Characterization of recombinant prolyl aminopeptidase from Aspergillus oryzae.

AIMS: Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides. The food-grade fungus Aspergillus oryzae produces this enzyme only in small amounts. In this paper, we present efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties. METHODS AND RESULTS: The gene encoding PAP was overexpressed as a His-tag fusion protein under a taka-amylase gene (amyB) promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich medium containing glucose (repressing condition) was twice that in starch (inducing condition). The enzyme prepared as cell-free extract was partially purified through two-step column chromatography. The PAP was estimated to be a hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l(-1). CONCLUSIONS: Aspergillus oryzae PAP was produced under the repressing condition of amyB promoter in a PAP-overexpressing strain and purified 1800-folds. Overproduction of PAP under promoter-inducing conditions led to an increase in inactive PAP, possibly because of irregular folding. SIGNIFICANCE AND IMPACT OF THE STUDY: PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.

Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae.

AIMS: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. METHODS AND RESULTS: The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co(2+)-containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. CONCLUSIONS: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co(2+)-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.

Salmonella prevalence in seafood imported into Japan.

A total of 353 samples of 29 types of seafood were tested for Salmonella prevalence and total microbial population. Salmonella enterica serotype Weltevreden was isolated from 2 of 47 black tiger prawn samples. The contamination levels of Salmonella were in a range of <30 to 40 most probable number per 100 g. In addition, one sample of black tiger prawns and two samples of white shrimp were positive for Salmonella invA gene on PCR assay. Although the mean aerobic bacterial count was greater than 4 log CFU/g in most of the sample types, those in the two Salmonella-isolated samples of black tiger prawn were 7.48 and 5.18 log CFU/g, respectively. These results indicate the possibility that shrimp and prawns contribute to foodborne infections. The improvement of seafood quality is an important issue, and the information on contamination by pathogens should be provided as feedback to the originating country, with the aim of increasing safety.

Salmonella prevalence and total microbial and spore populations in spices imported to Japan.

A total of 259 samples of 40 types of spices were tested for Salmonella prevalence and total microbial and spore populations. Salmonella enterica serotypes Weltevreden and Senftenberg were isolated from a black- and red-pepper sample, respectively. Because Salmonella was not detected by the most-probable-number method, it indicated that at least one cell of the microorganism was present in 25 g of sample. The mean aerobic bacterial count was greater than 5.39 log CFU/g in turmeric, garam masala, curry powder, and paprika. The mean bacterial spore counts were greater than 4.33 log CFU/g in turmeric and curry powder. The mean aerobic bacterial count in the two Salmonella-isolated samples was 6.93 log CFU/g. These results indicate that spices can be a source of contamination in the products where they are used as ingredients, and methods to reduce the microbial load in spices should be used.

Prevalence of Cryptosporidium parvum infections in weaned piglets and fattening porkers in Kanagawa Prefecture, Japan.

Fecal samples from 232 weaned piglets (1 and 3 months old) and 252 fattening porkers (6 months old) in 8 stock-raising farms located in Kanagawa Prefecture, Japan, from June 1998 to June 2000 were examined to determine the prevalence of Cryptosporidium infection. Detection of oocysts was performed using the ethyl acetate fecal concentration method and immunofluorescent staining. C. parvum oocysts were identified in 77 (33.2%) 1-3 months old weaned piglets from four farms. The odds of excreting among 1-3 months old piglets were more than 100 times greater than among 6 months old porkers (95% confidence interval: 17-902). This strongly suggests that weaned piglets are important reservoirs of pathogenic microbes whose potential contamination of drinking water has epidemiological implications for human health.

Occurrence of clostridia in glass bottled foods.

Forty-one marketed samples of imported and domestic glass bottled foods were tested for clostridia contamination. It was detected in nine (22%) samples. Clostridia were isolated from fish sauce (Nam pla, Nuoc-mam), dressing, mustard, hot and sour soup mix (Tom Yum), mysids boiled down in soy, and salmon flakes. The origin of all clostridia positive samples was Asia. Clostridium botulinum and C. perfringens were not detected. The frequency of occurrence was higher by enrichment broth culture detection methods than by agar plate or pouch methods. These findings suggest that the number of bacteria in most of these clostridia positive food samples is very low, and the use of enrichment methods for detection of clostridia is essential.

[A comparison of sensitivity between direct plate culture, immunomagnetic separation and polymerase chain reaction for the isolation of enterohemorrhagic Escherichia coli O157].

Sensitivities of direct plate culture (DPC) method, immunomagnetic separation (IMS) method, and polymerase chain reaction (PCR) assay for successful detection Escherichia coli O157 in the food samples were compared. Three lots of minced beef and three lots of radish sprout, both of which were commercially retailed, were enriched with non-selective broth media at 36 degrees C for 6 h. After enrichment, the cultures of the minced beef and those of the radish sprout were found to have background microflora at ca.10(5)-10(7) CFU/ml and ca.10(8) CFU/ml, respectively. The cultures were then experimentally inoculated with E. coli O157 strains at various final concentrations ranging from ca.10 to 10(7) CFU/ml. The samples thus prepared were subjected to the above three methods to evaluate their detection limits. For the samples of minced beef, the detection limits of the DPC method was 10(2) CFU/ml whilst that of the IMS method was ca.10 CFU/ml. For the samples of radish sprout, the detection limits of the DPC method, the IMS method, and the PCR assay were ca.10(4) CFU/ml, ca.10(2) CFU/ml, and ca.10(6) CFU/ml, respectively. There results strongly suggest that the IMS method is most sensitive method for the detection of O157 from food samples among the methods currently available.

Measurement of the albumin content of urinary protein using dipsticks.

An albumin selective urine strip based on bis (3',3''-diiodo-4', 4''-dihydroxy-5',5''-dinitrophenyl)-3,4,5,6-tetrabromo sulfonphthalein dye (DIDNTB) dye was examined in populations with clinical proteinuria. The relationship of albumin to the sum concentration of all protein in urine was found to vary widely even though the albumin concentration generally increased with the total protein concentration. The albumin reagent strips correlated well with immuno-nephrometric assays for albumin on specimens from hypertensives, diabetics, and renal disease which tended to have albumin contents of >/= 50.0%. High proteinuria concentrations of > 250 mg/l, with low albumin contents of

Practical synthesis of androgen: the efficient transformation of 17-oxo group to 17 alpha-hydroxy group.

The present report describes the improved transformation of the 17-oxo group in 3 beta-acetoxy-5 alpha-androstan-17-one to a 17 alpha-hydroxy group. A mixture of 17 alpha-acetoxy and 16-ene compounds, which are usually produced by the standard synthetic route, were treated with peracetic acid (epoxidation of the 16-ene compound) and then sodium borohydride-sodium hydroxide (reduction-hydrolysis) to give the desired 17 alpha-hydroxy compound in much better yield than that in previous reports. Recrystallization of the crude product with cyclohexane-methanol gave the pure compound in 54% yield (total yield from starting ketone).

Regulation by uniconazole-P and gibberellins of morphological and anatomical responses of Fraxinus mandshurica seedlings to gravity.

The present study deals with roles of gibberellins (GAs) in gravitropic responses of woody stems of horizontally-positioned, 2-year-old seedling of Fraxinus mandshurica Rupr. var. japonica Maxim. The application of uniconazole-P, an inhibitor of GAs biosynthesis, to stem nodes at various concentrations significantly inhibited not only righting the stems but also wood formation. The application of GAs (GA3 and GA4) in combination with uniconazole-P negated the inhibitory effect of the treatment of uniconazole-P alone. The GAs alone did not affect wood formation, however, both GAs increased wood formation on both the upper and lower sides of horizontal stems with an increase in the concentration of combined uniconazole-P. In all of the seedlings, tension wood was formed on the upper side of stems. The application of uniconazole-P alone at various concentrations did not inhibit the formation of tension wood fibres.

Signal analysis and compression performance evaluation of pathological microscopic images.

Digitizing high-quality microscopic images and developing input/output technology for displaying those results is critical to telepathology in which pathological microscopic images are transferred to remote locations where they are diagnosed by specialists. This paper will discuss the results achieved by directly digitizing (nonfilm process) pathological microscopic images at a 2k x 2k resolution, and then using a super-high-definition imaging system to analyze their signals and evaluate compression performance. We will start off by digitizing samples that a pathologist will actually use in making a diagnosis, and then analyze their color distribution and spatial frequencies characteristics by comparing them to general images. This will make it apparent that such pathological images characteristically contain high spatial frequency in their chrominance components. We will also discuss the evaluation results of color differences for L*a*b* space and compression ratios achieved when using JPEG to encode pathological images. We will also present a subjective evaluation of the influence subsampling of chrominance components has on image quality.

Contrast mapping and evaluation for electronic X-ray images on CRT display monitor.

Simple chest X rays on film are the most common type of image in medical diagnosis. However, amongst the various types of medical X-ray images, they require the highest level of display quality due to the fact that the body structures they capture on film have varying degrees of permeability to X rays. Conventional high-definition digital display technology has not always been able to match the quality of such film images. This has been a major impediment against progress toward the complete digitization of simple chest X rays. The intent of this paper is to examine that, when applied to medical diagnosis of chest X rays, super-high-definition (SHD) images (digital images with resolution exceeding that of HDTV) are capable of producing a level of quality of diagnostic accuracy on a par with conventional film images. We will start out by seeking out the overall transmission characteristics of a system that uses digital radiography and a film digitizer to digitize images. We will then derive gray-scale transform characteristics based on the luminance linear method for approximating, as closely as possible on a CRT, film images on a light box that have wide dynamic range and high luminance. Finally, we will present the results of image evaluation experiments using high-definition CRT monitors. These results indicate that conventional film images and those on super-high-definition CRT monitors have nearly the same quality. They will also show that the contrast mapping selected by radiologists and theoretical luminance linear characteristics were almost the same except in low-luminance regions. We will also discuss radiologists' comments on CRT monitors after they participated in the evaluation experiment.

A case of cutaneous T-cell lymphoma expressing gamma delta T-cell receptors.

Cutaneous T-cell lymphoma expressing gamma delta T-cell receptors (TCRs) is rare; only a few cases have been reported. We report another case of the disease that had a fatal outcome and differs from two previously reported cases in its clinical, histologic, and immunohistochemical aspects. The patient had multiple skin tumors with central crusts; she showed no response to chemotherapy and died within 1 year. Histologic findings included perivascular infiltrates in the dermis and subcutaneous tissue with no epidermotropism; the large lymphoid cells had a phenotype of CD1-, CD3+, CD4-, CD8-, CD25-/+, CD30-, CD38-/+, HLA-DR+, and gamma delta TCR (beta F1-, TCR delta 1+, delta TCS1-, Ti gamma A+, BB3-). The differences in the phenotype of gamma delta T-cell malignancies may reflect the uniqueness of its clinical and histologic features. A study of gamma delta T-cell malignancies may disclose important biologic features of gamma delta TCR+ cells.

Reduction of proteinuria by adrenalectomy and its restoration by glucocorticoids in rats bearing functioning pituitary tumor.

Intense proteinuria in rats bearing a functioning pituitary tumor MtT SA5 was considered to be evoked by overproduction of albumin due to elevated serum growth hormone (GH). The present study revealed a striking reduction of proteinuria by bilateral adrenalectomy. Supplementation for about 6 weeks with glucocorticoids to adrenalectomized tumor-bearing rats again induced marked proteinuria, but supplementation with mineralocorticoids failed to augment proteinuria. An analysis of systemic blood pressure and renal blood flow did not yield any conclusive result. In spite of an assumption that glucocorticoids enhance GH production of the tumor based on the presence of glucocorticoid receptor in the tumor tissue, no difference was found in serum GH levels between glucocorticoid- and mineralocorticoid-supplemented adrenalectomized rats. The mechanism remains to be clarified, but modification of glomerular permeability or a change in glomerular hydraulic pressure by glucocorticoids might be considered.

Assays of serum lipase by the "BALB-DTNB method" mechanized for use with discrete and continuous-flow analyzers.

We successfully adapted the dimercaprol (BAL) tributyrate-5,5'-dithiobis(2-nitrobenzoic acid) method (J. Biochem. 81: 361, 1977) for assay of lipase in human serum to a discrete analyzer (the TBA 880) (I) or a continuous-flow analyzer (AutoAnalyzer, Type II) (II). In both, BAL-tributyrate is used as substrate, in combination with serum esterase inhibitors and a chromogenic reagent for the SH group of the liberated BAL. Serum lipase activities of patients with pancreatic diseases, measured at 90 or 40 samples per hour by I or II, respectively, correlated well with those measured by the corresponding manual method or by Kaplan's radioassay (Anal. Biochem. 33: 213, 1970). The correlation coefficients were all greater than 0.95, and the coefficients of variation were less than 8%, showing the practical usefulness of these procedures.

Continuous-flow enzymic determination of creatine in urine.

A new enzymic method is described for the determination of creatine in urine by continuous-flow analysis. The measurement is accomplished by transforming creatine to formaldehyde in reactions catalyzed by creatinase (creatine amidinohydrolase) and sarcosine dehydrogenase. The formaldehyde is reacted with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole to form a purple product, which is measured colorimetrically. The method gives a linear standard curve for creatine concentrations up to 100 mg/L. Precision and analytical recovery are excellent, and results correlate well with those by the more-difficult Folin method, which currently is most often used for this analysis.

Clinical experiences with aclacinomycin-A.

Aclacinomycin is a new anthracycline analog of adriamycin and daunomycin. Aclacinomycin contains three sugars. The drug has been studied in 22 cases in a phase 1 type of trial on a schedule 20 mg i.v. every other day up to a total of 300 mg. Toxicity has consisted of myelosuppression, nausea and vomiting, and transient hepatic disturbances. Evidence of clinical activity was observed in several cases including a case of breast cancer and gastric cancer. Although no full patial remissions were recorded, further study is continuing.