Real time detection and quantification of inclusion bodies expressed in Escherichia coli by impedance measurements

Escherichia coli expressing human growth hormone as inclusion bodies was cultured in a fermenter. Real time, non-invasive detection and quantification of inclusion body protein expressed in E. coli was performed by impedance measurements at 50 MHz and 180 MHz. At 50 MHz rotation of dipoles of the protein and their proton fluctuation, i.e., -dispersion of protein aggregates formed inside the cell as a result of expressed protein, results in an additional decrease in impedance. At 180 MHz the impedance remained at a plateau. In a high cell density E. coli culture, after induction with IPTG, when the cell mass remained unchanged, an increase in the magnitude of -dispersion was observed at 50 MHz. This was due to the formation and subsequent increase in the concentration of r-human growth hormone which aggregate as inclusion bodies. The estimation of inclusion bodies by taking the ratio of impedance at 180 MHz and at 50 Mhz matched with the amount of protein estimated after extraction and purification (coefficient of correlation was 0.92). This is the first report of real time detection and monitoring of recombinant protein expressed as inclusion bodies by impedance measurements.     

Modulation of myosin phosphatase targeting subunit and protein phosphatase 1 in the heart

Myosin light chain 2 (LC2) phosphorylation is of both physiological and pathological importance to myocardial function. The phosphatase that directly dephosphorylates LC2 is a type 1 protein phosphatase (PP1) that contains a catalytic subunit that complexes with a myosin-binding phosphatase targeting subunit (MYPT). The goal of the present study was to examine the role of MYPT in the regulation of PP1 in ventricular myocytes. In the first part of the study, regional distribution of MYPT expression and phosphorylation were determined in unstimulated hearts. The pattern of MYPT phosphorylation was inversely related to the LC2 phosphorylation spatial gradient as described by Epstein and colleagues (Davis JS, Hassanzadeh S, Winitsky S, Lin H, Satorius C, Vemuri R, Aletras AH, Wen H, and Epstein ND. Cell 107: 631-641, 2001). In the second part of the study, adult rat isolated ventricular myocytes were exposed to an alpha-adrenergic receptor agonist, and properties of MYPT, PP1, and LC2 were studied. We found MYPT associates with cardiac myofilaments, and this association increases upon alpha-adrenergic receptor stimulation. Activation of alpha-adrenergic receptors also led to a decrease in the PP1-myofilament association. Furthermore, alpha-adrenergic receptor stimulation results in phosphorylation of MYPT and LC2 and an increase in myocyte Ca(2+) sensitivity of tension that all depend on Rho kinase activation. These data support the hypothesis that alpha-adrenergic receptor activation works through Rho kinase to phosphorylate MYPT, and phosphorylated MYPT dissociates from PP1 so that PP1 is no longer physically associated with LC2. Hence, we propose a pathway for the dynamic modulation of LC2 phosphorylation through receptor-dependent phosphorylation of MYPT, and a spatial gradient of LC2 phosphorylation under basal conditions that occurs due to varied levels of phosphorylation of MYPT in ventricles.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> SP5212

Summary Production of poly(3-hydroxybutyric acid) [P(3HB)] by Rhodopseudomonas palustris SP5212 isolated in this laboratory has been optimized under phototrophic microaerophilic conditions. Cells grown in malate medium accumulated 7.7% (w/w) P(3HB) of cellular dry weight at the early stationary phase of growth. The accumulated P(3HB) however, attained 15% (w/w) of cellular dry weight when acetate (1.0%, w/v) was used as the sole carbon source under nitrogen-limiting conditions. Synthesis and accumulation of polymer was favoured by sulphate-free conditions and at a phosphate concentration sub-optimal for growth. The polymer content of cells was increased drastically (34% of cellular dry weight) when the acetate containing medium was supplemented with n-alkanoic acids. Compositional analysis by H¹ NMR revealed that these accumulated polymers were composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (3HV). The contents of 3HV in these copolymers ranged from 14 to 38 mol%.     

Conservation categories of siluroid fishes in North-East Sundarbans,India

In stock fishery of North-East Sundarbans, siluroid fishes hold a remarkable position owing to the culture potentiality of many of them. Scales in fishes play significant role in protection and resistance against environmental stresses and the lack of scalation in case of siluroid fishes is supposed to be one of the reasons for their decline in eco-degraded aquatic habitats. 11 species of fishes belonging to 9 genera, 8 families under the order Siluriformes have been recorded from different water bodies of North-East Sundarbans, India. Fish Magnitude Value (FMV) of those catfishes were recorded especially by information harvested from local fishermen community and stakeholders as well as from market survey on fish landing. The extensive damage to the population of catfishes in the area has placed the fishes under threat categories. Following the IUCN guidelines and also through Participatory Rural Appraisal (PRA) methods, an attempt has been made to assess the status of such fishes. Trend analysis, Fish Magnitude Value (FMV) and Rank Based Quotient (RBQ) revealed 1 catfish as Endangered, 5 as Vulnerable and 5 as Near Threatened species. The situation warrants immediate attention of ecologists, administrators, managers and entrepreneurs to propose remedial measures for revival of the fishes. The study also delineates distribution, present status and conservation measures for revival of the fishes in North-East Sundarbans wet-land environment of West Bengal, India.     

Vitamin C transiently arrests cancer cell cycle progression in S phase and G2/M boundary by modulating the kinetics of activation and the subcellular localization of Cdc25C phosphatase

Regulation of cell cycle progression involves redox (oxidation-reduction)-dependent modification of proteins including the mitosis-inducing phosphatase Cdc25C. The role of vitamin C (ascorbic acid, ASC), a known modulator of the cellular redox status, in regulating mitotic entry was investigated in this study. We demonstrated that vitamin C inhibits DNA synthesis in HeLa cells and, mainly the form of dehydroascorbic acid (DHA), delays the entry of p53-deficient synchronized HeLa and T98G cancer cells into mitosis. High concentrations of Vitamin C caused transient S and G2 arrest in both cell lines by delaying the activation of the M-phase promoting factor (MPF), Cdc2/cyclin-B complex. Although vitamin C did not inhibit the accumulation of cyclin-B1, it may have increased the level of Cdc2 inhibitory phosphorylation. This was achieved by transiently maintaining Cdc25C, the activator of Cdc2, both in low levels and in a phosphorylated on Ser216 inactive form that binds to 14-3-3 proteins contributing thus to the nuclear exclusion of Cdc25C. As expected, vitamin C prevented the nuclear accumulation of Cdc25C in both cell lines. In conclusion, it seems that vitamin C induces transient cell cycle arrest, at least in part, by delaying the accumulation and the activation of Cdc25C.     

Enhanced erythrocytic lipid peroxides level in rabbits after repeated parental administration of iron

An experiment was conducted in rabbits to evaluate the possible involvement of oxidative stress in iron-overload animals. Ten adult female New Zealand white rabbits were divided into 2 equal groups with 5 animals each. Group II animals received intramuscular iron dextran injections (120 mg/kg body wt/day) on alternate day for 14 days (8 injections), while Group I animals did not receive any iron supplementation to serve as negative controls. The blood samples were collected by cardiac puncture before the start of iron dosing and thereafter, at weekly intervals for 28 days. The samples were processed to measure blood iron concentration, packed cell volume, erythrocytic lipid peroxide (LPO) level, superoxide dismutase (SOD) and catalase (CAT) activities. The blood iron concentration showed a rising trend following repeated iron administration, and the mean level recorded on day 14 was significantly higher than respective day 0 value. LPO level remained significantly higher from day 14 onwards till the end of the observation period of 14 more days after cessation of iron adminstration. Erythrocytic superoxide dismutase activities showed a transient significant rise on day 7, and thereafter, showed a declining trend, but remained statistically comparable to respective day 0 or corresponding value of the control animals.     

Density functional theory for the nonspecific binding of salt to polyelectrolytes: thermodynamic properties

The thermodynamics of the nonspecific binding of salt to a polyelectrolyte molecule is studied using a density functional approach. The polyelectrolyte molecule is modeled as an infinite, inflexible, and impenetrable charged cylinder and the counterions and co-ions are modeled as charged hard spheres of equal diameter. The density functional theory is based on a hybrid approach where the hard-sphere contribution to the one-particle correlation function is evaluated nonperturbatively and the ionic contribution to the one-particle correlation function is evaluated perturbatively. The advantage of the approach is that analytical expressions are available for all the correlation functions. The calculated single ion preferential interaction coefficients, excess free energy, and activity coefficients show a nonmonotonic variation as a function of polyion charge in the presence of divalent ions. These properties display considerable departure from the predictions of the nonlinear Poisson-Boltzmann (NLPB) equation, with qualitative differences in some cases, which may be attributed to correlation effects neglected in the NLPB theory.     

Oral rehydration formula containing alanine and glucose for treatment of diarrhoea: a controlled trial.

OBJECTIVE--To determine whether adding L-alanine to the glucose based oral rehydration solution recommended by the World Health Organisation would improve its efficacy in treating acute diarrhoea. DESIGN--Randomised double blind controlled trial of oral rehydration solution containing L-alanine and glucose. SETTING--Inpatient service of a hospital treating diarrhoea. PATIENTS--97 Male patients aged 6-59 years admitted to the hospital with acute and severe dehydration due to diarrhoea associated with Vibrio cholerae or enterotoxigenic Escherichia coli. Forty nine received the standard glucose based oral rehydration solution (control group) and 48 this solution with alanine added (study group). INTERVENTIONS--All of the patients received rapid intravenous acetate solution for the initial four hours after admission, which fully corrected the signs of dehydration. They were then admitted to the study and randomised. Immediately after the intravenous treatment oral rehydration treatment was started. All of the patients received oral tetracycline for 48 hours, starting 24 hours after start of the study. If signs of dehydration reappeared during oral treatment patients were given rapid intravenous acetate solution until they were fully corrected and then continued to take the assigned oral rehydration solution. END POINT--Passage of the last watery stool. MEASUREMENTS and MAIN RESULTS--The median stool output/kg body weight during the initial 24 hours of oral rehydration treatment and until diarrhoea stopped was reduced in the study group compared with the control group from 309 ml to 196 ml and from 393 ml to 236 ml respectively. Intake of oral rehydration solution and intravenous acetate solution was reduced from 455 ml to 308 ml and from 616 ml to 425 ml respectively. Two patients in the study group compared with 18 patients in the control group required unscheduled rapid intravenous acetate solution to correct signs of dehydration during oral rehydration treatment. CONCLUSION--Oral rehydration solution containing L-alanine was considerably better than standard oral rehydration solution at reducing the severity of symptoms and the need for fluid of male patients with diarrhoea associated with V cholerae and enterotoxigenic E coli.     

Experiences of a Community-Based Lymphedema Management Program for Lymphatic Filariasis in Odisha State,India: An Analysis of Focus Group Discussions with Patients,Families, Community Members and Program Volunteers

Background

Globally 68 million people are infected with lymphatic filariasis (LF), 17 million of whom have lymphedema. This study explores the effects of a lymphedema management program in Odisha State, India on morbidity and psychosocial effects associated with lymphedema.

Methodology/Principal Findings

Focus groups were held with patients (eight groups, separated by gender), their family members (eight groups), community members (four groups) and program volunteers (four groups) who had participated in a lymphedema management program for the past three years. Significant social, physical, and economic difficulties were described by patients and family members, including marriageability, social stigma, and lost workdays. However, the positive impact of the lymphedema management program was also emphasized, and many family and community members indicated that community members were accepting of patients and had some improved understanding of the etiology of the disease. Program volunteers and community members stressed the role that the program had played in educating people, though interestingly, local explanations and treatments appear to coexist with knowledge of biomedical treatments and the mosquito vector.

Conclusions/Significance

Local and biomedical understandings of disease can co-exist and do not preclude individuals from participating in biomedical interventions, specifically lymphedema management for those with lymphatic filariasis. There is a continued need for gender-specific psychosocial support groups to address issues particular to men and women as well as a continued need for improved economic opportunities for LF-affected patients. There is an urgent need to scale up LF-related morbidity management programs to reduce the suffering of people affected by LF.     

Effect of periphyton on growth performance of grey mullet, Mugil cephalus (Linn.), in inland saline groundwater ponds

The present study attempts to assess the potential of artificial substrates to enhance fish production in inland saline groundwater ponds through periphyton production. Grey mullet, Mugil cephalus, was cultured for 100 days in ponds with substrate (treatment ponds) and without substrate (control ponds). To enhance the surface area, bamboo poles were used as substrate. The periphyton population, pigment concentration and hydrobiological characteristics of pond water were monitored. The studies revealed little difference in most of the water quality parameters observed in the two treatments. However, turbidity (27.0 ± 0.1–35.0 ± 0.1 Nephalo Turbidity Unit (NTU)), chlorophyll ‘a’ (6.6 ± 0.6–7.6 ± 0.6 μg L^?1), plankton population (phytoplankton 8.4 × 10³–9.4 ×10³ numbers L^?1; zooplankton 4.0 × 10³–5.1 × 10³ numbers L^?1) and NH₄–N (2.0 ± 0.2–2.3 ± 0.1 mg L^?1) were high in the treatment with no additional substrate; however, in the treatment with substrate the total Kjeldahl nitrogen (9.8 ± 0.8–10.8 ± 0.7 mg L^?1) and o‐PO₄ (0.1 ± 0.01–0.1 mg L^?1) remained significantly (P < 0.05) higher. Highest periphyton biomass in terms of dry matter (DM) (0.8 ± 0.01–1.4 ±0.01 mg cm^?2), ash free DM (0.4 ± 0.0–0.6 ± 0.01 mg cm^?2), chlorophyll ‘a’ (3.1 ± 0.2–8.1 ± 0.8 μg cm^?2) and pheophytin ‘a’ (1.9 ± 0.4–3.9 ± 0.5 μg cm^?2) was observed at 50 cm depth in ponds provided with additional substrate. Fifteen plankton genera showing periphytic affinity colonized the bamboo substrates. Fish growth (mean fish weight 524.3 ± 8.7 g and SGR 2.5 ± 0.1) was significantly (P < 0.05) higher in ponds provided with additional substrate compared with control ponds (387.2 ± 6.0). Length–weight relationship (LWR) (W = cLⁿ) also showed that the exponential value (‘n’) of length was high in substrate‐supported ponds (n = 2.36) in comparison with controls (n = 1.09). These studies suggest that a periphyton‐supported aquaculture system can be used successfully for the culture of herbivorous brackishwater fish species like M. cephalus in inland saline groundwaters and thus could contribute to the development of sound and sustainable aquaculture technology.