Ultrasound efficacy of targeted-synthetic disease-modifying anti-rheumatic drug treatment in rheumatoid arthritis: a multicenter prospective cohort study in Japan.

OBJECTIVE: This study investigated the effectiveness of treatment with Janus kinase (JAK) inhibitors in rheumatoid arthritis (RA) assessed by ultrasonography (US) activity, and the influence of patient characteristics and previous treatments. METHOD: This prospective study assessed 60 treatment initiations among 53 Japanese patients diagnosed with RA who underwent treatment with JAK inhibitors during June 2013 to February 2020. Of the 53 patients, seven patients were enrolled in duplicate because they were treated with two different JAK inhibitors at different periods. For each case, the improvement rate on the power Doppler (PD) score was assessed at 6 month follow-up. Median improvement rate of PD score was used to classify cases as either US responders or non-responders, and patient characteristics were compared between the two groups. RESULTS: All indicators of clinical disease activity and US activity showed a significant improvement at 3 months compared with baseline. Although the JAK inhibitor-cycler group and the interleukin-6 (IL-6) inhibitor inadequate response (IR) group tended to show a later improvement for US activity, all indicators of clinical disease activity and US activity showed a significant improvement at 6 months compared with baseline for both groups. Multivariate analysis showed that concomitant methotrexate use and an IR to the previous biologic or targeted-synthetic disease-modifying anti-rheumatic drug (b/tsDMARD) treatment were independently and significantly associated with US responders. CONCLUSION: Use of a JAK inhibitor in combination with methotrexate and an absence of IR to any previous b/tsDMARDs demonstrated superior effectiveness for patients with RA.

IL-15 is a biomarker involved in the development of rapidly progressive interstitial lung disease complicated with polymyositis/dermatomyositis.

BACKGROUND: Polymyositis/dermatomyositis (PM/DM) is an autoimmune disease that is sometimes complicated with rapidly progressive interstitial lung disease (RPILD). However, serum and lung biomarkers that can predict RPILD development remain unclear. OBJECTIVES: To determine potential serum and lung biomarkers that can predict RPILD development in patients with PM/DM-ILD. METHODS: In total, 49 patients with PM/DM-ILD were enrolled. We measured the serum levels of 41 cytokines/chemokines, ferritin and anti-MDA5 antibody, compared them between the RPILD (n = 23) and non-RPILD (n = 26) groups, and ranked them by their importance through random forest analysis. To distinguish the two groups, we determined biomarker combinations by logistic regression analysis. We also measured the bronchoalveolar lavage fluid (BALF) levels of 41 cytokines/chemokines. Using immunohistochemistry, we examined IL-15 expression in lung tissues. The IL-15 production was also investigated using A549 and BEAS-2B cells. RESULTS: The RPILD group had significantly higher IL-15, IL-1RA, IL-6, CXCL10, VCAM-1, anti-MDA5 antibody and ferritin serum levels than the non-RPILD group, but it had a significantly low CCL22 level. Meanwhile, anti-MDA5 antibody, IL-15, CXCL8, CCL22, IL-1RA and ferritin were the best combination to distinguish the two groups. IL-15 and CCL22 were also predictive marker for RPILD development in anti-MDA5 antibody-positive patients. Additionally, the RPILD group had significantly high IL-15 levels in BALF. The lung tissues expressed IL-15, which increased after cytokine stimulation in the A549 cells. CONCLUSION: This study identified a combination of biomarkers predicting PM/DM-RPILD progression, and IL-15 is an important cytokine for predicting RPILD development and reflecting ILD severity.

Fluorescence optical imaging in patients with active rheumatoid arthritis: a comparison with ultrasound and an association with biomarkers.

Objectives: This study compared indocyanine green (ICG)-enhanced fluorescence optical imaging (FOI) and musculoskeletal ultrasound (MSUS), and explored the significance of the FOI findings based on the association between the FOI and MSUS findings and serum biomarkers in patients with rheumatoid arthritis (RA). The study also explored the association between the FOI findings and patients' joint destruction at the joint-area level.Method: We enrolled 50 consecutive patients with active RA from among the patients hospitalized from May 2014 to March 2016 at Nagasaki University Hospital, Japan. FOI images were acquired with the Xiralite® fluorescence imaging system and compared with the patients' clinical examination results and MSUS findings. On the same day, the patients' clinical disease activity and levels of serum biomarkers (including vascular endothelial growth factor) were obtained.Results: Although the FOI detected synovitis with high sensitivity, the frequency of positive findings and the diagnostic performance with MSUS as the reference standard for FOI differed considerably among the phases of FOI as well as among the affected joint regions. The FOI scores were positively correlated with clinical disease activity, MSUS scores, and serum biomarkers. The severity of FOI-proven synovitis was associated with the presence of MSUS-proven bone erosion.Conclusion: FOI is effective for detecting joint inflammation in RA patients, with high accuracy. The severity of the FOI score was closely associated with the joint destruction at the joint-area level. However, the significance of positive FOI findings differed depending on not only the phase of FOI but also the affected joint regions.

Significance of anti-Ro/SSA antibodies in the response and retention of abatacept in patients with rheumatoid arthritis: a multicentre cohort study.

Objective: To determine whether the positivity of baseline anti-Ro/Sjögren's syndrome antigen A (SSA) antibodies influences the response to abatacept, we compared therapeutic responses between anti-Ro/SSA antibody-negative and -positive patients with rheumatoid arthritis (RA) using a multicentre RA ultrasonography prospective cohort. Method: We reviewed Japanese patients with RA who started abatacept as the first biological disease-modifying anti-rheumatic drug between June 2013 and April 2018. We assessed 28-joint Disease Activity Score-erythrocyte sedimentation rate (DAS28-ESR) change between baseline and 6 or 12 months after treatment in RA patients treated with abatacept, and European League Against Rheumatism (EULAR) response at 6 and 12 months. The Global OMERACT-EULAR Synovitis Score (GLOESS) was calculated at baseline and at 6 and 12 months. Results: Overall, 51 patients were enrolled and divided into anti-Ro/SSA antibody-negative and -positive groups of 35 and 16, respectively. Median age at baseline was significantly higher in the anti-Ro/SSA antibody-negative group (p = 0.04). The retention rate and percentage of EULAR good responders at 12 months were significantly higher in the anti-Ro/SSA antibody-negative group (both p = 0.02). Anti-Ro/SSA antibody-negative patients exhibited larger decreases in both DAS28-ESR and DAS28-C-reactive protein at 12 months than anti-Ro/SSA antibody-positive patients (p = 0.02 and 0.04, respectively). GLOESS decreased significantly at 6 months in anti-Ro/SSA antibody-negative patients (p = 0.03). Multivariate analyses showed that anti-Ro/SSA antibody positivity was an independent factor associated with change in the DAS28-ESR at 6 months (p < 0.05). Conclusion: Anti-Ro/SSA antibody positivity predicts a poor response to abatacept and low retention rate.

Discrepancy between clinical and ultrasound remissions in rheumatoid arthritis: a multicentre ultrasound cohort study in Japan.

Objectives: Using multicentre ultrasound (US) cohort data among patients with rheumatoid arthritis (RA), we aimed to identify baseline factors that permit differentiation between two patient cohorts achieving US remission and clinical remission, and to determine the factors contributing to the discrepancy.Method: We reviewed 248 Japanese patients diagnosed with RA who underwent treatment with biological disease-modifying anti-rheumatic drugs at 13 centres. We performed US assessments of the synovia of 22 joints. We assessed the percentages of patients with clinical remission and US remission, defined as total power Doppler scores of 0 at 12 months.Results: The 87 patients who achieved US remission were divided into a group that achieved both clinical and US remission (n = 53) and a group that achieved US remission only (n = 34). Baseline factors that were significantly and independently associated with clinical remission at 12 months among patients who also achieved US remission included short disease duration, the presence of concomitant methotrexate use, and low patient global assessment score (p < 0.05, p < 0.05, and p < 0.005, respectively).Conclusions: RA patients with baseline high patient global assessment scores and long disease duration at baseline were unlikely to achieve clinical remission even after achieving US remission. Objective joint assessments using US provide additional information of potential importance for the management of RA.

Complement profile in microscopic polyangiitis and granulomatosis with polyangiitis: analysis using sera from a nationwide prospective cohort study.

OBJECTIVE: The complement cascade, especially the alternative pathway of complement, has been shown in basic research to be associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). We aimed to elucidate relationships between serum complement components and clinical characteristics in AAV. METHOD: In a nationwide prospective cohort study (RemIT-JAV-RPGN), we measured the serum levels of C1q, C2, C3, C3b/iC3b, C4, C4b, C5, C5a, C9, factor B, factor D, factor H, factor I, mannose-binding lectin, and properdin in 52 patients with microscopic polyangiitis (MPA) and 39 patients with granulomatosis with polyangiitis (GPA). RESULTS: The properdin level of MPA and GPA was significantly lower than that of healthy donors. The properdin level was negatively correlated with the Birmingham Vasculitis Activity Score (BVAS) (ρ = -0.2148, p = 0.0409). The factor D level at 6 months was significantly positively correlated with the Vasculitis Damage Index (VDI) at 6, 12, and 24 months (ρ = 0.4207, 0.4132, and 0.3115, respectively). Patients with a higher ratio of C5a to C5 had higher neutrophil percentage and serum immunoglobulin G levels, and significantly lower creatinine levels. Cluster analysis divided the MPA and GPA patients into three subgroups. A principal component (PC) analysis aggregated 15 types of complements into alternative pathway-related PC 1 and complement classical pathway and common pathway-related PC 2. CONCLUSIONS: The serum levels of properdin and factor D were correlated with the BVAS and the VDI in MPA and GPA, respectively. Our analyses suggested the pathological heterogeneity of MPA and GPA from the aspect of complement components.

Anti-citrullinated protein antibody titre as a predictor of abatacept treatment persistence in patients with rheumatoid arthritis: a prospective cohort study in Japan.

Objective: Successful rheumatoid arthritis (RA) outcome depends on treatment efficacy in the early stages of the disease and its sustainability. It is thus critical to identify factors predicting treatment persistence with biological agents, such as abatacept. We compared clinical profiles, including early changes in autoantibody titres at 3 months, between patients with RA demonstrating sustained persistence and those discontinuing abatacept treatment.Method: We prospectively enrolled 71 and 78 active RA patients treated with abatacept and tumour necrosis factor inhibitors (TNF-Is), respectively, who had previous disease-modifying anti-rheumatic drug) failure. Clinical characteristics were compared between non-continuation and continuation groups stratified according to abatacept or TNF-I persistence for at least 12 months from treatment initiation.Results: Significantly larger decreases in rheumatoid factor titre and anti-citrullinated protein autoantibody (ACPA) titre were observed in the continuation group of abatacept therapy at 3 months, and early reduction in ACPA titre remained a significant and independent predictor of sustained persistence with abatacept in multivariate analysis. In addition, we obtained the area under the receiver operator characteristics curve of 0.904 from a model including baseline ACPA titre and reduction of ACPA titre at 3 months. Sustained reduction of RA disease activity score at 12 months was significantly and independently associated with reduced ACPA titre at 3 months.Conclusions: Persistence with abatacept and sustained therapeutic response are associated with an early reduction in ACPA titre. Prediction of abatacept continuation and efficacy will facilitate the optimal design of therapy in the early stages of RA.

Complete renal response at 12 months after induction therapy is associated with renal relapse-free rate in lupus nephritis: a single-center, retrospective cohort study.

BACKGROUND: Lupus nephritis (LN) is a major risk factor for overall morbidity and mortality in systemic lupus erythematosus (SLE). METHODS: We retrospectively analyzed cases of proliferative and membranous LN patients who underwent a renal biopsy at our hospital in 1993-2016. We analyzed the association between complete renal response (CR) rates at 12 months after induction therapy and predictive factors for CR and their association with renal flares. RESULTS: Of the 95 cases analyzed, we were able to track the therapeutic responses of 81 patients at 12 months after their induction therapy. The median follow-up duration after renal biopsy was 51 months (interquartile range: 16.5-154.5 months). The Cox proportional hazards model showed that, compared to not attaining CR at 12 months, the attainment of CR at 12 months was correlated with being free from renal flares. The multivariate logistic analysis revealed that the predictive factors for CR at 12 months were the anti-La/SSB antibodies (U/ml) (odds ratio (OR) 1.22, 95% confidence interval (CI) 1.01-1.63, p = 0.0220), blood urea nitrogen (BUN) (OR 0.68, 95% CI 0.44-0.90, p = 0.00048) and serum ß2 microglobulin (MG) (OR 0.26, 95% CI 0.06-0.74, p = 0.00098) levels. CONCLUSIONS: Among LN patients, being free from renal flares was associated with attaining CR at 12 months after induction therapy. Anti-La/SSB antibodies were a positive predictive factor, and BUN and serum ß2MG levels were negative predictive factors of CR at 12 months.

Factors predictive of long-term mortality in lupus nephritis: a multicenter retrospective study of a Japanese cohort.

BACKGROUND: Lupus nephritis (LN) is a major determinant of mortality in systemic lupus erythematosus (SLE). Here we evaluated the association between complete renal response (CR) and mortality in LN. METHODS: We retrospectively analyzed the cases of 172 of 201 patients with LN for whom data on the therapeutic response at 6 and 12 months after induction therapy were available. The patients underwent a renal biopsy at Nagasaki University Hospital and community hospitals in Nagasaki between the years 1990 and 2016. We determined the CR rates at 6 and 12 months after induction therapy initiation and evaluated the predictive factors for CR and their relationship with mortality. We performed univariate and multivariable competing risks regression analyses to determine the factors predictive of CR. The patients' survival data were analyzed by the Kaplan-Meier method with a log-rank test. RESULTS: The median follow-up duration after renal biopsy was 120 months (interquartile range: 60.3-191.8 months). The 5-, 10-, 15- and 20-year survival rates of our cohort were 99.3, 94.6, 92.0 and 85.4%, respectively. During follow-up, nine patients (5.2%) died from cardiovascular events, infection, malignancy and other causes. The multivariate analysis revealed that the following factors were predictive of CR. At 6 months: male gender (odds ratio (OR) 0.23, 95% confidence interval (CI) 0.08-0.65, p = 0.0028), proteinuria (g/gCr) (OR 0.83, 95% CI 0.71-0.97, p = 0.0098) and index of activity (0-24) (OR 0.84, 95% CI 0.71-0.99, p = 0.0382). At 12 months: male gender (OR 0.25, 95% CI 0.09-0.67, p = 0.0043) and index of activity (0-24) (OR 0.82, 95% CI 0.69-0.98, p = 0.0236). The Kaplan-Meier analysis showed that compared to not achieving CR at 12 months, achieving CR at 12 months was significantly correlated with the survival rate (OR 0.18, 95% CI 0.04-0.92, p = 0.0339). CONCLUSIONS: Our results suggest that the survival rate of patients with LN is associated with the achievement of CR at 12 months after induction therapy, and that male gender and a higher index of activity (0-24) are the common predictive factors for failure to achieve CR at 6 and 12 months.

The efficacy of adjunct tacrolimus treatment in pregnancy outcomes in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) involves multiple organ systems and primarily affects women during their reproductive years. Pregnancy in a woman with SLE may lead to higher rates of disease flares. Little is known regarding which medications are safe to maintain remission and/or treat flares throughout such pregnancies. Here we retrospectively analyzed the efficacy of tacrolimus (TAC) in the pregnancy outcomes of SLE patients. We studied the 54 deliveries of 40 SLE patients over an eight-year period from 2008 to 2016. We used analyses of covariance with adjustments for the propensity score and inverse probability of treatment weights to compare the patient backgrounds between the TAC users and non-TAC users. TAC was administered to the patient in 15 of the 54 (27.8%) pregnancies, and these patients had a significantly higher dose of prednisolone, hypocomplementemia, lower estimated glomerular filtration rate, past history of lupus nephritis, and complication with antiphospholipid syndrome. In the adjusted background of the TAC deliveries, the risks of decreased fetal body weight, low birth weight infant, non-reassuring fetal status (NRFS), and preterm birth were not increased compared to the non-TAC deliveries. Thrombocytopenia and hypertension during the pregnancy were extracted as independent predictive risk factors for decreased fetal body weight and NRFS, respectively. We had anticipated that the maternal and fetal outcomes in the TAC-use deliveries would be poor before the analysis; however, the TAC-use group showed no significant difference in risks contributing to outcomes compared to the non-TAC group, suggesting that adjunct TAC treatment corrected various risk factors during the lupus pregnancies.

Sweet potato resistance to Euscepes postfasciatus (Coleoptera: Curculionidae): larval performance adversely effected by adult's preference to tuber for food and oviposition.

The preferences of the West Indian sweet potato weevil, Euscepes postfasciatus (Fairmaire), to tubers of sweet potato, Ipomoea batatas (L.), for food and for oviposition were evaluated, and correlated to sweet potato's resistance to immatures. Adults (parent) were released in a plastic box containing tubers of sweet potato cultivars and maintained for 5 d, after which the adults on each tuber were counted. All adults were then removed and each tuber was maintained separately. New adults that emerged from the tubers were counted. Cultivars were grouped by cluster analyses using the number of parent adults on the tubers and the number of new adults emerging from the tubers, adjusted for the weight of each tuber. Cultivars were divided into five groups: average level of preference, preferred, preferred for oviposition but not for food, preferred for food but not for oviposition, and not preferred. New adults from the first two groups took less time to eclose than those from the other groups, and their body size was smaller. In a second experiment, one to five cultivars were selected from each group and inoculated each tuber with 10 weevil eggs on each cultivar. Although the proportion of eclosed adults was not significantly different between cultivars, the time to eclosion was shorter and body size was smaller on preferred cultivars. The selection of tubers by parent adults was not linearly related with larval development, and did not reduce the survival of the immatures.

Q-band high-performance notch filters at 56 and 77 GHz notches for versatile fusion plasma diagnostics.

A six-pole Q-band waveguide filter with a notch frequency above the Q-band has been developed for plasma diagnostics. The previous paper [Nishiura et al., J. Instrum. 10, C12014 (2015)] reported that the notch frequency exists within the standard band. In this study, the newly required notch filter extends the function, which prevents a thorny wave from being mixed into an instrument beyond the standard bandwidth of the waveguide. The mode control technique for cavities realizes a deep and sharp filter shape for Q-band notch filters with 56 and 77 GHz notches, respectively. The former filter has an attenuation more than 50 dB at 56.05 GHz and a bandwidth of 1.1 GHz at -3 dB. The latter filter has an attenuation more than 55 dB at 76.95 GHz and a bandwidth of 1.6 GHz at -3 dB. The electron cyclotron emission imaging and the electron cyclotron emission (ECE) diagnostics for the Q-band implemented a pair of the fabricated filters and demonstrated the ECE measurement successfully in the intense stray radiation from a 56 GHz gyrotron.

Association of distinct clinical subsets with myositis-specific autoantibodies towards anti-155/140-kDa polypeptides, anti-140-kDa polypeptides, and anti-aminoacyl tRNA synthetases in Japanese patients with dermatomyositis: a single-centre, cross-sectional study.

OBJECTIVE: To determine the association of distinct clinical subsets with myositis-specific autoantibodies (MSAs) towards anti-155/140-kDa polypeptides [anti-155/140 antibodies (Abs)], anti-140-kDa polypeptides (anti-140 Abs), and anti-aminoacyl tRNA synthetases (ARS Abs) in Japanese patients with dermatomyositis (DM). METHODS: We compared the clinical features and short-term prognoses of 30 DM patients whose serological status included these MSAs. The MSAs were determined by immunoprecipitation. RESULTS: Anti-155/140 Abs (n = 5), anti-140 Abs (n = 8), and anti-ARS Abs (n = 7) did not overlap each other. All of the anti-155/140 Ab-positive patients (n = 5) were complicated by malignancies, as were all of the anti-140 Ab-positive patients (n = 8), who showed rapidly progressive interstitial lung disease (ILD). The survival rate at 6 months from the diagnosis of DM was significantly lower in the anti-140 Ab-positive patients than in the other patients. CONCLUSION: This is the first study to report, in a single cohort of DM patients, that distinct clinical subsets are distributed in an anti-155/140 Ab-positive group, an anti-140 Ab-positive group, or an anti-ARS Ab-positive group. Our data also confirm previous evidence that anti-155/140 Abs are involved in malignancies and that anti-140 Abs are involved in rapidly progressive ILD.

First Report of Root-Knot Nematode Meloidogyne enterolobii on Guava in Vietnam.

In southern Vietnam, citrus is interplanted with guava (Psidium guajava L.) for the management of citrus greening disease. Guava seedlings exhibited decline symptoms including leaf browning, growth inhibition, leaf drop, and death. These symptomatic seedlings were accompanied by severely galled roots, possibly attributable to root-knot nematodes. We collected second-stage juveniles from the soil and picked adult females from roots to perform morphological observations. The perineal patterns of most specimens were similar to those in the original description of Meloidogyne enterolobii (3). However, some were similar to that of M. incognita. Such variability among perineal patterns precluded unequivocal identification. Therefore, DNA was extracted from these nematodes and their sequences were compared with those in the DNA sequence database. For the comparison, we employed the primer sets of Powers and Harris (1) to amplify a region between cytochrome oxidase II and the 16S ribosomal DNA gene of mitochondria by PCR. An approximate 700-bp fragment was obtained and analyzed, revealing more than 99.6% homology to M. mayaguensis, a synonym of M. enterolobii (2), when aligned with sequence data of isolates from France (GenBank Accession No. AJ421396), the United States (GenBank Accession No. AY446978), and China (GenBank Accession No. AY831967). These results indicate that the nematode species responsible for guava damage in southern Vietnam is M. enterolobii. To our knowledge, this is the first report of M. enterolobii in Vietnam. References: (1) T. O. Powers and T. S. Harris. J. Nematol. 25:1, 1993. (2) J. Xu et al. Eur. J. Plant Pathol. 110:309, 2004. (3) B. Yang and J. D. Eisenback. J. Nematol. 15:381, 1983.

Role of alpha 2-adrenergic receptor in platelet activation during insulin-induced hypoglycemia in normal subjects.

The effects of alpha 2-adrenergic-receptor blocker mianserin on the responses of blood glucose, plasma beta-thromboglobulin (beta-TG), and various counterregulatory hormones to insulin-induced hypoglycemia were studied in nine healthy male subjects. The alpha 2-adrenoceptor-blocking action of mianserin was confirmed by its inhibitory effect on platelet activation in vitro. Mianserin was given orally 90 min before insulin injection; the same study without mianserin was performed on another day as the control study. The time courses of blood glucose and serum C-peptide (0, 20, 45, and 180 min after the insulin injection) were identical in both studies, indicating that mianserin has no effect on these parameters. However, a significant increase of beta-TG at 45 min after insulin injection was completely suppressed by the administration of mianserin (mean +/- SE, 68.5 +/- 6.0 vs. 28.8 +/- 7.6 ng/ml, n = 6, P less than .05). No significant differences were obtained between the two studies in the responses of plasma or serum catecholamines, cortisol, glucagon, growth hormone, thromboxane B2, and 6-ketoprostaglandin F1 alpha. These results suggest that epinephrine is responsible for some, if not all, of the beta-TG release from the platelets during insulin-induced hypoglycemia.

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity.

BACKGROUND: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Generally, it is proposed that GTs are two-domain proteins with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aimed at finding out the region responsible for determination of substrate specificities of these two urdamycin GTs. RESULTS: A series of 10 chimeric GT genes were constructed consisting of differently sized and positioned portions of urdGT1b and urdGT1c. Gene expression experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT1c-like activity. A 31 amino acid region (aa 52-82) located close to the N-terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chimeric gene product of the 10 was not functional. Targeted stepwise alterations of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues conserved among streptomycete GTs. Alterations G226S and G226R restored a weak activity, whereas G226SR showed an activity comparable with other functional chimeras. CONCLUSIONS: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it.

The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster.

BACKGROUND: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. RESULTS: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. CONCLUSIONS: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.

The granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22: sequence analysis and expression in a heterologous host.

BACKGROUND: The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. RESULTS: The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber T-22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber. Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. CONCLUSIONS: On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.

Increased adherence of T cells to human endothelial cells in patients with human T-cell lymphotropic virus type I-associated myelopathy.

We investigated the adherence of T cells to human umbilical vein endothelial cells in seven patients with human T-lymphotropic virus type I (HTLV-I)-associated myelopathy. The adherence of T cells to endothelial cells increased significantly in all the patients with HTLV-I-associated myelopathy when compared with the adherence in the seronegative controls (1.3- to 2.8-fold) and compared with the adherence in the anti-HTLV-I-seropositive non-HTLV-I-associated myelopathy carriers (1.4- to 2.8-fold). Prior treatment of the endothelial cell monolayer with recombinant interferon gamma (50 IU/mL) enhanced the T cell-endothelial cell adhesion in both the controls and patients with HTLV-I-associated myelopathy. However, values after prior treatment in the patients with HTLV-I-associated myelopathy were significantly higher than those in seronegative controls and carriers. The results suggest that the significantly increased T cell-endothelial cell adherence may be related to the initial stages of lymphocyte migration from the blood to the central nervous system in patients with HTLV-I-associated myelopathy.