Refolding of recombinant human granulocyte colony stimulating factor: effect of cysteine/cystine redox system.

Granulocyte colony-stimulating factor (G-CSF) is a multifunctional cytokine which is widely used for treating neutropenia in humans. Evaluation of alternative to expensive components of redox buffer (reduced and oxidized glutathione) is an important step in reducing the cost of production of human biotherapeutic proteins. In the present study, refolding of recombinant human G-CSF expressed as inclusion bodies (IBs) in E. coli was optimized using cysteine and cystine redox agents. The refolding to correct native form of G-CSF was assessed by reverse phase high performance liquid chromatography (RP-HPLC). The optimized concentrations of cysteine and cystine for correct refolding of G-CSF were found to be 2 mM and 1 mM, respectively. The correctly refolded G-CSF was detected as early as 4 h of incubation in renaturation buffer containing optimized concentrations of cysteine (2 mM) and cystine (1 mM) redox agents. Refolding of G-CSF in optimized redox system increased with increase in shuffling time. Overall, the results suggested the use of cysteine/cystine redox pair could be an alternative to the costlier redox pairs for successful refolding of G-CSF and possibly other human biotherapeutic proteins of importance.

Do gene polymorphism in IL-1ß, TNF-α and IL-6 influence therapeutic response in patients with drug refractory epilepsy?

PURPOSE: Pro-inflammatory cytokines may play an important pathophysiological role in patients with epilepsy. To understand the role of genes encoding pro-inflammatory cytokines in epilepsy, this study aimed to evaluate the polymorphisms of the promoter regions of IL-1ß-511C>T (rs16944), TNF-α-308G>A (rs1800629) and IL-6-174G>C (rs1800795) genes and to look into the interaction between these genes in influencing seizure susceptibility, seizure frequency and response to therapy. METHODS: The comparative frequency of polymorphism was determined in rs16944, rs1800629 and rs1800795 using PCR-RFLP in a group of 120 persons with epilepsy (PWE) and 110 ethnically matched healthy subjects of comparable age and sex in the North Indian population. RESULTS: Alleles and genotypes of rs16944, rs1800629 and rs1800795 were not found to influence the odds ratio of having susceptibility to epilepsy. Also gene-gene interaction of possible nine combinations of these genes did not show any positive association with epilepsy. The genotype and allelic frequency of rs1800795 showed a significant association (p<0.05) in seizure frequency (number of seizures/6-months) and drug refractory epilepsy. However, the genotype and allelic frequency of rs16944 and rs1800629 were not found to have such effect. CONCLUSION: This study demonstrates that the rs16944, rs1800629 and rs1800795 polymorphism does not act as a strong susceptibility factor for epilepsy in North Indian population. The genotypic association of rs1800795 with seizure frequency and drug-refractory epilepsy raises the issue that a specific set of polymorphic genes can influence seizures and therapeutic response in epilepsy.

Nucleotide sequences and chromosomal localization of 45S and 5S rDNA in Neolissochilus hexagonolepis (Pisces, Cyprinidae), using dual-color fish.

Dual-color fluorescence in situ hybridization (FISH) was performed to study the simultaneous localization of major (45S) and minor (5S) family ribosomal RNA genes on chromosomes of Neolissochilus hexagonolepis. The partial 45S (18S, ITS 1, 5.8S, ITS 2 and 28S) and complete 5S (coding and NTS) rDNA units were amplified, sequenced, analyzed, and mapped on the metaphase chromosomes. The complete 18S, 5.8S and partial 28S rDNAs were 1849 bp, 157 bp and 1819 bp long, respectively. Internal transcribed spacers, namely ITS 1 (828 bp) and ITS 2 (359 bp), showed significant nucleotide variations from other fish species listed in NCBI database. The 5S rDNA contained an identical coding region of 120 bp and a highly divergent, non-transcribed 81-bp spacer. The specimens of N. hexagonolepis showed six bright fluorescent signals of 18S, while the 5S signals were present only on one pair of chromosomes. Subsequent analyses between conventional Ag-NORs and 18S rDNA FISH strongly suggested the possible inactivation of one pair of NORs that was localized at a telomeric position of a submetacentric chromosome. The sequencing and chromosomal localization of 45S and 5S rDNAs may serve as a useful genetic marker in taxonomic classification as well as in phylogenetic and evolutionary studies.

Alu polymorphic insertions reveal genetic structure of north Indian populations.

The Indian subcontinent is characterized by the ancestral and cultural diversity of its people. Genetic input from several unique source populations and from the unique social architecture provided by the caste system has shaped the current genetic landscape of India. In the present study 200 individuals each from three upper-caste and four middle-caste Hindu groups and from two Muslim populations in North India were examined for 10 polymorphic Alu insertions (PAIs). The investigated PAIs exhibit high levels of polymorphism and average heterozygosity. Limited interpopulation variance and genetic flow in the present study suggest admixture. The results of this study demonstrate that, contrary to common belief, the caste system has not provided an impermeable barrier to genetic exchange among Indian groups.

In vitro clonal propagation of bael (Aegle marmelos Corr.) CV. CISH-B1 through enhanced axillary branching.

Rapid clonal micropropagation protocol of Aegle marmelos (L.) Corr. cv. CISH-B1 was achieved by nodal stem segment of mature bearing tree. Three centimeter long shoots having one axillary bud excised from 10-15th nodal region of shoots during September gave quick in vitro bud burst (5.33 days) when cultured on MS medium supplemented with BAP, 8.84 µM + IAA 5.7 µM. The maximum number of proliferated shoots (9.0/explant) were obtained on same medium supplemented with BAP 8.84 µM + IAA 5.7 µM. The micro shoots were rooted (100 %) on + IAA 5.7 µM. In vitro rooted plants were acclimatized on autoclaved coconut husk containing plant salt mixture and under shade net house (50 % shade 70-80 % RH). The plants were established in the field after acclimatization. The micropropagated plants were tested for its genetic fidelity using 13 RAPD, 3 ISSR and 2 DAMD primers. Profile obtained by all the three Single Primer Amplification Reaction (SPAR) technique from mother tree and micropropagated plants revealed genetic integrity of micropropagated plants with that of mother tree.