Structure,function and applications of carbonic anhydrase isozymes

The carbonic anhydrases enzymes (CAs, EC 4.2.1.1) are zinc containing metalloproteins, which efficiently catalyse the reversible conversion of carbon dioxide to bicarbonate and release proton. These enzymes are essentially important for biological system and play several important physiological and patho-physiological functions. There are 16 different alpha-carbonic anhydrase isoforms studied, differing widely in their cellular localization and biophysical properties. The catalytic domains of all CAs possess a conserved tertiary structure fold, with predominately β-strands. We performed an extensive analysis of all 16 mammalian CAs for its structure and function in order to establish a structure–function relationship. CAs have been a potential therapeutic target for many diseases. Sulfonamides are considered as a strong and specific inhibitor of CA, and are being used as diuretics, anti-glaucoma, anti-epileptic, anti-ulcer agents. Currently CA inhibitors are widely used as a drug for the treatment of neurological disorders, anti-glaucoma drugs, anti-cancer, or anti-obesity agents. Here we tried to emphasize how CAs can be used for drug discovery, design and screening. Furthermore, we discussed the role of CA in carbon capture, carbon sensor and metabolon. We hope this review provide many useful information on structure, function, mechanism, and applications of CAs in various discipline.     

Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells

Leptospermum flavescens Sm. (Myrtaceae), locally known as ‘Senna makki’ is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC₅₀ values of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml respectively. LF1 treated cells showed a sub-G₁ region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treatment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death.     

Impact of inflammatory cytokine and adipokine gene variations in the development of HIV-associated lipodystrophy

Cytokines affect lipid and glucose metabolism and also alter the body's habitus. They play a role in the development of lipodystrophy syndrome. Adipocytes secrete the pro-inflammatory cytokines IL-1, TNF-α and IL-6. The plasma cytokine concentration is associated with the percentage and distribution of fat tissue in the body. The metabolic disturbances are strongly associated with increased levels of pro-inflammatory cytokines (IL-1, IL-6 and TNF-α). Plasma levels of cytokines such as TNF-α, IL-6 and leptin were found to be increased while plasma resistin levels were found to be variable in patients suffering from obesity and type II diabetes mellitus. Until now, limited information has been available on the polymorphism of cytokine and adipokine genes in patients of HIV-associated lipodystrophy (HIVLD), which can contribute to individual variations in susceptibility to metabolic diseases, especially to HIVLD. Hence, we studied the association of cytokine and adipokine gene polymorphisms in various diseases and their impact on HIVLD. We carry out an extensive search using several databases, including PubMed, EMBASE and Google Scholar. The distribution of cytokine and adipokine gene polymorphisms and their expression levels varied among various populations. We examined the variants of cytokine and adipokine genes, which can contribute to individual variations in susceptibility to metabolic diseases, especially to HIVLD. In the current review, we present a brief account of the risk factors of HIVLD, the pathogenesis of HIVLD and the polymorphism of cytokine and adipokine genes in various diseases with special reference to their impact on HIVLD.     

Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae

Multidrug-resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an attempt to identify a homologue of the Escherichia coli TolC in V . cholerae , we isolated a DNA fragment (pVC) that enabled an E . coli tolC mutant to grow in the presence of 0.05% deoxycholate (DOC). However, other TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins respectively. The translational products of these two ORFs correlated closely with the molecular weights of the predicted proteins. The deduced amino acid sequences of ORF1 and ORF2 showed a high degree of similarity with conserved regions of the E . coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E . coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V . cholerae isogenic mutant defective in ORF2 was constructed by gene replacement. Characterization of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results suggest that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V . cholerae . In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.     

Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells

Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.     

Taxonomic character of plant species in absorbing and accumulating alkali and alkaline earth metals grown in temperate forest of Japan

Summary Absorption and accumulation of alkali (Li, Na, K, Rb, Cs) and alkaline earth (Mg, Ca, Sr, Ba) metals were investigated as taxonomic characteristics (in 62 plant species). Leaf and soil samples were collected from 9 sites in temperature forest in Japan and the above mentioned elements were analyzed. Considerable differences were found among species in their ability to accumulate alkali and alkaline earth metals. Very high concentrations of Li (45 ppm, D.W.), K (37×10³ ppm), Rb (159 ppm) and Cs (8.2 ppm) were detected inLastrea japonica which were about 412, 12, 27 and 6 times higher than those of the species with the lowest concentrations. Na content was high inAcer micranthum (358 ppm) which was 16 times higher than species with the lowest concentration. Other species containing high levels of alkali metals wereHydrangea macrophylla, Struthiopteris niponica, Clethra barbinervis. Mean discrimination ratio (D.R.) for all investigated plant species for Li, Na, Rb, and Cs to K were 1.7, 0.44, 0.9 and 1.8 respectively. High concentrations of alkaline earth metals Ca (36×10³ ppm), Sr (345 ppm), and Ba (241 ppm) were found in the leaves ofHydrangea paniculata which were about 31, 84, and 72 times higher than those for the species with the lowest concentration. Mg was very high inStruthiopteris niponica (83×10² ppm). Other species with high concentrations of alkaline earth metals belonged to the genus Viburnum. Mean D.Rs. for Mg, Sr, and Bavs Ca were 1.0, 0.7 and 0.08. Principal component analysis of interrelationships between the mineral content in leaf tissues indicated that these elements could be classified into 2 groups with respect to their accumulation behavior in plants. The alkali metals K, Li, Rb, and Cs behaved similarly in their accumulation in leaves but Na behaved independently. Alkaline earth metals Ca, Mg, Sr, and Ba were also found to behave similarly in their accumulation. Factors scores of 1st and 2nd components revealed three groups of plant species: alkaliphilic, alkaline earthphilic, and neutral (non-accumulators).     

Carbohydrate mediated recognition of lysosomal enzymes by cell surface receptors

The recognition of lysosomal enzymes by various carbohydrate specific cell surface receptors is reviewed. In particular the biosynthesis of mannose 6-phosphate residues in lysosomal enzymes and their role for targeting of lysosomal enzymes to lysosomes are discussed.     

Population structure and dynamics oi Juniperus excelsa in Balouchistan,Pakistan

Abstract. 60 monospecific stands of Juniperus excelsa were sampled at four locations in Balouchistan. Density, basal area and height of individuals were recorded. Soils were analysed for selected physical and chemical characteristics and the degree of disturbance due to logging and burning was noted. The density of juniper trees (> 6 cm dbh) ranged from 56 to 332 stems / ha (average 174 stems / ha). Higher densities were recorded for relatively undisturbed stands and on west facing slopes. Density of seedlings and saplings (< 6 cm dbh) was strongly correlated with tree density and tree basal area. Among the edaphic variables CaC0₃ was correlated with juniper density and basal area. Diameter distributions within stands were mostly skewed and unimodal with gaps appearing in large size classes. The male to female ratio was close to 1. Cross-sections of 16 trees were used to determine age and growth rate. Number of rings in trees with 20 to 30 cm dbh ranged from 95 to 221 (x = 160 ± 38). Diameter and age were not related. Mean annual diameter increment ranged from 6 to 16 yr / cm x = 10 ± 3 yr / cm). It is concluded that size class gaps and low seedling / sapling densities are the consequence of anthropogenic disturbance.     

Ca2+ and cell fusion during sexual development in liquid cultures of Dictyostelium discoideum

Cell fusion resulting in zygote giant cell formation is the first observable event of sexual development in D. discoideum. The results reported here show that this process is Ca²⁺-dependent and that by increasing the level of Ca²⁺ in the medium the number of cell fusions can be increased 57-fold over control cultures. The data also suggest that Ca²⁺ has both an early and late function in the development of zygotes and these functions are mediated at the cell surface. These results plus the availability of a liquid culture for generating large volumes of cells make sexual development in D. discoideum an excellent system for the analysis of membrane fusion in eukaryotes.     

Molecular analysis and characterization of the Cochliobolus heterostrophus beta-tubulin gene and its possible role in conferring resistance to benomyl

Cochliobolus heterostrophus Tub1 described here is the first beta-tubulin gene characterized from a naturally occurring benomyl-resistant ascomycete plant pathogen. The gene encodes a protein of 447 amino acids. The coding region of Tub1 is interrupted by three introns, of 116, 55, and 56 nt, situated after codons 4, 12, and 53, respectively. As a result of the preference for pyrimidines in the third position of the codons when a choice exists between purines and pyrimidines, codon usage in the Tub1 gene is biased. Tub1 shows high homology with beta-tubulin genes of other ascomycete species. However, Tub1 is exceptional in having Tyr(167), compared with Phe(167), possessed by beta-tubulin genes of other ascomycetes sequenced thus far. The Tyr(167) residue has been associated with benomyl resistance in other organisms. In contrast, all other benomyl-implicated residues of Tub1 correspond to sensitivity. Based on these results, we suggest that benomyl resistance in the fungus probably is attributed to Tyr(167).