Genetic Variability of Thermal <Emphasis Type="Italic">Nymphaea</Emphasis> (Nymphaeaceae) Populations Based on ISSR Markers: Implications on Relationships,Hybridization, and Conservation

The globally widespread genus Nymphaea exhibits a wide range of morphological and taxonomical diversity. The intrusion of a cultivated variety by progressive propagation and its affect on aquatic habitat is demonstrated in this case study. We have studied the genetic diversity, population, and stand structure of the neophyte Nymphaea × ‘Panama Pacific’ as well as other species found in Lake Hévíz and dikes nearby using inter-simple sequence repeat (ISSR) markers. The ISSR assay revealed a low genetic variability for the small populations of Nymphaea caerulea, Nymphaea lotus var. thermalis, and a medium level for Nymphaea alba, Nymphaea rubra var. longiflora, and Nymphaea × ‘Panama Pacific’. The evolutionary genetic status of individuals found in the overlapping cultivation area of Nymphaea × ‘Panama Pacific’ and N. caerulea was affirmed to be of hybrid origin by reticulate network analysis and with morphological parameters. The Bayesian analysis of hybrid classes and the segregation of the ISSR markers also confirmed the hybrid origin of the individuals in question and revealed that they are falling into F2 or latter genotype frequency classes, indicating the viability and fertility of the hybrids. The set of analyzed species by phylogenetic network analysis of ISSR data has been divided into three major groups according to their evolutionary patterns (subg. Barachyceras, Lotos, and Nymphaea). Our results are in accordance with these three major subgenera within Nymphaea.     

Structure and stability analysis of cytotoxic complex of camel α-lactalbumin and unsaturated fatty acids produced at high temperature

α-Lactalbumin α-La), together with oleic acid can be converted to a complex, which kills tumor cells selectively. Cytotoxic α-La -oleic acid and α-La -linoleic acid complexes were generated by adding fatty acid to camel holo α-La at 60 ° C (referred to as La-OA-60 and La-LA-60 state, respectively). Structural properties of these complexes were studied and compared to the camel α-La. The experimental results show that linoleic acid induces α-La partial unfolding but oleic acid does not change the protein structure significantly. Also the stability of La-OA-60 and La-LA-60 toward thermal denaturation was measured. The order of temperature at the transition midpoint is as follows: La-LA-60 < La-OA-60 < α-La. La-OA-60 complex inhibited tubulin polymerization in vitro. Although the structures of La-OA-60 and La-LA-60 were different, these two complexes had similar cytotoxic effect to DU145 human prostate cancer cells. Samples of La-OA-60 that have been renatured after denaturation lost the specific biological activity toward tumor cells.     

Hydroxyl-substituted sulfonylureas as potent inhibitors of specific [3H]glyburide binding to rat brain synaptosomes

We are seeking to discover potent CNS-active sulfonylureas with structural features that allow for the formation of several types of prodrugs. We report herein the syntheses of compounds comprising an initial series of hydroxyl-substituted analogues of the potent ATP-sensitive potassium channel blockers glyburide (glibenclamide) and gliquidone. Somewhat unexpectedly, several of the compounds were found to be comparably potent to glyburide as inhibitors of specific [(3)H]glyburide binding in rat brain preparations.     

In vitro propagation and the acclimatization effect on the synthesis of 2-hydroxy-4-methoxy benzaldehyde in Decalepis hamiltonii Wight and Arn.

The present study reports a high frequency in vitro propagation protocol through apical bud sprouting and basal organogenic nodule formation in shoot tip explants of Decalepis hamiltonii, an endemic and endangered medicinal liana. Among different combinations of plant growth regulators (PGRs) and growth additives, maximum of 8.20 shoots per explant with mean shoot length of 6.54 cm were induced on Murashige and Skoog’s medium (MS) supplemented with 5.0 µM 6-benzyladenine (BA) + 0.5 µM indole-3-acetic acid (IAA) + 30.0 µM adenine sulphate (ADS) through apical bud sprouting. On single cytokinin treatment explants did not exhibit good multiplication but showed nodulation (N₁) from the basal cut end similar to cytokinin–auxin combination (N₂). Between two types of nodular tissues, N₂ was proved to be better for maximum shoot regeneration (15.40 shoots per explant) and shoot length (4.56 cm) when cultured on MS medium supplemented with 5.0 µM BA, 0.5 µM IAA, 30.0 µM ADS and 1.0 µM gibberellic acid (GA₃). Microshoots were efficiently rooted on half-strength MS medium supplemented with 2.5 μM α-naphthalene acetic acid (NAA). After successful acclimatization in Soilrite, 95.10 % plantlets were survived in field conditions. Histological investigation proved useful in ascertaining the callogenic nature of the regenerating nodular tissue formed at the basal cut end of shoot tip explant. Acclimatized plantlets were studied for the estimation of chlorophyll and carotenoid content as well as the net photosynthetic rate (PN) during subsequent days of transfer to ex vitro condition. Moreover, acclimatization had a significant effect on biomass production and the synthesis of 2-hydroxy-4-methoxy benzaldehyde (2HMB). Maximum fresh weight (3.78 gm/plant), dry weight (0.39 gm/plant) of roots and 2HMB content (15.94 µg/ml of extract) were noticed after 8 weeks of acclimatization.     

Soluble T cell immunoglobulin and mucin domain (TIM)-1 and -4 generated by A Disintegrin And Metalloprotease (ADAM)-10 and -17 bind to phosphatidylserine

T cell immunoglobulin and mucin domain 1 and 4 (TIM-1 and -4) proteins serve as phosphatidylserine receptors to engulf apoptotic cells. Here we show that human TIM-1 and TIM-4 proteins are targets of A Disintegrin And Metalloprotease (ADAM)-mediated ectodomain shedding resulting in soluble forms of TIM-1 and TIM-4. We identified ADAM10 and ADAM17 as major sheddases of TIM-1 and TIM-4 as shown by protease-specific inhibitors, the ADAM10 prodomain, siRNA and ADAM10/ADAM17 deficient murine embryonic fibroblasts (MEFs). TIM-1 and TIM-4 lacking the intracellular domain were efficiently cleaved after ionomycin- and PMA-treatment, indicating that the intracellular domain was not necessary for ectodomain shedding. Soluble TIM-1 and -4 were able to bind to phosphatidylserine, suggesting that soluble TIM-1 and -4 might act as negative regulators of cellular TIM-1 and -4. In summary, we describe TIM-1 and TIM-4 as novel targets for ADAM10- and ADAM17-mediated ectodomain shedding.     

Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.     

Association of mutation and hypermethylation of p21 gene with susceptibility to breast cancer: a study from north India

p21 gene located at chromosome 6p21.2 is a possible tumour suppressor gene involved in the pathogenesis of breast cancer. Both genetic and epigenetic alterations in p21 have been implicated in breast carcinoma. In the present study, our main aim was to study the impact of these two kinds of alterations of p21 gene in Indian female breast cancer patients. A total of 150 female breast cancer patients of north India were screened by PCR-SSCP followed by direct sequencing and methylation specific PCR. Mutational screening of p21 gene revealed significant amount of mutations [32.66 % (49/150)] in exon 2, whereas p21 promoter was found hypermethylated in 42 of 150 (28 %) breast cancer patients in our population. The intriguing feature of the study was the G>T transition (GAG>TAG) at codon 107 and the A>C transition (AGC>CGC) at codon 146 possibly rendering p21 completely ineffective in its anti- proliferative activity. Our results suggest a significant association between the mutational and hypermethylation profile of p21 gene. Therefore, we show for the first time that the significant association of p21 mutation and hypermethylation leads to the complete inactivation of p21 gene in Indian female breast cancer patients. Complete silencing of the p21 gene seems to be the result not only of genetic alterations but also of epigenetic modification.     

Role of 677C→T polymorphism a single substitution in methylenetetrahydrofolate reductase (MTHFR) gene in North Indian infertile men

Failure or severe difficulty in conceiving a child is surprisingly common, worldwide problem. Half of these cases are due to male factors with defects in sperm (1 in 15 men) being the single most common cause. Also about 60–75 % of male infertility cases are idiopathic, since the molecular mechanisms underlying the defects remain unknown. DNA methylation is crucial for spermatogenesis and high methylenetetrahydrofolate reductase (MTHFR) activity in adult testis than other organs in mouse, signifies its critical role in spermatogenesis. According to recent findings there is a correlation of epigenetic regulation of several imprinted genes with disturbed spermatogenesis and fertility. Consequently any change in the MTHFR gene sequence can modify the spermatogenesis including transmission of infertility to the carriers. The aim of the study is to analyze the distribution of the single nucleotide polymorphism C677T in the MTHFR gene in 637 North Indian infertile patients and 364 fertile North Indian men as controls by using PCR–RFLP technique and Chi Square test for statistical analysis. The average MTHFR 677CC, 677CT, 677TT genotype frequencies of total infertile men were 70.17, 24.17, 5.65 % in infertile men and 75.27, 21.7, 2.74 % in controls, respectively. The average frequency of the MTHFR 677T allele was 17.73 % in infertile men as compared to 13.59 % in controls. The statistical difference was significant. Disease risk was found 2.27-folds increased in patients who were carrying T allele. We found an association of C677T polymorphism with male infertility and that it may be a genetic risk factor for male infertility in North Indian population.     

Expression line approach to recombinant human epidermal growth factor into the yeast,Pichia pastoris from Huh-7 cell line

Beta-urogastrone also known as human epidermal growth factor is a key member of epidermal growth factor family having role in cell proliferation and differentiation in vivo as well as in vitro. Human epidermal growth factor gene has been isolated from different tissues but the method of isolation is technically difficult and complicated as it deals with biopsies. Here we isolated mature partial human epidermal growth factor gene from Huh-7 cell line, amplified and abridged toward mature coding region with three steps PCR, sequenced for homology with wild type human epidermal growth factor gene, inbuilt with sites of interest and cloned in Pichia pastoris for expression study. Isolated mature human epidermal growth factor gene from Huh-7 cell line showed 100 % sequence homology to wild type human epidermal growth factor gene and gives the native expression for human epidermal growth factor peptide. In this study we report that Huh-7 cell line is an easy source for the particular gene of human epidermal growth factor isolation and we are also suggesting P. pastoris is an expression system to produce recombinant human epidermal growth factor of the therapeutic importance resembling to the natural human system.