Improvement of the expression level of β-glucosidase from Agrobacterium sp. in Escherichia coli by rare codon optimization

To achieve high level expression of β-glucosidase from Agrobacterium sp. (Abg) in an Escherichia coli expression system, 6 rare codons at +3, +11, +158, +308, +314, and +318 of abg gene were replaced with favored codons using site-directed mutagenesis. The rare codon replacements of +3 (CCC) and +11 (CCC) positions enhanced the expression level of Abg by 2- and 3.6-fold, respectively. The double mutant, Abg-DM, where both of +3 and +11 rare codon positions were modified showed a 5.2-fold higher expression level than the original abg gene. Circular dichroism (CD) spectra of the over-expressed Abg and original Abg enzymes were identical, indicating that Abg was properly folded during over-expression.     

Indicator to predict the movement of phosphorus from soil to subsurface flow

The movement of phosphorus (P) in subsurface flow can contribute to losses from agricultural land. This study aims to identify a soil P threshold above which P loss is likely to accelerate as a function of soil and management type. Lysimeters (25 cm i.d. by 30 cm deep) were taken of four soils from agricultural watersheds in Pennsylvania and New York. The soils had a range of Mehlich-3 extractable P (7-517 mg of P kg(-1)) in surface soil (0-7.5 cm for grassland and 0-23 cm for cultivated/arable) and reactive P, filtered <0.45 microm (RP(<0.45 microm) in subsurface flow (0.007-1.53 mg of P L(-1)). The loss of P from lysimeters increased greatly when Mehlich-3 extractable P was in excess of a mean concentration of 280 mg kg(-1), the degree of saturation of P sorption sites exceeded 38%, and the corrected P sorption strength (corrected for desorption) derived from the monolayer Langmuir equation was less than 0.07 L of P mg(-1). Of these variables, P sorption strength was most consistently related to RP(<0.45 microm) in subsurface flow across a range of soil managements. Use of the corrected Langmuir sorption strength parameter to estimate subsurface flow RP(<0.45 microm) derived from four U.S. soils was tested on four soils of different physiochemical characteristics (one calcareous) from the U.K. The U.K. soils showed similar concentration trends to the U.S. soils, with elevated RP(<0.45 microm) below a sorption strength of 0.07 L of P mg(-1). We propose that the P sorption strength derived from the monolayer Langmuir equation may be used to estimate the potential for P loss in subsurface flow, when simpler environmental tests such as P saturation derived from acid ammonium oxalate extraction are unclear or unsuitable.     

Glycosidase mechanisms: anatomy of a finely tuned catalyst

In order to accelerate the hydrolysis of glycosidic bonds by factors approaching 10(17)-fold, glycosidases have evolved finely tuned active sites optimally configured for transition-state stabilization. Structural analyses of various enzyme complexes representing stable intermediates along the reaction coordinate, in conjunction with detailed mechanistic studies on wild-type and mutant enzymes, have delineated the contributions of nucleophilic and general acid/base catalysis, as well as the roles of noncovalent interactions, to these impressive rate enhancements.     

跟踪式热平衡温度测量

介绍了跟踪式热平衡温度测量的原理：当检测头与被测体靠近时，通过对流换热形成梯度为零的温度场，因而二者温度相等。这种梯度为零的温度场是通过对检测头的一端制冷（或加热）跟踪被测体的温度形成的．最后给出了静态和动态实验的测试结果。     

A model to predict survival following liver retransplantation

In the current era of critical-organ shortage, one of the most controversial questions facing transplantation teams is whether hepatic retransplantation, which has historically been associated with increased resource utilization and diminished survival, should be offered to a patient whose first allograft is failing. Retransplantation effectively denies access to orthotopic liver transplantation (OLT) to another candidate and further depletes an already-limited organ supply. The study group was comprised of 1,356 adults undergoing hepatic retransplantation in the United States between 1990 and 1996 as reported to the United Network for Organ Sharing (UNOS). We analyzed numerous donor and recipient variables and created Cox proportional-hazards models on 900 randomly chosen patients, validating the results on the remaining cohort. Five variables consistently provided significant predictive power and made up the final model: age, bilirubin, creatinine, UNOS status, and cause of graft failure. Although both hepatitis C seropositivity and donor age were significant by univariate and multivariate analyses, neither contributed independently to the estimation of prognosis when added to the final model. The final model was highly predictive of survival (whole model chi2 = 139.63). The risk scores for individual patients were calculated, and patients were assigned into low-, medium-, and high-risk groups (P <.00001). The low degree of uncertainty in the probability estimates as reflected by confidence intervals, even in our high-risk patients, underscores the applicability of our model as an adjunct to clinical judgment. We have developed and validated a model that uses five readily accessible "bedside" variables to accurately predict survival in patients undergoing liver retransplantation.     

Pathologic parameters in the diagnosis and prognosis of primary cutaneous melanoma

Significant progress has been made in the last 10 years on the identification of histologic parameters that are independent predictors of melanoma prognosis, immunohistochemical markers of cells of melanocytic origin and changes in adhesion molecules, cytoskeletal proteins, growth factor receptors, cell signaling, and nuclear proliferation proteins associated with tumor progression. Histologic criteria may never be completely sufficient to predict behavior accurately, because the fundamental change that renders a cell aggressive may not be morphologically reflected and may require immunohistochemical or other molecular markers to establish behavior. To date, it is humbling that no immunohistochemical or molecular marker provides a greater predictable value for aggressive behavior than does the simple calibrated ocular micrometer to measure tumor thickness. Nevertheless, development of multiple histologic parameters with the concept of nontumorigenic RGP and tumorigenic VGP provides a reliable statistical model to predict metastases. Fortunately, nontumorigenic RGP melanomas with greater than 75% regression are rare. Thus, individual patients with melanoma without regression and without the tumorigenic VGP can be given reasonable assurance of 100% survival. Nevertheless, this assurance is based on a statistical model with a finite population studied. Additional studies are needed to confirm this model, as well as more definitive markers to precisely predict outcome for those individuals with tumorigenic VGP melanoma.     

The National Cancer Data Base report on the relationship of race and national origin to the histology of nasopharyngeal carcinoma

This study was undertaken to clarify several aspects of morphological and taxonomic characters of Physaloptera bispiculata Vaz and Pereira, 1935, a parasite of the water rat, Nectomys squamipes. The cephalic structures (including lips, papillae, teeth, amphids, and porous areas) and details of the posterior end of male and female adult worms were examined by scanning electron microscopy, leading to the addition of new taxonomic characters for this species. We consider P. bispiculata a valid species, based on a comparative analysis of the specific characters for P. bispiculata and P. getula Seurat, 1917, including the morphology and morphometry of body structures as well as number and disposition of caudal papillae of the males.     

Inappropriate use of daily mortality analyses to estimate longer-term mortality effects of air pollution

Identification of enterococci species by hybridization with recently designed species-specific and group-specific 23S rDNA-targeted oligonucleotide probes was superior to results obtained with a common biochemical test panel. Considering these findings, a molecular biological procedure for the detection of enterococci in water samples was developed. A short enrichment is followed by an amplification step and a hybridization reaction in microtiter plate format. The detection limit is about 1 CFU/ml, and results are available within 26 h.     

Toxicity of the styrene metabolite, phenylglyoxylic acid, in rats after three months' oral dosing

Male Wistar rats were dosed with 0, 1250, 3750 or 5000 mg/l of phenylglyoxylic acid (PGA) (CAS no. 611-73-4) in the drinking water ad libitum for 3 months. During the entire treatment period, there were no gross signs of toxicity related to PGA. No changes in neurobehavior were found after using a functional observational battery or radial arm maze. An increased relative kidney weight was seen in the highest dose-group (Controls: 0.504 +/- 0.031 g/100 g b.wt.; 5000 mg PGA/l: 0.579 +/- 0.033 g/100 g b.wt.; p<0.01). No other organ weights were affected. Histopathology revealed no change in kidney structure. No changes in clinical biochemistry. In the highest dose-group three animals out of ten showed reduction in peripheral nerve myelin sheath thickness. No such changes were seen in the control group. The study revealed no changes in auditory brain stem response but minor changes in electroretinography. The noradrenaline (NA) concentration decreased in pons and thalamus whereas it increased in medulla oblongata and whole brain. The dopamine (DA) concentration increased in cerebellum, hippocampus, pons, and whole brain. The most marked DA increase was seen in hippocampus (Controls: 0.56 +/- 0.10 nmol/g tissue; 5000 mg/l: 1.04 +/- 0.11 nmol/g tissue; p<0.001). The 5-hydroxytryptamine (5-HT) concentration decreased in cerebellum, cerebral cortex, hippocampus, and medulla oblongata, whereas it increased in thalamus. The yield of synaptosomal protein, synaptosomal NA, DA, and 5-HT concentrations, and DA uptake rate were not affected. When dosed males were mated with naive females, there were no differences between groups in the pregnancy rate, number of corpora luteae, implantations, live or dead fetuses, resorptions, preimplantation loss, or postimplantation loss. It is concluded that a part of the effects on kidney, peripheral nerves, and vision, which have previously been reported after exposure to styrene, might be induced by the styrene metabolite, PGA. If PGA has ototoxic effects in rats, the dosing in the present study is not sufficient to induce the necessary ototoxic concentration in blood. Alternatively, the ototoxicity of styrene, like toluene, may be caused the parent compound itself and not by a metabolite like PGA.     

Induction of neutralizing antibody in mice by immunization with recombinant 56 kDa protein of Orientia tsutsugamushi

Anti-oriential antibody inhibits Orientia tsutsugamushi attachment to, and penetration of, host cells. However, O. tsutsugamushi antigens that induce the production of a neutralizing antibody have not been identified. The authors immunized mice and rabbits with the recombinant 56 kDa protein of O. tsutsugamushi fused to the maltose binding protein of Escherichia coli (MBP-Bor56) and analysed their effect on O. tsutsugamushi attachment to or penetration of L929 cells. O. tsutsugamushi attachment and penetration were measured by using an indirect immunofluorescent antibody assay (IFA). O. tsutsugamushi growth in L929 cells was determined by [3H]thymidine uptake assay. By IFA, we observed a 96% reduction of attachment or penetration of O. tsutsugamushi treated with rabbit anti-MBP-Bor56 sera. [3H]thymidine uptake showed that mouse anti-MBP-Bor56 sera caused a 91% reduction in O. tsutsugamushi growth, when compared to mouse anti-MBP sera. These results suggest that the 56 kDa protein of O. tsutsugamushi plays an important role in O. tsutsugamushi attachment to or penetration of cells.