Defective expression of polarity protein PAR-3 gene (PARD3) in esophageal squamous cell carcinoma.

The partition-defective 3 (PAR-3) protein is implicated in the formation of tight junctions at epithelial cell-cell contacts. We investigated DNA copy number aberrations in human esophageal squamous cell carcinoma (ESCC) cell lines using a high-density oligonucleotide microarray and found a homozygous deletion of PARD3 (the gene encoding PAR-3). Exogenous expression of PARD3 in ESCC cells lacking this gene enhanced the recruitment of zonula occludens 1 (ZO-1), a marker of tight junctions, to sites of cell-cell contact. Conversely, knockdown of PARD3 in ESCC cells expressing this gene caused a disruption of ZO-1 localization at cell-cell borders. A copy number loss of PARD3 was observed in 15% of primary ESCC cells. Expression of PARD3 was significantly reduced in primary ESCC tumors compared with their nontumorous counterparts, and this reduced expression was associated with positive lymph node metastasis and poor differentiation. Our results suggest that deletion and reduced expression of PARD3 may be a novel mechanism that drives the progression of ESCC.

Role of miR-143 targeting KRAS in colorectal tumorigenesis.

Dysregulated expression of microRNAs (miRNAs) is associated with a variety of diseases, including colorectal cancer. By comparing more than 200 miRNAs in 13 pairs of matched colorectal cancer and normal adjacent tissue samples through qRT-PCR and microarray analysis, we found a widespread disruption of miRNA expression during colorectal tumorigenesis. In particular, among a panel of presumed targets generated by in silico analysis that may interact with these aberrantly expressed miRNAs, KRAS oncogene has been further experimentally validated as the target of miR-143. First, an inverse correlation between KRAS protein and miR-143 in vivo was found. Second, KRAS expression in Lovo cells was significantly abolished by treatment with miR-143 mimic, whereas miR-143 inhibitor increased KRAS protein level. Third, luciferase reporter assay confirmed that miR-143 directly recognize the 3'-untranslated region of KRAS transcripts. Four, Lovo cells treated with miR-143 inhibitor showed a stimulated cell proliferation, whereas miR-143 overexpression had an opposite effect. Finally, inhibition of KRAS expression by miR-143 inhibits constitutive phosphorylation of ERK1/2. Taken together, the present study provides the first evidences that miR-143 is significant in suppressing colorectal cancer cell growth through inhibition of KRAS translation.

IL-4-producing CD8(+) T cells may be an immunological hallmark of chronic GVHD.

Chronic graft-versus-host disease (cGVHD) occurs in approximately 60-80% of those who survive over 100 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the pathophysiology of cGVHD is poorly understood. To gain more insight into the immunological mechanism of cGVHD, we examine cytokine production of peripheral blood T cells from 19 patients in the chronic phase of allo-HSCT. The percentage of IFN-gamma-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with or without cGVHD than in normal control subjects (P<0.001). On the other hand, the percentage of IL-4-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with cGVHD (mean 3.3%; range 1.3-8.2%) than in patients without cGVHD (mean 1.2%; range 0.8-1.7%) and normal control subjects (mean 1.1%; range 0.1-1.6%) (both P<0.001). By contrast, the percentage of IL-4-producing CD4(+) T cells was not different among patients with and without cGVHD and normal controls. These findings suggest that IL-4-producing CD8(+) T cells may be an immunological marker of cGVHD.

Blood dendritic cells are decreased in acute graft-versus-host disease.

The recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) often develop acute graft-versus-host disease (aGVHD), which is closely related to morbidity and mortality. However, the essential part of the immune responses elicited in aGVHD remains largely unknown. We attempt to determine if peripheral blood dendritic cells (PBDCs) are altered in aGVHD, and find that the number of PBDCs (both myeloid and lymphoid DCs) is significantly decreased. Immunohistochemical staining of the biopsied skin from patients with aGVHD demonstrates that a number of fascin(+) cells with dendritic projections infiltrate the dermis of the skin. Based on these findings, we hypothesize that the PBDCs are recruited to the affected tissues and may thus play important roles in immune responses elicited in aGVHD.

Intra-arterial steroid-injection therapy for steroid-refractory acute graft-versus-host disease with the evaluation of angiography.

We treated three patients with steroid-refractory acute graft-versus-host disease (aGVHD) with intra-arterial steroid-injection therapy (IAST). Two patients with gut aGVHD received IAST into both superior and inferior mesenteric arteries, while one patient with liver aGVHD received IAST into the proper hepatic artery. The volume of stools and the bilirubin level improved soon after IAST. Angiography of the superior and inferior mesenteric arteries was performed in the two patients with steroid-refractory gut aGVHD, and identical abnormal findings were obtained. IAST might be an earlier option for steroid-refractory aGVHD.

Uncommon and dynamic changes detected by 123I-15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid myocardial single photon emission computed tomography in a stunned myocardium induced by coronary microvascular spasm.

A 55-yr-old man underwent surgery. Soon after the procedure was finished, the patient complained of chest pain, and the electrocardiogram showed increase in the ST-segment in some leads. Emergency angiography showed normal coronary arteries, but there was asynergy in the left ventricle, and delayed filling of contrast medium was observed in the LCA. An intracoronary infusion of isosorbide dinitrate did not improve the delayed filling of contrast medium or ST segment increase in the electrocardiogram. Soon after nicorandil was injected into the LCA, the patient's symptoms, electrocardiogram, and delayed filling of contrast medium dramatically improved. On the second day, initial imaging by 123I-BMIPP myocardial SPECT showed a moderate increase in tracer uptake in the apico-anteroseptal region and a moderate decrease in tracer uptake in the lateral region, in which the first left ventriculography showed akinesis, and delayed imaging revealed a moderate increase in tracer uptake in the apical region and a high washout of 123I-BMIPP in the anteroseptal and lateral regions. On the sixth day, initial imaging by 123I-BMIPP myocardial SPECT showed a moderate decrease in tracer uptake in the apical and lateral regions and a mild decrease in tracer uptake in the anteroseptal region, and delayed imaging revealed a moderate increase in tracer uptake in the apical region and a high washout of 123I-BMIPP in the anteroseptal and lateral regions. By the 30th day, 123I-BMIPP myocardial SPECT had normalized. We consider that these dynamic changes in 123I-BMIPP myocardial SPECT imaging may reflect metabolic changes in fatty acids in the ischemic state, the size of the triacylglycerol pool, and the degree of turnover in the triacylglycerol pool.

[Clinical usefulness of 123I-BMIPP myocardial SPECT in collagen disease].

This study was designed to evaluate the clinical usefulness of 123I-BMIPP myocardial SPECT for detecting cardiac involvement in patients with collagen disease. We studied 12 patients with systemic lupus erythematosus (SLE), 8 with progressive systemic sclerosis (PSS), 6 with polymyositis/dermatomyositis (PM/DM) and 3 with allergic granulomatosis and angiitis (AGA). A 111 MBq of 123I-BMIPP was intravenously injected at rest, and SPECT images were obtained at 15 min after the injection. Seven of 12 SLE, 6 of 8 PSS, 3 of 6 PM/DM and all 3 AGA patients showed an abnormal tracer uptake. The left ventricular ejection fraction was inversely correlated with a BMIPP abnormality. The regional wall motion abnormality was reduced in regions with reduced tracer uptake. These findings suggest that 123I-BMIPP imaging could be useful for assessment of cardiac involvement in patients with collagen disease.

[Bone marrow transplantation-associated thrombotic microangiopathy manifested by visual disturbance].

In October 1996, a 26-year-old woman was given a diagnosis of acute myeloblastic leukemia, FAB subtype M1. Treatment with combined chemotherapy achieved a complete remission (CR). In May 1997, the patient received an allogenic bone marrow transplant (BMT) from an HLA-identical sibling donor. Cyclosporine (CsA) and short-term methotrexate were given for graft-versus-host disease (GVHD) prophylaxis. Successful engraftment was obtained and signs of acute or chronic GVHD never developed. Five months after BMT, the patient experienced low-grade fever and blurred vision. Retinal examination demonstrated intraretinal hemorrhages, cotton-wool spots, and retinal detachments, which were presumably attributable to multiple thrombosis of retinal microvessels. The patient also exhibited hemolytic anemia with red cell fragmentation, thrombocytopenia, elevated lactate dehydrogenase, and renal impairment, and was thus given a diagnosis of BMT-associated thrombotic microangiopathy (BMT-TM). Discontinuation of CsA and administration of ticlopidine and prednisolone induced successful recovery from BMT-TM. Three months after the onset of BMT-TM, however, the patient experienced generalized clonic-tonic seizures with consciousness loss. Single-photon-emission computed tomography revealed blood-flow disturbances in the brain, suggesting the recurrence of microthrombosis. Accordingly, multiple transfusions of fresh frozen plasma were administered together with dipyridamole and aspirin. The patient gradually recovered and remained asymptomatic through the following 13 months. Currently, early diagnosis of BMT-TM is considered to be difficult. We suggest that careful examination of the ocular base may be useful for the early detection of BMT-TM.

Homology modeling of the insect chitinase catalytic domain--oligosaccharide complex and the role of a putative active site tryptophan in catalysis.

Knowledge-based protein modeling and substrate docking experiments as well as structural and sequence comparisons were performed to identify potential active-site residues in chitinase, a molting enzyme from the tobacco hornworm, Munduca sexta. We report here the identification of an active-site amino acid residue, W145. Several mutated forms of the gene encoding this protein were generated by site-directed mutagenesis, expressed in a baculovirus-insect cell-line system, and the corresponding mutant proteins were purified and characterized for their catalytic and substrate-binding properties. W145, which is present in the presumptive catalytic site, was selected for mutation to phenylalanine (F) and glycine (G), and the resulting mutant enzymes were characterized to evaluate the mechanistic role of this residue. The wild-type and W145F mutant proteins exhibited similar hydrolytic activities towards a tri-GlcNAc oligosaccharide substrate, but the former was approximately twofold more active towards a polymeric chitin-modified substrate. The W145G mutant protein was inactive towards both substrates, although it still retained its ability to bind chitin. Therefore, W145 is required for optimal catalytic activity but is not essential for binding to chitin. Measurement of kinetic constants of the wild-type and mutant proteins suggests that W145 increases the affinity of the enzyme for the polymeric substrate and also extends the alkaline pH range in which the enzyme is active.

NF-kappaB activation is required for human endothelial survival during exposure to tumor necrosis factor-alpha but not to interleukin-1beta or lipopolysaccharide.

In the presence of a protein synthesis inhibitor, cycloheximide, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), or lipopolysaccharide (LPS) induces human umbilical vein endothelial cells (HUVECs) to undergo apoptosis, suggesting that constitutive or inducible cytoprotective pathways are required for cell survival. We studied the correlation between nuclear factor-kappaB (NF-kappaB) activation and cell death induced by TNF-alpha, IL-1beta, or LPS. Adenovirus-mediated overexpression of a dominant-negative IkappaBalpha (inhibitor of kappaB) mutant blocked NF-kappaB activation by gel shift assay and blocked induction of vascular cell adhesion molecule-1 protein by TNF-alpha, IL-1beta, and LPS, a NF-kappaB-dependent response. In cells overexpressing the IkappaBalpha mutant, TNF-alpha induced cell death, whereas IL-1beta or LPS did not. We conclude that cell survival following TNF-alpha stimulation is NF-kappaB-dependent but that a constitutive or inducible NF-kappaB-independent pathway(s) protects IL-1beta- or LPS-treated HUVECs from cell death.

Proximity between Glu126 and Arg144 in the lactose permease of Escherichia coli.

Evidence has been presented [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] that Glu126 (helix IV) and Arg144 (helix V) which are critical for substrate binding in the lactose permease of Escherichia coli are charge paired and therefore in close proximity. To test this conclusion more directly, three different site-directed spectroscopic techniques were applied to permease mutants in which Glu126 and/or Arg144 were replaced with either His or Cys residues. (1) Glu126-->His/Arg144-->His permease containing a biotin acceptor domain was purified by monomeric avidin affinity chromatography, and Mn(II) binding was assessed by electron paramagnetic resonance spectroscopy. The mutant protein binds Mn(II) with a KD of about 40 microM at pH 7.5, while no binding is observed at pH 5.5. In addition, no binding is detected with Glu126-->His or Arg144-->His permease. (2) Permease with Glu126-->Cys/Arg144-->Cys and a biotin acceptor domain was purified, labeled with a thiol-specific nitroxide spin-label, and shown to exhibit spin-spin interactions in the frozen state after reconstitution into proteoliposomes. (3) Glu126-->Cys/Arg144-->Cys permease with a biotin acceptor domain was purified and labeled with a thiol-specific pyrene derivative, and fluorescence spectra were obtained after reconstitution into lipid bilayers. An excimer band is observed with the reconstituted E126C/R144C mutant, but not with either single-Cys mutant or when the single-Cys mutants are mixed prior to reconstitution. The results provide strong support for the conclusion that Glu126 (helix IV) and Arg144 (helix V) are in close physical proximity.

Lipopolysaccharide-induced NF-kappaB activation in human endothelial cells involves degradation of IkappaBalpha but not IkappaBbeta.

We studied the signal transduction pathways involved in NF-kappaB activation and the induction of the cytoprotective A20 gene by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC). LPS induced human A20 mRNA expression with a maximum level 2 h after stimulation. The proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and the tyrosine kinase inhibitor herbimycin A (HMA) blocked A20 mRNA expression and partially inhibited NF-kappaB DNA-binding activity induced by LPS treatment. LPS induced IkappaBalpha degradation at 30-60 min after treatment, but did not induce IkappaBbeta degradation up to 120 min. In contrast, TNF-alpha rapidly induced IkappaBalpha degradation within 5 min and IkappaBbeta degradation within 15 min. Cycloheximide did not prevent LPS-induced IkappaBalpha degradation, indicating that newly synthesized proteins induced by LPS were not involved in LPS-stimulated IkappaBalpha degradation. LPS-induced IkappaBalpha degradation was inhibited by ALLN, confirming that ALLN inhibits NF-kappaB activation by preventing IkappaBalpha degradation. Of note, HMA also inhibited LPS-induced IkappaBalpha degradation. However, tyrosine phosphorylation of IkappaBalpha itself was not elicited by LPS stimulation, suggesting that tyrosine phosphorylation of a protein(s) upstream of IkappaBalpha is required for subsequent degradation. We conclude that in HUVEC, LPS induces NF-kappaB-dependent genes through degradation of IkappaBalpha, not IkappaBbeta, and propose that this degradation is induced in part by HMA-sensitive kinase(s) upstream of IkappaBalpha.

Monocyte-derived macrophages prime peripheral T cells to undergo apoptosis by cell-cell contact via ICAM-1/LFA-1-dependent mechanism.

We designed the present study to clarify the mechanism of superantigen-induced apoptosis of human mature T cells and to elucidate the pivotal roles of monocyte-derived macrophages in induction of T cell apoptosis. Exposure of unfractionated human peripheral blood mononuclear cells to SEA, SEB or PHA elicited apoptosis in T cells after 5-day culture. In purified T cell preparations, SEB was unable to induce apoptosis, but was inductive when the purified T cells were cocultured with monocyte-derived macrophages adhering to plastic culture dishes. Placing the T cells in the insert wells which physically separated them from the adhering macrophages resulted in a complete loss of SEB-induced apoptosis. The addition of blocking antibodies against LFA-1, ICAM-1 and CD2 to the cocultures significantly inhibited the SEB-induced T cell apoptosis. We concluded therefore that direct contact of macrophages with T cells is critical in SEB-induced T cell apoptosis, and that adhesion molecules such as LFA-1/ICAM-1 and CD2 may be involved in the mechanism of this effect.

Cloning, expression, and hormonal regulation of an insect beta-N-acetylglucosaminidase gene.

Chitinolytic enzymes such as beta-N-acetylglucosaminidases are major hydrolases involved in insect molting. By screening a Manduca sexta (tobacco hornworm) cDNA library with an antibody against beta-N-acetylglucosaminidase from molting fluid of M. sexta pharate pupae, several putative cDNA clones for this enzyme were isolated. The longest of the cDNA clones has an insert of approximately 3 kb, and the complete nucleotide sequence was determined. Because this clone is missing the initiation codon and nucleotides corresponding to the leader peptide, the mRNA 5'-end sequence was determined by PCR (polymerase chain reaction) amplification and cycle sequencing. The sequence of the encoded protein from positions 23 to 35 is identical to the NH2-terminal sequence of one of the beta-N-acetylglucosaminidases isolated from pharate pupal molting fluid. The amino acid sequence is similar to those of silkworm, human, mouse, bacterial, and several other beta-N-acetylglucosaminidases. Two highly conserved regions in the amino acid sequence were found in all members of this family. Southern blot analysis suggested that the number of genes in the Manduca genome closely related to the cDNA clone may be as few as one. The beta-N-acetylglucosaminidase gene is expressed most abundantly in epidermal and gut tissues on days 6 and 7 of fifth instar larvae. Injection of 20-hydroxyecdysone induced expression of the beta-N-acetylglucosaminidase gene, whereas topical application of the juvenile hormone analog, fenoxycarb, suppressed the inductive effect of molting hormone.

Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains.

The lactose permease of Escherichia coli is a membrane transport protein containing 12 transmembrane hydrophobic domains connected by hydrophilic loops. Coexpression of lacY gene fragments encoding contiguous polypeptides corresponding to the first and second halves of the permease [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325-4329] or the first two transmembrane domains and the remainder of the molecule [Wrubel, W., Stochaj, U., Sonnewald, U., Theres, C., & Ehring, R. (1990) J. Bacteriol. 172, 5374-5381] leads to active lactose transport. It is shown here that contiguous permease fragments with discontinuities in loop 1 (periplasmic), loop 6 (cytoplasmic), or loop 7 (periplasmic) exhibit transport activity; however, fragments with discontinuities in transmembrane domains III or VII fail to do so. The results are consistent with the interpretation that contiguous permease fragments with discontinuities in hydrophilic loops form functional duplexes, while fragments with discontinuities in transmembrane alpha-helical domains do not. On the basis of this notion, a series of contiguous, nonoverlapping permease fragments with discontinuities at various positions in loop 6, putative helix VII, and loop 7 were coexpressed to approximate the boundaries of putative transmembrane domain VII. Contiguous fragments with a discontinuity between Leu222 and Trp223 or between Gly254 and Glu255 are functional, but fragments with a discontinuity between Cys234 and Thr235, between Gln241 and Gln242, or between Phe247 and Thr248 are inactive. Therefore, it is likely that Leu222 and Gly254 are located in hydrophilic loops 6 and 7, respectively, while Cys234, Gln241, and Phe247 are probably located within transmembrane domain VII.(ABSTRACT TRUNCATED AT 250 WORDS)

What's new with lactose permease.

The lactose permease of Escherichia coli is a paradigm for polytopic membrane transport proteins that transduce free energy stored in an electrochemical ion gradient into work in the form of a concentration gradient. Although the permease consists of 12 hydrophobic transmembrane domains in probable alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic "loops", little information is available regarding the folded tertiary structure of the molecule. In a recent approach site-directed fluorescence labeling is being used to study proximity relationships in lactose permease. The experiments are based upon site-directed pyrene labeling of combinations of paired Cys replacements in a mutant devoid of Cys residues. Since pyrene exhibits excimer fluorescence if two molecules are within about 3.5A, the proximity between paired labeled residues can be determined. The results demonstrate that putative helices VIII and IX are close to helix X. Taken together with other findings indicating that helix VII is close to helices X and XI, the data lead to a model that describes the packing of helices VII to XI.

Properties and purification of an active biotinylated lactose permease from Escherichia coli.

A simplified approach for purification of functional lactose permease from Escherichia coli is described that is based on the construction of chimeras between the permease and a 100-amino acid residue polypeptide containing the biotin acceptor domain from the oxaloacetate decarboxylase of Klebsiella pneumoniae [Cronan, J. E., Jr. (1990) J. Biol. Chem. 265, 10327-10333]. Chimeras were constructed with a factor Xa protease site and the biotin acceptor domain in the middle cytoplasmic loop (loop 6) or at the C terminus of the permease. Each construct catalyzes active lactose transport in cells and right-side-out membrane vesicles. Moreover, the constructs are biotinylated in vivo, and in both chimeras, the factor Xa protease site is accessible from the cytoplasmic surface of the membrane. Both biotinylated permeases bind selectively to immobilized monomeric avidin and are eluted with free biotin in a high state of purity, and the loop 6 chimera catalyzes active transport after reconstitution into proteoliposomes. The methodology described should be applicable to other membrane proteins.

Interferon-gamma suppresses PDGF production from THP-1 cells and blood monocyte-derived macrophages.

Involvement of the immunological mechanisms in atherogenesis has recently been suggested by immunohistological detection of macrophages and T lymphocytes in atherosclerotic lesions. In the present study, we have investigated the regulatory effect of interferon-gamma (IFN-gamma), a cytokine secreted by activated T cells, on the production and secretion of platelet-derived growth factor (PDGF) from macrophages in culture. The human monocytic leukemia cell line, THP-1, was treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce macrophage differentiation and PDGF production, and then various doses of recombinant human IFN-gamma (0-1000 I.U./ml) were added to the culture. After 48 h, the conditioned medium and the cells were harvested and analyzed for PDGF production. PDGF-dependent mitogenic activity in the conditioned medium, estimated by neutralization of mitogenic activity with anti-PDGF antibody, was suppressed by IFN-gamma treatment. Radioimmunoassays for PDGF also revealed a decrease in both PDGF-AA and -BB in the conditioned medium with IFN-gamma treatment, whereas neither total cell DNA as an indication of cell number nor overall protein synthesis based on [3H]leucine incorporation were decreased. Northern analysis of total RNA extracted from the cells demonstrated that IFN-gamma suppressed the level of PDGF mRNA. Analysis of mRNA degradation in the presence of actinomycin D demonstrated that the decrease in PDGF mRNA was not due to enhanced degradation of mRNA. A similar inhibitory effect of IFN-gamma on PDGF mRNA levels was also found in monocyte-derived macrophages cultured in the presence of granulocyte-macrophage colony stimulating factor. These results suggest that IFN-gamma modulates production and secretion of PDGF from macrophages and that the functions of macrophages in atherogenesis may be regulated by the cellular interactions between T cells and macrophages through the action of cytokines such as IFN-gamma.