Basic biology of Pneumocystis carinii: a mini review

Basic aspects of cell biology of Pneumocystis carinii are reviewed with major emphasis on its life cycle and the structural organization of the trophozoites and cyst forms. Initially considered as a protozoan it is now established that Pneumocystis belongs to the Fungi Kingdom. Its life cycle includes two basic forms: (a) trophozoites, which are haploid cells that divide by binary fission and may conjugate with each other forming an early procyst and (b) cysts where division takes place through a meiotic process with the formation of eight nuclei followed by cytoplasmic delimitation and formation of intracystic bodies which are subsequently released and transformed into trophozoites. Basic aspects of the structure of the two developmental stages of P. carinii are reviewed.     

Lethargic crab disease: multidisciplinary evidence supports a mycotic etiology

Although lethargic crab disease (LCD) is causing massive mortalities in populations of the mangrove crab Ucides cordatus of Northeastern Brazil, the identity of its etiological agent was hitherto unknown. In this study we provide robust evidence suggesting that LCD is caused by an anamorph Ascomycota (Fungi). We examined specimens of U. cordatus collected from stocks affected by LCD. Histological and TEM methods detected the presence of hyphae, conidia, and condiophores in several host tissues. Moreover, the abundance of fungal stages is negatively associated with crab health. Finally, DNA was isolated from the fungus and a region of its 18S ribosomal gene was sequenced Phylogenetic analyses not only confirm the diagnosis of the LCD fungus in crab tissues as an ascomycete, but also suggest a close relationship with members of the subphylum Pezizomycotina.     

Cloning and characterization of the phosphoglucomutase of Trypanosoma cruzi and functional complementation of a Saccharomyces cerevisiae PGM null mutant

Trypanosoma cruzi is the etiological agent of Chagas' disease, a chronic illness characterized by progressive cardiomyopathy and/or denervation of the digestive tract. The parasite surface is covered with glycoconjugates, such as mucin-type glycoproteins and glycoinositolphospholipids (GIPLs), whose glycans are rich in galactopyranose (Galp) and/or galactofuranose (Galf) residues. These molecules have been implicated in attachment of the parasite to and invasion of mammalian cells and in modulation of the host immune responses during infection. In T. cruzi, galactose (Gal) biosynthesis depends on the conversion of uridine diphosphate (UDP)-glucose (UDP-Glc) into UDP-Gal by an NAD-dependent reduction catalyzed by UDP-Gal 4-epimerase. Phosphoglucomutase (PGM) is a key enzyme in this metabolic pathway catalyzing the interconversion of Glc-6-phosphate (Glc-6-P) and Glc-1-P which is then converted into UDP-Glc. We here report the cloning of T. cruzi PGM, encoding T. cruzi PGM, and the heterologous expression of a functional enzyme in Saccharomyces cerevisiae. T. cruzi PGM is a single copy gene encoding a predicted protein sharing 61% amino acid identity with Leishmania major PGM and 43% with the yeast enzyme. The 59-trans-splicing site of PGM RNA was mapped to a region located at 18 base pairs upstream of the start codon. Expression of T. cruzi PGM in a S. cerevisiae null mutant-lacking genes encoding both isoforms of PGM (pgm1Delta/pgm2Delta) rescued the lethal phenotype induced upon cell growth on Gal as sole carbon source.     

Impurity analysis of retinoic acid samples

The structure of an impurity contained in samples of all trans-retinoic acid was established by means of NMR and MS spectra, and confirmed by X-ray diffraction analysis. The chemical structure of the impurity 2 was found to be strictly correlated to the synthetic procedure employed for the preparation of the retinoic acid samples. Single crystal analysis allowed us to characterise the molecular conformation and the crystal structure of 2.     

Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production

Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species.     

Novel and simple high-performance liquid chromatographic method for determination of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity

We present here a high-performance liquid chromatographic method for the evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. The automated method was applied to fungal and mouse liver extracts and validated by the addition of mevastatin to the reaction mixture and by several intra- and inter-day assays. This method offers important advantages over those previously reported because no radiolabeled substrates or expensive techniques such as mass spectrometry are required, and the time of analysis is relatively short. Moreover, the method can be successfully applied to different biological samples; hence, it should be very useful in evaluating potential inhibitors of the HMG-CoA enzyme and investigating cholesterol metabolism, cell growth and differentiation processes.     

A gene from the mesophilic bacterium Dehalococcoides ethenogenes encodes a novel mannosylglycerate synthase

Mannosylglycerate (MG) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes. In this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (MGSD) encoded in the genome of the bacterium Dehalococcoides ethenogenes. mgsD encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (MPGS, EC 2.4.1.217) and to mannosyl-3-phosphoglycerate phosphatase (MPGP, EC 3.1.3.70), which catalyze the consecutive synthesis and dephosphorylation of mannosyl-3-phosphoglycerate to yield MG in Pyrococcus horikoshii, Thermus thermophilus, and Rhodothermus marinus. The bifunctional MGSD was overproduced in Escherichia coli, and we confirmed the combined MPGS and MPGP activities of the recombinant enzyme. The optimum activity of the enzyme was at 50 degrees C. To examine the properties of each catalytic domain of MGSD, we expressed them separately in E. coli. The monofunctional MPGS was unstable, while the MPGP was stable and was characterized. Dehalococcoides ethenogenes cannot be grown sufficiently to identify intracellular compatible solutes, and E. coli harboring MGSD did not accumulate MG. However, Saccharomyces cerevisiae expressing mgsD accumulated MG, confirming that this gene product can synthesize this compatible solute and arguing for a role in osmotic adjustment in the natural host. We did not detect MGSD activity in cell extracts of S. cerevisiae. Here we describe the first gene and enzyme for the synthesis of MG from a mesophilic microorganism and discuss the possible evolution of this bifunctional MGSD by lateral gene transfer from thermophilic and hyperthermophilic organisms.     

Effect of indinavir used alone or in double or triple combination with AZT and ddC on human immune functions

Indinavir (IDV) is a potent and selective human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) widely used in antiretroviral therapy, but its effects on the immune system are relatively unknown. In this study we have investigated the in vitro effect of IDV on normal human peripheral blood mononuclear cells (PBMC). We used the drug alone or in double and triple combination with AZT and ddC to assess whether IDV interferes with the previously observed immunomodulatory effects induced by AZT and ddC. We found that proliferative response, induction of immunoglobulins (Ig) production and cytokine production was not modulated by IDV. More importantly, IDV used in double or triple combination with AZT and ddC, does not further strenghten the inhibition of proliferative response induced by AZT and is able to abrogate the inhibitory effect induced by ddC on proliferative response. Similarly, IDV/AZT, IDV/ddC and IDV/AZT/ddC combinations does not strenghten the modulation of TNF-alpha, IFN-gamma and IL-4 induced by AZT, ddC and AZT/ddC. On the other hand, IDV neutralizes the up-regulating effects of AZT on IL-2 production while the up-regulating effects of ddC on IL-2 production is not affected. These data suggest that IDV used in combination with AZT and ddC did not add any further immunotoxicity.