight peptides. The proteinase K isolated from the hydrolyzed product does not show enzymatic activity suggesting that the enzyme is inhibited. Furthermore, the hydrolysis experiments on N-lobe and C-lobe showed that the inhibitory fragment came from the C-lobe. The purified lactoferrin fragment was found to be a decapeptide with an amino acid sequence of H₂N-Val-Ala-Gln-Gly-Ala-Ala-Gly-Leu-Ala-COOH. The complex formed between proteinase K and lactoferrin fragment was crystallized by microdialysis. The crystals belonged to the monoclinic space group P2₁with cell dimensions a = 44.4 Å, b = 38.6 Å, c = 79.2 Å, β = 105.8^o and Z = 2. The crystal structure has been determined at 2.4 Å resolution. It has been refined to an R factor of 0.163 for 9044 reflections. The Lf-fragment forms several intermolecular interactions with proteinase K. The Ser-224 Oγ and His-57 Nϵ2 move away to a distance of 3.68 Å in the complex. In the crystal structure, Gln-3I (I indicates inhibitor i.e., lactoferrin fragment) is involved in a direct intermolecular interaction with a symmetry related proteinase K molecule through a strong hydrogen bond with Asp-254. The mode of intermolecular interactions in the complex conformational features of the enzyme and placement of the fragment with respect to the enzyme resemble with the molecular complex of proteinase K with its natural inhibitor PKI3 from wheat. Proteins 33:30–38, 1998. © 1998 Wiley-Liss, Inc.     

In vitro Hormonal Regulation of Laticifer Differentiation in Calotropis procera

Cultures of Calotropis procera were maintained on MS mediumsupplemented with 4·6 µM FAP and 3 µM NAA.Laticifer initials were observed in 2-week-old cultures whichwere converted into matured, branched, non-articulated laticifersin 4 weeks. It was observed that laticifer differentiation increasedwith the age of cultures, from 2·78% (in the second passage)to 15·11% (in the 12th passage). It has been establishedthat laticifer differentiation in vitro is a cytokinin-dependentprocess and among the cytokinins, FAP was more effective thanBA and 2-iP. But the type of auxin and its concentration alsoplay an important role in modifying the effect of cytokinin.Among the different auxins used IAA was more effective for laticiferdifferentiation than IBA and NAA, while 2,4D was inhibitory.Maximum laticifer differentiation (17·01% was observedon MS medium supplemented with 4·6 µM FAP and 1µM IAA.Copyright 1995, 1999 Academic Press Calotropis procera, callus culture, laticifer, differentiation, hormonal regulation     

Cardioprotection from ischemia and reperfusion injury by Withania somnifera: a hemodynamic, biochemical and histopathological assessment

The efficacy of Withania somnifera (Ws) to limit myocardial injury after ischemia and reperfusion was explored and compared to that of Vit E, a reference standard known to reduce mortality and infarct size due to myocardial infarction. Wistar rats (150-200 g) were divided into six groups and received orally saline (sham, control group), Ws-50/kg (Ws control and treated group) and Vit E-100 mg/kg (Vit E control and treated group) respectively for 1 month. On the 31st day, rats of the control, Vit E and Ws treated groups were anesthetized and subjected to 45 min occlusion of the LAD coronary artery followed by 60 min reperfusion. Hemodynamic parameters: systolic, diastolic and mean arterial pressure (SAP, DAP, MAP), heart rate (HR), left ventricular end diastolic pressure (LVEDP), left ventricular peak (+)LVdP/dt and (-)LVdP/dt were monitored. Hearts were removed and processed for histopathological and biochemical studies: Myocardial enzyme viz, creatin phosphokinase (CPK), and antioxidant parameters: malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx) were estimated. Postischemic reperfusion produced significant cardiac necrosis, depression of left ventricular functions (MAP, LVEDP, (+) and (-)LVdP/dt) and a significant fall in GSH (p < 0.01), SOD, CAT (p < 0.05), LDH and CPK (p < 0.01) as well as an increase in MDA level (p < 0.05) in the control group rats as compared to sham group. The changes in levels of protein and GPx was however, not significant. Ws and Vit E favorably modulated most of the hemodynamic, biochemical and histopathological parameters though no significant restoration in GSH, MAP (with Vit E) were observed. Ws on chronic administration markedly augmented antioxidants (GSH, GSHPx, SOD, CAT) while Vit E did not stimulate the synthesis of endogenous antioxidants compared to sham. Results indicate that Ws significantly reduced myocardial injury and emphasize the beneficial action of Ws as a cardioprotective agent.     

Both CAG repeats and inverted DNA repeats stimulate spontaneous unequal sister-chromatid exchange in Saccharomyces cerevisiae

Genomic regions containing trinucleotide repeats (TNRs) are highly unstable, as the repeated sequences exhibit a high rate of mutational change, in which they undergo either a contraction or an expansion of repeat numbers. Although expansion of TNRs is associated with several human genetic diseases, the expansion mechanism is poorly understood. Extensive studies in model organisms have indicated that instability of TNRs occurs by several mechanisms, including replication slippage, DNA repair and recombination. In all models, the formation of secondary structures by disease-associated TNRs is a critical step in the mutation process. In this report, we demonstrate that TNRs and inverted repeats (IRs) both of which have the potential to form secondary structures in vivo, increase spontaneous unequal sister-chromatid exchange (SCE) in vegetatively growing yeast cells. Our results also show that TNR-mediated SCE events are independent of RAD50, MRE11 and RAD51, whereas IR-stimulated SCEs are dependent on the RAD52 epistasis-group genes. We propose that many TNR expansion mutations occur by SCE.     

Inhibitory effect of vitamin E on proinflammatory cytokines-and endotoxin-induced nitric oxide release in alveolar macrophages

Nitric oxide is thought to be an important modulator of various functions in normal and inflamed airways. In the present study, we evaluated the effects of high vitamin E (250 mg and 1250 mg alpha-tocopheryl acetate (TA)/kg diet/10 days) on nitric oxide (NO(.)) release by alveolar macrophages (AMs) in response to lipopolysaccharide (LPS), interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF-alpha). LPS and IL-1beta treatment (1-10 microg/ml) enhanced NO(.) release in AMs from control animals fed on 50 mg vitamin E/kg diet in a concentration dependent manner. However, this enhancement of NO(.) was attenuated in the AMs of animals fed with 250 mg or 1250 mg vitamin E/kg diet. TNF-alpha had no effect in eliciting the release of NO(.) in AMs obtained either from control or from hyper vitamin E fed animals. Further, LPS (1-10 microg/ml) enhanced the inducible nitric oxide synthase (iNOS) activity of AMs of control group and TA-fed animals almost to equal extent. Similarly, LPS-induced formation of N-nitrosamine (N-nitroso-L-[(14)C]-proline) in AMs of control and TA-supplemented animals were not different statistically. On the other hand, in vitro addition of vitamin E (200 microM) in AMs of control animals, when triggered with 10 microg LPS/ml, caused a significant decrease in N-nitroso-L-[(14)C]-proline formation. It seems that high doses of TA in diet may play a role in reducing the lipopolysaccharide and proinflammatory cytokines-induced NO(.)-mediated damage by AMs.     

Immunosensors for pesticide analysis: antibody production and sensor development

Immunosensors, a type of affinity biosensor, are based on the binding interactions between an immobilized biomolecule (antibody/antigen) on the electronic transducer surface with the analyte of interest (antigen/antibody), resulting in a detectable signal. The sensor system takes advantage of the high selectivity provided by the molecular recognition characteristic of an antibody, which binds reversibly with a specific antigen. This review article presents the current status of immunosensors, highlighting their potential benefits and limitations for pesticide analysis. The basic criteria for generating specific antibodies against low-molecular-mass pesticides, which are usually nonimmunogenic in nature, are briefly discussed. The article also describes the fundamentals of important transducer technologies and their use in immunosensor development.