Inhibition of human leukocyte elastase by phosphate esters of N-hydroxysuccinimide and its derivatives: direct observation of a phosphorylated enzyme by 31P nuclear magnetic resonance spectroscopy

A series of phosphate esters derived from N-hydroxysuccinimide and 3-alkyl-N-hydroxysuccinimide have been synthesized and found to be potent time-dependent irreversible inhibitors of human leukocyte elastase (HLE). The observed inhibitory activity in this series of compounds correlated well with the known preference of HLE for substrates with small hydrophobic side chains. Maximum potency was reached when a favorable aromatic interaction involving a phenyl group present in the inhibitor and an aromatic residue located in the vicinity of the S2' subsite was operative. 31P NMR spectroscopy was used to probe the mechanism of action of these compounds. Direct evidence is presented in support of a mechanism involving phosphorylation of the active site serine. These compounds constitute a new class of hydrolytically stable phosphorylating agents.     

The Social Life of Infants in the Context of Infectious Disease Transmission; Social Contacts and Mixing Patterns of the Very Young

Insight into how humans interact helps further understanding of the transmission of infectious diseases. For diseases such as pertussis, infants are at particular risk for severe outcomes. To understand the contact pattern of infants, especially those too young to be vaccinated, we sent contact diaries to a representative sample of 1000 mothers in the United Kingdom. We received 115 responses with a total of 758 recorded contacts. The average number of daily contacts for an infant was 6.68 overall and 5.7 for those aged ≤10 weeks. Of the latter, 2.1 (37%) contacts were with non-household members and were >15 minutes duration, suggesting that a cocooning programme may miss a substantial proportion of exposures leading to disease transmission. The least contact was between adolescents and infants. Thus the impact of adolescent (pertussis) vaccination on infants would likely be limited, unless it reduces transmission to other age groups whose contact with infants is greater.     

An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: Molecular and functional characterization

APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPX1, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. Am-pAPX1 contains an ORF of 286 amino acids coding for a 31.4kDa protein. The C-terminal region of the Am-pAPX1 ORF has a putative transmembrane domain and a peroxisomal targeting signal (RKKMK), suggesting peroxisomal localization. The peroxisomal localization of Am-pAPX1 was confirmed by stable transformation of the GFP-(Ala)(10)-Am-pAPX1 fusion in tobacco. RNA blot analysis revealed that Am-pAPX1 is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron). The isolated genomic clone of Am-pAPX1 was found to contain nine exons. A fragment of 1616bp corresponding to the 5' upstream region of Am-pAPX1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative light and abiotic stress regulatory elements.     

Human papillomavirus (HPV) oncoprotein E6 facilitates Calcineurin-Nuclear factor for activated T cells 2 (NFAT2) signaling to promote cellular proliferation in cervical cell carcinoma

The calcineurin-NFAT signaling pathway regulates cell proliferation, differentiation, and development in diverse cell types and organ systems. Deregulation of calcineurin-NFAT signaling has been reported in leukaemias and few solid tumors such as breast and colon. In the present study, we found elevated calcineurin protein levels and phosphatase activity in cervical cancer cell lines and depletion of the same attenuated cell proliferation. Additionally, nuclear levels of NFAT2, a downstream target of calcineurin, viz, was found elevated in human papillomavirus (HPV) infected cells, HeLa and SiHa, compared to the HPV negative cells, HaCaT and C33A, indicative of its higher DNA binding activity. The nuclear levels of both NFAT1 and NFAT3 remain unaltered implicating they have little role in cervical carcinogenesis. Similar to the in vitro studies, the HPV infected human squamous cell carcinoma specimens showed higher NFAT2 levels compared to the normal cervical epithelium. Depletion of NFAT2 by RNAi attenuated growth of SiHa cells. Overexpression of HPV16 oncoproteins viz, E6 and E7 increased NFAT2 expression levels and DNA binding activity, while knockdown of E6 by RNAi decreased the same. Briefly, we now report an activation of calcineurin-NFAT2 axis in cervical cancer and a novel role of HPV oncoprotein in facilitating NFAT2 dependent cell proliferation.     

Decavanadate possesses α-adrenergic agonist activity and a structural motif common with trans-β form of noradrenaline

Decavanadate, an inorganic polymer of vanadate, produced contraction of rat aortic rings at a relatively high concentration compared to phenylephrine, an agonist of -adrenergic receptor. This effect was blocked by two known a-adrenergic receptor antagonists, prazosin and phenoxybenzamine. Decavanadate, formed by possible dimerization of V5 under acid conditions, possessed a structural feature of two pairs of unshared oxygen atoms at a distance of 3.12 Å, not found in its constituents of V4 or V5. A structural motif of O..O..O using such oxygen atoms is recognized in decavanadate. This matches with a similar motif of N..O..O that uses the essential amino and hydroxyl groups of the side-chain and the m-hydroxyl group in trans-b form of noradrenaline. The interaction of such a structural motif with the membrane receptor is likely to be the basis of the unusual noradrenaline-mimic action of decavanadate.     

Process simulation for recombinant protein production: cost estimation and sensitivity analysis for heparinase I expressed in Escherichia coli

Heparinase I from flavobacterium heparinum has several potential clinical applications; the resulting high demands on protein purity and quantity can be met by recombinant expression in Escherichia coli. Based on laboratory scale experiments with insoluble heparinase I expression followed by renaturation, a process for production of 3 kg/year of heparinase I was designed. We present a comparative analysis of the production costs of soluble and insoluble heparinase I expression, as well as a generalized approach to sensitivity analysis, based on perturbation around a base case design scenario. This may assist focusing further development on process steps for which improvements both are feasible and result in significant cost saving. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 575-582, 1997.     

Rod outer segment membrane guanylate cyclase type 1 (ROS-GC1) gene: Structure,organization and regulation by phorbol ester,a protein kinase C activator

At present there are two recognized members of the ROS-GC subfamily of membrane guanylate cyclases. They are ROS-GC1 and ROS-GC2. A distinctive feature of this family is that its members are not switched on by the extracellular peptide hormones; instead, they are modulated by intracellular Ca2+ signals, consistent to their linkage with phototransduction. An intriguing feature of ROS-GC1, which distinguishes it from ROS-GC2, is that it has two Ca2+ switches. One switch inhibits the enzyme at micromolar concentrations of Ca2+, as in phototransduction; the other, stimulates. The stimulatory switch, most likely, is linked to retinal synaptic activity. Thus, ROS-GC1 is linked to both phototransduction and the synaptic activity. The present study describes (1) the almost complete structural identity of 18.5 kb ROS-GC1 gene; (2) its structural organization: the gene is composed of 20 exons and 19 introns with classical GT/AG boundaries; (3) the activity of the ROS-GC1 promoter assayed through luciferase reporter in COS cells; and (4) induction of the gene by phorbol ester, a protein kinase C (PKC) activator. The co-presence of PKC and ROS-GC1 in photoreceptors suggests that regulation of the ROS-GC1 gene by PKC might be a physiologically relevant phenomenon.     

Exploiting Phosphate-Starved cells of <Emphasis Type="Italic">Scenedesmus</Emphasis> sp. for the Treatment of Raw Sewage

Phosphate depletion is one of the favorable ways to enhance the sewage water treatment with the algae, however, detailed information is essential with respect to internal phosphate concentration and physiology of the algae. The growth rate of the phosphate-starved Scenedesmus cells was reduced drastically after 48 h. Indicating cells entered in the stationary phase of the growth cycle. Fourier Transform Infrared analysis of phosphate-starved Scenedesmus cells showed the reduction in internal phosphate concentration and an increase in carbohydrate/phosphate and carbohydrate/lipid ratio. The phosphate-starved Scenedesmus cells, with an initial cell density of, 1 × 10⁶ cells mL^?1 shows 87% phosphate and 100 % nitrogen removal in 24 h. The normal Scenedesmus cells need approximately 48 h to trim down the nutrients from wastewater up to this extent. Other microalgae, Ankistrodesmus, growth pattern was not affected due to phosphate starvation. The cells of Ankistrodesmus was able to reduce 71% phosphate and 73% nitrogen within 24 h, with an initial cell density of, 1 × 10⁶ cells mL^?1.     

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.