Development and validation of an indirect competitive ELISA for quantification of recombinant staphylokinase in rabbit plasma: Application to pharmacokinetic study.

The relatively short circulatory half-life (2-3 min) of staphylokinase is a major drawback in the development of SAK- (staphylokinase) based thrombolytic drug. A rapid and sensitive method, based on indirect competitive ELISA, was developed and validated for quantitative determination of SAK in rabbit plasma. The dynamic range of the assay varied between 0.41 ± 0.16 µg/L and 9.03 ± 0.38 µg/L (R(2) = 0.98) for SAK in rabbit plasma. There were no dilution linearity issues apparent with this assay. The precision (% CV) ranged from 4.6-9.7% for the intraassay and from 17.1-19.3% for interassay. This validated method was successfully employed for evaluation of various pharmacokinetic parameters of SAK in rabbit.

Does gut brain axis has an impact on Parkinson's disease (PD)?

Parkinson's Disease (PD) is becoming a growing global concern by being the second most prevalent disease next to Alzheimer's Disease (AD). Henceforth new exploration is needed in search of new aspects towards the disease mechanism and origin. Evidence from recent studies has clearly stated the role of Gut Microbiota (GM) in the maintenance of the brain and as a root cause of various diseases and disorders including other neurological conditions. In the case of PD, with an unknown etiology, the GM is said to have a larger impact on the disease pathophysiology. Although GM and its metabolites are crucial for maintaining the normal physiology of the host, it is an undeniable fact that there is an influence of GM in the pathophysiology of PD. As such the Enteroendocrine Cells (EECs) in the epithelium of the intestine are one of the significant regulators of the gut-brain axis and act as a communication mediator between the gut and the brain. The communication is established via the molecules of neuroendocrine which are said to have a crucial part in neurological diseases such as AD, PD, and other psychiatry-related disorders. This review is focused on understanding the proper role of GM and EECs in PD. Here, we also focus on some of the metabolites and compounds that can interact with the PD genes causing various dysfunctions in the cell and facilitating the disease conditions using bioinformatical tools. Various mechanisms concerning EECs and PD, their identification, the latest studies, and available current therapies have also been discussed.

Statistical approach for the enhanced production of cold-active ß-galactosidase from Thalassospira frigidphilosprofundus: a novel marine psychrophile from deep waters of Bay of Bengal.

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active ß-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for ß-galactosidase production. Under optimal conditions, the production of ß-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. ß-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active ß-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active ß-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.

Cold active ß-galactosidase from Thalassospira sp. 3SC-21 to use in milk lactose hydrolysis: a novel source from deep waters of Bay-of-Bengal.

The cold active ß-galactosidase from psychrophilic bacteria accelerate the possibility of outperforming the current commercial ß-galactosidase production from mesophilic sources. The present study is carried out to screen and isolate a cold active ß-galactosidase producing bacterium from profound marine waters of Bay-of-Bengal and to optimize the factors for lactose hydrolysis in milk. Isolated bacterium 3SC-21 was characterized as marine psychrotolerant, halophile, gram negative, rod shaped strain producing an intracellular cold active ß-galactosidase enzyme. Further, based upon the 16S rRNA gene sequence, bacterium 3SC-21 was identified as Thalassospira sp. The isolated strain Thalassospira sp. 3SC-21 had shown the enzyme activity between 4 and 20 °C at pH of 6.5 and the enzyme was completely inactivated at 45 °C. The statistical method, central composite rotatable design of response surface methodology was employed to optimize the hydrolysis of lactose and to reveal the interactions between various factors behind this hydrolysis. It was found that maximum of 80.18 % of lactose in 8 ml of raw milk was hydrolysed at pH of 6.5 at 20 °C in comparison to 40 % of lactose hydrolysis at 40 °C, suggesting that the cold active ß-galactosidase from Thalassospira sp. 3SC-21 would be best suited for manufacturing the lactose free dairy products at low temperature.

Targeting delta-endotoxin (Cry1Ac) of Bacillus thuringiensis to subcellular compartments increases the protein expression, stability, and biological activity.

Evolving insect resistance to delta-endotoxins can be delayed by using a few strategies like high dosage, refugia, and gene stacking which require the expression of delta-endotoxins at sufficiently high levels to kill the resistant insects. In this study, we comparatively analyzed the efficacy of targeting truncated cry1Ac protein to the cytoplasm, endoplasmic reticulum (ER), and chloroplast to obtain high protein expression. mRNA and protein profiling of cry1Ac showed that both ER and chloroplast are efficient targets for expressing high levels of truncated cry1Ac. A maximum of 0.8, 1.6, and 2.0% cry1Ac of total soluble protein were obtained when the truncated cry1Ac was expressed in the cytoplasm, routed through ER, and targeted to the chloroplast. We further showed that not only the protein content but also the biological activity of truncated cry1Ac increases by sub-cellular targeting and the biological activity is slightly greater in the ER routed transgenic lines by conducting different bioassays on Helicoverpa armigera. Using native Western analysis, we demonstrated that the truncated cry1Ac protein could exist as oligomers in plant cells and this oligomerization capability is low in the cytoplasm. In conclusion, routing of delta endotoxins through ER is the first choice to obtain high protein expression and bioactivity.

Rifampicin Increases Expression of Plant Codon-Optimized Bacillus thuringiensis δ-Endotoxin Genes in Escherichia coli.

Transgenic crops expressing Cry δ-endotoxins of Bacillus thuringiensis for insect resistance have been commercialized worldwide with increased crop productivity and spectacular socioeconomic gains. To attain the enhanced level of protein expression, the cry genes have to be extensively modified for RNA stability and translation efficiency in the plant systems. However, such modifications in nucleotide sequences make it difficult to express the cry genes in Escherichia coli because of the presence of E. coli rare codons. Induction of gene expression through the T7 promoter/lac operator system results in high levels of transcription but limits the availability of activated tRNA corresponding to rare codons that leads to translation stalling at ribosomes. In the present study, an Isopropyl ß-D-1-thiogalactopyranoside (IPTG)/rifampicin combination-based approach was adopted to induce transcription of cry genes through T7 promoter/lac operator while simultaneously inhibiting the transcription of host genes through rifampicin. The results show that the IPTG/rifampicin combination leads to high-level expression of four plant codon-optimized cry genes (cry2Aa, cry1F, cry1Ac, and cry1AcF). Northern blot analysis of the cry gene expressing E. coli samples showed that the RNA expression level in the IPTG-induced samples was higher as compared to that in the IPTG/rifampicin-induced samples. Diet overlay insect bioassay of IPTG/rifampicin-induced Cry toxins with Helicoverpa armigera larvae showed bioactivity (measured as LC50) similar to the previous studies. The experiment has proved that recombinant synthetic gene (plant codon-optimized gene) with the combination of Rifampicin which inhibits DNA-dependent bacterial RNA polymerase and reduces the excessive baggage of translational machinery of the bacterial cell triggers the production of synthetic protein. Purification of protein using high pH buffer increases the solubility of the protein. Further, LC50 analysis shows no reduction of protein activity leads to protein stability. Further, purified cry toxin protein can be used for crop protection against pests and a purified form of the synthetic protein can be used for antibody production and perform the immunoassay for the identification of the transgenic plant. The crystallographic structure of synthetic protein could be used for interaction study with another insect to see insecticidal activity.

Role of heavy metals (copper (Cu), arsenic (As), cadmium (Cd), iron (Fe) and lithium (Li)) induced neurotoxicity.

Parkinson's disease (PD) is a neurodegenerative condition characterized by the dopamine (DA) neuronal loss in the substantia nigra. PD impairs motor controls symptoms such as tremor, rigidity, bradykinesia and postural imbalance gradually along with non-motor problems such as olfactory dysfunction, constipation, sleeping disorder. Though surplus of factors and mechanisms have been recognized, the precise PD etiopathogenesis is not yet implied. Reports suggest that various environmental factors play a crucial role in the causality of the PD cases. Epidemiological studies have reported that heavy metals has a role in causing defects in substantia nigra region of brain in PD. Though the reason is unknown, exposure to heavy metals is reported to be an underlying factor in PD development. Metals are classified as either essential or non-essential, and they have a role in physiological processes such protein modification, electron transport, oxygen transport, redox reactions, and cell adhesion. Excessive metal levels cause oxidative stress, protein misfolding, mitochondrial malfunction, autophagy dysregulation, and apoptosis, among other things. In this review, we check out the link between heavy metals like copper (Cu), arsenic (As), cadmium (Cd), iron (Fe), and lithium (Li) in neurodegeneration, and how it impacts the pathological conditions of PD. In conclusion, increase or decrease in heavy metals involve in regulation of neuronal functions that have an impact on neurodegeneration process. Through this review, we suggest that more research is needed in this stream to bring more novel approaches for either disease modelling or therapeutics.

Effect of alpha-ketoglutarate on neurobehavioral, neurochemical and oxidative changes caused by sub-chronic cyanide poisoning in rats.

Recent studies revealed that alpha-ketoglutarate (A-KG) alone or with sodium thiosulfate (STS) provide significant protection against acute and sub-acute cyanide poisoning in rodents. This study addresses the protective effect of A-KG and/or STS in sub-chronic (90 days) cyanide poisoning. Wistar rats were divided into seven groups (n = 10): Control animals, potassium cyanide (KCN) A-KG, STS, KCN + A-KG, KCN + STS and KCN + A-KG + STS. Spontaneous motor activity and motor coordination were recorded every 15th day. Lipid peroxidation (LPO), reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) in blood, brain, liver and kidney, and glutamate, aspartate and dopamine in discrete regions of brain were measured following 90 days exposure. Cyanide significantly decreased motor coordination, accompanied by increase in LPO (blood, brain and liver) and dopamine (corpus striatum and cerebral cortex) levels, and depletion in GSH (blood, brain and liver), GPx (brain and liver), SOD (brain and liver), and CAT (blood and brain) levels. Although treatment of A-KG and STS alone significantly blunted the toxicity of KCN, concomitant use of both afforded the maximum protection. This study shows a promising role of A-KG and STS as treatment regime for long term cyanide exposure.

A fatal case of primary melioidotic prostatic abscess: the peril of poor drug compliance.

Primary prostatic melioidosis is a rare presentation of melioidosis even in melioidosis endemic areas. We report a case of a 58-year-old man with underlying diabetes mellitus who presented with a 5-day history of high-grade fever associated with lower urinary tract symptoms. Suprapubic tenderness and tender prostatomegaly were noted on examination. An abdominal computed tomography (CT) scan confirmed the presence of a prostatic abscess. Both blood and prostatic pus cultures grew Burkholderia pseudomallei. He was initially started on intravenous ceftazidime, followed by an escalation to intravenous meropenem. He was discharged home with oral amoxicillin-clavulanate and doxycycline after completing 12 days of meropenem. Unfortunately, his compliance to oral antibiotic therapy was poor, and he succumbed to the disease.

Australia antigen (hepatitis B antigen): a conformational antigen dependent on disulfide bonds.

Reduction and alkylation of purified hepatitis-associated Australia antigen (hepatitis B antigen) resulted in a total loss of serologic activity. The reduced and alkylated protein formed a single band with a sedimentation coefficient of 31S on analytical ultracentrifugation, and no subunits were detected by Sephadex gel filtration. Although this preparation induced a delayed hypersensitivity response when injected into guinea pigs, it failed to stimulate humoral antibody formation. The data suggest that hepatitis B antigen is a conformational antigen critically dependent upon the disulfide bonds of the protein moiety.

Phonon Density of States and Specific Heat of Forsterite, Mg2SiO4.

The phonon density of states of the geophysically important mineral forsterite has been calculated with a rigid-ion model, which gives good agreement with an experimental measurement by inelastic neutron scattering. The density of states has been used to calculate the specific heat as a function of temperature, the results of which are in excellent agreement with calorimetrically measured values. The rigid-ion model takes account of the interatomic interactions and normal modes of vibration on a detailed microscopic basis, and is therefore more realistic than the Debye and other empirical models used previously.

Modulation of macronutrient metabolism in the offspring by maternal micronutrient deficiency in experimental animals.

Epidemiological evidence indicates that poor early growth is associated with increased susceptibility to visceral obesity, insulin resistance and associated diseases in adulthood. Studies in experimental animals have demonstrated a robust association between nutrient imbalance during foetal life and disease prevalence in their later life specially of those involving macronutrient metabolism. There is very little data on the role of maternal micronutrient deficiencies widely prevalent in India. This review focuses on different animal models of micronutrient restriction, mimicking human situations during pregnancy and lactation that cause aberrations in macronutrient metabolism in the offspring. These aberrations consist of altered body composition, dyslipidaemia and altered insulin sensitivity associated with modulated insulin production. These phenotypic changes were associated with altered lipid profile, fatty acid synthesis / transport, oxidative stress, glucose tolerance / tissue uptake. Further, these were also associated with altered myogenesis and insulin expression and secretion from pancreatic beta-islets. While these changes during in utero or early postnatal life serve as essential adaptations to overcome adverse conditions, these become maladaptive subsequently and set the stage for obesity and type 2 diabetes.

Hepatoprotective activity of six polyherbal formulations in paracetamol induced liver toxicity in mice.

BACKGROUND & OBJECTIVE: Polyherbal formulations available with a wide range of indications like protective to liver, appetite and growth promoters, gastrointestinal and hepatic regulator, as treatment for hepatic dysfunction, for hepatic regeneration as well as liver stimulant and tonic. Despite the widespread use, there is a lack of scientific evidence on their efficacy and safety. This study was undertaken to evaluate the hepatoprotective activity of six commercially available formulations, namely Liv 52, Livergen, Livokin, Octogen, Stimuliv and Tefroliv in acute liver toxicity in mice model induced by paracetamol (PCM). METHODS: Swiss albino mice of either sex were used, divided in 28 groups with six in each group. The dose of the polyherbal formulations was calculated from human dose (20 ml/day) using a standard conversion table. They were given as pretreatment (2.60 ml/kg/day) for 7 days by oral route twice a day prior to PCM administration. Hepatotoxicity was induced by administering a single oral dose of PCM (500 mg/kg bw) on day 8. The study parameters were conducted on day 9. The biochemical parameters included liver enzyme levels alanine tranaminases (ALT), aspartate transaminases (AST) and alkaline phosphatase (ALP). The pharmacological and pathological parameters were phenobarbitone sleeping time and macroscopic and microscopic changes of liver tissues respectively. RESULTS: PCM toxicity significantly increased ALT, AST and ALP (321.00 +/- 87.93, 273.17 +/- 45.68, 257.50 +/- 17.64 IU/l vs normal control, 33.33 +/- 0.61, 89.33 +/- 9.50, 152.17 +/- 11.40 IU/l respectively, P<0.05), prolonged phenobarbitone induced sleeping time (from 277.50 +/- 8.04 min to 335.83 +/- 7.00 min, P<0.05). When PCM higher dose (1g/kg p.o. single dose) was used, the liver tissue, in macroscopic appearance, showed extensive necrosis associated with haemorrhages. Low dose (500 mg/kg p.o. single dose) showed punctate haemorrhagic necrosis of liver tissue. In the microscopic studies, PCM induced toxicity showed haemorrhages, fatty changes and necrosis. The pretreatment in low doses (2.6 ml/kg/day) with liquid formulations of Liv 52 and Livergen reversed the PCM induced liver toxicity. At higher doses (5.2 ml/ kg/day), all the six herbal formulations conclusively showed marked beneficial effects in the studied pharmacological, biochemical and histological parameters. INTERPRETATION & CONCLUSION: The present findings demonstrated the efficacy of polyherbal liquid formulations at two dose levels in PCM induced hepatotoxicity in mice. However, it suggests that a dose adjustment may be necessary to optimize the effects in clinical settings.

Chaperonin GroEL: structure and reaction cycle.

The structure of Escherichia coli chaperonin GroEL was studied using various experimental tools. Such studies produced information about its structure with increasing details. Moreover, remarkable advances in experimental methods provided a step forward in understanding the reaction cycle involved in GroEL-mediated protein folding. In the current review we summarize recent progress, focus on the structure of GroEL and understand the mechanism involved in GroEL-mediated protein folding. This review is divided into the following sections: (i) Section 1 provides basic understanding on protein folding, (ii) Section 2 not only describes various tools used to elucidate the structural aspects of GroEL but also provides details about its structure with particular emphasis, (iii) Section 3 describes allosteric transitions and the reaction cycle involved in GroEL-mediated protein folding, (iv) Section 4 explains iterative annealing and smoothing of the energy landscape model and finally (v) Section 5 discusses applications and recent progress.

Studies on Copper and Aß1-16-Induced Conformational Changes in CAG/CTG Trinucleotide Repeats Sequence.

DNA conformation and stability are critical for the normal cell functions, which control many cellular processes in life, such as replication, transcription, DNA repair, etc. The accumulation of amyloid-ß peptide (Aß) and Copper (Cu) are the etiological factors for neurodegenerative diseases and hypothesized that they can cause DNA instability. In the current investigation, we studied copper and Aß1-16 induced conformation and stability changes in CAG/CTG sequences and found alterations from B-DNA to altered B-conformation. Further, the interaction of the copper and Aß1-16 with CAG/CTG sequences was studied by molecular docking modeling and results indicated that the interaction of copper and Aß1-16 was through the hydrogen bond formation between adenine, guanine, and cytocine. This study illustrates the role of the copper and Aß1-16 in modulating the DNA conformation and stability.

Peptide inhibitors of West Nile NS3 protease: SAR study of tetrapeptide aldehyde inhibitors.

A series of inhibitors related to the benzoyl-norleucine-lysine-arginine-arginine (Bz-nKRR) tetrapeptide aldehyde was synthesized. When evaluated against the West Nile virus (WNV) NS3 protease, the measured IC(50) ranges from approximately 1 to 200 microM. Concurrently, a modeling study using the recently published crystal structure of the West Nile NS3/NS2B protease complex (pdb code 2FP7) was conducted. We found that the crystal structure is relevant in explaining the observed SAR for this series of tetrapeptides, with the S1 and S2 pockets being the key peptide recognition sites. In general, a residue capable of both pi-stacking and hydrogen bonding is favored in the S1 pocket, while a positively charged residue is preferred in the S2 pocket. This study not only confirms the importance of the NS2B domain in substrate-based inhibitor binding of WNV, it also suggests that the crystal structure would provide useful guidance in the drug discovery process of related Flavivirus proteases, given the high degree of homology.

Sickle cell heart disease. Two-dimensional echo and Doppler ultrasonographic findings in the hearts of adult patients with sickle cell anemia.

Sickle cell anemia (SCA) is a commonly diagnosed condition in the black population in the United States. With better medical management, patients with SCA are living longer and what were previously uncommon sequelae are being recognized frequently, including those involving the cardiovascular system. Previous reports on the cardiovascular effects of SCA have focused predominantly on children, but the conclusions remain controversial. To study this question further, 40 adult patients with documented SCA were examined using two-dimensional and Doppler cardiac ultrasound. Valvular structures were normal despite an increased incidence of flow murmurs. Abnormalities were found more frequently in the left heart than the right, as manifested primarily by increased left ventricular mass, and left ventricular and left atrial dilatation with preservation of systolic function. Pulmonary hypertension, which was present in two thirds of the sample, was minimal to moderate as assessed by our Doppler technique. The effects of SCA on the heart seem to be minimal and similar to those of other anemias, predominantly confined to the left atrium and ventricle with passive elevation of pulmonary pressures. Clinical murmurs were most often physiologic; there was no association with myxomatous valvular degeneration or mitral valve prolapse.

Iron deficiency and sickle cell anemia.

In a patient with sickle cell anemia, iron deficiency was accompanied by hypochromic, microcytic RBCs, absence of bone marrow iron, and a low serum ferritin level. The mean corpuscular hemoglobin concentration (MCHC) was decreased (27.6 g/dL) and was associated with an extreme scarcity of sickled erythrocytes in blood smears. Iron therapy resulted in reticulocytosis and an increase in sickled erythrocytes. In vitro studies demonstrated a decrease in sickling of erythrocytes as a function of oxygen saturation of the blood when the patient was iron deficient. The whole blood oxygen dissociation curve showed a substantial decrease in oxygen pressure necessary to produce 50% saturation of hemoglobin at pH 7.4 and 37 degrees C (P50), indicating an increased oxygen affinity. These data suggest that a reduction of the MCHC induced by iron deficiency may ameliorate sickling.

A fatal case of primary melioidotic prostatic abscess: the peril of poor drug compliance

@#Primary prostatic melioidosis is a rare presentation of melioidosis even in melioidosis endemic areas. We report a case of a 58-year-old man with underlying diabetes mellitus who presented with a 5-day history of high-grade fever associated with lower urinary tract symptoms. Suprapubic tenderness and tender prostatomegaly were noted on examination. An abdominal computed tomography (CT) scan confirmed the presence of a prostatic abscess. Both blood and prostatic pus cultures grew Burkholderia pseudomallei. He was initially started on intravenous ceftazidime, followed by an escalation to intravenous meropenem. He was discharged home with oral amoxicillin-clavulanate and doxycycline after completing 12 days of meropenem. Unfortunately, his compliance to oral antibiotic therapy was poor, and he succumbed to the disease.