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#27154214   2016/07/14 Save this To Up

Selected metabolic biochemical and enzyme activities associated with Besnoitia besnoiti infection in dairy cattle.

The main aim of the study was to explore, compare, and identify whether there is an association between Besnoitia besnoiti seropositivity in apparently healthy dairy cows with some biochemical parameters, enzyme activities, and beta-hydroxybutyrate (BHBA). A total of 98 dairy cows were included in the study, of which there was 50 seropositive and 48 seronegative cows. Analysis of serum antibodies against B. besnoiti antibodies was performed using an indirect enzyme-linked immunosorbent assay kit. Student's independent t test showed that there was a significant difference in BHBA, albumin, and lactate dehydrogenase (LDH) between the seropositive and seronegative groups. Univariable regression analysis showed no significant association between seropositivity status with any of the evaluated parameters except BHBA level, mastitis, and abomasum displacement. Multivariable logistic regression analysis showed that there was a strong association between seropositivity with BHBA level. The significant association between BHBA and B. besnoiti seropositivity represents preliminary finding that needs further exploration.

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#26873635   2016/03/13 Save this To Up

In vitro three-dimensional coculturing poly3-hydroxybutyrate-co-3-hydroxyhexanoate with mouse-induced pluripotent stem cells for myocardial patch application.

Identifying a suitable polymeric biomaterial for myocardial patch repair following myocardial infarction, cerebral infarction, and cartilage injury is essential. This study aimed to investigate the effect of the novel polymer material, poly3-hydroxybutyrate-co-3-hydroxyhexanoate, on the adhesion, proliferation, and differentiation of mouse-induced pluripotent stem cells in vitro. Mouse-induced pluripotent stem cells were isolated, expanded, and cultured on either two-dimensional or three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films (membranes were perforated to imitate three-dimensional space). Following attachment onto the films, mouse-induced pluripotent stem cell morphology was visualized using scanning electron microscopy. Cell vitality was detected using the Cell Counting Kit-8 assay and cell proliferation was observed using fluorescent 4',6-diamidino-2-phenylindole (DAPI) staining. Mouse-induced pluripotent stem cells were induced into cardiomyocytes by differentiation medium containing vitamin C. A control group in the absence of an inducer was included. Mouse-induced pluripotent stem cell survival and differentiation were observed using immunofluorescence and flow cytometry, respectively. Mouse-induced pluripotent stem cells growth, proliferation, and differentiation were observed on both two-dimensional and three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Vitamin C markedly improved the efficiency of mouse-induced pluripotent stem cells differentiation into cardiomyocytes on poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Three-dimensional culture was better at promoting mouse-induced pluripotent stem cell proliferation and differentiation compared with two-dimensional culture.

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#25978442   2015/06/06 Save this To Up

Development of quantum dots-based biosensor towards on-farm detection of subclinical ketosis.

Early detection of dairy animal health issues allows the producer or veterinarian to intervene before the animals' production levels, or even survival, is threatened. An increased concentration of β-hydroxybutyrate (βHBA) is a key biomarker for diagnosis of subclinical ketosis (SCK), and provides information on the health stress in cows well before any external symptoms are observable. In this study, quantum dots (QDs) modified with cofactor nicotinamide adenine dinucleotide (NAD(+)) were prepared for the sensing event, by which the βHBA concentration in the cow's blood and milk samples was determined via fluorescence analysis of the functionalized QDs. The detection was performed on a custom designed microfluidic platform combining with a low cost and miniaturized optical sensor. The sensing mechanism was first validated by a microplate reader method and then applied to the microfluidic platform. Standard βHBA solution, βHBA in blood and milk samples from cows were successfully measured by this novel technology with a detection limit at a level of 35 µM. Side by side comparison of the developed microfluidic biosensor with a commercial kit presented its good performance.

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#22940329   2012/09/28 Save this To Up

Electrofiltration as a purification strategy for microbial poly-(3-hydroxybutyrate).

The biodegradable polyester poly-(3-hydroxybutyrate) (PHB), produced by Ralstonia eutropha in batch and fed-batch processes, was purified by electrofiltration. The protein film on PHB granules determines their high negative zeta potential, enabling the application of electrofiltration as an integrated technology in the downstream processing of PHB. In order to determine the optimal purification parameters, various pressure and electric field strength conditions were tested. Electrofiltration of PHB at 4bars and 4V/mm provided an up to four times higher concentration factor than conventional filtration. FT-Raman spectroscopy demonstrated that electrofiltration did not result in structural changes to the products. The study demonstrates the efficiency and practical advantages of electrofiltration as a promising downstream step in the PHB production technology.

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#17603262   2007/07/02 Save this To Up

[Biochemistry and structural biology of microbial enzymes and their medical applications].

Microbial enzymes were studied from two medicinal viewpoints. First, we examined proline-specific peptidases from pathogenic microorganisms. We found several proline-specific peptidases in pathogenic bacteria. Among them, prolyl tripeptidyl aminopeptidase from Porphylomonas gingivals and prolyl aminopeptidase from Serratia marcescens were crystallized. The complex structures of those enzymes and inhibitors were clarified in X-ray crystallography. Aminopeptidase N, which has wide specificity for amino acids, was distributed in the pathogens. The crystal structure of the aminopeptidase N elucidated the reasons for its wide substrate specificity but inertness to the X-Pro bond. It was also revealed that proline-specific peptidases and aminopeptidase N cooperatively degrade collagen for the uptake of amino acids as nutrition when these bacteria infect cells. Second, we applied enzymes from microorganisms to diagnostic analyses. We found a series of creatinine-metabolizing enzymes in Pseudomonas putida. Creatininase, creatinase, and sarcosine oxidase were coupled and have been developed for a diagnostic analysis kit that examines renal function. The structures of the native and the Mn2+-activated creatininases were determined in X-ray crystallography. Based on the structure, the activated enzyme was used for an improved assay kit. The structure of D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi was also clarified in crystallography. The enzyme is useful for diagnostic analysis of diabetes mellitus while monitoring ketone bodies.

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#17386920   2007/04/16 Save this To Up

Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyric acid.

Hypertension and related diseases afflict millions of individuals worldwide, and many investigations of angiotensin I-converting enzyme (ACE) activity have been carried out. Most of these have used hippuryl-histidyl-leucine (HHL) as a substrate for ACE reaction with considerable interferences. Here we propose the use of a new substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG) for the screening of ACE inhibitors. Under the actions of ACE and aminoacylase, 3HB-GGG is cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The assay conditions were optimized and applied to monitor the ACE inhibitory activity in terms of 3HB measured using an F-kit. Under the optimum assay parameters-ACE (0.2 U/ml) and aminoacylase (172 kU/ml) incubated with 3HB-GGG (3.4 mg/ml) at 37 degrees C for 30 min-the Gly-Gly and Gly cleaved from 3HB-GGG by enzymes was able to be identified, affirming the feasibility of substituting 3HB-GGG for the conventional substrate HHL. In addition, the current method was more sensitive, accurate, rapid, and convenient than the conventional method.

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#16202461   2006/01/27 Save this To Up

Incidence of subclinical ketosis in cows supplemented with a monensin controlled-release capsule in Holstein cattle, Florida, USA.

The objective of this study was to determine the effect of a monensin controlled-release capsule on the proportion of cows with subclinical ketosis (SK). During July to August 2001, 300 cows dried-off 50-70 days before expected parturition were randomly assigned to either a treatment (n = 150, oral capsule, 335 mg/d of monesin for 95 d) or control group (no capsule, n = 150). At 14 days postpartum, a milk sample was obtained and evaluated for beta-hydroxybutyrate (BHBA) using a semi-quantitative ketone test strip. In a sub-sample of 50 cows per group a blood sample was taken and analyzed for BHBA using an ELISA kit. Milk BHBA > or = 200 micromol/L was used as the cut-off value for diagnosis of SK. The incidence of SK based on the milk test was statistically different between groups (P < or = 0.05) with a value of 26.6% for control and 14.5% for cows treated with monensin, respectively. Cows treated with monensin were 0.68 times less likely to give a positive result for milk BHBA than non-treated cows (0.53-0.80; 95% CI). Serum BHBA concentrations did not differ between groups (0.81 +/- 0.09 mmol/L versus 0.70 +/- 0.07 mmol/L for controls and treated, respectively; P > 0.05). However, for each incremental increase in serum BHBA of 0.1 mmol/L occurrence of SK increased 52% (OR = 1.52; 1.21-1.91; 95% CI).

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#12696920   2003/04/16 Save this To Up

An automatic flow procedure for the determination of 3-hydroxybutyrate in animal serum and plasma.

An automatic flow procedure based on the multicommutation concept, comprising three-way solenoid valves, for the spectrophotometric determination of 3-hydroxybutyrate in animal serum and plasma is proposed. The 3-hydroxybutyrate was enzymatically converted to acetoacetate with the reduction of NAD+ to NADH monitored at 340 nm. It was possible to carry out up to 600 determinations without a significant decrease in the analytical signal, with 5 mg of 3-hydroxybutyrate dehydrogenase immobilized on porous silica beads and packed in a column. The system enabled 60 determinations/h of 3-hydroxybutyrate in the range of 10-150 mg L(-1), with a consumption of 0.9 mg of NAD+ and 200 microL of sample per determination. A detection limit of 2 mg L(-1) for both animal serum and plasma and coefficients of variation of 1.4% and 1.2% (n = 17), respectively, were determined. Animal serum and plasma samples were analyzed without previous treatment, the results of which agreed with those obtained using the conventional method (UV kit, Sigma).

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#1303814   1993/06/29 Save this To Up

[The kinetic observation of serum ketone body levels in surgical patients receiving MCT or LCT emulsions].

The changes of serum beta-hydroxybutyrate concentrations in patients after receiving medium chain triglycerides (MCT) or long chain triglycerides (LCT) were studied. The patients were divided into two groups: in Group 1, the dose of fat was 0.14 g/kg.h for fat emulsion clearance test: and in Group 2, the dose was 0.06 g/kg.h for total parenteral nutrition (TPN) support. The beta-hydroxybutyrate concentrations were estimated by an enzymatic method using Sigma's beta-hydroxybutyrate kit (Sigma Diagnostics, MO 63178, USA). The normal range of beta-hydroxybutyrate concentration in 30 healthy Chinese subjects was 1.04 +/- 1.42 mg/dl (range 0-3.88 mg/dl). In Group 1, we found that the concentrations of beta-hydroxybutyrate rose steadily after fat emulsion infusion. The concentrations in the medium chain triglyceride group were higher than those in long chain triglyceride group. The elevated concentrations of beta-hydroxybutyrate gradually returned to normal levels when the emulsion infusion was stopped. In Group 2, the concentrations of beta-hydroxybutyrate in the patients were within the normal range.

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#2685405   1989/12/29 Save this To Up

[Diagnosis and treatment of diabetes mellitus-recent advances].

In 1982, two years after the second report of the WHO Expert Committee on Diabetes Mellitus, new diagnostic criteria for diabetes mellitus were recommended by the Japan Diabetes Society and widely used in Japan. In the assessment of diabetic control, a blood ketone body measurement kit was developed and has been used for detecting its metabolic abnormality. Glycated hemoglobin has been introduced to clinical practice as an index of the past 1-2 months' diabetic control, fructosamine of the past 1-2 weeks' diabetic control and they are effectively used to achieve the good metabolic control. In the field of treatment, the extrapancreatic action of the sulfonylurea agent has been re-estimated and combination therapy with insulin is now being tested. In insulin treatment, conventional insulin injection (once a day) was not sufficient for preventing the chronic vascular complication of diabetes and intensive insulin therapy has been strongly recommended and is gradually coming into wide use. Since the development of an insulin injection pump, we have been able to mimic the normal insulin secretion pattern. With the increase of blood glucose self-monitoring, the sliding scale method can be used where the patient himself adjusts the insulin dose under certain circumstances.

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