Microbiological acceptance criteria, specifications of herbal drugs and herbal drug preparations in various pharmacopoeias: a global scenario.

PURPOSE: A pharmacopoeia is a compendium of guidelines and criteria for drug quality. It was established by a national or regional entity and has legal significance. This applies to administration of drugs in a particular nation or region. METHOD: In this study, the differences and similarities of microbiological acceptance criteria, specifications for microbial enumeration of herbal drugs and herbal drug preparations in 14 national and international pharmacopeias were investigated. RESULTS: It was found that 12 pharmacopeias have given separate microbial limits for total aerobic microbial count (TAMC) and total yeast and mold count (TYMC), and a list of specified microorganisms for which acceptance criteria are defined. However, similarities were noticed in Ph.Eur, Ph. Helv and, BP. Salmonella, and Escherichia coli are the most common pathogens specified for herbal preparations in which boiling water is added prior to use and for internal use in all Pharmacopoeias because they serve as indicators of potential contamination. CONCLUSION: From this study, it can be concluded that the differences in microbial limit tests and their acceptance criteria as specified in the various pharmacopoeias need to be harmonized. It will become a more convenient option for global drug manufacturers to import/export herbal drugs, and this would also eliminate the burden of performing various analytical methods and comply with different microbial acceptance criteria set by various pharmacopoeias. The comparative data obtained from this study will be used to develop strategies for revisions of pharmacopoeias in a harmonized manner with respect to microbiological acceptance criteria, specifications for microbial enumeration of herbal drugs and herbal drug preparations.

Overexpression of a conserved HSP40 chaperone reduces toxicity of several neurodegenerative disease proteins.

TDP-43 and FUS are DNA/RNA binding proteins associated with neuronal inclusions in amyotrophic lateral sclerosis (ALS) patients. Other neurodegenerative diseases are also characterized by neuronal protein aggregates, e.g. Huntington's disease, associated with polyglutamine (polyQ) expansions in the protein huntingtin. Here we discuss our recent paper establishing similarities between aggregates of TDP-43 that have short glutamine and asparagine (Q/N)-rich modules and are soluble in detergents, with those of polyQ and PIN4C that have large Q/N-rich domains and are detergent-insoluble. We also present new, similar data for FUS. Together, we show that like overexpression of polyQ or PIN4C, overexpression of FUS or TDP-43 causes inhibition of the ubiquitin proteasome system (UPS) and toxicity, both of which are mitigated by overexpression of the Hsp40 chaperone Sis1. Also, in all cases toxicity is enhanced by the [PIN+] prion. In addition, we show that the Sis1 mammalian homolog DNAJBI reduces toxicity arising from overexpressed FUS and TDP-43 respectively in human embryonic kidney cells and primary rodent neurons. The common properties of these proteins suggest that heterologous aggregates may enhance the toxicity of a variety of disease-related aggregating proteins, and further that chaperones and the UPS may be key therapeutic targets for diseases characterized by protein inclusions.

The chbG gene of the chitobiose (chb) operon of Escherichia coli encodes a chitooligosaccharide deacetylase.

The chb operon of Escherichia coli is involved in the utilization of the ß-glucosides chitobiose and cellobiose. The function of chbG (ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show that chbG encodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of the chb promoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations in chbR conferring the ability to grow on both cellobiose and chitobiose are independent of chbG function for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-ß-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence of chbG, suggesting an additional role of chbG in the hydrolysis of chitooligosaccharides. The homologs of chbG in metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function of E. coli chbG has a broader significance.

Phylogeny based on 16S rDNA and nifH sequences of Ralstonia taiwanensis strains isolated from nitrogen-fixing nodules of Mimosa pudica, in India.

Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence. Isolates from the nodules of M. pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates. Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R. taiwanensis strain isolated from M. pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha. Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to alpha-rhizobia, suggesting that nif genes in the beta-rhizobia might have been acquired from alpha-rhizobia via lateral transfer during co-occupancy of nodules by alpha-rhizobia and progenitors of R. taiwanensis, members of the beta-subclass of Proteobacteria. Immunological cross-reaction of the bacteroid preparation of M. pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R. taiwanensis grown microaerobically in minimal medium with and without NH4Cl. In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.

Inhibition of biosynthesis and activity of nitrogenase in Azospirillum brasilense Sp7 under salinity stress.

Azospirillum brasilense is a microaerophilic, plant growth-promoting bacterium, whose nitrogenase activity has been shown to be sensitive to salinity stress. Growth of A. brasilense in semi-solid medium showed that diazotrophic growth in N-free medium was relatively less sensitive to high NaCl concentrations (200-400 mM) than that in presence of NH4+. Increase in salinity stress to diazotrophic A. brasilense in the semi-solid medium led to the migration of the pellicle to deeper anaerobic zones. Assays of acetylene reduction and nifH- lacZ and nifA- lacZ fusions indicated that salinity stress inhibited nitrogenase biosynthesis more strongly than nitrogenase activity. Under salt stress, the amount of dinitrogenase reductase inactivated by ADP-ribosylation was strongly reduced, indicating that the dinitrogenase reductase ADP ribosyl transferase (DRAT) activity was also inhibited by increased NaCl concentrations. Movement of the pellicle to the anaerobic zone and inhibition of DRAT might be adaptive responses of A. brasilense to salinity stress under diazotrophic conditions. Supplementation of glycine betaine, which alleviates salt stress, partially reversed both responses.