Plasminogen activator: Isolation and purification from Lymphosarcoma of ascites bearing mice

Plasminogen activator secreted by lymphosarcoma (ascites) of mice was purified up to 163-fold by ammonium sulphate fractionation at 35% saturation and chromatography on p-aminobenzamidine-Sepharose 4B. The purified activator contained specific activity of 9980 IU/mg. The plasminogen activator displayed homogeneity by polyacrylamide slab gel electrophoresis and high performance liquid chromatography. The activator consisted of a single polypeptide chain with an apparent molecular weight of 66,000 daltons as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions as well as gel filtration on Sephadex G-100. Distinct differences between this activator and urokinase were discernible in respect of specific activities, fibrin affinity and immunochemical properties. The lymphosarcoma activator appears to be of tissue-type origin since it showed gross similarity to standard tissue plasminogen activator in terms of modes of binding to fibrin and immunological attributes.     

Regulation by testosterone of the glucosamine-6-phosphate synthase activity in the male reproductive organs of rat

Accessory reproductive organs of male rat were found to contain high activities of glucosamine-6-phosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19). Castration caused drastic reduction (75%) in the enzyme activity of ventral prostate. Testosterone propionate administration restored the enzyme activity while cortisol and estradiol-17β did not cause any effect. Cycloheximide blocked the stimulation caused by testosterone.     

Assessment of Mineral Pathophysiology in Patients with Diabetic Foot Ulcer

Biological Trace Element Research - Chronic non-healing diabetic foot ulcers (DFU) with a recurrence rate of over 50% in 3 years account for more than 1,08000 non-traumatic lower extremity...     

Atypical activation of signaling downstream of inactivated Bcr-Abl mediates chemoresistance in chronic myeloid leukemia

Chronic myeloid leukemia (CML) epitomises successful targeted therapy, where inhibition of tyrosine kinase activity of oncoprotein Bcr-Abl1 by imatinib, induces remission in 86% patients in initial chronic phase (CP). However, in acute phase of blast crisis, 80% patients show resistance, 40% among them despite inhibition of Bcr-Abl1 activity. This implies activation of either Bcr-Abl1- independent signalling pathways or restoration of signalling downstream of inactive Bcr-Abl1. In the present study, mass spectrometry and subsequent in silico pathway analysis of differentiators in resistant CML-CP cells identified key differentiators, 14–3-3ε and p38 MAPK, which belong to Bcr-Abl1 pathway. Their levels and activity respectively, indicated active Bcr-Abl1 pathway in CML-BC resistant cells, though Bcr-Abl1 is inhibited by imatinib. Further, contribution of these components to resistance was demonstrated by inhibition of Bcr-Abl1 down-stream signalling by knocking-out of 14–3-3ε and inhibition of p38 MAPK activity. The observations merit clinical validation to explore their translational potential.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00647-x.     

Cholesterol organization in membranes at low concentrations: effects of curvature stress and membrane thickness

Cholesterol is often found distributed nonrandomly in domains in biological and model membranes and has been reported to be distributed heterogeneously among various intracellular membranes. Although a large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol. Using a fluorescent cholesterol analog (25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol, or NBD-cholesterol), we have previously shown that cholesterol may exhibit local organization even at very low concentrations in membranes, which could possibly be attributable to transbilayer tail-to-tail dimers. This is supported by similar observations reported by other groups using cholesterol or dehydroergosterol, a naturally occurring fluorescent cholesterol analog which closely mimics cholesterol. In this paper, we have tested the basic features of cholesterol organization in membranes at low concentrations using spectral features of dehydroergosterol. More importantly, we have investigated the role of membrane surface curvature and thickness on transbilayer dimer arrangement of cholesterol using NBD-cholesterol. We find that dimerization is not favored in membranes with high curvature. However, cholesterol dimers are observed again if the curvature stress is relieved. Further, we have monitored the effect of membrane thickness on the dimerization process. Our results show that the dimerization process is stringently controlled by a narrow window of membrane thickness. Interestingly, this type of local organization of NBD-cholesterol at low concentrations is also observed in sphingomyelin-containing membranes. These results could be significant in membranes that have very low cholesterol content, such as the endoplasmic reticulum and the inner mitochondrial membrane, and in trafficking and sorting of cellular cholesterol.     

Effects of heavy water on mitochondrial respiration and oxidative phosphorylation

Studies on the influence of heavy water on mitochondrial respiration and oxidative phosphorylation revealed that both isotope and solvent effects, may be responsible for the observed changes. Although the two types of effects could not be totally delineated from each other by the experimental approaches employed, the isotope effect appeared to be primarily responsible for the uncoupling of oxidative phosphorylation, while the inhibition of respiration in the presence of ADP (State 3 respiration) could be a manifestation of the solvent effect.     

Bacillus thuringiensis crystal delta-endotoxin: role of proteases in the conversion of protoxin to toxin

The conversion of delta-endoprotoxins of Bacillus thuringiensis to active toxins is mediated by trypsin, insect gut (exogenous) and bacterial (endogenous) proteases. The biochemical aspects of exogenous and endogenous proteases involved in the conversion of protoxin to toxin are reviewed. Perhaps, these proteases also play a role in influencing the host range of toxin and in the development of resistance to toxin.     

A novel tunnel in mycobacterial type III polyketide synthase reveals the structural basis for generating diverse metabolites

The superfamily of plant and bacterial type III polyketide synthases (PKSs) produces diverse metabolites with distinct biological functions. PKS18, a type III PKS from Mycobacterium tuberculosis, displays an unusual broad specificity for aliphatic long-chain acyl-coenzyme A (acyl-CoA) starter units (C(6)-C(20)) to produce tri- and tetraketide pyrones. The crystal structure of PKS18 reveals a 20 A substrate binding tunnel, hitherto unidentified in this superfamily of enzymes. This remarkable tunnel extends from the active site to the surface of the protein and is primarily generated by subtle changes of backbone dihedral angles in the core of the protein. Mutagenic studies combined with structure determination provide molecular insights into the structural elements that contribute to the chain length specificity of the enzyme. This first bacterial type III PKS structure underlines a fascinating example of the way in which subtle changes in protein architecture can generate metabolite diversity in nature.     

MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule dynamics and intracellular transport

MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule stability and mitotic spindle positioning in mitotic cells. This study elucidates the effect of MGRN1‐mediated ubiquitination of α‐tubulin in interphase cells. Here, we show that MGRN1‐mediated ubiquitination regulates dynamics of EB1‐labeled plus ends of microtubules. Intracellular transport of mitochondria and endosomes are affected in cultured cells where functional MGRN1 is depleted. Defects in microtubule‐dependent organellar transport are evident in cells where noncanonical K6‐mediated ubiquitination of α‐tubulin by MGRN1 is compromised. Loss of MGRN1 has been previously correlated with late‐onset spongiform neurodegeneration. Mislocalised cytosolically exposed PrP (^CtmPrP) interacts with MGRN1 leading to its loss of function. Expression of ^CtmPrP generating mutants of PrP[PrP(A117V) and PrP(KHII)] lead to decrease in MGRN1‐mediated ubiquitination of α‐tubulin and intracellular transport defects. Brain lysates from PrP(A117V) transgenic mice also indicate loss of tubulin polymerization as compared to non‐transgenic controls. Depletion of MGRN1 activity may hamper physiologically important processes like mitochondrial movement in neuronal processes and intracellular transport of ligands through the endosomal pathway thereby contributing to the pathogenesis of neurodegeneration in certain types of prion diseases.      

Biological monitoring of bidi rollers with respect to genotoxic hazards of occupational tobacco exposure

Smokeless tobacco habits are associated with a high incidence of oropharyngeal cancer in India. Hence, the biological effects of occupational exposure to smokeless tobacco used for making bidis (the Indian version of cigarettes) were studied in 2 groups of bidi rollers designated BR-K and BR-S and in control subjects with no tobacco habits. Specific tobacco exposure and the electrophilic burden were determined by estimating urinary cotinine and thioethers respectively. Urine mutagenicity was tested with the Ames assay using Salmonella typhimurium strains TA98 and TA100. While cotinine was not detected in control samples, the mean cotinine levels (mmole/mole creatinine) in the BR-K and BR-S groups were 0.79 +/- 0.30 and 0.09 +/- 0.03 respectively. Urinary thioether excretion (mmole/mole creatinine) was significantly elevated in the BR-S group 4.59 +/- 0.52; p less than 0.001) but it was lower in the BR-K group (0.54 +/- 0.08; p less than 0.001) compared to the control (1.83 +/- 0.34). Furthermore, beta-glucuronidase-treated samples from both groups of bidi rollers exhibited increased mutagenicity to TA98 compared to the control group; in addition, BR-S samples exhibited direct mutagenicity to TA98. The results show that occupational tobacco exposure modulates the glutathione conjugation pathway and increases the mutagenic burden of bidi rollers.