Analysis of chromatin of the brain of young and old rats by micrococcal nuclease and DNase I

Micrococcal nuclease (MCN) and DNase I were used to study the conformational changes in chromatin of the brain of rats of different ages. Purified nuclei and chromatin were digested separately by MCN and DNase I. Kinetics of digestion of chromatin by MCN are similar for young, adult and old rats. Also agarose gel electrophoresis of DNA fragments do not show any differences. The kinetics of digestion with DNase I, on the other hand, are greater and faster for 20-week old rats than for 90-week old rats. High performance denaturing polyacrylamide gel electrophoresis reveals that a greater amount of smaller fragments of DNA are produced in the 20-week old rats than in the 90-week. These conformational changes occur in the chromatin during aging.     

Ascorbic acid and aging in the rat. Uptake of ascorbic acid by teeth and concentration of various forms of ascorbic acid in different organs

1. The uptake of ascorbic acid in vitro by the teeth of rats showed a gradual decrease with age, indicating that the uptake may be related to collagen synthesis as in bone. 2. The concentration of total free ascorbic acid in various organs declined with age, but the rate of decline was different in different organs. In the spleen, however, it increased until maturity and then declined. 3. This decrease may be due to one or both of the following reasons: (a) the permeability of different tissues may decrease at different rates for ascorbic acid, or (b) the requirement for ascorbic acid may decrease at different rates. 4. The bound ascorbic acid declined with age in the skin, kidney, liver and brain after the age of 10-12 weeks, and in the spleen after the age of 26 weeks. 5. The concentration of dehydroascorbic acid and dioxogulonic acid declined with age in the skin.     

Integration of Leprosy Elimination into Primary Health Care in Orissa,India

Background

Leprosy was eliminated as a public health problem (<1 case per 10,000) in India by December 2005. With this target in sight the need for a separate vertical programme was diminished. The second phase of the National Leprosy Eradication Programme was therefore initiated: decentralisation of the vertical programme, integration of leprosy services into the primary health care (PHC) system and development of a surveillance system to monitor programme performance.

Methodology/Principal Findings

To study the process of integration a qualitative analysis of issues and perceptions of patients and providers, and a review of leprosy records and registers to evaluate programme performance was carried out in the state of Orissa, India. Program performance indicators such as a low mean defaulter rate of 3.83% and a low-misdiagnosis rate of 4.45% demonstrated no detrimental effect of integration on program success. PHC staff were generally found to be highly knowledgeable of diagnosis and management of leprosy cases due to frequent training and a support network of leprosy experts. However in urban hospitals district-level leprosy experts had assumed leprosy activities. The aim was to aid busy PHC staff but it also compromised their leprosy knowledge and management capacity. Inadequate monitoring of a policy of ‘new case validation,’ in which MDT was not initiated until primary diagnosis had been verified by a leprosy expert, may have led to approximately 26% of suspect cases awaiting confirmation of diagnosis 1–8 months after their initial PHC visit.

Conclusions/Significance

This study highlights the need for effective monitoring and evaluation of the integration process. Inadequate monitoring could lead to a reduction in early diagnosis, a delay in initiation of MDT and an increase in disability rates. This in turn could reverse some of the programme''s achievements. These findings may help Andhra Pradesh and other states in India to improve their integration process and may also have implications for other disease elimination programmes such as polio and guinea worm (dracunculiasis) as they move closer to their elimination goals.     

Exogenously expressed human Ku70 stabilizes Ku80 in Xenopus oocytes and induces heterologous DNA-PK catalytic activity

The Ku protein is a heterodimer composed of 70 kD (Ku70) and 80 kD (Ku80) subunits. Ku is the regulatory component of the DNA-dependent protein kinase (DNA-PK) that has a catalytic subunit of ~460 kD (DNA-PK_cs). In this study, the two polypeptides (Ku80/Ku70) of the human Ku were expressed in Xenopus oocytes in order to investigate their over-expression, sub-cellular localization, and functional interaction with the Xenopus DNA-PK_cs. In vitro-transcribed mRNAs for Ku70 and Ku80 were obtained from the respective plasmid constructs. The exogenously expressed proteins from the injected mRNAs were immunoprecipitated using a specific anti-T7 Tag antibody. The T7 Tag epitope is present in the vector at the amino-terminus and is in-frame with the Ku cDNA sequences. While injected Ku70 mRNA translated to a full-length Ku70 polypeptide that translocated to the nucleus, injected Ku80 mRNA resulted in the expression of a truncated product that was retained in the cytoplasm. Although Ku80 mRNA was stable for a period of 18 h in the oocytes post-microinjection, the protein was only stabilized when co-expressed with Ku70, suggesting that Ku80 is susceptible to proteolytic degradation when not dimerized with Ku70. Furthermore, the immunocomplex was capable of phosphorylating the DNA-PK-specific substrate thereby indicating that the holoenzyme could functionally reconstitute in vivo in the oocytes by heterologous subunits thus demonstrating evolutionary conservation of the enzyme subunit structure and function among diverse species.     

Age-dependent Expression of S100β in the Brain of Mice

S100β is a soluble calcium binding protein released by glial cells. It has been reported as a neurotrophic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and in long term potentiation in the adult brain. The ability of S100β to modulate neuronal morphology raises the important question whether there is an age-related difference in the expression of S100β in the cerebral and cerebellar cortices of AKR strain mice and is this change is region specific. Our RT–PCR and Western blotting experiments show that the expression of S100β gene in the cerebral and cerebellar cortices starts from 0 day, peaks at about 45 days. However, in 70-week old mice its expression is significantly up-regulated as compared to that of 20-week old mice. S100β follows the same age-related pattern in both cerebral and cerebellar cortices. These results suggest that S100β is important for brain development and establishment of proper brain functions. Up-regulation of S100β in old age may have some role in development of age-related pathological systems in the brain.     

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator,Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys²⁴⁵–Ala²⁷⁷. p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1–42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.     

Review: Current status and future prospects of associative nitrogen fixation in rice

Biological sources of nutrients are gaining importance over the chemical and organic sources from the standpoint of environmental safety and quality, and sustainable agriculture. The nutrient input for a growing rice crop can largely be met by promoting the activities of physiologically diverse microorganisms in the aerobic, anaerobic and interface zones in the ecologically important flooded soils. Associative bacteria contribute from 10 to 80 kg N per hectare per cropping season depending upon the ecosystem, cultural practices and rice variety grown. In addition to N contribution, these bacterial associations can improve the nutrient transformations and contribute to plant growth-promoting effects. Current improved agronomic and crop production management systems greatly affect the contributions of biological sources to the overall soil nutrient status. Azospirillum and other associative bacterial systems have been intensively researched using various evaluation techniques to understand the diazotrophic rhizocoenosis. Researches clearly indicate that these associations are governed by several soil, water, nutrient, agrochemical, plant genotype and other biological factors. Considerable efforts have been made so far in selecting efficient bacterial strains as inoculants and identifying host genotypes which support maximum nitrogenase activity in addition to other beneficial traits of effective associative relationships. Knowledge gained so far on how the N2-fixing system in rice functions suggests the need for providing optimum management practices to ensure greater contribution from the plant-microbe associations. Holistic approaches integrating technological developments and achievements in biological sciences could lead to crop improvement. Research on extending nitrogen-fixing symbiosis to rice using molecular and genetic approaches is underway, albeit at a slow pace. The need for further fine-tuning and developing management practices, innovative approaches to improve rice-bacterial systems and the strategies to sustain the benefits from associative diazotrophy are discussed.     

Phosphorylation and acetylation of nonhistone chromosomal proteins of the brain of rats of various ages and their modulation by calcium and estradiol.

$\text{In}\text{vitro}$ phosphorylation and acetylation of nonhistone chromosomal (NHC) proteins and their modulation by Ca⁺⁺ and estradiol were studied by incubating slices of cerebral cortex of 2-, 15- and 84-week female rats with ³²Pi and ¹⁴C-Na-acetate. Phosphorylation pattern of NHC proteins is unique for each age. Ca⁺⁺ and estradiol stimulate phosphorylation of different NHC proteins which is also age-specific. Acetylation of NHC proteins decreases precipitously with age. No unique NHC protein is acetylated preferentially at any age, nor does Ca⁺⁺ stimulate acetylation. Estradiol, however, stimulates acetylation of a few NHC proteins. It is suggested that phosphorylation of NHC proteins and its modulation by effectors may be more important for gene expression than their acetylation.     

Modulation of acetylation of chromosomal proteins of the brain of rats of various ages by epinephrine and estradiol

During purification of branched chain ketoacid dehydrogenase from $\text{Pseudomonas}\text{putida}$, large losses in enzyme activity occurred. Much of the activity was restored by the addition of a heat-treated, soluble fraction. The factor had a molecular weight less than 1000 and, of several potential effectors tested, the branched chain amino acids were the only compounds which stimulated enzyme activity, with valine being the most effective. The concentration of valine in the heat-treated fraction was found to be sufficient to account for all of the stimulation produced by this fraction. Valine had no effect on either pyruvate or 2-ketoglutarate dehydrogenases.