Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis.

A critical function of tumor suppressor p53 is the induction of apoptosis in cells exposed to noxious stresses. We report a previously unidentified pro-apoptotic gene, Noxa. Expression of Noxa induction in primary mouse cells exposed to x-ray irradiation was dependent on p53. Noxa encodes a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family of proteins; this member contains the BH3 region but not other BH domains. When ectopically expressed, Noxa underwent BH3 motif-dependent localization to mitochondria and interacted with anti-apoptotic Bcl-2 family members, resulting in the activation of caspase-9. We also demonstrate that blocking the endogenous Noxa induction results in the suppression of apoptosis. Noxa may thus represent a mediator of p53-dependent apoptosis.

A case of minimal change nephrotic syndrome with acute renal failure complicating Hashimotoâs disease.

A 63-year-old man was admitted to our hospital for evaluation of generalized edema. Coexistence of severe hypothyroidism and nephrotic syndrome was detected by laboratory examination. High titer of both antimicrosomal antibody and antithyroid peroxidase antibody indicated Hashimotoâs disease. Renal biopsy showed minimal change glomerular abnormality, but no findings of membranous nephropathy. A series of medical treatments, including steroid therapy, thyroid hormone and human albumin replacement therapy, were administered. However, acute renal failure accompanied by hypotension, was not sufficiently prevented. After 9 sessions of plasmapheresis therapy, the severe proteinuria and low serum albumin levels were improved. Even after resting hypotension was normalized, neither renal function nor thyroid function were fully recovered. After discharge, renal function gradually returned to normal, and the blood pressure developed into a hypertensive state concomitant with the normalization of thyroid function. This report is a rare case of autoimmune thyroid disease complicated with minimal change nephrotic syndrome. In most cases of nephritic syndrome, acute renal failure (ARF) has been reported to coexist with hypertension. Although pseudohypothyroidism is well-known in nephrotic pathophysiology, complications of actual hypothyroidism are uncommon. It is suggested that the development of hypotension and ARF could be enhanced not only by hypoproteinemia, but also by severe hypothyroidism.

Fanconi's syndrome and distal (type 1) renal tubular acidosis in a patient with primary Sjögren's syndrome with monoclonal gammopathy of undetermined significance.

Tubulointerstitial nephritis is a well-recognized complication in primary Sjögrens syndrome. Fanconi's syndrome is a far less frequent complication compared with distal tubular dysfunction. We here describe a 49-year-old woman with primary Sjögren's syndrome. In 1997, she was diagnosed with primary Sjögren's syndrome with tubulointerstitial nephritis, and was then treated with oral prednisolone for the tubulointerstitial nephritis. In 2002, she was referred to our hospital because of progressive fatigue. At that time, biclonal spike on serum protein (IgG-kappa and IgA-kappa) and Bence-Jones protein in urine were found. Bone marrow aspiration showed 1.0% plasma cell infiltration. Thus, a diagnosis of monoclonal gammopathy of undetermined significance (MGUS) was made. In 2004, she was again admitted to our hospital because of mild renal dysfunction and hypokalemia. Laboratory evaluation showed inappropriate, alkaline urine in hyperchloremic metabolic acidosis and a positive urine anion gap, indicating the presence of distal (Type 1) renal tubular acidosis (RTA). The urine concentration defect was also found. Further studies revealed proximal tubular dysfunction, including renal glycosuria, generalized aminoaciduria, phosphaturia, uricosuria and proximal RTA. The kidney biopsy represented diffuse and severe tubulointerstitial nephritis with dense infiltrates of lymphocytes and IgA and K light chain-positive plasma cells. No findings of multiple myeloma or malignant lymphoma were observed. In conclusion, our patient had Sjögren's syndrome with MGUS and exhibited dysfunction of both proximal tubule (Fanconi's syndrome) and distal tubule, which may be attributed to diffuse tubulointerstitial nephritis.

Changes in osteoprotegerin and markers of bone metabolism during glucocorticoid treatment in patients with chronic glomerulonephritis.

It is well known that long-term glucocorticoid treatment causes osteoporosis, but the precise mechanism remains unclear. Recently, osteoprotegerin (OPG) has been identified as a cytokine that inhibits osteoclast differentiation. We have previously demonstrated that serum OPG is suppressed by glucocorticoids. Therefore, the present study was carried out to clarify the interrelationships between OPG and other markers of bone metabolism during glucocorticoid treatment. Thirteen patients (7 men, 6 women; 44.1 +/- 5.9 years old) with chronic glomerulonephritis who were to be treated with glucocorticoids for the first time were chosen for this study. Markers of bone metabolism, including serum OPG, osteocalcin (OC), bone-specific alkaline phosphatase activity (bAP), parathyroid hormone (PTH), tartrate-resistant acid phosphatase (TRAP), and bone mineral density (BMD), were measured before and during the treatment period. Glucocorticoids significantly reduced BMD of the lumbar spine in the 6 month treatment period (p < 0.01). Serum OPG was decreased significantly by glucocorticoids within 2 weeks (p < 0.001), and serum TRAP, a marker of bone resorption, was markedly increased (p < 0.001). On the other hand, there were no remarkable changes in serum PTH. Serum OC and bAP, markers of bone formation, were transiently reduced during the treatment period (p < 0.01). Furthermore, only serum OPG was positively and independently correlated with percentage BMD of age-matched reference (%AMR). These findings imply that glucocorticoid-induced bone loss develops rapidly via enhanced bone resorption and suppressed bone formation. Moreover, the increased bone resorption caused by glucocorticoids may be, at least in part, mediated by inhibition of OPG, not increment of PTH.

[Surgery of acoustic neurinoma in a hemodialyzed patient].

Successful surgical treatment of acoustic neurinoma in a case of hemodialyzed patient is reported. A 42-year-old female patient, who had been treated by hemodialysis, was diagnosed as having a C-P angle tumor by CT scan. She was refered to our clinic on June 24, 1983. Laboratory examinations on admission showed severe anemia and renal dysfunction. Every possible treatment was done in order to improve the laboratory data preoperatively. Another big problem in this case of hemodialyzed patient was brain edema and bleeding tendency. In order to cope with brain edema, intravenous administration of glycerol and slow hemodialysis for three days were performed preoperatively. These treatments were thought to be effective to reduce bleeding tendency also. During operation, however, heavy swelling of the cerebellum forced us to resect one third of the hemisphere to remove the tumor totally. In addition to this, postoperative mild bleeding in the cavity after tumor resection, subcortical hemorrhage around the shunt tube and oozing from the wound were observed. The patient was discharged from the hospital four months after surgery without any neurological deficit. The way of recovery, however, was not uneventful, because the patient developed various kinds of postoperative complications as mentioned above. In the postoperative managements, we felt almost as if we were treading on thin ice. Neurosurgical management in hemodialyzed patients is not yet very common. We should improve the postoperative management by adding new experience with similar cases.

Spinal evoked potentials following transcranial magnetic stimulation.

Motor evoked potentials by magnetic stimulation is less invasive and causes no pain as opposed to high current electric stimulation. However, the distribution of the magnetic field generated by the round coil has not been fully studied. In this report, we mapped the extent of the magnetic induction flux density, and then the evoked potentials from the spinal cord were investigated by transcranial magnetic stimulation. We also examined the origin of the evoked potentials obtained by the magnetic stimulation. The following results were obtained. The magnetic induction flux density was at its maximum at the edge of the coil. The potentials consisted of a first negative wave and subsequent multiphasic waves. The first negative wave was similar to a response of the subcorticospinal tract in the lower brain stem, while the subsequent multiphasic waves were similar to those of the pyramidal tract. Although magnetic stimulation has certain advantages over electric stimulation, several problems remain to be solved for the monitoring of motor functions in the clinical settings.

The evaluation of facial palsy by amount of feature point movements at facial expressions.

At present, in the medical field 40-point method and facial nerve grading system are generally used for evaluation of facial palsy. However, these methods have limitation in the precise evaluation, because of subjectivity in diagnosis. Purpose of This work is to propose quantitative evaluation of facial palsy based on the amount of movements of the feature point on the face. Facial nerve symptoms generally appear in either side of the face. In facial expression movement, the motion in the palsy side becomes smaller than that of the healthy side. We defined some indices of palsy grade obtained by the observation of the facial motion. Those indices showed the asymmetry of the facial motion quantitatively. We confirmed that our proposed method is valid for estimation of facial palsy from comparison with 40 points method.

Reprimo, a new candidate mediator of the p53-mediated cell cycle arrest at the G2 phase.

A novel gene, Reprimo, in which induction in cells exposed to X-irradiation is dependent on p53 expression, has been isolated. Ectopic p53 expression results in the induction of its mRNA. Reprimo is a highly glycosylated protein and, when ectopically expressed, it is localized in the cytoplasm and induces G(2) arrest of the cell cycle. In the arrested cells, both Cdc2 activity and nuclear translocation of cyclin B1 are inhibited, suggesting the involvement of Reprimo in the Cdc2.cyclin B1 regulation pathway. Thus, Reprimo may be a new member involved in the regulation of p53-dependent G(2) arrest of the cell cycle.

Analysis of cytokine mRNAs induced by the administration of a highly branched (1-->3)-beta-D-glucan, OL-2.

OL-2, a highly branched (1-->3)-beta-D-glucan, is an antitumor glucan showing strong hematopoietic activity with weaker adjuvant activity than schizophyllan (SPG), another antitumor glucan and one which is used clinically. This paper deals with the gene expression of cytokines in mice by OL-2 and SPG in order to characterize their immunopharmacological activity. Gene expression was examined by a reverse transcriptase-polymerase chain reaction method after intraperitoneal administration of OL-2 or SPG (250 micrograms/mouse). The OL-2 administered mice strongly expressed the interleukin 1 receptor antagonist (IL-1ra) gene but SPG administered mice did not. The difference would be strongly related to the antigen-specific response between OL-2 and SPG. In the genes related to haematopoiesis, OL-2 induced G-CSF and GM-CSF, but SPG induced IL-3. These differences would relate to the pattern of haematopoietic response. Comparing the cytokine gene expression in ICR and AKR mice by OL-2 administration, the changes in cytokine gene expression were less in AKR mice administered OL-2. These findings suggest that the immunopharmacological characteristics of OL-2 are closely related, at least in part, to the activation of the complement system. The data shown in this paper also suggest that cytokine gene expression by beta-glucan would be significantly affected by the structure of these glucans.

Expression of interleukin 1 family mRNAs by a highly branched (1-->3)-beta-D-glucan, OL-2.

OL-2, a highly branched (1-->3)-beta-D-glucan, is an antitumor glucan showing strong hematopoietic activity with weaker adjuvant activity than schizophyllan (SPG), also an antitumor glucan and one which is clinically used. This paper deals with the gene expression of the interleukin 1 (IL-1) family in mice by OL-2 and SPG in order to characterize the immunopharmacological activity. Gene expression was examined by reverse transcriptase-polymerase chain reaction method. Intraperitoneal administration of OL-2 (250 micrograms/mouse) expressed all three genes of IL-1 alpha, beta, and IL-1 receptor antagonist (IL-1ra) in the peritoneal exudate cells, while SPG induced a strength of IL-1 alpha mRNA comparable to that by OL-2 but a weaker level of IL-1 beta mRNA. SPG did not induce IL-1ra. Similar patterns were seen in spleen and liver by OL-2 or SPG administration. These findings suggest that the immunopharmacological characteristics of (1-->3)-beta-D-glucan are regulated under the gene expression of the IL-1 family.

Hyperosmolality stimulates Na-K-ATPase gene expression in inner medullary collecting duct cells.

Primary cultures of inner medullary collecting duct (IMCD) cells of rats were incubated in hyperosmotic media to determine the effects on Na-K-ATPase alpha 1- and beta 1-subunit mRNA expression. Osmolality of the incubation media was raised from 300 up to 500 mosmol/kgH2O by adding NaCl, mannitol, raffinose, or urea. Hyperosmotic media supplemented with NaCl, mannitol, or raffinose caused two- to fourfold increases in the alpha 1-subunit mRNA accumulation and five- to eightfold increases in the beta 1-subunit mRNA accumulation, with peak elevations of both subunits at 12 h after addition. In sharp contrast, hyperosmolar urea medium had no effect at any time. When NaCl or mannitol was added to the media in amounts ranging from 300 to 600 mosmol/kgH2O, the maximal effects on both alpha 1- and beta 1-subunit mRNA accumulation occurred at 500 mosmol/kgH2O. In urea-supplemented medium, however, there was no significant change at any level of osmolality. The upregulation of alpha 1- and beta 1-subunit mRNA induced by hyperosmotic mannitol- or raffinose-supplemented media was markedly inhibited by removal of Na from the culture medium. Furthermore, pretreatment with a protein synthesis inhibitor cycloheximide partially inhibited the upregulation of alpha 1- and beta 1-subunit mRNA in IMCD cells exposed to hyperosmotic media treated with NaCl or mannitol. When IMCD cells were incubated with hyperosmotic media (500 mosmol/kgH2O) supplemented with NaCl or mannitol for 24 h, Na-K-ATPase activity increased by 78.6 and 82.8%, respectively. In contrast, hyperosmolar urea medium had no significant effect on Na-K-ATPase activity. These results demonstrate that 1) hyperosmolality induced by the poorly permeating solutes (NaCl, mannitol, and raffinose) but not the rapidly permeating solute (urea) stimulates both alpha 1- and beta 1-subunit mRNA accumulations in IMCD cells in a time- and an osmolality-dependent manner, 2) the hyperosmolality-induced upregulation of alpha 1- and beta 1-subunit mRNA leads to an increase in Na- K -ATPase activity; and 3) the above upregulation of alpha1- and beta 1-subunit mRNA in response to hyperosmotic media requires, at least in part, the presence of Na in the extracellular medium and the de novo synthesis of intermediate proteins.

Intracellular Na+ directly modulates Na+,K+-ATPase gene expression in normal rat kidney epithelial cells.

BACKGROUND: In a wide variety of cell systems, increases in cell Na+ ([Na+]i) lead to an induction of N+,K+-ATPase mRNA expression. On the other hand, the increase in [Na+]i can also induce a rise in cell Ca2+ ([Ca2+]i) through a secondary inhibition of Na+/Ca2+ exchange and a decrease in cell pH (pHi) through a secondary inhibition of Na+/H+ exchange. It is not known whether [Na+]i, [Ca2+]i, and/or pHi directly modulate N+,K+-ATPase mRNA expression. METHODS: We used normal rat kidney epithelial cells (NRK) to examine the effects of ouabain on N+,K+-ATPase alpha1- and beta1-mRNA accumulation by Northern blot analysis and the relationship between the mRNA accumulation and [Na+]i, [Ca2+]i, or pHi. [Na+]i, [Ca2+]i, and pHi were measured using a Na+-sensitive fluorescent dye (SBFI), a Ca2+-sensitive fluorescent dye (Fura-2), and a pH-sensitive fluorescent dye (BCECF), respectively. RESULTS: Ouabain (1 mmol/L) significantly increased [Na+]i. Upon addition of ouabain, alpha1-mRNA levels increased to 2. 3 times the control level at three hours, with maximum 3.3-fold elevations at 12 hours. beta1-mRNA levels also increased to 2.4 times the control level at 3 hours, with a maximum 3.3-fold increase at 12 hours. The ouabain-mediated alpha1- and beta1-mRNA induction was inhibited by both the RNA transcription inhibitor (actinomycin D) and the protein synthesis inhibitor (cycloheximide). Ouabain at three hours caused an increase in [Ca2+]i. Similar increases in [Ca2+]i, which were elicited by the Ca2+ ionophore (ionomycin) in the presence of extracellular Ca2+, had no effect on alpha1- or beta1-mRNA levels. In Ca2+-free medium treated with EGTA, ouabain at three hours caused a significant increase in [Na+]i without any changes in [Ca2+]i, and also increased alpha1- and beta1-mRNA levels. Ouabain at three hours caused a significant decrease in pHi. Similar decreases in pHi, which were elicited by the specific inhibitor of Na+/H+ exchange (ethylisopropylamiloride), caused no effect on alpha1- or beta1-mRNA levels. Exposure of NRK to the Na+ ionophore (monensin) in the absence of extracellular Ca2+ increased [Na+]i and alpha1- and beta1-mRNA levels. The increases in alpha1- and beta1-mRNA levels upon addition of ouabain were associated with significant increases in alpha1- and beta1-subunit proteins. CONCLUSIONS: In NRK, ouabain causes an increase in [Na+]i, which directly modulates Na+,K+-ATPase alpha1- and beta1-mRNA accumulation.

Serum transcriptionally regulates Na(+)-K(+)-ATPase gene expression in vascular smooth muscle cells.

The present study was designed to examine the effects of serum on Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit gene expression in cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas. Addition of 10% serum to VSMC for 24 h increased Na(+)-K(+)-ATPase activity 1.5-fold and alpha 1- and beta 1-subunit protein levels 1.9-fold. Serum (10%) caused a 3.5-fold increase in alpha 1-mRNA levels and a 6.7-fold increase in beta 1-mRNA levels, with peak elevations at 12 h. The protein synthesis inhibitor cycloheximide abolished serum-mediated beta 1-mRNA induction but did not affect serum-mediated alpha 1-mRNA induction. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) significantly reduced serum-mediated beta 1-mRNA induction but had no effect on serum-mediated alpha 1-mRNA induction. Transfection experiments with the 5'-flanking sequences of the alpha 1- or beta 1-subunit genes linked to the luciferase reporter gene revealed that 10% serum caused 2.8- and 6.5-fold increases in luciferase activity, respectively. Among growth factors, only basic fibroblast growth factor (FGF) enhanced luciferase activities for the alpha 1- and beta 1-subunit genes. We conclude that 1) serum stimulates alpha 1- and beta 1-mRNA expression, alpha 1- and beta 1-subunit protein accumulation, and Na(+)-K(+)-ATPase activity; 2) serum-mediated beta 1-mRNA induction partly requires de novo synthesis of intermediate regulatory proteins and activation of PKC and TK, whereas serum-mediated alpha 1-mRNA induction occurs through PKC- and TK-independent mechanisms; 3) the 5'-flanking regions of the alpha 1- and beta 1-subunit genes are serum responsive; and 4) FGF mimics stimulatory effects of serum on promoter activities for the alpha 1- and beta 1-subunit genes.

Aldosterone activates Na+/H+ exchange in vascular smooth muscle cells by nongenomic and genomic mechanisms.

BACKGROUND: In vascular smooth muscle cells (VSMCs), Na+/H+ exchange (NHE) plays an important role in intracellular pH (pHi) regulation. Recently, nongenomic effect of aldosterone (ALDO) on NHE activity has been suggested in VSMCs. However, the nongenomic and genomic effects of ALDO on NHE and the intracellular signaling mechanisms for these effects have not fully been determined in VSMCs. METHODS: The effects of short- (3 hr) and long- (24 hr) term exposure to ALDO on NHE activity were examined in cultured VSMCs from rat thoracic aortae by using single-cell pHi measurement with the pH-sensitive dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The NHE activity was calculated from the initial rate of Na+-dependent pHi recovery after acid load. RESULTS: The NHE activity significantly increased after short- and long-term exposure of VSMCs to ALDO (10(-6) M). The inhibitors of gene transcription (actinomycin D) and of protein synthesis (cycloheximide) had no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. The antagonists of the mineralocorticoid receptor (MR) (spironolactone) and of the glucocorticoid receptor (GR) (RU38486) caused no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. Two protein kinase C (PKC) inhibitors (staurosporine A and calphostin C) and PKC down-regulation (24 hr pre-exposure to phobol 12-myristate 13-acetate, PMA) inhibited both the short- and long-term ALDO effects. Exposure of VSMCs to PMA for 3 hours mimicked the short-term effect of ALDO on NHE activity. ALDO significantly increased PKC activity in VSMCs. The short-term ALDO effect was inhibited by disruptors of microtubule (colchicine) and of filamentous-actin (cytochalasin B). Long-term exposure of ALDO caused a threefold increase in NHE (NHE-1) mRNA levels. CONCLUSIONS: The short-term effect of ALDO on NHE activity is not mediated through either MR or GR, occurs independent of gene transcription and protein synthesis, and occurs through a mechanism involving the structural elements of cytoskeleton. The long-term effect of ALDO on NHE activity occurs through both MR and GR and requires gene transcription and protein synthesis. Both short- and long-term effects of ALDO are mediated through PKC activation. Therefore, ALDO activates NHE by nongenomic and genomic mechanisms in VSMCs.

Corticosterone and 11-dehydrocorticosterone stimulate Na,K-ATPase gene expression in vascular smooth muscle cells.

BACKGROUND: In mineralocorticoid target tissues such as kidney and colon, the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta OHSD) catalizes the reversible conversion of corticosterone (CS) to inactive 11-dehydrocorticosterone (DHCS) in rats, and cortisol to inactive cortisone in humans. This enzyme is also expressed in vascular smooth muscle cells (VSMC). METHODS: In cultured VSMC from rat thoracic aortae, we examined the effects of CS and DHCS on Na,K-ATPase alpha 1- and beta 1-mRNA accumulation by Northern blot analysis, on alpha 1- and beta 1-subunit protein accumulation by Western blot analysis, and on Na,K-ATPase activity by the coupled assay method. RESULTS: In VSMC, CS and DHCS (10(-6) M) increased alpha 1-mRNA level 2.6- and 2.5-fold at 48 hours and beta 1-mRNA level 9.2- and 9.1-fold at 12 hours, respectively. The RNA transcription inhibitor (actinomycin D) abolished both CS- and DHCS-mediated alpha 1- and beta 1-mRNA induction. The glucocorticoid receptor antagonist (RU38486) and the mineralocorticoid receptor antagonists (ZK91587) inhibited both CS- and DHCS-mediated alpha 1- and beta 1-mRNA induction. The 11 beta OHSD inhibitor (carbenoxolone) inhibited DHCS-mediated alpha 1- and beta 1-mRNA induction, whereas it caused no effect on CS-mediated alpha 1- or beta 1-mRNA induction. The addition of CS or DHCS to VSMC significantly increased alpha 1- and beta 1-subunit protein levels and Na,K-ATPase activity. When adrenalectomized rats were treated with CS or DHCS for 12 hours, aorta alpha 1- and beta 1-mRNA levels increased 3.0- and 8.7-fold or 3.4- and 8.4-fold, respectively. CONCLUSIONS: In VSMC, both CS and DHCS stimulate Na,K-ATPase alpha 1- and beta 1-mRNA accumulation, alpha 1- and beta 1-subunit protein accumulation, and Na,K-ATPase activity. The CS-mediated alpha 1- and beta 1-mRNA induction occurs independently of 11 beta OHSD, whereas the DHCS-mediated alpha 1- and beta 1-mRNA induction occurs through 11 beta OHSD-dependent mechanisms, possibly via conversion of inactive DHCS into active CS.

Effects of hyperosmolality on Na, K-ATPase gene expression in vascular smooth muscle cells.

Cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas were exposed to hyperosmotic media to determine the effects on Na, K-ATPase alpha1- and beta1-mRNA expression. Hyperosmotic media (500 mOsm/kgH2O) supplemented with glucose or mannitol increased alpha1-mRNA levels threefold at 24 hr and beta1-mRNA levels sevenfold at 12 hr. In sharp contrast, hyperosmotic urea medium had no effect at any time. Both the protein synthesis inhibitor cycloheximide and the RNA transcription inhibitor actinomycin D reduced alpha1- and beta1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) had no effect on the alpha1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Hyperosmotic glucose or mannitol media (500 mOsm/kgH2O) significantly increased alpha1- and beta1-subunit protein levels and Na, K-ATPase activity, whereas hyperosmotic urea medium had no effect. Transfection experiments with the 5'-flanking sequences of the alpha1- or beta1-subunit genes linked to the luciferase reporter gene revealed that hyperosmolar glucose medium increased luciferase activity 2.9- and 3.7-fold, respectively. Similarly, hyperosmotic mannitol medium increased such activity 2.7- and 3.4-fold, respectively. These results demonstrate that: (i) hyperosmolality induced by the poorly permeating solutes (glucose and mannitol) stimulates alpha1- and beta1-mRNA accumulation, alpha1- and beta1-subunit protein accumulation, and Na, K-ATPase activity, whereas the rapidly permeating solute (urea) has no effect; (ii) the upregulation of alpha1- and beta1-mRNA in response to hyperosmotic glucose or mannitol media requires, at least in part, de novo synthesis of intermediate regulatory proteins; (iii) the hyperosmolality-induced alpha1-mRNA upregulation occurs through PKC- and TK-independent mechanisms, whereas the hyperosmolality-induced beta1-mRNA upregulation occurs through activation of PKC and TK; and (iv) hyperosmolality induced by glucose or mannitol increases promoter activities of the alpha1- and beta1-subunit genes.

Immunopharmacological characterization of a highly branched fungal (1-->3)-beta-D-glucan, OL-2, isolated from Omphalia lapidescens.

The immunopharmacological activities of a fungal (1-->3)-beta-D-glucan, OL-2, isolated from "Leiwan" Omphalia lapidescens were examined. Intraperitoneal (i.p.) administration of OL-2 to ICR mice induced a significant number of peritoneal exudate cells (PEC) and white blood cells over the period of a few days. Spleen cell numbers were also increased by i.p. administration of OL-2 at about a week. These changes reverted to the normal level within a month. Responses of spleen cells and bone marrow cells (BM) to colony stimulating factors (CSF) were augmented by OL-2 administration assessed by cell proliferation assay. Sera from OL-2 administered mice contained an increased concentration of colony stimulating activity. Gene expressions of interleukin-1 beta, interleukin-6, and tumor necrosis factor alpha in the spleen were also increased. These results suggested the activation of hematopoietic responses, and would well relate to the incremental increase in PEC, white blood cell and spleen cell numbers. OL-2 also increased the serum concentration of fibronectin and complement component C-3. However, OL-2 did not show adjuvant activity to SRBC and antitumor activity against the solid form of Sarcoma 180 by i.p. administration. Yet, OL-2 did not interfere with the antitumor activity of SSG against the same tumor system. These facts suggested that OL-2 could enhance nonspecific host defense mechanisms by enhancing hematopoietic responses, but would not enhance or inhibit the specific immunity mediated by lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of Na+-K+-ATPase gene expression by corticosteriods in vascular smooth muscle cells.

To determine whether gluco- and mineralocorticoids have specific actions on Na+-K+-ATPase gene expression in vascular tissue, we used Northern blot analysis to compare the effects of dexamethasone (Dex) and aldosterone (Aldo) on Na+-K+-ATPase alpha1 and beta1-subunit mRNA expression in cultured vascular smooth muscle cells from rat aortae. Dex at 10(-6)M increased alpha1 -mRNA level 2.5-fold at 24 h and beta1-mRNA level 9.9-fold at 12 h. Aldo at 10(-6)M increased alpha1-mRNA 2.7-fold at 48 h and beta1-mRNA level 10.9-fold at 6 h. The half-maximal stimulation of both alpha1 and beta1-mRNA levels occurred at a concentration of 5-7 X 10(-9)M Dex, whereas it occurred at a concentration of 2-3 X 10(-9)M Aldo. The glucocorticoid receptor antagonist RU-38486 inhibited both Dex- and Aldo-mediated induction of beta1-mRNA. The mineralocorticoid receptor antagonist spironolactone inhibited Aldo-mediated induction of beta1-mRNA, whereas it had no effect on Dex-mediated induction of beta1-mRNA. Removal of Na+ from the extracellular medium (isosmotic replacement with choline) caused no effect on Dex-mediated induction beta1-mRNA, whereas it inhibited Aldo-mediated induction of beta1-mRNA. Addition of a specific inhibitor of the Na+/H+ exchange, ethylisopropylamiloride, had no effect on Dex-mediated induction of beta1-mRNA, whereas it resulted in a significant inhibition of Aldo-mediated induction of beta1-mRNA. We conclude that 1) both Dex and Aldo induce Na+-K+-ATPase alpha1- and beta1-mRNA expression in a time- and dose-dependent manner; 2) Dex-mediated induction of beta1-mRNA occurs only through glucocorticoid receptors, whereas Aldo-mediated induction of beta1-mRNA occurs through both gluco- and mineralocorticoid receptors; and 3) Dex-mediated induction of beta1-mRNA occurs through Na+-independent mechanisms, whereas Aldo-mediated induction of beta1-mRNA, at least in part, occurs through Na+-dependent mechanisms, including stimulation of the Na+/H+ exchange.