[Genetic characterization of clinical Klebsiella isolates circulating in Novosibirsk]. / Генетическая Ñ Ð°Ñ€Ð°ÐºÑ‚ÐµÑ€Ð¸ÑÑ‚Ð¸ÐºÐ° ÐºÐ»Ð¸Ð½Ð¸Ñ‡ÐµÑÐºÐ¸Ñ Ð¸Ð·Ð¾Ð»ÑÑ‚Ð¾Ð² клебсиелл, Ñ†Ð¸Ñ€ÐºÑƒÐ»Ð¸Ñ€ÑƒÑŽÑ‰Ð¸Ñ Ð² Новосибирске.

72 clinical strains of Klebsiella spp. isolated from samples obtained from humans in Novosibirsk, Russia, were analyzed. Species identification of strains was performed using 16S rRNA and rpoB gene sequences. It was revealed that Klebsiella pneumoniae strains were dominant in the population (57 strains), while the remaining 15 strains were K. grimontii, K. aerogenes, K. oxytoca and K. quasipneumoniae. By molecular serotyping using the wzi gene sequence, K. pneumoniae strains were assigned to twenty-one K-serotypes with a high proportion of virulent K1- and K2-serotypes. It was found that K. pneumoniae strains isolated from the hospitalized patients had a higher resistance to antibiotics compared to the other Klebsiella species. Real-time PCR revealed that the population contained genes of the blaSHV, blaTEM, blaCTX families and the blaOXA-48 gene, which are the genetic determinants of beta-lactam resistance. It has been shown that the presence of the blaCTX sequence correlated with the production of extended-spectrum beta-lactamases, and phenotypic resistance to carbapenems is due to the presence of the blaOXA-48 gene. At the same time, the carbapenemase genes vim, ndm, kpc, imp were not detected. Among the aminoglycoside resistance genes studied, the aph(6)-Id and aadA genes were found, but their presence did not always coincide with phenotypic resistance. Resistance to fluoroquinolones in the vast majority of strains was accompanied by the presence of the aac(6')-IB-cr, oqxA, oqxB, qnrB, and qnrS genes in various combinations, while the presence of the oqxA and/or oqxB genes alone did not correlate with resistance to fluoroquinolones. Thus, the detection of blaCTX and blaOXA-48 can be used to quickly predict the production of extended-spectrum beta-lactamases and to determine the resistance of Klebsiella to carbapenems. The detection of the aac(6')-Ib-cr and/or qnrB/qnrS genes can be used to quickly determine resistance to fluoroquinolones.

ABCB4 gene mutations and single-nucleotide polymorphisms in women with intrahepatic cholestasis of pregnancy.

AIM: To evaluate the nature and frequency of ATP-binding cassette subfamily B member 4 (ABCB4) gene variants in a series of French patients with intrahepatic cholestasis of pregnancy (ICP). METHODS: In this prospective study, the entire ABCB4 gene coding sequence was analysed by DNA sequencing in 50 unrelated women with ICP defined by pruritus and raised serum alanine aminotransferase activity or bile acid concentration, with recovery after delivery. Genomic variants detected in patients with ICP were sought in 107 control pregnant women. Patients with ICP and controls were of Caucasian origin. RESULTS: Eight genomic variants were observed. One nonsense mutation (p.Arg144Stop) and two missense mutations (p.Ser320Phe and p.Thr775Met) were revealed each in one heterozygous patient. A third missense mutation (p.Arg590Gln) was detected in three heterozygous patients and in two homozygous patients also homozygous for a particular haplotype of three single-nucleotide polymorphisms (c.175C>T, c.504T>C, c.711A>T). The chromosomal frequency of the p.Arg590Gln variant was significantly different between the ICP and control group (7.0% vs 0.5%; p = 0.0017; OR 16.03, 95% CI 1.94 to 132.16). An association was also found between allele T of the c.504T>C silent nucleotide polymorphism and ICP (68.0% vs 53.7%; p = 0.017; OR 1.83, 95% CI 1.08 to 3.11). The chromosomal frequency of the p.Arg652Gly variant did not differ between the ICP and control group (p = 0.40). CONCLUSIONS: This study shows that 16% of Caucasian patients with ICP bear ABCB4 gene mutations, and confirms the significant involvement of this gene in the pathogenesis of this complex disorder.

Role of Gab proteins in phosphatidylinositol 3-kinase activation by thrombopoietin (Tpo).

In this study, we show that upon thrombopoietin (Tpo) stimulation the two adapter proteins Gab1 and Gab2 are strongly tyrosine phosphorylated and associated with Shc, SHP2, PI 3-kinase and Grb2 in mpl-expressing UT7 cells. Although Gab1 and Gab2 seem to mediate overlapping biological signals in many cells, only Gab1 is expressed and phosphorylated in response to Tpo in primary human megakaryocytic progenitors; furthermore, it associates with the same proteins. Although a low level of tyrosine phosphorylated IRS-2 protein is also detected in PI 3-kinase immunoprecipitates, Gab proteins are the essential proteins associated with PI 3-kinase after Tpo stimulation. We demonstrate that, albeit no association is detected between the Tpo receptor mpl and Gab proteins, Y112 located in the C-terminal cytoplasmic domain of mpl is required for Gab1/2 tyrosine phosphorylation. Gab proteins are not tyrosine phosphorylated after Tpo stimulation of UT-7 and Ba/F3 cells expressing a mpl mutant lacking Y112. Moreover, no activation of the PI 3-kinase/Akt pathway is observed in cells expressing this mpl mutant. Finally, we show that this mutant does not allow cell proliferation, thereby confirming that PI 3-kinase activation is required for Tpo-induced cell proliferation.

A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor.

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.

Coupling of heterotrimeric Gi proteins to the erythropoietin receptor.

To identify new proteins involved in erythropoietin (Epo) signal transduction, we purified the entire set of proteins reactive with anti-phosphotyrosine antibodies from Epo-stimulated UT7 cells. Antisera generated against these proteins were used to screen a lambdaEXlox expression library. One of the isolated cDNAs encodes Gbeta2, the beta2 subunit of heterotrimeric GTP-binding proteins. Gbeta and Galpha(i) coprecipitated with the Epo receptor (EpoR) in extracts from human and murine cell lines and from normal human erythroid progenitor cells. In addition, in vitro Gbeta associated with a fusion protein containing the intracellular domain of the EpoR. Using EpoR mutants, we found that the distal part of the EpoR (between amino acids 459-479) was required for Gi binding. Epo activation of these cells induced the release of the Gi protein from the EpoR. Moreover in isolated cell membranes, Epo treatment inhibited ADP-ribosylation of Gi and increased the binding of GTP. Our results show that heterotrimeric Gi proteins associate with the C-terminal end of the EpoR. Receptor activation leads to the activation and dissociation of Gi from the receptor, suggesting a functional role of Gi protein in Epo signal transduction.

Proteasomes regulate the duration of erythropoietin receptor activation by controlling down-regulation of cell surface receptors.

The binding of erythropoietin (Epo) to its receptor leads to the transient phosphorylation of the Epo receptor (EpoR) and the activation of intracellular signaling pathways. Inactivation mechanisms are simultaneously turned on, and Epo-induced signaling pathways return to nearly basal levels after 30-60 min of stimulation. We show that proteasomes control these inactivation mechanisms. In cells treated with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal (LLnL) or lactacystin, EpoR tyrosine phosphorylation and activation of intracellular signaling pathways (Jak2, STAT5, phosphatidylinositol 3-kinase) were sustained for at least 2 h. We show that this effect was due to the continuous replenishment of the cell surface pool of EpoRs in cells treated with proteasome inhibitors. Proteasome inhibitors did not modify the internalization and degradation of Epo.EpoR complexes, but they allowed the continuous replacement of the internalized receptors by newly synthesized receptors. Proteasome inhibitors did not modify the synthesis of EpoRs, but they allowed their transport to the cell surface. N-Ac-Leu-Leu-norleucinal, but not lactacystin, also inhibited the degradation of internalized Epo.EpoR complexes, most probably through cathepsin inhibition. The internalized EpoRs were not tyrosine-phosphorylated, and they did not activate intracellular signaling pathways. Our results show that the proteasome controls the down-regulation of EpoRs in Epo-stimulated cells by inhibiting the cell surface replacement of internalized EpoRs.

Erythropoietin induces glycosylphosphatidylinositol hydrolysis. Possible involvement of phospholipase c-gamma(2).

We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-gamma(2) in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-gamma(2) was time- and dose-dependent. By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor. Erythropoietin-activated PLC-gamma(2) hydrolyzed purified [(3)H]GPI indicating that GPI hydrolysis and PLC-gamma(2) activation under erythropoietin stimulation were correlated. Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-gamma(2), whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects. Thus, our results suggest that erythropoietin regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-gamma(2) activation.

Erythropoietin induces the tyrosine phosphorylation of GAB1 and its association with SHC, SHP2, SHIP, and phosphatidylinositol 3-kinase.

Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.

Tyrosine residues of the erythropoietin receptor are dispensable for erythroid differentiation of human CD34+ progenitors.

To study the role of the cytoplasmic domain and particularly the tyrosine residues of the erythropoietin receptor (EpoR) in erythroid differentiation of human primary stem cells, we infected cord blood-derived CD34+ cells with retroviruses encoding chimeric receptors containing the extracellular domain of the prolactin receptor (PRLR) and the cytoplasmic domain of either the normal EpoR or a truncated EpoR devoid of tyrosine residues. Erythroid differentiation of the infected progenitors could thus be studied after stimulation by PRL. The complete PRLR was used to assess its ability to substitute for EpoR in erythroid differentiation. Typical erythroid day-14 colonies were observed from CD34+ cells grown in PRL when infected with any of the three viral constructs. These results demonstrate that: (i) the activation of the virally transduced PRLR leads to erythroid colony formation showing that erythroid terminal differentiation can be induced by a non-erythroid receptor in human progenitors; (ii) a chimeric receptor PRLR/EpoR is able to transduce a signal leading to terminal erythroid differentiation of human CD34+ cells; (iii) in contrast to results previously reported in murine models, tyrosine residues of the EpoR are not required for growth and terminal differentiation of human erythroid progenitors.

Physical and functional interaction between p72(syk) and erythropoietin receptor.

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid cells through interaction with a cell surface receptor (EpoR) that belongs to the cytokine receptor family. The Jak2 tyrosine kinase was previously shown to bind to the EpoR, to be activated upon Epo stimulation, and to play a critical role in Epo-induced proliferation. However, little is known about the role of other tyrosine kinases in Epo signaling. In this paper, we examined whether Syk was involved in EpoR activation. Coimmunoprecipitation experiments showed that the phosphorylated EpoR was associated with the Syk kinase in activated UT7 cells. The interaction of Epo with its receptor led to an increased kinase activity. The use of recombinant Syk Src homology 2 (SH2) domains expressed in tandem or individually revealed that both N- and C-SH2 domains of Syk participated in EpoR binding with a major contribution of the C-terminal SH2 domain. Far Western blotting further indicated that Syk directly binds to the EpoR and that the interaction of Syk with EpoR only occurred after Epo activation. These data suggest that phosphorylation of EpoR on tyrosine residues may mediate Syk binding to the receptor through interaction between the two SH2 domains of Syk and tyrosines of the receptor. We propose that in addition to Jak2, Syk protein kinase may be a component of EpoR signaling.

Proteasomes regulate erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) activation. Possible involvement of the ubiquitinated Cis protein.

Cis is an Src homology 2 domain-containing protein, which binds to the erythropoietin receptor and decreases erythropoietin-stimulated cell proliferation. We show that Cis associates with the second tyrosine residue of the intracellular domain of the erythropoietin receptor (Tyr401). Two forms of Cis with molecular masses of 32 and 37 kDa were detected, and we demonstrate that the 37-kDa protein resulted from post-translational modifications of the 32-kDa form. Anti-ubiquitin antibodies recognized the 37-kDa form of Cis and the proteasome inhibitors N-acetyl-leucyl-leucyl-norleucinal and lactacystin inhibited its degradation, showing that the 37-kDa form of Cis is a ubiquitinated protein, which seems to be rapidly degraded by the proteasome. In erythropoietin-stimulated UT-7 cells, the activation of the erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) was transient and returned to basal levels after 30-60 min of erythropoietin stimulation. In contrast, these proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. These experiments demonstrate that the proteasomes are involved in the down-regulation of the erythropoietin receptor activation signals. Because the proteasome inhibitors induced the accumulation of both the ubiquitinated form of Cis and the Cis-erythropoietin receptor complexes, our results suggest that the ubiquitinated form of Cis could be involved in the proteasome-mediated inactivation of the erythropoietin receptor.

A sequence of the CIS gene promoter interacts preferentially with two associated STAT5A dimers: a distinct biochemical difference between STAT5A and STAT5B.

Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5.

Abnormal erythropoietin (Epo) gene expression in the murine erythroleukemia IW32 cells results from a rearrangement between the G-protein beta2 subunit gene and the Epo gene.

Abnormal production of erythropoietin (Epo) has been described in several human and murine erythroleukemia. The murine IW32 cell line is derived from an F-MuLV-induced erythroleukemia. An autocrine Epo production due to the rearrangement of one Epo allele has been previously described (Beru et al., 1989). However, the exact mechanism leading to the transcriptional activation of the abnormal Epo gene was unknown. In this study, we show that this deregulated expression results from a deletion within chromosome 5. The Epo gene in the abnormal allele is under the control of the G-protein beta2 subunit gene promoter and the expressed mRNA results from the fusion of the non coding exon 1 of the G-protein beta2 subunit gene to a truncated Epo exon 1 gene. This resulting abnormal cDNA allows the expression of a normal Epo protein.

Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation.

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine following treatment of UT-7 cells with erythropoietin. We have investigated the expression of IRS-1 and IRS-2 in several cell lines with erythroid and/or megakaryocytic features, and we observed that IRS-2 was expressed in all cell lines tested. In contrast, we did not detect the expression of IRS-1 in these cells. In response to erythropoietin, IRS-2 was immediately phosphorylated on tyrosine, with maximal phosphorylation between 1 and 5 min. Tyrosine-phosphorylated IRS-2 was associated with phosphatidylinositol 3-kinase and with a 140-kDa protein that comigrated with the phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase, SHIP. Moreover, IRS-2 was constitutively associated with the erythropoietin receptor. We did not observe the association of IRS-2 with JAK2, Grb2, or PTP1D. Using BaF3 cells transfected with mutated erythropoietin receptors, we demonstrate that neither the tyrosine residues of the intracellular domain nor the last 109 amino acids of the erythropoietin receptor are required for erythropoietin-induced IRS-2 tyrosine phosphorylation. Altogether, our results indicate that erythropoietin-induced IRS-2 tyrosine phosphorylation could account for the previously reported activation of phosphatidylinositol 3-kinase mediated by erythropoietin receptors mutated in the phosphatidylinositol 3-kinase-binding site (Damen, J., Cutler, R. L., Jiao, H., Yi, T., and Krystal, G. (1995) J. Biol. Chem. 270, 23402-23406; Gobert, S., Porteu, F., Pallu, S., Muller, O., Sabbah, M., Dusanter-Fourt, I., Courtois, G., Lacombe, C., Gisselbrecht, S., and Mayeux, P. (1995) Blood 86, 598-606).

Erythropoietin-induced erythroid differentiation of the human erythroleukemia cell line TF-1 correlates with impaired STAT5 activation.

The TF-1 cell line has been established from a patient with erythroleukemia. While various cytokines induce TF-1 cell proliferation, erythropoietin (Epo) only sustains the short-term growth of these cells and induces their differentiation along the erythroid lineage. A truncated Epo receptor (EpoR) is overexpressed in these cells. The truncation removed the 96 C-terminal amino acids, including seven tyrosine residues. An additional single mutation at position +3 of Tyr344 led to the replacement of leucine 347 by proline. Stimulation by Epo induced an impaired activation of the STAT5 transcription factor in these cells. The same defect in STAT5 activation was found in the murine FDCP-1 cell line transfected with a chimeric EpoR containing the abnormal TF-1 EpoR cytoplasmic domain. Infection of TF-1 cells with a retrovirus containing a normal murine EpoR was able to restore both Epo-induced STAT5 activity and cellular proliferation. In contrast, Epo-induced differentiation was reduced strongly in infected TF-1ER cells. These results suggest that Epo-induced differentiation correlates with impaired Epo-induced STAT5 activation.

Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.

FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.

A CCACC motif mediates negative transcriptional regulation of the human erythropoietin receptor.

We have previously shown that the +79 to +135 fragment of the human erythropoietin receptor (Epo-R) acts negatively on the transcriptional activity and confers erythroid specificity to the gene [Maouche, L., Cartron, J.-P. & Chrétien, S. (1994) Nucleic Acids Res. 22, 338-346]. In this work, we demonstrate that this effect is mediated by a CCACC motif that binds weakly to the simian virus 40 protein 1 (Sp1) factor and that the increase of the affinity for Sp1 augments transcription inhibition. The repression is not restricted to the human Epo-R promoter, although it seems more efficient on heterologous promoters of erythroid genes. In chloramphenicol acetyl transferase constructs containing the mouse Epo-R promoter, rearranged by retroviral long terminal repeat (LTR) insertion of murine erythroleukemia cell lines, we found that positioning the CCACC motif 3' to the LTR represses the transcriptional activity mediated by the LTR in non-erythroid cells. These results demonstrate that Epo-R gene expression is negatively regulated by a CCACC or a GC box-binding factor, which is most likely identical to the Sp1 transcription protein. Further data suggest that Sp1-mediated negative regulation is not the result of a direct competition between Sp1 and another DNA-binding protein.

Thrombopoietin is not responsible for the thrombocytosis observed in patients with acute myeloid leukemias and the 3q21q26 syndrome.

Patients with acute myeloblastic leukaemia (AML) and chromosomic abnormalities of the 3q21;q26 region have striking dysmegakaryopoiesis and normal or increased platelet counts. Leukaemic cells ectopically express the Evi-1 gene which maps to human chromosome 3q26:q27. Thrombopoietin (TPO) has been cloned recently and shown to be the major hormone stimulating both megakaryocytopoiesis and thrombopoiesis. The TPO gene maps to human chromosome 3q26. For this report we studied four patients with typical 3q21:q26 syndrome. Karyotype analysis showed inv(3)(q21;q26) in three cases and t(3:3)(q21;q26) in one case. Although high levels of Evi-1 transcripts could be detected in mRNA isolated from the bone marrow cells of these patients by Northern blot analysis, no TPO transcripts were detectable by RT-PCR technique on the same mRNA samples. These results demonstrate that TPO gene transcription is not activated in patients with 3q26 chromosomic abnormality, and that abnormal TPO production is not responsible for the observed thrombocytosis.

Efficacy and safety of controlled-release niacin in dyslipoproteinemic veterans.

OBJECTIVE: To evaluate the safety and efficacy of controlled-release niacin in patients with hyperlipoproteinemia. DESIGN: A retrospective cohort study. SETTING: A Department of Veterans Affairs Medical Center. PATIENTS: A consecutive sample of 969 predominantly elderly male veterans treated for dyslipoproteinemia with controlled-release niacin between October 1988 and October 1991. MAIN OUTCOME MEASURES: Primary outcomes were lipid levels and lipoprotein cholesterol response, alternations in levels of hepatic enzymes and blood chemistry test results, and characterization of niacin-induced hepatotoxicity abstracted from the patient's medical, laboratory, and pharmacy records. RESULTS: 93% (896 of 969) of the cohort was evaluable. Patients (age, 61.7 years [9.4 years], mean [SD]) were treated for 1 to 36 months (13.0 months [9.7 months]) with an average maintenance dose of 1.67 g/d (0.8 g/d). Niacin was discontinued in 48.5% (435 of 896) of the patients primarily because of adverse effects. Poor glycemic control led to discontinuation in 40.6% (43 of 106) of the patients with diabetes mellitus. The lipoprotein response was dose-related and favorable (levels of total cholesterol, -19.1%; low-density lipoprotein cholesterol, -24.0%; high-density lipoprotein cholesterol, +5.7%; and triglycerides, -32.5%). Statistically but not clinically meaningful dose-related increases were seen in levels of liver enzymes and serum glucose (aspartate aminotransferase, +29%; alanine aminotransferase, +23%; alkaline phosphatase, +25%; and glucose, +7%; P = 0.0001). Twenty of 896 (2.2%) and 42 of 896 (4.7%) patients met biochemical criteria for probable and for possible or probable niacin-induced hepatotoxicity, respectively. Predisposing factors included high dose, alcohol use, preexisting liver disease, and concurrent oral sulfonylurea therapy. CONCLUSIONS: Controlled-release niacin is effective in treating dyslipoproteinemia in selected middle-aged and elderly veterans, but approximately one half of patients discontinued the drug because of adverse effects or other causes including noncompliance. Niacin should be avoided in patients with hepatic dysfunction or a history of liver disease, patients with diabetes mellitus, and patients who abuse alcohol. Because controlled-release niacin seems to be more potent than crystalline niacin, product substitution without dose adjustment should be avoided.