Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.     

Encapsulation of interleukin-2 in murine erythrocytes and subsequent deposition in mice receiving a subcutaneous injection

Radiolabeled recombinant human interleukin-2 (IL-2) was successfully encapsulated in both mouse and sheep erythrocytes. Of the added IL-2, 70% was recovered bound to or encapsulated within the carrier cells. Erythrocytes containing IL-2 were stable in vitro and most of the IL-2 remained associated with the cells following a 16-h incubation at 37 degrees C. When carrier erythrocytes containing IL-2 were injected subcutaneously into mice, intact [35S]IL-2 was detectable in a number of tissues 3 days after injection.     

A continuous-flow high-yield process for preparation of lipid-free hemoglobin

Hypotonic hollow-fiber dialysis of bovine red blood cells followed by ultrafiltration through 0.1-micron pore hollow fibers provides a simple method for isolation of lipid-free hemoglobin. Hemoglobin (Hb) isolated by comparative techniques were all contaminated with membrane stroma. HPLC analysis of Hb revealed a protein peak of 99.6% purity and sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis revealed a single band. The process requires hypoosmotic dialysis of bovine RBC to a final 160-180 mosmol/kg osmotic pressure. Additional reduction in osmotic pressure causes irreversible cell lysis which leads to lipid contamination of the Hb. Processing of 1/2 liter of packed red blood cells requires 4-5 h, resulting in an average of 90% hemoglobin recovery.     

An evaluation of Ficoll density gradient centrifugation as a method for eliminating microbial contamination and purifying plant protoplasts

Ficoll density gradient centrifugation was used to generate sterile protoplasts from non-sterile Pteridium cultures. The method was found to drastically reduce levels of fungal and bacterial contamination introduced prior to centrifugation whilst concentrating and purifying protoplasts.     

An autosomal recessive nonsyndromic-hearing-loss locus identified by DNA pooling using two inbred Bedouin kindreds.

Autosomal recessive nonsyndromic hearing loss (ARNSHL) is the most common form of severe inherited childhood deafness. We present the linkage analysis of two inbred Bedouin kindreds from Israel that are affected with ARNSHL. A rapid genomewide screen for markers linked to the disease was performed by using pooled DNA samples. This screen revealed evidence for linkage with markers D9S922 and D9S301 on chromosome 9q. Genotyping of individuals from both kindreds confirmed linkage to chromosome 9q and a maximum combined LOD score of 26.2 (recombination fraction [theta] .025) with marker D9S927. The disease locus was mapped to a 1.6-cM region of chromosome 9ql3-q2l, between markers D9S15 and D9S927. The disease segregates with a common haplotype in the two kindreds, at markers D9S927, D9S175, and D9S284 in the linked interval, supporting the hypothesis that both kindreds inherited the deafness gene from a common ancestor. Although this nonsyndromic-hearing-loss (NSHL) locus maps to the same cytogenetic interval as DFNB7, it does not overlap the currently defined DFNB7 interval and may represent (1) a novel form of NSHL in close proximity to DFNB7 or (2) a relocalization of the DFNB7 interval to a region telomeric to its reported location. This study further demonstrates that DNA pooling is an effective means of quickly identifying regions of linkage in inbred families with heterogeneous autosomal recessive disorders.     

Binding of epidermal growth factor and transforming growth factor-alpha in mammalian preimplantation embryos

Preimplantation embryos of the pig (Days 11 to 15), cow (Days 14 to 16), sheep (Day 14) and pony (Day 16) bind epidermal growth factor (EGF) specifically. Binding was not detected in embryos of the rabbit at Day 5 or 6 or the hamster at Day 3. Transforming growth factor-alpha displaced [(125)I] EGF in pig, cow and pony embryos almost as much as unlabeled EGF. The binding affinities of EGF ranged from 12 to 233 pM in pig and cow embryos. The range of species and binding features indicate that the EGF family may play a significant role in mammalian preimplantation development.     

A denaturing gradient gel electrophoresis assay for sensitive detection of p53 mutations

p53 is a tumor suppressor gene located on 17p, a region of the human genome frequently deleted in tumors. Mutation of the p53 gene is an important step leading to development of many forms of human cancer. To simplify the analysis of tumors for p53 point mutations, we describe a GC-clamped denaturing gradient gel assay for detecting single-base substitutions within highly conserved regions of the p53 gene. This assay alows for efficient screening of tumors for single-base substitutions within the p53 gene and can be used to facilitate sequence analysis of p53 point mutations.     

An extensive review on antifungal approaches in the treatment of mucormycosis

During the period of COVID-19, the occurrences of mucormycosis in immunocompromised patients have increased significantly. Mucormycosis (black fungus) is a rare and rapidly progressing fungal infection associated with high mortality and morbidity in India as well as globally. The causative agents for this infection are collectively called mucoromycetes which are the members of the order Mucorales. The diagnosis of the infection needs to be performed as soon as the occurrence of clinical symptoms which differs with types of Mucorales infection. Imaging techniques magnetic resonance imaging or computed tomography scan, culture testing, and microscopy are the approaches for the diagnosis. After the diagnosis of the infection is confirmed, rapid action is needed for the treatment in the form of antifungal therapy or surgery depending upon the severity of the infection. Delaying in treatment declines the chances of survival. In antifungal therapy, there are two approaches first-line therapy (monotherapy) and combination therapy. Amphotericin B ( 1 ) and isavuconazole ( 2 ) are the drugs of choice for first-line therapy in the treatment of mucormycosis. Salvage therapy with posaconazole ( 3 ) and deferasirox ( 4 ) is another approach for patients who are not responsible for any other therapy. Adjunctive therapy is also used in the treatment of mucormycosis along with first-line therapy, which involves hyperbaric oxygen and cytokine therapy. There are some drugs like VT-1161 ( 5 ) and APX001A ( 6 ), Colistin, SCH 42427, and PC1244 that are under clinical trials. Despite all these approaches, none can be 100% successful in giving results. Therefore, new medications with favorable or little side effects are required for the treatment of mucormycosis.     

Immune complex glomerulonephritis in baboons (Papio cynocephalus) with indwelling intravascular catheters

Baboons with long term, indwelling, intravascular catheters developed clinical signs of renal and hepatic impairment. These included proteinuria and hypoalbuminemia without edema, and albumin to globulin ratios were reversed. Serum IgM, IgG, rheumatoid factor, and liver enzyme concentrations were above normal. Immunofluorescent staining of renal glomerular capillary loops was positive for IgG, IgM, B1c, and C4. Major microscopic lesions were membranoproliferative glomerulonephritis, chronic active hepatitis, degenerative arthritis, and chronic sialoadenitis. Electron microscopy of renal glomeruli demonstrated dense deposits in a variety of locations, mesangial cell interpositioning, and foot process fusion. These alterations, found in conjunction with the isolation of Staphylococcus aureus from the blood of affected baboons as well as the intravascular catheters, suggested that chronic bacterial infection was important in the pathogenesis of this disease.