Characterization and localization of fluorescent Pseudomonas cold shock protein(s) by monospecific polyclonal antibodies

Cold shock protein (CSP) from Pseudomonas fluorescens MTCC 103 and cold resistant protein (CRP) from its mutant CRPF8 of 14 and 35 kd, respectively were purified to homogeneity by HPLC. Polyclonal antibodies were raised against these proteins and the expression level was checked at different temperatures, i.e., 4, 10, 20, 30 and 37 C. Furthermore, morphological changes in P. fluorescens MTCC 103 and its mutant (CRPF8) were analyzed by transmission electron microscopy (TEM). Localization of CSP and CRP documented with immunoelectron microscopy, using colloidal gold particles conjugated with secondary antibodies being the probe were used. Nevertheless, the results of cytosolic localization of CSP and CRP were evident. Furthermore, the expression of CSP and CRP increased with decrease in temperature and the cell wall thickness of the mutant exhibited 2-fold increase, thus facilitating low temperature survival.     

Productivity modifications to flour beetles (Coleoptera: Tenebrionidae) by gamma radiation (60Co) of one or both sexes and by the addition of radiated males or females to population

The effects on productivity by the addition of various numbers of radiated adult males or females to populations of two densities were determined for two species of flour beetles. Modifications in productivity after exposure to gamma radiation from⁶⁰Co included:

1. females of T. confusum were sterilized by an exposure between 2620 to 5230 R.
2. females of T. castaneum were not sterilized by 10, 200 R.
3. males were not sterilized by 10, 200 R.
4. sterile females may serve as depositories for normal sperm thereby reducing the productivity. Addition of such females to populations of flour beetles did not reduce the numbers of progeny.
5. since sterile females did not suppress population development, and since they destroy grain and cereal products, they should be eliminated from control programs featuring the sterile insect release technique.
6. population density decreased the numbers of progeny.

The use of the sterile insect release technique to control insects of stored grain products does not seem likely with present day sanitary standards since large numbers of insects need be added to effect control.     

Immunodetection and localization of protein(s) related to retinal S-antigen (arrestin) in kidney.

S-antigen (arrestin) is a cytosolic protein which regulates phototransduction in retinal rods. A protein immunologically related to S-antigen was identified in fractions from soluble extract of bovine kidney enriched by gel filtration or by immunoaffinity chromatography using a polyclonal antibody to retinal S-antigen. On immunoblots, this protein was recognized by a panel of monoclonal antibodies (mAbs S2D2, S1A3 and S9E2) directed against different S-antigen epitopes and displayed the same apparent molecular mass (48 kDa) as retinal S-antigen. All three mAbs revealed a specific immunoreactivity by indirect immunocytochemical technique on rat kidney sections. The three mAbs recognized some but not all glomerular cells, identified as epithelial cells by immunoelectron microscopy using the mAb S9E2. Both mAbs S2D2 and S1A3 gave a diffuse cytoplasmic staining in all tubule cells. Proximal tubule cells exhibited a weak immunoreactivity, whereas distal and collecting tubule cells were strongly labeled. In contrast, the mAb S9E2 immunoreaction was restricted to a cell subpopulation from distal and collecting tubules corresponding to intercalated cells identified by immunoelectron microscopy. With the mAb S9E2, the labeling of proximal tubule cells was localized in the apical region of the cytoplasm. These results suggest that two or more 48-kDa proteins immunologically cross-reactive with retinal S-antigen are present in kidney. The observed pattern of distribution is in keeping with the hypothesis that such proteins could play a role in the regulation of G-protein-related receptors present in renal glomerulus and tubule epithelial cells.     

Inhibitor of Nrf2 (INrf2 or Keap1) protein degrades Bcl-xL via phosphoglycerate mutase 5 and controls cellular apoptosis

INrf2 (Keap1) is an adaptor protein that facilitates INrf2-Cul3-Rbx1-mediated ubiquitination/degradation of Nrf2, a master regulator of cytoprotective gene expression. Here, we present evidence that members of the phosphoglycerate mutase family 5 (PGAM5) proteins are involved in the INrf2-mediated ubiquitination/degradation of anti-apoptotic factor Bcl-xL. Mass spectrometry and co-immunoprecipitation assays revealed that INrf2, through its DGR domain, interacts with PGAM5, which in turn interacts with anti-apoptotic Bcl-xL protein. INrf2-Cul3-Rbx1 complex facilitates ubiquitination and degradation of both PGAM5 and Bcl-xL. Overexpression of PGAM5 protein increased INrf2-mediated degradation of Bcl-xL, whereas knocking down PGAM5 by siRNA decreased INrf2 degradation of Bcl-xL, resulting in increased stability of Bcl-xL. Mutation of PGMA5-E79A/S80A abolished INrf2/PGAM5/Bcl-xL interaction. Therefore, PGAM5 protein acts as a bridge between INrf2 and Bcl-xL interaction. Further studies showed that overexpression of INrf2 enhanced degradation of PGAM5-Bcl-xL complex, led to etoposide-mediated accumulation of Bax, increased release of cytochrome c from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. In addition, antioxidant (tert-butylhydroquinone) treatment destabilized the Nrf2-INrf2-PGAM5-Bcl-xL complex, which resulted in release of Nrf2 in cytosol and mitochondria, release of Bcl-xL in mitochondria, increase in Bcl-xL heterodimerization with Bax in mitochondria, and reduced cellular apoptosis. These data provide the first evidence that INrf2 controls Bcl-xL via PGAM5 and controls cellular apoptosis.     

Combination of 'omics' data to investigate the mechanism(s) of hydrazine-induced hepatotoxicity in Rats and to identify potential biomarkers

To gain novel insight into the molecular mechanisms underlying hydrazine-induced hepatotoxicity, mRNAs, proteins and endogenous metabolites were identified that were altered in rats treated with hydrazine compared with untreated controls. These changes were resolved in a combined genomics, proteomics and metabonomics study. Sprague-Dawley rats were assigned to three treatment groups with 10 animals per group and given a single oral dose of vehicle, 30 or 90 mg kg^-1 hydrazine, respectively. RNA was extracted from rat liver 48 h post-dosing and transcribed into cDNA. The abundance of mRNA was investigated on cDNA microarrays containing 699 rat-specific genes involved in toxic responses. In addition, proteins from rat liver samples (48 and 120/168 h post-dosing) were resolved by two-dimensional differential gel electrophoresis and proteins with changed expression levels after hydrazine treatment were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting. To elucidate how regulation was reflected in biochemical pathways, endogenous metabolites were measured in serum samples collected 48 h post-dosing by 600-MHz ¹H-NMR. In summary, a single dose of hydrazine caused gene, protein and metabolite changes, which can be related to glucose metabolism, lipid metabolism and oxidative stress. These findings support known effects of hydrazine toxicity and provide potential new biomarkers of hydrazine-induced toxicity.     

Combination of classical and statistical approaches to enhance the fermentation conditions and increase the yield of Lipopeptide(s) by Pseudomonas sp. OXDC12: its partial purification and determining antifungal property

Around 200 different lipopeptides (LPs) have been identified to date, most of which are produced via Bacillus and Pseudomonas species. The clinical nature of the lipopeptide (LP) has led to a big surge in its research. They show antimicrobial and antitumor activities due to which mass-scale production and purification of LPs are beneficial. Response surface methodology (RSM) approach has emerged as an alternative in the field of computational biology for optimizing the reaction parameters using statistical models. In the present study, Pseudomonas sp. strain OXDC12 was used for production and partial purification of LPs using Thin Layer Chromatography (TLC). The main goal of the study was to increase the overall yield of LPs by optimizing the different variables in the fermentation broth. This was achieved using a combination of one factor at a time (OFAT) and response surface methodology (RSM) approaches. OFAT technique was used to optimize the necessary parameters and was followed by the creation of statistical models (RSM) to optimize the remaining variables. Maximum mycelial growth inhibition (%) against the fungus Mucor sp. was 61.3% for LP. Overall, the combination of both OFAT and RSM helped in increasing the LPs yield by 3 folds from 367mg/L to 1169mg/L.     

Using the life history model to set the stage(s) of growth and senescence in bioarchaeology and paleodemography

Paleodemography, the study of demographic parameters of past human populations, relies on assumptions including biological uniformitarianism, stationary populations, and the ability to determine point age estimates from skeletal material. These assumptions have been widely criticized in the literature and various solutions have been proposed. The majority of these solutions rely on statistical modeling, and have not seen widespread application. Most bioarchaeologists recognize that our ability to assess chronological age is inherently limited, and have instead resorted to large, qualitative, age categories. However, there has been little attempt in the literature to systematize and define the stages of development and ageing used in bioarchaeology. We propose that stages should be based in the human life history pattern, and their skeletal markers should have easily defined and clear endpoints. In addition to a standard five-stage developmental model based on the human life history pattern, current among human biologists, we suggest divisions within the adult stage that recognize the specific nature of skeletal samples. We therefore propose the following eight stages recognizable in human skeletal development and senescence: infancy, early childhood, late childhood, adolescence, young adulthood, full adulthood, mature adulthood, and senile adulthood. Striving toward a better prediction of chronological ages will remain important and could eventually help us understand to what extent past societies differed in the timing of these life stages. Furthermore, paleodemographers should try to develop methods that rely on the type of age information accessible from the skeletal material, which uses life stages, rather than point age estimates.     

Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH₄)₂SO₄ to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca²⁺ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca²⁺ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca²⁺ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca²⁺ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca²⁺ uptake without appreciably influencing the apparent affinity of the transport system for Ca²⁺. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca²⁺ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca²⁺ uptake, CPF-I also inhibited the Ca²⁺-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca²⁺-preloaded sarcoplasmic reticulum vesicles did not promote Ca²⁺ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca²⁺ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca²⁺ pump, and therefore in determining the duration of Ca²⁺ signal in slow-contracting muscle fibers.     

The Responses of Pro‐ and Antioxidative Systems to Cold‐hardening and Pathogenesis Differ in Triticale (x Triticosecale Wittm.) Seedlings Susceptible or Resistant to Pink Snow Mould (Microdochium nivale Fr., Samuels & Hallett)

Two winter triticale (x Triticosecale Wittm.) cultivars, Magnat (susceptible to pink snow mould) and Hewo (relatively resistant), were used in a model system to test the effect of prehardening and different cold‐hardening regimes on pro‐ and antioxidative activity in seedling leaves. The concentration of hydrogen peroxide and the activity of total superoxide dismutase, catalase, peroxidase and ascorbic peroxidase were analysed spectrophotometrically. As there has been no previous analysis of the pro/antioxidative reaction of cereals to Microdochium nivale infection has been undertaken to‐date, this is the first in the series describing our results. We confirmed that both exposure to abiotic stress of low temperature and subsequent low light intensity, as well as biotic stress of M. nivale infection, change the pro‐ and antioxidative activity in model plants. Genotypes differed substantially in their hydrogen peroxide content: susceptible cv. Magnat generally showed higher levels during all the experiments. This result can lead to the conclusion that cv. Magnat is also more susceptible to low temperature and low light intensity than cv. Hewo. Simultaneous measurements of antioxidative activity indicated that the increased activity of catalases and peroxidases and the consequent lower H₂O₂ level are correlated with a higher resistance to low temperature, low light intensity and pink snow mould in triticale seedlings. The higher H₂O₂ level observed in the susceptible line is likely to be derived from the imbalance of reactive oxygen species production and consumption in this genotype under stress conditions.