Conformational analysis of a 20-membered cyclic peptide disulfide from Conus virgo with a WPW segment: evidence for an aromatic-proline sandwich.

A novel peptide containing a single disulfide bond, CIWPWC (Vi804), has been isolated and characterised from the venom of the marine cone snail, Conus virgo. A precursor polypeptide sequence derived from complementary DNA, corresponding to the M-superfamily conotoxins, has been identified. The identity of the synthetic and natural peptide sequence has been established. A detailed analysis of the conformation in solution is reported for Vi804 and a synthetic analogue, CI(D) WPWC ((D) W3-Vi804), in order to establish the structure of the novel WPW motif, which occurs in the context of a 20-membered macrocyclic disulfide. Vi804 exists exclusively in the cis W3P4 conformer in water and methanol, whereas (D) W3-Vi804 occurs exclusively as the trans conformer. NMR spectra revealed a W3P4 type VI ß turn in Vi804 and a type II' ß turn in the analogue peptide, (D) W3-Vi804. The extremely high-field chemical shifts of the proline ring protons, together with specific nuclear Overhauser effects, are used to establish a conformation in which the proline ring is sandwiched between the flanking Trp residues, which emphasises a stabilising role for the aromatic-proline interactions, mediated predominantly by dispersion forces.

Targeting mitochondrial dynamics and redox regulation in cardiovascular diseases.

Accumulating evidence highlights the pivotal role of mitochondria in cardiovascular diseases (CVDs). Understanding the molecular mechanisms underlying mitochondrial dysfunction is crucial for developing targeted therapeutics. Recent years have seen substantial advancements in unraveling mitochondrial regulatory pathways in both normal and pathological states and the development of potent drugs. However, specific delivery of drugs into the mitochondria is still a challenge. We present recent findings on regulators of mitochondrial dynamics and reactive oxygen species (ROS), critical factors influencing mitochondrial function in CVDs. We also discuss advancements in drug delivery strategies aimed at overcoming the technical barrier in targeting mitochondria for CVD treatment.

Neurogranin expression regulates mitochondrial function and redox balance in endothelial cells.

Endothelial dysfunction and endothelial activation are common early events in vascular diseases and can arise from mitochondrial dysfunction. Neurogranin (Ng) is a 17kD protein well known to regulate intracellular Ca2+-calmodulin (CaM) complex signaling, and its dysfunction is significantly implicated in brain aging and neurodegenerative diseases. We found that Ng is also expressed in human aortic endothelial cells (HAECs), and depleting Ng promotes Ca2+-CaM complex-dependent endothelial activation and redox imbalances. Endothelial-specific Ng knockout (Cre-CDH5-Ngf/f) mice demonstrate a significant delay in the flow-mediated dilation (FMD) response. Therefore, it is critical to characterize how endothelial Ng expression regulates reactive oxygen species (ROS) generation and affects cardiovascular disease. Label-free quantification proteomics identified that mitochondrial dysfunction and the oxidative phosphorylation pathway are significantly changed in the aorta of Cre-CDH5-Ngf/f mice. We found that a significant amount of Ng is expressed in the mitochondrial fraction of HAECs using western blotting and colocalized with the mitochondrial marker, COX IV, using immunofluorescence staining. Seahorse assay demonstrated that a lack of Ng decreases mitochondrial respiration. Treatment with MitoEbselen significantly restores the oxygen consumption rate in Ng knockdown cells. With the RoGFP-Orp1 approach, we identified that Ng knockdown increases mitochondrial-specific hydrogen peroxide (H2O2) production, and MitoEbselen treatment significantly reduced mitochondrial ROS (mtROS) levels in Ng knockdown cells. These results suggest that Ng plays a significant role in mtROS production. We discovered that MitoEbselen treatment also rescues decreased eNOS expression and nitric oxide (NO) levels in Ng knockdown cells, which implicates the critical role of Ng in mtROS-NO balance in the endothelial cells.

Conformational diversity in contryphans from Conus venom: cis-trans isomerisation and aromatic/proline interactions in the 23-membered ring of a 7-residue peptide disulfide loop.

Conformational diversity or "shapeshifting" in cyclic peptide natural products can, in principle, confer a single molecular entity with the property of binding to multiple receptors. Conformational equilibria have been probed in the contryphans, which are peptides derived from Conus venom possessing a 23-membered cyclic disulfide moiety. The natural sequences derived from Conus inscriptus, GCV(D)LYPWC* (In936) and Conus loroisii, GCP(D)WDPWC* (Lo959) differ in the number of proline residues within the macrocyclic ring. Structural characterisation of distinct conformational states arising from cis-trans equilibria about Xxx-Pro bonds is reported. Isomerisation about the C2-P3 bond is observed in the case of Lo959 and about the Y5-P6 bond in In936. Evidence is presented for as many as four distinct species in the case of the synthetic analogue V3P In936. The Tyr-Pro-Trp segment in In936 is characterised by distinct sidechain orientations as a consequence of aromatic/proline interactions as evidenced by specific sidechain-sidechain nuclear Overhauser effects and ring current shifted proton chemical shifts. Molecular dynamics simulations suggest that Tyr5 and Trp7 sidechain conformations are correlated and depend on the geometry of the Xxx-Pro bond. Thermodynamic parameters are derived for the cis↔trans equilibrium for In936. Studies on synthetic analogues provide insights into the role of sequence effects in modulating isomerisation about Xxx-Pro bonds.

Multi-modal fusion of deep transfer learning based COVID-19 diagnosis and classification using chest x-ray images.

COVID-19 pandemic has a significant impact on the global health and daily lives of people living over the globe. Several initial tests are based on the detecting of the genetic material of the coronavirus, and they have a minimum detection rate with a time-consuming process. To overcome this issue, radiological images are recommended where chest X-rays (CXRs) are employed in the diagnostic process. This article introduces a new Multi-modal fusion of deep transfer learning (MMF-DTL) technique to classify COVID-19. The proposed MMF-DTL model involves three main processes, namely pre-processing, feature extraction, and classification. The MMF-DTL model uses three DL models namely VGG16, Inception v3, and ResNet 50 for feature extraction. Since a single modality would not be adequate to attain an effective detection rate, the integration of three approaches by the use of decision-based multimodal fusion increases the detection rate. So, a fusion of three DL models takes place to further improve the detection rate. Finally, a softmax classifier is employed for test images to a set of six different. A wide range of experimental result analyses is carried out on the Chest-X-Ray dataset. The proposed fusion model is found to be an effective tool for COVID-19 diagnosis using radiological images with the average sens y of 92.96%, spec y of 98.54%, prec n of 93.60%, accu y of 98.80%, F score of 93.26% and kappa of 91.86%.

Nicotinamide nucleotide transhydrogenase (NNT) regulates mitochondrial ROS and endothelial dysfunction in response to angiotensin II.

Endothelial dysfunction is a critical, initiating step in the development of hypertension (HTN) and mitochondrial reactive oxygen species (ROS) are important contributors to endothelial dysfunction. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in the nicotinamide nucleotide transhydrogenase (Nnt) gene that are associated with endothelial dysfunction and increased risk for HTN. NNT is emerging as an important enzyme that regulates mitochondrial NADPH levels and mitochondrial redox balance by supporting the thiol dependent peroxidase systems in the mitochondria. We have previously shown that the absence of NNT in C57Bl/6J animals promotes a more severe hypertensive phenotype through reductions in •NO and endothelial dependent vessel dilation. However, the impact of NNT on human endothelial cell function remains unclear. We utilized NNT directed shRNA in human aortic endothelial cells to test the hypothesis that NNT critically regulates mitochondrial redox balance and endothelial function in response to angiotensin II (Ang II). We demonstrate that NNT expression and activity are elevated in response to the mitochondrial dysfunction and oxidative stress associated with Ang II treatment. Knockdown of NNT led to a significant elevation of mitochondrial ROS production and impaired glutathione peroxidase and glutathione reductase activities associated with a reduction in the NADPH/NADP+ ratio. Loss of NNT also promoted mitochondrial dysfunction, disruption of the mitochondrial membrane potential, and impaired ATP production in response to Ang II. Finally, we observed that, while the loss of NNT augmented eNOS phosphorylation at Ser1177, neither eNOS activity nor nitric oxide production were similarly increased. The results from these studies clearly demonstrate that NNT is critical for the maintenance of mitochondrial redox balance and mitochondrial function. Loss of NNT and disruption of redox balance leads to oxidative stress that compromises eNOS activity that could have a profound effect on the endothelium dependent regulation of vascular tone.

Stearic acid methyl ester affords neuroprotection and improves functional outcomes after cardiac arrest.

Cardiac arrest causes neuronal damage and functional impairments that can result in learning/memory dysfunction after ischemia. We previously identified a saturated fatty acid (stearic acid methyl ester, SAME) that was released from the superior cervical ganglion (sympathetic ganglion). The function of stearic acid methyl ester is currently unknown. Here, we show that SAME can inhibit the detrimental effects of global cerebral ischemia (i.e. cardiac arrest). Treatment with SAME in the presence of asphyxial cardiac arrest (ACA) revived learning and working memory deficits. Similarly, SAME-treated hippocampal slices after oxygen-glucose deprivation inhibited neuronal cell death. Moreover, SAME afforded neuroprotection against ACA in the CA1 region of the hippocampus, reduced ionized calcium-binding adapter molecule 1 expression and inflammatory cytokines/chemokines, with restoration in mitochondria respiration. Altogether, we describe a unique and uncharted role of saturated fatty acids in the brain that may have important implications against cerebral ischemia.

Bowman-Birk protease inhibitor from the seeds of Vigna unguiculata forms a highly stable dimeric structure.

Different protease inhibitors including Bowman-Birk type (BBI) have been reported from the seeds of Vigna unguiculata. Protease isoinhibitors of double-headed Bowman-Birk type from the seeds of Vigna unguiculata have been purified and characterized. The BBI from Vigna unguiculata (Vu-BBI) has been found to undergo self-association to form very stable dimers and more complex oligomers, by size-exclusion chromatography and SDS-PAGE in the presence of urea. Many BBIs have been reported to undergo self-association to form homodimers or more complex oligomers in solution. Only one dimeric crystal structure of a BBI (pea-BBI) is reported to date. We report the three-dimensional structure of a Vu-BBI determined at 2.5 A resolution. Although, the inhibitor has a monomer fold similar to that found in other known structures of Bowman-Birk protease inhibitors, its quaternary structure is different from that commonly observed in this family. The structural elements responsible for the stability of monomer molecule and dimeric association are discussed. The Vu-BBI may use dimeric or higher quaternary association to maintain the physiological state and to execute its biological function.

Ethnobotanical survey of plants used to treat asthma in Andhra Pradesh, India.

Tribal and non-tribal inhabitants of Andhra Pradesh used nearly 80 medicinal plants for treating asthma. The tribal people have a strong faith and belief in the traditional health care system, through herbal treatment. Plant species are generally used along with other materials and plant products in different combinations to effective cure. Herbalists reported that plant ingredients are used in the form of dry powder, decoction and juice in the treatment of asthma. The knowledge of most asthma drug plants used in herbal treatment and their method of using them are confined to some of the local healers. Some of the plants mentioned by local healers however, are extensively used nationally in the preparation of Ayurvedic medicines including those to treat asthma. Clinical and pharmacological data are available for these plants. Most of the plants used for treating asthma by local herbalists appear not to have been recorded hitherto.

Lipid composition and de novo cholesterogenesis in normal and neoplastic rat mammary tissues.

Female Wistar rats were given 5 mg of 7,12-dimethylbenz[a]anthracene (DMBA) and fed either a control diet (AIN), a 4% cholestyramine (CHST) diet, a 2% corn oil plus 18% coconut oil (saturated fat) diet, a 20% corn oil [unsaturated fatty acid (USF)] diet, or a USF + 4% cholestyramine (USF + CHST) diet. The mammary glands, tumors, livers, and sera were analyzed for lipids, de novo cholesterogenesis, and serum lecithin-cholesterol acyltransferase (LCAT) activity levels. Level and type of fat in the diet, DMBA, CHST, and length of feeding influenced the lipid composition of liver and mammary tissues. Stimulation of de novo cholesterogenesis in the mammary gland and depression in circulating LCAT activity levels correlated with the incidence and growth of mammary tumors, suggesting that stimulation of de novo cholesterogenesis plays an important role in mammary cancer development.

Acinar cell carcinoma of the rat pancreas grown in cell culture and in nude mice.

An acinar cell carcinoma of the pancreas, which was induced in a male Wistar rat by repeated injections of azaserine, was propagated in cell culture. Sheets of epithelial-like cells grew within 2 weeks and were subcultured serially. Initially acinar cell carcinoma in the culture medium produced a high level of amylase, but the secretion ceased rapidly as cells began to proliferate. Only negligible amounts of trypsinogen and chymotrypsinogen were detected in cell homogenates at passages 7 and 9. The chromosome distribution ranged from hypodiploid to hypertetraploid. When cultured cells were transplanted s.c. into nude mice, palpable tumors appeared within 4 weeks and could be transplanted serially. Histological examination of the tumor showed poorly differentiated carcinoma without acinar structures. Tumor homogenate contained amylase, trypsinogen, and chymotrypsinogen, and the electron microscopic examination revealed that many tumor cells contained zymogen-like granules. These results indicate that pancreatic acinar cell carcinoma in cell cultures, in which there was no differentiated function, can be activated to synthesize tissue-specific enzymes when transplanted into nude mice by yet undefined factors present in the host animals. The cell line and transplantable tumors of pancreatic acinar cell carcinoma may be useful in the analysis of the biological behavior of this type of tumor and in the study of the control mechanisms of the synthesis of tissue-specific products in cells.

Genetic control of susceptibility to diethylnitrosamine carcinogenesis in inbred ACP (grc+) and R16 (grc) rats.

The R16 strain, which carries the major histocompatibility complex-linked growth and reproduction complex (grc), and its normal counterpart, the ACP strain, were initiated at 8 wk of age with a single i.p. dose of diethylnitrosamine (DEN), and 2 wk later they were fed either a choline-deficient (CD) or a choline-supplemented (CS) diet. The rats were sacrificed 2, 4, 6, 10, and 12 mo later; complete autopsies were performed, and all of the tissues were examined histologically. Sections of the liver were also examined histochemically for gamma-glutamyl transpeptidase activity. Shortly after the administration of DEN, the R16 strain showed a significant increase in the number and size of gamma-glutamyl transpeptidase-positive foci and more severe histological changes (disruption of the lobular architecture, bile duct and oval cell proliferation, cellular atypia, and accumulation of fat) compared with the ACP strain. These changes occurred in animals fed either CD or CS diet, but they were much more extensive and severe in the animals on the CD diet. They did not occur in rats of either strain fed the diets alone. The first hepatocellular carcinoma appeared in the R16 rats on the CD diet at 4 mo after administration of the DEN and on the CS diet, at 10 mo. The only hepatocellular carcinoma that occurred in the ACP rats did so at 12 mo in one animal on the CD diet. Combining the data at 10 and 12 mo for the rats on the CD diet, 50% (20 of 40) of the R16 rats had hepatocellular carcinomas, whereas only 3% (one of 30) of ACP rats did. The R16 strain (22%, 9 of 40), but not the ACP strain (0 of 30), also had a variety of other malignancies: squamous cell carcinomas (8%); renal cell carcinomas (8%); lymphomas (5%); and pheochromocytoma (3%). A similar pattern of malignancies also occurred in the R16 rats on the CS diet, and there were no malignancies in the ACP rats. These observations indicate that the grc confers unusual susceptibility to the induction of cancer by the chemical carcinogen DEN and that this genetic susceptibility to cancer of the R16 strain extends beyond the primary target organ of the carcinogen used.

Studies on the enzymatic hydrolysis of polyglutamyl folates by chicken liver folyl poly-gamma-glutamyl carboxypeptidase. I. Intracellular localization, purification and partial characterization of the enzyme.

Intracellular distribution, purification and properties of a folyl poly-gamma-glutamyl carboxypeptidase (peptidyl-L-glutamate hydrolase, EC 3.4.12.10) from chicken liver have been investigated. The post-nuclear particulate and cell supernatant fractions showed activity. The particulate enzyme exhibited characteristics suggestive of its lysosomal origin; on solubilization, however, it cannot be distinguished from the cell cytosol activity. The bulk enzyme was purified about 80-fold to apparent homogeneity by 50--90% ammonium sulfate fractional precipitation, dialysis and chromatography on a 'mixed column' of Sephadex G-100 superimposed on CM-Sephadex C-50. The purified enzyme behaved homogeneously on Sephadex G-100 chromatography, sucrose density gradient centrifugation analyses and polyacrylamide gel electrophoresis. However, polyacrylamide gel electrophoresis in the presence of mercaptoethanol dissociated the native enzyme into two separable isoenzymic components. The enzyme exhibits two pH optima (4.1 and 5.2) and a temperature optimum of 35--40 degrees C. The reaction is linear for 20 min. The enzyme sequentially cleaves the terminal gamma-glutamyl residues of polyglutamylfolates, finally releasing a monoglutamyl end-product. An apparent Km value of 0.83 - 10(-6) M and a V of 1.50 mmol/min were determined for N5-methyltetrahydropteroyltetraglutamate with 0.20 mg enzyme protein/ml reaction. The enzyme is substantially stimulated in the presence of mercaptoethanol, Na+, Mn2+ and low concentrations of denaturing agents (urea). Citrate potently inhibits and phosphate inactivates the enzyme.

Studies on the enzymatic hydrolysis of polyglutamyl folates by chicken liver folyl poly-gamma-glutamyl carboxypeptidase. II. Structural studies.

Further studies on the purified chicken hepatic folyl poly-gamma-glutamyl carboxypeptidase (peptidyl-L-glutamate hydrolase, EC 3.4.12.10) have elucidated some of the structural characteristics of the enzyme. Various analytical studies described reveal 424 amino acid residues in the isolated native enzyme with molecular weight of around 57 900. beta-Mercaptoethanol (14.3 mM) activated the enzyme 2.2-fold and induced reductive cleavage of an interchain disulfide linkage resulting in the splitting of the native enzyme into two active polypeptides (molecular weights 43 000 and 18 000). The constituent polypeptides have identical NH2-terminal residues (valine) and exhibit a high degree of sequence homology as revealed by finger print analyses of their tryptic digests. The 10-fold greater sensitivity of the reductively cleaved enzyme to p-chloromercuribenzoate would imply that active site related sulfhydryl groups are not readily accessible in the native enzyme. Ionic strength effects in the presence of Mn2+ and Na+ and the presence of low urea concentration (0.55 M) result in a further up to 5-fold stimulation of reductively cleaved native enzyme. Citrate inhibited and phosphate induced autolytic degradation of the enzyme. The physiological role of gamma-glutamyl carboxypeptidase has been discussed.

Lipid composition and 3-hydroxy-3-methylglutaryl-CoA reductase activity of acinar cell carcinoma of rat pancreas.

The lipid composition and 3-hydroxy-3-methylglutaryl-CoA reductase activity of subcutaneous transplantable pancreatic acinar cell tumors on nude mice were compared with those of normal, regenerating, fetal and newborn rat pancreata. The tumors and also the fetal tissues showed decreased concentration in total lipids, increased concentration in sphingomyelin and an increase in cholesterol when compared to normal rat pancreas. The regenerating pancreas showed an intermediate elevation in these lipid parameters. Specifically, only tumor showed an increase in phosphatidylethanolamine/phosphatidylcholine ratio. The tumors and also the fetal tissues showed an increase in hydroxymethylglutaryl-CoA reductase activities, suggesting that the de novo synthesis of cholesterol is a requirement for cell proliferation. The cholesterol metabolism in normal tissues is under metabolic regulation as indicated by decreased hydroxymethylglutaryl-CoA reductase activities and decreased cholesterol concentration in postnatal tissues when compared with the fetal tissues. The fast growing AT3A tumor showed higher hydroxymethylglutaryl-CoA reductase activity when compared to the slow growing AT3B tumor, indicating that the differences in growth rate of the tumors may be related at least in part to differences in their cholesterol metabolism.

Biocompatible and Antibacterial SnO2 Nanowire Films Synthesized by E-Beam Evaporation Method.

In this work, the biocompatibility and antibacterial activities of novel SnO2 nanowire coatings prepared by electron-beam (E-Beam) evaporation process at low temperatures were studied. The nanowire coatings were characterized by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and X-ray diffraction (XRD) methods. The results of in vitro cytotoxicity and cell proliferation assays suggested that the SnO2 nanowire coatings were nontoxic and promoted the proliferation of C2C12 and L929 cells (> 90% viability). Cellular activities, cell adhesion, and lactate dehydrogenase activities were consistent with the superior biocompatibility of the nanowire materials. Notably, the nanowire coating showed potent antibacterial activity against six different bacterial strains. The antibacterial activity of the SnO2 material was attributed to the photocatalytic nature of SnO2. The antibacterial activity and biocompatibility of the newly developed SnO2 nanowire coatings may enable their use as coating materials for biomedical implants.

Novel euglycemic and hypolipidemic agents. 1.

A series of [[(heterocyclyl)ethoxy]benzyl]-2,4-thiazolidinediones have been synthesized by the condensation of corresponding aldehyde 1 and 2,4-thiazolidinedione followed by hydrogenation. Both unsaturated thiazolidinedione 2 and its saturated counterpart 3 have shown antihyperglycemic activity. Many of these compounds have shown superior euglycemic and hypolipidemic activity compared to troglitazone (CS 045). The indole analogue DRF-2189 (3g) was found to be a very potent insulin sensitizer, comparable to BRL-49653 in genetically obese C57BL/6J-ob/ob and 57BL/KsJ-db/db mice. Pharmacokinetic and tissue distribution studies conducted on BRL-49653 and DRF-2189 (3g) indicate that these drugs are well-distributed in target tissues. On the basis of euglycemic activity as well as enhanced selectivity against reduction of triglycerides in plasma, DRF-2189 (3g) has been selected for further evaluation.

Alterations in glycoproteins and lipids in azaserine-induced acinar cell carcinoma of rat pancreas.

Glycoproteins and lipids of rat pancreatic acinar cell carcinomas maintained in nude mice and in cell culture, were analyzed. The tumor contained significantly elevated levels of glycoproteins when compared with their normal counterparts. SDS-PAGE of tumor glycoproteins revealed that there were increased amounts of small molecular weight glycoproteins and the tumor also contained a 51,000 dalton glycoprotein which was not detected in the pancreas, liver or the sera of the control animals. The tumor in nude mice and cancer cells in culture had decreased lecithins and triglycerides, and increased amounts of free fatty acids, and both free and esterified cholesterols. The results indicate that altered glycoprotein and lipid compositions represent some of the characteristic features of the acinar cell carcinoma.

Biomarkers of liver regeneration allow early prediction of hepatic recovery after acute necrosis.

Acute toxic hepatic necrosis is common and may be fatal. Predicting clinical outcome may be aided by following serum markers that could indicate recovery or may signify massive (substantial) destruction of functional liver mass. Previously, in a published case of chloroform poisoning, we serially assayed serum biomarkers of hepatocellular necrosis (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase) and markers of hepatocellular regeneration (alpha-fetoprotein, retinol-binding protein, gamma-glutamyl transferase, and des-gamma-carboxyprothrombin). We noted a decline in necrotic markers and a synchronous elevation in regenerative markers, which could be suggestive of a favorable outcome in similar cases. We now report 6 Amanita mushroom poisonings with favorable outcome and 2 fatal acetaminophen poisonings in which the same markers were observed. Our results further support our hypothesis that a sustained decline in serum markers of hepatocyte necrosis with a concurrent elevation in regenerative markers could aid in prediction of favorable outcome in patients with acute liver injury.