Elevated urinary neopterin levels in patients with the acquired immunodeficiency syndrome (AIDS). A preliminary report

Neopterin excretion in urine of patients with AIDS was measured by high pressure liquid chromatography. Urinary neopterin levels in patients with generalized lymphadenopathy, which is considered to be part of the spectrum of AIDS, as well as in severe AIDS cases were significantly elevated, compared to normal controls. This finding may prove useful as a screening test for donors of blood products in order to prevent transmission of AIDS by this route.     

Hybridization screening of very short PCR products for paleoepidemiological studies of Chagas' disease

Single strands of very short PCR products can be covalently immobilized to a slide and then easily detected by probe hybridization. In this work, the PCR product was a 70-nucleotide segment of ancient DNA, representing a portion of repeat mini-circle DNA from the kinetoplast of Trypanosoma cruzi, the infectious agent of Chagas' disease (American Trypanosomiasis). The target segment was initially established to be present in soft tissue samples taken from four "naturally" mummified Andean bodies using PCR followed by cloning and sequencing. Hybridization screening of the covalently immobilized PCR products positively identified products from 25 of 27 specimens of different tissues from these four mummies. The method appears to be ideal for the purpose of screening a large number of specimens when the target PCR product is very short.     

植物提取物和常用药剂对蚜茧蜂的存活、羽化和寄生的影响

天然源药剂由于具有与环境良好的相容性而日益受到重视。研究测试苍耳XanthiumsibiricumPetr.etWidd.和白蝴蝶SyngoniumpodophyllumSchott的提取物以及几种药剂在田间常用浓度下对蚜茧蜂的影响。结果表明,几种药剂对蚜茧蜂成虫有明显杀伤力,毒性的大小次序是绿福(4.5%高效顺反氯氰菊酯乳油)(稀释3000倍)>0.3%印楝素乳油(稀释2000倍)>0.6%阿维菌素(稀释2000倍),2h内可致半数以上的试虫死亡,2.5%鱼藤酮精(稀释800倍)次之,99.9%机油乳剂(稀释400倍)和两种植物提取物(0.04gDW·ml-1)对成蜂的存活无显著影响;处理僵蚜的结果表明,印楝素乳油和机油乳剂影响较明显,僵蚜羽化率分别比对照降低14.67%和18.67%,而两种植物提取物和其余药剂无显著影响;鱼藤精和印楝素乳油处理后,蚜茧蜂寄生蚜虫的效能明显降低,而机油乳剂和两种植物提取物对蚜茧蜂的寄生作用无明显影响。可以看出,目前使用的一些天然源药剂虽然对害虫有较好的控制作用,但在害虫天敌的某些生长期,其仍存在不利影响,因此应根据田间害虫和天敌的发生情况和不同发育期,有选择性地合理用药;同时,筛选对害虫特异性强,对害虫天敌不利影响小的作用物质,是当前乃至将来筛选和应用天然源物质的目标。     

Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.     

LCA of food products and production systems

This article is a summary of my dissertation in which LCA was applied to food products and production systems. The overall objectives were: (1) to learn more about the feasibility and limitations of LCAs of systems for the production and consumption of foods (food systems); and (2) to generate information on the environmental impact of such systems. Case studies of tomato ketchup and white bread were carried out. The main conclusion is that LCA is very valuable for incorporating environmental aspects in the development of more sustainable food systems. One of the major problems encountered was the great scarcity of environmental data. It was found that there is a need for simplified methods that can be used as a compass to show the direction towards sustainability. Accordingly, the feasibility of combinng the concept of sustainabiliry principles and LCA for product development was examined and discussed. This combination was found to yield a simplified method well suited for screening analysis and product development.     

New aspects of natural products in drug discovery

During the past 15 years, most large pharmaceutical companies have decreased the screening of natural products for drug discovery in favor of synthetic compound libraries. Main reasons for this include the incompatibility of natural product libraries with high-throughput screening and the marginal improvement in core technologies for natural product screening in the late 1980s and early 1990 s. Recently, the development of new technologies has revolutionized the screening of natural products. Applying these technologies compensates for the inherent limitations of natural products and offers a unique opportunity to re-establish natural products as a major source for drug discovery. Examples of these new advances and technologies are described in this review.     

The global HIV vaccine enterprise.

AIDS, which twenty-five years ago no one even knew it existed, has become the most serious infectious disease worldwide. The development of an HIV vaccine is one of the most difficult challenges that modern biomedical science is confronting. To address this challenge, scientists may need to organize themselves in a more intense, targeted, and collaborative effort, such as the one proposed by the Global HIV/AIDS Vaccine Enterprise. The enterprise concept proposes to complement the creativity of individual investigators with a collaborative system that ensures a more effective use of human and financial resources to produce new scientific knowledge. It also implies that the scientific knowledge can be harnessed in a targeted way to develop practical solutions to urgent global health problems, including explicit product development activities. Different modalities of the enterprise concept are being explored for the development of drugs to treat tuberculosis and vaccines to prevent malaria.     

Scottish attitudes to blood donation and AIDS.

OBJECTIVE--To see whether the issue of AIDS has influenced the observed decline in blood donation in Scotland. DESIGN--Two methods: a quantitative survey using personal interviews based on a questionnaire and a qualitative survey based on group discussions. SETTING--Interviews based on the questionnaire were conducted in the respondents'' homes. The group discussions were held in the homes of professional market research interviewers. PARTICIPANTS--For the quantitative survey a representative sample of 976 Scottish adults was selected by multistage sampling. In the qualitative survey 16 groups of five to eight respondents assigned according to donating experience and sociodemographic criteria took part. MAIN RESULTS--AIDS was not mentioned as a discouraging factor in donation, and off putting aspects identified before AIDS became a public issue remained salient--for example, fear of needles. Many (75%) thought it unlikely that donation entailed a risk of developing AIDS. Nevertheless, respondents were reluctant to consider the AIDS issue personally. Being asked to do so, as in the routine screening of donors, aroused fears and resentment. CONCLUSIONS--The issue of AIDS, including fear of infection, has not directly influenced the declining numbers of donors, but the unpleasant associations of AIDS have had an indirect effect, particularly in undermining the emotional benefits of giving blood. For example, the screening process, which requires potential donors to consider their personal risk from AIDS, had had the effect of discouraging donors in general. Redressing the balance is difficult as screening must be universally applied. Rather than minimising the issue of AIDS, publicity needs urgently to reassert the positive benefits of and rewards from giving blood.     

Virally induced CD4+ T cell depletion is not sufficient to induce AIDS in a natural host

Peripheral blood CD4+ T cell counts are a key measure for assessing disease progression and need for antiretroviral therapy in HIV-infected patients. More recently, studies have demonstrated a dramatic depletion of mucosal CD4+ T cells during acute infection that is maintained during chronic pathogenic HIV as well as SIV infection. A different clinical disease course is observed during the infection of natural hosts of SIV infection, such as sooty mangabeys (Cercocebus atys), which typically do not progress to AIDS. Previous studies have determined that SIV+ mangabeys generally maintain healthy levels of CD4+ T cells despite having viral replication comparable to HIV-infected patients. In this study, we identify the emergence of a multitropic (R5/X4/R8-using) SIV infection after 43 or 71 wk postinfection in two mangabeys that is associated with an extreme, persistent (>5.5 years), and generalized loss of CD4+ T cells (5-80 cells/microl of blood) in the absence of clinical signs of AIDS. This study demonstrates that generalized CD4+ T cell depletion from the blood and mucosal tissues is not sufficient to induce AIDS in this natural host species. Rather, AIDS pathogenesis appears to be the cumulative result of multiple aberrant immunologic parameters that include CD4+ T cell depletion, generalized immune activation, and depletion/dysfunction of non-CD4+ T cells. Therefore, these data provide a rationale for investigating multifaceted therapeutic strategies to prevent progression to AIDS, even following dramatic CD4 depletion, such that HIV+ humans can survive normal life spans analogous to what occurs naturally in SIV+ mangabeys.     

结直肠癌无创筛查技术研究进展

结直肠癌和高危腺瘤的早期诊断及治疗,能够大大降低其死亡率。因而,在临床普及结直肠癌筛查有助于遏制该疾病的危害。目前,结肠镜检查是结直肠癌诊断的金标准,但该方法需要肠道准备,且具有侵入性,易造成肠道穿孔等缺点,导致患者依从性差,不利于其作为大规模筛查技术推广实施。近年来非侵入性的结直肠癌诊断方法发展迅速,这类方法主要通过检测粪便或血液样本中与结直肠癌发生相关的生物标志物,为结直肠癌的无创筛查提供了可能。但由于粪便、血液样本的成分复杂,对其中生物标志物的检测技术仍然在不断研究和完善中。本文分别从基于粪便样本和血液样本的检测技术两方面入手,探讨了近年来结直肠癌无创筛查技术的研究进展。     

Pharmacogenetics place in modern medical science and practice

Pharmacogenetic evidence-based treatment strategies will have major implications for all aspects of the product pipeline, including drug discovery, high throughput target screening protocols, lead optimization, and drug formulation to produce series of medicines for a particular disease which will meet the efficacy needs of the majority of patients. The initial proof of principle experiments involves whole genome screening for DNA variants and determination of specific patterns of variants associated with adverse events of marketed products [SNP Print(sm)]. Pharmacogenetics has the potential of changing the pipeline model of drug discovery, clinical development, and mass customization marketing.     

Soyasaponin I, a potent and specific sialyltransferase inhibitor

A growing number of reports demonstrate that hypersialylation, which is observed in certain pathological processes, such as oncogenic transformation, tumor metastasis, and invasion, is associated with enhanced sialyltransferase (ST) activity. There is therefore a need for the development of ST inhibitors to modulate ST activity and thus alleviate the disease processes caused by STs. In the present study, soyasaponin I had been discovered to be a potent and specific ST inhibitor by screening strategy from 7500 samples including micribial extracts and natural products. Kinetic analysis shows that it is a CMP-Neu5Ac competitive inhibitor with for ST3Gal I with an inhibition constant (K(i)) of 2.1 microM. In addition, it is only active against ST, but not against the other tested glycosyltransferases and glycosidases. Our study is the first report to discover ST inhibitor by screening method and also to provide the new chemical structure information that should be useful in the development of other novel ST inhibitors.     

Genomic instability and AIDS]

The biochemical mechanisms underlying blood lymphoid cell genome destabilization in patients with HIV infection have been analyzed. Lymphocytes from HIV patients are characterized by increasing intensity of free radical oxidation together with activation of the xanthine oxidase D-form conversion into the O-form, enhanced activity of UV-endonuclease, and intensification of prooxidant-induced proteolysis. These changes increasing with the progress of the disease with a maximum at the AIDS stage form a metabolic basis for labilization of the lymph cell genome. The degree of biochemical manifestations of genome instability (levels of chromatin degradation products and intensity of formation of one-filament nicks of DNA) increase in the dynamics of HIV-infection. The data obtained are discussed in terms of the author's conception on the origin of AIDS from retroposons (retrotransposons?). A hypothesis is postulated on accumulation of autonomous genetic information on the basis of genome labilization under the influence of genotoxic factors. Clinico-biochemical data on the appearance of HIV proteins (p17, p24) in the blood of patients (previously negative for all HIV markers) in the presence of transfusions of HIV-negative blood and UV-irradiation of the autoblood are also discussed from this standpoint.     

Plant natural products: Back to the future or into extinction?

Natural product substances have historically served as the most significant source of new leads for pharmaceutical development. However, with the advent of robotics, bioinformatics, high throughput screening (HTS), molecular biology-biotechnology, combinatorial chemistry, in silico (molecular modeling) and other methodologies, the pharmaceutical industry has largely moved away from plant derived natural products as a source for leads and prospective drug candidates. Can, or will, natural products ever recapture the preeminent position they once held as a foundation for drug discovery and development? The challenges associated with development of natural products as pharmaceuticals are illustrated by the Taxol^® story. Several misconceptions, which constrain utilization of plant natural products, for discovery and development of pharmaceuticals, are addressed to return natural products to the forefront.     

Blood transfusion and acquired immunodeficiency syndrome (AIDS)

This report was presented at the June 1983 meeting of the Blood Transfusion National Consultative Commission at the French Ministry of Health. Clinical and epidemiological data on AIDS as well as problems raised by the lack of specific tests for screening of blood donors were briefly summarized. Out of 49 AIDS patients recorded in France up to April 1983, only one had a history of previous blood transfusion given in Haiti, 4 years before the clinical onset of the disease. Blood donors, all Haitians, had no sign and symptom of AIDS. Retrospective review of 2 300 hemophiliacs followed up in France until April 1983 disclosed no AIDS. However, in 6 patients, the following features, more or less associated, were found to be present: thrombocytopenia, neutropenia, micropolyadenopathy, splenomegaly, hypergammaglobulinemia and low OKT4/OKT8 ratio. No clear correlation could be found between these abnormalities and the origin, commercial or national, of the coagulation factor concentrates used for the treatment. Three main recommendations were proposed: -- information of blood donors and experimental evaluation of some non specific screening tests, in the at risk donor population. -- more cautious use of coagulation factor concentrates -- reduction of importations aiming at complete national self sufficiency concerning factor VIII concentrates.     

Combinatorial synthesis of natural products

Combinatorial syntheses allow production of compound libraries in an expeditious and organized manner immediately applicable for high-throughput screening. Natural products possess a pedigree to justify quality and appreciation in drug discovery and development. Currently, we are seeing a rapid increase in application of natural products in combinatorial chemistry and vice versa. The therapeutic areas of infectious disease and oncology still dominate but many new areas are emerging. Several complex natural products have now been synthesised by solid-phase methods and have created the foundation for preparation of combinatorial libraries. In other examples, natural products or intermediates have served as building blocks or scaffolds in the synthesis of complex natural products, bioactive analogues or designed hybrid molecules. Finally, structural motifs from the biologically active parent molecule have been identified and have served for design of natural product mimicry, which facilitates the creation of combinatorial libraries.     

Design and synthesis of substrates for newborn screening of Maroteaux–Lamy and Morquio A syndromes

In continued efforts to develop enzymatic assays for lysosomal storage diseases appropriate for newborn screening laboratories we have synthesized novel and specific enzyme substrates for Maroteaux–Lamy (MPS VI) and Morquio A (MPS IVA) diseases. The sulfated monosaccharide derivatives were found to be converted to product by the respective enzyme in blood from healthy patients but not by blood from patients with the relevant lysosomal storage disease. The latter result shows that the designed substrates are highly selective for the respective enzymes.     

Directed Evolution of Toluene Dioxygenase from Pseudomonas putida for Improved Selectivity Toward cis-Indandiol during Indene Bioconversion

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S, 2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S, 2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.