Synthesis and antimicrobial activities of structurally novel S,S'-bis(heterosubstituted) disulfides

The central focus of this study is on the antibacterial and antifungal properties of synthetically produced S,S'-bis(heterosubstituted) disulfides as a means to control the growth of various infection-causing pathogens. Staphylococcus aureus, Francisella tularensis and Candida albicans were each found to be highly susceptible to several of these compounds by agar or broth dilution and Kirby-Bauer diffusion assays. These structurally simple, low molecular weight disulfides have shown promising bioactivities and may serve as leads to the development of effective new antibacterials for pathogenic bacteria such as methicillin-resistant S. aureus and F. tularensis.     

Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy

Sphingolipids are comprised of a backbone sphingoid base that may be phosphorylated, acylated, glycosylated, bridged to various headgroups through phosphodiester linkages, or otherwise modified. Organisms usually contain large numbers of sphingolipid subspecies and knowledge about the types and amounts is imperative because they influence membrane structure, interactions with the extracellular matrix and neighboring cells, vesicular traffic and the formation of specialized structures such as phagosomes and autophagosomes, as well as participate in intracellular and extracellular signaling. Fortunately, "sphingolipidomic" analysis is becoming feasible (at least for important subsets such as all of the backbone "signaling" subspecies: ceramides, ceramide 1-phosphates, sphingoid bases, sphingoid base 1-phosphates, inter alia) using mass spectrometry, and these profiles are revealing many surprises, such as that under certain conditions cells contain significant amounts of "unusual" species: N-mono-, di-, and tri-methyl-sphingoid bases (including N,N-dimethylsphingosine); 3-ketodihydroceramides; N-acetyl-sphingoid bases (C2-ceramides); and dihydroceramides, in the latter case, in very high proportions when cells are treated with the anticancer drug fenretinide (4-hydroxyphenylretinamide). The elevation of DHceramides by fenretinide is befuddling because the 4,5-trans-double bond of ceramide has been thought to be required for biological activity; however, DHceramides induce autophagy and may be important in the regulation of this important cellular process. The complexity of the sphingolipidome is hard to imagine, but one hopes that, when partnered with other systems biology approaches, the causes and consequences of the complexity will explain how these intriguing compounds are involved in almost every aspect of cell behavior and the malfunctions of many diseases.     

VILIP-1 Expression In Vivo Results in Decreased Mouse Skin Keratinocyte Proliferation and Tumor Development

VILIP-1, a member of the neuronal Ca²⁺ sensor protein family, is able to act as a tumor suppressor in carcinoma cells by inhibiting cell proliferation and migration. In order to study the role of VILIP-1 in skin carcinogenesis we generated transgenic mice overexpressing VILIP-1 in epidermis under the control of the bovine keratin K5 promoter (K5-VILIP-1). We studied the susceptibility of FVB wild type and VILIP-1 transgenic mice to chemically mediated carcinogenesis. After 30 weeks of treatment with a two-stage carcinogenesis protocol, all animals showed numerous skin tumors. Nevertheless, K5-VILIP-1 mice showed decreased squamous cell carcinoma (SCC) multiplicity of ∼49% (p<0.02) with respect to the corresponding SCC multiplicity observed in wild type (WT) mice. In addition, the relative percentage of low-grade cutaneous SCCs grade I (defined by the differentiation pattern according to the Broders grading scale) increased approximately 50% in the K5-VILIP1 mice when compared with SCCs in WT mice. Similar tendency was observed using a complete carcinogenesis protocol for skin carcinogenesis using benzo(a)pyrene (B(a)P). Further studies of tumors and primary epidermal keratinocyte cultures showed that matrix metalloproteinase 9 (MMP-9) levels and cell proliferation decreased in K5-VILIP-1 mice when compared with their wild counterparts. In addition tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was higher in K5-VILIP-1 keratinocytes. These results show that VILIP-1 overexpression decreases the susceptibility to skin carcinogenesis in experimental mouse cancer models, thus supporting its role as a tumor suppressor gene.     

Nested and TaqMan® probe based quantitative PCR for the diagnosis of Ca. Phytoplasma in coconut palms

The root (wilt) disease caused by phytoplasma (Ca. Phytoplasma) is one of the major and destructive occurs in coconut gardens of Southern India. As this organism could not be cultured in vitro, the early detection in the palm is very much challenging. Hence, proper early diagnosis and inoculum assessment relay mostly on the molecular techniques namely nested and quantitative PCR (qPCR). So, the present study qPCR assay conjugated with TaqMan^® probe was developed which is a rapid, sensitive method to detect the phytoplasma. For the study, samples from different parts of infected coconut palms viz., spindle leaflets, roots and the insect vector—leaf hopper (Proutista moesta) were collected and assessed by targeting 16S rRNA gene. Further, nested PCR has been carried out using p1/p7 and fU5/rU3 primers and resulted in the amplification product size of 890 bp. From this amplified product, specifically a target of 69 bp from the 16S rRNA gene region has been detected through primers conjugated with Taqman probe in a step one instrument. The results indicated that the concentration of phytoplasma was more in spindle leaflets (8.9?×?10⁵ g of tissue) followed by roots (7.4?×?10⁵ g of tissue). Thus, a qPCR approach for detection and quantification of coconut phytoplasma was more advantageous than other PCR methods in terms of sensitivity and also reduced risk of cross contamination in the samples. Early diagnosis and quantification will pave way for the healthy coconut saplings selection and management under field conditions.

     

PCL-Based Composite Scaffold Matrices for Tissue Engineering Applications

Biomaterial-based scaffolds are important cues in tissue engineering (TE) applications. Recent advances in TE have led to the development of suitable scaffold architecture for various tissue defects. In this narrative review on polycaprolactone (PCL), we have discussed in detail about the synthesis of PCL, various properties and most recent advances of using PCL and PCL blended with either natural or synthetic polymers and ceramic materials for TE applications. Further, various forms of PCL scaffolds such as porous, films and fibrous have been discussed along with the stem cells and their sources employed in various tissue repair strategies. Overall, the present review affords an insight into the properties and applications of PCL in various tissue engineering applications.     

Chronically elevated glucose-induced apoptosis is mediated by inactivation of Akt in cultured Müller cells

Hyperglycemia induces apoptotic cell death in a variety of cell types in diabetes, and the mechanism remains unclear. We report here that culture of rat retinal glial Müller cells in 25 mM glucose for 72 h significantly inactivated Akt and induced apoptosis. Likewise, hyperglycemia caused a significant dephosphorylation of Akt at serine-473 in Müller cells in the retina of streptozotocin-induced diabetic rats. Inactivation of Akt was associated with dephosphorylation of BAD, increased cytochrome c release, and activation of caspase-3 and caspase-9. Upregulation of Akt activity by overexpression of constitutively active Akt inhibited elevated glucose-induced apoptosis, whereas downregulation of Akt activity by overexpression of dominant negative Akt exacerbated elevated glucose-induced apoptosis, as assessed by caspase activity and nucleic acid staining. These data suggest that apoptosis induced by chronically elevated glucose is at least in part mediated by downregulation of Akt survival pathway in cultured Müller cells. It has been reported that antiapoptotic effect of Akt requires glucose in growth factor withdrawal-induced apoptosis. Our data suggest that although acutely elevated glucose may be beneficial to the cell survival, chronically elevated glucose can cause apoptosis via downregulation of Akt survival signaling.     

Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells

Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G(1) to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E(276-395) (where cyclin E(276-395) is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E(1-275), cyclin E(276-395) deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E(276-395), but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.     

Anti-C5 antibody treatment ameliorates weakness in experimentally acquired myasthenia gravis

Myasthenia gravis (MG) is a neuromuscular transmission disorder in which damage to acetylcholine receptors (AChR) on motor endplates by autoantibody-induced complement attack causes muscle weakness. To determine whether and, if so, to what extent, blockade of complement cascade at the C5 step ameliorates disease, we evaluated the effect of administering a functionally blocking anti-C5 mAb in passive experimental MG in Lewis rats induced with AChR Ab McAb-3. In contrast to uniform severe weakness at 24 h requiring euthanasia in untreated animals, anti-C5 mAb-pretreated rats showed no weakness at 48 h. Anti-C5 mAb treatment 24 h after disease induction restored strength in two-thirds of the rats. Immunofluorescence staining of endplates from the treated animals showed that C9 deposition at AChR was reduced and ultrastructural analyses showed that endplates were intact. The results argue that targeting C5 may warrant testing in MG patients and that this approach may be particularly valuable for myasthenic crisis.     

Tissue-specific expression of PPAR mRNAs in diabetic rats and divergent effects of cilostazol

Cilostazol and ligands of peroxisome proliferator-activated receptors (PPARs) have been effectively used to alleviate diabetic complications, but the common and tissue-specific expression patterns of PPARs in different tissues in diabetic patients and those treated with cilostazol have not been reported. Here, we aimed to assess the effects of diabetes and cilostazol on mRNA expression of PPARalpha and PPARgamma in the aorta, renal cortex, and retina of diabetic rats treated with cilostazol for 8 weeks. PPARalpha mRNA expression showed uniform downregulation in all these tissues in diabetic rats, and this effect was reversed by cilostazol treatment. Surprisingly, PPARgamma mRNA expression was reduced in the renal cortex and retina, yet increased in the aorta of diabetic rats, although cilostazol still reversed these changes. Interestingly, cilostazol, a well-known phosphodiesterase 3 inhibitor and cAMP elevator, augmented cAMP content only in the aorta, but showed no significant effects in the renal cortex of diabetic rats. In conclusion, mRNA expression of PPARs is tissue-specific in diabetes and may be differently affected by cilostazol, possibly because of its tissue-specific effects on cAMP content.