Purification and characterization of the carbonic anhydrase enzyme from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney and inhibition effects of some metal ions on enzyme activity

In this study, the carbonic anhydrase (CA) enzyme was purified from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney with a specific activity of 603.77 EU/mg and a yield of 35.5% using Sepharose-4B-l-tyrosine- sulphanilamide affinity column chromatography. For determining the enzyme purity and subunit molecular mass, sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and single band was observed. The molecular mass of subunit was found approximately 29.71 kDa. The optimum temperature, activation energy (E_a), activation enthalpy (ΔH) and Q₁₀ values were obtained from Arrhenius plot. K_m and V_max values for p-nitrophenyl acetate of the purified enzyme were calculated from Lineweaver-Burk graphs. In addition, the inhibitory effects of different heavy metal ions (Fe²⁺, Pb²⁺, Co²⁺, Ag⁺ and Cu²⁺) on Black Sea trout kidney tissue CA enzyme activities were investigated by using esterase method under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC₅₀) were obtained. Finally K_i values and inhibition types were calculated from Lineweaver-Burk graphs.     

Identification of polcalcin as a novel allergen of Amaranthus retroflexus pollen

IntroductionAmaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients’ sera.MethodsAllergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract.ResultsOf 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10 kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10 kDa protein in crude extract. These results were confirmed by inhibition methods, too.ConclusionThe recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart.     

低分子量聚合物电解质的合成与性能

研究了低分子量梳状聚合物电解质的合成方法及结构，性能。首先合成了不同分子量的甲基丙烯酸聚乙二醇单甲醚酯，并进一步合成了分子量一万左右的梳状聚合物电解质，结果表明：反应严格按照反应方程进行，精制产物是非晶的梳状聚合物，本聚合物体系均存在两个玻璃化转变温度，一个在100℃左右，归属为梳状聚合物主链的玻璃化转变，另一个在－20摄氏度以下，归属于侧链玻璃化转变，在室温下侧链可以运动，有利于电活性物质的迁移和扩散，并用超微电极研究了该电解质的行为。     

Melatonin Receptors on Ovine Pars Tuberalis: Characterization and Autoradiographicai Localization

The functional significance of the pars tuberalis (PT) of the mammalian adenohypophysis has remained an enigma (1, 2). One view of its function is that it acts as an auxiliary gland to support the endocrine role of the pars distalis (PD) (2), as it has been shown to contain immunocytochemically identifiable thyrotrophs and gonadotrophs (1). Many of the cells of the PT are, however, ultrastructurally unique suggesting an independent function for this tissue. Our recent demonstration that the PT of the rat is a major binding site for the ligand iodomelatonin lends further support to this idea (3). We have utilized the highly specific ligand [¹²⁵l]melatonin, and have demonstrated that it binds exclusively, with very high affinity, to the PT but not the PD of the adult sheep adenohypophysis. These findings support the conclusion that the PT has a distinct role in relation to melatonin action and seasonal reproduction.     

Surface modification of titanium by etching in concentrated sulfuric acid

OBJECTIVE: The purpose of this study was to characterize the etching behavior of titanium in concentrated sulfuric acid and discuss its application on surface modification of titanium for biological use. METHODS: Commercially pure titanium (cpTi) plate was etched in 48% H2SO4 at RT -90 degrees C for 0.25-8 h. The weight loss was derived from the weight differences before and after etching. The surfaces after etching were characterized by surface roughness, X-ray diffractometry, and scannning electron spectroscopy. The apparent activation energy of the dissolution of cpTi into acid was derived from an Arrhenius plot of the rate of weight loss versus the acid temperature. RESULTS: The surface roughness of cpTi increased with the acid temperature and etching time. The surface roughness was strongly related to the weight loss. The weight loss increased drastically with the acid temperature after an initial period, which shortened with increasing acid temperature. The apparent activation energy for the dissolution of cpTi in H2SO4 was derived as 67.8 kJ/mol. SIGNIFICANCE: This study indicates that etching with concentrated sulfuric acid is an effective way to modify the surface of titanium for biological applications.     

Hematopoietic reconstitution after bone marrow transplantation: Assessment with MR imaging and H-1 localized spectroscopy

Magnetic resonance (MR) studies were performed in 14 patients as early as possible (21–110 days) after bone marrow transplantation (BMT). MR characteristics of lumbar vertebral bone marrow were studied with T1-weighted spin-echo imaging, water- and fatselective imaging with a frequency-selective excitation technique, and point-resolved spatially localized proton spectroscopy. Signals from water and fat protons and their T1 and T2 values were analyzed. Water proton signal intensity correlated well with cellularity within bone marrow, as determined with parallel iliac crest biopsies. The fraction of signal from water in red bone marrow of patients with allogeneic transplants from siblings (four cases) was significantly higher than in four patients with autologous transplants. The latter showed very low cellularity in the period of about 4 weeks after BMT because of the cytotoxic pretreatment of the bone marrow. The MR results in six patients with allogeneic transplants from unrelated donors ranged widely, depending on the complications after BMT. Analysis of data obtained with the different techniques showed that water- and fat-selective MR imaging and spectroscopic methods are useful for noninvasive monitoring of hematopoietic reconstitution after BMT.     

Precise and accurate measurement of proton T1 in human brain in vivo: Validation and preliminary clinical application

Precise and accurate inversion-recovery (PAIR) magnetic resonance (MR) measurements of T1 were obtained in eight brain regions and cerebrospinal fluid of 26 healthy volunteers. Accuracy of the technique was assessed by measuring T1 in small fluid volumes with the PAIR technique and with two independent spectroscopic techniques. The mean difference between T1 measured with PAIR and with the two spectroscopic techniques was 3.1% ± 1.3. The precision (reproducibility) of measurements with the PAIR technique was excellent. The coefficient of variation (CV) across 16 measurements in a head phantom was 2.0%, compared with a CV of 2.7% across 45 separate measurements in a single subject. The within-subject CV was 1.8% ± 0.6 in white matter and 1.4% ± 1.0 in basal ganglia. The between-subject CV in 26 healthy volunteers was 3.6% ± 0.6 in white matter and 4.1% ± 1.9 in basal ganglia. Comparison between a patient with an active recurrent brain tumor and an agematched patient with an inactive brain tumor showed that T1 was significantly elevated throughout the brain of the active-tumor patient, especially in white matter tracts, even though no tumor or edema was detected in the white matter on standard MR images. Comparisons between five brain tumor patients and four healthy volunteers of similar age showed that T1 was significantly and substantially elevated throughout the white matter tracts and in the caudate nucleus, putamen, and thalamus. These results are consistent with the hypothesis that white matter tracts are selectively vulnerable to edema and that T1 increases in white matter are a sensitive indicator of patient status or tumor aggressiveness.     

Time after excision and temperature alter ex vivo tissue relaxation time measurements

Previously unreported effects of tissue storage were recently observed in the authors' experimental magnetic resonance (MR) studies. To evaluate the effect of elapsed time after excision and storage temperature on tissue relaxation time measurements, tissue samples from the liver, pancreas, kidney, testis, spleen, and brain were obtained in rats. T1 and T2 were first measured within 5 minutes of excision, and between subsequent measurements, tubes were kept in a water bath at 40°C, at room temperature (28°C), or in an ice bath (4°C). Cellular and organellar integrity was assessed with electron microscopy and correlated with the MR findings. At 40°C (20-MHz spectrometer), the T1 of liver decreased from 280 msec ± 8 to 212 msec ± 10 during the first 60 minutes; the T1 of pancreas decreased from 276 msec ± 3 to 208 msec ± 2. Other tissues showed less than a 5% decrease in T1. T2 changes were smaller than T1 changes in all tissues. Electron microscopy of pancreatic acinar cells showed postmortem changes in mitochondria evolving over the first 60 minutes after death. Manganese loading experiments implicated mitochondrial manganese stores in the observed enhanced postmortem decrease in T1. This study calls into question reported relaxation time data for liver and pancreas. MR studies of excised tissues must account for time and temperature to prevent systematic experimental errors.