Cloning and sequencing of a new gro transcript from activated human monocytes: expression in leukocytes and wound tissue.

Using cDNA cloning, we have identified a new member of the gro/KC inflammatory protein superfamily (gro-beta) produced by activated human monocytes and neutrophils and expressed at sites of inflammation in vivo. The open reading frame encodes a protein with 91% sequence homology to the human gro protein. Interestingly, some of the amino acid substitutions are located at regions where proteolytic cleavage occurs in the gro protein generating biologically active peptides (neutrophil activating peptide III). These changes would likely alter the processing of the gro-beta peptide and suggest that peptides with biological activities different from those produced by gro-alpha are encoded by this transcript.

Facial emphysema caused by cheek bite.

Biting of the buccal mucosa is very frequent injury, whereas facial emphysema caused by cheek bite is rare. We report a case of facial emphysema causing puffing of the cheek through a self-inflicted bite of the buccal mucosa.

Monoclonal antibodies directed to a disulfated glycosphingolipid, SB1a (GgOse4Cer-II3IV3-bis-sulfate), associated with human hepatocellular carcinoma.

Two murine monoclonal antibodies, 2H6G5 (IgM) and 4A9E10 (IgG3), were obtained by using either cultured human hepatocellular carcinoma cells (PLC/PRF/5) or the acidic glycolipid mixture prepared from the same cells as immunogens. The antigen in PLC/PRF/5 cell membranes recognized by both antibodies was identified as a disulfated acidic glycolipid, GgOse4Cer-II3IV3-bis-sulfate (SB1a). Both antibodies reacted specifically with SB1a, and no significant reactivity was noted with other sulfated glycolipids or gangliosides except that the antibody 2H6G5 showed a weak cross-reactivity with LacCer-II3-sulfate (SM3), another sulfated glycolipid which partly shares the same carbohydrate structure as SB1a. The SB1a antigen is a relatively minor glycolipid in PLC/PRF/5 cells, but it was strongly expressed at the surface of PLC/PRF/5 cells as ascertained by cytofluorometry using both antibodies. A significant amount of SB1a antigen was present in 3 of the acidic glycolipid fractions isolated from 15 human hepatocellular carcinoma tissues as well as in the acidic glycolipid fraction prepared from PLC/PRF/5 cells, while all the acidic glycolipid fractions prepared from cirrhotic livers and a normal liver were essentially negative for SB1a, as ascertained by both solid phase enzyme immunoassay and the thin-layer chromatography-immunostaining method. These results strongly suggest that the SB1a antigen as defined by these new monoclonal antibodies is associated with human hepatocellular carcinoma.

A new process of cancer prevention mediated through inhibition of tumor necrosis factor alpha expression.

Mechanisms of cancer prevention were studied using structurally different cancer-preventive agents, sarcophytol A, canventol, (-)-epigallo-catechin gallate, and tamoxifen, based on our evidence that tumor necrosis factor alpha (TNF-alpha) acts as an endogenous tumor promoter relevant to human carcinogenesis. Pretreatment with the four preventive agents commonly inhibited TNF-alpha mRNA expression and TNF-alpha release in BALB/3T3 cells induced by a tumor promoter, okadaic acid, whereas the expression of early response genes (c-jun, junB, c-fos, and fosB) was enhanced. These results strongly suggest that inhibition of TNF-alpha mRNA expression and its release is a new process of cancer prevention.

Nodularin, a potent inhibitor of protein phosphatases 1 and 2A, is a new environmental carcinogen in male F344 rat liver.

Nodularin and microcystin-LR are cyanobacterial toxins and environmental hazards. Nodularin inhibits protein phosphatases 1 and 2A with the same potency as does microcystin-LR, which has recently been identified as a potent tumor promoter in rat liver. Our results suggested that nodularin is also a new tumor promoter in rat liver. A two-stage carcinogenesis experiment in rat liver initiated with diethylnitrosamine and without partial hepatectomy revealed that nodularin stimulated glutathione S-transferase placental form-positive foci in rat liver more effectively than did microcystin-LR, and that nodularin alone induced glutathione S-transferase placental form-positive foci as well as did diethylnitrosamine alone. Thus, nodularin itself is a new liver carcinogen, and microcystin-LR is a tumor promoter rather than a carcinogen. Nodularin induced hyperphosphorylation of cytokeratin peptides 8 and 18 in primary cultured rat hepatocytes 20% more effectively than did microcystin-LR, suggesting that nodularin penetrates more easily into the hepatocytes than does microcystin-LR. Nodularin up-regulated induction of c-jun, jun-B,jun-D,c-fos,fos-B, and fra-1 mRNA transcripts in rat liver after i.p. administration, and the accumulation of the mRNA transcripts was sustained for over 9 h after treatment. The environmental hazards of cyanobacterial toxins are discussed in relation to human primary liver cancer in Qidong county in the People's Republic of China. Our results support this hypothesis and indicate the need for prevention measures against cyanobacterial toxins.

Identification of bladder tumor-derived hyaluronidase: its similarity to HYAL1.

The glycosaminoglycan hyaluronic acid (HA) and its degrading enzyme, hyaluronidase, are intricately associated with tumor metastasis and angiogenesis. HA promotes tumor cell adhesion and migration, whereas its small fragments stimulate angiogenesis. Such small HA fragments are generated from the degradation of HA by hyaluronidase. We have previously shown (V. B. Lokeshwar et al., Cancer Res., 57: 773-777, 1997) that the HA levels are elevated in the urine and tumor tissues of bladder cancer patients regardless of the tumor grade (G). The hyaluronidase levels were found to be elevated in the urine and tumor tissues of G2 and G3 bladder cancer patients. Furthermore, angiogenic HA fragments were isolated from the urine of G2/G3 bladder cancer patients, which stimulated endothelial cell proliferation, a key event in angiogenesis. In this study, we characterized the bladder tumor-derived hyaluronidase. Analysis of hyaluronidase activity in the culture-conditioned media (CM) of 11 bladder cancer cell lines, using an ELISA-like assay and a substrate (HA)-gel technique, showed that the invasive bladder cancer cell lines secrete elevated levels of a Mr approximately 60,000 hyaluronidase. Reverse transcription-polymerase chain reaction, cloning, and sequence analyses revealed the expression of an HYAL1 transcript in bladder cancer lines. HYAL1 encodes for a hyaluronidase that is present in serum. Immunoblot analysis using an anti-HYAL1 peptide IgG confirmed the presence of a Mr approximately 60,000 HYAL1-related protein in the CM of bladder cancer cell lines, in the urine specimens from G2 and G3 bladder cancer patients, and in the partially purified preparations of bladder tumor-derived hyaluronidase. No HYAL1-related protein was detected in urine specimens from normal individuals, G1 bladder cancer patients, and patients with a history of bladder cancer but no disease at the time of testing. The bladder tumor-derived hyaluronidase present in CM and partially purified preparations was found to have maximum activity at a pH range of 4.1-4.3. The identification of bladder tumor-derived hyaluronidase should help in elucidating its role in bladder tumor progression.

Requirement of Ras for the activation of mitogen-activated protein kinase by calcium influx, cAMP, and neurotrophin in hippocampal neurons.

Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term potentiation both in vitro and in living animals. MAP kinase is activated in response to a broad range of stimuli, including calcium influx through NMDA receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and cAMP response element-binding protein (CREB) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase-activating protein, Gap1(m), or dominant negative Ras by means of adenovirus vectors. Gap1(m) expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed similar effects. On the other hand, Rap1GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. In contrast to MAP kinase activation, the inactivation of Ras activity did not inhibit significantly NMDA-induced CREB phosphorylation, whereas BDNF-induced CREB phosphorylation was inhibited almost completely. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons and further suggest that CREB phosphorylation depends on multiple pathways.

A new splice variant of the inositol-1,4,5-triphosphate (IP3) receptor.

In this study we have identified a new splice variant of the IP3 receptor (IP3R) transcript in a number of mouse cell lines (e.g. mouse T-lymphoma cells, mouse splenic lymphocytes and mouse NIH 3T3 fibroblast cell lines) using the reverse transcriptase-polymerase chain reaction. This variant IP3 receptor (designated as IP3RV-S2, approximately 453 bp) is larger than the non-neuronal form (402 bp) but smaller than the neuronal form (522 bp) of the IP3 receptors. Nucleotide sequencing data indicate that this new isoform (IP3RV-S2) contains a 51 nucleotide insertion within the non-neuronal form of IP3R at the S2 splice site. During mitogenic stimulation by Con A, the ratio between IP3R (non-neuronal form) and IP3RV-S2 (variant isoform) in mouse splenic T-lymphocytes increases approximately 1.5-fold. The change in relative amounts of these two IP3 receptor isoforms during mitogenic-stimulation suggests that T-lymphocytes may have different requirements for the IP3 isoforms in order to control intracellular calcium mobilization. The selective expression of these two IP3R isoforms (IP3RV-S2 and non-neuronal IP3R) may be critically important for the onset of signal transduction and cell activation.

Different flow regulation mechanisms between celiac and mesenteric vascular beds in conscious rats.

The aims of this study were to elucidate the vasoconstrictor mechanism that mediates the changes in celiac and mesenteric vascular resistances during vasoconstriction and hypertension induced by ganglionic blockade and to explore the preferential mechanism that contributes to the elevation of arterial pressure in conscious spontaneously hypertensive rats (SHR). In conscious SHR and normotensive control rats, blood flow and arterial pressure were measured with an implanted electromagnetic flow probe and an indwelling arterial catheter. Peripheral vascular resistance was calculated as arterial pressure divided by regional flow. Celiac contribution to the hypertension in SHR was below average for the entire body and was smaller than that from the superior mesenteric bed. The increase of mesenteric resistance with arterial pressure elevation after ganglionic blockade suggests that mesenteric blood flow is regulated by a stretch-dependent myogenic mechanism, whereas celiac blood flow is regulated preferentially by the sympathetic neural mechanism. It is speculated that the flow superregulation in the mesenteric bed in SHR is due to the enhanced myogenic response and contributes to the early stage of hypertension.

A new CD44V3-containing isoform is involved in tumor cell growth and migration during human breast carcinoma progression.

CD44 isoforms belong to a family of cell adhesion molecules expressed on the cell surface of many tumor cells during human breast cancer progression. In this study we have analyzed the expression of CD44v3-containing isoforms [containing heparan sulfate addition sites for growth factor binding] in primary breast tumors, axillary nodal metastases and normal breast tissue. Using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot, cloning, nucleotide sequencing and RT-in situ-PCR analyses, we have found that at least two CD44v3-containing isoforms, including one new species of CD44v2,deltav3-10 (deltav3 defined as a v3 exon lacking the first 24 base pairs) and another previously reported CD44v3,8-10 are preferentially expressed in human primary breast tumor and axillary nodal metastases but not in normal breast tissues. These finding suggest that these CD44v3-containing isoforms are closely associated with breast cancer metastasis.

Discrete roles of cytokines, TNF-alpha, IL-1, IL-6 in tumor promotion and cell transformation.

Based on our previous results, which pointed to tumor necrosis factor-alpha (TNF-alpha) as the essential cytokine in tumor promotion in mouse skin, we present here three principal findings related to the specific roles of TNF-alpha, interleukin-1 (IL-1) and IL-6 in tumor promotion (using TNF-alpha- and IL-6-deficient mice) and in BALB/3T3 cell transformation: i) The previously reported residual tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in TNF-/- mice was confirmed by experiments with TNF+/+ and TNF-/- 129/Svj mice of the same strain, using two-stage carcinogenesis experiments. TPA produced tumors in 100% of TNF+/+ and 78% of TNF-/- mice at 20 weeks, and the average number of tumors per mouse was 11.1 in the former group and 2.1 in the latter. Judging from the expression of various inflammatory cytokine genes in TNF+/+ and TNF-/- mice, the residual tumor promoting activity of TPA in TNF-/- mice may be dependent on expression of IL-1alpha and IL-1beta genes. ii) Tumor promotion by TPA and okadaic acid in IL-6+/+ and IL-6-/- C57/BL6 mice was studied, with TPA producing tumors in 57.1% of IL-6+/+ and 40.0% of IL-6-/- mice at 20 weeks, and okadaic acid in 40.0% of IL-6+/+ and 53.3% of IL-6-/- mice. Thus, there was no significant difference between TPA or okadaic acid tumor promotion in either group. In addition, expression of IL-6 gene in skin of both types of mice suggested that IL-6 is not the essential cytokine in tumor promotion, since it can be replaced by other cytokines. iii) In transformed clones of BALB/3T3 cells induced by TNF-alpha alone, IL-1alpha gene expression was induced after transformation by TNF-alpha had occurred, which did not occur in parental cells. Expression patterns of TNF-alpha, IL-1beta, IL-6 and IL-10, along with TGF-beta, were similar in both parental and transformed cells. Considering all these results, we conclude that various cytokines have discrete roles in tumor promotion and cell transformation.

A possible pathogenic role of CD8+ T cells and their derived cytokine, IL-16, in SLE.

Current investigations into the role of CD8+ T cells and their derived cytokine, interleukin (IL)-16, in the induction of CD4+ T cell abnormalities in systemic lupus erythematosus (SLE) were reviewed and discussed on the basis of results mainly obtained in our laboratory.

Purification, molecular cloning, and genomic organization of human brain long-chain acyl-CoA hydrolase.

An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g. a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.

A polymorphism of DRD2 gene and brain atrophy in methamphetamine psychosis.

Our group, Ujike et al., recently reported that the A1 allele of TaqI A polymorphism of the dopamine receptor D2 (DRD2) gene, associated with transient psychosis, significantly differs from that of patients with prolonged psychosis in methamphetamine psychosis. Therefore, we examined the association between the TaqI A polymorphism of the DRD2 gene and the brain MRI view for patients with methamphetamine psychosis. The subjects underwent brain MRI scans using the FLAIR method. Genotyping was performed by PCR-RFLP methods using genomic DNA extracted from peripheral blood by the phenol method. Ten subjects had the A1/A2 genotype, eleven subjects had the A2/A2 genotype, and no subject had the A1/A1 genotype. The domain size, including the thalamus and basal ganglia that were inside each side of the putamens, did not differ between the three groups (the A1/A2-group, the A2/A2-group, and the young healthy person group). In the comparison based on this domain, the temporal lobe tended to narrow in the A2/A2-group compared to the A1/A2-group (P = .06). The other domain (cerebrum, corpus callosum, etc.) showed no difference between the A1/A2-group and the A2/A2-group. It is suggested that in methamphetamine psychosis the TaqI A polymorphism not only regulates prolongation of psychosis symptoms but also influences the form of the temporal lobe.

Pharmacokinetics of ofloxacin in severe chronic renal failure.

The pharmacokinetics of ofloxacin were studied in patients with severe renal impairment. In five healthy subjects and nine patients with chronic renal failure (CRF) (creatinine clearance [CCR] less than 20 ml/min) receiving 100 mg of ofloxacin orally in the fasted state, the serum concentration was measured over the subsequent 48 hours. Intracorporeal dynamics were examined, employing a one-compartment open model. Ofloxacin levels were measured using the paper disk method. The half-life of ofloxacin was markedly prolonged, to 23.1 +/- 7.0 hr in the CRF group versus 2.9 +/- 0.5 hr for healthy subjects. In the CRF group, the maximum concentration and area under the curve were greater than in healthy subjects. There were no significant differences in volume of distribution between the two groups. The renal clearance of ofloxacin decreased from 261.0 +/- 46.6 ml/min in healthy subjects to 8.0 +/- 4.7 ml/min in the CRF group and correlated significantly with CCR in the CRF group (r = .88, P less than 0.01). There were no significant differences in nonrenal clearance between the two groups. The 24-hour renal excretion of ofloxacin averaged 91.9 +/- 5.4% and 14.1 +/- 5.5% of the dose in the healthy and CRF groups, respectively. In three hemodialysis patients on the regular hollow-fiber dialyser, the dialysance of ofloxacin was about 50% that of creatinine. Based on these findings, a reduction in the dose of ofloxacin is necessary in patients with CRF. In the hemodialyzed patients, its high dialyzability should be considered when deciding dose regimens.

Allergic diseases in systemic lupus erythematosus: prevalence and immunological considerations.

We attempted to obtain a deeper understanding of the relationship between systemic lupus erythematosus (SLE) and allergic diseases through comparative studies. Accordingly, we reviewed the association of both disorders and compared their immunological features based on the literature and our own findings. Recent studies (including ours) have indicated that the risk of IgE-mediated and/or associated allergic diseases is not markedly increased in SLE patients despite their more allergic family history when compared with controls, in contrast with earlier studies. This may be related to a change of the environmental factors contributing to allergy. In addition, assessment of the immunological similarities and differences between SLE and various allergic diseases seems to be useful for understanding the relationship between them.

Systemic lupus erythematosus-like autoimmune abnormalities induced by bacterial infection.

Recent evidence has revealed that bacterial DNA can promote several of the autoimmune abnormalities observed in systemic lupus erythematosus (SLE), and a possible pathogenic role in the induction of SLE has been highlighted. We have recently encountered patients in whom bacterial infection (septicemia) triggered the production of several autoantibodies. This seems to be interesting with respect to the consideration of the relationship between SLE and bacterial infection.

Hemophagocytosis in autoimmune disease.

Reactive hemophagocytic syndrome (HPS) is known to be associated with various autoimmune diseases, as well as infection and/or malignancy. Here we review the features of autoimmune-associated HPS and describe the possible role of autoantibodies, especially antiphospholipid antibodies (aPL), in HPS based on data obtained from our own patients.

Cytomegalovirus infection in patients with systemic lupus erythematosus.

Cytomegalovirus (CMV) infection is known to induce several autoimmune abnormalities in mice that resemble those found in systemic lupus erythematosus (SLE). In addition, a potential role for CMV in the development and/or progression of SLE has been suggested. In order to further clarify this issue, we reviewed the relationship between SLE and CMV infection on the basis of the clinical and immunological features of cases reported in the literature and our own patients.

HLA-DRB1 alleles and beta 2 glycoprotein I-dependent anticardiolipin antibodies in Japanese patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the association of HLA DRB1 alleles with beta 2 glycoprotein I (beta 2 GPI)-dependent anticardiolipin antibodies (aCL) in Japanese patients with systemic lupus erythematosus (SLE). METHODS: One hundred and forty-five Japanese patients with SLE were studied. beta 2 GPI-dependent aCL was measured by enzyme-linked immunosorbent assays. DNA typing of the DRB1 alleles was performed by the polymerase chain reaction sequence specific oligonucleotide probe method. RESULTS: beta 2 GPI-dependent aCL was positive in 29 (20.0%) out of 145 SLE patients. SLE patients with beta 2 GPI-dependent aCL had a significantly higher frequency or one or more of the clinical manifestations assumed to be associated with aCL, compared to those without beta 2 GPI-dependent aCL (p < 0.05). The frequency of DRB1*0901 was lower in SLE patients than in healthy subjects. SLE patients with beta 2 GPI-dependent aCL were significantly associated with DRB1*0901 as compared to those without beta 2 GPI-dependent aCL (41.4% vs 15.5%, p < 0.005, R.R. = 3.8), although the corrected P value was not significant. CONCLUSION: A possible association of DRB1*0901 with Japanese SLE patients with beta 2 GPI-dependent aCL was found. This association indicates an association between the disease and the HLA-DR53 (DRB4)-bearing haplotypes in different ethnic groups.