HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys4 of histone H3) and H3K9ac (acetylated Lys9 of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys14 of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production.

BackgroundSerratia marcescens, a Gram-negative nosocomial pathogen secretes a 50 kDa multi-domain zinc metalloprotease called serratiopeptidase. Broad substrate specificity of serratiopeptidase makes it suitable for detergent and food processing industries The protein shows potent anti-inflammatory, anti-edemic, analgesic, antibiofilm activity and sold as an individual or fixed-dose enteric-coated tablets combined with other drugs. Although controversial, serratiopeptidase as drug is used in the treatment of chronic sinusitis, carpal tunnel syndrome, sprains, torn ligaments, and postoperative inflammation. Since the native producer of serratiopeptidase is a pathogenic microorganism, the current production methods need to be replaced by alternative approaches. Heterologous expression of serratiopeptidase in E. coli was tried before but not found suitable due to the limited yield, and other expression related issues due to its inherent proteolytic activity such as cytotoxicity, cell death, no expression, minimal expression, or inactive protein accumulation.ResultsRecombinant expression of mature form serratiopeptidase in E. coli seems toxic and resulted in the failure of transformation and other expression related issues. Although E. coli C43(DE3) cells, express protein correctly, the yield was compromised severely. Optimization of protein expression process parameters such as nutrient composition, induction point, inducer concentration, post-induction duration, etc., caused significant enhancement in serratiopeptidase production (57.9 ± 0.73% of total cellular protein). Expressed protein formed insoluble, enzymatically inactive inclusion bodies, and gave 40–45 mg/l homogenous (> 98% purity) biologically active and conformationally similar serratiopeptidase to the commercial counterpart upon refolding and purification.ConclusionExpression of mature serratiopeptidase in E. coli C43(DE3) cells eliminated the protein expression associated with toxicity issues. Further optimization of process parameters significantly enhanced the overexpression of protein resulting in the higher yield of pure and functionally active recombinant serratiopeptidase. The biological activity and conformational features of recombinant serratiopeptidase were very similar to the commercially available counterpart suggesting it-a potential biosimilar of therapeutic and industrial relevance.

The purpose of this study was to investigate the presence of phytoconstituents in the extracts of Convolvulus microphyllus, Acacia nilotica, Withania somnifera, Tribullus terrestris, and Citrullus colocynthis, and to test their anti-microbial activity against the most common strains causing bacterial urinary tract infection (UTI). The dried powdered plant parts were extracted using five different solvents in a Soxhlet apparatus for 48 h at a temperature ranging from 60 to 80°C. These extracts were used for preliminary phytochemical and antimicrobial analyses. Fourier-transform infrared spectroscopy (FTIR) with a scan range of 450 to 4000/cm and 4/cm resolution and GC-MS analysis were carried out to analyse the samples for the presence of phytochemicals having antimicrobial activity. The methanolic extract of Tribulus terrestris was found to have the most promising antibacterial properties. The results revealed that the methanolic extract of Tribullus terrestris displayed significant antimicrobial activity against the uropathogens, especially against Escherichia coli, Pseudomonas aeruginosa and Candida albicans. Phytochemicals like steroids, glycosides and alkaloids were found in the methanolic extracts of T. terrestris. FTIR analysis and GC-MS revealed the presence of 50 compounds. We conclude that the methanolic extract of T. terrestris fruit has more potent antimicrobial activity than other extracts and standard antibiotics used in this study. In the future, additional investigation will be required to isolate the active compound, undertake toxicological investigations, and start a clinical trial.