Hepato and nephroprotective activity of methanol extract of Hygrophila spinosa and its antibacterial potential against multidrug resistant Pandoraea sputorum.

This research was aimed to evaluate the phytochemical profile, bactericidal activity of Hygrophila spinosa against multidrug resistant Pandoraea sputorum and assess their antioxidant competence against various radicals and studied their hepatoprotective and nephroprotective activity on HepG2 and HEK 293 cell line. The results showed that the methanol extract has various phytochemical components with reasonable quantity. Fortunately, the multidrug-resistant P. sputorum was sensitive (22.8 ± 0.2 mm of the zone of inhibition) at 15 mg mL-1 concentration of methanol extract. The higher concentration of phenolic and other phytochemical components, showed significant antioxidant activity against ferric, DPPH, hydroxyl, and ABTS radicals, with IC50 values of 71.09, 64.333, 91.157, and 104.931 g mL-1, respectively. Surprisingly, the methanol extract possesses hepato and nephroprotective activity against CCl4 and cisplatin-induced cytotoxicity on HepG2 and HEK 293 cell lines, respectively. It maintains the cell viability as up to 90.48% and 90.35% of HepG2 and EK 293 cell line at the concentration of 20 µg mL-1. The FTIR analysis states that the methanol extract possesses a significant functional group responsible for these multi-potential activities. These results suggest that, the methanol extract of H. spinosa might contain the most significant bioactive components with outstanding medicinal properties.

A novel assay method for calcium calmodulin dependent phosphatase from bovine brain extract.

Calcium calmodulin dependent protein ser/thr phosphatase, also referred to as protein phosphatase 2B (PP2B), is rich in neural tissue, and plays an important role in the overall function of the nervous system. Routinely phosphatase assay employs, para-Nitrophenlylphosphate (p-NPP), as a substrate, is also extended to assay PP2B. However, in the present study, the differential spectral characterstic property of tyrosine and phopshotyrosine has been exploited to employ the latter as a candidate substrate for the PP2B assay. The specific activity of PP2B using phosphortyrosine in bovine Bos Taurus indicus brain extract (Bos Taurus indicus), was measured in presence of different metal ions like Ca(2+), Mn(2+) and Mg(2+). Further modulators like dithiothreitol (DTT), calmodulin (CaM) and metal chelators such as EGTA and EDTA were applied to confirm the role of divalent cations and to determine calcium calmodulin dependent phoshphatase activity. PP2B activity was higher with phosphotyrosine in presence of Ca(2+) than with p-NPP. Further experiments, involving calmodulin as a modulator, confirmed phosphotyrosine as a better substrate over p-NPP. Calmodulin further enhanced the effect of phosphotyrosine as a potential substrate confirming calcium calmodulin dependent phosphatase activity. Phosphotyrosine is proposed as a better substrate in assaying calcium dependent phosphatase activity when compared to para-nitrophenylphosphate.

Heterologous Expression and Structural Elucidation of a Highly Thermostable Alkaline Serine Protease from Haloalkaliphilic Actinobacterium, Nocardiopsis sp. Mit-7.

A highly thermostable alkaline serine protease gene (SPSPro, MN429015) obtained from haloalkaliphilic actinobacteria, Nocardiopsis sp. Mit-7 (NCIM-5746), was successfully cloned and overexpressed in Escherichia coli BL21 under the control of the T7 promoter in the pET Blue1 vector leading to a 20-kDa gene product. The molecular weight of the recombinant alkaline protease, as determined by SDS-PAGE and the Mass Spectrometer (MALDI-TOF), was 34 kDa. The structural and functional attributes of the recombinant thermostable alkaline serine protease were analyzed by Bioinformatic tools. 3D Monomeric Model and Molecular Docking established the role of the amino acid residues, aspartate, serine, and tryptophan, in the active site of thealkaline protease.The activity of the recombinant alkaline protease was optimal at 65 °C, 5 °C higher than its native protease. The recombinant protease was also active over a wide range of pH 7.0-13.0, with a maximal activity of 6050.47 U/mg at pH 9. Furthermore, the thermodynamic parameters of the immobilized recombinant alkaline protease suggested its reduced vulnerability against adverse conditions under which the enzyme has to undergo varied applications.

Solvent tolerant enzymes in extremophiles: Adaptations and applications.

Non-aqueous enzymology has always drawn attention due to the wide range of unique possibilities in biocatalysis. In general, the enzymes do not or insignificantly catalyze substrate in the presence of solvents. This is due to the interfering interactions of the solvents between enzyme and water molecules at the interface. Therefore, information about solvent-stable enzymes is scarce. Yet, solvent-stable enzymes prove quite valuable in the present day biotechnology. The enzymatic hydrolysis of the substrates in solvents synthesizes commercially valuable products, such as peptides, esters, and other transesterification products. Extremophiles, the most valuable yet not extensively explored candidates, can be an excellent source to investigate this avenue. Due to inherent structural attributes, many extremozymes can catalyze and maintain stability in organic solvents. In the present review, we aim to consolidate information about the solvent-stable enzymes from various extremophilic microorganisms. Further, it would be interesting to learn about the mechanism adapted by these microorganisms to sustain solvent stress. Various approaches to protein engineering are used to enhance catalytic flexibility and stability and broaden biocatalysis's prospects under non-aqueous conditions. It also describes strategies to achieve optimal immobilization with minimum inhibition of the catalysis. The proposed review would significantly aid our understanding of non-aqueous enzymology.

Acute nicotine changes dynorphin and prodynorphin mRNA in the striatum.

RATIONALE: Nicotine displays rewarding and aversive effects, and while dopamine has been linked with nicotine's reward, the neurotransmitter(s) involved with aversion remains speculative. The kappa-dynorphinergic system has been associated with negative motivational and affective states, and whether dynorphin (Dyn) contributes to the behavioral pharmacology of nicotine is a pertinent question. OBJECTIVE: We determined whether administration of a single dose of nicotine alters the biosynthesis of Dyn in the striatum of mice. RESULTS: Nicotine free base, 1 mg/kg, sc, induced a biphasic, protracted increase of striatal Dyn, an initial rise by 1 h, which declined to control levels by 2 h, and a subsequent increase, between 6 and 12 h, lasting over 24 h. At 1 h, the nicotine effect was dose dependent, with doses>or=0.5 mg/kg inducing a response. Prodynorphin mRNA increased by 30 min for over 24 h, and in situ hybridization demonstrated elevated signal in caudate/putamen and nucleus accumbens. The nicotinic antagonist mecamylamine prevented the Dyn response, and a similar effect was observed with D1- and D2-like dopamine receptor antagonists, SCH 23390, sulpiride, and haloperidol. The glutamate NMDA receptor antagonist MK-801 reversed the nicotine-induced increase of Dyn, while the AMPA antagonist NBQX had a marginal effect. CONCLUSIONS: We interpret our findings to indicate that acute nicotine enhances the synthesis and release of striatal Dyn. We propose that nicotine influences Dyn primarily through dopamine release and that glutamate plays a modulatory role. A heightened dynorphinergic tone may contribute to the aversive effects of nicotine in naive animals and first-time tobacco smokers.

Lack of pathologic Q waves: a specific marker of viability in myocardial hibernation.

BACKGROUND: The present study evaluates the lack of Q waves on the electrocardiogram (ECG) in the prediction of myocardial viability compared with dobutamine stress echocardiography (DSE) and rest-redistribution thallium-201 (Tl-201) scintigraphy. HYPOTHESIS: The lack of pathologic Q waves (NoQ) may be a readily available and specific marker for the presence of viability. METHODS: Sixty four patients with stable coronary artery disease (CAD) and ventricular dysfunction underwent rest ECG, DSE, and Tl-201 scintigraphy before revascularization, and a repeat rest 2-Dimensional (2-D) echocardiogram more than 3 mo later. RESULTS: Total viability at baseline (% of total segments) was higher in the NoQ group by Tl-201 scintigraphy (87 +/- 19% versus 70 +/- 20%, p = 0.008) and by DSE (81 +/- 20% versus 65 +/- 24%, p = 0.013). As expected, the sensitivity of NoQ waves was low in predicting recovery of function (23%), and inferior to Tl-201 (82%) and DSE (84%) (p<0.08). However, specificity of NoQ waves for predicting recovery of global function was high (72%); higher than Tl-201 (50%) and DSE (45%). Positive predictive values were comparable among all modalities. Results were similar if the data were analyzed regionally for viability. CONCLUSION: Lack of pathologic Q waves is a specific and readily available marker of myocardial viability in patients with chronic CAD, which should alert the clinician for myocardial hibernation.

Heeding clues to metformin-associated lactic acidosis: prompt response can save life.

The case history is presented of a patient who developed metformin-associated lactic acidosis. The patient made a complete recovery with supportive care. Recognition of metformin-associated lactic acidosis requires a high index of suspicion as presentation can be very subtle.

Unusual lung mass: benign or malignant?

Primary adenoid cystic carcinoma of lung is a rare tumour. It is a slowly growing, indolent tumour. Average time that elapses before diagnosis is reported to be two years. We report the case of a patient who remained well inspite of harbouring primary adenoid cystic carcinoma of lung for 15 years.

Implant-retained auricular prostheses: a clinical challenge.

Microtia, malformation, deformity, and partial or complete loss of the pinna may be due to various congenital or acquired factors. In adult patients, surgical reconstruction of the missing pinna is difficult and the results are often far from satisfactory. An implant-retained auricular prosthesis is a suitable alternative. A retrospective study of eight patients treated with implant-retained auricular prostheses was performed. For each missing pinna, three titanium implants were placed in the temporal bone. After 6 months of osseointegration, the implants were loaded. Four cases were rehabilitated with a magnet-retained prosthesis and the remaining four with a bar and clip retained prosthesis. There were six male and two female patients with an average age of 30.62 years. Seven patients had unilateral absence of the pinna and one had bilateral absence. The loss was due to trauma in four patients and to burn in one patient, and three had congenital absence. A total 27 implants were placed, 12 on the right side and 15 on the left. The average post-rehabilitation follow-up was 21 months. Peri-implant tissue reactions were observed at two sites. The implant-retained auricular prosthesis is an alternative treatment approach with good retention and patient satisfaction. Long-term follow-up is required to assess delayed complications.

Hospital-based surveillance of invasive pneumococcal disease and pneumonia in South Bangalore, India.

OBJECTIVE: To estimate the incidence of invasive pneumococcal disease and pneumonia, distribution of pneumococcal serotypes, and antibiotic susceptibility in children aged 28 days to <60 months. DESIGN: Hospital-based surveillance. SETTING: South Bangalore, India. PARTICIPANTS: 9950 children aged 28 days to <60 months with clinical suspicion of invasive pneumococcal disease or pneumonia. RESULTS: The estimated at-risk population included 224,966 children <5 years of age. Forty cases of invasive pneumococcal disease were identified. Estimated invasive pneumococcal disease incidence was 17.8/100,000 with incidence being highest among children aged 6 months to <12 months (49.9/100,000). Clinical pneumonia syndrome was the most frequent diagnosis (12.5/100,000). Pneumococcal serotypes included: 6A (n=6, 16.7%); 14 (n=5, 13.9%); 5 (n=4, 11.1%); 6B (n=4, 11.1%); 1, 18C, and 19A (n=3 each, 8.3%); 9V (n=2, 5.6%); and 3, 4, 10C, 18A, 18F, and 19F (n=1 each, 2.8%). Serotypes 6A, 14, 6B, 1, 18C, 19A, 9V, 4, 10C, and 18A showed antibiotic resistance. Clinical pneumonia incidence was 2109/100,000, with incidence being highest among children aged 28 days to <6 months (5033/100,000). Chest radiograph-confirmed pneumonia incidence was 1114/100,000, with incidence being highest among children aged 28 days to <6 months (2413/100,000). CONCLUSIONS: Invasive pneumococcal disease and pneumonia were found to be common causes of morbidity in young children living in South Bangalore, India.

Met-enkephalin and preproenkephalin mRNA changes in the striatum of the nicotine abstinence mouse.

We studied the changes of met-enkephalin (Met-Enk) content and preproenkephalin (PPE) mRNA in the striatum in a mouse model of nicotine abstinence. Nicotine, 2 mg/kg, s.c., was administered four times daily for 14 days and Met-Enk and PPE mRNA evaluated at various times (4-96 h) following drug discontinuation. Met-Enk, assayed by radioimmunoassay, was increased in the ventral (nucleus accumbens) but not dorsal (putamen/caudate) striatum, while PPE mRNA, assayed in whole striatum by Northern blotting was elevated. Both changes were seen early during withdrawal and lasted over 72 h. In situ hybridization revealed enhanced signal in the dorsal striatum, mostly laterally, and smaller increases in the rostral pole, core and shell of the nucleus accumbens. These observations indicate that during nicotine withdrawal, striatal enkephalinergic neurons undergo adaptative responses, which might contribute to the abstinence behavioral syndrome.

Cutaneous analgesia, hemodynamic and respiratory effects, and beta-endorphin concentration in spinal fluid and plasma of horses after acupuncture and electroacupuncture.

OBJECTIVE: To determine cutaneous analgesia, hemodynamic and respiratory effects, and beta-endorphin concentration in spinal fluid and plasma of horses after acupuncture and electroacupuncture (EA). ANIMALS: 8 healthy 10- to 20-year-old mares that weighed between 470 and 600 kg. PROCEDURE: Each horse received 2 hours of acupuncture and 2 hours of PAES at acupoints Bladder 18, 23, 25, and 28 on both sides of the vertebral column as well as sham needle placement (control treatment). Each treatment was administered in a random order. At least 7 days elapsed between treatments. Nociceptive cutaneous pain threshold was measured by use of skin twitch reflex latency (STRL) and avoidance to radiant heat (< or = 50 degrees C) in the lumbar area. Skin temperature, cardiovascular and respiratory variables, and beta-endorphin concentration in spinal fluid (CSF-EN) and plasma (plasma-EN) were measured. RESULTS: Acupuncture and PAES significantly increased STRL and skin temperature. The CSF-EN was significantly increased from baseline values 30 to 120 minutes after onset of PAES, but it did not change after acupuncture and control treatments. Heart and respiratory rates, rectal temperature, arterial blood pressure, Hct, total solids and bicarbonate concentrations, base excess, plasma-EN, and results of blood gas analyses were not significantly different from baseline values after acupuncture, PAES, and control treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of PAES was more effective than acupuncture for activating the spinal cord to release beta-endorphins into the CSF of horses. Acupuncture and PAES provided cutaneous analgesia in horses without adverse cardiovascular and respiratory effects.

Heeding clues to metformin-associated lactic acidosis: prompt response can save life.

The case history is presented of a patient who developed metformin-associated lactic acidosis. The patient made a complete recovery with supportive care. Recognition of metformin-associated lactic acidosis requires a high index of suspicion, as presentation can be very subtle.

Dynorphin and prodynorphin mRNA changes in the striatum during nicotine withdrawal.

Nicotine withdrawal causes somatic and negative affective symptoms that contribute to relapse and continued tobacco smoking. So far, the neuronal substrates involved are not fully understood, and an opioid role has been suggested. In this regard, the opioid dynorphin (Dyn) is of interest as it produces aversive states and has been speculated to play a role in the nicotine behavioral syndrome. These studies explore whether Dyn metabolism is altered during withdrawal following chronic administration of nicotine. Mice were administered nicotine, 2 mg/kg, s.c., four times daily for 14 days, and Dyn and prodynorphin (PD) mRNA estimated in selective brain regions at various times (30 min to 96 h) following drug discontinuation. The content of Dyn, estimated by RIA, was decreased in the striatum for a protracted time, from 30 min to over 72 h. In contrast, the mRNA for PD, evaluated by Northern blot, was elevated, appearing by 8 h and lasting over 96 h. Dyn was decreased in both ventral and dorsal striatum, and PD mRNA was differentially increased in the two striatal compartments as demonstrated by in situ hybridization. PD message was predominantly augmented in the nucleus accumbens, rostral pole, core, and shell, and the medial aspects of caudate/putamen. We interpret these data to indicate increased activity of striatal, particularly accumbal, dynorphinergic neurons during nicotine withdrawal resulting in enhanced peptide release and compensatory synthesis. Heightened dynorphinergic tone might be responsible, in part, for the emergence of the negative affective states observed during nicotine withdrawal.

The role of spinal opioid receptors in antinociceptive effects produced by intrathecal administration of hydromorphone and buprenorphine in the rat.

UNLABELLED: The intrathecal administration of morphine has been the standard therapy to control long-term intractable pain. Recently, a panel of pain therapy experts suggested that because of the lack of efficacy or because of the side effects produced by morphine in some patients, other drugs, such as hydromorphone and buprenorphine, should be investigated for their analgesic properties. We designed this study to compare the efficacy of intrathecal hydromorphone and buprenorphine to suppress thermal nociception in male Sprague-Dawley rats. An additional objective was to understand whether hydromorphone and buprenorphine bind and act as agonists to mu-, delta-, and kappa-spinal opioid receptors. Intrathecally-administered hydromorphone and buprenorphine produced a dose- and time-dependent increase in the tail-flick response latency in rats. The 50% effective dose value for the antinociceptive effect of buprenorphine and hydromorphone were 4 and 69.5 nmol/L, respectively. Both drugs act as agonists to mu-opioid receptors, as determined by their ability to displace [(3)H]-DAMGO from the spinal opioid receptors and by the ability of an opioid receptor antagonist, naloxone, to reverse their antinociceptive effects. Buprenorphine also has an agonistic effect on the kappa-opioid receptors. For the first time, we report that intrathecal buprenorphine is approximately 17 times more effective than hydromorphone in inhibiting thermal pain, and buprenorphine produces its antinociceptive effect by acting as an agonist at both mu- and kappa-spinal opioid receptors. Naloxone administered intrathecally was effective in preventing the antinociceptive effects of subsequent intrathecal injections of buprenorphine. IMPLICATIONS: Hydromorphone and buprenorphine are two important drugs used for pain relief. We observed that intrathecal buprenorphine is 17 times more potent than hydromorphone to inhibit pain in rats. Both drugs exert their effects through specific spinal opioid receptors.