Comparison of four enrichment broths for the detection of non-O157 Shiga-toxin-producing Escherichia coli O91, O103, O111, O119, O121, O145 and O165 from pure culture and food samples.

AIMS: We compared the efficiency of universal pre-enrichment broth (UPB), modified Escherichia coli broth containing novobiocin (mEC + n), modified Tryptic Soy Broth (mTSB) and mTSB with novobiocin (mTSB + n) for the enrichment of non-O157 Shiga-toxin-producing E. coli (STEC). METHODS AND RESULTS: Freeze-injured and control non-O157 STEC (O91, O103, O111, O119, O121, O145 and O165) strains were used to artificially contaminate beef and radish sprout samples, which were then cultivated in each of the four enrichment media. After incubation, STEC strains were detected by loop-mediated isothermal amplification (LAMP) and plating assays. Enrichment in mEC + n was least effective for facilitating the detection of uninjured STEC strains in radish sprouts, while mTSB + n was least effective for enriching freeze-injured non-O157 STEC strains from beef samples for detection by LAMP assay. UPB and mTSB were superior to mEC + n and mTSB + n for the enrichment of non-O157 STEC from food samples. CONCLUSIONS: The enrichment of non-O157 STEC was negatively affected by the addition of novobiocin to enrichment broths. SIGNIFICANCE AND IMPACT OF THE STUDY: Novobiocin should not be added to media used for the enrichment of non-O157 STEC in food when cell injury is anticipated.

Simple and rapid detection of Campylobacter spp. in naturally contaminated chicken-meat samples by combination of a two-step enrichment method with an immunochromatographic assay.

A simple and rapid method to detect Campylobacter spp. in chicken-meat samples was established. This method consisted of a combination of a two-step enrichment method with a commercially available immunochromatographic assay, named NH Immunochromato Campylobacter (NH IC Campy, Nippon Meat Packers, Ibaraki, Japan), which is able to detect Campylobacter antigen in an enrichment culture within 15 min. The enrichment method did not require much blood or a particular system of generating a microaerobic atmosphere, in contrast to the standard method of enriching Campylobacter spp. in chicken-meat samples. The sensitivity of a combination of the two-step enrichment method with NH IC Campy for detection of non- and freeze-stressed Campylobacter spp. in spiked chicken meat was determined using bacterial cells of Campylobacter jejuni and Campylobacter coli. The detection sensitivities for non-stressed C. jejuni and C. coli were found to range from 5.5 to 1.3x10(1) CFU per 25 g of chicken meat, and those for freeze-stressed C. jejuni and C. coli were found to range from 9.2x10(1) to 1.5x10(2) CFU per 25 g of chicken meat. When a total of 68 chicken-meat samples were tested, the combination method determined that 61 samples were positive for Campylobacter spp. This method was more sensitive than a bacterial culture test, which consists of standard enrichment culturing and plating onto selective agars. Because the combination could be conducted in approximately 48 h, from the beginning of the enrichment culture to final determination, it was more rapid than the bacterial culture test, which requires four to five days. Moreover, the combination was simple to perform. These results suggest that combining the two-step enrichment method with NH IC Campy is useful as a simple and rapid alternative to the conventional bacterial culture test for detecting Campylobacter spp. in naturally contaminated chicken meat samples.

Increased basal phosphorylation of mitogen-activated protein kinases and reduced responsiveness to inflammatory cytokines in neutrophils from patients with rheumatoid arthritis.

OBJECTIVE: We studied the functions of peripheral blood (PB) and synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA), focusing the molecular basis for the activated state and the functional responsiveness of RA neutrophils to inflammatory cytokines. METHODS: Paired samples of PB neutrophils and SF neutrophils from the inflamed knee joint were obtained from 18 RA patients (5 males and 13 females). RESULTS: RA neutrophils exhibited increased spontaneous superoxide (O2-) release and adherence, increased basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase, accelerated spontaneous apoptosis, and enhanced O2- release in response to N-formyl-methionyl-leucyl-phenylalanine as compared with healthy normal PB neutrophils. When challenged with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) or tumor necrosis factor alpha (TNF-alpha), RA neutrophils exhibited reduced responses to these cytokines, which included O2- release, adherence, priming for enhanced O2- release, and phosphorylation of ERK and p38. The functional alterations were greater in SF neutrophils than in PB neutrophils from RA. Reduced responsiveness to cytokines in RA neutrophils was closely associated with increased serum and SF levels of GM-CSF and TNF-alpha. RF and RAHA titers were closely correlated with increased TNF-alpha level in SF. CONCLUSION: These findings indicate that RA neutrophils are in the activated state with increased basal phosphorylation of ERK and p38, and exhibit reduced responsiveness to inflammatory cytokines (G-CSF, GM-CSF and TNF-alpha) and accelerated spontaneous apoptosis.

Increased incidence of vertebral fracture in older female hemodialyzed patients with type 2 diabetes mellitus.

Bone disease in hemodialysis (HD) patients with type 2 diabetes mellitus (DM) is characterized by low bone turnover (Inaba M, et al. Am J Kidney Dis 2002; 39:1261-1269), although their bone quality is yet to be determined. The present study was designed to examine whether the prevalence of vertebral fracture in female HD patients with type 2 DM, age 65 years and older, might be increased, and the relation of this fracture to bone mineral density (BMD) determined by dual X-ray absorptiometry (DXA), since few data are available on the effect of DM on bone strength at lumbar spine. The prevalence of vertebral fracture in type 2 DM HD patients was 32.3%, which was greater than that of non-DM HD patients (13.3%) when adjusted for age and HD duration. Logistic regression analysis elucidated the presence of DM and age as independent risk factors for an increased prevalence of vertebral fracture in HD patients. In non-DM HD patients, those with vertebral fracture showed age significantly higher and BMD in either lumbar spine or distal one third of radius significantly lower than the respective value in those without fracture. However, in DM HD patients, neither BMD in lumbar spine nor distal one third of radius was significantly lower in those with vertebral fracture than in those without. Furthermore, age did not differ significantly between DM HD patients with and without fracture. In conclusion, female type 2 DM HD patients, age 65 years and older, showed significantly higher incidence of vertebral fracture than non-DM HD patients. Although age and low BMD emerged as independent risk factors for vertebral fracture in non-DM HD patients, those factors failed to be a risk factor in DM HD patients, suggesting that BMD determined by DXA might not be reliable in assessing bone strength in DM HD patients.

An extensive outbreak of staphylococcal food poisoning due to low-fat milk in Japan: estimation of enterotoxin A in the incriminated milk and powdered skim milk.

An extensive outbreak of staphylococcal food poisoning occurred in Kansai district in Japan. As many as 13,420 cases frequently ingested dairy products manufactured by a factory in Osaka City. The main ingredient of these dairy products was powdered skim milk manufactured by a factory in Hokkaido. Staphylococcal enterotoxin A (SEA) (< or = 0.38 ng/ml) was detected in low-fat milk and approx. 3.7 ng/g in powdered skim milk. The total intake of SEA per capita was estimated mostly at approx. 20-100 ng. The assumed attack rate was considerably lower than those reported in previous outbreaks. SEA exposed at least twice to pasteurization at 130 degrees C for 4 or 2 s retained both immunological and biological activities, although it had been partially inactivated. The present outbreak was unusual in that the thermal processes had destroyed staphylococci in milk but SEA had retained enough activity to cause intoxication.

Increased stiffness in common carotid artery in hyperthyroid Graves' disease patients.

The present study was undertaken to assess the effect of hyperthyroidism on stiffness in the common carotid artery (CCA) in patients with Graves' disease (GD) and elucidate the mechanism by which arterial stiffness is increased in hyperthyroidism. The arterial stiffness index beta (stiffness beta) was evaluated in the CCA using an ultrasonic phase-locked echo-tracking system. Stiffness beta was defined as the logarithm of the ratio of systolic to diastolic blood pressure divided by the fractional diameter increase during the cardiac cycle and thus established as a measure of arterial stiffness uninfluenced by the change in blood pressure. Seventy euthyroid GD patients were measured for CCA stiffness beta to determine its relationship to retinal blood flow and plasma levels of vascular injury markers. To investigate the effect of hyperthyroidism, 27 GD patients were measured for changes during antithyroid drug (ATD) therapy in stiffness beta and in hemodynamic parameters, retinal blood flow and plasma vascular injury markers. In euthyroid GD patients, stiffness beta in the CCA showed a significant and positive correlation with systolic blood pressure and pulse pressure, but not with peripheral blood flow in the central retinal artery. ATD therapy significantly reduced stiffness beta from 5.23 +/- 2.10 to 4.36 +/- 1.48. The fractional reduction of stiffness beta during ATD therapy correlated significantly with reductions in pulse pressure and retinal blood flow, but not with the reductions in systolic and mean blood pressure, or any of the plasma injury markers. In summary, the significant increase in stiffness beta in the hyperthyroid state may reflect the harmful effect of hyperthyroidism on the arterial wall, which may in turn result from increased stroke volume.

Impaired secretion of parathyroid hormone is coherent to diabetic hemodialyzed patients.

Diabetic bone disease is characterized by low bone turnover resulting from impaired secretion of parathyroid hormone (PTH). However, it was suggested that the difference in duration of hemodialysis (HD) therapy and age of patients between HD patients with and without diabetes mellitus (DM) may be responsible for a significant reduction in serum intact PTH (iPTH) level in HD patients with DM. The present study showed that although such major factors affecting PTH secretion as age, sex, HD duration, and serum calcium, phosphate, and magnesium levels did not differ significantly between HD patients with and without DM, serum iPTH levels were still significantly lower in HD patients with than without DM. Among biochemical markers for bone metabolism, serum levels of intact osteocalcin (iOC) and deoxypyridinoline (DPD) were significantly lower in HD patients with than without DM, whereas serum bone-specific alkaline phosphatase, pyridinoline, and beta-crosslaps did not differ significantly between the two groups of patients. In summary, our findings indicate that PTH secretion may be significantly impaired in HD patients with DM compared with those without DM, and serum iOC and DPD are bone markers sensitive enough to detect low bone turnover in HD patients with DM.

Heteroduplex panel analysis, a novel method for genetic identification of Aspergillus Section Flavi strains.

For genetic identification of Aspergillus Section Flavi isolates and detection of intraspecific variation, we developed a novel method for heteroduplex panel analysis (HPA) utilizing fragments of the internal transcribed spacer (ITS) regions (ITS1-5.8S-ITS2) of the rRNA gene that was PCR amplified with universal primers. The method involves formation of heteroduplexes with a set of reference fragments amplified from Aspergillus flavus, A. parasiticus, A. tamarii, and A. nomius and subsequent minislab vinyl polymer gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1, and N-1) generated by pairwise reannealing among four reference fragments. Of 90 test panels, 89 succeeded in identifying the species and 74 were identical to one of the four standard panels. Of the 16 new panels, 11 A. flavus/A. oryzae panels were identical and typed as F-2 and 4 of 5 A. nomius panels were typed as N-2 or N-3. The other strain, A. nomius IMI 358749, was unable to identify the species because no single bands were formed with any of the four reference strains. DNA sequencing revealed that our HPA method has the highest sensitivity available and is able to detect as little as one nucleotide of diversity within the species. When Penicillium or non-Section Flavi Aspergillus was subjected to HPA, the resulting bands of heteroduplexes showed apparently lower mobility and poor heteroduplex formation. This indicates that HPA is a useful identification method without morphological observation and is suitable for rapid and inexpensive screening of large numbers of isolates. The HPA typing coincided with the taxonomy of Section Flavi and is therefore applicable as an alternative to the conventional methods (Samson, R. A., E. S. Hoekstra, J. C. Frisvad, and O. Filtenborg, p. 64-97, in Introduction to Food- and Airborne Fungi, 6th ed., 2000).

Increased retinal blood flow in patients with Graves' disease: influence of thyroid function and ophthalmopathy.

OBJECTIVE: Graves' ophthalmopathy (GO), resulting from the inflammation of retro-orbital tissue, is one of the major complications of Graves' disease (GD). We investigated the clinical usefulness of the measurement of retinal blood flow (RBF) in the evaluation of GO and its activity. MEASUREMENT: RBF was quantitated by pulsed Doppler mode at just below the branch of central retinal artery, from which the resistance index (RI) was calculated. PATIENTS: Forty-seven euthyroid GD patients and 70 gender- and age-matched normal controls were measured for RI to investigate the effect of GO on RBF. To investigate the effect of hyperthyroidism, 20 GD patients were measured for RI changes during antithyroid drug (ATD) therapy. Furthermore, 17 GD patients with clinically overt GO were measured for RI changes during treatment with glucocorticoid plus retro-orbital radiation. RESULTS: RI and exophthalmos showed a significant positive correlation in 47 treated euthyroid GD patients without clinically overt GO (r=0.307, P<0.05), but not in 70 age- and sex-matched normal subjects (r=0.185, P=0.161). Furthermore, RI, but not exophthalmos, significantly correlated with serum TSH receptor antibodies, an indicator for the disease activity of GO. ATD therapy significantly reduced RI in GD patients from 0.719+/-0.041 in the hyperthyroid state to 0.661+/-0.051 in the euthyroid state, but not to the levels observed in normal subjects having the similar exophthalmos (0.640+/-0.049). The fractional reduction of RI during ATD therapy significantly correlated with those of pulse pressure and ultrasonographic distensibility in carotid artery, but not with those of serum vascular injury markers. In 17 GD patients with clinically overt GO, all four patients having adipose tissue enlargement but not extraocular muscle hypertrophy (inactive GO) showed RI within the mean +/- 1 s.d. for treated GD patients without GO. In the other 13 GD patients having extraocular muscle hypertrophy (active GO), four and eight patients showed RI outside mean +/- 2 s.d. and mean +/- 1 s.d. respectively. Treatment with glucocorticoid plus radiation moved RI in 8 out of 10 patients toward the mean values of GD patients without GO, in spite of little improvement of exophthalmos. CONCLUSIONS: It was suggested that GD patients showed altered retinal hemodynamics, possibly resulting either from the cardiovascular effect of hyperthyroidism or from retro-orbital inflammation, particularly in extraocular muscle.

Persistent increase in bone turnover in Graves' patients with subclinical hyperthyroidism.

Hyperthyroid patients exhibit accelerated bone loss by increased bone turnover, and normalization of thyroid function is associated with a significant attenuation of increased bone turnover, followed by an increase in bone mineral density. However, of patients with Graves' disease (GD) maintained on antithyroid drug (ATD) treatment, some exhibit persistent suppression of TSH long after normalization of their serum free T3 (FT3) and free T4 (FT4) levels. The aim of this study was to examine whether bone metabolism is still enhanced in TSH-suppressed premenopausal GD patients with normal FT3 and FT4 levels after ATD therapy (n = 19) compared with that in TSH-normal premenopausal GD patients (n = 30), and to evaluate the relationship between serum TSH receptor antibody (TRAb), an indicator of disease activity of GD, and various biochemical markers of bone metabolism. No difference was found between the two groups in serum Ca, phosphorus, or intact PTH, or in urinary Ca excretion. Serum bone alkaline phosphatase (B-ALP), bone formation markers, and urinary excretions of pyridinoline (U-PYD) and deoxypyridinoline (U-DPD), which are bone resorption markers, were significantly higher in the TSH-suppression group than in the TSH-normal group (B-ALP, P < 0.05; U-PYD, P < 0.001; U-DPD, P < 0.001). For the group of all GD patients enrolled in this study, TSH, but neither FT3 nor FT4, exhibited a significant negative correlation with B-ALP (r = -0.300; P < 0.05), U-PYD (r = -0.389; P < 0.05), and U-DPD (r = -0.446; P < 0.05), whereas TRAb exhibited a highly positive and significant correlation with B-ALP (r = 0.566; P < 0.0001), U-PYD (r = 0.491; P < 0.001), and U-DPD (r = 0.549; P < 0.0001). Even in GD patients with normal TSH, serum TRAb was positively correlated with B-ALP (r = 0.638; P < 0.001), U-PYD (r = 0.638; P < 0.001), and U-DPD (r = 0.641; P < 0.001). In conclusion, it is important to achieve normal TSH levels during ATD therapy to normalize bone turnover. TRAb was not only a useful marker for GD activity, but was also a very sensitive marker for bone metabolism in GD patients during ATD treatment.

Antifungal activity of Hinokitiol-related compounds on wood-rotting fungi and their insecticidal activities.

Hinokitiol (beta-thujaplicin), beta-dolabrin and gamma-thujaplicin isolated from Thujopsis dolabrata SIEB. et ZUCC var hondai MAKINO showed antifungal activities against all of the wood-rotting fungi examined. The antifungal activity of three compounds on Daedalea dickinsii IFO-4979 was especially strong, their minimum inhibitory concentration (MIC) values being 0.2 microg/ml. Their antifungal activities on D. dickinsii IFO-4979 were as high as that of amphotericin B used as a positive control. Three compounds had strong insecticidal activities on Tyrophagus putrescentiae [50%-lethal concentration (LC50 : g/m2) 0.25 in hinokitiol, 0.02 in beta-dolabrin and gamma-thujaplicin. Their insecticidal activities were higher than that of N,N-diethyl-m-toluamide (DEET, LC50 : 1.46 g/m2) used as a positive control. Three compounds also showed strong insecticidal activity on Coptotermes formosanus [LC50 (g/m2) 0.07 in hinokitiol, 0.05 in beta-dolabrin and gamma-thujaplicin], although their insecticidal activities were much lower than that of commercial chloropyrifos (LC50 : 0.00016 g/m2).

Levels of serum and synovial fluid pyridinium crosslinks in patients with rheumatoid arthritis.

OBJECTIVE: To elucidate the major source of pyridinium crosslinks in rheumatoid arthritis (RA). METHODS: Serum samples were collected from 75 patients with RA and 41 healthy controls, and synovial fluid (SF) samples were collected from 20 patients with RA and 13 with osteoarthritis (OA). Paired samples of serum and SF were collected at the same time from 26 patients with RA. Levels of pyridinium crosslinks were determined by a recently developed high sensitivity assay method using high pressure liquid chromatography. RESULTS: The levels of serum pyridinoline (PYD) and serum deoxypyridinoline (DPD) were significantly higher in patients with RA than in healthy controls, and significantly correlated with laboratory variables indicating disease activity and severity. The levels of SF DPD, but not SF PYD, were significantly higher in patients with RA than in patients with OA. The levels of SF PYD and SF DPD both showed a significantly positive correlation with those of either SF interleukin 1beta or SF interleukin 6 in patients with RA. Finally, the levels of PYD, but not DPD, were higher in SF than in serum in all paired RA samples collected at the same time, with significant correlation between the members of each pair. CONCLUSION: These observations suggest than an increase of PYD in RA serum may originate mostly from affected joints and that an increase of DPD in RA serum may be influenced more by systemic bone resorption.

Antimicrobial activity and metalloprotease inhibition of hinokitiol-related compounds, the constituents of Thujopsis dolabrata S. and Z. hondai MAK.

Gamma-thujaplicin, beta-dolabrin and hinokitiol(beta-thujaplicin), hinokitiol-related compounds isolated from the wood of Thujopsis dolabrata S. and Z. hondai MAK have antimicrobial activity. In particular, strong antibacterial activity of hinokitiol and beta-dolabrin on Staphylococcus epidermidis IFO-12993 was found, with a minimum inhibitory concentration (MIC) of 0.2 microg/ml. This activity was higher than that of gentamicin, used as a positive control, and so the strong antibacterial activity of both compounds on this bacterium is of considerable interest. Of the three compounds, gamma-thujaplicin showed the strongest antifungal activity and its MIC was found to be around 1.5 microg/ml. The three compounds also inhibited metalloproteases. The inhibitory activity of hinokitiol on carboxypeptidase A was especially strong, its 50%-inhibitory concentration (IC50) being 2.76x10(-6) M. Considering that metalloproteases are involved in inflammation, the strong inhibitory activity of hinokitiol could be important. On the other hand, hinokitiol-acetate did not show any antimicrobial activity and metalloprotease inhibition, suggesting that at least part of the activity is due to metal chelation between the carbonyl group at C-1 and the hydroxyl group at C-2 in the tropolone skeleton.

Remission of insulin autoimmune syndrome in a patient with Grave's disease by treatment with methimazole.

The patient, a 24-year-old man, had suffered from hunger, sweating, tachycardia and palpitation for three years. He was diagnosed as having Graves' disease (GD) and treated with methimazole (MMI) for 3 months. He noted that palpitation and perspiration seemed to particularly occur when he was hungry, and thus he was examined to determine whether these symptoms were caused by hypoglycemia. As a markedly elevated immunoreactive insulin level and the presence of insulin antibody in serum were found, he was diagnosed as having insulin autoimmune syndrome (IAS). HLA typing revealed the patient to be positive for group Bw62/Cw4/DR4, which is reportedly a specific HLA type in MMI-treated euthyoroid GD patients with IAS. In spite of the continuation of MMI treatment, the % binding of IRI decreased and the hypoglycemic episode disappeared. In contrast to the previously reported MMI induced IAS in GD cases, MMI is unlikely to have exacerbated IAS in the present case, although his HLA combination is identical to that of the previous cases.

Poor glycemic control impairs the response of biochemical parameters of bone formation and resorption to exogenous 1,25-dihydroxyvitamin D3 in patients with type 2 diabetes.

Osteoblast deficit plays a principal role in the development of diabetic osteopenia. We have previously reported that high glucose conditions impair the function of osteoblast-like MG-63 cells. This study was performed to assess the sensitivity of osteoblasts to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in patients with type 2 diabetes without insulin deficiency or overt diabetic complications. During stimulation with 1,25(OH)2D3 at 2.0 micrograms/day for 6 consecutive days in 9 type 2 diabetic patients, serum levels of bone alkaline phosphatase (BALP), osteocalcin (OC) and the carboxyterminal propeptide of type 1 procollagen, and the urinary excretion of pyridinoline and deoxypyridinoline (DPYR), were monitored. As parameters of glycemic control, the mean level of fasting plasma glucose (mFPG) throughout the 1,25(OH)2D3 stimulation test and the level of HbA1C were used. 1,25(OH)2D3 increased serum 1,25(OH)2D significantly by day 2, which was followed by a significant reduction in the serum level of intact parathyroid hormone. The maximal increment of serum OC adjusted for that of 1,25(OH)2D was negatively correlated with both mFPG and HbA1C levels (p < 0.05). Furthermore, the magnitude of 1,25(OH)2D3-induced bone resorption, as reflected by the maximal increase in urinary DPYR excretion, was negatively correlated with the mFPG level (p < 0.05). Basal BALP tended to be negatively correlated with HbA1C, although not to a significant extent. In conclusion, our findings would indicate that poor glycemic control impairs the responses of osteoblasts and osteoclasts to 1,25(OH)2D3 in normo-insulinemic type 2 diabetic patients.

Insecticidal and antifungal activities of aminorhodanine derivatives.

Aminorhodanine (1) showed strong insecticidal activity against Culex pipiens pallens and Musca domestica, with respective LD50 values of 0.21 microgram/insect and 0.87 microgram/insect. Compound 1 had antifungal activity against Aspergillus niger ATCC-16404, Trichophyton mentagrophytes IFO-32412, Candida albicans ATCC-10231, Hansenula anomala OPS-308 and Penicillium expansum IFO-8800. In particular, 1 had potent antifungal activity against Aspergillus niger ATCC-16404, its minimal inhibitory concentration (MIC) being 6.25 micrograms/ml. Both activities of 1 were much higher than those of rhodanine (4), suggesting that the introduction of an amino group into N-3 of 4 plays an important role in the biological activity of rhodanine-related compounds. On the other hand, N-acetylaminorhodanine (2) and N-benzoylaminorhodanine (3) did not show either activity, suggesting that the free amino group at N-3 of 1 is closely related to the inhibitory activity of rhodanine derivatives.

[Secondary osteoporosis and its treatment--diabetes mellitus].

Japanese epidemiological study showed higher frequency of osteopenia/osteoporosis in diabetic patients as compared with sex- and age-matched control. The mechanism by which bone loss occurs in diabetic patients could be explained by a reduction of insulin/insulin-like growth factor-I action, sustained hyperglycemic state, a generation of advanced glycosylation end-products, and diabetic complication such as neuropathy, nephropathy and myopathy. Osteoblast deficit is hypothesized to play a major role in the occurrence of diabetic osteopenia. Besides the deficiency of insulin and insulin-like growth factor-I, we demonstrated that sustained hyperglycemia alone causes suppression of osteoblast proliferation and its response to parathyroid hormone and 1 alpha, 25-dihydroxyvitamin D, Hyporesponse of osteoblast to 1 alpha, 25-dihydroxyvitamin D, was also confirmed in diabetic patients as reflected by a reduction in an incremental response of serum osteocalcin during 1 alpha, 25-dihydroxyvitamin D administration. The regimens having stimulatory effect on bone turnover, such as intermittent PTH therapy and vitamin D, are recommended to treat diabetic osteopenia, besides improvement of diabetic control state.

Single-strand conformation polymorphism analysis of PCR-amplified ribosomal DNA internal transcribed spacers to differentiate species of Aspergillus section Flavi.

A novel genetic approach for classifying the species of Aspergillus Section Flavi is described here. This approach consists of PCR amplification of the 5.8S ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II) with universal primers and of analysis of the PCR product by the principle of single-strand conformation polymorphism (SSCP). The approximately 570- to 590-bp PCR products were denatured and subjected to electrophoresis on a polyacrylamide gel supplemented with 20% formamide. The SSCP patterns of these species became more distinct by the addition of formamide to the gel and by visualization with ethidium bromide staining. A little interspecific length polymorphism among amplified ribosomal DNAs was enhanced to be detected by PCR-SSCP analysis. This analysis was capable of classifying 67 of the 68 Aspergillus Section Flavi strains tested into the following four groups, regardless of origin: A. flavus/A. oryzae, A. parasiticus/A. sojae, A. tamarii, and A. nomius. The results of restriction fragment length polymorphism analysis with PCR products of the ITS regions were consistent with those of PCR-SSCP analysis, except for A. nomius, which was not clearly differentiated from A. parasiticus/A. sojae. Nonradiolabelled PCR-SSCP analysis is inexpensive and practical to perform without special apparatus or skill and should assist in fungal morphological identification.

Aspergillus ochraceus Production of 5,6-Dihydropenicillic Acid in Culture and Foods.

The production of 5,6-dihydropenicillic acid (DHPA) and penicillic acid (PA) by Aspergillus ochraceus was studied. DHPA has been revealed to be genotoxic in the Drosophila DNA-repair test in our previous report. The two compounds were produced by various type strains of A. ochraceus and related strains of Aspergillus in nutrient cultures. When A. ochraceus cells were inoculated into nutrient medium, PA was rapidly produced followed by a steady increase of DHPA and a decrease in the PA level. On the contrary, A. ochraceus produced almost exclusively PA in potato dextrose agar medium, which is low in nutrients. A. ochraceus cells produced DHP A when inoculated into grain, especially in rice flour at 20 to 24°C, and the DHPA level increased as the incubation was prolonged. These results suggested that PA was converted to DHPA under highly nutritive conditions. In vitro mutagenicity tests, the Ames test and the umu test, showed that DHPA was not mutagenic.

Mutagenicity of 5,6-Dihydropenicillic Acid (DHPA) in Drosophila melanogaster and Bacterial Conversion of Penicillic Acid to DHPA.

We studied the mutagenicity of 5,6-dihydropenicillic acid (DHPA) by means of the Drosophila wing-spot test. DHPA (10 mg/g of medium) significantly increased the number of small single and twin spots. Since twin spots were exclusively caused by mitotic crossing-over, the results demonstrated that DHPA can induce chromosome recombination in Drosophila somatic cells. Penicillic acid (PA) was converted to DHPA by an incubation with Agrobacterium radiobactor . The conversion occurred with other spoilage bacteria, such as Pseudomonas aeruginosa and P. cepacia . PA was completely converted to DHPA within a 24-h incubation with P. aeruginosa or P. cepacia in medium containing yeast extract. The results suggested that mutagenic DHPA is produced by environmental bacteria when foods are contaminated by fungi which produce PA.