Interaction of aldehydes with collagen: effect on thermal, enzymatic and conformational stability

Stabilization of type I rat tail tendon (RTT) collagen by various aldehydes, viz. formaldehyde, gluteraldehyde, glyoxal and crotanaldehyde was studied to understand the effect of each on the thermal, enzymatic and conformational stability of collagen. The aldehydes have been found to increase the heat stability of rat tail tendon collagen fibres from 62 to 77-86 degrees C. The increase in thermal stability was found to be in a species dependent manner. The variation in the thermal stability of collagen brought about by aldehydes was in the order of formaldehyde > gluteraldehyde > glyoxal > crotanaldehdye. The aldehydes also impart a high degree of stability to collagen against the activity of the degrading enzyme, collagenase. The order of enzymatic stability brought about by aldehydes follows the same trend as the thermal stability brought about by them. This shows that the number of cross-links formed influence both the thermal and enzymatic stability in the similar manner. The effect of various aldehydes on the secondary structure of collagen was studied using circular dichroism and it was found that the aldehydes lead to changes in the amplitude of the circular dichroic (CD) spectrum but did not alter the triple helical conformation of collagen. The secondary structure of collagen is not significantly altered on interaction with different aldehydes.     

Production and partial purification of invertase using <Emphasis Type="Italic">Cympopogan caecius</Emphasis> leaf powder as substrate

The present study investigates the efficiency of Aspergillus niger to produce invertase, an industrially important enzyme by using powdered stem of Cympopogan caecius (Lemon grass) as sole substrate and sole carbon source for the microorganism. The molecular weight of invertase was estimated to be 66–70 kDa by sodium do decyl sulphate poly acrylamide gel electrophoresis (SDS PAGE). The production of the enzyme was studied at different pH scales ranging from pH 4.0 to 7.0 at a constant temperature of 30°C and 2% substrate concentration. The maximum production of invertase (specific activity −0.0516 μk/mg protein) was obtained at pH 5.5 at 30°C temperature, and incubation for 48 h. The activity was found to be stable at pH 5.5 for 30 min. The enzyme was found to be stable in the temperature range of 20–55°C. The effect of divalent metal ions Cu²⁺, Fe²⁺, Co²⁺ on the activity of the enzyme invertase showed that these ions affected the activity by a certain factor. The study can be further industrially exploited in a country-like India where lemon grass is found in plenty and can be used as substrate for enzyme production. Moreover, the preparation of the substrate is also a simple process.     

Deletion of the aceE gene (encoding a component of pyruvate dehydrogenase) attenuates Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major food-borne pathogen. From a transposon insertion mutant library created previously using S. Enteritidis 10/02, one of the mutants was identified to have a 50% lethal dose (LD(50) ) at least 100 times that of the parental strain in young chicks, with an attenuation in a poorly studied gene encoding a component of pyruvate dehydrogenase, namely the aceE gene. Evaluation of the in vitro virulence characteristics of the ΔaceE∷kan mutant revealed that it was less able to invade epithelial cells, less resistant to reactive oxygen intermediate, less able to survive within a chicken macrophage cell line and had a retarded growth rate compared with the parental strain. Young chicks vaccinated with 2 × 10(9) CFU of the ΔaceE∷kan mutant were protected from the subsequent challenge of the parental strain, with the mutant colonized in the liver and spleen in a shorter time than the group infected with the parental strain. In addition, compared with the parental strain, the ΔaceE∷kan mutant did not cause persistent eggshell contamination of vaccinated hens.     

Development and validation of functional marker targeting an InDel in the major rice blast disease resistance gene Pi54 (Pik h )

Rice blast is one of the most devastating diseases affecting the rice crop throughout the world. In molecular breeding for host plant resistance, functional markers are very useful for enhancing the precision and accuracy in marker-assisted selection (MAS) of target gene(s) with minimum effort, time and cost. Pi54 (which was earlier known as Pik ^h ) is one of the major blast resistance genes and has been observed to show resistance against many isolates of the blast pathogen in India. The gene has been cloned through map-based strategy and encodes a nucleotide-binding site?Cleucine-rich repeat (NBS?CLRR) domain-containing protein. In the present study, we carried out allele mining for this gene and identified a 144-bp insertion/deletion (InDel) polymorphism in the exonic region of the gene. A PCR-based co-dominant molecular marker targeting this InDel, named Pi54 MAS, was developed. Pi54 MAS was observed to perfectly co-segregate with blast resistance in a mapping population with no recombinants. Validation of this marker in 105 genotypes which are either susceptible or resistant to rice blast disease showed that the marker is polymorphic in most of the resistant?Csusceptible genotype combinations and is more accurate than the earlier reported markers for Pi54. Hence this functional, co-dominant marker is suggested for routine deployment in MAS of Pi54 in breeding programs.     

Predicting the clinical lethality of osteogenesis imperfecta from collagen glycine mutations

Osteogenesis imperfecta (OI), or brittle bone disease, often results from missense mutation of one of the conserved glycine residues present in the repeating Gly-X-Y sequence characterizing the triple-helical region of type I collagen. A composite model was developed for predicting the clinical lethality resulting from glycine mutations in the alpha1 chain of type I collagen. The lethality of mutations in which bulky amino acids are substituted for glycine is predicted by their position relative to the N-terminal end of the triple helix. The effect of a Gly --> Ser mutation is modeled by the relative thermostability of the Gly-X-Y triplet on the carboxy side of the triplet containing the substitution. This model also predicts the lethality of Gly --> Ser and Gly --> Cys mutations in the alpha2 chain of type I collagen. The model was validated with an independent test set of six novel Gly --> Ser mutations. The hypothesis derived from the model of an asymmetric interaction between a Gly --> Ser mutation and its neighboring residues was tested experimentally using collagen-like peptides. Consistent with the prediction, a significant decrease in stability, calorimetric enthalpy, and folding time was observed for a peptide with a low-stability triplet C-terminal to the mutation compared to a similar peptide with the low-stability triplet on the N-terminal side. The computational and experimental results together relate the position-specific effects of Gly --> Ser mutations to the local structural stability of collagen and lend insight into the etiology of OI.     

Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics

Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing in vivo articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in the percentage of collagen degradation and the release of glycosaminoglycans against collagenase digestion, indicating the increase physical integrity and resistance of polyphenol crosslinked cartilage to enzymatic digestion. To examine the in vivo cartilage protective effects, polyphenols were injected intra-articularly before (prophylactic) and after (therapeutic) the induction of collagen-induced arthritis in rats. The hind paw volume and histomorphological scoring was done for cartilage damage. The intra-articular injection of epigallocatechin gallate and tannic acid did not significantly influence the time of onset or the intensity of joint inflammation. However, histomorphological scoring of the articular cartilage showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, indicating that intra-articular injections of polyphenols bind to articular cartilage and making it resistant to degradation despite ongoing inflammation. These studies establish the value of intra-articular injections of polyphenol in stabilization of cartilage collagen against degradation and indicate the unique beneficial role of injectable polyphenols in protecting the cartilage in arthritic conditions.     

Chronic estradiol exposure induces oxidative stress in the hypothalamus to decrease hypothalamic dopamine and cause hyperprolactinemia

Estrogens are known to cause hyperprolactinemia, most probably by acting on the tuberoinfundibular dopaminergic (TIDA) system of the hypothalamus. Dopamine (DA) produced by TIDA neurons directly inhibits prolactin secretion and, therefore, to stimulate prolactin secretion, estrogens inhibit TIDA neurons to decrease DA production. However, the mechanism by which estrogen produces this effect is not clear. In the present study, we used a paradigm involving chronic exposure to low levels of estradiol-17β (E(2)) to mimic prolonged exposures to environmental and endogenous estrogens. We hypothesized that chronic exposure to low levels of E(2) induces oxidative stress in the arcuate nucleus (AN) of the hypothalamus that contains TIDA neurons and causes nitration of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA. This results in a significant decrease in DA and consequently, hyperprolactinemia. To investigate this, adult, intact female cycling rats were implanted with slow-release E(2) pellets (20 ng/day) for 30, 60, or 90 days and were compared with old (16-18 mo old) constant estrous (OCE) rats. Chronic E(2) exposure significantly increased the expression of glial fibrillary acidic protein and the concentrations of interleukin-1β (IL-1β) and nitrate in the AN that contains perikarya of TIDA neurons and increased nitration of TH in the median eminence (ME) that contains the terminals. These levels were comparable to those seen in OCE rats. We observed a significant decrease in DA concentrations in the ME and hyperprolactinemia in an exposure-dependent manner similar to that seen in OCE rats. It was concluded that chronic exposure to low levels of E(2) evokes oxidative stress in the AN to inhibit TIDA neuronal function, most probably leading to hyperprolactinemia.