Cloning of human keratolinin cDNA: keratolinin is identical with a cysteine proteinase inhibitor, cystatin A, and is regulated by Ca2+, TPA, and cAMP.

Keratolinin has been described as one of the precursor proteins of cornified cell envelope of keratinocytes. Using rabbit polyclonal anti-human keratolinin antibody, we isolated a cDNA clone of human keratolinin gene from a human Agt11 cDNA expression library that was constructed by random priming from poly(A)+RNA extracted from cultured normal human keratinocytes. Screening by rabbit anti-human keratolinin antibody detected one positive clone (HKL-1). The recombinant 12.5-kDa protein constructed from the clone reacted specifically with the anti-human keratolinin antibody. DNA sequence analysis revealed that HKL-1 clone was 448 bp long, and its putative amino acid sequence was identical with that of a human cysteine proteinase inhibitor, cystatin A. Western blot analysis showed that the commercially available recombinant cystatin A also reacted specifically with the anti-human keratolinin antibody. Northern blot analysis indicated that HKL-1 clone hybridizes with mRNA of about 0.5 kb, consistent with the size of the HKL-1 clone. The keratolinin mRNA was highly expressed in cultured human keratinocytes in high Ca2+ (1 mM); in low Ca2+ (0.05 mM), the keratolinin mRNA expression was significantly lower. Using SV40-transformed human keratinocytes (SVHK cells), we further analyzed the regulation of keratolinin mRNA. In low Ca2+ (0.05 mM), keratolinin mRNA in SVHK cells was marginally detectable. Upon shift to 1 mM calcium, keratolinin mRNA was markedly increased. The upregulation of keratolinin mRNA was also observed by the treatment of SVHK cells with 10 ng TPA per ml or 100 microM forskolin under low calcium conditions (0.05 mM). Our results indicate that keratolinin is identical with cystatin A, a cysteine proteinase inhibitor, and its expression is positively regulated by Ca2+, TPA, and forskolin.

The alpha and eta isoforms of protein kinase C stimulate transcription of human involucrin gene.

Involucrin is one of the precursor proteins of the cornified cell envelope that is formed beneath the cell membrane during terminal differentiation of keratinocytes. 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a potent protein kinase C (PKC) activator, induces terminal differentiation of keratinocytes. We previously demonstrated that involucrin promoter activity is stimulated by TPA in cultured fetal rat skin keratinocytes. PKC is a large family of proteins and keratinocytes containing five PKC isozymes: alpha, delta, epsilon, eta, and zeta. In order to determine the role of the PKC isozyme(s) on involucrin gene expression, we constructed the chloramphenicol acetyl transferase (CAT)-involucrin promoter expression vector by connecting the 5'-upstream region of the human involucrin gene containing the untranslated first exon to the CAT reporter gene. The CAT-involucrin promoter expression vector was transfected with various PKC isozyme expression vectors into SV40-transformed human keratinocytes (SVHK cells). Transfection of the CAT-involucrin promoter expression vector with PKC-alpha or PKC-eta expression vectors resulted in a significant increase in the TPA-dependent involucrin promoter activity. The PKC inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride, inhibited the promoter activity stimulated by TPA. Transfection of PKC-delta, -epsilon, and -zeta had no effect on the involucrin-promoter activity. Although the promoter activity was stimulated by transfection of PKC-gamma, TPA did not enhance the promoter activity in the PKC-gamma-transfected SVHK cells. Previously we showed three AP-1 binding sites (AP1-1, -2, and -3) on the involucrin promoter region. Both the basal and the TPA-stimulated involucrin promoter activities were suppressed by deleting the AP1-1 site (-119 to -113) that is the most proximal to the transcription start site. The deletion of AP1-2 (-297 to -303) or AP1-3 (-447 to -453) did not affect the involucrin promoter activity. Gel retardation analyses disclosed that TPA stimulated the specific DNA binding of the nuclear protein(s) of control, PKC-alpha, or PKC-eta-transfected SVHK cells, but not of PKC-gamma-transfected cells. Addition of anti-c-Jun and anti-c-Fos antibodies decreased the specific protein-DNA complex band with a concomitant appearance of supershifted bands. These results indicate that PKC, specifically PKC-alpha and PKC-eta, mediates the TPA-dependent activation of involucrin gene expression of SVHK cells. PKC-gamma, which is not present in keratinocytes, also induces involucrin gene expression in a TPA-independent manner, when introduced into SVHK cells.

Mutant loricrin is not crosslinked into the cornified cell envelope but is translocated into the nucleus in loricrin keratoderma.

Loricrin is a major constituent of the epidermal cornified cell envelope. We have recently identified heterozygous loricrin gene mutations in two dominantly inherited skin diseases, the ichthyotic variant of Vohwinkel syndrome and progressive symmetric erythrokeratoderma, collectively termed loricrin keratoderma. In order to see whether the mutant loricrin molecules predicted by DNA sequencing are expressed in vivo and to define their pathologic effects, we raised antibodies against synthetic peptides corresponding to the mutated sequences of loricrin. Immunoblotting of horny cell extracts from loricrin keratoderma patients showed specific bands for mutant loricrin. Immunohistochemistry of loricrin keratoderma skin biopsies showed positive immunoreactivity to the mutant loricrin antibodies in the nuclei of differentiated epidermal keratinocytes. The immunostaining was localized to the nucleoli of the lower granular cell layer. As keratinocyte differentiation progressed the immunoreactivity moved gradually into the nucleoplasm leaving nucleoli mostly nonimmunoreactive. No substantial staining was observed along the cornified cell envelope. This study confirmed that mutant loricrin was expressed in the loricrin keratoderma skin. Mutant loricrin, as a dominant negative disrupter, is not likely to affect cornified cell envelope crosslinking directly, but seems to interfere with nuclear/nucleolar functions of differentiating keratinocytes. In addition, detection of the mutant loricrin in scraped horny layer could provide a simple noninvasive screening test for loricrin keratoderma. J Invest Dermatol 115:1088-1094 2000

Analysis of codon usage diversity of bacterial genes with a self-organizing map (SOM): characterization of horizontally transferred genes with emphasis on the E. coli O157 genome.

With increases in the amounts of available DNA sequence data, it has become increasingly important to develop tools for comprehensive systematic analysis and comparison of species-specific characteristics of protein-coding sequences for a wide variety of genomes. In the present study, we used a novel neural-network algorithm, a self-organizing map (SOM), to efficiently and comprehensively analyze codon usage in approximately 60,000 genes from 29 bacterial species simultaneously. This SOM makes it possible to cluster and visualize genes of individual species separately at a much higher resolution than can be obtained with principal component analysis. The organization of the SOM can be explained by the genome G+C% and tRNA compositions of the individual species. We used SOM to examine codon usage heterogeneity in the E. coli O157 genome, which contains 'O157-unique segments' (O-islands), and showed that SOM is a powerful tool for characterization of horizontally transferred genes.

Interferon-gamma-dependent induction of manganese superoxide dismutase activity of SV40-transformed human keratinocytes by anti-Fas antibody and by TNF-alpha.

It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF-alpha on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF-alpha in the presence of interferon-gamma (IFN-gamma) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN-gamma, no increase in Mn-SOD activity was detected. The induction of IFN-gamma-dependent Mn-SOD activity by anti-Fas antibody or TNF-alpha was concentration-dependent; the maximal effect was observed at 1-10 micrograms/ml and 5-10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF-alpha in the presence of IFN-gamma resulted in an additive increase in Mn-SOD activity. Although the addition of 12-o-tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN-gamma-dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF-alpha. The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN-gamma and that TPA augments the process through the activation of protein kinase C.

Copper, zinc-superoxide dismutase protects from ultraviolet B-induced apoptosis of SV40-transformed human keratinocytes: the protection is associated with the increased levels of antioxidant enzymes.

It has been reported that cellular oxidative stress induces apoptosis. Ultraviolet radiation that generates reactive oxygen intermediates (ROIs) also induces apoptosis. Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Mammalian cells express two isozymes of SOD, copper, zinc-SOD (Cu, Zn-SOD) and manganese-SOD (Mn-SOD). Using SV40-transformed human keratinocytes (SVHK cells), we investigated the role of SODs in the ultraviolet B (UVB) irradiation-induced apoptosis. UVB irradiation decreased transiently Cu, Zn- and Mn-SOD activities and their protein levels, with subsequent recovery to the basal levels by 24 h. The UVB-induced decrease in SOD activity was dose-dependent and the maximal effect was obtained at 75 mJ/cm(2). The decrease in Cu, Zn-SOD was more marked than that in Mn-SOD. The cell death assay, annexin-V/propidium iodide flow cytometry, and DNA fragmentation analysis revealed that UVB irradiation induces apoptosis in SVHK cells. The UVB-induced apoptosis was suppressed by the treatment of antioxidants, catalase, glutathione, and alpha-tochopherol. The stable transfection of Cu, Zn-SOD expression vectors into SVHK cells was accompanied by the increased activities of antioxidant enzymes, catalase, and glutathione reductase, as well as glutathione and the cells were shown to be more resistant to UVB-induced apoptosis. In contrast, the transfection of Mn-SOD affected neither activities of antioxidant enzymes nor the UVB-induced apoptosis. The transfection of Cu, Zn-SOD antisense oligomers but not sense oligomers into SVHK or Cu, Zn-SOD cDNA-transfected SVHK (C2) cells significantly decreased the antioxidant enzyme activities and increased the UVB-induced apoptosis. On the other hand, the transfection of Mn-SOD antisense oligomers did not affect the UVB-induced apoptosis. These results suggest that the transfection of Cu, Zn-SOD expression vector, which is accompanied by the increased level of antioxidant enzymes, suppresses the UVB-induced apoptosis of SVHK cells.

Differential phosphorylation of mitogen-activated protein kinase families by epidermal growth factor and ultraviolet B irradiation in SV40-transformed human keratinocytes.

SV-40 transformed human keratinocytes (SVHK cells) were stimulated with epidermal growth factor (EGF) and ultraviolet B (UVB) irradiation. Following the stimulation, cell growth, apoptosis, and the activities of mitogen-activated protein (MAP) kinase families were analyzed. EGF (100 ng/ml) increased SVHK cell number compared with control cells cultured in serum-free DMEM medium. The EGF-stimulated cells did not show DNA fragmentation. In contrast, UVB irradiation (40 mJ/cm(2)) markedly decreased viable cell number that was accompanied with DNA fragmentation. EGF stimulated extracellular signal-regulated kinase (ERK) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Following the EGF stimulation, phosphorylated ERK and JNK were detected by phospho-p42/44 MAP kinase antibody and phospho-SAPK/JNK antibody, respectively. On the other hand, UVB irradiation stimulated the phosphorylation of p38 and JNK but not of ERK. The stimulation of ERK and JNK induced by EGF was observed earlier than the stimulation of p38 and JNK induced by UVB. PD98059, a specific MAP kinase kinase (MAPKK) 1 (also referred to as MEK1) inhibitor, inhibited EGF-dependent cell proliferation, that was associated with the inhibition of ERK and JNK phosphorylation. In contrast, UVB-induced overall cell death was not significantly affected by PD98059, that inhibited phosphorylation of JNK but not of p38. PD98059, however, significantly augmented UVB-induced cell death earlier time points (30 min--2 h). These results indicate that ERK and JNK are activated following EGF stimulation that might be associated with cell proliferation. On the other hand, UVB-induced apoptosis seems to be mostly associated with the activation of p38. JNK stimulation might provide an anti-apoptotic tonus during the UVB-induced, p38-associated SVHK cell death.

Adenylate cyclases in keratinocytes: FRSK cells express types I, II, III, IV, VI and VIII, and 1,25(OH)2D3, retinoic acid and TPA augment forskolin-induced cyclic AMP accumulation in the absence of altered isozyme expression.

Molecular cloning analysis has detected at least nine adenylate cyclase isozymes in mammalian tissues. Using fetal rat skin keratinocytes (FRSK), we investigated adenylate cyclase expression and its modulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), a retinoid (Ro10-1670), and 12-O-tetradecanoylphorbol-13-acetate (TPA). Reverse transcription polymerase chain reaction (RT-PCR) indicated that FRSK contain adenylate cyclases I, II, III, IV, VI and VIII. Treatment with 1,25(OH)2D3 (1 x 10(-7) M), Ro10-1670 (1 x 10(-6) M), and TPA (100 ng/ml) resulted in increased forskolin-induced cyclic AMP accumulation by FRSK cells and normal human keratinocytes (NHK). Quantitative RT-PCR and Western blot analysis, however, detected no alteration in mRNA and protein levels of each adenylate cyclase isozyme for at least 48 h. These results indicate that FRSK contain at least six (I, II, III, IV, VI and VIII) adenylate cyclase isozyme mRNAs, suggesting a complex regulatory mechanism of cyclic AMP generation in keratinocytes. Although 1,25(OH)2D3, Ro10-1670, and TPA augmented forskolin-induced cyclic AMP accumulation, they do not seem to affect the expression of specific adenylate cyclase isozymes by FRSK cells.

[Right ventricular outflow tract reconstruction with PTFE mono-cusped transannular patch for tetralogy of Fallot].

Conotruncal repair for tetralogy of Fallot consists of (1) precise closure of the VSD with the membranous flap and (2) reconstruction of the right ventricular outflow tract (RVOT) by a short transannular patch (< 30% of the RV length) with a wide PTFE monocusp. This report describes the mid-term results in 46 patients with tetralogy of Fallot who underwent conotruncal repair with PTFE monocusped transannular patch and have been followed up for 4 years or more. There was no early and late death and no patient required reoperation. No patient has had a significant residual defect or tricuspid regurgitation (> II). The right and left ventricular pressure ratio was 0.41 +/- 0.12 and the pressure gradient across RVOT was 10.5 +/- 5.9 mmHg, signifying sufficient relief of RVOT obstruction. The mobility of the PTFE monocusp was echocardiographically detected in 86% over a mean follow-up period of 84 +/- 34 months and % freedom from pulmonary regurgitation (> II) was 85.9% at 10 years postoperatively. Excellent long-term durability of the PTFE monocusp provided the normal right vent performance with RVEDV of 91.8 +/- 29.5% of normal and a central venous pressure of 5 +/- 1 mmHg. In conclusion, conotruncal repair with a wide and short transannular patch has provided good mid-term results with the excellent long-term durability of PTFE monocusp.

Interleukin-1beta-converting enzyme and CPP32 are involved in ultraviolet B-induced apoptosis of SV40-transformed human keratinocytes.

Interleukin-1beta-converting enzyme (ICE) and CPP32 are cysteine proteinases, that are involved in apoptotic process in various cell systems. We investigated the effects of ICE on ultraviolet B (UVB) induced-apoptosis in SV40-transformed human keratinocytes (SVHK cells). The ICE inhibitor (Z-Val-Ala-Asp-CH2F) and CPP32 inhibitor (Z-Asp-Glu-Val-Asp-CH2F) blocked the apoptotic cell death caused by UVB irradiation. The addition of both ICE and CPP32 inhibitors to the incubation medium resulted in neither an additive nor a synergistic suppression of UVB-induced apoptosis. Reverse transcription and polymerase chain reaction (RT-PCR) analysis indicated that SVHK cells expressed ICE-alpha, and beta mRNAs. UVB irradiation increased the mRNA of both isoforms and Western blot analysis confirmed that UVB increased an active form of ICE protein, p20, that is generated by autoproteolytic cleavage of inactive 45 kDa proenzyme derived from both mRNAs. Transfection of ICE expression vector into SVHK cells resulted in apoptosis in a dose dependent manner and UVB-irradiation further augmented the ICE expression vector-induced apoptosis. These results indicate that ICE plays an important role in UVB-induced apoptosis of SVHK cells.

Self-association of purine base, 6-methylpurine, in water - organic component mixtures.

Thermodynamic quantities of the self-association of 6-methylpurine in water (1)-dimethylsulfoxide (DMSO) (2) mole fraction, x2 less than 0.1) and water (1) - N,N-dimethylformamide (DMF) (2) (x2 less than or equal to 1.0) mixed solvents have been obtained through heat of dilution measurements, at 25 degrees C. In the water-DMSO solvent system, the standard enthalpy and entropy changes, delta Ho and delta So, of the association exhibited an abrupt behavior. They decreased remarkbly with the the mole fraction of DMSO until about x2 = 0.012 and after that, they increased steeply. In the case of water-DMF solvent system, the values of delta Ho and delta So didn't show the abrupt behavior. They decreased steeply until about x2 = 0.1 and, at higher mole fractions, became relatively constant. These behaviors are rationalized on the basis of solvent structural effects and solvation in these association systems.

Epidermal adenylate cyclase system is regulated by diacylglycerol-protein kinase C signal, but not by calcium signal.

The breakdown of inositol phospholipids is an important transmembrane signalling system that is composed of two kinds of signals: the diacylglycerol-protein kinase C signal, and the inositol trisphosphate-Ca2+ signal. Using membrane-permeable diacylglycerol, I-oleoyl-2-acetylglycerol (OAG), and calcium ionophore, A-23187, the effects of these chemicals on the epidermal adenylate cyclase system were investigated. OAG increased forskolin- and cholera toxin-induced cyclic AMP accumulations, but receptor adenylate cyclase responses were markedly decreased by treatment with OAG. The effects of OAG were inhibited by the protein kinase C inhibitor, H-7. Calcium ionophore, A-23187, had no effect on the epidermal adenylate cyclase responses. Combinations of OAG and A-23187 (as well as the calcium chelator, EGTA), showed that the action of OAG was mostly unaffected by the modulation of intracellular and extracellular Ca2+ concentrations. The results suggest that among the signals triggered by the breakdown of inositol phospholipids, only diacylglycerol-protein kinase C signal is involved in the regulation of the epidermal adenylate cyclase system.

Decreased beta 2-adrenergic receptor-mRNA and loricrin-mRNA, and increased involucrin-mRNA transcripts in psoriatic epidermis: analysis by reverse transcription-polymerase chain reaction.

Psoriatic hyperproliferative epidermis is characterized by a decreased beta 2-adrenergic adenylate cyclase response as well as by altered differentiation markers that include decreased loricrin and increased involucrin. Using a quantitative reverse transcription-polymerase chain reaction, we analysed the expression of beta 2-adrenergic receptor-mRNA, loricrin-mRNA, and involucrin-mRNA in the epidermis of five patients with psoriasis vulgaris. The mRNAs of the beta 2-adrenergic receptor and loricrin in the involved epidermis were significantly decreased, by 0.35-fold (P < 0.01) and 0.55-fold (P < 0.05) respectively, compared with uninvolved epidermis. In contrast, the involucrin mRNA expression of the involved epidermis was significantly increased, by 3.77-fold (P < 0.01). No significant difference in beta-actin mRNA transcripts was detected between the involved and the uninvolved epidermis. These results indicate that the altered expression of the beta 2-adrenergic receptor, loricrin, and involucrin, in the psoriatic involved epidermis, is associated with different amounts of each mRNA transcripts.

Fas antigen modulates ultraviolet B-induced apoptosis of SVHK cells: sequential activation of caspases 8, 3, and 1 in the apoptotic process.

Interferon-gamma (IFN-gamma) induces various apoptosis-related proteins, including Fas antigen (Fas) in keratinocytes. Ultraviolet B (UVB) irradiation produces "sunburn cells," a specific type of apoptosis. Previously, we reported that IFN-gamma augments Fas-dependent apoptosis of SV40-transformed human keratinocytes (SVHK cells). Caspases are a new class of cysteine proteinases that play an important role in apoptosis. We investigated the mechanism of UVB-induced apoptosis by examining activation of the caspase cascade. UVB irradiation of SVHK cells increased the activities of caspases 1, 3, and 8, which were detected at 3 h, and peak activities occurred at 6 h. Pretreatment of SVHK cells with IFN-gamma significantly increased the activity of caspases 1, 3, and 8. UVB-induced caspase 8 stimulation was significantly suppressed only by caspase 8 inhibitor, while inhibitors of caspases 1, 3, and 8 significantly suppressed UVB-induced caspase 1 stimulation. Caspase 3 and 8 inhibitors, but not caspase 1 inhibitor, significantly suppressed UVB-induced caspase 3 activity, suggesting sequential activation of caspases 8, 3, and 1 in UVB-irradiated SVHK cells. Cross-linking and immunoprecipitation analyses showed multimerization of Fas antigen following UVB irradiation of SVHK cells. Pretreatment of SVHK cells with IFN-gamma significantly augmented UVB-induced apoptosis that was accompanied by increased Fas expression. The susceptibility to UVB-induced apoptosis was also increased in Fas-transfected SVHK cells (F2 cells). Neutralizing anti-Fas antibody significantly suppressed caspase activation and Fas-dependent apoptosis of SVHK cells and F2 cells. In contrast, UVB-induced caspase activation and apoptosis were not inhibited by neutralizing anti-Fas antibody in both cell lines. Our results suggest that UVB directly activates Fas and subsequent caspase cascade resulting in apoptosis of SVHK cells. Furthermore, the expression level of Fas antigen in keratinocytes influenced their susceptibility to UVB-induced apoptosis.

Structure and transcriptional regulation of the human cystatin A gene. The 12-O-tetradecanoylphorbol-13-acetate (TPA) responsive element-2 site (-272 to -278) on cystatin A gene is critical for TPA-dependent regulation.

Cystatin A, a cysteine proteinase inhibitor, is one of the precursor proteins of cornified cell envelope of keratinocytes and is expressed during the late stage of keratinocyte differentiation. We have isolated and characterized the human cystatin A gene. The cystatin A gene consists of three exons and two introns. The first, the second, and the third exons consist of coding sequences that are 66, 102, and 126 base pairs in length, respectively. The first and the second introns consist of 14 and 3.6 kilobase pairs, respectively. The transcription initiation site was located 55 base pairs upstream from the first translation site. The fragment, +77 to -2595 in the 5'-flanking region of the human cystatin A gene, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector, p2672CAT, produced a significant CAT activity in transiently transfected SV40-transformed human keratinocytes (SVHK cells), that were further stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator. Sequence analysis of the gene detected three TPA responsive elements (TRE-1, TRE-2, and TRE-3) and one AP-2 site on the 5' upstream promoter region. Deletion analyses of the p2672CAT vector demonstrated that TRE-2, which was located between -272 and -278, was critical for the regulation by TPA. Gel shift analyses revealed that c-Jun, JunD, and c-Fos bound to the TRE-2 region and that the p2672CAT activity level was elevated by co-transfection with c-Jun and c-Fos or with JunD and c-Fos expression vectors. Furthermore, co-transfection of SVHK cells with the protein kinase C-alpha expression vector and the p2672CAT expression vector also resulted in an increased CAT activity. These results indicate that the 5'-flanking region of the human cystatin A gene confers promoter activity and contains a TRE (TRE-2) that mediates, at least in part, the enhanced expression of this gene by TPA.

Codon usage and tRNA genes in eukaryotes: correlation of codon usage diversity with translation efficiency and with CG-dinucleotide usage as assessed by multivariate analysis.

The species-specific diversity of codon usage in five eukaryotes (Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and Homo sapiens) was investigated with principal component analysis. Optimal codons for translation were predicted on the basis of tRNA-gene copy numbers. Highly expressed genes, such as those encoding ribosomal proteins and histones in S. pombe, C. elegans, and D. melanogaster, have biased patterns of codon usage which have been observed in a wide range of unicellular organisms. In S. pombe and C. elegans, codons contributing positively to the principal component with the largest variance (Z1-parameter) corresponded to the optimal codons which were predicted on the basis of tRNA gene numbers. In D. melanogaster, this correlation was less evident, and the codons contributing positively to the Z1-parameter corresponded primarily to codons with a C or G in the codon third position. In X. laevis and H. sapiens, codon usage in the genes encoding ribosomal proteins and histones was not significantly biased, suggesting that the primary factor influencing codon-usage diversity in these species is not translation efficiency. Codon-usage diversity in these species is known to reflect primarily isochore structures. In the present study, the second additional factor was explained by the level of use of codons containing CG-dinucleotides, and this is discussed with respect to transcription regulation via methylation of CG-dinucleotides, which is observed in mammalian genomes.

Satisfactory remission achieved by PUVA therapy in a case of crisis-type adult T-cell leukaemia/lymphoma with generalized cutaneous leukaemic cell infiltration.

We used PUVA therapy in a patient with crisis-type adult T-cell leukaemia/lymphoma and generalized cutaneous leukaemic cell infiltration. PUVA proved very effective in reducing leukaemic cells and in clearing the eruption. To understand the way in which PUVA produced a reduction in the number of leukaemic cells, we examined peripheral blood cells by light and electron microscopy. Light microscopy was of little help, but electron microscopy revealed that PUVA induced apoptosis-like changes in circulating leukaemic cells. This suggests that apoptosis-like changes in leukaemic cells might be the reason for the success of this treatment.

Introduction of antisense CD44S CDNA down-regulates expression of overall CD44 isoforms and inhibits tumor growth and metastasis in highly metastatic colon carcinoma cells.

We created antisense CD44 transfectants using LS174T, a colon adenocarcinoma cell line and assessed the effects of overall CD44 down-regulation on colorectal tumor growth and metastasis. The expression of antisense CD44s (the standard form of CD44) cDNA markedly inhibited the overall expression of CD44 variants. In vitro studies showed a significantly reduced ability of the stable antisense transfectants (LS174TAS1 and LS174TAS2) to bind hyaluronate and osteopontin, ligands for CD44. These cells developed tumors more slowly than controls (parental LS174T and mock transfectants) when the cells were subcutaneously injected into SCID mice. However, in vitro proliferation assays demonstrated no significant difference between the antisense transfectants and the controls on a hyaluronate-coated surface, suggesting the participation of ligands other than hyaluronate in tumor growth in vivo. Intrasplenic injection of parental LS174T cells produced colonies in the liver in 10 of 11 mice, whereas mice injected with the antisense transfectants were completely free of metastasis. In peritoneal dissemination, the weight of nodules and amount of ascites were significantly reduced in LS174TAS1 and AS2 compared with the controls. In vitro adhesion assays between the transfectants or controls and human peritoneal mesothelial cells revealed that the binding of LS174T cells to mesothelial cells was partly mediated by CD44-hyaluronate interaction. These data suggest that CD44-hyaluronate interaction plays a crucial role in peritoneal dissemination in colorectal carcinoma. The results of our study demonstrate the possible application of antisense CD44s to the treatment of colorectal carcinoma.

Proteome analysis of Oncorhynchus species during embryogenesis.

To understand the molecular mechanisms underlying normal and abnormal development of two salmonids, masu salmon (Oncorhynchus masou) and rainbow trout (O. mykiss), we used two-dimensional (2-D) electrophoresis to construct a series of 2-D maps during the embryonic period. We identified all visible protein spots on the 2-D map by assigning numbers for masu salmon and rainbow trout, and we determined N-terminal sequences of proteins for one hundred of the spots, that appear at very high concentrations in the whole embryos of masu salmon and rainbow trout. We also characterized embryonic stages according to the periods of appearance of spots. Most of the N-terminal sequences were identical or at least highly similar to partial sequences reported for vitellogenin (Vtg) of O. mykiss. A potential proteolytic processing of Vtg for rainbow trout is discussed in relation to the time of appearance and relative position of Vtg fragments within the complete protein sequence.