Ternary Cobalt (II)-Metformin-Glycine/Histidine/Proline Complexes: Multispectroscopic DNA, HSA, and BSA Interaction and Cytotoxicity Studies.

The synthesized water-soluble ternary complexes [Co(met)(gly)(Cl)2] (1), [Co(met)(hist)(Cl)2] (2), and [Co(met)(pro)(Cl)2] (3), (met = metformin, gly = glycine, hist = histidine, and pro = proline) were evaluated using spectro-analytical techniques, and the stereochemistry of the complexes was determined to be octahedral. UV-Vis absorption, competitive DNA-binding experiments using ethidium bromide (EB) by fluorescence, fluorescence emission studies, viscosity studies, and gel electrophoresis techniques were all employed to explore the binding characteristics of the cobalt (II) complexes with CT-DNA and groove-binding mechanism established. The salt-dependent association of the complexes to CT-DNA was investigated using UV-Vis spectrophotometric analysis. The association of the cobalt (II) complexes with BSA and HSA was explored by utilizing UV-Vis absorption and fluorescence spectroscopy approaches. The findings show that the complexes exhibit adequate capacity to quench BSA and HSA fluorescence and that the binding response is mostly a static quenching mechanism. The cytotoxicity of the complexes has also been appraised with the human breast adenocarcinoma cell lines (MCF-7) and (MDA-MB-231) by utilizing the MTT assay. For each cell line, the IC50 values were computed. In both cell lines, all the complexes were active.

Metformin-Derived Water-Soluble Cobalt Complexes: Thermal, Spectroscopic, DNA Interaction, and Molecular Docking Studies.

Novel three water-soluble cobalt (II) complexes of type [Co(metf)(o-phen)2]Cl2 (1), [Co(metf)(opda)2]Cl2 (2), and [Co(metf)(2-2'bipy)2]Cl2 (3) (Metf, metformin; o-phen, ortho-phenanthroline; opda, ortho-phenylenediamine; 2,2'-bipy, 2,2'-bipyridine) were synthesized and characterized by various analytical and spectral techniques. Based on these studies, octahedral geometry is assigned to these complexes. The stability of the complexes has been calculated from quantum chemical parameters using HOMO-LUMO energies. Thermal degradation pattern of the compounds was studied and Coats-Redfern method is used to determine kinetic parameters for complexes 1, 2, and 3 from thermal studies. The DNA interaction of these complexes was investigated by absorption, emission, and viscosity studies. From the spectral data, it was concluded that the complexes bind to DNA through groove mode of binding. The intrinsic binding constants (Kb) from absorption spectroscopy were 2.49 × 104, 2.48 × 104, and 2.64 × 104 M-1 for 1, 2, and 3, respectively, and Stern-Volmer quenching constants (Ksv) from emission spectroscopy were 0.21, 0.20, and 0.13, respectively. These complexes were screened for nuclease activity of pUC19 DNA, in the presence of H2O2. Discovery studio 2.1 software was used to evaluate binding affinity and interaction pattern of complexes with B-DNA receptor protein and the maximum dock score is seen for complex 2.

Nickel-Metformin Ternary Complexes: Geometrical, Thermal, DNA Binding, and Molecular Docking Studies.

Novel three nickel(II) complexes of type [Ni(metf)(o-phen)2]Cl2 (1), [Ni(metf)(opda)2]Cl2 (2), [Ni(metf)(2-2'bipy)2]Cl2 (3), (Metf = metformin, o-phen = ortho-phenanthroline, opda = ortho-phenylenediamine, 2-2' bipy = 2-2' bipyridyl) were synthesized and characterized by various analytical and spectral techniques. Based on these studies, octahedral geometry is assigned to these complexes. The DNA binding properties of these complexes were investigated by absorption, emission, and viscosity studies. From the spectral data, it was concluded that the complexes bind to DNA through groove mode of binding. The intrinsic binding constants (Kb) from absorption spectroscopy were 1.60 × 104, 3.57 × 104, and 5.70 × 104 M-1 for 1, 2, and 3, respectively, and Stern-Volmer quenching constants (Ksv) from emission spectroscopy were 0.11, 0.87, and 0.24, respectively. Thermal degradation pattern of the compounds was studied and Coats-Redfern method is used to determine kinetic parameters for complexes 1, 2, and 3 from thermal studies. The software Discovery Studio 2.1 was used to assess the binding affinity and interaction pattern of complexes with the B-DNA receptor protein and complex 1 has the highest dock score.

Effectiveness of structured teaching programme on transmission and prevention of human immunodeficiency virus/acquired immune deficiency syndrome among adolescent girls in Lowry Memorial High School, Bengaluru.

BACKGROUND: Adolescence is a critical stage in human development. Most young people become sexually active during adolescence and are more likely to have multipartner and unprotected sex with high-risk behavior that predisposes them to sexually transmitted infections such as human immunodeficiency virus (HIV). OBJECTIVES: The objective of the study was to evaluate the effectiveness of a structured teaching programme on transmission and prevention of HIV/acquired immune deficiency syndrome (HIV/AIDS) among adolescent girls. METHODS: An evaluative research approach was adopted, in which a preexperimental, one group pre- and post-test research design was used to evaluate the effectiveness of the structured teaching programme on transmission and prevention of HIV/AIDS among adolescent girls studying at Lowry Memorial High School, Bengaluru. A self-administered structured questionnaire was used for data collection. Data were presented in frequency tables and statistical graphs (bar charts) and analyzed using descriptive statistics (mean, standard deviation) and inferential statistical methods (Chi-square and paired "t"-tests) using SPSS version 21. RESULTS: The findings of the study revealed that the mean percentage difference in the pre- and post-test knowledge scores was statistically significant at 5% level (P < 0.05). The overall mean post-test knowledge score of adolescent girls on transmission and prevention of HIV/AIDS was 88.83%. It is apparently higher compared to the pretest knowledge score, which was 67.67% with enhancement of 21.16%. This implies that the structured teaching programme was effective in gaining knowledge of adolescent girls regarding transmission and prevention of HIV/AIDS. CONCLUSION: Our study suggests that structured teaching programme enhanced the knowledge of the adolescent girls on transmission and prevention of HIV/AIDS. We, therefore, recommend that structured teaching programmes on transmission and prevention of HIV/AIDS should be encouraged among adolescents and youths to reduce the spread of HIV infection.

Human intestinal capillariasis: a rare case report from non-endemic area (Andhra Pradesh, India).

Human intestinal capillariasis is caused by Capillaria philippinensis. This disease is endemic in Philippines and Thailand. To the best of our knowledge, we report the third case of human intestinal capillariasis from India and the first case from Andhra Pradesh, which is a non-endemic area. A 40-year-old female presented with diarrhoea, vomiting, decreased urinary output, ascitis, pedal oedema, hypoalbuminemia, and electrolyte imbalance. Microscopic examination of stool sample revealed the presence of ova, larvae, and adult worms of C. philippinensis. Patient recovered from the disease after taking albendazole 400 mg daily for 1 month along with supportive treatment.

A fluorescent method to determine vitamin K-dependent gamma-glutamyl carboxylase activity.

The gamma (γ)-glutamyl carboxylase is a key enzyme in vitamin K-dependent carboxylation of proteins involved in hemostasis and inflammation. It is an endoplasmic enzyme posttranslationally converting glutamic acid residues into γ-carboxyglutamic acid residues in proteins. The activity of tissue derived γ-glutamyl carboxylase is commonly assayed by incorporation of H¹4CO3⁻ into synthetic peptides and subsequent quantification using liquid scintillation counting. We present a nonradioactive assay using a fluorescein isothiocyanate-labeled short peptide that can be readily detected in its unmodified and γ-glutamyl carboxylated form by reversed-phase HPLC. This method offers a convenient alternative to the established radioactive labeling techniques.

Morphological and Ginsenoside Differences among North American Ginseng Leaves.

Leaf characteristics of mature 2, 3 and 4-year-old North American ginseng (Panax quinquefolius L.) leaves on fruiting and non-fruiting (NF) plants were studied. Leaflets of the 2-year-old plants had the lowest fresh and dry weight, area, volume and internal gas volume. Inflorescence removal in 3-year-old plants did not affect leaf characteristics or ginsenoside concentration but in 4-yearold plants it increased leaf fresh (38.6%) and dry (43.9%) weight, leaf area (29.1%), specific leaf mass (11.4%), leaf volume (43.1%), and leaf thickness (12.1%), and decreased leaf water content (6.2%). Cultivated ginseng, although an understorey plant, had the specific leaf mass, 35.6 g m(-2) (range, 36 to 39 g m(-2)) and a chlorophyll a/b ratio of 2.40 to 2.61, both suggesting the ability to perform like a sunny habitat plant. Also, specific leaf mass of 35.6 g m(-2) is similar to that reported for perennial plants, 36.8 g m(-2), rather than that for annuals, 30.9 g m(-2).

Effect of vitamin K-dependent protein precursor propeptide, vitamin K hydroquinone, and glutamate substrate binding on the structure and function of {gamma}-glutamyl carboxylase.

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO(3), on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation.

Rho GTPase/Rho kinase inhibition as a novel target for the treatment of glaucoma.

Rho kinase (ROCK1 and ROCK2) is a serine/threonine kinase that serves as an important downstream effector of Rho GTPase, and plays a critical role in regulating the contractile tone of smooth muscle tissues in a calcium-independent manner. Several lines of experimental evidence indicate that modulating ROCK activity within the aqueous humor outflow pathway using selective inhibitors could achieve very significant benefits for the treatment of increased intraocular pressure in patients with glaucoma. The rationale for such an approach stems from experimental data suggesting that both ROCK and Rho GTPase inhibitors can increase aqueous humor drainage through the trabecular meshwork, leading to a decrease in intraocular pressure. In addition to their ocular hypotensive properties, inhibitors of both ROCK and Rho GTPase have been shown to enhance ocular blood flow, retinal ganglion cell survival and axon regeneration. These properties of the ROCK and Rho GTPase inhibitors indicate that targeting the Rho GTPase/ROCK pathway with selective inhibitors represents a novel therapeutic approach aimed at lowering increased intraocular pressure in glaucoma patients.

alpha-Crystallin localizes to the leading edges of migrating lens epithelial cells.

alpha-crystallin (alphaA and alphaB) is a major lens protein, which belongs to the small heat-shock family of proteins and binds to various cytoskeletal proteins including actin, vimentin and desmin. In this study, we investigated the cellular localization of alphaA and alphaB-crystallins in migrating epithelial cells isolated from porcine lens. Immunofluorescence localization and confocal imaging of alphaB-crystallin in confluent and in migrating subconfluent cell cultures revealed a distinct pattern of subcellular distribution. While alphaB-crystallin localization was predominantly cytoplasmic in confluent cultures, it was strongly localized to the leading edges of cell membrane or the lamellipodia in migrating cells. In accordance with this pattern, we found abundant levels of alphaB-crystallin in membrane fractions compared to cytosolic and nuclear fractions in migrating lens epithelial cells. alphaA-crystallin, which has 60% sequence identity to alphaB-crystallin, also exhibited a distribution profile localizing to the leading edge of the cell membrane in migrating lens epithelial cells. Localization of alphaB-crystallin to the lamellipodia appears to be dependent on phosphorylation of residue serine-59. An inhibitor of p38 MAP kinase (SB202190), but not the ERK kinase inhibitor PD98059, was found to diminish localization of alphaB-crystallin to the lamellipodia, and this effect was found to be associated with reduced levels of Serine-59 phosphorylated alphaB-crystallin in SB202190-treated migrating lens epithelial cells. alphaB-crystallin localization to the lamellipodia was also altered by the treatment with RGD (Arg-Ala-Asp) peptide, dominant negative N17 Rac1 GTPase, cytochalasin D and Src kinase inhibitor (PP2), but not by the Rho kinase inhibitor Y-27632 or the myosin II inhibitor, blebbistatin. Additionally, in migrating lens epithelial cells, alphaB-crystallin exhibited a clear co-localization with the actin meshwork, beta-catenin, WAVE-1, a promoter of actin nucleation, Abi-2, a component of WAVE-1 protein complex and Arp3, a protein of the actin nucleation complex, suggesting potential interactions between alphaB-crystallin and regulatory proteins involved in actin dynamics and cell adhesion. This is the first report demonstrating specific localization of alphaA and alphaB-crystallins to the lamellipodia in migrating lens epithelial cells and our findings indicate a potential role for alpha-crystallin in actin dynamics during cell migration.

Impaired cytoskeletal organization and membrane integrity in lens fibers of a Rho GTPase functional knockout transgenic mouse.

To investigate the effects of Rho GTPase inactivation on lens fiber cell cytoskeletal and morphological integrity, a transgenic mouse model expressing C3-exoenzyme (a bacterial toxin) in a lens-specific manner was utilized. Cryosections of whole eyes from C3 transgenic mice and littermate controls were stained for F-actin with rhodamine-phalloidin or immunostained for beta-catenin, aquaporin-0 or connexin-50, and confocal images were recorded. Lens fiber cell morphology was examined at both light and electron microscopic levels. To investigate the influence of Rho GTPase inactivation on the profiles of gene expression, cDNA libraries generated from transgenic and littermate control mouse lenses were screened by cDNA microarray analysis. In contrast to the wild-type lens, fiber cells of the transgenic lens were grossly swollen and disorganized, with abnormal membrane architecture. Staining of F-actin, beta-catenin, aquaporin-0 and connexin-50 was reduced dramatically in the C3 transgenic lens as compared to controls. Western blot analysis and cDNA microarray analysis did not reveal any noticeable decreases in actin, beta-catenin and aquaporin-0 protein levels or expression in C3 transgenic lenses, indicating that altered cytoskeletal organization in response to Rho GTPase inactivation might underlie the noted changes in staining for these proteins. Additionally, cDNA microarray analysis of C3 lens revealed altered expression (at least two-fold, compared to littermate controls) of 44 genes. These include genes encoding extracellular matrix and basement membrane proteins, cell survival and apoptotic pathways, and ion and protein transport. These data indicate that disruption of Rho GTPase function in the developing mouse lens results in abnormal cytoskeletal organization, fiber cell interactions, impaired lens fiber cell morphology and altered gene expression of cellular proteins involved in diverse functions. This work reveals that the morphological and cytoskeletal abnormalities triggered upon Rho GTPase inactivation in lens could be one of the important insults associated with cataract formation in C3 transgenic mouse lens.

A conserved region of human vitamin K-dependent carboxylase between residues 393 and 404 is important for its interaction with the glutamate substrate.

Certain individuals with combined deficiencies of vitamin K-dependent proteins have a mutation, L394R, in their gamma-glutamyl carboxylase causing impaired glutamate binding. The sequence surrounding Leu394 is similar in all known carboxylases, suggesting that the region is functionally important. To test this hypothesis we made the following mutant enzymes: W390A, Y395A, S398A, W399A, and H404A. We purified the enzymes and corrected the activity measurements for active enzyme concentration. Carboxylases W390A, S398A, and H404A had activities similar to that of wild type; however, Y395A and W399A had lower activities than did wild type. In the following descriptions we include our previously reported results for L394R. Kinetic studies with the substrate FLEEL, revealed Km values of 0.5 (wild type), 6.5 (L394R), 15 (Y395A), and 24 (W399A) mm. The kcat values relative to wild type were 51% (L394R), 1% (Y395A), and 2% (W399A). The kcat/Km values were 24-fold (L394R) and >2000-fold lower for Y395A and W399A than for wild-type carboxylase. Inhibition of FLEEL carboxylation by the competitive inhibitor, Boc-mEEV, gave Ki values of 0.013 (wild type), 1.4 (L394R), 2.1 (Y395A), and >5 (W399A) mm. The Y395A propeptide affinity was similar to that of wild type, but those of L394R and W399A were 16-22-fold less than that of wild type. Results of kinetic studies with a propeptide-containing substrate were consistent with results of propeptide binding and FLEEL kinetics. Although propeptide and vitamin K binding in some mutants were affected, our data provide compelling evidence that glutamate recognition is the primary function of the conserved region around Leu394.

Determination of disulfide bond assignment of human vitamin K-dependent gamma-glutamyl carboxylase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Vitamin K-dependent gamma-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to gamma-carboxyglutamate. Carboxylase has ten cysteine residues, but their form (sulfhydryl or disulfide) is largely unknown. Pudota et al. in Pudota, B. N., Miyagi, M., Hallgren, K. W., West, K. A., Crabb, J. W., Misono, K. S., and Berkner, K. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13033-13038 reported that Cys-99 and Cys-450 are the carboxylase active site residues. We determined the form of all cysteines in carboxylase using in-gel protease digestion and matrix-assisted laser desorption/ionization mass spectrometry. The spectrum of non-reduced, trypsin-digested carboxylase revealed a peak at m/z 1991.9. Only this peak disappeared in the spectrum of the reduced sample. This peak's m/z is consistent with the mass of peptide 92-100 (Cys-99) disulfide-linked with peptide 446-453 (Cys-450). To confirm its identity, the m/z 1991.9 peak was isolated by a timed ion selector as the precursor ion for further MS analysis. The fragmentation pattern exhibited two groups of triplet ions characteristic of the symmetric and asymmetric cleavage of disulfide-linked tryptic peptides containing Cys-99 and Cys-450. Mutation of either Cys-99 or Cys-450 caused loss of enzymatic activity. We created a carboxylase variant with both C598A and C700A, leaving Cys-450 as the only remaining cysteine residue in the 60-kDa fragment created by limited trypsin digestion. Analysis of this fully active mutant enzyme showed a 30- and the 60-kDa fragment were joined under non-reducing conditions, thus confirming Cys-450 participates in a disulfide bond. Our results indicate that Cys-99 and Cys-450 form the only disulfide bond in carboxylase.