A Novel Trehalose Synthase for the Production of Trehalose and Trehalulose.

A novel putative trehalose synthase gene (treM) was identified from an extreme temperature thermal spring. The gene was expressed in Escherichia coli followed by purification of the protein (TreM). TreM exhibited the pH optima of 7.0 for trehalose and trehalulose production, although it was functional and stable in the pH range of 5.0 to 8.0. Temperature activity profiling revealed that TreM can catalyze trehalose biosynthesis in a wide range of temperatures, from 5°C to 80°C. The optimum activity for trehalose and trehalulose biosynthesis was observed at 45°C and 50°C, respectively. A catalytic reaction performed at the low temperature of 5°C yielded trehalose with significantly reduced by-product (glucose) production in the reaction. TreM displayed remarkable thermal stability at optimum temperatures, with only about 20% loss in the activity after heat (50°C) exposure for 24 h. The maximum bioconversion yield of 74% trehalose (at 5°C) and 90% trehalulose (at 50°C) was obtained from 100 mM maltose and 70 mM sucrose, respectively. TreM was demonstrated to catalyze trehalulose biosynthesis utilizing the low-cost feedstock jaggery, cane molasses, muscovado, and table sugar. IMPORTANCE Trehalose is a rare sugar of high importance in biological research, with its property to stabilize cell membrane and proteins and protect the organism from drought. It is instrumental in the cryopreservation of human cells, e.g., sperm and blood stem cells. It is also very useful in the food industry, especially in the preparation of frozen food products. Trehalose synthase is a glycosyl hydrolase 13 (GH13) family enzyme that has been reported from about 22 bacterial species so far. Of these enzymes, to date, only two have been demonstrated to catalyze the biosynthesis of both trehalose and trehalulose. We have investigated the metagenomic data of an extreme temperature thermal spring to discover a novel gene that encodes a trehalose synthase (TreM) with higher stability and dual transglycosylation activities of trehalose and trehalulose biosynthesis. This enzyme is capable of catalyzing the transformation of maltose to trehalose and sucrose to trehalulose in a wide pH and temperature range. The present investigation endorses the thermal aquatic habitat as a promising genetic resource for the biocatalysts with high potential in producing high-value rare sugars.

Biotransformation of hydroquinone into α-arbutin by transglucosylation activity of a metagenomic amylosucrase.

Arbutin is a naturally occurring glycosylated product of hydroquinone. With the ability to interrupt melanin biosynthesis in epidermal cells, it is a promising cosmetic ingredient. In this study, a novel amylosucrase, Asmet, identified from a thermal spring metagenome, has been characterized for arbutin biosynthesis. Asmet was able to catalyze transglucosylation of hydroquinone to arbutin, taking sucrose as glycosyl donor, in the temperature range of 20 °C to 40 °C and pH 5.0 to 6.0, with the relative activity of 80% or more. The presence of chloride salts of Li, K, and Na at 1 mM concentration did not exhibit any notable effect on the enzyme's activity, unlike Cu, Ni, and Mn, which were observed to be detrimental. The hydroquinone (20 mM) to sucrose ratio of 1:1 to 1:10 was appropriate for the catalytic biosynthesis of arbutin. The maximum hydroquinone to arbutin conversion of 70% was obtained in 24 h of Asmet led catalysis, at 30 °C and pH 6.0. Arbutin production was also demonstrated using low-cost feedstock, table sugar, muscovado, and sweet sorghum stalk extract, as a replacement for sucrose. Whole-cell catalysis of hydroquinone to arbutin transglucosylation was also established.

Characterization of a novel amylosucrase gene from the metagenome of a thermal aquatic habitat, and its use in turanose production from sucrose biomass.

Turanose is a natural isomer of sucrose. It is an emerging functional sweetener of the next generation. Turanose is catalytically synthesized from the sucrose biomass by employing amylosucrase enzyme. In this study, a novel gene encoding amylosucrase (Asmet) has been identified from the metagenome of a thermal aquatic habitat. Asmet exhibits 37-55% identity at the protein level with the known amylosucrases characterized till date. Asmet was cloned and expressed in Escherichia coli, followed by protein purification, and characterization. Asmet protein exhibited the maximum total activity at 9.0 pH and 60 °C temperature, whereas, 8.0 pH and 50 °C temperature were found optimum for transglycosylation activity. Asmet showed fairly high thermal tolerance at 50 °C. The conjugation of Asmet protein with functionalized iron nanoparticles significantly improved its thermal tolerance, showing hardly any loss in the enzyme's activity even after 72 h of heat (50 °C) exposure. The turanose yield of about 47% was achieved from 1.5 M sucrose, containing 0.5 M fructose in the reaction. Turanose was purified (˜95%) via a bio-physical process, and characterized by TLC, HPLC, and NMR. The novel amylosucrase gene was demonstrated to be a potential candidate for turanose production, utilizing various sucrose containing feedstocks.

Kinetic characterization of laccase from Bacillus atrophaeus, and its potential in juice clarification in free and immobilized forms.

In the present study, a laccase gene (BaLc) from a lignin degrading bacterium, Bacillus atrophaeus, has been cloned and expressed in Escherichia coli. The optimal catalytic activity of the protein was achieved at 5.5 pH and 35°C temperature, measured by oxidation of ABTS. The Km and Vmax values were determined as 1.42 mM and 4.16 µmole/min, respectively. To achieve the enzyme recovery, the biocatalyst (BaLc) was covalently attached onto the functionalized iron magnetic-nanoparticles. The nanoparticles were characterized by zeta-potential and FTIR analyses. The immobilized BaLc enzyme was physico-kinetically characterized, exhibiting retention of 60% of the residual activity after ten reaction cycles of ABTS oxidation. The immobilized biocatalyst system was tested for its biotechnological exploitability in plant juice processing, achieving 41-58% of phenol reduction, 41-58% decolorization, 50-59% turbidity reduction in the extracts of banana pseudo-stem and sweet sorghum stalk, and apple fruit juice. This is the first study to demonstrate the use of nanoparticle-laccase conjugate in juice clarification. The findings suggest that B. atrophaus laccase is a potential catalytic tool for plant juice bioprocessing activities.

Catalytic biosynthesis of levan and short-chain fructooligosaccharides from sucrose-containing feedstocks by employing the levansucrase from Leuconostoc mesenteroides MTCC10508.

Levansucrase gene (LmLEVS) was cloned from Leuconostoc mesenteroides MTCC 10508. The heterologous expression and purification of the truncated (TrLmLEVS) gene, lacking the N-terminal signal peptide, was performed in Escherichia coli. The recombinant enzyme (TrLmLEVS) was physico-kinetically characterized using sucrose as substrate. TrLmLEVS exhibited the maximum activity at pH 6 and temperature 30 °C. Thin layer chromatography and high performance liquid chromatography analyses unveiled the biosynthesis of fructooligosaccharides and levan by TrLmLEVS using sucrose as substrate. The catalytically synthesized polymer was characterized by Fourier-Transform Infrared Spectroscopy and Nuclear Magnetic Resonance analyses, confirming it as levan. TrLmLEVS was capable of catalyzing the transformation of raffinose-derived molecules, besides sucrose, into fructans. Further, TrLmLEVS was employed for the genesis of non-digestible fructans from sucrose-containing feedstocks like table sugar, jaggery, cane molasses, and sweet sorghum juice. The results suggest that Leu. mesenteroides MTCC 10508 levansucrase is a potential candidate for the production of levan-type biomolecules in plant-based food products.

Evaluation of sacral rhomboid dimensions to predict contracted pelvis: a pilot study of Indian primigravidae.

OBJECTIVE: This prospective study aimed to evaluate dimensions of sacral rhomboid (quadrangular area at lower back) as predictors of Contracted pelvis (CP). METHODS: In 300 uncomplicated primigravidae after 37 weeks gestation, transverse and vertical diagonal (TD and VD) of sacral rhomboid were recorded. Post- delivery, patients fell into two groups: Normal Deliveries (group 1) and CP (group 2, i.e., LSCS for contracted pelvis, instrumental delivery for Non Progress of Labor (NPOL) or birth mishaps). Analysis was done with SPSS version 10.5 and cross tabulation. RESULTS: Rhomboid dimensions were smaller in CP group (P value 0.003 and 0.012. On univariate analysis, 10th percentile cutoff of TD was the most significant risk factor with Odds ratio of 7.5 (95% CI 3.5-16.2). VD and height below 10th percentile had Odds ratio of 2.7 (95% CI 1.2-6.1) and 2.99 (95% CI 1.2-7.3), respectively. Both dimensions of sacral rhomboid below 10th percentile increased risk of CP by 13 times (OR 13, 95% CI 3.5-48.6) and detected 2 out of 3 at risk women. CONCLUSION: Smaller dimensions of sacral rhomboid are promising screening parameter for contracted pelvis which can be used in community to pick up high risk primigravidae.

Elevated urinary catecholamines and adrenal haemorrhage mimicking phaeochromocytoma.

A 51-year-old woman was admitted with left-sided flank pain initially thought to be renal colic. However, a CT urogram was normal. During the course of the admission the pain persisted and she developed severe sustained hypertension. A repeat CT scan of the abdomen revealed a 5×3 cm left adrenal abnormality consistent with haemorrhage, not seen on the original scan. Further assessment revealed elevated urine catecholamines and a short synacthen test showed a suboptimal cortisol response. The diagnosis was initially considered as a phaeochromocytoma, she received phenoxybenzamine with good resolution of hypertension and was referred for surgical opinion. However, serial urinary catecholamine concentrations returned to within the normal range and the diagnosis was revised to adrenal infarction and haemorrhage due to antiphospholipid syndrome. This case illustrates the importance of recognising adrenal infarction as a potential cause of 'pseudophaeochromocytoma'.

Metformin reduces arterial stiffness and improves endothelial function in young women with polycystic ovary syndrome: a randomized, placebo-controlled, crossover trial.

CONTEXT: Patients with polycystic ovary syndrome (PCOS) have an increased prevalence of insulin resistance and display subclinical evidence of early cardiovascular disease. Metformin improves insulin sensitivity and circulating markers of cardiovascular risk in patients with PCOS, but it is unclear whether this translates into improvements in vascular function. OBJECTIVE: Our objective was to evaluate the effects of metformin on arterial stiffness and endothelial function in women with PCOS. DESIGN AND INTERVENTION: Thirty women with PCOS were assigned to consecutive 12-wk treatment periods of metformin or placebo in a randomized, double-blind, crossover design separated by an 8-wk washout. MAIN OUTCOME MEASURES: The primary outcome measures were assessments of arterial stiffness [augmentation index (AIx), central blood pressure, and brachial and aortic pulse wave velocity (PWV)] and endothelial function. Anthropometry, testosterone, and metabolic biochemistry (lipids, homeostasis model of assessment for insulin resistance, high-sensitivity C-reactive protein, adiponectin, and plasminogen activator inhibitor-1) were also assessed. RESULTS: Metformin improved AIx [-6.1%; 95% confidence interval (CI) for the difference -8.5 to -3.5%; P < 0.001], aortic PWV (-0.76 m/sec; 95% CI for the difference -1.12 to -0.4 m/sec; P < 0.001), brachial PWV (-0.73 m/sec; 95% CI for the difference -1.09 to -0.38; P < 0.001), central blood pressure (P < 0.001), and endothelium-dependent (AIx after albuterol; P = 0.003) and endothelium-independent (AIx after nitroglycerin; P < 0.001) vascular responses. Metformin also reduced weight (P < 0.001), waist circumference (P < 0.001), and triglycerides (P = 0.004) and increased adiponectin (P = 0.001) but did not affect testosterone or other metabolic measures. CONCLUSIONS: Short-term metformin therapy improves arterial stiffness and endothelial function in young women with PCOS.

Effects of dehydroepiandrosterone replacement on vascular function in primary and secondary adrenal insufficiency: a randomized crossover trial.

CONTEXT: Patients with Addison's disease and hypopituitarism have increased mortality, chiefly related to vascular disease. Both diseases are characterized by dehydroepiandrosterone (DHEA) deficiency, yet this is not usually corrected. It is unclear whether treatment of these conditions with DHEA improves cardiovascular risk. OBJECTIVE: The aim of the study was to evaluate the effects of DHEA on arterial stiffness and endothelial function in subjects with Addison's disease and hypopituitarism. DESIGN AND INTERVENTION: Forty subjects (20 with Addison's disease, 20 with panhypopituitarism) were assigned to consecutive 12-wk treatment periods of DHEA 50 mg or placebo in a randomized, double-blind, crossover design separated by an 8-wk washout. MAIN OUTCOME MEASURES: Primary outcome parameters were measures of arterial stiffness [augmentation index, central blood pressure, brachial and aortic pulse wave velocity (PWV)] and endothelial function. Serum androgens, anthropometry, and metabolic biochemistry (lipids, homeostasis model of assessment for insulin resistance, high sensitivity C-reactive protein, adiponectin, plasminogen activator inhibitor-1) were also assessed. RESULTS: Despite normalization of DHEA sulfate, androstenedione, and testosterone (females), DHEA replacement did not affect augmentation index, aortic PWV, brachial PWV, central blood pressure, or endothelial function. DHEA did not affect any anthropometric or metabolic measures, apart from a small reduction in high-density lipoprotein cholesterol (-0.08 mmol/liter; P = 0.007; 95% confidence interval for the difference, -0.13 to -0.02 mmol/liter). CONCLUSIONS: Short-term DHEA supplementation does not significantly affect measures of arterial stiffness or endothelial function in patients with adrenal insufficiency.

Role of misoprostol in overcoming an unsatisfactory colposcopy: a randomized double-blind placebo-controlled clinical trial.

OBJECTIVE: To assess the effectiveness of vaginal misoprostol in overcoming an unsatisfactory colposcopy and to analyse the factors that might influence the response to misoprostol, i.e. age, menopausal status, duration of menopause and cervical characteristics. STUDY DESIGN: A randomized double-blind placebo-controlled clinical trial. METHODS: Forty patients with unsatisfactory colposcopy were recruited from the colposcopy clinic and were randomly allocated to receive either 400 microg misoprostol or similar-looking placebo tablets vaginally. Repeat colposcopy was performed after 6 h noting the side effects, if any. RESULTS: One patient was excluded from the misoprostol group because postdrug colposcopic examination could not be done due to a technical fault. Of the 19 patients in the misoprostol group, 15 (78.9%) had satisfactory examination compared to only 6 of the 20 (30%) patients in the placebo group. This effect of misoprostol was statistically significant (p = 0.004). Misoprostol in the present study averted 3 cone biopsies and 12 endocervical curettages. The side effects of misoprostol were comparable in both groups. The slit-like external os and the posterior cervical lip as the site of unsatisfactory colposcopy had better conversion rates compared to pinhole os (66.6 vs.81.2%) and anterior lip involvement (85.7 vs.100%). CONCLUSION: Four hundred micrograms of intravaginal misoprostol is an effective and safe method to convert an unsatisfactory colposcopy into a satisfactory one.

Role of uterine artery velocimetry using color-flow Doppler and electromyography of uterus in prediction of preterm labor.

AIM: To evaluate the role of Doppler waveforms of uterine vessels and electromyography (EMG) of the uterus in predicting preterm labor. METHODS: One hundred ante-natal women at >or=24 weeks of gestation who fulfilled the selection criteria were included in the study. A single Doppler recording for bilateral uterine vessels was taken at >or=26 weeks gestation using the transvaginal route. Transabdominal EMG of the uterus was recorded serially at 4-week intervals from 24 weeks until delivery or until 37 weeks were completed. RESULTS: Women with a sensation of heaviness in the lower abdomen during pregnancy had a significant association with preterm delivery. Of the three Doppler indices, the systolic/diastolic ratio and the resistivity index of uterine vessels were found to have a sensitivity as high as 83-95% for the prediction of preterm labor. The electrical activity of uterine musculature, as recorded using EMG, showed that there was a statistically significant difference between the electrical activity recorded for the two groups at 31 weeks and later, while the duration of burst activity was significantly longer in the preterm group at 26 weeks and later. CONCLUSIONS: Patients with a dull aching sensation in the lower abdomen should not be neglected. Doppler of the uterine vessels and EMG of the uterus could prove to be a good predictor of preterm labor.

Evaluation of endometrial steroid receptors and cell mitotic activity in women using copper intrauterine device: Can Cu-T prevent endometrial cancer?

AIMS: To study changes in endometrial proliferative indices (i.e. cell mitosis and Ki67), endometrial steroid (estrogen and progesterone) receptors, and serum estradiol and progesterone level in women using copper intrauterine device (IUD) after 6 months of use. METHODS: Fifteen healthy women were recruited having no contra indication to IUD or endometrial biopsy. Endometrial biopsies and serum samples were taken on 10/11th day of menstrual cycle at recruitment and after six months. The endometrial samples were subjected to: (i) steroid receptor estimation by immunohistochemistry; (ii) cell mitosis and Ki67 estimation by morphology and proliferative indices. RESULTS: The mean glandular mitotic index was 1.69 +/- (0.39) at first visit and 0.35 +/- (0.15) at second visit after 6 months. Similarly, Ki-67 count in glands was 11.79 +/- (1.59) at first visit and 2.40 +/- (0.54) at second visit. In the stroma, Ki67 decreased from a mean of 0.85 to 0.01 in six months. This suggested statistically significant reduction in proliferation and mitotic activity in endometrial glands and the stroma of women who used Cu-T continuously for a long period. There was a significant reduction of estrogen receptor concentration in glands (from a mean of 51.67 down to 11.67) and the stroma (from a mean of 33.00 to 8.00) over this 6-month period. The observed fall in progesterone receptor counts was less compared to other parameters studied, being marginally significant in stroma (from a mean of 32.00 down to 14.33), but not significant enough in glands (from a mean of 45.00 down to 25.00). No changes were observed in serum hormone levels in these two visits. CONCLUSION: Continuous use of copper intrauterine device causes significant reduction in endometrial mitotic activity as well as estrogen receptor concentration without affecting serum estradiol or progesterone levels. This finding infers that Cu-T may have a protective role against endometrial cancer.

'Quick course' neoadjuvant chemotherapy with cisplatin, bleomycin and vincristine in advanced cervical cancer.

To evaluate the response and safety of 'quick course' neoadjuvant chemotherapy, 30 patients with advanced squamous cell carcinoma of cervix were given cisplatin, bleomycin, and vincristine weekly for 3 courses. The response was evaluated by subjective parameters and by standard response criteria. In addition to the marked improvement in symptoms, the overall objective response was 60% with a complete pathological response of 6.6%. Tumor volume decreased significantly (p=0.002) after chemotherapy. Patients with stage IB and 27% (3 of 11) of patients with stage II disease who became technically stage IB (stage reduction) after chemotherapy underwent surgery. Radiotherapy was given to the remaining patients. All patients tolerated the chemotherapy.

Dilantin as anticonvulsant in eclampsia.

Sixty-six cases of eclampsia amongst 9178 deliveries were managed from July, 1997 to December, 1998 in the department of obstetrics and gynaecology at UCMS & GTB Hospital, Delhi. The aim was to evaluate the changing trends in patients of eclampsia and to assess the efficacy of dilantin in its treatment. The incidence recorded was 1 in 139 deliveries (0.7%). Majority (90.91%) were unsupervised in antenatal period and 68.18% were primigravidae. Eclampsia developed at < 28 weeks of pregnancy in 3.03% of patients. All the patients had hypertension and proteinuria at the time of admission and 51.52% showed hypertensive changes on fundus examination. While single anticonvulsant therapy in the form of dilantin was used to manage 57.57% of patients, 13.64% required intubation and positive pressure ventilation. Fit recurrence on treatment was noticed in 40.91% of patients. Lower segment caesarean section was conducted in 18.18% of patients. Three maternal deaths (4.56%) attributed to ventricular tachycardia, aspiration and intracerebral haemorrhage were recorded. Morbidity was frequent in the form of status eclampticus (n = 5), postpartum psychosis (n = 5), retinal detachment (n = 1), coagulation abnormality (n = 11). The perinatal mortality rate was 30.43%. The study concludes that eclampsia is still rampant in India and despite better medical facilities, maternal and perinatal mortality remains high. The efficacy of dilantin as anti-eclamptic needs to be reviewed.

Del Castello syndrome--an unusual presentation.

Del Castello syndrome in a 28-year-old female, characterised by bilateral galactorrhoea, amenorrhoea and hyperinvoluted uterus, has been described. She had hyperprolactinaemia without any demonstrable pituitary tumour. She was successfully treated with two short courses of bromocriptine and was spontaneously cured after her second conception. The case is discussed with a brief review of the literature.