Bactericidal activity of different oxovanadium(IV) complexes with Schiff bases and application of chelation theory

Oxovanadium(IV) complexes have been synthesized and characterized the general composition [VOL(A)], where H₂L = salicylidene-o-aminothiophenol A¹ = bis(benzylidene)ethylenediamine, A² = bis(acetophenone)ethylenediamine, A³ = 2,2′-bipyridylamine, A⁴ = bis(benzylidene) ? 1,8-diaminonaphthalene, A⁵ = thiophene-o-carboxaldeneaniline and A⁶ = thiophene-o-carboxaldene-p-anisidine. Spectral studies indicate that the oxovanadium(IV) complexes assume a six-coordinate octahedral geometry. The antibacterial activities of the complexes against Salmonella typhi, Escherichia coli and Serratia mercescens are higher as compared to the free ligands, vanadyl sulphate, and the control (DMSO) but of moderate activity as compared to the standard drug (tetracycline).     

An innovative approach for the characterization of the isoforms of a monoclonal antibody product

Protein biopharmaceuticals, such as monoclonal antibodies (mAbs) are widely used for the prevention and treatment of various diseases. The complex and lengthy upstream and downstream production methods of the antibodies make them susceptible to physical and chemical modifications. Several IgG1 immunoglobulins are used as medical agents for the treatment of colon, breast and head and neck cancers, and at least four to eight isoforms exist in the products. The regulatory agencies understand the complex nature of the antibody molecules and allow the manufactures to set their own specifications for lot release, provided the safety and efficacy of the products are established in animal models prior to clinical trials. During the manufacture of a mAb product, we observed lot-to-lot variability in the isoform content and, although the variability is within the set specifications for lot release, made attempts to gain mechanistic insight by isolating and characterizing the individual isoforms. Matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography (LC)/mass spectrometry (MS)/MS analyses of the isolated isoforms indicate that this variability is caused by sialic acid content, as well as truncation of C-terminal lysine of the individual isoforms. Sialidase and carboxypeptidase treatment of the product confirm the observations made by MALDI and LC/MS/MS.Key words: IgG1, isoforms, charge heterogeneity, monoclonal antibody, glycosylation, silaic acidMonoclonal antibodies (mAbs) are used as medical agents to treat a variety of diseases including cancer, cardiovascular diseases and blood disorders.^1–3 Although a few IgG2 (e.g., panitumumab, denosumab) and IgG4 antibody molecules are in the market, most of the approved products are IgG1 molecules. IgG1 antibodies are glycoproteins with a conserved N-glycosylation site at Asn 297. Glycosylation influences the biological functions, such as antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) of the antibodies. The oligosaccharides present in the IgG1 molecules are heterogeneous due to the presence of various sugar residues, including sialic acid, galactose, N-acetylglucasmine and fucose residues. Molecular alterations in antibodies can take place at every stage of manufacturing: upstream and downstream processing, formulation and storage. These alterations can take place enzymatically or non-enzymatically and may produce charge or size heterogeneity. Deamidation, proteolytic fragmentation, oxidation, disulfide bond shuffling and glycosylation are the most common modifications that occur during the production of protein therapeutics.^4–7 These modifications can reduce the biological activity and may induce immunogenicity in patients. Hence, the regulatory agencies require a comprehensive characterization of the structural integrity, purity and stability of the protein therapeutics.⁸To date, eight chimeric, humanized and human IgG1 mAbs have been approved in the United States, Europe, as well as other countries, for the treatment of several types of cancers.^9–12 One such molecule produced at ImClone has two N-glycosylation sites and at least six to eight isoforms with isoelectric points (pIs) between 7.9–8.9 are present in this product. Although techniques such as ion exchange chromatography (IEX) and capillary isoelectic focusing (IEF) are available for the separation and characterization of charge varients,^13,14 we were not successful in separating the individual isoforms with these techniques from the IgG1 product used in this investigation. The peaks from IEX showed the presence of multiple bands on IEF. Hence, an alternative approach was used to isolate each isoform of this IgG1 product, and we demonstrated the involvement of sialic acid and C-terminal lysine as the root causes for lot-to-lot variation observed during the production of this molecule. The method is fast and very effective in separating isoforms with a difference in the pI values < 0.1.     

Invariant natural killer T cells: bridging innate and adaptive immunity

Cells of the innate immune system interact with pathogens via conserved pattern-recognition receptors, whereas cells of the adaptive immune system recognize pathogens through diverse, antigen-specific receptors that are generated by somatic DNA rearrangement. Invariant natural killer T (iNKT) cells are a subset of lymphocytes that bridge the innate and adaptive immune systems. Although iNKT cells express T cell receptors that are generated by somatic DNA rearrangement, these receptors are semi-invariant and interact with a limited set of lipid and glycolipid antigens, thus resembling the pattern-recognition receptors of the innate immune system. Functionally, iNKT cells most closely resemble cells of the innate immune system, as they rapidly elicit their effector functions following activation, and fail to develop immunological memory. iNKT cells can become activated in response to a variety of stimuli and participate in the regulation of various immune responses. Activated iNKT cells produce several cytokines with the capacity to jump-start and modulate an adaptive immune response. A variety of glycolipid antigens that can differentially elicit distinct effector functions in iNKT cells have been identified. These reagents have been employed to test the hypothesis that iNKT cells can be harnessed for therapeutic purposes in human diseases. Here, we review the innate-like properties and functions of iNKT cells and discuss their interactions with other cell types of the immune system.     

Synthesis and biological evaluation of 4-styrylcoumarin derivatives as inhibitors of TNF-α and IL-6 with anti-tubercular activity

A series of 4-styrylcoumarin have been synthesized by Knoevenagel condensation between substituted 4-methylcoumarin-3-carbonitrile and different heterocyclic or aromatic aldehydes. 4-Methylcoumarin-3-carbonitrile has been synthesized by the base catalyzed reaction between substituted 2-hydroxyacetophenone and ethyl cyanoacetate. The structures of the newly synthesized compounds were confirmed by ¹H NMR, IR and mass spectral analysis. All the compounds were evaluated for their anti-inflammatory activity (against TNF-α and IL-6) and anti-tubercular activity. Compounds 6a, 6h and 6j exhibited promising activity against IL-6 with 72-87% inhibition and compound 6v showed potent activity against TNF-α with 73% inhibition at 10 μM concentration. Whereas compounds 6n, 6o, 6r and 6u showed very good anti-tubercular activity against Mycobacterium tuberculosis H37Rv strain at <6.25 μM.     

Regulation of store-operated calcium channels by the intermediary metabolite pyruvic acid

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses, including neurotransmitter release, muscle contraction, and cell growth and proliferation. A major route for Ca(2+) influx is through store-operated Ca(2+) channels. One important intracellular target for Ca(2+) entry through store-operated channels is the mitochondrion, which increases aerobic metabolism and ATP production after Ca(2+) uptake. Here, we reveal a novel feedback pathway whereby pyruvic acid, a critical rate-limiting substrate for mitochondrial respiration, increases store-operated entry by reducing inactivation of the channels. Importantly, the effects of pyruvic acid are manifest at physiologically relevant concentrations and membrane potentials. The reduction in the inactivation of calcium release-activated calcium (CRAC) channels by pyruvate is highly specific in that it is not mimicked by other intermediary metabolic acids, does not require its metabolism, is independent of its Ca(2+) buffering action, and does not involve mitochondrial Ca(2+) uptake or ATP production. These results reveal a new and direct link between intermediary metabolism and ion-channel gating and identify pyruvate as a potential signaling messenger linking energy demand to calcium-channel function.     

Historical frequency of wind dispersal events and role of topography in the dispersal of anostracan cysts in a semi-arid environment

Propagules of several freshwater invertebrates are passively dispersed by wind, but the importance of wind as a dispersal vector remains poorly understood. We examined the historical frequency of wind dispersal events in cysts of Artemia franciscana. A threshold wind speed of ~5 km/h was required to begin dispersing surface-resting cysts, beyond which cyst dispersal increased rapidly with increasing wind speed. The analysis of wind speed and cyst dispersal data from our field experiment and wind data for the last 45 years from a nearby airport revealed that wind events strong enough to disperse most cysts at a wind-exposed site are common, occurring on ~16 snow-free days per year and occurring consistently over long time periods. In a topographically uneven landscape, however, wind speeds vary substantially from one location to another; wind speeds at our exposed site were 69% higher than in a nearby wind-sheltered depression. Accordingly, winds strong enough to disperse most cysts from this sheltered site occurred on only 5 days of the 45 year period. Cysts are thus less likely to disperse from, and more likely to settle in, sheltered depressions than wind-exposed areas. Sheltered areas could thus function as traps for wind-dispersing propagules.     

Glucose Directly Promotes Antifungal Resistance in the Fungal Pathogen,Candida spp.

Effects of glucose on the susceptibility of antifungal agents were investigated against Candida spp. Increasing the concentration of glucose decreased the activity of antifungal agents; voriconazole was the most affected drugs followed by amphotericin B. No significant change has been observed for anidulafungin. Biophysical interactions between antifungal agents with glucose molecules were investigated using isothermal titration calorimetry, Fourier transform infrared, and ¹H NMR. Glucose has a higher affinity to bind with voriconazole by hydrogen bonding and decrease the susceptibility of antifungal agents during chemotherapy. In addition to confirming the results observed in vitro, theoretical docking studies demonstrated that voriconazole presented three important hydrogen bonds and amphotericin B presented two hydrogen bonds that stabilized the glucose. In vivo results also suggest that the physiologically relevant higher glucose level in the bloodstream of diabetes mellitus mice might interact with the available selective agents during antifungal therapy, thus decreasing glucose activity by complex formation. Thus, proper selection of drugs for diabetes mellitus patients is important to control infectious diseases.     

GSK-3β Protein Phosphorylates and Stabilizes HLXB9 Protein in Insulinoma Cells to Form a Targetable Mechanism of Controlling Insulinoma Cell Proliferation

Insulinomas (pancreatic islet β cell tumors) are the most common type of functioning pancreatic neuroendocrine tumors that occur sporadically or as a part of the MEN1 syndrome that is caused by germ line mutations in MEN1. Tissue-specific tumor predisposition from germ line mutations in ubiquitously expressed genes such as MEN1 could occur because of functional consequences on tissue-specific factors. We previously reported the proapoptotic β cell differentiation factor HLXB9 as a downstream target of menin (encoded by MEN1). Here we show that GSK-3β inactivates the proapoptotic activity of HLXB9 by phosphorylating HLXB9 at Ser-78/Ser-80 (pHLXB9). Although HLXB9 is found in the nucleus and cytoplasm, pHLXB9 is predominantly nuclear. Both pHLXB9 and active GSK-3β are elevated in β cells with menin knockdown, in MEN1-associated β cell tumors (insulinomas), and also in human sporadic insulinomas. Pharmacologic inhibition of GSK-3β blocked cell proliferation in three different rodent insulinoma cell lines by arresting the cells in G₂/M phase and caused apoptosis. Taken together, these data suggest that the combination of GSK-3β and pHLXB9 forms a therapeutically targetable mechanism of insulinoma pathogenesis. Our results reveal that GSK-3β and pHLXB9 can serve as novel targets for insulinoma treatment and have implications for understanding the pathways associated with β cell proliferation.