p51/p63 Inhibits ultraviolet B-induced apoptosis via Akt activation.

The epidermis must be protected against excess apoptotic cell death in response to ultraviolet-B (UV-B) irradiation. p53 is known to be critical for this protection. Although the p53 family member DeltaNp51B/DeltaNp63alpha (an N terminal-deleted form of p51/p63) is abundantly expressed in keratinocytes, its contribution to UV-B-dependent apoptosis is largely unknown. We found that, after a transient increase, DeltaNp51B is downregulated in UV-B-irradiated keratinocytes undergoing apoptosis, whereas p53 is upregulated with delayed kinetics. Furthermore, the reduction of DeltaNp51B by small interfering RNAs augmented UV-B-dependent apoptosis in keratinocytes, indicating that DeltaNp51B blocks keratinocyte apoptosis. Although the exogenous expression of DeltaNp51B in keratinocytes did not further block the UV-B-dependent apoptosis, to our surprise the expression of TAp51B (an isoform with a full NH(2)-terminal transactivation domain that is structurally and functionally similar to p53) decreased apoptosis significantly. The blockade of keratinocyte apoptosis by the p51 was dependent on the phosphorylation of Akt, resulting in the activation of a survival pathway. Thus, in addition to its indispensable roles in epithelial development, p51 acts in adult cells to protect the epidermis against UV-B irradiation by preventing excess depletion of keratinocytes.

p53 homologue, p51/p63, maintains the immaturity of keratinocyte stem cells by inhibiting Notch1 activity.

p53 homologue, p51/p63, predominantly expressed in keratinocyte stem cells, is indispensable for the formation of epidermis. Notch1, another such gene indispensable for the process, induces growth arrest and differentiation in keratinocytes. We found that exogenous expression of DeltaNp51B (DeltaNp63alpha), one of the isoforms of p51 specifically expressed in basal keratinocytes, blocked Notch 1-dependent growth arrest and differentiation in mouse keratinocytes by inhibiting p21 expression and maintaining integrins expression. Furthermore, DeltaNp51B by itself was found to have ability to induce expression of integrin alpha6beta4, which promotes attachment of basal cells to basal membrane thereby keeping the cells in immature state. Therefore, we conclude that DeltaNp51B expression warrants integrin expression even under the influence of Notch1 and that DeltaNp51B is a long-sought factor required to maintain basal cell keratinocytes immaturity by inhibiting Notch1 activity. We will postulate a plausible model explaining the maintenance of the squamous epithelium architectures as well as offering mechanistic explanations for pathological features of skin diseases, including cancers, psoriasis along with physiological wound healings.

[Effective clinical information system closely connected with clinical laboratory data for infectious diseases].

Nowadays, information on clinical laboratory tests for infectious disease is almost supported by information on such as clinical, identical and susceptible microbiological tests. However, information support necessary for clinical diagnosis and therapy with clinically valuable data remains unsatisfactory. To offer more useful microbiological information to support clinics from clinical laboratory division, exchange of clinical information between clinical divisions, integrated systems of clinical information in the district and establishment of domestic and international information network et al are required. Establishment of these advanced total information network systems for clinical microbiological tests for infectious diseases may exceedingly contribute to earlier diagnosis, control and prevention of various infectious diseases.

The involvement of L-type Ca(2+) channels in the relaxant effects of the ATP-sensitive K(+) channel opener ZD6169 on pig urethral smooth muscle.

1. The effects of ZD6169, a novel ATP-sensitive K(+) channel (K(ATP) channel) opener, were investigated on membrane currents in isolated myocytes using patch-clamp techniques. Tension measurement was also performed to study the effects of ZD6169 on the resting tone of pig urethral smooth muscle. 2. Levcromakalim was more potent than ZD6169 in lowering the resting urethral tone. Relaxation induced by low concentrations of ZD6169 (< or =3 microM) was completely suppressed by additional application of glibenclamide (1 microM). In contrast, glibenclamide (1-10 microM) only partially inhibited the relaxation induced by higher concentrations of ZD6169 (> or = microM). 3. Bay K8644 (1 microM) reduced the maximum relaxation produced by ZD6169 (> or =10 microM). 4. In whole-cell configuration, ZD6169 suppressed the peak amplitude of voltage-dependent Ba(2+) currents in a concentration- and voltage-dependent manner, and at 100 microM, shifted the steady-state inactivation curve of the voltage-dependent Ba(2+) currents to the left at a holding potential of -90 mV. 5. In cell-attached configuration, open probability of unitary voltage-dependent Ba(2+) channels (27 pS, 90 mM Ba(2+)) was inhibited by 100 microM ZD6169 and by 10 microM nifedipine. 6. Reverse transcriptase-polymerase chain reaction (RT - PCR) analysis revealed the presence of the transcript of the alpha(1C) subunit of L-type Ca(2+) channels in pig urethra. 7. These results demonstrate that ZD6169 causes urethral relaxation through two distinct mechanisms, activation of K(ATP) channels at lower concentrations and inhibition of voltage-dependent Ca(2+) channels at higher concentrations (about 10 microM).

Cerebral vascular resistance assessed by transcranial color Doppler ultrasonography in patients with chronic liver diseases.

BACKGROUND AND AIM: Cerebral hemodynamic derangement is well known in patients with liver cirrhosis. The advent of transcranial Doppler enables a non-invasive observation of cerebral hemodynamics. To evaluate the clinical usefulness we examined cross-sectionally and longitudinally cerebral hemodynamic parameters in patients with cirrhosis. METHODS: The subjects of the cross-sectional study were 117 patients with cirrhosis, 15 patients with chronic hepatitis and 25 healthy controls. The longitudinal study included 26 cirrhotic patients without encephalopathy, and 27 cirrhotic patients with encephalopathy. The pulsatility and resistive indices of the right middle cerebral artery were used as parameters of cerebral hemodynamics. RESULTS: Cerebral pulsatility and resistive indices were significantly higher in patients with cirrhosis (1.05 +/- 0.23, P < 0.0001 and 0.63 +/- 0.07, P < 0.0001, respectively) than in the controls (0.75 +/- 0.11 and 0.55 +/- 0.05, respectively) and patients with chronic hepatitis (0.81 +/- 0.11 and 0.52 +/- 0.05, respectively). Cerebral pulsatility and resistive indices were significantly related with the severity of liver cirrhosis. Patients with encephalopathy had higher cerebral pulsatility and resistive indices than patients without encephalopathy. In the longitudinal studies, cerebral pulsatility and resistive indices were changed in parallel with the severity of cirrhosis and encephalopathy. Cerebral pulsatility and resistive indices were significantly correlated with the blood ammonia level and serum levels of bilirubin and albumin. CONCLUSION: These cross-sectional and longitudinal studies showed that cerebral vascular resistance indices measured by using transcranial Doppler were increased in association with the severity of cirrhosis and encephalopathy. Cerebral pulsatility and resistive indices are real-time and useful parameters to assess and monitor cirrhotic patients.

Homologous pairing promoted by the human Rad52 protein.

The Rad52 protein, which is unique to eukaryotes, plays important roles in the Rad51-dependent and the Rad51-independent pathways of DNA recombination. In the present study, we have biochemically characterized the homologous pairing activity of the HsRad52 protein (Homo sapiens Rad52) and found that the presynaptic complex formation with ssDNA is essential in its catalysis of homologous pairing. We have identified an N-terminal fragment (amino acid residues 1-237, HsRad52(1-237)) that is defective in binding to the human Rad51 protein, which catalyzed homologous pairing as efficiently as the wild type HsRad52. Electron microscopic visualization revealed that HsRad52 and HsRad52(1-237) both formed nucleoprotein filaments with single-stranded DNA. These lines of evidence suggest the role of HsRad52 in the homologous pairing step of the Rad51-independent recombination pathway. Our results reveal the striking similarity between HsRad52 and the Escherichia coli RecT protein, which functions in a RecA-independent recombination pathway.

Effect of solvent on the cis-trans conformational equilibrium of a proline imide bond of short model peptides in solution.

Proton NMR spectra of proline-containing short peptides with N-terminal sequences of N-acetyl-prolyl- (Ac-Pro-) N-tert-butoxycarbonyl-phenylalanyl-prolyl- (Boc-Phe-Pro-) and N-tert-butoxycarbonyl-leucyl-prolyl- (Boc-Leu-Pro-) were measured in mixed solvents of hexadeuterodimethylsulfoxide and deuterochloroform (CDCl3). Population ratios of cis and trans conformers with respect to the proline imide bond and chemical shifts of NH protons were obtained as a function of a CDCl3 fraction of solvent. With increasing fraction of CDCl3, the trans percentages of the Ac-Pro-imide bonds increased. On the other hand, those of Boc-Phe-Pro- decreased, and those of Boc-Leu-Pro- exhibited middle tendency. From the solvent-dependent variation of the chemical shifts of the NH protons, intramolecular hydrogen bonds that stabilize the trans form of Ac-Pro- and the cis form of Boc-Phe-Pro- were discussed. For the Ac-Pro- peptides, only the trans forms are found to the compatible with 7-, 10-, and 13-membered hydrogen-bonded rings that would be similar to the ordinary secondary structures, gamma- and beta-turns and alpha-helix, respectively. For the cis form of Boc-Phe-Pro-R (R = O-methyl or glycyl-O-ethyl), the hydrogen-bonded structure is found to be similar to the type-VIa beta-turn. On the other hand, for Boc-Phe-Pro-Pro-Leu-Gly-NH2, it has been suggested that two different hydrogen bonds, which are different from that of the type-VIa beta-turn, support each other and cooperatively stabilize the cis form.

Mutation analysis of the p51 gene and correlation between p53, p73, and p51 expressions in prostatic carcinoma.

BACKGROUND: p73 and p51 are genes possessing amino-acid similarities to p53. We previously found no mutation in p73 in prostatic carcinoma, but did find abnormal expression of the gene. Involvement of these genes in prostatic carcinogenesis is still poorly understood. METHODS: Mutation analysis of the p51 gene and allelotyping of 3q28, on which p51 lies, were performed. Expression of p53, p73, and p51 was examined using reverse transcription-polymerase chain reaction, and expression levels were compared. RESULTS: No mutation in p51 was found (0/55 cases). Loss of heterozygosity at 3q28 was detected in 6 of 28 cases (21.8%). By expression analysis we found that in p53, 4 of 38 cases (10.5%) showed downregulation. No cases showed upregulation of p53. In contrast, p73 and p51 were downregulated in 42.1 and 39.5% of cases, respectively, and upregulated in 31.5 and 34.2% of cases, respectively. Expression levels of p51 corresponded with those of p73 in 25 of 38 cases (65.8%). CONCLUSIONS: Somatic mutations in p73 and p51 are not important in prostatic carcinogenesis. These genes may be associated with tumors by expression levels and may have roles in addition to tumor suppression.

Homologous-pairing activity of the human DNA-repair proteins Xrcc3.Rad51C.

The human Xrcc3 protein is involved in the repair of damaged DNA through homologous recombination, in which homologous pairing is a key step. The Rad51 protein is believed to be the only protein factor that promotes homologous pairing in recombinational DNA repair in mitotic cells. In the brain, however, Rad51 expression is extremely low, whereas XRCC3, a human homologue of Saccharomyces cerevisiae RAD57 that activates the Rad51-dependent homologous pairing with the yeast Rad55 protein, is expressed. In this study, a two-hybrid analysis conducted with the use of a human brain cDNA library revealed that the major Xrcc3-interacting protein is a Rad51 paralog, Rad51C/Rad51L2. The purified Xrcc3.Rad51C complex, which shows apparent 1:1 stoichiometry, was found to catalyze the homologous pairing. Although the activity is reduced, the Rad51C protein alone also catalyzed homologous pairing, suggesting that Rad51C is a catalytic subunit for homologous pairing. The DNA-binding activity of Xrcc3.Rad51C was drastically decreased in the absence of Xrcc3, indicating that Xrcc3 is important for the DNA binding of Xrcc3.Rad51C. Electron microscopic observations revealed that Xrcc3.Rad51C and Rad51C formed similar filamentous structures with circular single-stranded DNA.

Atomic force microscopy of RecA--DNA complexes using a carbon nanotube tip.

We report high resolution images of RecA-double stranded (ds) DNA complexes obtained by atomic force microscopy (AFM). When a carbon nanotube (CNT) tip was used, AFM images visualized the 10-nm pitch of RecA-dsDNA complexes and RecA filaments as three-dimensional surface topography without reconstruction analysis. The depth of the notch between two pitches was less than 1 nm. When adsorbed on a soft surface covered with proteins, naked DNA, RecA monomers, RecA hexamers, and short RecA filaments were all clearly resolved in one image. The high resolution images with a CNT tip provided valuable information on the initiation process of RecA-dsDNA complex formation.

A factor XI deficiency associated with a nonsense mutation (Trp501stop) in the catalytic domain.

We identified a novel mutation in an asymptomatic 65-year-old Japanese man with severe factor XI deficiency. Sequence analysis after polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) analysis of his factor XI gene revealed a G-->A transition in codon 501 of exon 13, resulting in a substitution of Trp501 (TGG) by a stop codon (TAG) in the catalytic domain. This mutation abolished a FokI restriction site. The PCR product from normal subjects was digested with FokI and yielded two fragments, one of 223 bp and one of 47 bp. The PCR product from the patient gave a single 270-bp fragment, demonstrating possible homozygosity.

Liver perforation: a serious complication of percutaneous acetic acid injection for hepatocellular carcinoma.

A 75-year-old cirrhotic man, after undergoing percutaneous acetic acid injection therapy for hepatocellular carcinoma, presented high fever and right hypochondralgia. Computed tomography disclosed an area of low attenuation in the liver and a crescent-shaped intraperitoneal lesion adjacent to it. We diagnosed liver perforation and localized peritonitis due to the leakage of acetic acid following acetic acid injection. It is important to consider this serious complication when evaluating the indications for percutaneous acetic acid injection for hepatocellular carcinoma.

The functions of exogenous and endogenous laminin-5 on corneal epithelial cells.

Recent evidence suggests that the basement membrane not only separates basal cells from Bowman's layer, but also has a crucial role in the proliferation, differentiation and migration of corneal epithelial cells. The basement membrane is composed of a mixture of matrix components including collagens, laminins and heparan sulfate proteoglycans. In these extracellular matrixes, laminin is a major component of the basement membrane. Of 11 laminin isoformes, laminin-5 is a variant, composed of three nonidentical subunits alpha3, beta3, gamma2 and is a major component of the corneal basement membrane. However, little is known about the interactions of laminin-5 with corneal epithelial cells. In this study, we investigated the functions of laminin-5 on SV-40 transfected human corneal epithelial cells (HCE cells). We also revealed different functions between exogenous and endogenous laminin-5 on HCE cells. Laminin-5 is synthesized initially as a 490 kDa molecule that undergoes specific processing to cleavaged isoforms after being secreted. The alpha3 subunit is processed from 200-190 kDa to 160 kDa/145 kDa. The gamma2 subunit is processed from 150 kDa to 105 kDa/80 kDa. The beta3 subunit (140 kDa) is not processed. Exogenously added laminin-5 (soluble form) in this study was purified from a serum-free, conditioned medium of a human gastric carcinoma cell line STKM-I. This soluble laminin is a processed isoform containing alpha3 (160 kDa), beta3 (140 kDa) and gamma2 (105 kDa) chains. On the other hand, immunocytochemical analysis showed that HCE cells themselves secreted laminin-5 endogenously. Western blotting analysis revealed that HCE cells initially produced unprocessed isoform containing 190 kDa alpha3, 140 kDa beta3 and 150 kDa gamma2 chains and that after being secreted, the alpha3 chain was processed to 160 kDa/145 kDa and the gamma2 chain was processed to 105 kDa. Initially we investigated the functions of exogenous (processed) laminin-5 on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion via alpha3beta1 integrin, cell spreading, assembly of hemidesmosomes and mildly inhibited cell migration. Next we estimated the effect of endogenous (unprocessed) laminin-5 on HCE cells. Using an anti laminin-5 monoclonal antibody (mAb) or anti integrin alpha3beta1 mAbs, the blocking of the interaction between endogenously secreted laminin-5 and HCE cells caused strong inhibition of cell migration. Integrin alpha3beta1 and alpha6beta4 were expressed in HCE cells. These integrins are receptors of laminin-5. But, anti integrin alpha6beta4 mAbs did not have any blocking ability against cell migration. These results indicated that endogenous (unprocessed) laminin-5 has a crucial role in cell migration on HCE cells via alpha3beta1 integrin. In conclusion, structural differences between exogenous (processed) and endogenous (unprocessed) laminin-5 regulated their functions on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion, cell spreading and assembly of hemidesmosomes. Endogenously secreted laminin-5 had a crucial role in cell migration. In the future, processed soluble laminin-5 could be a useful drug for the prevention of recurrent corneal erosion, and unprocessed soluble laminin-5 could be applied for the treatment of prolonged corneal epithelial defects.

Specific defects in double-stranded DNA unwinding and homologous pairing of a mutant RecA protein.

The DNA molecules bound to RecA filaments are extended 1.5-fold relative to B-form DNA. This extended DNA structure may be important in the recognition of homology between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). In this study, we show that the K286N mutation specifically impaired the dsDNA unwinding and homologous pairing activities of RecA, without an apparent effect on dsDNA binding itself. In contrast, the R243Q mutation caused defective dsDNA unwinding, due to the defective dsDNA binding of the C-terminal domain of RecA. These results provide new evidence that dsDNA unwinding is essential to homology recognition between ssDNA and dsDNA during homologous pairing.

Allele frequency of human endothelial nitric oxide synthase gene polymorphism in abdominal aortic aneurysm.

OBJECTIVE: This study was performed to examine the role of the endothelial constitutive NO synthase (ecNOS) gene in patients with abdominal aortic aneurysm (AAA). METHODS: We determined the distributions of polymorphism in intron 4 of the ecNOS (ecNOS4) gene, amplified by polymerase chain reaction, and compared the allele frequencies between subjects with abdominal aortic aneurysms (AAAs) and healthy individuals. PATIENTS: Fifty-eight patients with AAAs and 410 race-matched healthy controls were studied. RESULTS: Two alleles of the ecNOS4 gene, containing 4 (a-allele) and 5 (b-allele) repeats, were identified. We found that the a-allele frequency of this gene was significantly higher in the surgical than in the non-surgical group. CONCLUSION: The results of this study suggest that the a-allele of the ecNOS4 gene is indicative of the need for surgery for AAA. Analysis of the alleles of the ecNOS4 gene polymorphism could provide useful information concerning the clinical course of AAA progression.

p51A (TAp63gamma), a p53 homolog, accumulates in response to DNA damage for cell regulation.

p51A, or TAp63gamma, a translation product of gene p51, or p63, was identified as a homolog of p53 in its primary structure and transactivating function. p53 plays a decision-making role in inducing either cell cycle arrest or apoptosis in response to DNA damage, and thereby preserves genome integrity of living cells. To compare the biological activities between p51A and p53, cell lines with low-level, constitutive expression of each protein were obtained by cDNA transfection of mouse erythroleukemic cells. Production of p51A with an apparent molecular mass of 57-kilodalton (kD) accompanied induction of p21waf1 and appearance of hemoglobin-producing cells. After DNA-damaging treatment either with ultraviolet light (UV) irradiation or with actinomycin D, the p51A protein accumulated in time courses corresponding to those of wild-type p53, and caused an increase in the hemoglobin-positive cell count. In contrast, p53-accumulated cells underwent apoptosis without exhibiting the feature of erythroid differentiation. The mode of p21waf1 and Bax-alpha upregulations varied between p51A- and p53-expressing cells and between the types of DNA damage. These results suggest the possibility that p51A induces differentiation under genotoxic circumstances. There may be cellular factors that control p51A protein stability and transactivating ability.

A common polymorphism acts as an intragenic modifier of mutant p53 behaviour.

The p73 protein, a homologue of the tumour-suppressor protein p53, can activate p53-responsive promoters and induce apoptosis in p53-deficient cells. Here we report that some tumour-derived p53 mutants can bind to and inactivate p73. The binding of such mutants is influenced by whether TP53 (encoding p53) codon 72, by virtue of a common polymorphism in the human population, encodes Arg or Pro. The ability of mutant p53 to bind p73, neutralize p73-induced apoptosis and transform cells in cooperation with EJ-Ras was enhanced when codon 72 encoded Arg. We found that the Arg-containing allele was preferentially mutated and retained in squamous cell tumours arising in Arg/Pro germline heterozygotes. Thus, inactivation of p53 family members may contribute to the biological properties of a subset of p53 mutants, and a polymorphic residue within p53 affects mutant behaviour.