Mossbauer investigation of shocked and unshocked iron meteorites and fayalite.

Mössbauer spectra of several iron meteorites have been measured by a resonant scattering technique rather than by the conventional transmission method, thereby eliminating the necessity for the preparation of thin samples. No significant differences were observed in the spectra of specimens of mechanically deformed, shocked, and unshocked iron meteorites, nor in the absorption spectra of artificially shocked and unshocked fayalite.

The use of mucolysed induced sputum for the identification of pulmonary pathogens associated with human immunodeficiency virus infection.

We describe a system for diagnosis of pulmonary disease in the human immunodeficiency virus-infected patient using induced sputum and other diagnostic procedures. This system has been successfully used at San Francisco (Calif) General Hospital for more than 2 years. It utilizes outpatient facilities and reduces the need for bronchoscopy. Sputum induced by inhalation of 3% saline mist, mucolysed, concentrated by centrifugation, and stained by a rapid modified Giemsa stain was the first diagnostic specimen examined in 404 episodes of suspected human immunodeficiency virus-associated pulmonary disease in 358 patients. Pneumocystis carinii was found in 222 (55%) sputum specimens. In 118 episodes in which the sputum did not contain P carinii, bronchoscopy with transbronchial biopsy and/or bronchoalveolar lavage was performed and P carinii was found in 50 (42%). These 118 bronchoscopy results, as well as evaluation of the subsequent clinical course of those patients who accounted for 64 episodes of lung disease and who did not have bronchoscopy following examination of nondiagnostic induced sputum, indicated a range of sensitivity for detection of P carinii in induced sputum of 74% to 77% and a negative predictive value of 58% to 64%. Mycobacteria were recovered from 11 (6%) of the induced sputum and 6 (12%) of the bronchoscopic specimens containing P carinii. However, only oral or environmental fungi were recovered from P carinii-containing induced sputum or bronchoscopic specimens. For those patients in whom P carinii was not detected, only the bronchoscopic specimens were cultured for Mycobacteria and fungi. Potentially pathogenic Mycobacteria and fungi were recovered from 16 (23.5%) and 34 (50%), respectively, of these P carinii-negative specimens. Analysis of these results, obtained under routine practice conditions, indicates that bronchoscopy should be reserved for those patients whose induced sputum examinations do not show P carinii and that mycobacterial and fungal cultures be performed only on bronchoscopic specimens in which P carinii is not detected.

Fatty acid synthesis in protozoan parasites: unusual pathways and novel drug targets.

Fatty acid biosynthesis pathways in protozoan parasites are reviewed with a view to targeting this metabolism for drug therapy. The type II fatty acid biosynthesis pathways derived from bacteria in protozoan relict plastids and mitochondria are examined in different groups with emphasis on apicomplexa. The suitability of different enzymes from the type II fatty acid biosynthesis pathway for drug intervention, and the state-of-play with known and potential inhibitors is explored. The type I acid biosynthesis pathways that occur in select protozoan parasites and their potential for inhibition using anti-tumour and obesity management compounds currently in development are also examined. Pathways used by parasites to scavenge and modify host lipids are also described briefly and their potential for therapeutics discussed.

Fatty acid biosynthesis as a drug target in apicomplexan parasites.

Apicomplexan parasitic diseases impose devastating impacts on much of the world's population. The increasing prevalence of drug resistant parasites and the growing number of immuno-compromised individuals are exacerbating the problem to the point that the need for novel, inexpensive drugs is greater now than ever. Discovery of a prokaryotic, Type II fatty acid synthesis (FAS) pathway associated with the plastid-like organelle (apicoplast) of Plasmodium and Toxoplasma has provided a wealth of novel drug targets. Since this pathway is both essential and fundamentally different from the cytosolic Type I pathway of the human host, apicoplast FAS has tremendous potential for the development of parasite-specific inhibitors. Many components of this pathway are already the target for existing antibiotics and herbicides, which should significantly reduce the time and cost of drug development. Continuing interest--both in the pharmaceutical and herbicide industries--in fatty acid synthesis inhibitors proffers an ongoing stream of potential new anti-parasitic compounds. It has now emerged that not all apicomplexan parasites have retained the Type II fatty acid biosynthesis pathway. No fatty acid biosynthesis enzymes are encoded in the genome of Theileria annulata or T. parva, suggesting that fatty acid synthesis is lacking in these parasites. The human intestinal parasite Cryptosporidium parvum appears to have lost the apicoplast entirely; instead relying on an unusual cytosolic Type I FAS. Nevertheless, newly developed anti-cancer and anti-obesity drugs targeting human Type I FAS may yet prove efficacious against Cryptosporidium and other apicomplexans that rely on this Type I FAS pathway.

AN9, a petunia glutathione S-transferase required for anthocyanin sequestration, is a flavonoid-binding protein.

AN9 is a glutathione S-transferase from petunia (Petunia hybrida) required for efficient anthocyanin export from the site of synthesis in the cytoplasm into permanent storage in the vacuole. For many xenobiotics it is well established that a covalent glutathione (GSH) tag mediates recognition of molecules destined for vacuolar sequestration by a tonoplast-localized ATP-binding cassette pump. Here we inquired whether AN9 catalyzes the formation of GSH conjugates with flavonoid substrates. Using high-performance liquid chromatography analysis of reaction mixtures containing enzyme, GSH, and flavonoids, including anthocyanins, we could detect neither conjugates nor a decrease in the free thiol concentration. These results suggest that no conjugate is formed in vitro. However, AN9 was shown to bind flavonoids using three assays: inhibition of the glutathione S-transferase activity of AN9 toward the common substrate 1-chloro 2,4-dinitrobenzene, equilibrium dialysis, and tryptophan quenching. We conclude that AN9 is a flavonoid-binding protein, and propose that in vivo it serves as a cytoplasmic flavonoid carrier protein.

Identification and analysis of a class 2 alpha-mannosidase from Aspergillus nidulans.

A Class 2 alpha-mannosidase gene was cloned and sequenced from the filamentous fungus Aspergillus nidulans. A portion of the gene was amplified using degenerate oligonucleotide primers which were designed based on similarity between the Saccharomyces cerevisiae vacuolar and rat ER/cytosolic Class 2 protein sequences. The PCR amplification product was used to isolate the full length gene, and DNA sequencing revealed a 3383 bp coding region containing three introns. The predicted 1049 amino acid reading frame contained six potential N-glycosylation sites and encoded a protein of 118 kDa. The protein sequence did not appear to encode a typical fungal signal sequence or membrane spanning domain. Although the cellular location of the A.nidulans mannosidase was not determined, experimental evidence suggested that it was located within a subcellular organelle. The Matchbox sequence similarity matrix indicated that the A.nidulans protein sequence was more highly similar to the rat ER/cytosolic (Rij = 0.33) and S.cerevisiae vacuolar alpha-mannosidases (Rij = 0.43) than the rat and yeast sequences were to each other (Rij = 0.29). These three enzymes were found to be distantly related to other Class 2 sequences, and compose a third subgroup of Class 2 alpha-mannosidases, as shown by ClustalW sequence alignment.

Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases.

Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1.

Evaluation of an indirect fluorescent-antibody stain for detection of Pneumocystis carinii in respiratory specimens.

Two prospective studies were undertaken to evaluate a commercial indirect fluorescent-antibody (IFA) stain for the detection of Pneumocystis carinii in respiratory specimens from individuals at risk for or with the acquired immunodeficiency syndrome. The first study compared IFA with Diff-Quik (DQ; a rapid Giemsa-like stain) for detecting P. carinii in 95 induced sputa obtained from 77 asymptomatic patients who had survived one previous episode of P. carinii pneumonia and who were being treated prophylactically with aerosolized pentamidine. Only one induced sputum specimen was found to contain P. carinii; organisms were detected by both stains. The second study compared the performance of the IFA stain versus DQ, modified toluidine blue O, and Gomori methenamine silver stains for detecting P. carinii in symptomatic individuals at risk for or with acquired immunodeficiency syndrome. Of 182 specimens examined, P. carinii was detected in 105 by one or more stains; the DQ stain detected 73 (70%), the modified toluidine blue O stain detected 75 (71%), the Gomori methenamine silver stain detected 76 (72%), and the IFA stain detected 95 (90%). The IFA stain was more sensitive (P less than 0.01) than the other traditional stains for detecting P. carinii; however, a subsequent clinical evaluation revealed that a subset of IFA-positive-only specimens were from patients whose clinical symptoms resolved without specific anti-P. carinii therapy.