Purification and identification of lactoperoxidase in milk basic proteins as an inhibitor of osteoclastogenesis.

A milk protein fraction with alkaline isoelectric points (milk basic protein, MBP) inhibits both bone resorption and osteoclastogenesis for in vitro models. We previously identified bovine angiogenin as a component of MBP that inhibits bone resorption. However, purified angiogenin had no effect on osteoclastogenesis, suggesting that MBP contains unidentified component(s) that inhibit osteoclast formation. In this study, we purified lactoperoxidase (LPO) as the predominant inhibitor of osteoclastogenesis in MBP. The LPO treatment downregulated levels of reactive oxygen species in osteoclasts. Signaling by receptor activator of NF-kappa-B ligand/receptor activator of NF-kappa-B (RANKL/RANK) was downregulated in LPO-treated cells, and, in particular, the ubiquitination of tumor necrosis factor receptor associate factor 6 (TRAF6) and activation of downstream signaling cascades (JNK, p38, ERK, and NFκB) were suppressed. Ultimately, LPO treatment led to decreased expression of c-Fos and NFAT2. These results suggest that MBP contains at least 2 components that independently suppress bone resorption through a unique mechanism: angiogenin inhibits bone resorption and LPO inhibits RANKL-induced osteoclast differentiation. These data explain many of the positive aspects of milk consumption on bone health.

The role of endothelin in the pathophysiology of renal impairment during acute liver rejection.

We assessed the role of endothelin in the development of renal dysfunction during acute rejection by examining the effect of a selective endothelin A (ETA) receptor antagonist BQ-123 in rats with acute liver rejection. Serum endothelin levels and endogenous creatinine clearance (Ccr) were monitored on days 1, 3, 5, 7, and 9 postoperatively. As indicators of renal hemodynamics, the estimated hemoglobin concentration of renal tissue (IHb) and the oxygen saturation of hemoglobin in renal blood (ISO2) were determined by reflectance spectrophotometry. In addition, the clearance of inulin and P-aminohippurate were determined, and the renal tissue blood flow was estimated by laser-Doppler flowmetry (LDF). As a model of allograft rejection, Lewis rats were transplanted orthotopically with DA rat livers. The serum endothelin level of allografted rejectors was significantly (P < 0.05) higher than that of isografted controls (Lewis rats with Lewis livers) on postoperative day 5, and it increased to a maximum of 5.38 +/- 0.95 pg/ml on day 9 (versus 1.23 +/- 0.18 pg/ml preoperatively). The values of Ccr, IHb, and ISO2 were all significantly (P < 0.05) lower in allografted rejectors than in isografted controls on day 5, and subsequently declined to a minimum on day 9 (P < 0.01). Treatment of allografted rejectors with BQ-123 markedly improved the renal parameters to levels similar to those in the isografted controls. These results strongly suggest that endogenous endothelin may play an important role in the development of renal impairment during acute liver rejection by reducing renal blood flow through binding with ETA receptor.

Hepatic release of endothelin-1 after warm ischemia. Reperfusion injury and its hemodynamic effect.

This study investigated the release of endothelin (ET)-1 from the liver after warm ischemia/reperfusion (I/R) injury. Wistar rats were subjected to 120 min of warm hepatic ischemia by clamping the hepatic hilum under porto-jugular shunting. Reperfusion was performed by unclamping. The rats were divided into 2 groups receiving intravenous treatment with an anti-ET-1 mAb before ischemia (AET group) and with mouse immunoglobulin G (sham group). Hepatic blood flow was assessed by laser-Doppler flowmetry and reflectance spectrophotometry and was compared between the 2 groups along with the bile flow rate. The ET-1 concentrations of hepatic venous and portal blood were determined in the sham group, and the portal blood endotoxin levels were assayed in both groups. Both groups developed transient hypotension after reperfusion, but hepatic blood flow subsequently showed a significant improvement in the AET group. Hepatic congestion was detected in the sham group by both reflectance spectrophotometry and histological examination. After reperfusion, bile flow was significantly greater in the AET group. The portal endotoxin concentration showed no increase in both groups, and the hepatic venous blood ET-1 level in the sham group was significantly higher until 3 hr after reperfusion compared to the portal blood level. The 30-day survival rate was 50% in the AET group, whereas all the sham rats died within 12 hr. ET-1 was released from the liver after I/R injury and apparently participated in systemic and local hemodynamic changes that affected survival.

The roles of platelet-activating factor and endothelin-1 in renal damage after total hepatic ischemia and reperfusion.

BACKGROUND: This study was designed to verify the involvement of platelet-activating factor (PAF) in renal damage associated with hepatic ischemia and reperfusion (HIR) injury through the release of endothelin (ET)-1 and to determine the modulating effect of a specific PAF receptor antagonist on these insults in rats. METHODS: Male rats pretreated with either normal saline as a vehicle (NS group) or intravenous TCV-309, a PAF receptor antagonist (TCV group), were subjected to 120 min of total hepatic ischemia under an extracorporeal portosystemic shunt. Plasma aspartate transaminase, creatinine, blood urea nitrogen, and ET-1 levels and the relative renal wet weight were determined under nonischemic conditions and at 1, 3, and 6 hr of reperfusion after hepatic ischemia. Changes in mean arterial blood pressure and renal tissue blood flow measurements in the kidney were determined throughout the experiment. RESULTS: Increased plasma aspartate transaminase, creatinine, blood urea nitrogen, and ET-1 levels and the relative renal wet weight after HIR in the NS group were significantly suppressed by TCV-309 pretreatment. Mean arterial blood pressure and renal tissue blood flow after HIR in the TCV group were significantly improved when compared with those in the NS group. These effects resulted in attenuation of structural hepatic and renal damage with the improvement of 7-day survival (62%). CONCLUSIONS: The present study demonstrates that renal damage as well as critical liver injury is produced after reperfusion following 120 min of total hepatic ischemia. A PAF receptor antagonist may be therapeutically useful to protect against these types of damage via indirect modulation of plasma ET-1 levels.

A new approach to three-dimensional reconstructed imaging of hormone-secreting cells and their microvessel environments in rat pituitary glands by confocal laser scanning microscopy.

There has been considerable interest in the relationship between hormone- secreting endocrine cells and their microvessels in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). This study was designed to visualize and to reveal the relationship between hormone-secreting endocrine cells and their microvessel environment in 3D, using rat pituitary glands under various (hyper/hypo) experimental conditions by confocal laser scanning microscopy (CLSM). Female adult Wistar rats were used after bilateral adrenalectomy or ACTH administration for 2 weeks. Clear 3D reconstructed images of ACTH cells, the microvessel network and counterstained nuclei were obtained at a maximal focus depth of 1 mm by CLSM without any background noise. In the hyperfunctional state, slender cytoplasmic processes of hypertrophic stellate ACTH cells frequently extended to the microvessels. In the hypofunctional state, ACTH cells appeared atrophic and round with scanty cytoplasm, and cytoplasmic adhesions to microvessel network patterns were inconspicuous. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways.

Hepatopancreatoduodenectomy for advanced gallbladder carcinoma.

OBJECTIVE: To assess the effectiveness of hepatopancreatoduodenectomy (HPD) in patients with advanced gallbladder carcinoma directly invading the liver and pancreas, generally considered to be nonresectable. PATIENTS AND METHODS: Sixty patients with gallbladder carcinoma admitted to our hospital from 1978 to 1992, of whom 55 had Nevin stage V carcinoma and 21 had resectable tumors. Of these patients, seven underwent HPD. The remaining 34 patients had nonresectable tumors. The outcomes of patients undergoing HPD and those with nonresectable tumors were compared and the effect on their quality of life was also analyzed. RESULTS: Postoperative complications occurred in five of the seven patients after HPD, but there were no operative deaths. The 1- and 2-year survival rates were 57% and 28.6%, respectively, with a median survival time of 12 months. In contrast, the 1- and 2-year survival rates of the 34 patients with nonresectable tumors were both 5.8%, and the median survival time was 2 months. The median and mean durations of home stay after HPD were 6 and 10.5 months, respectively. CONCLUSION: Hepatopancreatoduodenectomy has the potential to improve both survival and the quality of life for carefully selected patients with advanced gallbladder carcinoma.

Three-dimensional imaging of hormone-secreting cells and their microvessel environment in estrogen-induced prolactinoma of the rat pituitary gland by confocal laser scanning microscopy.

This study focused on the three-dimensional imaging of hormone-secreting cells and their microvascular environment in estrogen-induced prolactinoma of the rat pituitary gland. Adult female Wistar-Imamichi rats were injected with estradiol dipropionate and killed 7 weeks later. Some rats given estrogen for 7 weeks also were injected with bromocriptine before killing. To obtain a detailed three-dimensional image of microvessels, dialyzed fluorescein isothiocyanate (FITC)-conjugated gelatin was injected into the left ventricle of the rat heart. After the perfusion, the pituitary glands were resected and subjected to immunohistochemistry (IHC). To evaluate the effects of estrogen and bromocriptine, IHC was performed with antibodies against prolactin (PRL), adrenocorticotropic hormone (ACTH), and growth hormone (GH). With the combination, microvessels and cells containing PRL, ACTH, and GH could be clearly identified by confocal laser scanning microscopy (CLSM). The PRL cells increased in number and became hypertrophic after prolonged exposure to estrogen. With bromocriptine administration after estrogen treatment, however, PRL cells decreased in number and became atrophic. The current study revealed that estrogen and bromocriptine had significant effects on PRL secretion and the microvascular environment. Therefore, this technique (FITC injection and IHC) with CLSM is suitable for the three-dimensional imaging of hormone-secreting mechanisms under various conditions.

Clinical outcome in localized renal cell carcinomas related to immunoexpression of proliferating cell nuclear antigen, Ki-67 antigen, and tumor size.

This study was designed to clarify the relationship between clinical outcome and immunoexpression of proliferating cell nuclear antigen (PCNA) and Ki-67 antigen (Ki-67) in 71 localized renal cell carcinomas (RCCs) of Robson stage I and II, related to the disease recurrence and tumor size. PCNA and Ki-67 expressions showed significant differences between non-recurring and recurring groups and more variability in stage II than in stage I. Recurrence rates according to tumor size were 0% for /=5.0 cm. It was concluded that PCNA and Ki-67 expression profiles were considered to be closely related to tumor stage and showed some promise for predicting the disease recurrence.

Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies.

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY) family of bacterial toxins. Four monoclonal antibodies (mAbs) to PLO were prepared for the analysis of functional domains of this toxin. Two (mAbs S and H) of these markedly inhibited the hemolytic activity of PLO, but the inhibiting activity of the other two antibodies (mAbs C and G) was weaker. Subsequently, nine truncated PLOs were derived from recombinant Escherichia coli by various deletions from the N-terminus. Strong hemolytic activity was recognized in truncates of PLO following the deletion of 30 or 55 amino acids, but not in the truncate with deletion of 74 residues. Truncated PLOs were used in immunoblotting experiments to locate the epitopes for the mAbs. The epitope for mAbs C and G lies within the undecapeptide region (amino acids 487-505) of the C-terminus of PLO, which seems to be the binding site to erythrocytes. In contrast, the epitopes for mAbs S and H, which showed strong neutralizing activity, were found to lie in the N-terminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. From these results, it seems that the N-terminal region of PLO, in particular, the region of amino acids 55-74 is important for hemolytic activity.

Hepatopancreatoduodenectomy for superficially spreading bile duct carcinoma: a report of two 5 year survivals.

We have treated two bile duct carcinoma patients using HPD and they have both survived for more than 5 years postoperatively. In this article, we report on the details of these two cases and discuss the indications for HPD in the treatment of bile duct carcinoma.

Cultural perceptions and health behaviors related to safe motherhood among village women in Eastern Sudan: Ethnographic study.

BACKGROUND: Meeting the health needs of Sudanese women, especially those living in village areas, is imperative and cannot be accomplished without understanding the cultural perceptions and health behaviors related to safe motherhood. Nevertheless, there is little literature exploring these perspectives through qualitative study, as most of the studies performed in Sudan applied quantitative methods and focused on urban areas. OBJECTIVE: This study aims to explore cultural perceptions and behaviors relevant to safe motherhood among Sudanese village women. DESIGN/METHOD: A qualitative method using an ethnographic approach was applied for the study. Semi-structured in-depth interviews were conducted with six village women of reproductive age living in a village in Gadarif State, Eastern Sudan. FINDINGS: The thematic content analysis revealed socio-economic factors, religious values and local beliefs shaping the village women's perceptions of their behaviors related to motherhood safety. Particular concerns included responses to health problems, preference for birth with traditional birth attendants, female genital mutilation/female genital cutting and a lack of utilizing family planning. CONCLUSIONS: An implication arising from this study is that maternal services should develop a collaboration between village midwives and traditional birth attendants. This study further suggests that educational messages must be delivered to family relatives with consideration of the cultural influences highlighted by the village women.

Enhanced polymer one-step staining (EPOS) for proliferating cell nuclear antigen (PCNA) and Ki-67 antigen: application to intra-operative frozen diagnosis.

Enhanced polymer one-step staining (EPOS) is a novel, highly sensitive one-step immunostaining method. This simple and rapid technique was applied to intra-operative frozen diagnosis. The markers of choice were proliferating cell nuclear antigen (PCNA) and Ki-67 antigen. These cell proliferation markers were both identifiable in fresh frozen sections of the human tonsil in approximately 7 min. The suitable staining sequences are as follows. Frozen sections prepared using 3-aminopropyltrimethoxysilane-coated glass slides are immediately fixed, without air drying, for 15 s in a mixture of 50% formalin and 50% methanol for PCNA, and in 10% formalin for Ki-67 antigen. After a brief rinse in phosphate-buffered saline (PBS), sections are incubated with the EPOS antibody for 3 min, followed by PBS rinse for 1 min. The peroxidase activity is visualized in diaminobenzidine-H2O2 solution containing 10 mmol/L imidazole for 2 min. After a light rinse in tap water, the nuclei are briefly counterstained with 5% methyl green. When necessary, endogenous peroxidase blockage in 1% periodic acid solution for 1 min is added before the EPOS antibody incubation. This procedure is applicable to frozen sections of gastric cancers, malignant lymphomas, and brain, liver and peritoneal lesions in which differential diagnosis between benignancy and malignancy was required.

Localized amyloidosis of the seminal vesicle: identification of lactoferrin immunoreactivity in the amyloid material.

Three specimens of localized amyloidosis of the seminal vesicle surgically removed for prostatic cancer were immunohistochemically analyzed to clarify the nature of the permanganate-sensitive congophilic subepithelial deposition. A variety of known amyloidogenic substances and secretory products in the seminal fluid were screened using the indirect immunoperoxidase method. In addition to reactivities with antibodies to amyloid P component and human seminal plasma, the amyloid material was immunoreactive for lactoferrin using a rabbit antiserum and two of three mouse monoclonal antibodies. All the antibodies labeled some of the normal seminal vesicle epithelial cells for this ironbinding, bacteriostatic glycoprotein. In the prostate without accompanying amyloid deposition, a considerable proportion of the glandular epithelium and secretory material were positive for lactoferrin. Pre-embedding immunoelectron microscopy showed lactoferrin immunoreactivity on the amyloid fibrils. Focal staining of the amyloid for gross cystic disease fluid protein-15 was also observed in two lesions. These findings strongly suggest that lactoferrin is the major constituent in localized senile amyloidosis of the seminal vesicle.

Involvement of platelet-activating factor in cytokine production and neutrophil activation after hepatic ischemia-reperfusion.

Although platelet-activating factor (PAF) is implicated as an important mediator in the pathogenesis of hepatic ischemia-reperfusion (IR) injury, the precise mechanism of its action has not been studied. We examined the hypothesis that PAF may influence neutrophils by promoting the production of tumor necrosis factor alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant (CINC), a member of the interleukin-8 (IL-8) family, and may be associated with liver and lung injury during the early phase of reperfusion after total hepatic ischemia. Rats pretreated with a specific PAF receptor antagonist exhibited suppression of the increase in plasma TNF-alpha and CINC levels, as well as the priming of peripheral neutrophils for superoxide production after reperfusion when compared with animals pretreated with physiological saline. These effects resulted in a reduction of plasma liver enzymes and of hepatic and pulmonary neutrophil sequestration, as well as an increased survival rate. There was a strong correlation between the time course of CINC release and hepatic or pulmonary neutrophil sequestration. We concluded that PAF activates neutrophils, either directly or by promoting the production of TNF-alpha and CINC, and is involved in hepatic IR injury.

Characterization of a mitochondrial metallopeptidase reveals neurolysin as a homologue of thimet oligopeptidase.

We have isolated a metallopeptidase from rat liver. The peptidase is primarily located in the mitochondrial intermembrane space, where it interacts non-covalently with the inner membrane. The enzyme hydrolyzes oligopeptides, the largest substrate molecule found being dynorphin A1-17; it has no action on proteins, and does not interact with alpha 2-macroglobulin, and can therefore be classified as an oligopeptidase. We term the enzyme oligopeptidase M. Oligopeptidase M acts similarly to thimet oligopeptidase (EC 3.4.24.15) on bradykinin and several other peptides, but hydrolyzes neurotensin exclusively at the -Pro+Tyr- bond (the symbol + is used to indicate a scissile peptide bond) rather than the -Arg+Arg- bond. The enzyme is inhibited by chelating agents and some thiol-blocking compounds, but differs from thimet oligopeptidase in not being activated by thiol compounds. The peptidase is inhibited by Pro-Ile, unlike thimet oligopeptidase, and the two enzymes are separable in chromatography on hydroxyapatite. The N-terminal amino acid sequence of rat mitochondrial oligopeptidase M contains 19 out of 20 residues identical with a segment of rabbit microsomal endopeptidase and 17 matching the corresponding segment of pig-soluble angiotensin II-binding protein. Moreover, the rat protein is recognized by a monoclonal antibody against rabbit soluble angiotensin II-binding protein, all of which is consistent with these proteins being species variants of a single protein that is a homologue of thimet oligopeptidase. The biochemical properties of the mitochondrial oligopeptidase leave us in no doubt that it is neurolysin (EC 3.4.24.16), for which no sequence has previously been reported, and which has not been thought to be mitochondrial.

Heat-induced antigen retrieval of proliferating cell nuclear antigen and p53 protein in formalin-fixed, paraffin-embedded sections.

Immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA) and p53 protein is important, particularly for the surgical diagnosis of neoplastic disorders. An effective, simple and reproducible method was established for observing the expression of these intranuclear antigens in routinely processed, formalin-fixed paraffin-embedded sections. Dramatic improvement of the antigenicity was obtained when the deparaffinized sections were heated in a hot water bath at 90 degrees C for 120 min in 0.01 mol/L citrate buffer, pH 6.0, for PCNA and in 0.01 mol/L phosphate-buffered saline, pH 7.2, for p53 protein. These reliable pretreatments are useful for the detailed comparative analysis of the expression of PCNA and p53 protein and fine histologic architecture and for retrospective study using a large number of archival specimens.

Degenerative processes of elastic fibers in sun-protected and sun-exposed skin: immunoelectron microscopic observation of elastin, fibrillin-1, amyloid P component, lysozyme and alpha1-antitrypsin.

Degenerative processes of elastic fibers in sun-protected and sun-exposed skin were analyzed by light and electron microscopic (post-embedding) immunocytochemistry using antisera to elastin, fibrillin-1, amyloid P component, lysozyme and alpha1-antitrypsin. To assess the effect of aging and sun exposure, biopsy specimens of sun-protected skin (back) and severely and moderately sun-exposed skin (face and forearms) were obtained from a young age group (1-27 years), an adult group (31-56 years) and an old aged group (61-100 years). Elastin and fibrillin-1 were the essential components of elastic fibers; elastin being localized in the electron-lucent matrix and fibrillin-1 in the dense microfibrillar strands. Aging and sun exposure provoked degenerative condensed spots, which represented widened dense microfibrillar strands, in the matrix of altered elastic fibers in the reticular dermis. Amyloid P component was first deposited on the peripheral microfibrils, and then in the intermediate density zone of the spots. Lysozyme was observed in both the electron-dense core and in the intermediate density zone of the spots. Deposition of lysozyme correlated with basophilic degeneration of the elastic fibers. In the severely photodamaged facial skin of the aged, which showed solar elastosis in the upper reticular dermis, fibrillin-1 immunoreactivity was lost from the thickened and vacuolated elastic fibers that lacked condensed spots, and amyloid P component, lysozyme and alpha1-antitrypsin were diffusely deposited in the elastin-positive matrix. It seemed that amyloid P component deposition on the elastic fibers was closely associated with aging, while immunoreactive lysozyme was related to sun exposure. Vertically oriented, thin, elastic (oxytalan) fibers in the papillary dermis tended to decrease with age, with frequent deposition of amyloid P component but no lysozyme. In the facial skin of the aged, dermal papillae disappeared, with the formation of degenerative elastic globules beneath the dermal-epidermal junction. The present study demonstrated an intimate relationship between ultrastructural alterations and deposition of exogenous substances on the degenerative elastic fibers in sun-exposed and/or aged skin.