Constitutive and interleukin-1 (IL-1)-inducible factors interact with the IL-1-responsive element in the IL-6 gene.

The interleukin-6 (IL-6) promoter is rapidly and transiently activated with other cytokines, including IL-1, tumor necrosis factor, and platelet-derived growth factor, as well as phorbol esters and agents that increase intracellular cyclic AMP. In this study, we have investigated cis-acting regulatory elements and trans-acting factors responsible for IL-1-induced IL-6 gene expression. Studies on the 5' deletion mutants of the human IL-6 gene suggested that the IL-1-responsive element was mapped within the IL-6 promoter region (-180 to -123) which was homologous to the c-fos serum-responsive enhancer element. Gel retardation assay identified two types of nuclear factors that bound to this region, one constitutive and the other inducible. These two factors recognized a 14-base-pair (bp) palindromic sequence, ACATTGCACAATCT. Furthermore, three copies of this 14-bp palindrome conferred IL-1 responsiveness to the basal enhancerless IL-6 promoter, indicating that a 14-bp-dyad symmetry sequence was an IL-1-responsive element in the IL-6 gene.

Action of crystalline acid carboxypeptidase from Penicillium janthinellum.

Acid carboxypeptidase (EC 3.4.12.-) crystallized from culture filtrate of Penicillium janthinellum has been investigated for its use in carboxy-terminal sequence determination of Z-Gly-Pro-Leu-Gly, Z-Gly-Pro-Leu-Gly-Pro, angiotensin I, native lysozyme, native ribonuclease T1, and reduced S-carboxy-methyl-lysozyme. The examination indicated that proline and glycine were liberated from Z-Gly-Pro-Leu-Gly-Pro. At high enzyme concentration, the enzyme catalyzed complete sequential release of amino acids from the carboxy-terminal leucine to the amino-terminal aspartic acid of angiotensin I. The enzyme released the carboxy-terminal leucine from native lysozyme, however, no release of the threonine from native ribonuclease T1 was observed after a prolonged period of incubation with the enzyme. The sequence of the first nine carboxy-terminal residues of denatured lysozyme, leucine, arginine, S-carboxymethyl-cysteine, glycine, arginine, isoleucine, tryptophane, alanine, and glutamine, could be deduced unequivocally from a time release plot of an incubation mixture with the enzyme.

A patient with anaplastic large cell lymphoma (Ki-1 lymphoma) showing clonal integration of HTLV-1 proviral DNA.

We encountered a patient with anaplastic large cell lymphoma (Ki-1 lymphoma) that originated in the stomach and showed histiocytic lymphoma-like morphology. CD43 antigen was positive, and rearrangement of TCR-beta gene was observed. The lymphoma was the T-cell type. Though no atypical lymphocytes or histological images specific to adult T-cell leukemia were observed, clonal integration of HTLV-1 proviral DNA was noted. Viruses such as HTLV-1 appear to be involved in the development of some anaplastic large cell lymphomas.

Molecular heterogeneity of the cDNA encoding a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A.

Two cDNAs for possible splicing variants of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A, were isolated. These variants were identified from human cerebral cortex by library screening and PCR, and designated delta1 and delta3 isoforms, while the previously reported isoform [Tanabe et al. (1996) FEBS Lett. 379, 107-1111 was designated delta2. Compared with the delta2 isoform, the delta1 isoform contained a 32-residue insertion beginning at residue 84, and consisted of 602 amino acids in all. The delta3 isoform lacked a 74-residue sequence corresponding to residues 1083 of the delta2 isoform, and consisted of 496 amino acids. Using isoform-specific antipeptide antisera, the 74-kDa subunit (B" or delta) originally purified from human erythrocytes was identified as the delta1 isoform.

Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B'' (delta) regulatory subunit in vitro and identification of the phosphorylation sites.

Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B'' (delta) subunit, was phosphorylated at serine residues of B'' in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 microM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B'', respectively. The Km value of A-kinase for CAB'' was 0.17 +/- 0.01 microM in the presence of OA. The major in vitro phosphorylation sites of B'' were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B'' did not dissociate B'' from CA, and stimulated the molecular activity of CAB'' toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a.

Molecular cloning of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A.

Based on amino acid sequence data of a 74-kDa regulatory subunit (B" or delta) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library. The cDNA had an open reading frame encoding an M(r) 66,138 protein of 570 amino acids. Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74-kDa subunit. The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72. Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, and SH3 accessible proline-rich domain, and a unique PQ repeat were found in the sequence. The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues.

Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one.

A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.

Interconversion of Mn(2+)-dependent and -independent protein phosphatase 2A from human erythrocytes: role of Zn(2+) and Fe(2+) in protein phosphatase 2A.

Human erythrocyte Mn(2+)-dependent (C'A') and -independent (CA) protein-serine/threonine phosphatase (PP) 2A are composed of 34-kDa catalytic C' and C subunits, in which the metal dependency resides, and 63-kDa regulatory A' and A subunits, respectively. Each catalytic and regulatory subunit gave the same V8- and papain-peptide maps, respectively. Stoichiometric zinc and substoichiometric iron were detected in CA but not in C'A' [Nishito et al. (1999) FEBS Lett. 447, 29-33]. The Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity of C'A' was about 70-fold higher than that of CA. Pre-incubation of CA with 25 mM NaF changed CA to a Mn(2+)-dependent form with higher PTP activity. The same NaF treatment had no effect on C'A'. Pre-incubation of C'A' with ZnCl(2), zinc-metallothionein, or FeCl(2) activated the Mn(2+)-independent PP activity, but pre-incubation with FeCl(3) did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl(2). Pre-incubation of C'A' with 5 microM ZnCl(2) and 15 microM FeCl(2) in the presence of 1 mM ascorbate synergistically stimulated the Mn(2+)-independent PP activity, with concomitant suppression of the Mn(2+)-dependent PP and PTP activities. The PP and PTP activities of CA were unaffected by the same zinc and/or iron treatment. Micromolar concentrations of vanadate strongly inhibited the Mn(2+)-dependent PP activity of C'A' but only slightly inhibited the PP activity of CA. Using the distinct effect of vanadate as an indicator, the interconversion between CA and C'A' with the above mentioned treatments was proved. These results support the notion that Mn(2+)-independent CA is a Zn(2+)- and Fe(2+)-metalloenzyme, whose apoenzyme is Mn(2+)-dependent C'A'.

Tissue and subcellular distributions, and characterization of rat brain protein phosphatase 2A containing a 72-kDa delta/B" subunit.

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/B". Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 +/- 0.3 S and the Stokes radius of 5.5 +/- 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10(-9) M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.

Identification of autophosphorylation sites in c-Yes purified from rat liver plasma membranes.

c-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNEYTAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-16 through Lys-46, YTPENPTEPVNTSAGHYGVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.

Studies on functions of the 63-kDa A- and 74-kDa B'(delta)-regulatory subunits in human erythrocyte protein phosphatase 2A: dissociation and reassociation of the subunits.

A heterodimeric form, CA, of protein-serine/threonine phosphatase (PP) 2A purified from human erythrocytes was dissociated into a 34-kDa catalytic subunit C and 63-kDa inactive subunit A by Sephacryl S-200 gel filtration in the presence of 6 M urea. Reassociation of the C- and A-subunits in the absence of urea suppressed the PP activity of the C subunit toward phosphorylase a, P-H2B histone, and P-H1 histone in the presence or absence of 20 mM MnCl(2) or 50 mM Mg(CH(3)COO)(2), but stimulated the PP activity toward P-H1 histone in the presence of 200 mM NaCl and the Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity toward P-Tyr-Glu copolymers. The 74-kDa inactive B'(delta) subunit was isolated from a heterotrimeric form, CAB'(delta), of PP2A partially purified from human erythrocytes, by heparin-Sepharose column chromatography. The B'(delta) subunit reassociated with CA and suppressed the PP- and PTP-activities of CA. The B'(delta) subunit did not associate with the isolated C subunit directly, and had no effect on the activities of the C subunit, indicating that the A subunit is essential for the association of the B'(delta) subunit with CA and the resulting suppression of the PP- and PTP-activities.

A possible mechanism of inability of immunoglobulin heavy-chain production in Bence-Jones type myeloma cells.

To clarify the mechanism of the inability of immunoglobulin heavy (IgH) chain production in Bence-Jones (BJ) type myeloma cells, we examined the rearrangement of the IgH gene, the expression of IgH mRNA and the presence of functional nuclear factors that bind to the IgH gene enhancer in myeloma cells from 6 cases of BJ-type myelomas, and three myeloma cell lines and one Epstein-Barr virus-transformed B cell line having no IgH chain production. All BJ-type myeloma cells we examined showed rearranged bands by Southern blotting using the JH probe, and the rearrangement of VDJ segments in these cells was confirmed by PCR amplification of VDJ segments with consensus VH and JH primers. However, the expressions of IgH mRNA were not detected by Northern blotting in any of the BJ-type myeloma cells or the three non-producer myeloma cell lines. These findings suggested that the transcription of the IgH gene ceased in these cells, and alteration of this transcriptional apparatus may explain this inability. The functional nuclear factors that bind to IgH gene enhancers (HE2 and HE3) were absent in the myeloma cells from one case of BJ-type myelomas among these BJ-type myeloma cells and myeloma cell lines. This suggests that alteration of the enhancer binding factors may be one of the reasons why BJ-type myeloma cells are unable to produce the IgH chain.

[Study on the therapy of multiple myelomas--initial induction therapy (MP, IFN alpha, steroid pulse) and maintenance therapy (VMP, MP continuous, VEP, MCNU)].

Untreated twenty patients of multiple myeloma were treated with the chemotherapy protocol as follows: Initial induction therapy;MP continuous or MP intermittent----IFN alpha----steroid pulse. Maintenance therapy;alkylating agents which have no cross resistance were used ((V) MP----(MP)----(V) EP----MCNU). Remission rate (CR+PR) after the initial MP therapy was 45%, and that after including IFN alpha and steroid pulse therapy was 50%, Fifty percent survival rate was almost as same as those reported previously (34M). Our protocol presented here was based on the idea that, initially, myeloma cells with proliferative activity could be affected by MP therapy, and subsequent IFN alpha therapy would have effect even on the residual myeloma cells. Serial checks of 3H-TdR uptake of myeloma cells during the therapy supported this idea. During the maintenance therapy, clinical responses to the initial induction therapy were not aggravated in the responded cases when evaluated by the variation of serum M-protein level. We propose that considering from a point of proliferative activity of myeloma cells is important for designing therapeutic protocols for multiple myeloma.

[A case of small cell carcinoma originating at the site of pneumoplication of giant bulla five years later].

A 64 year-old man was performed pneumoplication for his left-sided giant bulla on December 15, 1983. After five years, consciousness loss and right hemiplegia occurred. Brain CT revealed a cystic lesion with enhancement in the left occipital lobe. Brain biopsy disclosed metastatic small cell carcinoma. Lung biopsy showed the same histopathology, so primary lesion was considered to be from the site of the previous pneumoplication. Lung cancer associated with emphysematous bulla have been mostly characterized by tumor shadow or niveáu in the bulla. In this case, the postoperative infiltrating shadow of pneumoplication had made early diagnosis difficult.

[Mortality associated with influenza epidemics in Japan--analysis for viral type, age and risk groups].

In this report, mortality with influenza in Japan was analyzed. The data covered mainly the period 1975-94. I tried to analyze these data for cause of death, viral types, age groups and three categories of diseases in order to determine distinctive risk factors for influenza-associated mortality. To summarize, influenza is a common disease which could lead especially for the elderly and those who have increased risk factors (cardiovascular diseases, cerebrovascular diseases and pulmonary complications) to severe complications and death. The cause of death is thought to be mainly influenza related pneumonia. Elderly and high-risk persons should be vaccinated.

[Structure and expression of the human neutrophil elastase gene--regulatory mechanism and its relevance to the respiratory diseases].

Human neutrophil elastase is a 29 kDa, 220-residue single chain glycoprotein which functions as a powerful serine protease. Because NE is capable of destroying a broad range of substrates including cross-linked elastin and the major forms of collagen as well as the cell walls of gram-negative bacilli, it possesses the two-edged sword property that is required for normal tissue turnover and host defense, yet potentially harmful in its ability to destroy normal tissues simultaneously. In this regard, NE plays a central role in the pathogenesis of pulmonary emphysema by destroying the alveolar walls of the lung in the conditions that antiproteases in the lung such as alpha 1-antitrypsin (alpha 1-AT) are inactivated-e.g., cigarette smoking, or alpha 1-AT deficiency caused by mutations of the alpha 1-AT gene-resulting in excess burden of NE in the lung. The gene encoding the NE protein has 5 exons and is located at chromosome 19p13.3. Expression of the NE gene is tightly controlled mainly at the transcriptional level, and limited to the early stage of myeloid cell differentiation in bone marrow cells, mostly in promyelocytes. The knowledge on the modulation of lineage- and differentiation-specific NE gene expression could offer the possible therapeutic strategy to the diseases such as pulmonary emphysema.