PEX5 binds the PTS1 independently of Hsp70 and the peroxin PEX12

Most peroxisomal enzymes are targeted to peroxisomes by virtue of a type-1 peroxisomal targeting signal (PTS1) at their extreme C terminus. PEX5 binds the PTS1 through its C-terminal 40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Here we examined the PTS1-binding activity of purified, recombinant, full-length PEX5 using a fluorescence anisotropy-based assay. Like its C-terminal fragment, full-length tetrameric PEX5 exhibits high intrinsic affinity for the PTS1, with a K(d) of 35 nm for the peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO(-). The specificity of this interaction was demonstrated by the fact that PEX5 had no detectable affinity for a peptide in which the Lys was replaced with Glu, a substitution that inactivates PTS1 signals in vivo. Hsp70 has been found to regulate the affinity of PEX5 for a PTS1-containing protein, but we found that the kinetics of PEX5-PTS1 binding was unaffected by Hsp70, Hsp70 plus ATP, or Hsp70 plus ADP. In addition, we found that another protein known to interact with the PTS1-binding domain of PEX5, the PEX12 zinc RING domain, also had no discernable effect on PEX5-PTS1 binding kinetics. Taken together, these results suggest that the initial step in peroxisomal protein import, the recognition of enzymes by PEX5, is a relatively simple process and that Hsp70 most probably stimulates this process by catalyzing the folding of newly synthesized peroxisomal enzymes and/or enhancing the accessibility of their PTS1.     

Mapping the Fibrinogen-Binding Domain of Serum Opacity Factor of Group A Streptococci

Serum opacity factor (SOF) is a large, extracellular, and cell-bound protein of group A streptococci that has two known functions, opacification of serum and binding of fibronectin. Herein, we describe a new function of SOF, the binding of fibrinogen. Utilizing purified, truncated recombinant SOF proteins, the fibrinogen-binding domain was localized to a region in the C-terminus of SOF encompassing amino acid residues 844–1047. Western-blot analysis revealed that SOF bound primarily to the β subunit of fibrinogen. A SOF-negative mutant bound 50% less fibrinogen than did its wild-type parent. Furthermore, fibrinogen blocked the binding of SOF to fibronectin. These data suggest that fibrinogen and fibronectin bind to the same domain within SOF. It remains to be determined whether the binding of fibrinogen to SOF contributes to the virulence of group A streptococci. Received: 13 June 2001 / Accepted: 20 July 2001     

Osmosensitive Release of Neurotransmitter Amino Acids: Relevance and Mechanisms

Hyposmolarity activates amino acid efflux as part of the corrective volume process in a variety of cells. This review discusses the mechanism of amino acid release in brain cells preparations. Results present evidence of substantial differences between the efflux of taurine and that of GABA and glutamate, which besides a possible role as osmolytes, have a main function as synaptic transmitters. The differences found concern the efflux time course, the sensitivity to Cl^– channel blockers, the modulation by tyrosine kinases, the influence of PKC and the effect of cytoskeleton disruptive agents. While taurine efflux features fit well with the mechanisms so far described in most cell types, the efflux of GABA and glutamate does not. Alternate mechanisms for the release of these two amino acids are discussed, including a PKC-modulated, actin-dependent exocytosis.     

In vitro and in vivo assays of 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 micro g/ml for macrophages and a few of them maintained this cytotoxicity even at 10 microg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1microg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.     

In vitro and in vivo anti-Trypanosoma cruzi activity of a novel nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.     

Origin of human immunodeficiency virus type 1 quasispecies emerging after antiretroviral treatment interruption in patients with therapeutic failure

The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1 pol and env were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of pol revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from env sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.     

The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins

Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.     

Morphological Observations on Brevipalpus phoenicis (Acari: Tenuipalpidae) Including Comparisons with B. californicus and B. obovatus

The genus Brevipalpus has over 300 species worldwide. The three most important agricultural pest species in the genus, Brevipalpus californicus (Banks), B. obovatus Donnadieu, and B. phoenicis (Geijskes), have been consistently confused and misidentified for more than 50 years. The present study provides a discussion of the characters and character states used to separate these mites. Low-temperature scanning electron microscopy and traditional light microscopy techniques were used to illustrate the subtle morphological differences between these three species. Morphology of the dorsal propodosoma, opisthosoma, and leg chaetotaxy of all three species was examined and compared. The number of dorsal setae, the number of solenidia (omega) on tarsus II, and dorsal cuticular patterns were the most important characters in the identification of Brevipalpus species. B. phoenicis is similar to B. californicus in having two omega on tarsus leg II and different from B. obovatus which has only one omega on tarsus leg II and similar to B. obovatus in having only one pair of F setae (f3), but differing from B. californicus which has two pairs of F setae (f2-3). The dorsal opisthosomal and propodisomal cuticular patterns frequently used to distinguish between these three species are useful but one must be aware that age, feeding, and mounting techniques can affect the appearance of these characters.