Preferential activation of phospholipase A2 by low concentrations of phosphatidic acid with long-chain fatty acids in rabbit platelets.

The role of phosphatidic acid (PA) in the signal transduction system of platelets was studied using 1-stearoyl 2-arachidonoyl PA (PASA). When PASA was added to rabbit platelets, aggregation occurred. BW755C, a dual inhibitor of cyclooxygenase and lipoxygenase, as well as p-bromophenacyl bromide and mepacrine, inhibitors of phospholipase A2, inhibited the aggregation induced by low concentrations of PASA, but not that induced by high concentrations. PASA also stimulated, in a dose-dependent manner, arachidonic acid liberation, lysophosphatidylcholine and diacylglycerol formation, and mobilization of intracellular Ca2+; all of which were dependent on the presence of Ca2+ in the outer medium. The arachidonic acid liberation was inhibited by p-bromophenacyl bromide or mepacrine, while diacylglycerol formation by low concentrations of PASA was inhibited by BW755C. With platelet membrane fractions or with the platelets made permeable to Ca2+ by pretreatment with ionomycin, PASA caused arachidonic acid liberation in the presence of Ca2+. Furthermore, PASA enhanced the activity of phospholipase A2 partially purified from platelet cytosol acting on 1-palmitoyl-2-[14C]arachidonoyl-glycerophosphoethanolamine. These results provide evidence that PASA preferentially potentiates the activation of phospholipase A2 in cooperation with Ca2+, suggesting that PA acts as a positive feedback regulator to potentiate the activation of phospholipase A2 and contributes to the amplification of platelet activation.     

Pan-African sanctuary alliance: status and range of activities for great ape conservation

While wild populations continue to decrease, the number of orphaned primates, sanctuaries, and attempts to reintroduce primates back to the natural environment are increasing. An umbrella organization called the Pan-African Sanctuary Alliance (PASA) was formed in 2000 and recently the IUCN Reintroduction Specialist Group developed a set of specific policy guidelines for primates (2002). Data presented in this report are based upon questionnaire responses by managers from 17 African facilities that have become members of PASA (membership in PASA is defined by attendance at an annual PASA workshop). These PASA facilities house over 500 great apes. (There may be other facilities not represented here simply because their managers did not attend a PASA workshop.) The majority of the apes arrived at the sanctuaries when they were less than 4 years old and half were confiscated. Over 40% were found awaiting sale, and 30% had been previously kept as pets. Common ailments upon arrival included internal parasites, behavioral abnormalities, and malnutrition; 20% of the total sanctuary population died prematurely. Most sanctuaries use a combination of enclosures surrounded by electric fencing and cages to accommodate the apes. Sanctuaries actively participate in conservation education, habitat protection, tourism, scientific data collection, local development, and reintroduction. The median total facility operating cost was 65,000 US dollars per annum. The median facility cost per ape was 2,222 US dollars per annum. Most funding comes from overseas nongovernmental agencies. Discussion focuses on evaluating the present status of sanctuaries, the problems facing them, and their potential role in African conservation issues.     

From molecular variant to disease: initial steps in evaluating the association of transthyretin M119 with disease.

Traditionally, clinical research has sought to determine the molecular basis of clinical signs and symptoms. Increasingly, the traditional process will be reversed, as many structural protein variants are elucidated as a result of powerful PCR-based methods. Herein we describe a variant of transthyretin (TTR) found by direct genomic sequencing and illustrate the utility of PASA (PCR amplification of specific alleles) in the initial characterization of such variants. TTR is an intriguing protein of unknown function, but deposition of mutant TTR produces familial amyloidotic polyneuropathy (FAP). We identify a carrier of a variant TTR in which threonine119 is changed to methionine (T119----M). T119 is invariant in five mammalian species, suggesting that this residue is important for normal protein function. To determine the frequency of the M119 variant, individuals of northern- and western-European descent were rapidly screened by generating a PASA assay for the sequence change. Four additional individuals were found to be heterozygous for the mutation, for a total of five M119 alleles in 1,666 genes (1/333). Clinical records, initial clinical interviews, and family history of these patients hint at a high frequency of early-onset venous insufficiency and perhaps mild renal dysfunction. Haplotype analysis on the heterozygotes could be performed, despite the absence of samples from relatives, by performing "double PASA." The haplotype data suggest that the M119 variant derives from a common ancestor. The putative functional deficiency caused by TTR M119 should be most marked in the homozygotes, who can be calculated to occur in 1/100,000 conceptions. If viable, these individuals may provide important clues about the physiological role of TTR. Although the nature (if any) of disease caused by TTR M119 remains to be defined, the genetic and clinical data indicate that this mutation does not cause FAP. Future family studies can determine whether the heterozygous state for TTR M119 cosegregates with a disease or trait.     

Composition Patterns of Caribbean Limestone Forests: Are Parsimony,Classification, and Ordination Analyses Congruent?1

This study presents an analysis of floristic composition patterns for limestone vegetation from three Caribbean islands: Hispaniola, Mona, and Puerto Rico. The physical setting of these communities varies from very dry to wet climates, and from ridgetops, slopes, to plateaus. Consequently, vegetation communities have variable species composition. The questions addressed were: (1) What are the patterns of species composition among limestone vegetation types based on a parsimony analysis of species assemblages (PASA) and how congruent are they widi geography, climate, topography, and vegetation physiognomy? and (2) How do PASA patterns compare to floristic patterns obtained with a classification (UPGMA) and an ordination (NMS)? The main distinction of floristic categories was between communities of humid and dry climates, followed by a finer separation of communities congruent with topography; vegetation physiognomy corresponded with both. PASA, UPGMA, and NMS produced highly coincident floristic groups; however, affinities among groups were different. Advantages of PASA over UPGMA and NMS are that PASA produces groupings strictly based on the shared species and provides different measures of support for floristic groups. The three techniques indicated that the humid and dry limestone areas of Puerto Rico had floristically divergent dry‐type vegetation, even when they were structurally and physiognomically convergent. Also, floristic affinities of dry communities in Puerto Rico were stronger with dry communities on other islands than to more humid communities on the same island; thus, humidity regime is stronger than geography in promoting floristic links among limestone vegetation communities of die Caribbean. Almost every conclusion obtained from UPGMA and NMS was also taken from PASA, and so all diree techniques are compatible. The methodological, theoretical, and interpretive simplicity of PASA is what makes it an attractive procedure for studies that examine composition patterns.     

Binding site analysis of cellulose binding domain CBD(N1) from endoglucanse C of Cellulomonas fimi by site-directed mutagenesis

Endoglucanase C (CenC), a beta1,4 glucanase from the soil bacterium Cellulomonas fimi, binds to amorphous cellulose via two homologous cellulose binding domains, termed CBD(N1) and CBD(N2). In this work, the contributions of 10 amino acids within the binding cleft of CBD(N1) were evaluated by single site-directed mutations to alanine residues. Each isolated domain containing a single mutation was analyzed for binding to an insoluble amorphous preparation of cellulose, phosphoric acid swollen Avicel (PASA), and to a soluble glucopyranoside polymer, barley beta-glucan. The effect of any given mutation on CBD binding was similar for both substrates, suggesting that the mechanism of binding to soluble and insoluble substrates is the same. Tyrosines 19 and 85 were essential for tight binding by CBD(N1) as their replacement by alanine results in affinity decrements of approximately 100-fold on PASA, barley beta-glucan, and soluble cellooligosaccharides. The tertiary structures of unbound Y19A and Y85A were assessed by heteronuclear single quantum coherence (HSQC) spectroscopy. These studies indicated that the structures of both mutants were perturbed but that all perturbations are very near to the site of mutation.     

Analysis of early and late results of progressive Graves-Basedow ophthalmopathy treatment with different methods]

The early (immediately after the end of treatment) and late (from 12 to 91, mean 46.3 months after the end of treatment) results of progressive Graves-Basedow ophthalmopathy treatment were evaluated in 71 patients (57 women and 14 men, aged 25-66, mean 47.3 years). In all patients the thorough ophthalmological evaluation was performed early and late after treatment and the abnormalities found were classified according to Werner's method and Donaldson's ophthalmopathy index. The patients were divided into 7 groups according to different methods of treatment. Groups I-V consisted of 31 patients treated with glucocorticoids or glucocorticoids with azathioprine (Imuran) in the first stage of medication (30 patients). Plasmapheresis (7 patients) or tele-cobalt irradiation (3 patients) was applied as the second stage of treatment when the first stage was ineffective. In one person plasmapheresis and tele-cobalt irradiation was applied without previous glucocorticoid therapy. Two patients were treated successively by glucocorticoids, plasmapheresis and tele-cobalt irradiation. Very good and good late results of treatment were found in 95% of patients out of 22 reexamined persons of groups 1-5. Group 6 consisted of 7 patients treated with cobalt irradiation alone, in all 6 patients evaluated late results were good or very good. Out of 33 patients of group 7 treated by combination of glucocorticoids and telecobalt irradiation 23 were evaluated late and the results were found good or very good in 96%. The results suggest that combined treatment of progressive endocrine ophthalmopathy with glucocorticoids and cobalt irradiation is the most effective method of treatment at present. The affectivity of stage treatment is comparable with combined treatment but lasts longer and usually is more expansive.(ABSTRACT TRUNCATED AT 250 WORDS)     

与拟除虫菊酯抗性相关的烟粉虱钠通道基因突变及其检测

通过RT-PCR克隆了烟粉虱Bemisia tabaci (Gennadius) 南京种群(B-生物型)的钠离子通道结构域ⅡS4-6 cDNA片段,证实了与拟除虫菊酯抗性相关的是位于第925位亮氨酸到异亮氨酸的突变(L925I),并建立了L925I突变的PASA检测技术。与SUD-S敏感品系相比,2002年采自南京棉花上的烟粉虱种群对氯氰菊酯具有77倍的抗性,用氯氰菊酯对该种群进行多次筛选后,该种群对氯氰菊酯的抗药性提高到227倍。PASA检测结果表明筛选后的南京种群中100%个体都具有L925I突变(61.1%的个体为L925I突变纯合子,38.9%的个体为杂合子),而未筛选的南京种群只有75%个体具有L925I突变(35%个体为L925I突变纯合子,40%的个体为杂合子,25%的个体为野生型)。该结果表明了烟粉虱钠离子通道L925I突变与对拟除虫菊酯抗性密切相关。还讨论了烟粉虱对拟除虫菊酯抗性的代谢机理。     

Prion disease detection, PMCA kinetics, and IgG in urine from sheep naturally/experimentally infected with scrapie and deer with preclinical/clinical chronic wasting disease

Prion diseases, also known as transmissible spongiform encephalopathies, are fatal neurodegenerative disorders. Low levels of infectious agent and limited, infrequent success of disease transmissibility and PrP(Sc) detection have been reported with urine from experimentally infected clinical cervids and rodents. We report the detection of prion disease-associated seeding activity (PASA) in urine from naturally and orally infected sheep with clinical scrapie agent and orally infected preclinical and infected white-tailed deer with clinical chronic wasting disease (CWD). This is the first report on PASA detection of PrP(Sc) from the urine of naturally or preclinical prion-diseased ovine or cervids. Detection was achieved by using the surround optical fiber immunoassay (SOFIA) to measure the products of limited serial protein misfolding cyclic amplification (sPMCA). Conversion of PrP(C) to PrP(Sc) was not influenced by the presence of poly(A) during sPMCA or by the homogeneity of the PrP genotypes between the PrP(C) source and urine donor animals. Analysis of the sPMCA-SOFIA data resembled a linear, rather than an exponential, course. Compared to uninfected animals, there was a 2- to 4-log increase of proteinase K-sensitive, light chain immunoglobulin G (IgG) fragments in scrapie-infected sheep but not in infected CWD-infected deer. The higher-than-normal range of IgG levels found in the naturally and experimentally infected clinical scrapie-infected sheep were independent of their genotypes. Although analysis of urine samples throughout the course of infection would be necessary to determine the usefulness of altered IgG levels as a disease biomarker, detection of PrP(Sc) from PASA in urine points to its potential value for antemortem diagnosis of prion diseases.     

Detection of a new polymorphism of the human prothrombin (F2) gene by combination of PASA and mutated primer-mediated PCR-RFLP

A new polymorphism of the human prothrombin (F2) gene was detected by a combination of polymerase chain reaction (PCR) amplification of specific alleles (PASA) and mutated primer-mediated PCR restriction fragment length polymorphism (PCR-RFLP). The method is simple and useful for detecting polymorphisms and mutations. The new polymorphism of C1 and C2 examined by this method is highly heterozygous and serves as a good human DNA marker.     

PASA – A Program for Automated Protein NMR Backbone Signal Assignment by Pattern-Filtering Approach

We present a new program, PASA (Program for Automated Sequential Assignment), for assigning protein backbone resonances based on multidimensional heteronuclear NMR data. Distinct from existing programs, PASA emphasizes a per-residue-based pattern-filtering approach during the initial stage of the automated ¹³C^α and/or ¹³C^β chemical shift matching. The pattern filter employs one or multiple constraints such as ¹³C^α/C^β chemical shift ranges for different amino acid types and side-chain spin systems, which helps to rule out, in a stepwise fashion, improbable assignments as resulted from resonance degeneracy or missing signals. Such stepwise filtering approach substantially minimizes early false linkage problems that often propagate, amplify, and ultimately cause complication or combinatorial explosion of the automation process. Our program (http://www.lerner.ccf.org/moleccard/qin/) was tested on four representative small-large sized proteins with various degrees of resonance degeneracy and missing signals, and we show that PASA achieved the assignments efficiently and rapidly that are fully consistent with those obtained by laborious manual protocols. The results demonstrate that PASA may be a valuable tool for NMR-based structural analyses, genomics, and proteomics. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

青龙胶丸治疗血管性痴呆的临床研究

本研究用中国蛇协研制的青龙胶丸治疗血管性痴呆４０例。经二个月的治疗后,显效４例,有效１６例,改善１７例,总有效率达９２．５％。随着患者智能的改善,有８６％的病例脑电图好转。其表现为枕部α指数增加,异常慢波减少等。３６例红细胞Ｃ３ｂ受体花环形成率检测结果表明,治疗前明显低于正常值,治疗后显著提高并恢复至接近正常人的水平。提示青龙胶丸可能通过神经一内分泌－免疫网络各环节的同时改善,提高机体的红细胞免疫     

Role of the Per/Arnt/Sim domains in ligand-dependent transformation of the aryl hydrocarbon receptor

The aryl hydrocarbon receptor (AhR) mediates the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. In a process termed transformation, ligand binding converts the AhR into its high affinity DNA binding form that represents a dimer of the AhR and Arnt, a closely related nuclear protein. During transformation, protein chaperone Hsp90 is thought to be replaced by Arnt in overlapping binding sites in the basic helix loop helix and PASB domains of the AhR. Here, analysis of AhR variants containing a modified PASB domain and AhR PASA-PASB fragments of various lengths revealed (i) an inhibitory effect on transformation concomitant with Hsp90 binding in the PASB domain, (ii) an ability of the PASA-PASB fragment of the AhR to reproduce key steps in the transformation process, and (iii) a ligand-dependent conformational change in the PASA domain consistent with increased PASA exposure during AhR transformation. Based on these results, we propose a new mechanism of AhR transformation through initiation of Arnt dimerization and Hsp90 displacement in AhR PASA/B domains. This study provides insights into mechanisms of AhR transformation, dimerization of PAS domain proteins, and Hsp90 dissociation in activation of its client proteins.     

Orally administered HSV-specific transfer factor (TF) prevents genital or labial herpes relapses

Forty-four patients suffering from genital (22) and labial (22) herpes were orally treated with HSV-1/2-specific transfer factor(TF). TF was obtained by in vitro replication of a HSV-1/2-specific bovine dialysable lymphocyte extract. Treatment was administered bi-weekly the first 2 weeks, and then weekly for 6 months, most patients received 2–3 courses. The total observation period for all patients before treatment was 26660 days, with 544 relapses, and a relapse index of 61.2, whereas the cumulative observation period during and after treatment was 16945 days, with a total of 121 relapsing episodes and a cumulative RI of 21.4 (P<0.0001). Results were equally significant when the 2 groups of patients (labial and genital) were considered separately. These observations confirm previous results obtained with bovine HSV-specific TF, and warrant further studies to establish HSV-specific TF as a choice of treatment for preventing herpes recurrences.     

Predictors of Weight Loss in Adults with Topiramate‐Treated Epilepsy

Objective: We examined predictors of weight loss with topiramate, an anticonvulsant associated with weight loss in adults. Research Methods and Procedures: In this uncontrolled, prospective clinical trial, topiramate was added to existing anticonvulsants in adults (40 to 110 kg) with partial‐onset seizures. Primary measurements were change from baseline weight after 3 months and 1 year in patients completing 1 year of topiramate treatment (N = 38). Physiological and metabolic measures were analyzed for correlation with weight loss during topiramate treatment. Results: In patients who completed 1 year of topiramate treatment, baseline weight was reduced in 82% at 3 months and in 86% at 1 year. Mean body weight was reduced 3.0 kg (3.9% of baseline) at 3 months and 5.9 kg (7.3%) at 1 year. In obese patients [body mass index (BMI) ≥ 30 kg/m²], mean weight loss was 4.2 kg (4.3%) at 3 months and 10.9 kg (11.0%) at 1 year. Weight loss was primarily caused by reduction in body fat mass. For all patients, weight loss at 3 months correlated most strongly with reduced caloric intake (p = 0.02). At 1 year, caloric intake had returned to baseline levels; weight loss correlated most strongly with higher baseline BMI (p = 0.0007). Discussion: Our results suggest that weight loss occurs in most adults treated with topiramate and is sustained for at least 1 year. Reduced caloric intake may account, in part, for weight loss during early treatment. The pattern of weight loss differs according to baseline BMI, with obese patients experiencing greater weight loss during continued therapy.     

Efficacy and tolerance of Harpagophytum procumbens versus diacerhein in treatment of osteoarthritis.

In a double-blind, randomized, multicentre clinical study, the efficacy and tolerance of a herbal medicine product, Harpadol (6 capsules/day, each containing 435 mg of powdered cryoground powder Harpagophytum procumbens), was compared with diacerhein 100 mg/day in the treatment, for 4 months, of 122 patients suffering from osteoarthritis of the knee and hip. Assessments of pain and functional disability were made on a 10 cm horizontal visual analogue scale; severity of osteoarthritis was evaluated by Lequesne's index. Spontaneous pain showed a significant improvement during the course of the study and there was no difference in the efficacy of the two treatments. Similarly, there was a progressive and significant reduction in the Lequesne functional index and no statistical difference was found between Harpadol and diacerhein. At completion of the study, patients taking Harpadol were using significantly less NSAIDs and antalgic drugs. The frequency of adverse events was significantly lower in the Harpadol group. The most frequent event reported was diarrhea, occurring in 8.1% and 26.7% of Harpadol and diacerhein patients respectively. The global tolerance assessment by patients at the end of treatment favoured Harpadol. The results of this study demonstrate that Harpadol is comparable in efficacy and superior in safety to diacerhein.     

Education Program Evaluation at Multiple Primate Sanctuaries in Equatorial Africa

The past decade has witnessed a shift in the role of primate sanctuaries in Africa from warehouses of salvaged animals to proactive conservation organizations. As part of this new role, the Pan African Sanctuary Alliance (PASA) has conducted annual training workshops for educators from member sanctuaries. These workshops have included content on program development, interpretive techniques, and evaluation techniques. Here we use meta-analytic techniques to examine data from the resulting education evaluation programs for trends in knowledge acquisition across PASA sanctuaries. Question content strongly impacted the evaluation results of the conservation education program. In addition, effect size and final performance scores were independent of one another and provide different perspectives as to the effectiveness of a conservation education program. These results highlight the importance of using both final performance scores and effect size measures in conservation education program evaluation. They also underscore the importance of evaluating knowledge transfer as part of the larger picture of evaluating pro-environmental behavior change and conservation impact.     

Mapping chicken genes using preferential amplification of specific alleles

To map the chicken genome, an international reference population was developed at our laboratory (East Lansing, MI) using an F2 backcross between inbred jungle fowl (JF) and inbred white leghorns (WL). To augment the number of type I genes on the East Lansing (E) map, segregation of the JF-specific allele was followed using preferential amplification of specific alleles (PASA) in polymerase chain reactions (PCR). Among 15 functional genes that were added to the E map, agrin and mannose-6-phosphate receptor genes were found to occur in conserved syntenic groups. Using this PCR-based approach, six conserved groups spanning more than 243 centimorgans (cM) in the chicken were syntenic with human and mouse.     

Analysis of early results of combined treatment with glucocorticoids and telecobalt therapy for Graves' disease ophthalmopathy]

Early results of combined use of glucocorticoid administration and irradiation with radioactive cobalt for treatment of oedematous-infiltrative ophthalmopathy associated with Graves' disease have been analyzed in a group of 33 patients including 28 women and 5 men of age between 25 and 66 years (mean age 47.3 years). The combined therapy was a modification of the original method of Bartalena et al. which consisted in the gradual increase of the initial dose of glucocorticoids and prolongation of the period of administration of the drug. The ophthalmic lesions were assessed by thorough ophthalmologic examination and classified according to Werner. The ophthalmopathy index was calculated according to Donaldson. Satisfactory results of treatment have been obtained in 32 patients, with 9 patients being completely relieved from any objective or subjective ophthalmic symptoms (very good results), and 23 patients having still small afflictions originating from the soft tissues of the eye socket, exophthalmos, diplopia during marginal vision and a decreased visual acuity (good results). The clinical recovery was mostly connected with the improvement in the condition of soft tissues of the eye socket, cornea and external ocular muscles and, to a smaller extent, exophthalmos which persisted to some degree and acuity of vision not always attaining normal values. In one person the results of treatment were unsatisfactory despite some improvement. Very good and good results obtained in 97% of patients indicate that the administration of glucocorticoids combined with the irradiation of retrobulbar tissues with radioactive cobalt can now be regarded as the most effective method of treatment of the progressive oedematous-infiltrative ophthalmopathy.