Intermolecular disulfide bond to modulate protein function as a redox-sensing switch.

Recently, redox-regulated biological reactions have been elucidated. In the regulation of these reactions, redox-sensing molecular switches function as unique biological machineries that modulate the functional proteins present in enzymes, transcriptional factors, sensor proteins, and transcriptional factor modulators. The redox-sensing cysteine residues and the disulfide bond formed between these cysteine residues serve as redox-sensing molecular switches; these switches sense cellular oxidizing factors such as oxygen, reactive oxygen species, and cellular reducing factors such as thioredoxin (Trx), glutathione (GSH), and their family molecules. Depending on the redox status, the switch directly modulates the protein function via the "locking and unlocking" of the critically functional residue or indirectly modulates the protein function via "protein conformational changes," which affects the functioning of a distantly located critical residue in an allostery-like fashion or a topology change. Redox-sensing switches can be classified into two types-intramolecular (intrasubunit) and intermolecular (intersubunit) ones. Further, depending on the sensing specificity to reducing factors, the switch subtype is classified into Trx, GSH, or their family molecules-specific type. This review focused on the intermolecular redox-sensing switches found in various proteins.

Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation.

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys(223) is transferred in the active center to be coordinated by Cys(122), Cys(124), and Cys(132) (Sawada et al. in J Biol Inorg Chem 10:199-207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f (0). It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys(122), Cys(124), and Cys(132) and coordination with zinc ion is closely linked to change in the oligomeric state.

Frequent beta-catenin mutation and cytoplasmic/nuclear accumulation in pancreatic solid-pseudopapillary neoplasm.

Significance of Wnt signaling with beta-catenin mutations on solid-pseudopapillary neoplasm (SPN) of the pancreas was studied by immunohistochemistry and molecular analysis. On immunohistochemistry, all 18 SPNs tested showed diffuse cytoplasmic/nuclear positivity for beta-catenin. Upon direct DNA sequencing of exon 3 of the beta-catenin gene, 15 (83%) of the 18 SPNs showed 1-bp missense mutation in codons 32 (5 cases), 33 (3 cases), 34 (3 cases), 37 (3 cases), and 41 (1 case). Immunoreactivity for cyclin D1, one of the intranuclear targets of beta-catenin complexes, was found in tumor cells of more than half the tumor cells of all of the 18 SPNs. The present study strongly suggested a significant role of Wnt signaling, mostly associated with beta-catenin mutations in the tumorigenesis of SPN.

Two patients with chromosome 6q terminal deletions with breakpoints at q24.3 and q25.3.

We report on 2 patients with de novo terminal deletion of 6q. The first was a 4-month-old boy whose karyotype was 46,XY,del(6)(q24.3); the second a 2-year-old girl whose karyotype was 46, XX, del(6)(q25.3). The main anomalies in both patients included mental retardation, minor craniofacial and cerebral anomalies, and cardiac defects. The characteristic manifestations were imperforate anus in the first patient, and retinitis proliferans and a triatrial heart in the other. Comparison of clinical findings of our 2 patients with those of 18 previously reported patients with similar phenotypes suggests that terminal deletion of the 6q23 or 6q25 band is critical in producing the main anomalies of del(6q) syndrome.

Granulocytosis associated with malignant neoplasms: a clinicopathologic study and demonstration of colony-stimulating activity in tumor extracts.

We studied seven cases of malignant neoplasms, taken from various sites in the body, that were associated with marked granulocytosis. The seven cases were characterized clinically by marked granulocytosis with mature neutrophils, nonspecific inflammatory signs, and a rapid and progressive fatal course of the disease. The elevation of granulocyte count generally paralleled the increase in tumor size. Postmortem examination revealed no evidence of extensive bone marrow metastases or significant suppuration in any case. The bone marrows showed varying degrees of granulocytic hyperplasia with or without a shift to the left in the maturation series. Erythroid cell hyperplasia was observed in some cases, and in one instance there was an increase in immature eosinophils. The spleen showed various degrees of infiltration by neutrophils, from minimal to extremely marked; some spleens had foci of extramedullary hemopoiesis. Colony-stimulating activity was demonstrated in tumor extracts from three of these cases and from the serum in another case. Thus, it is suggested that marked granulocytosis in these patients was caused, at least in part, by colony-stimulating factor produced by the neoplastic cells.

Ewing's sarcoma family of tumor arising in the adrenal gland--possible diagnostic pitfall in pediatric pathology: histologic, immunohistochemical, ultrastructural, and molecular study.

We present an adrenal Ewing's sarcoma family of tumor (ESFT) arising in an 11-year-old Japanese boy. Although intensive chemoradiotherapy and radical surgery were performed, the patient died of obstinate disease 1 year and 3 months after the initial presentation. The primary site (adrenal gland) with radiologic findings (with foci of calcification), high titer of serum neuron specific enolase, and sheets of monotonous primitive rounded cells on histology mostly favored neuroblastoma. However, a diagnosis of ESFT was confirmed by immunohistochemical profile, including MIC2-positivity and molecular study disclosing EWS-FLI1 chimera gene verified by direct sequencing. Recognition of adrenal ESFT and use of newly developed diagnostic techniques are required for differential diagnosis of undifferentiated small round cell tumor of the adrenal gland.

Mercaptopyruvate sulfurtransferase as a defense against cyanide toxication: molecular properties and mode of detoxification.

In cyanide poisoning, metalloproteins and carbonyl groups containing proteins are the main target molecules of nucleophilic attack by cyanide. To defend against this attack, cyanide is metabolized to less toxic thiocyanate via transsulfuration. This reaction is catalyzed by rhodanese and mercaptopyruvate sulfurtransferase (MST). Rhodanese is a well characterized mitochondrial enzyme. On the other hand, little was known about MST because it was unstable and difficult to purify. We first purified MST to homogeneity and cloned MST cDNA from rat liver to characterize MST. We also found that MST was an evolutionarily related enzyme of rhodanese. MST and rhodanese are widely distributed in rat tissues, and the kidney and liver prominently contain these enzymes. Immunohistochemical study revealed that MST is mainly distributed in proximal tubular epithelial cells in the kidney, pericentral hepatocytes in the liver, the perinuclear area of myocardial cells in the heart, and glial cells in the brain, and immunoelectron microscopical study concluded that MST was distributed in both cytoplasm and mitochondria, so that MST first detoxifies cyanide in cytoplasm and the cyanide which escapes from catalysis due to MST enters mitochondria. MST then detoxifies cyanide again in cooperation with rhodanese in mitochondria. Tissues other than the liver and kidney are more susceptible to cyanide toxicity because they contain less MST and rhodanese. Even in the same tissue, sensitivity to cyanide toxicity may differ according to the kind of cell. It is determined by a balance between the amount of proteins to be attacked and that of enzymes to defend.

Evaluation of oral mucoadhesive microspheres in man on the basis of the pharmacokinetics of furosemide and riboflavin, compounds with limited gastrointestinal absorption sites.

When sustained-release adhesive and non-adhesive microspheres which release the same drugs at similar rates are administered orally, drug absorption after administration of adhesive microspheres should, if the gastrointestinal residence of adhesive microspheres is prolonged as a result of mucoadhesion, be higher than that after administration of non-adhesive microspheres. The gastrointestinal transit of oral adhesive microspheres in man has been evaluated pharmacokinetically using furosemide and riboflavin, compounds with limited absorption sites in the upper small intestine. In a preliminary experiment with fasted rats it was confirmed that a higher percentage of the drug remained in the stomach and that plasma drug levels were higher when furosemide was administered in the form of adhesive rather than non-adhesive microspheres. Two kinds of sustained-release microsphere, adhesive and non-adhesive, containing furosemide and riboflavin in hard gelatin capsules were prepared and orally administered to 10 healthy fasted volunteers in a cross-over design. Areas under the plasma concentration-time curves (AUC) were 1.8 times larger for furosemide and urinary recovery was 2.4 times higher for riboflavin when adhesive microspheres rather than when non-adhesive microspheres were used. When adhesive microspheres containing riboflavin were administered to fed volunteers, urinary recovery was 2.1 times higher and mean residence time (MRT) was more prolonged than when the microspheres were administered to fasted volunteers. Adhesive microspheres were found to adhere to the gastric or intestinal mucosa with high affinity in man and rats, resulting in prolonged gastrointestinal residence.

Gastrectomized patients are in a state of chronic protein malnutrition analyses of 23 amino acids.

BACKGROUND/AIMS: Malnutrition is one of the major postoperative complications of radical subtotal or total gastrectomy for gastric cancer. This study was conducted to clarify the nutritional consequences of radical gastrectomy with respect to protein metabolism. METHODOLOGY: To evaluate the nutritional status and the abnormalities in protein metabolism in such cases, serum concentrations of 23 amino acids were measured by high performance liquid chromatography in 40 patients who had undergone either subtotal (n = 20) or total (n = 20) gastrectomy more than 6 months prior to this analysis. RESULTS: Serum concentrations of total amino acids and nonessential amino acids were the same between gastrectomized patients and healthy controls (n = 50). However, concentrations of essential amino acids, essential amino acid/nonessential amino acid and branched-chain amino acid/total amino acid ratios were significantly lower in patient groups than in normal controls. Each essential amino acid was decreased and concentrations of glutamate and citrulline were increased in both patient groups compared with controls. The major differences between patients with subtotal and total gastrectomies included an increased ornithine and a decreased arginine concentration in patients with subtotal gastrectomy. CONCLUSIONS: These changes suggest that malabsorption of protein from the intestinal tract causes persistent proteolysis in the skeletal muscle for long periods of time after surgery in these patients and that changes in ornithine and citrulline levels may reflect more severe alterations in those with total gastrectomy.

A spigelian hernia in an infant.

The case of a two-month old Japanese girl with a left side Spigelian hernia that developed during the course of recovery from the surgical treatment of a meningomyelocele and ventriculo-peritoneal shunting (V-P shunting) is reported on. At this time, to our knowledge, 20 cases involving infants and children have been reported on in world-wide medical literature. Of these cases, 4 cases were caused by trauma, 1 by abdominal surgery, and the others had no evident causes. Our patient had a meningomyelocele, and underwent a V-P shunting. However, a cause and effect relationship between the Spigelian hernia and the treatment of the meningomyelocele followed by V-P shunting is inconceivable. We doubt any relationship between the preceding neurosurgery and the development of the hernia. This is the first case of infantile Spigelian hernia reported on in Japan.

[Prevention of hepatitis B--HLA DR/DQ in HBs Ag nonresponder].

To evaluate the immunological background in HBs Ag nonresponders against hepatitis B vaccine, the lymphocyte surface marker and HLA-DR/DQ antigen were determined on hospital personnel, 70 males and 256 females, injected hepatitis B vaccine for three times. The vaccine made from the plasma of HBs Ag carriers was injected at the first and second vaccination and recombinant hepatitis B vaccine was injected at the third vaccination. A month after the third vaccination, blood was withdrawn for HBs antigen test by RIA. Border line cases (cut off index 1.0-1.9) are included in nonresponder (cut off index less than 0.9). Both nonresponders and low responders (cut off index 2-49) are more often seen in males than females and high responder (cut off index 50 or more) are seen more often in young females than males. Lymphocyte surface markers were studied by flow cytometry using the following monoclonal antibodies; OKT 3, 4, 8, DR, NK, Ia 1 and B7. No differences between lymphocyte surface markers of nonresponders and responders were noted. HLA-DR/DQ antigens were studied by the cytotoxicity test using Locus DR/DQ, Terasaki Second DR W-60 Tray and using following antibodies; DR 1, DR 2, DRW 15, DR 4, DR 5, DR 7, DR 9, DRW 10, DRW 8, DRW 12, DRW 13, DRW 6, DRW 52, DRW 53, DQW 1, DQW 6, DQW 2, DQW 3, DQW 7 and DQW 4. No significant differences between HLA-DR/DQ of nonresponders and responders were noted.

[Campylobacter pylori and diseases of the stomach].

In spite of extensive clinical studies the pathogenicity of Campylobacter pylori in various diseases of the stomach is still not fully elucidated. In peptic ulcer Campylobacter pylori has not provided a reasonable explanation for the localized and solitary development of ulcer lesion which is one of characteristic features of this clinical entity. Our previous clinical study revealed that relapse occurs mostly at or near the scar of the preceding ulcer lesion and occurs more often in red scar than in white scar in peptic ulcer. Therefore, the scar was chosen to investigate whether there is a correlation between Campylobacter pylori and relapse. Twenty patients with healed gastric ulcer were biopsied in duplicate at 4 different sites, namely at the center of and near the scar of the preceding ulcer lesion, in the body and pyloric antrum. One of the duplicate specimen was stained, and the other cultured. Number of the colony observed on the plate was highest at the scar center followed by near the scar or in the body, and the pyloric antrum showed the lowest. Campylobacter pylori was detected in 46.7% of red scar, and 20% of white scar. Campylobacter pylori may play a pathogenic role in some patients of peptic ulcer, but not in all. Namely Campylobacter pylori is one of factors which may cause in imbalance between offensive and defensive powers at gastric mucosa and may lead to development of ulcer lesion. In case of positive bacteriological test, therefore, as to presence of Campylobacter pylori, the preventive measure for relapse such as bismuth preparation may be considered.

[Present status of neuroblastoma mass screening in Japan. Neuroblastoma-Committee of the Japanese Childhood Cancer Society].

In 1973, mass screening program for 6-month old infants for early detection of neuroblastoma using a VMA spot test of a urine sample was initiated in Kyoto. In 1985, nation wide mass screening was initiated throughout the entire country and the Government has given the financial support to each district. In 1988, the Government recommended the institution of mass screening by quantitative measurements of VMA, HVA and creatinine using HPLC (high performance liquid chromatography), instead of the qualitative test of VMA alone. From 1974, at the time of initiation of mass screening for neuroblastoma to the end of October, 1989, 383 cases with this tumor have been discovered throughout the screening program. Three hundreds eighty three cases (88%) of them had been registered to the Neuroblastoma Committee of the Japanese Society of Pediatric Oncology. In this paper, the mass screening program was introduced and the 337 cases with this tumor detected by 6-month old screening were analyzed their clinical symptoms, findings, urinary VMA and HVA levels, primary sites, weights of primary tumor, histology, stages at diagnosis, metastatic sites, and the results of the treatment. Three hundreds twenty eight cases (97%) of them are expected to be cured. And we discussed clinical problems related to mass screening program for neuroblastoma, such as an increase of the incidence of infantile neuroblastomas detected by this program and the spontaneous regression.

Protein cysteine modifications: (1) medical chemistry for proteomics.

Protein cysteines (cysteinyl residues) play critical roles in biological processes. In the course of protein evolution under oxidizing atmosphere of the Earth, organisms have utilized highly reactive cysteines in many proteins essential for maintenance of life, i.e. enzymes, transcriptional factors, cytoskeletons, and receptors. In some enzymes, sophistical cysteine modification characterizes each catalytic mechanism. In vivo modification of protein cysteines with natural chemical compounds modulates protein functions as a molecular switch. Oxidation/reduction, thiol-disulfide exchange, nitrosylation, sulfuration, thiolation, acylation and prenylation are involved. Some protein cysteines coordinate metals or metal cofactors such as a heme or an iron sulfur cluster to form metalloproteins, serving as sensor proteins, metalloenzymes or transcriptional factors. Information on the in vitro chemical modifications and their reaction specificities of protein cysteines are essential for the investigation of the mechanisms and functions of in vivo protein cysteine modifications. In this review, we also mention historically important knowledge other than recent results on protein cysteine modification and modulation of protein function to fertilize medical proteomics.

A pathological study of nephroblastoma with congenital aniridia.

A pathological study was conducted on four patients with nephroblastoma associated with aniridia. The age at diagnosis. was one year in three cases and 4 years in one case. Chromosomal analysis was performed in three cases, and showed 11p13 deletion in all. Each nephroblastoma consisted of metanephric blastemal, epithelial, and mesenchymal cells. All the tumors were centrilobar, and in one case an additional polypoid tumor grew into the renal pelvis. Histological examination showed conspicuous heterotopic cells, such as striated muscle and adipose tissue. Striated muscle was seen in all four cases, while both adipose tissue and smooth muscle were seen in two cases. Squamous epithelium was present in two cases. A literature survey revealed a high incidence of bilateral occurrence in cases of nephroblastoma associated with aniridia. Intralobar nephroblastomatosis was observed in all four cases, and it is proposed that this is the precursor lesion of nephroblastoma associated with aniridia.

Role of amino acid residues in the active site of rat liver mercaptopyruvate sulfurtransferase. CDNA cloning, overexpression, and site-directed mutagenesis.

A complete amino acid structure of rat liver mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) was determined by sequence analysis of cDNA and purified enzyme. The enzyme consists of 296 amino acid residues with a calculated molecular mass of 32,808 Da. Sequence identity in cDNA and the deduced amino acid sequence are 65 and 60% respectively, between rat MST and rhodanese. By their entire sequence similarity MST and rhodanese are confirmed to be evolutionarily related enzymes (Nagahara, N., Okazaki, T., and Nishino, T. (1995) J. Biol. Chem. 270, 16230-16235). The conversion of MST to rhodanese was attempted, and the role of amino acid residues was studied by site-directed mutagenesis with the isolated cDNA of rat liver MST. There is a strong possibility that Cys247 is a catalytic site of MST. Arg187 is suggested to be a binding site of both mercaptopyruvate and thiosulfate in MST. Arg196, which is missed in rhodanese, is important for catalysis in MST. On the other hand, the substitution of Arg for Gly248 or Lys for Ser249 facilitates catalysis of thiosulfate in MST. It is concluded that Arg187 and Arg196 of rat MST are critical residues in determining substrate specificity for mercaptopyruvate. On the other hand, Arg185, Arg247, and Lys248 of rat rhodanese are critical residues in determining substrate specificity for thiosulfate.