Design and study of peptide-based inhibitors of amylin cytotoxicity

The incidence of type II diabetes is on the increase each year and the World Health Organisation (WHO) predicts there to be over 360 million diabetic patients worldwide by the year 2030. Deposits consisting mainly of a small protein, called islet amyloid polypeptide (amylin), which aggregates into oligo-/polymeric beta sheet structures is responsible for cytoxicity to the pancreatic β-cells, thus inhibition of this process has been explored as a potential prevention or treatment. N-Methylated and non N-methylated peptides spanning the length of amylin_1–37 were synthesised and evaluated for their inhibition of full length amylin mediated cytoxicity to RIN-5F cells. The non N-methylated peptides were very effective in inhibiting the cytotoxicity while the N-methylated peptides were not. Both the N-methylated and non N-methylated versions of the 29-34 region were equally effective.     

Biofilm productivity and concomitant cell autolysis in a membrane bioreactor

Phanerochaete chrysoporium morphology and manganese peroxidase (MnP) productivity was characterised in a scalable, modularised 1145 cm³ membrane gradostat reactor in response to switching between an enhanced production medium and a nutrient limited feed (50% C and N reduction). Irrespective of the feed composition used nutrients permeating from the lumen of the ultrafiltration membrane matrix established nutrient gradients across the immobilised biofilm with distinct primary, stationary and decline growth phases observed. Severe nutrient C and N limitation did not change the cyclic nature of enzyme production (MnP_max = 189.5 U l⁻¹) but did reduce the overall bioreactor efficiency from 32 to 22 U l⁻¹ day⁻¹. Stress induced secondary metabolism resulted in concomitant cell autolysis causing biomass loss and increased operational flux after 20 days in the 33 day bioreactor operation cycle.     

PFGE and RAPD profiling differentiates closely related isolates of Ancylobacter aquaticus

1,2-dichloroethane (DCA) is a toxic synthetic haloalkane produced annually in excess of 20 billion tons. Five bacterial isolates capable of complete mineralization of DCA have recently been isolated from wastewater treatment facilities in South Africa. Pulsed field gel electrophoresis (PFGE) and random amplification of polymorphic DNA (RAPD) analysis were employed in this study to identify phylogenetic differences between these closely-related bacteria. Analysis of the 16S rDNA sequences of the selected isolates revealed similarities to previously characterised isolates of Ancylobacter aquaticus. It has been previously shown that all isolates follow the same catabolic pathway and possess an identical hydrolytic dehalogenase (DhlA) involved in the initial carbonchlorine bond cleavage. Analysis of homology matrices deduced from RAPD and restriction profiles, constructed using the GelCompar software package, revealed that although some of the isolates possessed identical profiles using one primer or restriction endonuclease, differences were observed when a different primer was used. Furthermore, the results obtained indicate that the previously characterised isolate A. aquaticus AD25 is significantly different from the isolates used in this study. PFGE was also able to show that isolates of A. aquaticus do not possess the 200 kb plasmid containing the hydrolytic dehalogenase gene previously identified in the DCA-degrading bacterium Xanthobacter autotrophicus GJ10. This study has been able to demonstrate that RAPD and PFGE analysis are suitable molecular tools for the differentiation of closely-related A. aquaticus isolates and may be routinely used in the differentiation of environmentally important bacteria.     

The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate K(m) of 7.5 μm and V(max) of 1800 nmol min(-1) mg(-1) of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg(2+) or Mn(2+). Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains.     

Microreactors for peptide synthesis: looking through the eyes of twenty first century !!!

The twenty first century has witnessed several advances in synthetic chemistry, among them microreactors. It is expected that these devices will have a considerable impact on synthetic organic chemistry since they offer a wide range of applications in various fields. Perhaps the synthesis of peptides deserves mention in this regard as these molecules are emerging as therapeutics and offer several advantages over the so-called small molecules. This minireview does not aim to address microreactors in detail, but explains various peptide synthesis methods that involve microfluidic techniques, highlighting the need for further improvement and expansion of microdevices/microreactors.     

Prospective monitoring reveals dynamic levels of T cell immunity to Mycobacterium tuberculosis in HIV infected individuals

Monitoring of latent Mycobacterium tuberculosis infection may prevent disease. We tested an ESAT-6 and CFP-10-specific IFN-γ Elispot assay (RD1-Elispot) on 163 HIV-infected individuals living in a TB-endemic setting. An RD1-Elispot was performed every 3 months for a period of 3-21 months. 62% of RD1-Elispot negative individuals were positive by cultured Elispot. Fluctuations in T cell response were observed with rates of change ranging from -150 to +153 spot-forming cells (SFC)/200,000 PBMC in a 3-month period. To validate these responses we used an RD1-specific real time quantitative PCR assay for monokine-induced by IFN-γ (MIG) and IFN-γ inducible protein-10 (IP10) (MIG: r?=?0.6527, p?=?0.0114; IP-10: r?=?0.6967, p?=?0.0056; IP-10+MIG: r?=?0.7055, p?=?0.0048). During follow-up 30 individuals were placed on ARVs and 4 progressed to active TB. Fluctuations in SFC did not correlate with CD4 count, viral load, treatment initiation, or progression to active TB. The RD1-Elispot appears to have limited value in this setting.     

Reproductive biology of longnose trevally (Carangoides chrysophrys) in the Arabian Sea, Oman

The reproductive pattern of longnose trevally (Carangoides chrysophrys) in the Arabian Sea, off the coast of Oman, is described. Samples were collected on a monthly base between April 2005 and September 2006 from two landing sites (ALlakbi and Raysuot). A single peak of the gonadosomatic index (GSI) started from September and ended in February signalling the spawning period. Values of the relative condition factor (K _n ) were fairly constant throughout the year for both males and females and no significantly difference was found in the average K _n values between males and females during sampling period. Male and female lengths-at 50% maturity were 46.90 and 42.08 cm, respectively and were significantly different. This study thus increases our understanding of the reproductive biology of an important commercial and artisanal fish species in one of its major distribution areas.     

Synthesis and NMR elucidation of novel tetrapeptides

The synthesis and NMR elucidation of Ala‐Val‐Pro‐Ile and five novel peptide‐based derivatives are reported. These peptides mimic the natural second mitochondria‐derived activator of caspase (Smac) protein. Purification was achieved using preparative HPLC and the NMR elucidation of all compounds is reported for the first time. A series of overlapping signals were observed in the 1D NMR spectra thus making assignment a difficult task to undertake. The use of 2D NMR techniques with the inclusion of efficient adiabatic symmetrized ROESY proved to be an effective tool in overcoming these difficulties. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.