Cold-tolerant phosphate-solubilizing Pseudomonas strains promote wheat growth and yield by improving soil phosphorous (P) nutrition status.

It is well-known that phosphate-solubilizing bacteria (PSB) promote crop growth and yield. The information regarding characterization of PSB isolated from agroforestry systems and their impact on wheat crops under field conditions is rarely known. In the present study, we aim to develop psychrotroph-based P biofertilizers, and for that, four PSB strains (Pseudomonas sp. L3, Pseudomonas sp. P2, Streptomyces sp. T3, and Streptococcus sp. T4) previously isolated from three different agroforestry zones and already screened for wheat growth under pot trial conditions were evaluated on wheat crop under field conditions. Two field experiments were employed; set 1 includes PSB + recommended dose of fertilizers (RDF) and set 2 includes PSB - RDF. In both field experiments, the response of the PSB-treated wheat crop was significantly higher compared to the uninoculated control. In field set 1, an increase of 22% in grain yield (GY), 16% in biological yield (BY), and 10% in grain per spike (GPS) was observed in consortia (CNS, L3 + P2) treatment, followed by L3 and P2 treatments. Inoculation of PSB mitigates soil P deficiency as it positively influences soil alkaline phosphatase (AP) and soil acid phosphatase (AcP) activity which positively correlated with grain NPK %. The highest grain NPK % was reported in CNS-treated wheat with RDF (N-0.26%, P-0.18%, and K-1.66%) and without RDF (N-0.27, P-0.26, and K-1.46%), respectively. All parameters, including soil enzyme activities, plant agronomic data, and yield data were analyzed by principal component analysis (PCA), resulting in the selection of two PSB strains. The conditions for optimal P solubilization, in L3 (temperature-18.46, pH-5.2, and glucose concentration-0.8%) and P2 (temperature-17°C, pH-5.0, and glucose concentration-0.89%), were obtained through response surface methodology (RSM) modeling. The P solubilizing potential of selected strains at <20°C makes them a suitable candidate for the development of psychrotroph-based P biofertilizers. Low-temperature P solubilization of the PSB strains from agroforestry systems makes them potential biofertilizers for winter crops.

Clinical Profile and Outcome of Children Admitted with Multisystem Inflammatory Syndrome (MIS-C) in a Tertiary Hospital.

Background Most children infected with SARS-CoV-2 infection, are asymptomatic or develops mild to moderate symptoms. Few weeks later, few children develops delayed hyper inflammatory syndrome known as Multisystem inflammatory syndrome in children (MIS-C). Objective To describe various demographic features of children with Multisystem inflammatory syndrome in children. To analyze common clinical presentation, clinical and laboratory markers of severity and outcome of children with Multisystem inflammatory syndrome. Method This study was prospective observational study conducted on children with Multisystem inflammatory syndrome in children. This was conducted in Department of Pediatrics of Nobel Medical College during 12 months period from July 2021 to June 2022. Basic demographic features, common clinical presentation in children with Multisystem inflammatory syndrome in children and its severity and outcome were analyzed. Independent sample t-test and chi square test was used for comparison of means and categorical variables. Logistic regression was done to assess the relationship between clinical variables and outcome. Result A total of 36 children were included in our study. Maximum number of cases were male (61.11%) and age group > 10 years (58.33%). Fever, gastrointestinal symptoms, shock and renal dysfunction were common clinical features. Children requiring mechanical ventilation had higher C-reactive protein (CRP), lower platelets, higher d-Dimer and lower ejection fraction. Vasoactive Inotropic score (VIS > 10) was associated with higher chances of mechanical ventilation and prolonged pediatric intensive care unit (PICU) stay. Mortality rate in our study was 5.55% and three children developed coronary aneurysm.

Influence of Temperature and Humidity on Pregnancy Rate of Murrah Buffaloes under Subtropical Climate.

Heat stress has adverse effects on fertility of dairy animals. Decline in fertility is linearly associated with an increase in combination of both temperature and humidity. The purpose of this study was to investigate the relationship between temperature humidity index (THI) and the pregnancy rate of Murrah buffaloes in a subtropical climate. The effects of genetic and non-genetic factors viz., sire, parity, period of calving and age group at first calving were found non-significant on pregnancy rate. The effect of THI was found significant (p<0.001) on pregnancy rate of Murrah buffaloes calved for first time and overall pregnancy rate. The threshold THI affecting the pregnancy rate was identified as THI 75. The months from October to March showed THI<75 and considered as non heat stress zone (NHSZ), while months from April to September were determined as heat stress zone (HSZ) with THI≥75. The lowest overall pregnancy rate (0.25) was obtained in July with THI 80.9, while the highest overall pregnancy rate (0.59) was found in November with THI 66.1. May and June were identified as critical heat stress zone (CHSZ) within the HSZ with maximum decline (-7%) in pregnancy rate with per unit increase in THI. The highest overall pregnancy rate was estimated as 0.45 in NHSZ with THI value 56.7 to 73.2. The pregnancy rate was found to have declined to 0.28 in HSZ with THI 73.5 to 83.7. However, the lowest pregnancy rate was estimated as 0.27 in CHSZ with THI value 80.3 to 81.6.

PHYTO-PHARMACOGNOSTICAL INVESTIGATION AND EVALUATION OF ANTI-INFLAMMATORY AND SEDATIVE HYPNOTIC ACTIVITY OF THE LEAVES OF ERYTHRINA INDICA Lam.

Pharmacognostical investigations were carried out on the Erythrina indica leaves, followed by phytochemical investigation. On the methanolic extract of leaves, TLC was performed and indole alkaloids were identified with selected solvent system. The UV analysis was also performed on the components confirming the presence of the indole nucleus. Anti-inflammatory activity was carried out on albino rats. Further, anti-inflammatory activity was compared to that of the standard drug indomethacin and percent inhibition of oedema was determined. Sedative hypnotic activity was also evaluated using pentobarbital which showed mild sedation.

The role of Rho in G protein-coupled receptor signal transduction.

Low molecular weight G proteins of the Rho subfamily are regulators of actin cytoskeletal organization. In contrast to the heterotrimeric G proteins, the small GTPases are not directly activated through ligand binding to G protein-coupled receptors (GPCRs). However, a subset of GPCRs, including those for lysophosphatidic acid and thrombin, induce stress fibers, focal adhesions, and cell rounding through Rho-dependent pathways. C3 exoenzyme has been a useful tool for demonstrating Rho involvement in these and other responses, including Ca2+ sensitization of smooth muscle contraction, cell migration, transformation, and serum response element-mediated gene expression. Most of the GPCRs that induce Rho-dependent responses can activate Gq, but this is not a sufficient signal. Recent data demonstrate that G alpha 12/13 can induce Rho-dependent responses. Furthermore, G alpha 12/13 can bind and activate Rho-specific guanine nucleotide exchange factors, providing a mechanism by which GPCRs that couple to G alpha 12/13 could activate Rho and its downstream responses.

The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element.

The low-molecular-weight GTP-binding protein RhoA mediates hypertrophic growth and atrial natriuretic factor (ANF) gene expression in neonatal rat ventricular myocytes. Neither the effector nor the promoter elements through which Rho exerts its regulatory effects on ANF gene expression have been elucidated. When constitutively activated forms of Rho kinase and two protein kinase C-related kinases, PKN (PRK1) and PRK2, were compared, only PKN generated a robust stimulation of a luciferase reporter gene driven by a 638-bp fragment on the ANF promoter. This ANF promoter fragment contains a proximal serum response element (SRE) and an Sp-1-like element required for the transcriptional response to phenylephrine (PE). This response was inhibited by dominant negative Rho. The ability of dominant negative Rho to inhibit the response to PE and the ability of PKN to stimulate ANF reporter gene expression were both lost when the SRE was mutated. Mutation of the Sp-1-like element also attenuated the response to PKN. A minimal promoter driven by ANF SRE sequences was sufficient to confer Rho- and PKN-mediated gene expression. Interestingly, PKN preferentially stimulated the ANF versus the c-fos SRE reporter gene. Thus PKN and Rho are able to regulate transcriptional activation of the ANF SRE by a common element that could implicate PKN as a downstream effector of Rho in transcriptional responses associated with hypertrophy.

Cardiac-specific overexpression of RhoA results in sinus and atrioventricular nodal dysfunction and contractile failure.

RhoA is a low-molecular-weight GTPase that has been implicated in the regulation of hypertrophic cardiac muscle cell growth. To study the role of RhoA in control of cardiac function in vivo, transgenic mice expressing wild-type and constitutively activated forms of RhoA under the control of the cardiac-specific alpha-myosin heavy chain promoter were generated. Transgene-positive mice expressing high levels of either wild-type or activated RhoA showed pronounced atrial enlargement and manifested a lethal phenotype, often preceded by generalized edema, with most animals dying over the course of a few weeks. Echocardiographic analysis of visibly healthy wild-type RhoA transgenic mice revealed no significant change in left ventricular function. As their condition deteriorated, significant dilation of the left ventricular chamber and associated decreases in left ventricular contractility were detected. Heart rate was grossly depressed in both wild-type and activated RhoA-expressing mice, even prior to the onset of ventricular failure. Electrocardiography showed evidence of atrial fibrillation and atrioventricular block. Interestingly, muscarinic receptor blockade with atropine did not elicit a positive chronotropic response in the transgenic mice. We suggest that RhoA regulates cardiac sinus and atrioventricular nodal function and that its overexpression results in bradycardia and development of ventricular failure.

Enhanced Galphaq signaling: a common pathway mediates cardiac hypertrophy and apoptotic heart failure.

Receptor-mediated Gq signaling promotes hypertrophic growth of cultured neonatal rat cardiac myocytes and is postulated to transduce in vivo cardiac pressure overload hypertrophy. Although initially compensatory, hypertrophy can proceed by unknown mechanisms to cardiac failure. We used adenoviral infection and transgenic overexpression of the alpha subunit of Gq to autonomously activate Gq signaling in cardiomyocytes. In cultured cardiac myocytes, overexpression of wild-type Galphaq resulted in hypertrophic growth. Strikingly, expression of a constitutively activated mutant of Galphaq, which further increased Gq signaling, produced initial hypertrophy, which rapidly progressed to apoptotic cardiomyocyte death. This paradigm was recapitulated during pregnancy in Galphaq overexpressing mice and in transgenic mice expressing high levels of wild-type Galphaq. The consequence of cardiomyocyte apoptosis was a transition from compensated hypertrophy to a rapidly progressive and lethal cardiomyopathy. Progression from hypertrophy to apoptosis in vitro and in vivo was coincident with activation of p38 and Jun kinases. These data suggest a mechanism in which moderate levels of Gq signaling stimulate cardiac hypertrophy whereas high level Gq activation results in cardiomyocyte apoptosis. The identification of a single biochemical stimulus regulating cardiomyocyte growth and death suggests a plausible mechanism for the progression of compensated hypertrophy to decompensated heart failure.

Tyrosine kinase and c-Jun NH2-terminal kinase mediate hypertrophic responses to prostaglandin F2alpha in cultured neonatal rat ventricular myocytes.

Myocardial infarction results in focal areas of ischemia, hypoxia, necrosis, and decreased contractile function. To compensate for loss of contractile function, remaining viable myocytes undergo hypertrophic growth. Prostaglandin F2alpha (PGF2alpha), which is released from cells of the myocardium during periods of stress such as hypoxia or ischemia/reperfusion, has recently been shown to stimulate hypertrophic growth in neonatal rat ventricular myocytes. In the present study, we determine which growth-related intracellular pathways are required for PGF2alpha to induce morphological and genetic features characteristic of the hypertrophic phenotype. In cardiomyocytes, PGF2alpha increases the hydrolysis of inositol phosphates and induces the translocation of protein kinase C epsilon to the myocyte membrane, consistent with PGF2alpha receptor coupling to Gq. PGF2alpha also activates the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase pathways. Surprisingly, studies using pharmacological inhibitors and transfection of dominant-interfering proteins demonstrate that PGF2alpha-induced myocyte hypertrophy occurs independent of either PKC, p38, or ERK pathways. Additional studies demonstrate that PGF2alpha stimulates protein tyrosine phosphorylation and activates c-Jun NH2-terminal kinase and suggest that these pathways mediate hypertrophic growth in response to PGF2alpha.

The low molecular weight GTPase Rho regulates myofibril formation and organization in neonatal rat ventricular myocytes. Involvement of Rho kinase.

The assembly of contractile proteins into organized sarcomeric units is one of the most distinctive features of cardiac myocyte hypertrophy. In a well characterized in vitro model system using cultured neonatal rat ventricular myocytes, a subset of G protein-coupled receptor agonists has been shown to induce actin-myosin filament organization. Pretreatment of myocytes with C3 exoenzyme ADP-ribosylated Rho and inhibited the characteristic alpha1-adrenergic receptor agonist-induced myofibrillar organization, suggesting involvement of the Rho GTPase in cardiac myofibrillogenesis. We used adenoviral mediated gene transfer to examine the effects of activated Rho and inhibitory mutants of one of its effectors, Rho kinase, in myocytes. Rho immunoreactivity was increased in the particulate fraction of myocytes infected with a recombinant adenovirus expressing constitutively activated Rho. Rho-infected cells demonstrated a striking increase in the assembly and organization of sarcomeric units and in the expression of the atrial natriuretic factor protein. These Rho-induced responses were markedly inhibited by co-infection with adenoviruses expressing putative dominant negative forms of Rho kinase. A parallel pathway involving Ras-induced myofibrillar organization and atrial natriuretic factor expression was only minimally affected. alpha1-Adrenergic receptor agonist-induced myofibrillogenesis was inhibited by some but not all of the Rho kinase mutants. Our data demonstrate that activated Rho has profound effects on myofibrillar organization in cardiac myocytes and suggest that Rho kinase mediates Rho-induced hypertrophic responses.

Cardiac hypertrophy induced by mitogen-activated protein kinase kinase 7, a specific activator for c-Jun NH2-terminal kinase in ventricular muscle cells.

Activation of stress-activated protein kinases, including the p38 and the c-Jun NH2-terminal kinases (JNK), have been associated with the onset of cardiac hypertrophy and cell death in response to hemodynamic overload and ischemia/reperfusion injury. Upon infection of cultured neonatal rat cardiac myocytes with recombinant adenoviral vectors expressing a wild type and a constitutively active mutant of MKK7 (or JNKK2), JNK was specifically activated without affecting other mitogen-activated protein kinases, including extracellular signal-regulated protein kinases and p38. Specific activation of the JNK pathway in cardiac myocytes induced characteristic features of hypertrophy, including an increase in cell size, elevated expression of atrial natriuretic factor, and induction of sarcomere organization. In contrast, co-activation of both JNK (by MKK7) and p38 (by MKK3 or MKK6) in cardiomyocytes led to an induction of cytopathic responses and suppression of hypertrophic responses. These data provide the first direct evidence that activation of JNK alone is sufficient to induce characteristic features of cardiac hypertrophy, thereby supporting an active role for the JNK pathway in the development of cardiac hypertrophy. The cytopathic response, as a result of co-activation of both JNK and p38, may contribute to the loss of contractile function and viability of cardiomyocytes following hemodynamic overload and cardiac ischemia/reperfusion injury.

Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family.

p38 mitogen-activated protein (MAP) kinase activities were significantly increased in mouse hearts after chronic transverse aortic constriction, coincident with the onset of ventricular hypertrophy. Infection of cardiomyocytes with adenoviral vectors expressing upstream activators for the p38 kinases, activated mutants of MAP kinase kinase 3b(E) (MKK3bE) and MAP kinase kinase 6b(E) (MKK6bE), elicited characteristic hypertrophic responses, including an increase in cell size, enhanced sarcomeric organization, and elevated atrial natriuretic factor expression. Overexpression of the activated MKK3bE in cardiomyocytes also led to an increase in apoptosis. The hypertrophic response was enhanced by co-infection of an adenoviral vector expressing wild type p38 beta, and was suppressed by the p38 beta dominant negative mutant. In contrast, the MKK3bE-induced cell death was increased by co-infection of an adenovirus expressing wild type p38 alpha, and was suppressed by the dominant negative p38 alpha mutant. This provides the first evidence in any cell system for divergent physiological functions for different members of the p38 MAP kinase family. The direct involvement of p38 pathways in cardiac hypertrophy and apoptosis suggests a significant role for p38 signaling in the pathophysiology of heart failure.

The MEKK-JNK pathway is stimulated by alpha1-adrenergic receptor and ras activation and is associated with in vitro and in vivo cardiac hypertrophy.

In neonatal rat ventricular myocytes, stimulation of the alpha1-adrenergic receptor (alpha1-AdrR) activates a program of genetic and morphological changes characterized by transcriptional activation of the atrial natriuretic factor (ANF) gene and enlargement (hypertrophy) of the cells. The low molecular weight GTPase Ras has been established as an important regulator of hypertrophy both in vitro and in vivo. Ras activates a kinase cascade involving Raf, the mitogen-activated protein kinase kinase (MEK), and the extracellular signal-regulated protein kinase (ERK). However, the extent of involvement of this pathway in regulating hypertrophic responses is controversial. We demonstrate here that both alpha1-AdrR stimulation and Ras can also activate the c-Jun NH2-terminal kinase (JNK) in cardiomyocytes. The alpha1-AdrR effect on JNK occurs through a pathway requiring Ras and MEK kinase (MEKK). A constitutively activated mutant of MEKK that preferentially activates JNK, stimulates ANF reporter gene expression, while a dominant negative MEKK mutant inhibits ANF expression induced by PE. Furthermore, JNK activity is increased in the ventricles of mice overexpressing oncogenic Ras, whereas ERK activity is not. These results suggest that the alpha1-AdrR mediates ANF gene expression through a Ras-MEKK-JNK pathway and that activation of this pathway is associated with in vitro and in vivo hypertrophy.

Pathways and roadblocks in muscarinic receptor-mediated growth regulation.

In some cell systems muscarinic receptor stimulation can induce proliferation or transformation. This phenomenon is subtype-specific (only m1 and m3 receptors are effective) and cell type dependent. In 1321N1 astrocytoma cells activation of m3 receptors stimulates phospholipase C, but does not induce DNA synthesis. In contrast the thrombin receptor, which also couples to phospholipase C, is strongly mitogenic and induces AP-1-dependent gene expression. Various experimental findings indicate that this discrepancy is not due to muscarinic receptor desensitization or blockade of growth stimulatory pathways. Muscarinic receptor number may be limiting, in particular for receptor coupling to the pertussis toxin-insensitive G-protein G12. This G-protein is required for thrombin-induced mitogenesis in 1321N1 cells and may couple selectively to the thrombin versus muscarinic receptor. In cardiomyocytes hypertrophic cell growth is induced by heterologously expressed m1 or m3 receptors but not by the endogenous m2 receptors. Studies using chimeric receptors confirm that induction of hypertrophy requires signalling through phospholipase C, but indicate that additional signals are needed to induce the morphological features of this response. We suggest that small G-proteins of the Rho subfamily, in addition to G12, mediate growth responses to G-protein-coupled receptors.

Rho is required for Galphaq and alpha1-adrenergic receptor signaling in cardiomyocytes. Dissociation of Ras and Rho pathways.

G protein-coupled receptor agonists initiate a cascade of signaling events in neonatal rat ventricular myocytes that culminates in changes in gene expression and cell growth characteristic of hypertrophy. These responses have been previously shown to be dependent on Gq and Ras. Rho, a member of the Ras superfamily of GTPases, regulates cytoskeletal rearrangement and transcriptional activation of the c-fos serum response element. Immunofluorescence staining of cardiomyocytes shows that Rho is present and predominantly cytosolic. We used two inhibitors of Rho function, dominant negative N19RhoA and Clostridium botulinum C3 transferase, to examine the possible requirement for Rho in alpha1-adrenergic receptor-mediated hypertrophy. Both inhibitors markedly attenuated atrial natriuretic factor (ANF) reporter gene expression induced by alpha1-adrenergic receptor stimulation with phenylephrine, and virtually abolished the increase in ANF reporter gene expression induced by GTPase-deficient Galphaq. These effects were reproduced with the myosin light chain-2 reporter gene. Notably, N19RhoA did not block the ability of activated Ras to induce ANF and myosin light chain-2 reporter gene expression. Furthermore, activation of the extracellular signal-regulated kinase by phenylephrine was not blocked by N19RhoA, nor was it stimulated by an activated mutant of RhoA. Since activated RhoA and Ras produce a large synergistic effect on ANF-luciferase gene expression, we conclude that Rho functions in a pathway separate from but complementary to Ras. Our results provide direct evidence that Rho is an effector of Galphaq signaling and suggest for the first time that a low molecular weight GTPase other than Ras is involved in regulating myocardial cell growth and gene expression in response to heterotrimeric G protein-linked receptor activation.

Altered cell cycle kinetics, gene expression, and G1 restriction point regulation in Rb-deficient fibroblasts.

Fibroblasts prepared from retinoblastoma (Rb) gene-negative mouse embryos exhibit a shorter G1 phase of the growth cycle and smaller size than wild-type cells. In addition, the mutant cells are no longer inhibited by low levels of cycloheximide at any point in G1 but do remain sensitive to serum withdrawal until late in G1. Certain cell cycle-regulated genes showed no temporal or quantitative differences in expression. In contrast, cyclin E expression in Rb-deficient cells is deregulated in two ways. Cyclin E mRNA is generally derepressed in mutant cells and reaches peak levels about 6 h earlier in G1 than in wild-type cells. Moreover, cyclin E protein levels are higher in the Rb-/- cells than would be predicted from the levels of its mRNA. Thus, the selective growth advantage conferred by Rb gene deletion during tumorigenesis may be explained in part by changes in the regulation of cyclin E. In addition, the mechanisms defining the restriction point of late G1 may consist of at least two molecular events, one cycloheximide sensitive and pRb dependent and the other serum sensitive and pRb independent.

A subset of p53-deficient embryos exhibit exencephaly.

Defects in neural tube formation are among the most common malformations leading to infant mortality. Although numerous genetic loci appear to contribute to the defects observed in humans and in animal model systems, few of the genes involved have been characterized at the molecular level. Mice lacking the p53 tumour suppressor gene are predisposed to tumours, but the viability of these animals indicates that p53 function is not essential for embryonic development. Here, we demonstrate that a fraction of p53-deficient embryos in fact do not develop normally. These animals display defects in neural tube closure resulting in an overgrowth of neural tissue in the region of the mid-brain, a condition known as exencephaly.