The subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

Incubation of subcellular fractions isolated from rat cerebral cortex with [-³²P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes.A preliminary account of part of this work has been published (1).     

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.     

Correction to: Microhabitats of sharknose goby (Elacatinus evelynae) cleaning stations and their links with cleaning behaviour

Coral Reefs - A correction to this paper has been published: https://doi.org/10.1007/s00338-021-02111-z     

Early Life Stress and Post-Weaning High Fat Diet Alter Tyrosine Hydroxylase Regulation and AT1 Receptor Expression in the Adrenal Gland in a Sex Dependent Manner

Previous studies have shown that early life stress induced by maternal separation or non-handling can lead to behavioural deficits in rats and that these deficits can be alleviated by providing palatable cafeteria high-fat diet (HFD). In these studies we investigated the effects of maternal separation or non-handling and HFD on tyrosine hydroxylase (TH) protein and TH phosphorylation at Ser40 (pSer40TH) and the expression of angiotensin II receptor type 1 (AT1R) protein in the adrenal gland as markers of sympatho-adrenomedullary activation. After littering, Sprague–Dawley rats were assigned to short maternal separation, S15 (15 min), prolonged maternal separation, S180 (180 min) daily from postnatal days 2–14 or were non-handled (NH) until weaning. Siblings were exposed to HFD or chow from day 21 until 19 weeks when adrenals were harvested. Maternal separation and non-handling had no effects on adrenal TH protein in both sexes. We found an effect of HFD only in the females; HFD significantly increased TH levels in NH rats and pSer40TH in S180 rats (relative to corresponding chow-fed groups), but had no effect on AT1R expression in any group. In contrast, in male rats HFD had no effect on TH protein levels, but significantly increased pSer40TH across all treatment groups. There was no effect of HFD on AT1R expression in male rats; however, maternal separation (for 15 or 180 min) caused significant increases in AT1R expression (relative to NH group regardless of diet). This is the first study to report that early life stress and diet modulate TH protein, pSer40TH and AT1R protein levels in the adrenal gland in a sex dependent manner. These results are interpreted in respect to the potential adverse effects that these changes in the adrenal gland may have in males and females in adult life.     

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.     

Tyrosine Hydroxylase Dephosphorylation by Protein Phosphatase 2A in Bovine Adrenal Chromaffin Cells

This study was undertaken to characterise the protein phosphatases in bovine adrenal chromaffin cells acting on tyrosine hydroxylase. Cells were pre-labelled with ³²P_i and permeabilized with digitonin. The extent of dephosphorylation of Ser-8, Ser-19, Ser-31 and Ser-40 on tyrosine hydroxylase was found to be 30%, 38%, 37% and 71% respectively over 5 min. For Ser-19, Ser-31 and Ser-40 the dephosphorylation was entirely due to protein phosphatase 2A, as the dephosphorylation could be completely blocked by microcystin, but not by the protein phosphatase 1 inhibitory peptide. Permeabilization did not change the distribution of protein phosphatase 2A or tyrosine hydroxylase, or the activity of PP2A, from that occurring in intact cells. The dephosphorylation of Ser-8 was not altered by any inhibitor, suggesting the involvement of other protein phosphatases. The method developed here can be used to determine the protein phosphatases acting on substrates in conditions closely approximating those in situ, including the endogenous state of substrate phosphorylation and phosphatase location.     

Synthesis and biological evaluation of a novel phenyl substituted sydnone series as potential antitumor agents

A series of compounds containing an N-(4'-substituted-3'-nitrophenyl)sydnone moiety with potential antitumor activity was prepared based on active analogues. The rationale behind the design of these compounds is presented along with the 4-step synthetic route to the derivatives in the 4'-position of the phenyl sydnone framework. Out of the six novel compounds, the 4'-fluoro derivative has an improved activity against all three cell lines as compared to the earlier leads.