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Purpose: To establish correlations between intraocular pressure (IOP) measurements obtained with the ocular response analyzer (ORA) and the Goldmann applanation tonometer (GAT). The effects of central corneal thickness on the measures obtained were also examined., Methods: This was a cross-sectional study. IOP was determined in 48 eyes of 48 patients with glaucoma In all patients, central corneal thickness (CCT) was measured by ultrasound pachymetry., Results: ORA readings were consistently higher than GAT measurements (Goldmann-correlated IOP - IOP GAT mean difference, 7.2 +/- 3.5 mm Hg; corneal-compensated IOP - IOP GAT mean difference, 8.3 +/- 4.0 mm Hg) However, differences were not constant and increased with increasing IOP GAT readings, both with respect to Goldmann-correlated IOP (slope = 0.623, P < 0.0001) and corneal-compensated IOP (slope = 0.538, P < 0.0001). Both pressure measurements provided by the ORA showed significant correlation with CCT (CCT versus Goldmann-correlated IOP: r = 0.460, P = 0.001; CCT versus corneal-compensated IOP: r = 0.442, P = 0.001). No significant effects of corneal curvature or refraction on any of the pressures were observed., Conclusions: The ORA significantly overestimates IOP compared with the GAT. Differences between both sets of measures increase as the GAT-determined IOP increases. ORA readings seem to be affected by central corneal thickness.

Background: Low back pain and its associated incapacitating effects constitute an important healthcare and socioeconomic problem, as well as being one of the main causes of disability among adults of working age. The prevalence of non-specific low back pain is very high among the general population, and 60-70% of adults are believed to have suffered this problem at some time. Nevertheless, few randomised clinical trials have been made of the efficacy and efficiency of acupuncture with respect to acute low back pain. The present study is intended to assess the efficacy of acupuncture for acute low back pain in terms of the improvement reported on the Roland Morris Questionnaire (RMQ) on low back pain incapacity, to estimate the specific and non-specific effects produced by the technique, and to carry out a cost-effectiveness analysis., Methods/design: Randomised four-branch controlled multicentre prospective study made to compare semi-standardised real acupuncture, sham acupuncture (acupuncture at non-specific points), placebo acupuncture and conventional treatment. The patients are blinded to the real, sham and placebo acupuncture treatments. Patients in the sample present symptoms of non specific acute low back pain, with a case history of 2 weeks or less, and will be selected from working-age patients, whether in paid employment or not, referred by General Practitioners from Primary Healthcare Clinics to the four clinics participating in this study. In order to assess the primary and secondary result measures, the patients will be requested to fill in a questionnaire before the randomisation and again at 3, 12 and 48 weeks after starting the treatment. The primary result measure will be the clinical relevant improvement (CRI) at 3 weeks after randomisation. We define CRI as a reduction of 35% or more in the RMQ results., Discussion: This study is intended to obtain further evidence on the effectiveness of acupuncture on acute low back pain and to isolate the specific and non-specific effects of the treatment.

The Young's double slit experiment is recreated using intense and short laser pulses. Our experiment evidences the role of the non-linear Kerr effect in the formation of interference patterns. In particular, our results evidence a mixed mechanism in which the zeroth diffraction order of each slit are mainly affected by self-focusing and self-phase modulation, while the higher orders propagate linearly. Despite of the complexity of the general problem of non-linear propagation, we demonstrate that this experiment retains its simplicity and allows for a geometrical interpretation in terms of simple optical paths. In consequence, our results may provide key ideas on experiments on the formation of interference patterns with intense laser fields in Kerr media.

Background: Although the painful shoulder is one of the most common dysfunctions of the locomotor apparatus, and is frequently treated both at primary healthcare centres and by specialists, little evidence has been reported to support or refute the effectiveness of the treatments most commonly applied. According to the bibliography reviewed, physiotherapy, which is the most common action taken to alleviate this problem, has not yet been proven to be effective, because of the small size of sample groups and the lack of methodological rigor in the papers published on the subject. No reviews have been made to assess the effectiveness of acupuncture in treating this complaint, but in recent years controlled randomised studies have been made and these demonstrate an increasing use of acupuncture to treat pathologies of the soft tissues of the shoulder. In this study, we seek to evaluate the effectiveness of physiotherapy applied jointly with acupuncture, compared with physiotherapy applied with a TENS-placebo, in the treatment of painful shoulder caused by subacromial syndrome (rotator cuff tendinitis and subacromial bursitis)., Methods/design: Randomised controlled multicentre study with blind evaluation by an independent observer and blind, independent analysis. A study will be made of 465 patients referred to the rehabilitation services at participating healthcare centres, belonging to the regional public health systems of Andalusia and Murcia, these patients presenting symptoms of painful shoulder and a diagnosis of subacromial syndrome (rotator cuff tendinitis and subacromial bursitis). The patients will be randomised into two groups: 1) experimental (acupuncture + physiotherapy); 2) control (TENS-placebo + physiotherapy); the administration of rescue medication will also be allowed. The treatment period will have a duration of three weeks. The main result variable will be the change produced on Constant's Shoulder Function Assessment (SFA) Scale; as secondary variables, we will record the changes in diurnal pain intensity on a visual analogue scale (VAS), nocturnal pain intensity on the VAS, doses of non-steroid anti-inflammatory drugs (NSAIDs) taken during the study period, credibility scale for the treatment, degree of improvement perceived by the patient and degree of improvement perceived by the evaluator. A follow up examination will be made at 3, 6 and 12 months after the study period has ended. Two types of population will be considered for analysis: per protocol and per intention to treat., Discussion: The discussion will take into account the limitations of the study, together with considerations such as the choice of a simple, safe method to treat this shoulder complaint, the choice of the control group, and the blinding of the patients, evaluators and those responsible for carrying out the final analysis.

The angucycline antibiotic family of the landomycins displays potent antitumor activity. To elucidate early post polyketide synthase (PKS) tailoring steps of the landomycin E biosynthetic pathway in Streptomyces globisporus 1912, the mutant S. globisporus M12 was prepared through gene replacement experiment of lndM2. It encodes an enzyme with putative oxygenase and reductase domains, according to sequencing of the gene and its counterpart lanM2 from S. cyanogenus S136 landomycin A biosynthetic gene cluster. The isolation of the novel shunt products 11-hydroxytetrangomycin and 4-hydroxytetrangomycin along with the well-known angucyclines tetrangomycin and tetrangulol from the culture of S. globisporus M12 provides evidence for the involvement of lndM2 in the early biosynthetic pathway of the landomycins, in particular in the formation of the alicyclic 6-hydroxy function of the landomycin aglycon. We therefore propose LndM2 to be responsible for both hydroxylation of the 6-position and its subsequent reduction. These reactions are necessary before the glycosylation reactions can occur. The results are in agreement with the originally published structure of landomycin but do not support the recently suggested revised structure.

Objective: Esophago-gastro-duodenoscopy (EGD) is frequently performed in patients with heartburn. However, the benefit of an EGD is unclear. The objective is to determine the clinical impact of performing an EGD in the management of GERD., Methods: Patients with alarm symptoms or persistent heartburn from primary care and gastroenterology clinics were invited to participate. Patients with known reflux complications or an EGD procedure within 3 yr were excluded. Endpoints for EGD-improving management were prospectively defined: change in medical therapy, dilating esophageal stricture, identifying Barrett's esophagus (BE), finding severe esophagitis (Hetzel-Dent grade 3 or 4), or finding cancer., Results: Two hundred and six patients (59 M/147 F; median age, 48 yr) were enrolled, in which, 124 had alarm symptoms and 82 had persistent heartburn. EGD improved the overall management in 40% of patients with alarm symptoms and 29% with persistent heartburn (p= NS). Medical therapy was altered in only 5% of patients, and no cancer was identified. EGD was more likely to improve management in males with alarm symptoms., Conclusions: It was uncommon for an EGD to alter antireflux therapy. The management was improved in approximately one-third of the patients by dilating esophageal stricture, finding BE to initiate surveillance, or finding severe esophagitis. EGD should be performed when alarm symptoms are present, especially in males.

Cosmomycin D (CosD) is the major constituent fraction isolated from a culture of Streptomyces olindensis ICB20. The ability of this compound to intercalate with double-stranded DNA was studied by gel mobility shift assays and electrospray ionization mass spectrometry (ESI-MS). ESI-MS experiments showed that the complex of CosD with 16-mer double-stranded DNA was at least as stable as a complex of daunorubicin with the same DNA sequence. This is the first study showing DNA binding properties of an anthracycline containing a beta-rhodomycinone aglycone chromophore O-linked to two trisaccharide chains.

Purpose: To compare the efficacy and safety of the concomitant administration of travoprost 0.004% once daily and brinzolamide 0.1% twice daily with those of a fixed combination of latanoprost 0.005%/timolol 0.5% once daily., Research, Design and Methods: Forty-four patients with primary open-angle glaucoma or ocular hypertension with elevated IOP insufficiently responsive to monotherapy were randomly assigned to one of the two treatment groups: concomitant administration of travoprost 0.004% once daily and brinzolamide 0.1% twice daily (TB group: 22 patients) or latanoprost 0.005% plus timolol 0.5% once daily (LT group: 22 patients). Visits were undertaken at screening (current ocular hypotensive therapy was discontinued), baseline (randomization), and after 2 weeks, 1 month, 2 months and 3 months of therapy., Main Outcome Measures: IOP was determined at 9 a.m., 12 p.m. and 4 p.m. at each study visit, and diurnal IOP was calculated as the mean of these recordings. Adverse events were recorded at each visit., Results: IOP at the baseline visit was similar in both groups. Overall mean IOP was significantly lower in the TB as compared to the LT group after 1 month, 2 month and 3 month follow-up; only 9 a.m. measurements were significantly different, reaching a maximum difference (16.9 +/- 0.9 mmHg vs 18.4 +/- 1.8 mmHg, p < 0.001) at the 3 month check. The percentage of responders (IOP decrease > or = 30%) was higher in the TB group. Both treatments were well tolerated and there were no cases of withdrawal from treatment., Conclusions: Travoprost 0.004% and brinzolamide 0.1% concomitant therapy showed a greater efficacy than the fixed latanoprost 0.005%/timolol 0.5% combination in terms of absolute IOP decreases. Travoprost/brinzolamide therapy also offered the advantages of a greater percentage of responders.

Purpose: To report bioultrasonic findings in Listeria monocytogenes-induced endophthalmitis (LMIE) that have not been described previously., Design: Interventional case report., Methods: To rule out intraocular tumor, ultrasound biomicroscopy was performed in a patient referred for a 2-day history of uveitis with elevated intraocular pressure, dark hypopyon, and pigment dispersion in the anterior chamber., Results: Ultrasound biomicroscopy examination showed increased iris thickness with rarefaction of tissue and irregular echogenicity as well as iris pigment epithelial detachment. A small choroidal detachment was also detected. The anterior chamber and vitreous sample confirmed the LMIE diagnosis., Conclusions: The detection of both pigment epithelial detachment and changes in the iris tissue could explain why black hypopyon frequently develops in LMIE with significant pigment dispersion in some cases.

Background/aims: Chronic hepatitis C patients infected by genotype 1 are the least responsive to combination therapy and therefore monitoring response is important in identifying non-responders quickly, permitting therapy discontinuation and avoiding side effects and costs. We examined the usefulness of measuring total HCV Core Ag in early treatment with peginterferon alpha-2b and ribavirin in genotype 1 patients in the prediction of response and compared the results with those from HCV RNA quantification., Methods: Two hundred and sixty-eight serum samples from 46 genotype 1 patients receiving combination therapy were examined for HCV Core Ag and quantitative HCV RNA., Results: At baseline, mean HCV RNA and HCV Core Ag concentrations were significantly lower in sustained virologic responders than in non-responders. The negative predictive value of HCV Core Ag testing in predicting non-response at week 12 is 100%, and for a 2 log drop in HCV RNA, using two quantitative tests, it is 88%., Conclusions: HCV Core Ag determination allows the identification of non-responders with only one test at week 12 and permits stopping therapy in these patients. HCV Core Antigen testing is cheaper and easier to perform than HCV RNA quantification.

Mild cognitive impairment (MCI) is increasingly being conceptualized in the literature as a cognitive disturbance representing a transitional phase between normal aging and dementia. The operational definitions of MCI provide an opportunity for neuropsychologists to detect subtle deficit and monitor cognitive status sequentially in order to determine rate and degree of progression. More importantly, clinical and neuropsychological studies are needed that can better characterize which MCI patients are at greatest risk for conversion to dementia. Preliminary data has also designated MCI as a potential indicator for initiation of pharmacotherapy, with the objective of decelerating rate of progression to dementia. Current criteria and clinical issues related to MCI are discussed, with the objective of better familiarizing clinicians with this syndrome and fostering ongoing investigations.

Objective: The role of prolactin (PRL) in the pathogenesis of systemic lupus erythematosus (SLE) is controversial. The effect of conventional treatment (steroids, antimalarials, immunosuppressor drugs) on PRL concentrations is unclear. We investigated correlation of PRL levels with lupus activity in patients at entry and after 6 months of conventional treatment., Methods: We studied 43 female patients with active SLE, who were divided in 2 groups; Group 1: 16 patients with minor organ involvement (cutaneous and articular involvement), and Group 2: 27 patients with major organ involvement (glomerulonephritis). Controls were 36 healthy individuals. PRL levels were determined by an immunoradiometric assay at entry and after 6 months of treatment. PRL levels were correlated with SLE Disease Activity Index (SLEDAI) score., Results: Mild hyperprolactinemia (HPRL, 20-40 ng/ml) was found in 30/43 (69.7%) SLE patients. After 6 months of treatment a reduction in PRL levels was found in both groups: Group 1: 24.3 +/- 10.8 to 16.96 +/- 10.87 ng/ml (p < 0.001); and Group 2: 23.6 +/- 5.7 to 12.07 +/- 11.13 ng/ml (p < 0.001). The SLEDAI score also decreased after treatment: Group 1: 16.5 +/- 5.9 to 2.1 +/- 1.3 (p < 0.001); Group 2: 16.8 +/- 5.4 to 1.6 +/- 1.4 (p < 0.001). At entry and after treatment, a significant correlation between PRL levels and SLEDAI score was found in all patients (r = 0.4946, p = 0.0007, and r = 0.9086, p = 0.0001, respectively)., Conclusion: HPRL was associated with SLE disease activity. Conventional immunosuppressive therapy decreased PRL levels in direct correlation with decreased SLE activity. This finding emphasizes that PRL may play a role in the pathogenesis and clinical expression of SLE.

Alcoholic liver disease (ALD) remains a major cause of morbidity and mortality worldwide. For example, the Veterans Administration Cooperative Studies reported that patients with cirrhosis and superimposed alcoholic hepatitis had a 4-year mortality of >60%. Interactions between acetaldehyde, reactive oxygen and nitrogen species, inflammatory mediators and genetic factors appear to play prominent roles in the development of ALD. The cornerstone of therapy for ALD is lifestyle modification, including drinking and smoking cessation and losing weight, if appropriate. Nutrition intervention has been shown to play a positive role on both an inpatient and outpatient basis. Corticosteroids are effective in selected patients with alcoholic hepatitis and pentoxifylline appears to be a promising anti-inflammatory therapy. Some complementary and alternative medicine agents, such as milk thistle and S-adenosylmethionine, may be effective in alcoholic cirrhosis. Treatment of the complications of ALD can improve quality of life and, in some cases, decrease short-term mortality.

The regulatory genes lanI and lndI have been cloned from the landomycin A (LaA) producer Streptomyces cyanogenus S136 and from the landomycin E (LaE) producer Streptomyces globisporus 1912, respectively and both have been sequenced. A culture of S. globisporus I2-1 carrying a disrupted lndI gene did not produce LaE and other related intermediates. Complementation of S. globisporus I2-1 with either the lndI or lanI gene reconstituted LaE production indicating that LanI and LndI are involved in activation of structural genes in the respective clusters. Structural features of these regulatory genes are discussed.

Background: Our in vivo model of tolerance, sublethal hemorrhage (SLH), alters cytokine production, nuclear factor-kappaB mobilization, mitogen-activated protein (MAP) kinase activity, and makes rats tolerant to shock. Heat shock protein (HSP) protects animals from stress. This study investigated if SLH induces in vivo HSP72 expression and whether in vitro HSP72 induction by sodium arsenite (NaArs) alters intracellular signal transduction and cytokine production similar to SLH., Methods: Sprague-Dawley rats were made tolerant by SLH (MAP = 30 mmHg for 15 min, shed blood returned) and given lipopolysaccharide (LPS; 40 mg/kg i.p.) 24 h later. Lung was harvested 1, 12, and 24 h after SLH (n = 4) and 1 h after LPS (n = 8). Other rats underwent bronchoalveolar lavage 24 h after SLH, and macrophages (mphi) were treated with LPS (10 microg/ml). The NR8383 alveolar mphi cell line was treated with 50 microM NaArsx 12 h and LPS. Reverse transcription polymerase chain reaction, Western blots, and enzyme-linked immunosorbent assay were performed for gene, MAPK, and protein expression (tumor necrosis factor [TNF], HSP, p38)., Results: SLH induced significantly more lung HSP72 mRNA and protein. SLH mphi had more HSP72 protein before and after LPS compared with shams. NaArs induced HSP72 mRNA and protein in NR8383 mphi, and these cells made less TNF compared with controls. NaArs significantly increased p38 activation vs control. SB203580 inhibition of p38 activity did not affect HSP72 expression, or reverse NaArs inhibition of LPS induced TNF production., Conclusion: SLH induces HSP72 in vivo. In vitro HSP72 induction is associated with increased p38 phosphorylation. Like SLH, mphi with induced HSP72 expression, have an attenuated TNF response. HSP72 acts independently from p38 in inducing tolerance.

Background: We have shown that SLH induces tolerance to endotoxin in vivo and in vitro, and is associated with alterations in MAP kinase (p38, p44/42, and SAPK/JNK) activation and TNF production. This study investigates the effect of sublethal hemorrhage (SLH) on cecal ligation and puncture (CLP) polymicrobial sepsis and examined the effect of the bioflavinoid, curcumin, a MAP kinase inhibitor, on this relationship., Materials and Methods: Sprague-Dawley rats underwent SLH (hemorrhage and MAP = 30 mm Hg for 15 min, with shed blood returned) or sham operation. After 24 h, rats had CLP (cecal base ligation with double puncture). Survival was determined +/- curcumin pretreatment (n = 10/group). Lung tissue, serum, and bronchoalveolar lavage (BAL) fluid were obtained 30 min after SLH and 4 and 12 h after CLP (n = 8/group). Lung tissue was analyzed for p38, p44/42 SAPK/JNK, and HSP-70 phosphorylation (Western). Lung myeloperoxidase (MPO) activity was measured as an index of neutrophil infiltration. TNF ELISA was performed on serum and BAL sample., Results: SLH significantly improved survival after CLP (21.5 vs. 7.5 h vs. sham, p = 0.008), and curcumin reversed this benefit (7.3 h, p = 0.0007 vs. SLH + CLP). MAP kinase activity was significantly greater in SLH rats 4 h post-CLP (p38: 720 vs. 331, p = 0.03, p44/42: 2759 vs. 1295, p = 0.006, SAPK: 413 vs. 254). Curcumin significantly inhibited MAPK activity both 30 min after SLH (p38: 297 vs. 3260, p44/42: 370 vs. 2628, SAPK: 748 vs. 1764, all p < 0.01 vs. SLH 30 min) and 4 h post CLP (p38: 146 vs. 720, p44/42: 616 vs. 2759, all p < 0.01 vs. SLH + CLP4 h). Four hours after CLP, SLH rats expressed more HSP72. Lung MPO activity was significantly lower in SLH + CLP rats at both 4 h (9.5 vs. 15.6, p = 0.02 vs. sham) and 12 h (18.1 vs. 37.5, p = 7 x 10(-5), vs. sham). Serum and BAL TNF levels were higher in SLH rats initially (serum: 145 vs. 28 pg/mL, p = 2 x 10(-5) BAL: 83 vs. 57 vs. sham + CLP4h); however, BAL TNF was significantly lower in SLH animals 12 h post-CLP (37 vs. 72.7 pg/mL, p = 0.003 vs. sham + CLP12h)., Conclusion: SLH induces tolerance to CLP. This tolerance is dependent on early MAP kinase activation, since the survival benefit is reversed by curcumin. Decreases in tissue cytokine levels and neutrophil infiltration are potential mechanisms by which SLH induces tolerance to CLP (polymicrobial sepsis), attenuates acute lung injury, and improves survival.

Barrett's esophagus is the transformation of normal esophageal squamous epithelium to specialized intestinal metaplasia (SIM). Among the Barrett's specialized cells, those that can develop protective mechanisms against apoptosis may have potential to become malignant. Studies have shown that overexpression of metallothionein (MT), low molecular protein that protects cells from apoptotic stimuli, appears to be associated with more advanced, highly malignant tumors. We thus investigated the relationship between MT expression and apoptosis in different stages of Barrett's carcinogenesis. Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling and immunohistochemical dual-staining assay were performed in human biopsy samples of normal, SIM, dysplasia, and adenocarcinoma. Apoptotic index and MT expression were quantified by using an image system to analyze the converted digital data. A negative correlation between MT expression and apoptotic index was found. MT expression was significantly increased along with the histologic progression towards adenocarcinoma. This study thus suggests that MT may contribute to cytoprotection, thereby inhibiting apoptosis and leading to carcinogenesis of Barrett's esophageal cells.

Alcoholic liver disease (ALD) is a serious and potentially fatal consequence of alcohol use. The diagnosis of ALD is based on drinking history, physical signs and symptoms, and laboratory tests. Treatment strategies for ALD include lifestyle changes to reduce alcohol consumption, cigarette smoking, and obesity; nutrition therapy; and pharmacological therapy. The diagnosis and management of the complications of ALD are important for alleviating the symptoms of the disease, improving quality of life, and decreasing mortality.

Exposure to sublethal hemorrhage (SLH) makes rats tolerant to subsequent hemorrhagic or septic shock. We have shown that this tolerance leads to alterations in cytokine production, macrophage NF-kappaB activation and p38 MAP-kinase activity. The purpose of this study was to explore whether changes in p44/42 and SAPK/JNK MAP kinase activity also occur after the induction of tolerance by SLH. Rats were made tolerant by SLH (mean arterial pressure = 30 mmHg for 15 min with shed blood returned). Shams had anesthesia and instrumentation only. Twenty-four hours after SLH or sham operation, LPS was given (40 mg/kg intraperitoneal). Lung, liver, and splenic tissues were harvested 15, 30, and 45 min following sham, SLH, or LPS. Protein was isolated from tissues for determination of p44/42 and SAPK/JNK phosphorylation by Western blot analysis. Phosphorylation of p44/42 and SAPK/JNK was detected in all tissues following both sham and SLH, and this effect was significantly more pronounced following SLH (P < 0.05). However, activation of both p44/42 and SAPK/JNK in response to LPS, was significantly lower in the SLH rats when compared to shams. Peak activation was seen 30 min after SLH and peak attenuation, 30 min after LPS. The amount of nonphosphorylated protein was comparable in all groups. The induction of tolerance by SLH leads to phosphorylation of both p44/42 and SAPK/JNK MAP-kinases. However, the activation of these same kinases is attenuated in response to LPS in animals made tolerant by SLH.

A Phase I clinical trial was performed on patients with solid tumors refractory to conventional therapy. Crotoxin was administered i.m. for 30 consecutive days at doses ranging from 0.03 to 0.22 mg/m(2). Patients entered the study after providing a written informed consent. Although 26 patients were entered only 23 were evaluated. Reversible, nonlimiting neuromuscular toxicity evidenced as diplopia because of pareses of the external ocular muscles was present in 13 patients. It started at doses of 0.18 mg/m(2) and lasted from 2 to 6 h. These episodes did not require dose adjustment and disappeared in 1-3 weeks of treatment. Three patients experienced palpebral ptosis, nystagmus (grade 2), and anxiety (grade 2-3) at the dose-limiting toxicity of 0.22 mg/m(2). Also at dose-limiting toxicity, 1 patient showed nystagmus (grade 2) and anxiety (grade 3) without evidence of palpebral ptosis. Transient increases (grades 1-3) in the levels of creatinine kinase, aspartate aminotransferase, and alanine transaminase attributed to crotoxin myotoxicity were observed but returned to normal by the last week of treatment. At 0.21 mg/m(2) there was a case of grade-3 anaphylactic reaction on day 31, which required treatment. Hypersensitivity was regarded as an adverse drug-related reaction, and the patient was removed from the protocol. Two patients at different doses (0.12 mg/m(2) and 0.22 mg/m(2)) had sialorrhea. Four patients had asymptomatic transient increase in blood pressure (up to 20 mm Hg) 12 h after the first injection, which lasted 24 h. No treatment was required and toxicity did not reappear. Six patients experienced slight eosinophilia during the first 2 weeks. The maximum tolerated dose was set at 0.21 mg/m(2). Objective measurable partial responses (>50% reduction of tumor mass) were noted in 2 patients treated at 0.21 mg/m(2) and 1 at 0.12 mg/m(2). One patient (at 0.21 mg/m(2)) presented a complete response on day 110. Crotoxin pharmacokinetics showed rapid absorption from the injection site to blood (t(1/2 A) = 5.2 +/- 0.6 min). Plasma concentration reached a peak (C(max) = 0.79 +/- 0.1 ng/ml) at tau(max) = 19 +/- 3 min. The half-life of the distribution (alpha) phase is 22 +/- 2 min. Starting at 1.5 h after injection, the decrease in plasma concentration becomes slower, reaching 14 +/- 3 pg/ml 24 h after injection. The profile is dominated by the elimination (beta) phase with a half-life of 5.2 +/- 0.6 h. Consequently, 24 h after the injection ( approximately 5 half-life) 97% of the product was eliminated. The area under plasma concentration versus time curve was 0.19 +/- 0.05 microg/min/ml. Assuming availability (F) approximately 1, the clearance is C(L) = 26.3 +/- 7 ml/min, and the apparent volume of distribution is V(d) = 12 +/- 3 liter/kg. The recommended dose for a Phase II study is 0.18 mg/m(2).

Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase-like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ-containing loading modules are shown here to contain a key arginine residue at their active site. Site-specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13-methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex-tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1-TE, in which the 4'-phosphopante-theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro-ducts in detectable amounts.

We previously identified a novel p53-induced mouse gene, wig-1, that encodes a 290 amino acid zinc finger protein (Varmeh-Ziaie et al., 1997). Here we have identified and characterized the human homolog of mouse wig-1. The human wig-1 protein is 87% identical to the mouse protein and contains three zinc finger domains and a putative nuclear localization signal. Human wig-1 mRNA and protein is induced following activation of wild type p53 expression in our BL41-ts p53 Burkitt lymphoma cells. Wig-1 is also induced in MCF7 cells following treatment with the DNA-damaging agent mitomycin C. Northern blotting detected low levels of wig-1 mRNA in normal human tissues. Fluorescence in situ hybridization mapped wig-1 to human chromosome 3q26.3-27. FLAG-tagged human wig-1 localizes to the nucleus. Ectopic overexpression of human wig-1 inhibits tumor cell growth in a colony formation assay. These results suggest that human wig-1 has a role in the p53-dependent growth regulatory pathway.

We report herein the results of the cross-cultural adaptation and validation into the Chilean language of the parent's version of two health related quality of life instruments. The Childhood Health Assessment Questionnaire (CHAQ) is a disease specific health instrument that measures functional ability in daily living activities in children with juvenile idiopathic arthritis (JIA). The Child Health Questionnaire (CHQ) is a generic health instrument designed to capture the physical and psychosocial well-being of children independently from the underlying disease. The Chilean CHAQ-CHQ were derived from the European Spanish version with changing of the few words whose use is different in the 2 countries. A total of 126 subjects were enrolled: 72 patients with JIA (29% systemic onset, 39% polyarticular onset, 4% extended oligoarticular subtype, and 28% persistent oligoarticular subtype) and 54 healthy children. The CHAQ clinically discriminated between healthy subjects and JIA patients, with the JIA patients having a higher degree of disability, pain, and a lower overall well-being when compared to their healthy peers. Also the CHQ clinically discriminated between healthy subjects and JIA patients, with the JIA patients having a lower physical and psychosocial well-being when compared to their healthy peers. In conclusion the Chilean version of the CHAQ-CHQ is a reliable, and valid tool for the functional, physical and psychosocial assessment of children with JIA.

The anthracycline-like polyketide drug elloramycin is produced by Streptomyces olivaceus Tü2353. Elloramycin has antibacterial activity against Gram-positive bacteria and also exhibits antitumor activity. From a cosmid clone (cos16F4) containing part of the elloramycin biosynthesis gene cluster, three genes (elmMI, elmMII, and elmMIII) have been cloned. Sequence analysis and data base comparison showed that their deduced products resembled S-adenosylmethionine-dependent O-methyltransferases. The genes were individually expressed in Streptomyces albus and also coexpressed with genes involved in the biosynthesis of l-rhamnose, the 6-deoxysugar attached to the elloramycin aglycon. The resulting recombinant strains were used to biotransform three different elloramycin-type compounds: l-rhamnosyl-tetracenomycin C, l-olivosyl-tetracenomycin C, and l-oleandrosyl-tetracenomycin, which differ in their 2'-, 3'-, and 4'-substituents of the sugar moieties. When only the three methyltransferase-encoding genes elmMI, elmMII, and elmMIII were individually expressed in S. albus, the methylating activity of the three methyltransferases was also assayed in vitro using various externally added glycosylated substrates. From the combined results of all of these experiments, it is proposed that methyltransferases ElmMI, ElmMII, and ElmMIII are involved in the biosynthesis of the permethylated l-rhamnose moiety of elloramycin. ElmMI, ElmMII, and ElmMIII are responsible for the consecutive methylation of the hydroxy groups at the 2'-, 3'-, and 4'-position, respectively, after the sugar moiety has been attached to the aglycon.

Introduction: The present study was designed to examine the extent to which differences in input resistance contribute to the heterogeneity of excitability in canine endocardium., Methods and Results: The experiments were performed in isolated canine right anterior papillary muscles. The preparations were superfused with oxygenated Tyrode's solution at 36 degrees to 37 degrees C. Conventional methods were used for recording and stimulation. To obtain pertinent data, two microelectrodes were inserted into the same cell or into two contiguous cells. One microelectrode injected intracellular hyperpolarizing current to polarize the membrane; the second microelectrode recorded the changes in transmembrane potential. The two micreoelectrodes were separated by 8 to 10 microm. The procedure used provided input resistance and threshold current values for the doubly impaled cell or the two contiguous cell units. The same procedure was repeated at different sites of the preparation. The plot of input resistance versus threshold current showed a good fit (R2 = 0.97) between the experimental data and the curve for the rectangular hyperbola XY = 30 mV; therefore, the input resistance and the threshold current are inversely related., Conclusion: The results indicate that the canine cardiac syncytium is nonhomogeneous with respect to input resistance and that input resistance is inversely related to the minimal current needed to reach threshold. Accordingly, the electrical excitability of the cells studied also is inhomogeneous.

Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 microg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 microg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA.

Background: Select pancreatic enzymes, primarily elastase, precipitate pulmonary injury similar to pancreatitis-associated adult respiratory distress syndrome and stimulate leukocyte cytokine production in vitro via nuclear factor kappa B (NF-kappaB) activation. This study explores the effect of systemic pancreatic enzymes on pulmonary NF-kappaB and inhibitory kappa B (IkappaB) proteins and their role in enzyme-induced pulmonary injury., Methods: Mice received pancreatic elastase, amylase, lipase, or trypsin intraperitoneally. Bronchoalveolar lavage IkappaBalpha/IkappaBbeta proteins were measured by immunoblot. Pulmonary NF-kappaB activation, tumor necrosis factor (TNF) gene expression, and neutrophil infiltration (myeloperoxidase) were determined and myeloperoxidase experiments repeated in p55 TNF receptor-deficient (TNF KO) animals. Additional animals received pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB activation, and TNF protein and pulmonary microvascular permeability were measured after elastase administration., Results: Pancreatic elastase induced pulmonary IkappaBalpha/IkappaBbeta degradation (30 minutes), NF-kappaB activation (60 minutes), and TNF gene expression (60 minutes) with subsequent neutrophilic inflammation (4 hours) and microvascular leakage (24 hours), whereas amylase, lipase, and trypsin did not. Furthermore, lung injury was markedly reduced in TNF KO animals and PDTC significantly attenuated TNF production and pulmonary microvascular leakage., Conclusions: Pancreatic elastase induces cytokine-mediated lung injury and this pathway involves the NF-kappaB second messenger system, further supporting elastase as a factor linking pancreatic inflammation to systemic illness during severe acute pancreatitis.

The triggering events by which mononuclear cells throughout the body are induced to produce large amounts of cytokines during acute pancreatitis are unclear. However, recent work in our laboratory demonstrated that three specific pancreatic enzymes (elastase, carboxypeptidase A, and lipase) induced dramatic tumor necrosis factor-alpha (TNF-alpha) protein production from macrophages, whereas all others could not. This series of experiments was designed to examine the second messenger system by which this occurs. The rat macrophage cell line NR8383 was incubated for 3 hours with elastase, carboxypeptidase A, lipase, trypsin, or lipopolysaccharide (positive control). Activation of nuclear factor kappa B (NF-kappa B) was demonstrated by electrophoretic mobility shift assay, presence of inhibitory kappa B alpha and beta (I kappa B-alpha and I kappa B-beta) by Western blot analysis, and TNF-alpha protein production by enzyme-linked immunosorbent assay. Elastase, carboxypeptidase A, and lipase induced degradation of I kappa B-beta (but not I kappa B-alpha), activation of NF-kappa B, and production of TNF-alpha protein, whereas inhibition of I kappa B with pyrrolidine dithiocarbamate attenuated this response. Trypsin was unable to elicit any of these responses. Macrophages can be induced by specific activated pancreatic enzymes-elastase, carboxypeptidase A, and lipase-to produce TNF-alpha. This process is dependent on I kappa B-beta degradation and NF- kappa B activation, suggesting that these enzymes trigger this second messenger system through specific membrane-bound receptors.

Background: Exposure to sublethal hemorrhage (SLH) makes rats tolerant to subsequent hemorrhagic or septic shock and is associated with altered NF-kappaB activity. The purpose of this study was to explore whether changes in p38 mitogen-activated protein (MAP) kinase activity also occur in the induction of tolerance by SLH., Methods: Rats were made tolerant by SLH or sham operation. Twenty-four hours later rats were exposed to lipopolysaccharide (LPS) or had peritoneal macrophages (Mphi) isolated. CNI-1493, a p38 MAP kinase inhibitor, or saline was given prior to SLH. Lungs were harvested 1 h after SLH or LPS and total protein was extracted. Peritoneal Mphi were stimulated with LPS (10 microg/ml) and total protein was isolated 1 h later. Active, dually phosphorylated p38 MAP kinase was determined by Western blot. Tumor necrosis factor (TNF) was measured in Mphi supernatants by enzyme-linked immunosorbent assay (ELISA) 18 h after LPS., Results: SLH activated p38 MAP kinase in the lung and this was inhibited by CNI-1493. Twenty-four hours later, lung p38 MAP kinase activity increased to the same degree in tolerant and sham rats following LPS, but much more prominently in the CNI-1493 treated rats. There was no p38 activity in peritoneal Mphi at baseline, and similar to lung p38, LPS led to increased p38 activity which was most significant in Mphi from rats that received CNI-1493 prior to SLH. TNF production by tolerant Mphi in response to LPS was significantly (P < 0.05, t test) decreased and p38 inhibition with CNI-1493 at the time of SLH reversed the inhibitory effects of tolerance on TNF production., Conclusions: TNF production by tolerant Mphi following a second insult (LPS) is attenuated despite preservation of normal p38 MAP kinase activity. However, activation of this intracellular second messenger is a necessary step in the "cellular reprogramming" that occurs during the induction of tolerance by SLH., (Copyright 2000 Academic Press.)

Corticotropin signal transduction pathway involves serine/threonine protein phosphorylation. Recent reports suggest that protein tyrosine dephosphorylation may also be an integral component of that pathway. The present study was performed to investigate the role played by protein tyrosine phosphatases (PTPs) on acute response to corticotropin and the hypothetical regulation of PTPs by this hormone. We have used two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), in order to examine the relevance of PTP activity on hormone-stimulated and 8-bromo-adenosine 3',5'-phosphate (8Br-cAMP is a permeant analogue of adenosine 3',5'-phosphate)-stimulated steroidogenesis in adrenal zona fasciculata (ZF) cells. In both cases, PAO and PV inhibited the steroid production in a dose-dependent fashion, and had no effect on steroidogenesis supported by a permeant analogue of cholesterol. The effect of hormonal stimulation on PTP activity was analyzed in rat adrenal ZF. In vivo corticotropin treatment reduced phosphotyrosine content in endogenous proteins and produced a transient increase of PTP activity in the cytosolic fraction, reaching a maximum (twofold) after 15 min. Incubation of adrenal ZF with 8Br-cAMP also produced PTP activation, suggesting that it can be mediated by cAMP-dependent protein kinase (PKA)-dependent phosphorylation. Detection of PTP activity in an in-gel assay showed three corticotropin-stimulated soluble PTPs with molecular masses of 115, 80 and 50 kDa. In summary, we report for the first time a hormone-dependent PTP activation in a steroidogenic tissue and provide evidence that PTP activity plays an important role in corticotropin signal pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane.

Objective: To measure patient satisfaction with pregnancy and newborn care., Study Design: To develop our survey, we reviewed domains of care that had been identified by national groups working to develop measures of satisfaction with care. Within these domains of care, items pertinent to pregnancy and newborn care were written, reviewed, and pilot tested in focus groups of women who had recently delivered a baby. A 24-item survey instrument was the result of this process., Patients and Methods: We sent our survey to 2337 female members of the Southern California Kaiser Permanente Medical Care Program approximately 8 weeks after they had delivered a baby. The demographic and clinical characteristics of the respondents were compared with those of the nonrespondents., Results: A total of 1017 women (44%) completed and returned the survey. Eighty-eight percent of the women who responded were very or somewhat satisfied with the care they received during their pregnancy and the newborn period. Women who responded differed from those who did not respond on infant birthweight, maternal age and education, and other variables. The results of our survey were forwarded to local quality managers and physicians., Conclusion: Satisfaction with care is an important measure of quality of care. Many changes in the delivery system were made in response to the information provided by survey respondents, including offering alternative appointment times and scheduling postpartum appointments before hospital discharge. Our survey instrument, which assesses satisfaction with pregnancy and newborn care, might be useful for other health maintenance organizations for identifying areas where improvement in care is needed.

The need for increased antibody production by hybridomas has been approached by the addition to cell cultures of different growth factors; in vitro addition of estradiol-17beta (E2) to human blood lymphocytes increases the accumulation of plasma-blasts and Ig-secreting cells. Four different murine-murine hybridomas secreting different monoclonal antibodies (MAbs) were treated with E2. Specific antibody concentration was measured by enzyme-linked immunoadsorbent assay (ELISA) in culture supernatants whereas expression of E2-receptor in the hybridoma cells was determined by polymerase chain reaction (PCR). When E2 was added as a growth supplement to alpha-estrogen receptor positive murine-murine hybridomas it enhanced MAb secretion by as much as 255%, in a dose-dependant manner. This effect lasted for as long as the alpha-estrogen receptor was detected in the hybridoma cells, was inhibited by tamoxifen and was not observed in alpha-estrogen receptor negative hybridomas. The synthetic estrogen analogue diethylstilbestrol had no effect. Estradiol-17beta should be added to the list of hybridoma-inducing growth factors.

Background: Despite the advent of dialysis, survival with acute renal failure when associated with multiorgan failure is poor. The development of lung injury after shock or visceral ischemia has been shown; however, the effects of isolated renal ischemia/reperfusion injury (IRI) on the lungs are unclear. We hypothesized that isolated renal IRI could alter pulmonary vascular permeability (PVP) and that macrophages could be important mediators in this response., Methods: Rats (N = 5 per group) underwent renal ischemia for 30 minutes, followed by reperfusion. Lung vascular permeability was evaluated by quantitation of Evans blue dye extravasation from vascular space to lung parenchyma at 1, 24, 48, or 96 hours after reperfusion. Serum was collected for blood urea nitrogen and creatinine at each time point. To examine the role of the macrophage, the macrophage pacifant CNI-1493, which inhibits the release of macrophage-derived inflammatory products, was administered in a blinded fashion during renal IRI., Results: PVP was significantly (P < 0.05) increased at 24 hours and peaked at 48 hours after IRI compared with shams as well as baseline levels. PVP after IRI became similar to shams after 96 hours. This correlated with increases in blood urea nitrogen and creatinine at similar time points. At 48 hours, CNI-1493 significantly abrogated the increase in PVP compared with IRI alone. However, CNI-1493 did not alter the course of the acute renal failure. Pulmonary histology demonstrated interstitial edema, alveolar hemorrhage, and red blood cell sludging after renal IRI, which was partially attenuated by CNI-1493., Conclusions: Increased PVP develops after isolated renal IRI, and macrophage-derived products are mediators in this response. These findings have implications for understanding the mechanisms underlying respiratory dysfunction associated with acute renal failure.

Objective: To determine if cross-tolerance to septic shock could be induced by a previous insult with sublethal hemorrhage (SLH) and to characterize the mechanisms involved in this induced protective response., Background Data: It is possible to condition animals by prior SLH such that they tolerate an otherwise lethal hemorrhage. It is also possible to condition animals with low doses of lipopolysaccharide (LPS) so that they survive a "lethal" septic insult. However, a paucity of information exists on cross-tolerance between hemorrhage and sepsis., Methods: Rats were made tolerant by conditioning SLH or sham operation. Twenty-four hours later, tolerant and sham rats were exposed to a lethal dose of LPS. To explore the mechanism of tolerance induction, rats were given the macrophage (Mphi) inhibitor CNI-1493 or saline carrier before SLH. Survival and pulmonary vascular injury were determined after LPS. Serum tumor necrosis factor (TNF) levels and splenic Mphi TNF gene expression were measured at several time points., Results: Prior SLH indeed made rats tolerant and imparted a significant survival benefit and reduction in pulmonary vascular injury after LPS. The tolerance induced by SLH was reversed by Mphi inhibition. Tolerant animals had low serum TNF levels immediately after SLH and reduced circulating TNF levels after LPS. SLH, however, did not inhibit the augmentation of TNF gene expression after LPS., Conclusions: Sublethal hemorrhage bestows protection against a lethal LPS challenge. Inhibition of the Mphi attenuated the benefit of the tolerance induced by SLH. Circulating TNF but not TNF gene after LPS is lessened by SLH. This implicates changes in Mphi intracellular signaling in induction of the tolerant state.

Background: Tolerance to hemorrhagic or endotoxic shock can be induced by prior sublethal hemorrhage (SLH). The purpose of this study was to explore whether alterations in signal transduction pathways involving NF-kappaB occur in macrophages (Mphi) following induction of tolerance by SLH., Methods: Using a model of SLH previously shown in our lab to impart a survival benefit to subsequent hemorrhagic or endotoxic shock, rats (n = 30) were conditioned by SLH. Peritoneal Mphi were harvested 24 h after conditioning and stimulated with lipopolysaccharide (LPS) (10 microg/mL). Nuclear and cytosolic proteins were isolated 1 h later for determination of NF-kappaB activation by gel-shift assay and IkappaB-alpha by Western blot. TNF mRNA gene expression was measured 4 h after LPS stimulation by reverse transcription/polymerase chain reaction (RT/PCR). TNF protein levels were measured in cellular supernatants by enzyme-linked immunosorbent assay (ELISA) 18 h after LPS. RESULTS. LPS stimulation of sham Mphi increased NF-kappaB activation with corresponding loss of its inhibitor IkappaB-alpha. In contrast, IkappaB-alpha was not detectable following conditioning, and conditioned Mphi had NF-kappaB activation at baseline which increased minimally with LPS stimulation. LPS increased TNF gene expression and significantly increased protein production by both sham and conditioned Mphi, but this increase was greater in the sham-conditioned group., Conclusions: The ability of Mphi from animals made tolerant by SLH to produce TNF in vitro is conserved. Nevertheless, these same Mphi exhibit alterations in TNF gene induction and expression as well as signal transduction, specifically, changes in IkappaB-alpha and NF-kappaB activation. This suggests a role for activation of NF-kappaB in the induction of tolerance., (Copyright 1999 Academic Press.)

The N-terminal amino acid sequences of two peptides derived from a Streptomyces coelicolor ribosomal protein were determined and degenerate oligonucleotide primers derived from these sequences were used as probes for the screening of a chromosomal DNA library of S. coelicolor. Two positive clones were isolated and DNA sequencing of a 1740-bp region of these clones that hybridised with the probes revealed the presence of four genes, two of them incomplete. The deduced products of the two complete genes. rplM and rpsI, showed clear similarities to L13 and S9 ribosomal proteins from various organisms. Promoter-probe and primer extension experiments suggest that the two genes form a single transcriptional unit. The specific rate of synthesis of both proteins was high at early stages of growth but decreased later.

Background: Mithramycin, nogalamycin and aclacinomycins are aromatic polyketide antibiotics that exhibit antitumour activity. The precursors of these antibiotics are formed via a polyketide biosynthetic pathway in which acetate (for mithramycinone and nogalamycinone) or propionate (for aklavinone) is used as a starter unit and nine acetates are used as extender units. The assembly of building blocks is catalyzed by the minimal polyketide synthase (PKS). Further steps include regiospecific reductions (if any) and cyclization. In the biosynthesis of mithramycin, however, ketoreduction is omitted and the regiospecificity of the first cyclization differs from that of anthracycline antibiotics (e.g. nogalamycin and aclacinomycins). These significant differences provide a convenient means to analyze the determinants for the regiospecificity of the first cyclization step., Results: In order to analyze a possible role of the minimal PKS in the regiospecificity of the first cyclization in polyketide biosynthesis, we expressed the mtm locus, which includes mithramycin minimal PKS genes, in Streptomyces galilaeus, which normally makes aclacinomycins, and the sno locus, which includes nogalamycin minimal PKS genes, in Streptomyces argillaceus, which normally makes mithramycin. The host strains are defective in the minimal PKS, but they express other antibiotic biosynthesis genes. Expression of the sno minimal PKS in the S. argillaceus polyketide-deficient strain generated mithramycin production. Auramycins, instead of aclacinomycins, accumulated in the recombinant S. galilaeus strains, suggesting that the mithramycin minimal PKS is responsible for the choice of starter unit. We also describe structural analysis of the compounds accumulated by a ketoreductase-deficient S. galilaeus mutant; spectroscopic studies on the major polyketide compound that accumulated revealed a first ring closure which is not typical of anthracyclines, suggesting an important role for the ketoreductase in the regiospecificity of the first cyclization., Conclusions: These experiments clearly support the involvement of ketoreductase and a cyclase in the regiospecific cyclization of the biosynthetic pathway for aromatic polyketides.

This study was designed to determine whether the prevalences of the DSM-III alcohol abuse/dependence symptoms in 87 early and 73 late onset male alcoholics differ from one another. The authors administered a 19-item alcohol abuse/dependence symptom checklist with items based on the DSM-III criteria. Nine of the 19 symptoms were reported significantly more often in the early than in the late onset alcoholics. Antisocial behaviors were reported to have been particularly frequent in the early onset group.

Objective: To determine the effects of an immune-enhancing experimental diet (XD = supplemental arginine, trace elements, and increased omega-3 fatty acids) versus standard diet (SD), on immune cell function and clinical outcome of critically injured patients., Design: Prospective randomized clinical trial of patients admitted to the surgical intensive care unit after trauma (Injury Severity Score > 13)., Materials and Methods: Patients received early enteral nutrition with either XD or SD for a minimum of 5 days., Measurements: Mortality, intensive care unit, ventilator, and hospital days, as well as incidence of adult respiratory distress syndrome (ARDS) and infectious complications were recorded. Nutritional parameters were also studied. Peripheral blood leukocytes were isolated from normal volunteers and from patients on days 1, 6, and 10 of feeding., Main Results: Demographics and injury severity were similar in both groups. Both SD (n = 21) and XD (n = 22) groups revealed depressed monocyte function (tumor necrosis factor, prostaglandin E2, and procoagulant activity) on day 1 compared with a reference group (p < 0.05). However, monocytes from XD patients began to "normalize" their response (tumor necrosis factor, prostaglandin E2, and procoagulant activity) by day 6. Although ARDS occurred more frequently in the XD group (45 vs. 19%), the majority of ARDS in both groups occurred very early, with only three patients in the XD (13.6%) and one patient in the SD (4.7%) groups developing ARDS after study entry. XD patients remained on the ventilator longer (16.4 vs. 9.7 days) and in the hospital longer (32.9 vs. 22 days) compared with the SD group, but overall mortality was nearly identical (4.5 vs. 5%)., Conclusion: The exact role and timing for diets with immune-enhancing effects has yet to be defined.

Endotoxin (lipopolysaccharide) stimulation of macrophages (M phi) induces the generation of toxic reactive oxygen intermediates (ROI); however, recent studies implicate intracellular redox changes in signal transduction pathways for cytokines. To test whether oxidant stress modulates M phi activation, rabbit alveolar M phi were exposed to the following: diamide (oxidizes intracellular glutathione); glucose oxidase (generates hydrogen peroxide); or xanthine oxidase (generates superoxide), before lipopolysaccharide. Supernatants were assayed for tumor necrosis factor (TNF) and cell lysates were assayed for procoagulant activity (PCA). TNF mRNA was analyzed by Northern blot. M phi exposure to diamide and glucose oxidase augmented TNF production, PCA expression, and TNF mRNA accumulation; however, xanthine oxidase exposure inhibited TNF production while augmenting PCA expression. M phi signal transduction can be enhanced by increasing cellular oxidant stress. The differential response of TNF versus PCA suggests the existence of distinct redox-sensitive signal transduction pathways. These data define a mechanism by which oxidants generated during inflammation may modulate M phi function.

Dietary nutrients may have pharmacological value in modulating the immune system. We studied the effects of two enteral diets, which differed in their content of arginine, fat source, and select trace elements, on immune function in critically injured patients. Leukocytes were isolated from healthy volunteers and severely injured (ISS > 13) patients on the first, sixth, and tenth day of receiving either a standard diet or experimental diet. Monocytes were assayed for tumor necrosis factor, procoagulant activity, and prostaglandin E2 following endotoxin exposure. Neutrophil oxidant production and lymphocyte blastogenesis was assessed. Leukocyte function was uniformly depressed compared to normal patients on day 1. The response of leukocytes from patients receiving experimental diet improved or "normalized" by day 6, while remaining depressed in patients receiving standard diet. Dietary nutrient modification can effect cellular immune responses to inflammatory stimuli in severely injured patients.

Background: Mithramycin, chromomycin, and olivomycin belong to the aureolic acid family of clinically important anti-tumor agents. These natural products share a common aromatic aglycone. Although isotope labeling studies have firmly established the polyketide origin of this aglycone, they do not distinguish between alternative biosynthetic models in which the aglycone is derived from one, two or three distinct polyketide moieties. We set out to determine the biosynthetic origin of this moiety using a recombinant approach in which the ketosynthase and chain-length factor proteins from the antibiotic-producer strain, which determine the chain length of a polyketide, are produced in a heterologous bacterial host., Results: The ketosynthase and chain-length factor genes from the polyketide synthase gene cluster from the mithramycin producer, Streptomyces argillaceus ATCC 12956, and the acyl carrier protein and ketoreductase genes from the actinorhodin polyketide synthase were expressed in Streptomyces coelicolor CH999. The recombinant strain produced a 20-carbon polyketide, comprising the complete backbone of the aglycone of mithramycin., Conclusions: The aglycone moieties of mithramycin, chromomycin, and olivomycin are derived from a single polyketide backbone. The nascent polyketide backbone must undergo a series of regiospecific cyclizations to form a tetracenomycin-like tetracyclic intermediate. The final steps in the aglycone biosynthetic pathway presumably involve decarboxylation and oxidative cleavage between C-18 and C-19, followed by additional oxidation, reduction, and methylation reactions.

Introduction: The purpose of this investigation was to study the properties of junction and action potentials elicited by nerve stimulation in the absence of the cardiac ganglion in crayfish myocardium., Methods and Results: The cardiac ganglion was surgically removed in isolated crayfish hearts. Electrical stimulation of one of the peripheral anterolateral nerves provided isolated junction potentials and action potentials free of the usual postspike bursts of junction potentials. The single junction potentials displayed amplitudes of up to 25 mV and slow exponential decay; the mean time constant was 170 +/- 13 msec (approximately x +/- SD). In fully recovered tissue, the junction potentials triggered action potentials free of repetitive subthreshold discharges. Tetrodotoxin did not alter the amplitude or shape of action potentials initiated by direct electrical stimulation of the muscle cells. Calcium channel blocking agents such as Cd2+ and Ni2+ eliminated the action potentials but not the junction potentials. Tetraethylammonium markedly prolonged the action potential duration., Conclusions: Our data suggest that: (1) A slow decay of the junction potentials may result from the disappearance of the neurotransmitter; this process also accounts for the late slow repolarization of the final part of the action potentials; (2) The equilibrium potential of the junction potentials is close to 0 mV; (3) The upstroke of the action potentials is carried by calcium currents; (4) The fast repolarization phase of the action potentials is likely caused by the delayed rectifier; and (5) The refractory phase outlasts the duration of the action potential.

In response to bacterial lipopolysaccharides (LPS; endotoxin), endothelial cells are converted to an activation phenotype expressing both proinflammatory and procoagulant properties that include the induction of leukocyte adhesion molecules and tissue factor expression. LPS-induced endothelial cell activation requires a soluble form of the monocyte LPS receptor, sCD14. We evaluated the capacity of multiple strains of gram-negative and gram-positive bacteria to induce endothelial E-selectin and tissue factor expression through sCD14-dependent pathways with cultured human umbilical vein endothelial cells (HUVE). Both viable and heat-killed gram-negative bacteria (Bacteroides fragilis, Enterobacter cloacae, Haemophilus influenzae, and Klebsiella pneumoniae) but not viable or heat-killed gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae) induced prominent E-selectin surface expression detected by enzyme-linked immunosorbent assay. Tissue factor activity on HUVE, indicated by factor X activation, was induced in response to gram-negative bacteria but not in response to gram-positive bacteria. Gram-negative bacteria induced transcriptional activation in HUVE, indicated by the appearance of E-selectin-specific mRNA and by the demonstration of activation of NF-kappa B, a trans-activating factor necessary for E-selectin and tissue factor gene transcription. In contrast, neither E-selectin mRNA nor activation of NF-kappa B was detected in HUVE treated with gram-positive bacteria. Endothelial cell activation by gram-negative bacteria in each of these assays was inhibited with a monoclonal antibody (60bd) against CD14. Furthermore, CHO-K1 cells, transfected with human recombinant CD14, responded to all strains of gram-negative bacteria (viable or heat killed), indicated by CHO-K1 NF-kappa B activation. We conclude that gram-negative bacteria induce endothelial cell activation through a common sCD14-dependent pathway.

Background: Endotoxin (lipopolysaccharide [LPS]) stimulation of tissue-fixed macrophages induces the generation of toxic oxidants. However, recent studies also implicate redox changes in both the signal transduction pathways for cytokine genes and the generation of physiologically active arachidonic acid metabolites. Because cytokines and arachidonic acid metabolites initiate and perpetuate deleterious systemic inflammatory responses, we tested whether macrophage activation could be modulated by antioxidants., Methods: Rabbit alveolar macrophages were obtained by bronchoalveolar lavage, isolated, treated with the antioxidants vitamin E or N-acetylcysteine (NAC), and stimulated with an optimal dose of LPS (10 ng/ml). Assays were performed for tumor necrosis factor (TNF), procoagulant activity, and prostaglandin E2. Total cellular RNA was extracted for Northern blot analysis of TNF messenger RNA., Results: Exposure of the macrophage to the antioxidants vitamin E and NAC inhibited TNF production, accumulation of TNF messenger RNA, procoagulant activity expression, and prostaglandin E2 production., Conclusions: Macrophage signal transduction of LPS is dependent on the generation of reactive oxygen intermediates that can be blocked both at the level of the lipid membrane (vitamin E) and at the intracellular level (NAC). This suggests a potential therapeutic role for antioxidants is disease states such as adult respiratory distress syndrome and multiple organ failure syndrome, which are characterized by excessive macrophage activation.

Objective: To determine the accuracy of diagnostic peritoneal lavage (DPL) for the evaluation of intraabdominal injury in patients with a pelvic fracture as a result of blunt trauma., Design: Retrospective cohort analysis., Setting: Level I trauma center in metropolitan Seattle, Wash., Patients: Four hundred ninety-seven consecutive patients admitted with pelvic fractures following blunt trauma during a 60-month period., Outcome Measures: Positive results of DPL, defined by one of the following: an immediate aspiration of more than 10 mL of gross blood; a red blood cell count of more than 0.0001 x 10(12)/L; a white blood cell count greater than 0.0005 x 10(9)/L; an elevated amylase, bilirubin, or creatinine level; or organic particles or bacteria in the effluent returned after installation of 1 L of crystalloid fluid lavaged in the peritoneal cavity., Results: Two hundred eighty-six patients underwent DPL. For 80 patients (28.0%), results of DPL were positive and for 194 patients (67.8%) the results of DPL were negative. For two patients (0.7%), results of DPL were false positive for a sensitivity of 94%. For another two patients (0.7%), the results of DPL were false negative for a specificity of 99%. As a diagnostic modality, DPL has a positive predictive value of 98% and a negative predictive value of 97%., Conclusions: Diagnostic peritoneal lavage is a reliable method for the evaluation of intra-abdominal injury and should remain a standard component in the evaluation of patients following blunt injury with or without pelvic fractures.

Fungal polysaccharides are cell wall components which may act as antigens or as structural substrates. As antigens, the role of mannans in Saccharomyces cerevisiae and Candida albicans, and of glycoproteins in Aspergillus fumigatus are discussed. Analyses on beta-glucan synthetase in Paracoccidioides brasiliensis and the inhibitory effect of Hansenula mrakii killer toxin on beta-glucan biosynthesis are also considered.

Cell extracts of Streptomyces antibioticus, an oleandomycin producer, can inactivate oleandomycin in the presence of UDP-glucose. The inactivation can be detected through the loss of biological activity or by alteration in the chromatographic mobility of the antibiotic. This enzyme activity also inactivates other macrolides (rosaramicin, methymycin, and lankamycin) which contain a free 2'-OH group in a monosaccharide linked to the lactone ring (with the exception of erythromycin), but not those which contain a disaccharide (tylosin, spiramycin, carbomycin, josamycin, niddamycin, and relomycin). Interestingly, the culture supernatant contains another enzyme activity capable of reactivating the glycosylated oleandomycin and regenerating the biological activity through the release of a glucose molecule. It is proposed that these two enzyme activities could be an integral part of the oleandomycin biosynthetic pathway.

Influence of 5,5-diphenylhydantoin (phenytoin; PHT) on the cytoplasmic free Ca2+ concentration, [Ca2+]i, was studied in fura 2 loaded adherent monolayers of human gingival fibroblasts derived from three patients before and after 9 months of PHT therapy. In the patient where gingival overgrowth developed during PHT medication (responder), addition of PHT to gingival fibroblasts derived before PHT medication induced a transient extracellular Ca2+ dependent increase in [Ca2+]i. In a non-responder patient, where gingival overgrowth did not develop during the same period of PHT therapy, addition of PHT to gingival fibroblasts derived before the start of medication did not significantly affect [Ca2+]i. Under extracellular Ca2+ deficient conditions, addition of PHT to serum-starved fibroblasts derived from the two categories of patients before the medication resulted in an increase in [Ca2+]i. In fibroblasts derived from the responder patient during PHT medication, in contrast to those from the non-responders (n = 2), the basal level of [Ca2+]i was significantly decreased. The results indicate that, in the cases studied, there is a relationship between PHT induced alterations in [Ca2+]i in gingival fibroblasts and the clinical development of gingival overgrowth.