Characterization of a GTP-binding protein in the ADP-ribosylation factor subfamily from Leishmania tarentolae

We report the cloning and characterization of a gene, LtARL, which encodes a small GTP-binding protein, from the protozoan Leishmania tarentolae. Hybridization analysis of genomic DNA under high stringency conditions indicates the single-copy nature of LtARL. LtARL is transcribed and yields a approximately 0.9 kb mRNA that is processed at the 5' end by trans-splicing. When expressed in Escherichia coli, LtArl binds GTP with a low stoichiometry and in a phospholipid-independent manner. Based on the greatest sequence identity with Homo sapiens Arl3 and lipid-independent binding of guanine nucleotides we designate this gene LtARL and the encoded protein LtArl.     

RNA-protein interactions in the ribonucleoprotein T-complexes in a mitochondrial extract from Leishmania tarentolae

We have investigated protein-RNA interactions and the incorporation of [alpha-32P]UTP into the guide RNA and mRNA components of the 'T-complexes' in a mitochondrial extract from Leishmania tarentolae. The terminal uridylyl transferase-containing complex T-IV is probably involved in the maturation of the 3'-oligo(U) tail of the gRNAs, but the biological function and biochemical nature of the remaining T-complexes is not known. We have found that the relative extent of labeling of the RNA components is dependent on the UTP concentration: at low levels, the main endogenous RNA components labeled are the gRNAs in T-IV; at higher levels, the mRNAs in all of the T-complexes are preferentially labeled. We also show a tentative correlation in the migration pattern of UTP-labeled T-complexes and complexes which bind exogenous labeled RNA. The relative extent of binding to specific complexes is dependent upon the type of RNA. Most of the interactions between the labeled RNAs and proteins can be disrupted by heparin or a large excess of rRNA, but two labeled complexes were resistant to competition. Most of the binding of labeled exogenous gRNA is disrupted by competition with a large excess of rRNA, but predigestion of the extract with micrococcal nuclease and saturation with rRNA uncovered a high affinity complex, which involves at least two proteins interacting with the bound gRNAs. A knowledge of the RNA and protein components may aid in understanding the biological roles of these RNP complexes.     

Purification and characterization of MAR1. A mitochondrial associated ribonuclease from Leishmania tarentolae

A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mitochondrial extract of Leishmania tarentolae and the gene cloned. The purified nuclease has a Km of 100-145 +/- 33 nM and a Vmax of 1.8-2.9 +/- 2 nmol/min, depending on the RNA substrate, and yields a 3'-OH and a 5'-phosphate. Cleavage was limited to several specific sites in the substrate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was affinity-purified. The cleavage specificity of the recombinant enzyme from L. tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820-kilobase pair chromosome and contains an open reading frame of 579 nucleotides. The 18-amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Data base searching revealed two homologues of MAR1 corresponding to unidentified open reading frames in Caenorhabditis elegans (GenBankTM accession number Z69637) and Archaeoglobus fulgidus (GenBankTM accession number AE000943). The function of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be determined.     

An ATP-dependent As(III)-glutathione transport system in membrane vesicles of Leishmania tarentolae

Membrane preparations enriched in plasma membrane vesicles prepared from promastigotes of Leishmania tarentolae were shown to accumulate thiolate derivatives of 73As(III). Free arsenite was transported at a low rate, but rapid accumulation was observed after reaction with reduced glutathione (GSH) conditions that favor the formation of As(GS)3. Accumulation required ATP but not electrochemical energy, indicating that As(GS)3 is transported by an ATP-coupled pump. Pentostam, a Sb(V)-containing drug that is one of the first-line therapeutic agents for treatment of leishmaniasis, inhibited uptake after reaction with GSH. Vesicles prepared from a strain in which both copies of the pgpA genes were disrupted accumulated As(GS)3 at wild-type levels, demonstrating that the PgpA protein is not the As(GS)3 pump. These results have important implications for the mechanism of drug resistance in the trypanosomatidae, suggesting that a plasma membrane As(GS)3 pump catalyzes active extrusion of metal thiolates, including the Pentostam-glutathione conjugate.     

Sequence of a 39,411 bp DNA fragment covering the left end of chromosome VII of Saccharomyces cerevisiae

We have sequenced a DNA fragment of 39,411 bp which includes part of the left telomere of chromosome VII of Saccharomyces cerevisiae. We have identified 19 open reading frames (ORFs); six correspond to known yeast genes (ADH4, FZF1, HKB, RTG2, HFM1 and PDE1), nine have similarity with other genes and four exhibit no significant similarity with any known gene. The average size of these ORFs seems to be related to their location, the eight ORF's nearest the telomere being shorter than the 11 others. These two groups of genes are separated by a region of 4.5 kb devoid of significant ORFs. One ORF, NRF120, is a new member of the seripauperine family, represented once in all sequenced yeast chromosomes, in a subtelomeric location.     

In vivo import of unspliced tRNATyr containing synthetic introns of variable length into mitochondria of Leishmania tarentolae

The mitochondrial genomes of trypanosomatids lack tRNA genes. Instead, mitochondrial tRNAs are encoded and synthesized in the nucleus and are then imported into mitochondria. This also applies for tRNATyr, which in trypanosomatids contains an 11 nt intron. Previous work has defined an exon mutation which leads to accumulation of unspliced precursor tRNATyr. In this study we have used the splicing-deficient tRNATyr as a vehicle to introduce foreign sequences into the mitochondrion of Leishmania tarentolae. The naturally occurring intron was replaced by synthetic sequences of increasing length and the resulting tRNATyr precursors were expressed in transgenic cell lines. Whereas stable expression of precursor tRNAsTyr was obtained for introns up to a length of 76 nt, only precursors having introns up to 38 nt were imported into mitochondria. These results demonstrate that splicing-deficient tRNATyr can be used to introduce short synthetic sequences into mitochondria in vivo. In addition, our results show that one factor which limits the efficiency of import is the length of the molecule.     

Role of intrinsic width in fragment momentum distributions in heavy ion collisions

It is demonstrated that the intrinsic widths incorporating correlations in conjunction with dynamical contributions give better agreement with experiments for collisions in the energy range of 200A MeV to 2A GeV than using only intrinsic widths without correlations. The sensitivity of the intrinsic width decreases with increasing projectile mass. A simple recipe for calculating intrinsic width with correlations is presented.     

The reaction kinetics of gastric intrinsic factor and IgG intrinsic factor blocking antibody: a soluble system

Three cases of high jugular bulb are presented. On of these occurred as a mass in the external auditory canal simulating an osteoma, and to our knowledge is the first reported case of a high jugular bulb presenting in the external canal. The clinical manifestations are described, and the value of polytomography as an aid to diagnosis is stressed.     

Role of the position of unsaturation on the phase behavior and intrinsic curvature of phosphatidylethanolamines

The bilayer-to-hexagonal phase transition temperatures (T(H)) of di-18:1(C) phosphatidylethanolamine with double bonds at positions 6, 9, and 11 are 37 degrees C, 8 degrees C, and 28 degrees C, respectively, as measured by differential scanning calorimetry and x-ray diffraction. Thus T(H) exhibits a minimum when the C=C is around position 9, similar to what has been found for the gel-to-liquid crystalline phase transition temperature in other lipids. Factors that may contribute to the dependence of T(H) on double bond position were studied by x-ray diffraction of the hexagonal phases in the presence and absence of added alkane, with or without the osmotic stress of polyethylene glycol, and over a wide temperature range. The lattice dimensions show that the intrinsic radius of lipid monolayer curvature increases as the double bond is moved toward the tail ends. A measure of the bending moduli of these lipid monolayers shows a higher value for the 9 position, and lower values for the other two. Consideration of the bilayer-to-hexagonal transition in terms of bending and interstitial energies provides a rationale for the relative values of T(H).     

Activity of pentamidine-loaded methacrylate nanoparticles against Leishmania infantum in a mouse model

Polyvinylpyrrolidone (PVP), formerly a plasma expander, has continued to be inappropriately used in Taiwan for intravenous injection as a "blood tonic." Five cases of PVP storage disease with cutaneous involvement were studied. Two patients presented with cutaneous eruptions mimicking collagen vascular disease and chronic pigmented purpuric dermatosis. Two other cases were found incidentally: one was with a metastatic tumor and the other in a pemphigus lesion. The fifth case was seen in a blind skin biopsy specimen taken to exclude Niemann-Pick disease after hematologic examination of a bone marrow smear. The latter patient and the patient with a collagen vascularlike disease also had severe anemia and serious orthopedic and neurologic complications due to massive infiltration of PVP-containing cells in the bone marrow with destruction of the bone. Severe irreversible anemia due to PVP storage disease has not been reported before. Three patients admitted having a history of receiving intravenous injection of PVP. The samples obtained from two of them indeed contained 5% PVP as determined by chemical analysis. PVP storage disease can be diagnosed by its histopathologic features. The skin biopsy specimens all showed a variable number of characteristic blue-gray vacuolated cells around blood vessels and adnexal structures with positive tinctorial reactions to mucicarmine, colloidal iron, and alkaline Congo red and negative to periodic acid-Schiff (PAS) and alcian blue. The PVP storage cells were shown to be CD68+ macrophages. The presence of PVP in the skin induced little or no inflammatory reaction. Only the pelvic mass in one patient had a foreign body granuloma formation. Our study showed that systemic parenteral administration of PVP preparation could result in the accumulation of PVP storage cells in the skin, with or without clinical eruptions. The diagnosis of systemic PVP storage disease can be established by performing a skin biopsy for pathologic study. It is important for pathologists and clinicians to be aware of this iatrogenic storage disease to avoid misdiagnosis for hereditary storage disease, osteomyelitis, or signet-ring cell carcinoma. Serious hematologic and orthopedic complications can be caused by repeated massive intravenous injection of PVP. Therefore, PVP preparations should be strictly prohibited for systemic administration.     

Computational analysis of flow in a curved tube model of the coronary arteries: effects of time-varying curvature

The flow through a curved tube whose radius of curvature varies with time was studied in order to better understand flow patterns in coronary arteries. A computational flow model was constructed using commercially available software. The artery model featured a uniform circular cross section, and the curvature was assumed to be constant along the tube, and in one plane. The computational model was verified with the use of a dynamically similar in vitro apparatus. A steady uniform velocity was prescribed at the entrance at a Reynolds number of 300. Two sets of results were obtained: one in which the curvature was held constant at the mean, maximum and minimum radii of curvature (quasistatic), and another in which the curvature was varied sinusoidally in time at a frequency of I Hz (dynamic). The results of the dynamic analysis showed that the wall shear rates varied as much as 52% of the static mean wall shear rate within a region of 10 tube diameters from the inlet. The results of the dynamic analysis were within 6% of the quasistatic predictions. Realistic modeling of the deforming geometry is important in determining which locations in the coronary arteries are subjected to low and oscillating wall shear stresses, flow patterns that have been associated with atherogenesis.     

"Break copy" duplication: a model for chromosome fragment formation in Saccharomyces cerevisiae

Scores on burnout among 631 Canadian and 1,180 Dutch teachers were compared with various demographic variables (sex and age) and factors related to work (experience in teaching, type of school, and number of hours employed). Burnout was assessed with the Maslach Burnout Inventory of three dimensions, Emotional Exhaustion, Depersonalization, and Personal Accomplishment. Analysis indicated that, over-all, Canadian teachers reported higher scores on Emotional Exhaustion and Depersonalization than their Dutch peers. Differences in the number of hours employed were also significant: full-time Canadian teachers scored higher on Depersonalization than their Dutch colleagues. Across countries, sex and type of school appeared significantly related to burnout. Male teachers rated higher on Emotional Exhaustion and Depersonalization than the women. Especially with regard to the attitudinal components of burnout, i.e., Depersonalization and Personal Accomplishment, secondary school teachers reported higher scores than elementary school teachers. Age was not significantly related to measures.     

A 42 bp fragment of the pmas1'' promoter containing an ocs-like element confers a developmental, wound- and chemically inducible expression pattern

The infusion of cytotoxic drugs in cancer patients is often accompanied by posttreatment nausea (PN). In addition, patients complain about nausea prior to an infusion [i.e., anticipatory nausea (AN)]. AN is mainly explained by classical conditioning, with the infusion as the unconditioned stimulus (US) and with the stimuli signaling the infusion as conditioned stimuli (CS). Despite this conditioning etiology, a specifically derived therapy to attenuate the CS-US contingency is missing. The purpose of this study is to develop and to test an overshadowing procedure for prevention of AN, and also for the modification of PN intensity. Sixteen cancer patients were assigned to one of two groups: Overshadowing+ (OV+) and Overshadowing- (OV-). At the start of all infusions of two consecutive chemotherapy cycles A and B (acquisition), OV+ subjects drank a saliently tasting beverage (the overshadowing CS), whereas group OV- drank water. All patients received water in cycle C (test). Self-reported symptoms and heart rates were recorded. As expected, in cycle C (test), no patient of group OV+ showed AN, whereas two patients of group OV- developed AN. There was a tendency for a reduction of the intensity of PN, in terms of duration and latency after overshadowing, in cycle C: OV+ patients tended to show a shorter duration and a longer latency between end of infusion and PN onset. In OV-, there was a significantly larger heart rate deceleration in both measurement periods, in the anticipatory and the posttreatment measurement period. Data suggest to apply overshadowing for prevention of AN and modification of PN. Physiological markers of conditioned nausea are revealed. After its procedural implementation, the technique can be used in larger samples now.