Apoptosis: mode of cell death induced in T cell leukemia lines by dexamethasone and other agents

Glucocorticoids can mediate the destruction of thymocytes and T cell-derived leukemia cells through a mechanism known as apoptosis. The characteristic feature of apoptosis is fragmentation of DNA at internucleosomal linkers through the activity of a specific endonuclease. In this study, an attempt was made to compare dexamethasone-induced apoptosis in two T cell-derived human leukemia lines (CEM-C1 and CEM-C7) to the cell killing brought about by selected cytotoxic agents. In the CEM-C7 cell line (dexamethasone-sensitive), apoptosis was induced not only by dexamethasone but by actinomycin D, cycloheximide, and 25-OH cholesterol. In the CEM-C1 cell line (dexamethasone-resistant) cycloheximide, 25-OH cholesterol, or cell starvation could induce apoptosis. It appears that in leukemic cells apoptosis may be induced by a variety of unrelated toxic agents and is not limited to glucocorticoids.     

Cardioprotective effect of lycopene in the experimental model of myocardial ischemia-reperfusion injury

The continuous advancements in cancer research have contributed to the overwhelming evidence of the presence of telomerase in primary and secondary tumours together with hsp90 and c-Myc. This review will discuss the important role of telomerase together with hsp90 and c-Myc within the initiation and progression of gliomas. Also it will review the differential expression of these genes in the different grades of gliomas and the possibility of new treatments targeting these specific genes.     

Lipid phenotyping of lung epithelial lining fluid in healthy human volunteers

Background

Lung epithelial lining fluid (ELF)—sampled through sputum induction—is a medium rich in cells, proteins and lipids. However, despite its key role in maintaining lung function, homeostasis and defences, the composition and biology of ELF, especially in respect of lipids, remain incompletely understood.

Objectives

To characterise the induced sputum lipidome of healthy adult individuals, and to examine associations between different ELF lipid phenotypes and the demographic characteristics within the study cohort.

Methods

Induced sputum samples were obtained from 41 healthy non-smoking adults, and their lipid compositions analysed using a combination of untargeted shotgun and liquid chromatography mass spectrometry methods. Topological data analysis (TDA) was used to group subjects with comparable sputum lipidomes in order to identify distinct ELF phenotypes.

Results

The induced sputum lipidome was diverse, comprising a range of different molecular classes, including at least 75 glycerophospholipids, 13 sphingolipids, 5 sterol lipids and 12 neutral glycerolipids. TDA identified two distinct phenotypes differentiated by a higher total lipid content and specific enrichments of diacyl-glycerophosphocholines, -inositols and -glycerols in one group, with enrichments of sterols, glycolipids and sphingolipids in the other. Subjects presenting the lipid-rich ELF phenotype also had significantly higher BMI, but did not differ in respect of other demographic characteristics such as age or gender.

Conclusions

We provide the first evidence that the ELF lipidome varies significantly between healthy individuals and propose that such differences are related to weight status, highlighting the potential impact of (over)nutrition on lung lipid metabolism.

     

The multiple-cluster mutation complex in mutagenesis with higher plants

Summary After treatment of dry and pre-soaked seeds of barley with gamma-rays, EMS, NEU and EI, the frequency of multiple mutations (multimutations) was higher with EMS and NEU treatment, while cluster mutations appeared in greater numbers following treatment with gamma rays and NEU. Pre-soaking the seeds led to a reduction in the frequency of total mutations, cluster mutations and multimutations. This has been explained as a result of the application of lower doses and the induction of mutations at a relatively later stage in ontogenetic development in the case of pre-soaked seeds.Some new mutation types in barley have been described and some of the old types have been given names representing the mutation characters more precisely.The compound mutation frequency of different seedling mutation types, when taken separately, was found to be independent of the mutagen employed and the stage of treatment. The size of mutated chimeras in M ₁ plants, as indicated by the segregation ratio of mutants in M ₂, was largest in albina, xantha, chlorina, albina-tigrina, chl-terminalis and eceriferum, and lowest in viridis, viridoalbina etc. This could be expected if the unstable premutations induced by mutagenic treatment are resolved into mutations at different intervals after their initiation, or it can be explained by the induction of dominant mutations, or lethal changes together with visible mutations.     

Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.     

QbD-Oriented Development and Characterization of Effervescent Floating-Bioadhesive Tablets of Cefuroxime Axetil

The objective of the present studies was systematic development of floating-bioadhesive gastroretentive tablets of cefuroxime axetil employing rational blend of hydrophilic polymers for attaining controlled release drug delivery. As per the QbD-based approach, the patient-centric target product profile and quality attributes of tablet were earmarked, and preliminary studies were conducted for screening the suitability of type of polymers, polymer ratio, granulation technique, and granulation time for formulation of tablets. A face-centered cubic design (FCCD) was employed for optimization of the critical material attributes, i.e., concentration of release controlling polymers, PEO 303 and HPMC K100 LV CR, and evaluating in vitro buoyancy, drug release, and ex vivo mucoadhesion strength. The optimized formulation was embarked upon through numerical optimization, which yield excellent floatation characteristic with drug release control (i.e., T _60%?>?6 h) and bioadhesion strength. Drug-excipient compatibility studies through FTIR and P-XRD revealed the absence of any interaction between the drug and polymers. In vivo evaluation of the gastroretentive characteristics through X-ray imaging and in vivo pharmacokinetic studies in rabbits revealed significant extension in the rate of drug absorption (i.e., T _max, K _a, and MRT) from the optimized tablet formulation as compared to the marketed formulation. Successful establishment of various levels of in vitro/in vivo correlations (IVIVC) substantiated high degree of prognostic ability of in vitro dissolution conditions in predicting the in vivo performance. In a nutshell, the studies demonstrate successful development of the once-a-day gastroretentive formulations of cefuroxime axetil with controlled drug release profile and improved compliance.     

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.     

CD11b+/Gr-1+ myeloid suppressor cells cause T cell dysfunction after traumatic stress

T cell dysfunction that occurs after surgery or trauma is associated with a poor clinical outcome. We describe that myeloid suppressor cells expressing CD11b(+)/Gr-1(+) markers invade the spleen after traumatic stress and suppress T cell function through the production of arginase 1. We created a consistent model of traumatic stress in C57BL/6 mice to perform this work. A significant number of CD11b(+)/Gr-1(+) cells expressing arginase 1 accumulated in T cell zones around the germinal centers of the white pulp of the spleen within 6 h of trauma and lasted for at least 72 h. Increased arginase activity and arginase 1 expression, along with increased [(3)H]arginine uptake, l-arginine depletion, and l-ornithine accumulation in the culture medium, were observed exclusively in CD11b(+)/Gr-1(+) cells after traumatic stress. Flow cytometry revealed CD11b(+)/Gr-1(+) as a heterogeneous myeloid suppressor cell also expressing low levels of MHC class I and II, CD80, CD86, CD31, and others. When compared with controls, trauma-induced CD11b(+)/Gr-1(+) cells significantly inhibited CD3/CD28-mediated T cell proliferation, TCR zeta-chain expression, and IL-2 production. The suppressive effects by trauma CD11b(+)/Gr-1(+) cells were overcome with the arginase antagonist N-hydroxy-nor-l-arginine or extrasupplementation of medium with l-arginine. Poor Ag-presenting capacity of control and trauma-induced CD11b(+)/Gr-1(+) cells was detected in allogeneic murine leukocyte reaction. This study demonstrates that CD11b(+)/Gr-1(+) cells invade the spleen following traumatic stress and cause T cell dysfunction by an arginase-mediated mechanism, probably that of arginine depletion. Understanding the mechanism of immune suppression by these cells has important clinical implications in the treatment of immune dysfunction after trauma or surgery.