Direct correlation between the circadian sleep-wakefulness rhythm and time estimation in humans under social and temporal isolation

Several bodily functions in humans vary on a 24 h pattern and most of these variations persist with a circadian period ofca 25 h when subjects are studied under conditions of social and temporal isolation. We report in this paper that the estimates of short time intervals (TE) of 2 h are strongly coupled to the circadian rhythm in sleepwakefulness. There is a linear correlation between the number of hours humans stay awake (α) and their estimation of 2 h intervals. The coupling of TE to α appears to obtain only under conditions of physical well-being.     

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.     

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.     

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.     

Adenosine A(2A) receptor mRNA regulation by nerve growth factor is TrkA-, Src-, and Ras-dependent via extracellular regulated kinase and stress-activated protein kinase/c-Jun NH(2)-terminal kinase

We have shown previously that nerve growth factor (NGF) down-regulates adenosine A(2A) receptor (A(2A)AR) mRNA in PC12 cells. To define cellular mechanisms that modulate A(2A)AR expression, A(2A)AR mRNA and protein levels were examined in three PC12 sublines: i) PC12nnr5 cells, which lack the high affinity NGF receptor TrkA, ii) srcDN2 cells, which overexpress kinase-defective Src, and iii) 17.26 cells, which overexpress a dominant-inhibitory Ras. In the absence of functional TrkA, Src, or Ras, NGF-induced down-regulation of A(2A)AR mRNA and protein was significantly impaired. However, regulation of A(2A)AR expression was reconstituted in PC12nnr5 cells stably transfected with TrkA. Whereas NGF stimulated the mitogen-activated protein kinases p38, extracellular regulated kinase 1 and 2 (ERK1/ERK2), and stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) in PC12 cells, these kinases were activated only partially or not at all in srcDN2 and 17.26 cells. Inhibiting ERK1/ERK2 with PD98059 or inhibiting SAPK/JNK by transfecting cells with a dominant-negative SAPKbeta/JNK3 mutant partially blocked NGF-induced down-regulation of A(2A)AR expression in PC12 cells. In contrast, inhibiting p38 with SB203580 had no effect on the regulation of A(2A)AR mRNA and protein levels. Treating SAPKbeta/JNK3 mutant-transfected PC12 cells with PD98059 completely abolished the NGF-induced decrease in A(2A)AR mRNA and protein levels. These results reveal a role for ERK1/ERK2 and SAPK/JNK in regulating A(2A)AR expression.     

Modeling the binding affinities of beta-secretase inhibitors: application to subsite specificity

A new linear binding affinity model has been developed for hydroxyethylene based inhibitors of beta-secretase (BACE). This model is an improvement over a previously published model, and has been applied to a series of analogs not included in the training set. The linear model has been used to study subsite specificity for the P(2) through P(2)' positions, and to evaluate a small number of C-terminal analogs. The predicted rankings are in good agreement with experiment and support using this model for structure-based design of BACE inhibitors.     

Pulmonary inflammation and edema induced by phospholipase A2: global gene analysis and effects on aquaporins and Na+/K+-ATPase

Victims of snakebite quickly succumb to severe respiratory failure, which can be fatal if left untreated. One of the most toxic components of snake venom is phospholipase A2 (PLA2; EC 3.1.1.4). PLA2 isolated from the elapid, Naja sputatrix, induced pulmonary inflammation and edema when administered intravenously and intratracheally to rats. Analysis of pulmonary gene expression profiles using oligonucleotide microarrays revealed 60 genes whose expression was altered by at least 3-fold in response to intratracheal instillation of PLA2 for 3 h as compared with controls. In addition to genes encoding cytokines and chemokines responsible for inflammatory processes, the Na+/K+-ATPase gene has been found to be involved in edema formation. Real-time PCR, Western blot, and immunohistochemical analyses confirmed that the expression of AQP1 and AQP5 mRNAs and proteins was decreased. Besides providing an experimental model for studies on the pathophysiology of the lung, this investigation yields a clue to the mechanisms by which endogenous PLA2s could mediate inflammation in conditions such as allergy and rheumatoid arthritis.     

Protein O-Glucosyltransferase 1 (POGLUT1) Promotes Mouse Gastrulation through Modification of the Apical Polarity Protein CRUMBS2

Crumbs family proteins are apical transmembrane proteins with ancient roles in cell polarity. Mouse Crumbs2 mutants arrest at midgestation with abnormal neural plate morphology and a deficit of mesoderm caused by defects in gastrulation. We identified an ENU-induced mutation, wsnp, that phenocopies the Crumbs2 null phenotype. We show that wsnp is a null allele of Protein O-glucosyltransferase 1 (Poglut1), which encodes an enzyme previously shown to add O-glucose to EGF repeats in the extracellular domain of Drosophila and mammalian Notch, but the role of POGLUT1 in mammalian gastrulation has not been investigated. As predicted, we find that POGLUT1 is essential for Notch signaling in the early mouse embryo. However, the loss of mouse POGLUT1 causes an earlier and more dramatic phenotype than does the loss of activity of the Notch pathway, indicating that POGLUT1 has additional biologically relevant substrates. Using mass spectrometry, we show that POGLUT1 modifies EGF repeats in the extracellular domain of full-length mouse CRUMBS2. CRUMBS2 that lacks the O-glucose modification fails to be enriched on the apical plasma membrane and instead accumulates in the endoplasmic reticulum. The data demonstrate that CRUMBS2 is the target of POGLUT1 for the gastrulation epithelial-to-mesenchymal transitions (EMT) and that all activity of CRUMBS2 depends on modification by POGLUT1. Mutations in human POGLUT1 cause Dowling-Degos Disease, POGLUT1 is overexpressed in a variety of tumor cells, and mutations in the EGF repeats of human CRUMBS proteins are associated with human congenital nephrosis, retinitis pigmentosa and retinal degeneration, suggesting that O-glucosylation of CRUMBS proteins has broad roles in human health.     

Lack of Association of a Spontaneous Mutation of the Chrm2 Gene with Behavioral and Physiologic Phenotypic Differences in Inbred Mice

The nucleotide substitution C797T in the Chrm2 gene causes substitution of leucine for proline at position 266 (P266L) of the CHRM2 protein. Because Chrm2 codes for the type 2 muscarinic receptor, this mutation could influence physiologic and behavioral phenotypes of mice. Chrm2 mRNA was not differentially expressed in 2 brain regions with high cholinergic innervation in a mouse strain that does (BALB/cByJ) or does not (C57BL/6J) have the mutation. In addition, strains of mice with and without the C797T point mutation in Chrm2 did not differ significantly in muscarinic binding properties. Variation across strains was detected in terms of acoustic startle, prepulse inhibition, and the physiologic effects of the muscarinic agonist oxotremorine. However, interstrain differences in these measures did not correlate with the presence of the mutation. Although we were unable to associate a measurable phenotype with the Chrm2 mutation, assessment of the mutation on other genetic backgrounds or in the context of other traits might reveal differential effects. Therefore, despite our negative findings, evaluation of characteristics that involve muscarinic function should be undertaken with caution when comparing mice with different alleles of the Chrm2 gene.Abbreviations: M2R, type 2 muscarinic receptor; NMS, N-methylscopolamine; OXO, oxotremorine; PPI, prepulse inhibition; RI, recombinant inbredAcetylcholine, a crucial neurotransmitter in both the central and peripheral nervous systems, acts through 2 major types of receptors: muscarinic and nicotinic. Muscarinic acetylcholine receptors are members of the superfamily of G protein-coupled receptors.¹⁷mRNA and protein for the type 2 muscarinic receptor (M2) are present in many peripheral and central sites in the nervous system and peripheral target organs. M2R mediates a complex combination of postsynaptic and presynaptic events in noncholinergic and cholinergic neurons, respectively.⁹The M2R is encoded by the gene Chrm2. The proline at position 266 and surrounding residues of the Chrm2 gene are relatively conserved across several species, including human, rat, mouse, and swine (http://www.ncbi.nlm.nih.gov/). However, a nucleo­tide substitution (C797T) has been identified in several strains of inbred mice (Mouse Genome Informatics SNP query for Chrm2; http://www.informatics.jax.org/searches). This nucleotide substitution results in an amino acid substitution, P266L, in the protein. Proline is the only amino acid that contains a secondary amino group and forms tertiary peptide bonds. Because of this attribute, substitution of leucine for proline could cause alloste­ric alterations in proteins, with potential structural or functional consequences.Allosteric modulation is a recognized regulatory mechanism of muscarinic receptors.^17,21 For example, introduction of a point mutation (Asn to Tyr) at position 410 (the junction of transmembrane domain 6 and the 3rd intracellular loop) of the human M2R generated a constitutively active receptor with altered receptor–G-protein coupling in response to agonist administration.²³ Single-nucleotide polymorphisms in the human Chrm2 gene are implicated in responses to visual stimuli requiring attention, working memory, and response selection.^8,15,16 In addition, a common Chrm2 polymorphism has been associated with major depression in women in some studies^6,35 but not others.⁵ Furthermore, Chrm2 has been implicated in nicotine addiction; Chrm2 single-nucleotide polymorphisms may be associated with the general possibility of becoming addicted, personality traits that predispose the person to becoming addicted, or altered regulation of cholinergic systems that affect the smoker''s response to nicotine and its addictive properties.²²These reports suggest that mouse strains that bear the Chrm2 mutation, as compared with strains that do not, potentially provide a unique model for exploring mechanisms by which Chrm2 variants may affect cholinergic mechanisms and associated physiologic processes in both brain and the periphery. We report here on studies conducted to determine whether the C797T Chrm2 mutation confers a detectable phenotypic difference in M2R-related processes in mice.