Acyl-chlorodeoxy-glycerophosphorylcholines,Part II: Total synthesis of “cyclic lysolecithin”

Reaction of 1-fattyacyl-sn-glycero-3-phosphorylcholine with triphenylphosphine — carbon tetrachloride gave 3-fattyacyl-2-chloro-2-deoxy-sn-glycero-1-phosphorylcholine together with small amounts of other chlorodeoxy isomers. 1-Chloro-1-deoxy-2-palmitoyl-rac-glycero-3-phosphorylcholine was prepared by total synthesis from 3-chloro-2-iodopropyl palmitate. The main step in the synthesis involves the nucleophilic displacement of iodide at C-2 with dibenzyl phosphate anion, which proceeds with an acyloxy migration, leading to the key intermediate 1-chloro-1-deoxy-2-palmitoyl-rac-glycero-3-(dibenzyl phosphate). Hydrogenolysis of this phosphate triester, followed by esterification with choline acetate gave the final product. The properties of the products support an earlier conclusion that the so-called “cyclic lysolecithin” is a mixture of isomeric acyl-chloro-deoxy-glycero-phosphorylcholines in which 1-chloro-1-deoxy-2-acyl-glycero-3-phosphorylcholine is the major component.     

Role of Host Immune Response and Viral Load in the Differential Outcome of Pandemic H1N1 (2009) Influenza Virus Infection in Indian Patients

Background

An unusually high number of severe pneumonia cases with considerable mortality is being observed with the pandemic H1N1 2009 virus infections globally. In India, all mild as well as critically ill cases were admitted and treated in the government hospitals during the initial phase of the pandemic. The present study was undertaken during this early phase of the pandemic.

Methodology

The role of viral load and host factors in the pathogenesis were assessed by examining 26 mild (MP), 15 critically ill patients (CIP) and 20 healthy controls from Pune, India. Sequential blood and lung aspirate samples were collected from CIP. Viral load and cytokines/chemokine levels were determined from the plasma and lung aspirates of the patients. TLR levels were determined by staining and FACS analysis. Gene profiling was done for both cells in the lung aspirates and PBMCs using TaqMan Low Density arrays. Antibody titres and isotyping was done using HA protein based ELISAs.

Principal Findings

13/15 critically ill patients expired. All plasma samples were negative for the virus irrespective of the patient''s category. Sequential lung samples from CIP showed lower viral loads questioning association of viral replication with the severity. Anti-rpH1N1-09-HA-IgG titres were significantly higher in critically ill patients and both categories circulated exclusively IgG1 isotype. Critically ill patients exhibited increase in TLR-3, 4, 7 and decrease in TLR-2 expressions. The disease severity correlated with increased plasma levels of IL1RA, IL2, IL6, CCL3, CCL4 and IL10. Majority of the immune-function genes were down-regulated in the PBMCs and up-regulated in the cells from lung aspirates of critically ill patients. No distinct pattern differentiating fatal and surviving patients was observed when sequential samples were examined for various parameters.

Conclusions

Disease severity was associated with pronounced impairment of host immune response.     

Identification of Thr29 as a Critical Phosphorylation Site That Activates the Human Proton Channel Hvcn1 in Leukocytes

Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pH_i regulation. The “enhanced gating mode” appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-δ phosphorylation sites in the human voltage-gated proton channel, H_V1 (Hvcn1), Thr²⁹ and Ser⁹⁷, both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-δ. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H_V1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT H_V1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr²⁹ is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.     

Diversity of plant cell wall esterases in thermophilic and thermotolerant fungi

Fourteen thermophilic and thermotolerant fungal strains isolated from composting soils produced plant cell wall-acting esterases in a medium containing corn cobs and oat spelt xylan. The concentrated and dialyzed protein extracts of these fungi were fractionated using isoelectric-focusing, gels sliced and eluted protein in each slice was assayed for esterase activity against p-nitrophenyl acetate. A total of 84 esterases detected on the basis of pI were found to show distinct preferential substrate specificities towards p-nitrophenyl acetate, p-nitrophenyl ferulate and p-nitrophenyl butyrate, and were putatively classified as acetyl esterases and esterases types I and II. None of the esterases were active against p-nitrophenyl myristate. In addition, these esterases were characterized as acid, neutral or alkaline active.     

Thiophilic interaction chromatography of prostate-specific antigen

Prostate-specific antigen (PSA) protein and complexes of PSA with α₁-antichymotrypsin (PSA-ACT) or α₂-macroglobulin (PSA-A₂M) prepared in vitro, have strong affinity for different thiophilic gels (T-gel). Free PSA, and these PSA complexes can be isolated due to their affinity for T-gels. The average recovery of PSA from several of the T-gels, based upon ELISA measurements, was 84 to 94%. The data suggest that T-gel affinity can be explored for the purification of free and complexed PSA from various biologic fluids.     

Identification of a glycosyltransferase from Mycobacterium marinum involved in addition of a caryophyllose moiety in lipooligosaccharides

Deletion of Mycobacterium marinum MMAR2333 resulted in the loss of three of four subclasses of lipooligosaccharides (LOSs). The mutant was unable to extend an intermediate (LOS-II*) by addition of caryophyllose. These data and the predicted domain structure suggest that MMAR2333 is a glycosyltransferase involved in the generation of a lipid-linked caryophyllose donor.