Characterization of the ornithine aminotransferase from Plasmodium falciparum.

The ornithine aminotransferase from Plasmodium falciparum 3D7 was cloned, functionally expressed, and characterized. The gene exists as a single copy in the malarial genome and is located on chromosomes 6/7/8. The deduced amino acid sequence was found to be 85% identical to a similar sequence discovered in Plasmodium yoelii, 82% identical to a partial sequence from Plasmodium vivax, and 42-53% identical to ornithine aminotransferases from other eukaryotes. The enzyme had a very narrow substrate specificity, and could only catalyze the transamination of alpha-ketoglutarate with ornithine or N-acetylornithine, and of glutamate-5-semialdehyde with glutamate and alanine. The aminooxy analogue of ornithine, canaline, was found to inhibit the ornithine aminotransferase uncompetatively with a Ki of 492+/-98 nM. As the enzyme effectively catalyzed both ornithine catabolism and formation, its potential role in ornithine biosynthesis from glutamine, via glutamate, glutamate-5-phosphate, and glutamate-5-semialdehyde, was examined. Over the course of a 3.5 h incubation, P. falciparum converted 34% of exogenous, radiolabeled glutamine to glutamate and 0.68% to ornithine. This low level of conversion suggests that the parasite may have alternative mechanisms for obtaining ornithine for polyamine biosynthesis.

Methionine regeneration and aspartate aminotransferase in parasitic protozoa.

Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549-561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a K(i) of 27 microm.

Growth control mechanisms in multiple myeloma.

Interleukin-6 (IL-6) is the major growth factor for the malignant plasma cell clone in patients with multiple myeloma (MM). Although interferon-alpha (IFN-alpha) has been widely used as maintenance therapy in MM, controversy exists as to its clinical utility. This review summarizes data showing that cell growth arrest brought about by type I (IFNs-alpha/beta) and type II (IFN-gamma) IFNs occurs in part through utilization/modification of various components of the otherwise stimulatory Jak-STAT and Ras signaling pathways triggered by IL-6. Recent experimental results indicating that IFN-alpha acts as a survival factor for certain myeloma cell lines and frequently induces endogenous IL-6 expression may help to explain the conflicting clinical findings obtained in this heterogeneous disease with this usually potent growth inhibitor. By comparison, consistent antiproliferative activity exhibited by IFN-gamma on IL-6-dependent myeloma cell lines and primary myeloma cells from patients suggests that further investigation of the possible value of this cytokine in the treatment of MM may be warranted.

Interferon-beta interrupts interleukin-6-dependent signaling events in myeloma cells.

Type I interferons (IFNs-alpha and IFN-beta) bind to a common receptor to exert strong antiproliferative activity on a broad range of cell types, including interleukin-6 (IL-6)-dependent myeloma cells. In this study, we investigated the effect of IFN-beta pretreatment on IL-6-stimulated mitogenic signaling in the human myeloma cell line U266. IL-6 induced transient tyrosine phosphorylation of the IL-6-receptor signal-transducing subunit gp130, the gp130-associated protein tyrosine kinases Jak1,Jak2, and Tyk2, the phosphotyrosine phosphatase PTP1D/Syp, the adaptor protein Shc and the mitogen-activated protein kinase Erk2, and accumulation of GTP-bound p21ras. Prior treatment of U266 cells with IFN-beta downregulated IL-6-induced tyrosine phosphorylation of gp130, Jak2, PTP1D/Syp, Shc, and Erk2, and GTP-loading of p21ras. Further analysis indicated that treatment with IFN-beta disrupted IL-6-induced binding of PTP1D/Syp to gp130 and the adaptor protein Grb2; IFN-beta pretreatment also interfered with IL-6-induced interaction of Shc with Grb2 and a 145-kD tyrosine-phosphorylated protein. These results suggest a novel mechanism whereby type I IFNs interrupt IL-6-promoted mitogenesis of myeloma cells in part by preventing the formation of essential signaling complexes leading to p21ras activation.

Dose-dependent activation of p21ras by IFN-beta.

Recent studies have demonstrated that interferon-beta (IFN-beta) activates extracellular regulated kinases (ERKs) in myeloma cells and have revealed a link between ERKs and the Jak/Stat pathway. The upstream components of the IFN-beta pathway involved in activation of ERKs are unknown. As p21ras is often an upstream component of the ERK pathway, we have investigated p21ras activity following IFN-beta treatment of the human myeloma cell line, U266. IFN-beta was found to strongly activate p21ras at relatively low doses and to exert a negative effect at the higher doses normally employed in signaling studies. There was no direct correlation between p21ras and ERK activity, suggesting that p21ras plays an alternate role in the IFN-beta signaling pathway. These results imply a unique integration of p21ras signaling within the milieu of IFN-induced cytoplasmic signaling events.

Interaction between T antigen and TEA domain of the factor TEF-1 derepresses simian virus 40 late promoter in vitro: identification of T-antigen domains important for transcription control.

The large tumor antigen (TAg) of simian virus 40 regulates transcription of the viral genes. The early promoter is repressed when TAg binds to the origin and DNA replication begins, whereas the late promoter is activated by TAg through both replication-dependent and -independent mechanisms. Previously it was shown that activation is diminished when a site in the viral enhancer to which the factor TEF-1 binds is disrupted. We show here that the NH2-terminal region of TAg binds to the TEA domain of TEF-1, a DNA binding domain also found in the Drosophila scalloped and the Saccharomyces cerevisiae TEC1 proteins. The interaction inhibits DNA binding by TEF-1 and activates transcription in vitro from a subset of naturally occurring late start sites. These sites are also activated by mutations in the DNA motifs to which TEF-1 binds. Therefore, TEF-1 appears to function as a repressor of late transcription, and its involvement in the early-to-late shift in viral transcription is discussed. The mutation of Ser-189 in TAg, which reduces transformation efficiency in certain assays, disrupts the interaction with TEF-1. Thus, TEF-1 might also regulate genes involved in growth control.

Expression of cyclooxygenase types 1 and 2 in human fetal membranes at term.

OBJECTIVE: Our purpose was to compare the level of expression of both type 1 and type 2 cyclooxygenase genes before and after labor and to localize their expression within the fetal membranes. STUDY DESIGN: The sites of type 2 and type 1 cyclooxygenase messenger ribonucleic acid synthesis were identified with in situ hybridization. Expression of both types 1 and 2 cyclooxygenase was studied by reverse transcriptase polymerase chain reaction. RESULTS: Cyclooxygenase type 2 and type 1 expression was localized within the amniotic epithelium and amniotic mesoderm. Type 1 but not type 2 enzyme was also expressed in the chorionic mesoderm. Expression of the type 2 enzyme was significantly increased with the onset of labor. Type 1 enzyme expression did not significantly change with labor. CONCLUSION: It is most likely that it is the inducible type 2 cyclooxygenase enzyme that mediates the increase in prostaglandin synthesis in amnion with the onset of labor.

Tyrosine phosphorylation of JAK-TYK kinases in malignant plasma cell lines growth-stimulated by interleukins 6 and 11.

The pleiotropic cytokine interleukin (IL-6) is a major growth factor for murine plasmacytomas/hybridomas and human myeloma cells. Here we report that IL-6 stimulated different patterns of tyrosine phosphorylation of JAK-TYK kinases in IL-6-responsive murine (B9E and T10D) and human (ANBL-6 and OCI-My4) plasma cell tumor lines. Interestingly, the Stat91 transcription factor essential for interferon signaling mediated by JAK-TYK kinases was significantly tyrosine phosphorylated in response to IL-6 in ANBL-6 cells but not in the other cell lines. We further show that IL-11, a cytokine that signals via the gp130 subunit of the IL-6 receptor, induced similar profiles of JAK-TYK tyrosine phosphorylation as IL-6 in B9E and T10D cells. These results suggest that functionally redundant JAK-TYK kinase cascades triggered through gp130 are involved in the growth regulation of plasma cell neoplasms.

Activity of cationically substituted bis-benzimidazoles against experimental Pneumocystis carinii pneumonia.

On the basis of a previously observed correlation between the antimicrobial activity and DNA binding strength of dicationic molecules, a series of 10 dicationically substituted bis-benzimidazoles were tested for activity in the rat model of Pneumocystis carinii pneumonia. One of the compounds, 1,4-bis[5-(2-imidazolinyl)-2-benzimidazolyl]butane, was found to be more potent and less toxic than pentamidine.

Isolation and identification of six Pneumocystis carinii genes utilizing codon bias.

Pneumocystis carinii pneumonia (PCP) is a leading cause of death among AIDS patients in the United States. Our analysis of P. carinii protein-coding genes has revealed a significant A + T codon bias. Polymerase chain reaction (PCR) was utilized to isolate and identify the genes encoding calmodulin, beta-tubulin, DNA polymerase II, and RNA polymerases I, II and III from P. carinii. Primer pairs were designed to incorporate P. carinii codon preference to known conserved protein regions from other organisms. This strategy should be useful for a large variety of P. carinii genes and assist in the comprehensive analysis of the genomic structure of this important pathogen.

Cloning and characterization of the calmodulin-encoding gene from Pneumocystis carinii.

Complete cDNA and genomic clones for the CaM gene encoding calmodulin (CaM) from Pneumocystis carinii have been isolated from rat and characterized. The nucleotide (nt) sequence contains an open reading frame interrupted by three introns, that encodes a protein of 152 amino acids. The predicted CaM protein of P. carinii shares a high degree of homology with other known CaM proteins.

Translation of poly(A)-binding protein mRNA is regulated by growth conditions.

Translational efficiency of a minor group of mRNAs is regulated by serum levels in 3T6 fibroblasts. Included within this group is the poly(A)-binding protein (PABP) mRNA. We analyzed the distribution of PABP mRNA in polysome profiles and found a large percentage of this mRNA to be translationally repressed in both actively growing (approximately 60%) and resting cells (approximately 70%). Elevated serum levels induced a distinct bimodal distribution of this mRNA between actively translated and repressed fractions. Similarly, treatment of cells with low doses of cycloheximide also generated a partial shift of repressed PABP mRNA into the actively translated fraction. In an attempt to characterize the factors which regulate PABP mRNA translation we have identified the proteins which bind to this mRNA in vitro. Sequences within the 5' untranslated region were found to be sufficient for binding of all proteins to this mRNA. We suggest that this region and the proteins associated with it may be essential for translation control of PABP mRNA.

Modification of mRNA-associated proteins during changes in growth conditions.

Following serum stimulation of quiescent 3T6 cells, an elevated in vivo rate of translation was observed. These studies were designed to identify the proteins associated with polysomal mRNA under different growth conditions in an attempt to establish a relationship between translational rate and the mRNA-associated proteins. Ultraviolet cross-linking of proteins to mRNA was employed to ensure that only genuine mRNA-associated proteins were investigated. Our results revealed little change in the population of mRNA-binding proteins, although minor variations in the synthesis of several proteins, most notably a 32 kilodalton species, were observed during growth transitions. These investigations demonstrate further that most of the mRNA-binding proteins were phosphorylated with the degree of phosphorylation of several proteins influenced by growth conditions.

Identification of proteins associating with poly(A)-binding-protein mRNA.

Synthesis of poly(A)-binding protein is regulated at the translational level. We have investigated the binding of proteins to this mRNA on the premise that the protein(s) of the mRNP complex may be involved in regulating the expression of the mRNA. We found the first 243 nucleotides of the 5' untranslated region to contain sequences essential for RNP formation. A large, single-stranded bulge structure encompassing stretches rich in adenine nucleotides and a potential stem-loop domain appear to be the primary sites for protein binding. Removal of the 243-nucleotide segment results in a drastic reduction in protein binding and a concomitant increase in translational efficiency in vitro. We suggest that proteins binding to this region, including poly(A)-binding protein itself, may be essential for regulating translation of this mRNA.

Timing of the quantitative recovery in the regenerating rat lung.

Growing rats 23 days of age were subjected to the resection of the upper and middle lobes of the right lung (25% of total lung volume). On postoperative days 1, 4, 6, 9, 12, 18, and 30, the lungs of 5 animals were fixed by intratracheal instillation of glutaraldehyde, their volumes measured by water displacement, and the lung tissue processed for quantitative light (LM) and electron microscopic (EM) investigations. For each group, 3 age-matched sham-operated, and 4 normal rats served as controls. Sham operation consisted in entering the pleural space, and collapsing and ventilating the lungs. In all animals, the following parameters were measured by means of point and intersection counting stereology at either LM or EM-levels: volume densities of lung parenchyma and of its components (airspaces, tissue, capillary blood) and surface densities of airspaces and of capillaries. From these data absolute parameter values were calculated for each lung. The results showed that after bilobectomy the remaining lung re-expanded rapidly first by an overinflation of the airspaces and after Day 4 by an increase in tissue mass and capillary volume. On Days 9 and 12 the operated lungs did not differ quantitatively from control lungs. Later, however, further sequels of the bilobectomy were detected: On Day 18, lobectomy-lungs were smaller than controls, and on Day 30 lungs, the left lung was significantly different in structure from the right lung and from control lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphometric analysis of adult rat lung after bilobectomy.

Female rats (mean body weight, 245 g) were subjected to resection of the upper and middle lobes of the right lung (25% of the total lung volume). Forty-five days after the surgery, the animals were killed and their lungs were fixed and processed for quantitative light and electron microscopic investigation. Two groups of animals, sham-operated and normal rats with the same body weight, served as controls. At death the resected animals had the same body weight and lung volumes as the control animals. The remaining tissue of the right lung had increased in volume by 40%, the left lung by 26%. In light microscopic morphometry, volume densities of parenchyma, air spaces, and septums were all close to control values. Nonparenchymal structures, bronchi, and larger blood vessels also contributed to the normalization; they were not significantly different from control values. Electron microscopic quantitation performed on the lung parenchyma revealed the volume densities of capillaries and tissue as well as the surface densities of air spaces and capillaries to be in the range of control data, both in the right and left lung. For total lung the absolute volumes of air spaces, capillaries, and tissue, the alveolar and capillary surface areas, and the arithmetic and harmonic mean thicknesses of the air-blood barrier were practically identical in resected and control animals. The morphometrically calculated DLO2 was restored to normal values. This adaptive response resembled closely that obtained in growing rats, despite the comparatively limited growth potential of the animals in this study.

Reactive changes in pulmonary parenchyma after bilobectomy: a scanning electron microscopic investigation.

This study describes the morphology of the adaptive response of the pulmonary parenchyma after the resection of lung tissue. Rats aged 23 days were subjected to the bilobectomy of the right upper and middle lobes representing 25% of the total lung volume. On the postoperative Days 1, 4, 6, 9, 12, 18, and 30, the lungs were fixed by standardized intratracheal instillation of fixative, their volume determined by water displacement, and pieces of the left lung prepared for scanning electron microscopy (SEM). Age-matched normal and sham-operated animals served as controls. Lung volumes of operated rats rapidly matched control values. In SEM, the volume gain appeared to be achieved by a widening of the airspaces affecting primarily the alveolar ducts on postoperative Day 1 and shifting later to the alveolar level (Day 4). On Days 4 and 6, the interalveolar septa were slightly thickened, presumably due to a proliferative response in the tissue, which is well documented in the literature. From Day 9 onwards, morphology of bilobectomized lungs did not differ from normal. At no instances we could detect clues for the formation of new alveoli. Several possible explanations for this structural recovery of the pulmonary parenchyma are discussed. Among these, a model is presented illustrating how the parenchymal morphology can be apparently normalized by the tissue proliferation, which renders the original widening of the airspaces unobtrusive on random sections through the respiratory tissue.