NADPH-binding component of the respiratory burst oxidase system: studies using neutrophil membranes from patients with chronic granulomatous disease lacking the beta-subunit of cytochrome b558.

The NADPH-binding site of the respiratory burst oxidase system of neutrophils has been proposed to be either at a cytosolic component or at the beta-subunit of cytochrome b558. In this study, affinity labeling of resting and stimulated membranes, the latter having been assembled by all of the oxidase components from both membrane and cytosol, was carried out using [32P]NADPH dialdehyde (oNADPH). Stimulation of human neutrophils with PMA greatly increased O2(-)-generating activity and caused considerable translocation of the cytosolic components p47phox and p67phox. Nevertheless, PMA stimulation did not produce a labeled band which included positions at 47, 67, and approximately 32 kD. The most intense band reflected a molecular mass of 84 kD regardless of the state of activation, but a labeled band was never found near the beta-subunit (91 kD) of cytochrome b558. This 84-kD protein was further confirmed in neutrophils of 14 patients with gp91phox-deficient X-linked chronic granulomatous disease. These results indicate that the NADPH-binding component is not recruited from the cytosol, and also, that a membranous redox component besides cytochrome b558 must be involved in the NADPH oxidase system.

Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox.

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.

Development of pancreatic hyperplasia in female SHN mice receiving ectopic pituitary isografts.

The long-term effects on pancreas of ectopic pituitary grafting at various sites were studied in female SHN mice. Hyperprolactinemia induced by pituitary grafting resulted in an increase in the pancreatic weights, mainly due to hyperplastic proliferation of the pancreatic acinar glands. In addition, islet-cell hyperplasia and adenoma were found in the pituitary-grafted mice. Hyperplastic nodules resembling adenoma of the pancreatic acinar cells were also induced by pituitary grafting. The pancreata of mice with pituitary grafts frequently adhered to the uterus, ovary, and other organs; some of the pancreata further invaded these organs. Control mice bearing no pituitary grafts showed no pancreatic lesions. These results indicate the participation of prolactin in pancreatic tumorigenesis.

Nucleoside-triphosphate binding of the two cytosolic components of the respiratory burst oxidase system: evidence for its inhibition by the 2',3'-dialdehyde derivative of NADPH and desensitization in their translocated states.

Affinity labeling of the two cytosolic components of the respiratory burst oxidase system, p49-phox and p63-phox, from resting porcine neutrophils was carried out with [32P]NADPH dialdehyde (oNADPH), [32P]oGTP and [32P]oATP. p49-phox and p63-phox showed 10-times higher affinities for both oGTP and oATP than for oNADPH, suggesting that they are nucleoside triphosphate (NTP)-binding proteins, rather than the NADPH-binding site of the oxidase. In addition, oNADPH markedly inhibited the affinity labeling of p49-phox with [32P]oGTP and [32P]oATP, well reflecting its inhibitory effect on the oxidase activity in the cell-free system, which was previously reported to propose the NADPH-binding site in a cytosolic component. Stimulation of porcine neutrophils with either myristic acid or phorbol myristate acetate resulted in great enhancement of the oxidase activity, and in considerable translocation of p49-phox and p63-phox. Nevertheless, the affinity labeling of the stimulated cell membranes in both cases revealed no labeled bands corresponding to molecular masses of 49 kDa and 63 kDa. p49-phox derived from the stimulated membranes had lost its [32P]oGTP binding ability in contrast with that from resting cytosol, suggesting that the NTP-binding sites of the two cytosolic components may be desensitized on NTP binding in their translocated states.

CPP32 activation during dolichyl phosphate-induced apoptosis in U937 leukemia cells.

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre-CPP32 to generate p17. Peptide inhibitors YVAD-cmk and Z-Asp-CH2-DCB (specific to ICE) and DEVD-CHO (specific to CPP32) blocked the dolichyl phosphate-induced apoptosis. The dolichyl phosphate-induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2',5'-dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.

Spongy degeneration of the CNS: an instance of the rare juvenile form.

To our knowledge, the juvenile form of spongy degeneration of the CNS (SD-CNS); van Bogaert-Bertrand disease) has been described previously only three times. We report the case of 21 1/4-year-old Japanese woman who was first seen at the age of 11 with growth retardation, ptosis, and ophthalmoplegia. Her progressive neurodegenerative disease included retinitis pigmentosa, blindness, partial deafness, cerebellar dysfunction, hyporeflexia, and muscle wasting. Simultaneous endocrine defects were diabetes mellitus and probable hyperaldosteronism. Heart block developed later. She died of bronchopneumonia. Autopsy showed CNS stigmas typical of spongy degeneration. Additional findings included peripheral nerve demyelination, neurogenic muscle atrophy, pituitary and pancreatic atrophy, right adrenal agenesis, and a left adrenal coritcal lipid-cell adenoma. To our knowledge, our patient was the oldest survivor, the first patient of Japanese ancestry, and had a unique concurrence of certain oculoendocrine defects.

Sclerosing mesenteritis. Report of two cases.

Two cases of benign tumor-like mesenteric lesions are presented. The limited literature on comparable and similar lesions is reviewed, and the histologic findings are correlated. The lesions are composed of chronically inflamed adipose and fibrous tissue in various proportions. They probably represent different stages of a reparative process initiated by damage of the mesenteric adipose tissue of various etiologies. Whereas lesions in the younger age groups (mean 39.9 years) are predominatly characterized the fibrosis, those in the older age groups (mean 55.8 years) usually show a chronic inflammatory cell infiltrate rather than fibrosis. More than a dozen terms have been used for these lesions. The summarizing term "sclerosing mesenteritis" is proposed.

Analyses of virus-induced homomeric and heteromeric protein associations between IRF-3 and coactivator CBP/p300.

Cellular genes including the type I interferon genes are activated in response to viral infection. We previously reported that IRF-3 (interferon regulatory factor 3) is specifically phosphorylated on serine residues and directly transmits a virus-induced signal from the cytoplasm to the nucleus, and then participates in the primary phase of gene induction. In this study, we analyzed the molecular mechanism of IRF-3 activation further. The formation of a stable homomeric complex of IRF-3 between the specifically phosphorylated IRF-3 molecules occurred. While virus-induced IRF-7 did not bind to p300, the phosphorylated IRF-3 complex formed a stable multimeric complex with p300 (active holocomplex). Competition using a synthetic phosphopeptide corresponding to the activated IRF-3 demonstrated that p300 directly recognizes the structure in the vicinity of the phosphorylated residues of IRF-3. These results indicated that the phosphorylation of serine residues at positions 385 and 386 is critical for the formation of the holocomplex, presumably through a conformational switch facilitating homodimer formation and the generation of the interaction interface with CBP/p300.

Palladium-catalyzed intramolecular delta-lactam formation of aryl halides and amide-enolates: syntheses of cherylline and latifine.

[reaction: see text] Palladium-catalyzed intramolecular carbon-carbon bond formation of aryl halides and amide-enolates gave 4-arylisoquinoline derivatives in good yields, which were further converted into the isoquinoline alkaloids cherylline and latifine.

Interrelationship between pituitary and ovarian hormones in normal and neoplastic growth of mammary glands of mice.

While prolactin is a key hormone for normal and neoplastic mammary gland growth, the participation of ovarian estrogen and progesterone is essential for these processes under the normal physiological conditions. Prolactin exerts its influence directly to the glands and indirectly through its luteotropic effects by stimulation of ovarian progesterone secretion. Furthermore, the action of prolactin, whether in promoting normal growth and function or enhancing the progression of neoplastic foci, depends upon the circulating levels of other mammotropic hormones as well as the level of prolactin itself. Presence of estrogen and prolactin is essential for manifestation of progesterone effects on mammary gland growth. Estrogen acts on the mammary glands directly by modulating mammary cell responsiveness to prolactin and indirectly by stimulating pituitary prolactin secretion. While data have been accumulated on the effects of growth hormone on mammary gland growth, the significance of these findings is still unknown. Neoplastic potential of mammary cells is largely dependent upon the susceptibility of the cells to mammotropic hormones.

Comparison of nonmetal and metal hydrophilic photosensitizer, ATX-S10 (Na) and ATN-2, binding with human serum proteins using spectrophotometry.

Intermolecular interactions of human serum proteins with a hydrophilic nonmetalloporphyrin, 13,17-bis(1-carboxypropionyl)carbomoylethyl-8-ethenyl-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetramethylporphyrin sodium salt (ATX-S10 (Na)), or a hydrophilic gallium-metalloporphyrin, diethylenetriamine pentaacetic acid ester of 2-[1-(2-hydroxy-ethoxy)ethyl]-4-vinyl-deuteroporphyrin (IX) Ga complex (ATN-2), were investigated using spectrophotometry. ATX-S10 (Na) caused a bathochromic shift with albumin, high-density lipoprotein and low-density lipoprotein, but little or no shift was observed with hemopexin, transferrin and immunoglobulin G. In contrast, ATN-2 displayed a bathochromic shift only with hemopexin. These results suggest that the association energy of ATX-S10 (Na) with albumin might be slightly greater than that with lipoproteins and that of ATN-2 with hemopexin might be greater than that with other serum proteins.

Mammary gland growth and serum levels of prolactin and growth hormone in four strains of male mice in comparison with females.

Male mammary gland growth in 4 strains of mice (SHN/Mei, SLN/Mei, GR/AMei and C3H/HeMei) was studied and compared to that in females of the same strains. The area of the mammary glands in female mice was greater in SHN and SLN, as compared to GR/A and C3H/He strains on both 20 and 60 days of age. A similar trend was seen in mammary gland growth in males on day 20. However, on day 60, there was a marked growth in SLN and GR/A strains, slight growth in SHN mice and no glands were found in C3H/He males. These patterns of mammary gland growth were not related to circulating levels of prolactin or growth hormone (GH) or to body growth in either sex. These findings provide the first description of these characteristic patterns of mammary gland growth in male mice.

A growth-promoting factor existing on the body surface of rats.

Effects of water-soluble matter adhering to rat hairs on fibroblasts were examined. The dialysate of the wash water of rat hairs significantly enhanced the cell proliferation of both diploid human dermal fibroblasts (DHDF) and diploid rat fibroblasts (DRDF). The cell growth-promoting activity was partially purified by a gel filtration column chromatography. The activity permeates through a ultrafiltration membrane (M.W. cut off: 500). Analyses of its chemical nature show that it is soluble in water, dimethyl sulfoxide or acetonitrile, insoluble in other organic solvents examined, stable to heat or pH shock, and resistant to a bacterial protease.

Effect of Harderian gland-derived growth factor on the growth of cornea stromal cells.

To investigate the physiological role of Harderian gland-derived growth factor (HGDGF), the effects of HGDGF and various other growth factors on the growth of cultured guinea pig cornea stromal cells were examined. HGDGF increased the incorporation of [3H]thymidine to 150% of the control (5% fetal calf serum). The combination of HGDGF with fibroblast growth factors (FGFs) or platelet-derived growth factor enhanced the cell growth over that of either growth factor alone, increasing the incorporation of [3H]thymidine to 180 and 190% of the control, respectively. The combination of HGDGF with transforming growth factor (TGF-beta) decreased the growth to 60% of the control, and epidermal growth factor had no effect on the activity of HGDGF. The growth-stimulating activity of HGDGF was inhibited by suramin in a different manner from that of FGFs. These findings suggest that HGDGF binds a specific cell-surface receptor and plays a role in the repair of injured parts of the cornea and in the maintenance of the cornea stromal cells.

Geographic variations in the histological characteristics of the gastric mucosa.

Sections from 65 gastrectomy specimens obtained from operations performed for peptic ulcer or adenocarcinoma in Caucasian patients living in Hawaii were retrieved from the files of the Queen's Medical Center (n = 45) and the Kuakini Medical Center (n = 20). The sections were examined under high-power light microscopy for the presence of ciliated gastric epithelial cells in nondiseased areas of the gastric mucosa. Ciliated cells were found in 14 of the 65 specimens (21.5%) and were seen more often in elderly patients. The percentage of specimens with ciliated cells at the Kuakini Hospital was 35, which was very similar to the percentage found in a previous survey (40.5) in Japanese patients at the same hospital or in Japanese living in Japan (35%). In previous studies we found that ciliated gastric cells are rarely present in indigenous populations in Scandinavia, the U.S. mainland, Mexico, and Spain. Thus, our results seem to be a further indication that local environmental factors may trigger histological changes in the gastric mucosa and that those changes may be unrelated to the ethnic origin of the patient.

Canal diameter, anteroposterior compression ratio, and spondylotic myelopathy of the cervical spine.

Nine patients with cervical spondylotic myelopathy, diagnosed during life, were subjected to detailed clinicopathologic study. The degree of cord destruction was in good correlation with the ratio of the anteroposterior diameter to the transverse diameter, designated as an anteroposterior compression ratio. Within the factors responsible for decrease in the ratio, developmental narrowing of the spinal canal was the most significant, and multiplicity of spondylotic protrusion less so. The former resulted in an extensive demyelination of the posterolateral funiculus and infarction of the gray matter. Recurrent trauma proved to cause distinct manifestations and cord pathology. Clinicopathologic correlations were also examined from the neurologic findings at the terminal stage.

Effects of 2-(hydroxyphenyl) indole on mammary gland growth and uterine adenomyosis in four strains of mice.

Subcutaneous implantation of a silastic tube containing the compound 4b, one of 2-(hydroxyphenyl) indoles, enhanced, while treatment with the compound diluted with cholesterol at the concentration of 1/40 inhibited the formation of preneoplastic mammary hyperplastic alveolar nodules in four strains of mice (SHN/Mei, SLN/Mei, GR/AMei and C3H/HeMei). However, there was an apparent strain-difference in mammary susceptibility to 4b. Furthermore, the development of uterine adenomyosis in response to 4b varied from strain to strain. These results suggest that more attention should be paid to the choice of specific mouse strains for the study of related fields.

Expression of beta 1 integrins (very late antigens-4 and -5) on myeloma cells and clinical correlates in patients with multiple myeloma.

beta 1 Integrins are considered to be essential for the differentiation of bone-marrow B cells through an interaction with fibronectin-expressed bone-marrow stromal cells. The expression of very late antigens-4 (VLA-4) and -5 (VLA-5) by CD38bright bone-marrow cells in patients with multiple myeloma was measured by flow cytometry using specific monoclonal antibodies. The percentage of CD38bright bone-marrow cells appeared to correlated with that of bone-marrow plasma cells as judged by examination of bone-marrow smears (r = 0.911, P < 0.0001). Expression of VLA-4 and VLA-5 by CD38bright cells varied between patients, but the expression of VLA-4 was always equal to or greater than that of VLA-5. The ratio of VLA-4 to VLA-5 expression (VLA-4:VLA-5 ratio) was calculated and compared with the clinical features of the myeloma patients. A high VLA-4:VLA-5 ratio (> 2.0) was associated with the presence of plasmacytomas and urinary Bence-Jones protein was more common in this group. No other correlations between the clinical features of the disease and the expression of beta 1 integrins were found.

Suppression of spontaneous development of uterine adenomyosis and mammary hyperplastic alveolar nodules by Chinese herbal medicines in mice.

The effects of traditional Chinese herbal remedies, Shakuyaku-kanzo-to (SKT) and Hachimijiou-gan (HJG), on the spontaneous development of uterine adenomyosis and mammary hyperplastic alveolar nodules (HAN) were examined in an experimental animal model using SHN strain of mice. Female mice were provided with the chow containing 1% of SKT or HJG during 25-150 days of age. At 150 days of age, SKT treatment showed significantly lower incidence of adenomyosis, and HJG treatment resulted in a significantly lower incidence of HAN when compared to a control chow containing no medicines. Long-term exposure to these herbal medicines affected little serum prolactin (PRL) level, estrous cycle, food intake and body growth. Thus, the present mouse data suggest that the oral administration of these herbal medicines is a useful tool for the treatment of uterine adenomyosis or mammary disorder such as cystic mastitis.

Effects of Chinese herbal medicines on DNA-synthesizing enzyme activities in mammary tumors of mice.

Sho-saiko-to (SST) and Juzen-taiho-to (JTT), Japanese modified Chinese herbal prescriptions, suppressed the activities of thymidylate synthetase and thymidine kinase involved in de novo and salvage pathways for pyrimidine nucleotide synthesis, respectively, in mammary tumors of SHN mice with the reduction of serum prolactin level. These results indicate that SST and JTT may have the antitumor effects on mammary tumors.