A novel steroid inhibitor for a rat 20alpha-hydroxysteroid dehydrogenase isozyme

20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD, E.C.1.1.1.149) in rat luteal tissue, which catalyzes conversion of progesterone to a biologically inactive steroid, 20alpha-hydroxypregn-4-ene-3-one (20alpha-OHP), suppresses progesterone secretion into the circulation. An increase in 20alpha-HSD activity in luteal tissue in rats is a prerequisite for functional corpus luteum regression. This study was undertaken to find a steroid inhibitor for ovarian cytosolic 20alpha-HSD activity among derivatives based on progesterone structure. A derivative designated as STZ26 (D-homo-16-oxa-4-androstene-3,16alpha-dione) was found to inhibit potently 20alpha-HSD activity in cultured luteal cells. Ovarian 20alpha-HSD activity consists of two isoforms (HSD1 and HSD2). Kinetic analyses of STZ26 for HSD1 and HSD2 showed that the compound suppressed only HSD1 activity by competitive inhibition. Pseudopregnant rats were treated with STZ26 from 13 to 19 days after cervical stimulation. Either an elevation of peripheral 20alpha-OHP levels or a concomitant depletion of peripheral progesterone levels at the end of pseudopregnancy was considerably inhibited in treated animals, although not completely. The results showed that STZ26 is a biologically active inhibitor for HSD1 activity in the luteal tissue and suggested that the depletion of progesterone levels toward the end of pseudopregnancy is not solely due to the elevation of HSD1 activity.     

Structure and expression of a murine homologue of sky receptor tyrosine kinase gene

We examined effects of adenine nucleotide on ischemic myocardial stunning in dogs. Pentobarbitalanesthetized open-chest dogs were subjected to 20-min ligation of the left anterior descending coronary artery (LAD), followed by reperfusion for 30 min. Either saline, 5 mM 8-bromo-5'-AMP (tributyryl-AMP), or 30 mM N6, 2', 3'-tributyryl-5'-AMP (tributyryl-AMP), 5 mM 5-amino-4-imidazole carboxamide riboside (AICAr) as a positive reference, was infused at 0.1 ml/kg/min in the left femoral vein throughout the experiment. The myocardial contractile function was measured by ultrasonometry. The tissue levels of high-energy phosphates in the reperfused heart were determined. Myocardial contractile function assessed by % segment shortening (%SS) in the saline-infused group decreased during ischemia and returned toward the preischemic level during reperfusion but incompletely. A significant improvement in the %SS during reperfusion was observed in the 8-bromo-AMP- and AICAr-infused groups but not in the tributyryl-AMP-infused group. The magnitude of the protective effect of the drugs on myocardial contractility during reperfusion was 8-bromo-AMP > AICAr > tributyryl-AMP = saline. Only in the 8-bromo-AMP-infused group were the levels of ATP, ADP, and total adenine nucleotides in the reperfused heart significantly higher than those in the saline-infused group. The present result indicates that 8-bromo-AMP improves the ability of the heart to recover from ischemia and reperfusion associated with a significant restoration of ATP.     

Phosphotyrosine phosphatase and tyrosine kinase inhibition modulate airway pressure-induced lung injury

We determined whether drugs which modulate the state of protein tyrosine phosphorylation could alter the threshold for high airway pressure-induced microvascular injury in isolated perfused rat lungs. Lungs were ventilated for successive 30-min periods with peak inflation pressures (PIP) of 7, 20, 30, and 35 cmH2O followed by measurement of the capillary filtration coefficient (Kfc), a sensitive index of hydraulic conductance. In untreated control lungs, Kfc increased by 1.3- and 3.3-fold relative to baseline (7 cmH2O PIP) after ventilation with 30 and 35 cmH2O PIP. However, in lungs treated with 100 microM phenylarsine oxide (a phosphotyrosine phosphatase inhibitor), Kfc increased by 4.7- and 16.4-fold relative to baseline at these PIP values. In lungs treated with 50 microM genistein (a tyrosine kinase inhibitor), Kfc increased significantly only at 35 cmH2O PIP, and the three groups were significantly different from each other. Thus phosphotyrosine phosphatase inhibition increased the susceptibility of rat lungs to high-PIP injury, and tyrosine kinase inhibition attenuated the injury relative to the high-PIP control lungs.     

Protein kinase A-directed antisense restrains cancer growth: sequence-specific inhibition of gene expression

Increased expression of the RI alpha subunit of cAMP-dependent protein kinase type I has been shown in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. The sequence-specific inhibition of RI alpha gene expression by an antisense oligodeoxynucleotide results in the differentiation of leukemia cells and growth arrest of cancer cells of epithelial origin. A single-injection RI alpha antisense treatment in vivo also causes a reduction in RI alpha expression and inhibition of tumor growth. Tumor cells behave like untransformed cells by making less protein kinase type I. The RI alpha antisense, which produces a biochemical imprint for growth control, requires infrequent dosing to restrain neoplastic growth in vivo.     

EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation

Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.     

12alpha- and 7alpha-hydroxysteroid dehydrogenase activities from Fusobacterium spp

On screening fecal organisms for hydroxysteroid dehydrogenase activities applicable to bile acid metabolism studies, we have isolated a gram negative "Bacteroides-like" anaerobe which yields both 12alpha- and 7alpha-hydroxysteroid dehydrogenase (HSDH) activities in cell-free preparation. At the optimal harvest time of 36 hours, approximately 4500 units 12alpha-HSDH and 360 units 7alpha-HSDH were produced per 10(10) viable cells. The two enzymes appear to be separate entities in the basis of their stabilities on freezing, and prolonged storage at room temperature and elution volumes on Sephadex G 200. Thin layer chromatography studies on oxidation products confirmed the respective sites of oxidation to be the 12alpha-OH and 7alpha-OH position. No 3alpha-OH oriented activity was measurable. Preliminary kinetic studies of the 12alpha-HSDH revealed a broad pH curve with optimal activity at pH 9.5. Michaelis constants for glycodeoxycholate and NADP were estimated at 1.5 x 10(-4)M and 3.3 x 10(-5)M respectively.     

Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain

During development of the vertebrate hindbrain regulatory gene expression is confined to precise segmental domains. Studies of cell lineage and gene expression suggest that establishment of these domains may involve a dynamic regulation of cell identity and restriction of cell movement between segments. We have taken a dominant negative approach to interfere with the function of Sek-1, a member of the Eph-related receptor tyrosine kinase family expressed in rhombomeres r3 and r5. In Xenopus and zebrafish embryos expressing truncated Sek-1, lacking kinase sequences, expression of r3/r5 markers occurs in adjacent even-numbered rhombomeres, in domains contiguous with r3 or r5. This disruption is rescued by full-length Sek-1, indicating a requirement for the kinase domain in the segmental restriction of gene expression. These data suggest that Sek-1, perhaps with other Eph-related receptors, is required for interactions that regulate the segmental identity or movement of cells.     

Selective inhibition of Src protein tyrosine kinase by analogues of 5-S-glutathionyl-beta-alanyl-L-dopa

Twelve analogues of the antibacterial phenolic peptide 5-S-glutathionyl-beta-alanyl-L-dopa (5-S-GA-L-D: 1) were synthesized via orthoquinones using tyrosinase. Several synthesized compounds inhibited the v-Src autophosphorylation tyrosine kinase reaction with an IC50 value comparable to that of herbimycin. The inhibition of c-Src substrate phosphorylation was much less active than v-Src autophosphorylation inhibition. The analogues showed no effects on substrate phosphorylation by epidermal growth factor receptor (EGFR), and this selectivity is the most characteristic feature of the analogues (1-12).     

Activation of human liver 3alpha-hydroxysteroid dehydrogenase by clofibrate derivatives

The NADP+-dependent dehydrogenase activity of a predominant isoenzyme of human liver 3alpha-hydroxysteroid dehydrogenase was activated by antihyperlipidemic drugs, such as bezafibrate and clinofibrate, and by clofibric acid and fenofibric acid (active metabolites of clofibrate and fenofibrate, respectively). The optimal pH of the activation by the drugs was about 7.5, and the concentrations giving maximum stimulation (1.8- to 2.4-fold) were 100, 50, 400 and 50 microM for bezafibrate, clinofibrate, clofibric acid and fenofibric acid, respectively. Clofibrate and fenofibrate acted as weak inhibitors, and the clofibric acid derivatives that lack the chloro group, methyl group on the alpha-carbon or carboxyl group greatly decreased the stimulatory effects. The activation by the drugs increased both Km and kcat (turnover number) values for the coenzyme and substrates. Kinetic analysis with respect to NADP+ showed that bezafibrate, clinofibrate, clofibric acid and fenofibric acid were nonessential activators showing dissociation constants of 32, 6, 103 and 11 microM, respectively. The combined activators experiments with one of the above drugs and sulfobromophthalein, a known activator specific for this enzyme, and comparison of their effects on the activities of mutant enzymes (with Met replacing Lys-270 or Arg-276) indicated that sulfobromophthalein and the drugs bind to an identical site on the enzyme. These results suggest that the long-term therapy with the antihyperlipidemic drugs influences the metabolism of steroid hormones, bile acids and several ketone-containing drugs mediated by the enzyme.     

Prostaglandin-E2 9-reductase from corpus luteum of pseudopregnant rabbit is a member of the aldo-keto reductase superfamily featuring 20 alpha-hydroxysteroid dehydrogenase activity

The prostaglandin-E2 9-reductase (PGE2 9-reductase) activity in the corpus luteum of rabbits corresponds to a cytosolic, NADPH-dependent enzyme with a molecular mass of 36 kDa. This enzyme was purified from corpora lutea on day 12 of pseudopregnancy with a 266-fold enrichment. The main purification step was affinity chromatography using Red Sepharose CL-6B. The efficiency of this column was improved by elution with 1 mM NADH prior to elution of the active fractions with 1 mM NADPH. Amino acid sequence data demonstrate that the rabbit luteal PGE2 9-reductase has to be classified as a member of the aldo-keto reductase superfamily. The enzyme revealed a wide substrate specificity comprising the reduction of aldehydes, ketones, and quinones. Apparent kinetic constants were determined using methylglyoxal, DL-glyceraldehyde, and 9,10-phenanthrenquinone as substrates. The fully purified enzyme showed two catalytic activities of particular interest: PGE2 9-reductase and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activities. The competitive inhibition of 20 alpha-HSD activity by PGE2 indicates that progesterone and PGE2 are substrates for the same enzyme. From these results, we conclude that prostaglandin and steroid metabolism are tightly linked to each other. For this reason the aldo-keto reductase could be a key enzyme in the cascade of events leading to the regression of the corpus luteum in the rabbit.     

Receptor tyrosine kinase expression in human bone marrow stromal cells

A case of crossed aphasia is presented in a strongly right-handed 77-year-old white female without history of familial sinistrality or prior neurological illness. She developed a right middle cerebral artery infarction documented by CT and accompanied by obvious clinical signs of a conduction aphasia with some resolution but continuing obvious language defect after 9 weeks in rehabilitation. Comprehensive neuropsychological and aphasia testing suggested anomalous lateralization of phonologic-output aspects of language, emotional prosody, motor planning and body schema modules with usual lateralization of lexical-semantic aspects of language and visuo-spatial functions. Experimental validation of the uncrossed lexical-semantic aspects of language using tachistoscope methods found support for the Alexander-Annett theory that different aspects of language can be dissociated in their lateralization. The subject had difficulty identifying a semantic associate of a picture presented to the left visual field (7 errors out of 10) relative to right visual field presentation (2 errors out of 10). Bilateral free naming errors (6 and 5 errors in the left and right visual fields, respectively) occurred consistent with the aphasic presentation, suggesting phonologic-output dysfunction from the right cerebral vascular accident. Implications of the results for aphasia classification are discussed.     

Inhibitors of tyrosine kinase

BACKGROUND AND OBJECTIVES: Neurolytic superior hypogastric plexus block has been shown to be safe and effective in selected cancer patients. A large cohort of patients was studied to evaluate the continued efficacy and safety of this block in cancer patients with advanced disease. METHODS: A total of 227 pelvic pain patients with gynecological, colorectal, or genitourinary cancer who experienced poor pain control due to either progression of disease or to untoward side effects were enrolled in this study during a 3-year period. All pain patients receiving oral opioids were eligible to participate. A bilateral percutaneous neurolytic superior hypogastric plexus block with 10% phenol was performed 1 day after a successful diagnostic block with 0.25% bupivacaine. RESULTS: All patients reported a visual analog scale (VAS) pain score of 7-10/10 before the block. A positive response to a diagnostic block was obtained in 159 patients (79%). Overall, 115 patients of the 159 patients who responded to a diagnostic block (72%, 95% confidence interval of 65-79%) had satisfactory pain relief (VAS < 4/10), 99 (62%) after one block, and 16 (10%) after a second block. The remaining 44 patients (28%) had moderate pain control (VAS 4-7/10) after two blocks and received oral pharmacological therapy and epidural analgesic therapy with good results. Both groups experienced significant reductions in oral opioid therapy after the neurolytic blocks. No additional blocks were required by patients who had a good response during a follow-up period of 3 months. No complications related to the block were detected. CONCLUSIONS: Neurolytic superior hypogastric plexus block provided both effective pain relief and a significant reduction in opioid usage (43%) in 72% of the patients who received a neurolytic block. Overall, this represents 51% of the patients enrolled in the study. Poor results should be expected in patients with extensive retroperitoneal disease overlying the plexus because of inadequate spread of the neurolytic agent.     

11-beta-hydroxysteroid dehydrogenase activity and gene expression in the hypertensive Bianchi-Milan rat

OBJECTIVE: 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD), by converting the active steroids cortisol and corticosterone to their inactive metabolites, regulates steroid exposure to the mineralocorticoid and glucocorticoid receptors. We explored the hypothesis that a defect in 11 beta-HSD could result in overstimulation of either the mineralocorticoid or glucocorticoid receptors with subsequent hypertension in an established animal model of hypertension, the Bianchi-Milan hypertensive (BMH) rat. DESIGN AND METHODS: Groups of BMH rats with established hypertension (42-46 days old) and prehypertensive rats (22 days old) were compared with age-matched normotensive control rats. Kidney and liver 11 beta-HSD and glucocorticoid receptor messenger RNA (mRNA) levels were assessed by Northern and dot-blot analyses, and 11 beta-HSD activity as percentage conversion of [3H]-corticosterone to [3H]11-dehydrocorticosterone by tissue homogenate. RESULTS: Hepatic 11 beta-HSD activity and gene expression were significantly reduced in the hypertensive BMH rat compared with its normotensive genetic control. 11 beta-HSD activity was also reduced in the prehypertensive BMH rat (aged 25 days) from hypertensive parents, excluding hypertension per se as the cause of the abnormality. Plasma corticosterone was higher in the hypertensive rats. There was no difference in renal 11 beta-HSD activity or gene expression between hypertensive and normotensive BMH rats, or in glucocorticoid receptor gene expression in the liver or kidney. CONCLUSIONS: Normal levels of renal 11 beta-HSD mRNA and activity are found in the BMH rat. However, the hypertensive BMH rat does demonstrate impaired hepatic 11 beta-HSD activity which occurs at a pretranslational level, although it is not clear how this relates to the pathogenesis of hypertension in this model.     

Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition

In the present open study, the long-term safety, tolerability, and efficacy of citalopram in the treatment of elderly people with emotional disturbances were studied. One hundred twenty-three elderly patients with symptoms of depression-anxiety were included. Most of the patients (76%) were demented. Fifty-two patients completed a 12-month treatment. Irritability, depressed mood, anxiety, restlessness, and fear-panic were significantly reduced. The severity of illness from baseline to Month 9 was rated as significantly improved. The side effects were infrequent and mostly mild.     

IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance

Tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance in obesity and diabetes through its ability to decrease the tyrosine kinase activity of the insulin receptor (IR). Treatment of cultured murine adipocytes with TNF-alpha was shown to induce serine phosphorylation of insulin receptor substrate 1 (IRS-1) and convert IRS-1 into an inhibitor of the IR tyrosine kinase activity in vitro. Myeloid 32D cells, which lack endogenous IRS-1, were resistant to TNF-alpha-mediated inhibition of IR signaling, whereas transfected 32D cells that express IRS-1 were very sensitive to this effect of TNF-alpha. An inhibitory form of IRS-1 was observed in muscle and fat tissues from obese rats. These results indicate that TNF-alpha induces insulin resistance through an unexpected action of IRS-1 to attenuate insulin receptor signaling.