Activation of pigeon erythrocyte adenylate cyclase by cholera toxin partial purification of an essential macromolecular factor from horse erythrocyte cytosol

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43 000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13 000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD⁺, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.     

Demonstration of choleragen-dependent ADP-ribosylation in whole cells and correlation with the activation of adenylate cyclase

3T3C₂ mouse fibroblasts rendered permeable to (α^?32P)NAD⁺ show cholera toxin-dependent labeling of a 45,000 m.w. protein and of a doublet of polypeptides around 52,000 m.w. These same bands are ADP-ribosylated in broken cells. Membranes prepared from pigeon erythrocytes pretreated with choleragen show a decrease in subsequent cholera toxin-specific ADP-ribosylation of a 43,000 m.w. polypeptide. Both whole cell and broken cell adenylate cyclase activation and toxin-specific ADP-ribosylation are reversed specifically by low pH and high concentrations of toxin and nicotinamide in all systems. Thus ADP-ribosylation appears to be relevant to the molecular action of choleragen in whole cells as well as in broken cells.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

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.     

Effects of serial subculture in vitro on the endogenous levels of indole-3-acetic acid and abscisic acid and rootability in microcuttings of ‘Jonathan’ apple

Proliferating axillary shoots of the difficult-to-root apple cultivar Jonathan acquired an enhanced ability to form adventitious roots with increasing number of subcultures in vitro. The transition between the difficult-to-root and the easy-to-root condition occurred at the fourth subculture.Endogenous levels of free IAA and ABA in shoot tissues were analysed by gas chromatography/mass spectrometry/single ion monitoring (GC/MS/SIM) using negative ion chemical ionisation. Tissues from the mother plants grown in the glasshouse contained more IAA and ABA than those from tissue-culture material. After establishment in vitro there was no variation in the IAA content throughout the subcultures but a decrease in ABA content was observed after the fourth transfer. The IAA/ABA ratio increased from 0.2 in difficult-to-root shoots from the initial culture up to 0.7 in easy-to-root shoots from the long-term subculture.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Characterisation of two ornithine-containing lipids from Erwina aroideae

An apparently pure ornithine-containing lipid (OCL) was isolated from Erwina aroideae by solvent extraction and thin-layer chromatography (TLC). However, selective hydrolysis of the lipid under acidic and basic conditions and analysis of hydrolysates by gas chromatography-mass spectrometry (GCMS) showed that two structurally similar OCL were in fact present. These lipids both contained a 3-hydroxyhexadecanoic acid moiety which was linked to ornithine by an amide group formed between the 2-amino group of ornithine and the carboxyl group of the acid. The two lipids, however, differ in the nature of the fatty acid bound through an ester linkage to the hydroxyl group of the 3-hydroxyhexadecanoic acid moiety. One lipid is the ester of hexadecanoic acid whereas the other lipid is the ester of octadecenoic acid. These lipids are present in approximately equal amounts.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Assembly and plasma membrane targeting of recombinant immunoglobulin chains in plants with a murine immunoglobulin transmembrane sequence

The cDNA encoding a full-length murine immunoglobulin 1 heavy chain with its native leader sequence, transmembrane and intracellular domains was introduced into transgenic plants. Transformed plants expressed the recombinant polypeptide, but, in contrast to plants expressing the heavy chain without transmembrane sequence, the protein appeared to be associated with a plant cell membrane. Extraction of the membrane-associated heavy chain required the presence of a non-ionic detergent, and immunofluorescence studies of protoplasts demonstrated surface expression of membrane Ig heavy chain on up to 40% of the cells from a transgenic leaf. In plants expressing both the membrane Ig heavy chain and its partner light chain, functional antibody was also localised to the plant cell membrane and retention of the heavy chain at this site appeared to have no effect on the efficiency of antibody assembly. This approach of localising and accumulating recombinant antibody in cell membranes may have a number of applications, including passive immunisation against plant pathogens.     

Arterial retention of apolipoprotein B(48)- and B(100)-containing lipoproteins in atherogenesis

PURPOSE OF REVIEW: The "response to retention" hypothesis of atherosclerosis suggests that the arterial deposition of cholesterol is directly proportional to the concentration of circulating plasma lipoproteins. However, there is increasing evidence to support the concept that specific lipoproteins may be preferentially retained within the arterial wall, possibly as a result of greater affinity for cell surface and extracellular matrices. RECENT FINDINGS: Recently, key studies have provided insight into mechanisms involved in the interaction of apolipoprotein B (apoB)-containing lipoproteins with extracellular matrices. In addition, novel methods and innovative experimental design has enabled us to differentiate between the delivery, retention and efflux of apoB(48)- and apoB(100)-containing lipoproteins. Other studies have demonstrated a relationship between extracellular matrix proteoglycan expression and the development of atherosclerosis. Discussion in the present review also extends to the mechanisms that are involved in the relative intimal retention of apoB(48)- and apoB(100)-containing lipoproteins in order to explain the atherogenicity of these macromolecules. SUMMARY: The perspective of this review is to highlight recent advances in the area of arterial lipoprotein retention and the physiological significance these processes may have in the aetiology of cardiovascular disease. Importantly, an understanding of the mechanisms responsible for the retention of apoB(48)/B(100)-containing lipoproteins will enable new strategies to be developed for the future management of cardiovascular disease.