Gallbladder aspiration for acute cholecystitis in average-surgical-risk patients.

We conducted a retrospective case note review to assess whether or not gallbladder aspiration can be applied as a temporary measure for the treatment of acute cholecystitis in average-surgical-risk patients. Gallbladder aspiration was performed in 79 consecutive average-surgical-risk patients with acute cholecystitis, who had no indications of emergent surgery and who complained of severe colicky pain. Elective surgery became possible in 92% of patients by gallbladder aspiration. The percentage reached 97 when percutaneous cholecystostomy was added (four patients). Emergent surgery was needed in one patient suffering bile leakage following gallbladder aspiration. Colicky pain was controlled soon after the procedure in most cases. Neither major complications nor mortalities were observed in the following surgical therapies. It is suggested that gallbladder aspiration might be applied as a temporary measure for acute cholecystitis in average-surgical-risk patients, although early surgery should remain the primary choice of therapy in such patients.

Activation of WNT family expression and signaling in squamous cell carcinomas of the oral cavity.

The WNT family activates an oncogenic signaling mediated through beta-catenin and is up-regulated in a variety of malignant neoplasms. The signaling translocates beta-catenin into the nucleus and stimulates carcinoma cells in the epithelial-mesenchymal transition (EMT). However, WNT expression and signaling in oral carcinomas have not been examined. The present study focused on unveiling the involvement of WNTs in oral carcinomas, and showed that carcinoma cells express 11 of 19 WNT family members by reverse-transcription/PCR. WNT-expressing carcinoma cells exhibited increased beta-catenin levels in the cytoplasmic pool and translocation to the nucleus. The activation state of signaling correlated with the expression of membrane-type 1 matrix metalloproteinase, which degrades territorial matrices in carcinoma invasion. Immunohistochemistry disclosed that WNT3 expression and nuclear localization of beta-catenin were predominant in carcinoma cells at the invasive front. These results suggest that enhanced WNT expression and signaling accelerate the progression of carcinomas via activating EMTs and local invasiveness.

The LIM-only protein, LMO4, and the LIM domain-binding protein, LDB1, expression in squamous cell carcinomas of the oral cavity.

Carcinoma cells can lose their epithelial cell characteristics and dedifferentiate into a fibroblast-like cell during progression of a neoplasm. Aberrant expression of oligomeric transcriptional complexes contributes to progression of carcinomas. Although individual transcription factors initiating progression remain unknown, LIM-only protein (LMO) and LIM-domain binding protein (LDB) negatively regulate breast carcinoma cell differentiation. In this study, we investigated the expression of LMO4 and LDB in squamous cell carcinomas of the oral cavity. LMO4 mRNA was amplified in four of six carcinoma tissues and eight of 12 carcinoma cell lines, and LDB1 in three carcinoma tissues and 11 cell lines examined. Immunoprecipitation studies revealed that LMO4 and LDB1 interact with each other in the nuclear milieu of the carcinoma cells indicating the presence of an LMO4-LDB1-mediated transcription complex. Both LMO4 and LDB1 proteins were preferentially localised in the nuclei of carcinoma cells at the invasive front and the immunoreactivity was increased in less-differentiated carcinoma tissues (P<0.01). Carcinoma cells metastasised to the cervical lymph nodes with increased immunoreactivity compared to the primary site of neoplasm (P<0.05). These data suggest that the LMO4-LDB1 complexes may be involved in carcinoma progression possibly through dedifferentiation of squamous carcinoma cells of the oral cavity.

Circadian and photic regulation of MAP kinase by Ras- and protein phosphatase-dependent pathways in the chick pineal gland.

Chick pineal mitogen-activated protein kinase (MAPK) exhibits circadian activation and light-dependent deactivation at nighttime. Here we report that, in the chick pineal gland, levels of active forms of MAPK, MEK, Raf-1 and Ras exhibited synchronous circadian rhythms with peaks during the subjective night, suggesting a sequential activation of components in the classical Ras-MAPK pathway in a circadian manner. In contrast, the light-dependent deactivation of MAPK was not accompanied by any change of MEK activity, but it was attributed to the light-dependent activation of protein phosphatase dephosphorylating MAPK. These results indicate that the photic and clock signals regulate MAPK activity via independent pathways, and suggest a pivotal role of MAPK in photic entrainment and maintenance of the circadian oscillation.

Correlation between the expression of methionine adenosyltransferase and the stages of human colorectal carcinoma.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet) from ATP and L-methionine. AdoMet is the major methyl donor in most transmethylation reactions in vivo, and it is also the propylamino donor in the biosynthesis of polyamines. In the present study, we assessed MAT activity in human colons with colorectal carcinoma and the values were compared with those of morphologically normal adjacent mucosa. Higher levels of MAT activity were observed in the colorectal carcinoma than in the normal colon. The ratio of MAT activity in tumor tissue versus normal tissue seemed to be correlated well will the stage of the colorectal tumor. Furthermore, immunoblot analysis showed that the high levels of MAT activity observed in colorectal carcinoma were due to the increased amounts of MAT protein. Immunohistochemical analysis revealed that MAT was most abundant in goblet cells, particularly in granules in the supranuclear area of these cells. In the colorectal carcinoma tissues, MAT was strongly stained in the cancerous cells and localized in granules in the supranuclear region. The results of this preliminary study suggest that determination of the relative ratio of MAT activity in both normal and tumor regions in human colorectal carcinoma could be a clinically useful tool for determining the stage of malignancy of colorectal carcinomas.

Doxorubicin preconditioning: a protection against rat hepatic ischemia-reperfusion injury.

Doxorubicin produces clinically useful responses in a variety of human cancers. However, the toxicity of doxorubicin has limited its usefulness. This side effect is mainly due to the doxorubicin-mediated free radical formation. Administration of doxorubicin (10 mg/kg body weight) to rats intravenously induces heme oxygenase-1 (HO-1) in the liver. The levels of HO-1 protein were first detected at 6 hours and peaked at about 18 to 24 hours after the injection. It is known that HO-1 plays a protective role against the oxidative injury. Therefore, we have examined the protective effect of doxorubicin preconditioning against the hepatic ischemia-reperfusion injury. Partial hepatic ischemia was produced in the left and medium lobes for 45 minutes followed by 120 minutes reperfusion. When low doses of doxorubicin (1 mg/kg body weight) was intravenously administered to rats 2 days before the ischemia, the serum alanine transaminase (ALT) levels in the preconditioning rat were clearly improved compared with those in the rat without preconditioning. Under this situation, zinc-protoporphyrin IX, a specific inhibitor of HO-1, was injected subcutaneously to rats at 3 and 16 hours before the ischemia, the ALT levels were not improved by doxorubicin preconditioning. Histopathologic examination also supported these results. Although the HO-1 protein level was fairly low 2 days after the doxorubicin administration, significant amounts of HO-1 protein were detected. Our results indicated that the induction of HO-1 played a protective role against hepatic ischemia-reperfusion injury and that doxorubicin preconditioning is more clinically useful than other preconditioning methods.

[Combination therapy with 5'-DFUR, MMC and CDDP in patient with far advanced gastric cancer: report of a case].

A 48-year-old man was referred to our hospital for a Borrmann 3 type advanced gastric cancer. Endoscopic biopsy disclosed poorly differentiated adenocarcinoma. Ultrasonography and CT scan revealed left hydronephrosis. Endoscopic retrograde cholangiography detected a stenosis of common bile duct at the hepatic hilum due to lymph nodal metastasis, and laparoscopy revealed peritoneal dissemination. Because the tumor was diagnosed as not for curative resection, the patient was treated by 4 courses of combination therapy with 5'-DFUR, MMC and CDDP. No adverse effect of chemotherapy was observed. As a result, lymph nodal metastasis and peritoneal dissemination were reduced. Curative intent total gastrectomy was performed, together with pancreatico-splenectomy, left hemicolectomy, cholecystectomy, and extended lymph nodal dissection. The patient is well and alive with no sign of recurrence 2 years after operation.

Cyst-like lesion of a developing tooth induced by mandibular fracture.

A dentigerous cyst-like formation in the lower canine region caused by mandibular fracture in a 10-year-old boy is reported. His medical history revealed that he had been unconscious for about 2 weeks after traumatic head injuries sustained in a traffic accident, and a complicated mandibular fracture had been left untreated until his dentist diagnosed the lesion. Eleven months after trauma, a dentigerous cyst measuring 20 mm in diameter was found in the fracture area. The lesion was enucleated and the boy's postoperative recovery was uneventful. The mass completely enveloped the developing canine, and epithelial cells proliferated into the connective tissue. However, there was no distinct epithelial lining. Small round cell infiltrations and several vessels with thrombosis were noted in the cyst wall. The cause of cyst formation was considered to be infection of the canine tooth bud and the surrounding soft tissue.

Calcium-bound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation.

In rod photoreceptor cells, Ca2+-bound recoverin associates with disk membranes and inhibits light-dependent phosphorylation of rhodopsin. However, the functional significance of Ca2+-induced membrane association of recoverin has not been fully evaluated. We found that Ca2+-bound recoverin forms a complex with rhodopsin kinase preferentially at the membrane surface. Addition of increasing amounts of membranes promoted the membrane association of recoverin, and remarkably suppressed rhodopsin kinase activity. It was concluded that the Ca2+-recoverin-rhodopsin kinase complex is stabilized by membrane association, leading to effective suppression of the kinase activity.

Role of heterogeneous N-terminal acylation of recoverin in rhodopsin phosphorylation.

Recoverin, a new member of the EF-hand superfamily, plays a critical role in the light/dark adaptation of retinal rods by regulating rhodopsin phosphorylation in a Ca(2+)-dependent manner. Recoverin is composed of four isoforms, each of which is modified at its N terminus by myristate (C14:0) or its structurally related fatty acid (C12:0, C14:2, or C14:1). Although the N-fatty acylation is implicated in protein-membrane and protein-protein interactions, the functional difference among the recoverin isoforms and the significance of the heterogeneous acylation have not been defined. Here we separated the heterogeneous recoverin into three fractions, C14:0-recoverin, C14:1-recoverin, and a mixture of C14:2- and C12:0- (C14:2/C12:0-) recoverin to evaluate the individual properties. Recoverin in every fraction bound Ca2+ as assessed by fluorescence spectroscopy and inhibited the light-dependent rhodopsin phosphorylation in the same range of free Ca2+ concentration (0.3-0.8 microM). However, the magnitude of the inhibition at higher Ca2+ concentration was different among the isoforms and ranked in the same order of the hydrophobicity of the N-fatty acyl groups: C14:0 > C14:1 > C14:2/C12:0. These results indicate that the diverged hydrophobicity of the recoverin N terminus plays an important role in the interaction with the membranes and/or its target protein but not with Ca2+.

Genetic polymorphisms of the CST2 locus coding for cystatin SA.

A new genetic polymorphism of cystatin SA has been identified in human submandibular-sublingual saliva by means of basic gel electrophoresis and immunoblotting with anti-cystatin S. Two proteins, SA1 and SA2, are given by two alleles of CST2, viz., CST2*1 and CST*2. Inheritance is controlled by two codominant alleles at an autosomal locus. This hypothesis is supported by studies of 16 families 32 children. Gene frequencies for CST2*1 and CST2*2 are 0.935 and 0.065, respectively (n = 341). Eighteen amino acids determined among 20 N-terminal residues of cystatin SA2 are identical with the sequence encoded by CST2. Three forms of cystatin S (mono-phosphorylated cystatin S, di-phosphorylated cystatin S, and non-phosphorylated cystatin S) are present in the 341 saliva samples tested.

Biochemical and clinical factors influencing oral malodor in periodontal patients.

The amounts of volatile sulfur compounds (VSC) and methyl mercaptan/hydrogen sulfide ratio in mouth air from patients with periodontal involvement were 8 times greater than those of control subjects. Our studies demonstrated that, in patients with periodontal disease: 1) the concentration of disulfide, which is converted to VSC, increased in proportion to the total pocket depth; 2) 60% of the VSC was produced from the tongue surface; 3) the amount of tongue coating was 4 times greater than in control subjects; and 4) VSC production and the methyl mercaptan/hydrogen sulfide ratio of the tongue coating were increased. 2-Ketobutyrate, which is a byproduct of the metabolism of methionine to methyl mercaptan, was higher in the saliva of patients with periodontal disease. This implies that metabolism of methionine to methyl mercaptan increases in the oral cavity of patients with periodontal pockets. Since free L-methionine, rather than protein, is the main source for methyl mercaptan, we estimated the methionine supply from the gingival fluid into the oral cavity of patients with periodontal involvement. The results showed that the ratio of methionine to whole free amino acids was significantly higher than that of cysteine. Our studies suggest that not only microorganisms, but also the tongue coating and gingival fluid are factors which enhance VSC production in patients with periodontal disease.

Effects of a two-phase oil-water mouthwash on halitosis.

Many oral microorganisms possess hydrophobic outer surfaces. A two-phase, oil-water mouthwash has, therefore, recently been developed to remove such oral microorganisms. The oil phase consists of olive oil and other essential oils. The aqueous phase includes cetylpyridinium chloride, which is a disinfectant that promotes the adhesion of microorganisms to oil droplets. This study determined the effects of this mouthwash on the production of volatile sulfide in vivo and in vitro. Neither rinsing with water nor brushing teeth decreased the concentration of sulfide in mouth air at 3.5 h after treatment. A reduction of only 30% of sulfide was observed when a commercial mouthwash was used. However, this study demonstrated that use of the two-phase mouthwash led to approximately 80% reduction of sulfide. Furthermore, volatile sulfide and 2-ketobutyrate productions from methionine in a saliva putrefaction system were completely inhibited by the two-phase mouthwash; and consumption of methionine was decreased by 65 percent. It is concluded that the two-phase mouthwash strongly inhibits the production of volatile sulfide.

Characterization of two members (CST4 and CST5) of the cystatin gene family and molecular evolution of cystatin genes.

Two members (CST4 and CST5) of the cystatin gene family have been characterized partially by DNA analysis. The CST4 clone contained the gene coding for the precursor form(141 amino acids) of cystatin S, and its exon-intron organization is the same as that of other members (the cystatin SN gene at the CST1 locus, the cystatin SA gene at the CST2 locus, the cystatin C gene at the CST3 locus and a cystatin pseudogene at the CSTP1 locus). The second cystatin pseudogene was elucidated in the clone, CST5, and it was assigned to the CSTP2 locus. Alignment of DNA sequences of cystatin genes with other genes suggested that the genes for cystatins, kininogens, and Bowman-Birk type inhibitors have evolved from an ancient ribonuclease-like gene.

Identification of full-sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva and determination of phosphorylation sites of cystatin S.

Our recent work on the gene structures for human salivary (S-type) cystatins [Saitoh, E. et al. (1987) Gene 61, 329-338] has suggested that the structures of cystatins which we determined previously at the protein level lack N-terminal peptide portions of the full-sized intact forms. In the present study, attempts were made to isolate full-sized S-type cystatins by introducing methanol fractionation into the purification steps to suppress the enzymatic activity present in saliva. Full-sized cystatin SN and two phosphorylated forms of full-sized cystatin S were thus isolated. Analysis of one fraction indicated that this was a mixture of full-sized cystatin SA and non-phosphorylated cystatin S. The phosphorylation sites of cystatin S were determined to be Ser-Ser-Ser1(P)-Lys-Glu-Glu- for monophosphorylated cystatin S and Ser1(P)-Ser-Ser3(P)-Lys-Glu-Glu- for diphosphorylated cystatin S. Immunoblotting analysis with anti-cystatin S antiserum revealed that tears and seminal plasma also contained S-type cystatins, but diphosphorylated cystatin S was detected neither in tears nor in seminal plasma and no cystatin SN was found in seminal plasma. These data indicate that S-type cystatins are secreted into the oral cavity without significant degradation in salivary glands or ducts and that they are expressed tissue specifically.

Cystatins of family II are harboring two domains which retain inhibitory activities against the proteinases.

Two cyclic peptides, Ac-CTKSQPNLDTC-NH2 (SA-LOOP1) and Ac-CSFQIYEVPWE DRMSLVNSRC-NH2 (SA-LOOP2) were prepared. These sequences are respectively found in the second and third exons of cystatin SA and are well conserved among the cystatins of family II. In addition, these sequences are extremely homologous to the inhibitory regions of several serine-proteinase inhibitors. The peptides were assayed for their inhibiting properties towards serine- and cysteine-proteinases. SA-LOOP1 inhibited porcine pancreatic trypsin (Ki = 370 microM), but did not inhibit cysteine-proteinases. SA-LOOP2 inhibited not only porcine pancreatic alpha-chymotrypsin (Ki = 23 microM) but also papain (Ki = 24 microM) and ficin (Ki = 52 microM). These data indicate that the exon-intron organization of the cystatin genes coinside with the structural and/or functional domains of the protein, and may have significant implications for understanding the active sites of cystatins.

The human cystatin gene family: cloning of three members and evolutionary relationship between cystatins and Bowman-Birk type proteinase inhibitors.

Three genes from the human cystatin gene family have been isolated from a bacteriophage lambda library containing Hind III digests of human genomic DNA. The cloned genes were identified with three DNA probes each containing exon 1, exon 2 and exon 3 of the CST1 gene for cystatin SN. The genes, which we name CST2B, CST4, and CST5, are 6.8 kb, 5.4 kb and 12.5 kb in size, respectively. Statistical analysis of DNA sequence homology elucidated that the second and third exons of cystatin (family II) genes and three cystatin (family II) gene like segments in the kininogen (family III) genes are significantly homologous to the gene segments coding for the inhibitory domains of Bowman-Birk type proteinase inhibitors.

The human cystatin C gene (CST3) is a member of the cystatin gene family which is localized on chromosome 20.

The fourth gene from the human cystatin gene family of salivary-type cysteine-proteinase inhibitors has been isolated and partially characterized by DNA analysis. The gene, which we name CST3, codes for human cystatin C, and has the same organization as the CST1 gene for cystatin SN and the CST2 gene for cystatin SA. Southern analysis of EcoR I digested DNAs from 32 independent somatic cell hybrid clones hybridized to a probe from CST1 demonstrated that all members of the cystatin gene family segregate with human chromosome 20. These results indicate that the genes for salivary-type cystatins and cystatin C are members of a multigene family--the cystatin gene family.