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Stimulation of transfected HepG2 cells (TFG2) with the alpha(1)-adrenergic agonist phenylephrine (PE) significantly activated p21(waf1/cip1) gene expression without affecting p53 gene expression. Northern blotting and reporter assay demonstrated that this induction was due to PE stimulation of p21(waf1/cip1) mRNA stability. To further define the underlying mechanism, we prepared a chloramphenicol acetyltransferase (CAT)-p21(waf1/cip1) 3'-untranslated region (3'-UTR) hybrid construct by inserting the 3'-UTR of p21(waf1/cip1) mRNA just downstream from the CAT coding sequence and transfected it into TFG2 cells. PE treatment enhanced the activity of this construct by 6-fold. Deletion analyses indicated that an AU-rich element (AURE) located between 553 to 625 within the p21(waf1/cip1) 3'-UTR was required for this induction. RNA gel shift assays demonstrated that this AURE bound an RNA-binding protein. This protein has been purified 5000-fold from PE-treated TFG2 cells by heparin-Sepharose and RNA affinity chromatography. SDS-polyacrylamide gel electrophoresis, UV cross-linking, and Northwestern analyses indicated the molecular mass of this protein as 24 and 52 kDa. Finally, PE treatment markedly enhanced this RNA-protein binding by a p42/44 mitogen-activated protein kinase-dependent mechanism. These data suggest that the AURE located between 553 and 625 within the p21(waf1/cip1) mRNA 3'-UTR, which binds an RNA-binding protein, is responsible for PE-induced p21(waf1/cip1) mRNA stability.

We have positionally cloned and characterized a new calcium channel auxiliary subunit, alpha(2)delta-2 (CACNA2D2), which shares 56% amino acid identity with the known alpha(2)delta-1 subunit. The gene maps to the critical human tumor suppressor gene region in chromosome 3p21.3, showing very frequent allele loss and occasional homozygous deletions in lung, breast, and other cancers. The tissue distribution of alpha(2)delta-2 expression is different from alpha(2)delta-1, and alpha(2)delta-2 mRNA is most abundantly expressed in lung and testis and well expressed in brain, heart, and pancreas. In contrast, alpha(2)delta-1 is expressed predominantly in brain, heart, and skeletal muscle. When co-expressed (via cRNA injections) with alpha(1B) and beta(3) subunits in Xenopus oocytes, alpha(2)delta-2 increased peak size of the N-type Ca(2+) currents 9-fold, and when co-expressed with alpha(1C) or alpha(1G) subunits in Xenopus oocytes increased peak size of L-type channels 2-fold and T-type channels 1.8-fold, respectively. Anti-peptide antibodies detect the expression of a 129-kDa alpha(2)delta-2 polypeptide in some but not all lung tumor cells. We conclude that the alpha(2)delta-2 gene encodes a functional auxiliary subunit of voltage-gated Ca(2+) channels. Because of its chromosomal location and expression patterns, CACNA2D2 needs to be explored as a potential tumor suppressor gene linking Ca(2+) signaling and lung, breast, and other cancer pathogenesis. The homologous location on mouse chromosome 9 is also the site of the mouse neurologic mutant ducky (du), and thus, CACNA2D2 is also a candidate gene for this inherited idiopathic generalized epilepsy syndrome.

When Hsc70 uncoats clathrin-coated vesicles in an auxilin- and ATP-dependent reaction, a single round of rapid uncoating occurs followed by very slow steady-state uncoating. We now show that this biphasic time course occurs because Hsc70 sequentially forms two types of complex with the dissociated clathrin triskelions. The first round of clathrin uncoating is driven by formation of a pre-steady-state assembly protein (AP)-clathrin-Hsc70-ADP complex. Then, following exchange of ADP with ATP, a steady-state AP-clathrin-Hsc70-ATP complex forms that ties up Hsc70, preventing further uncoating. This steady-state complex forms only during uncoating in the presence of APs; in the absence of APs, Hsc70 rapidly dissociates from the uncoated clathrin and continues to carry out uncoating. Whether it is complexed with ATP or ADP, the steady-state complex has very different properties from the pre-steady-state complex in that it cannot be immunoprecipitated by anti-clathrin antibodies and is readily dissociated by fast protein liquid chromatography. Remarkably, when the steady-state complex is incubated with uncoated vesicle membranes in ATP, the pre-steady-state complex reforms, suggesting that the clathrin triskelions in the steady-state complex rebind to the membranes and are again uncoated by Hsc70. We propose that Hsc70 not only uncoats clathrin but also chaperones it to prevent it from inappropriately polymerizing in the cell cytosol and primes it to reform clathrin-coated pits.

Cannabinoid CB1 receptor mRNA was detected using reverse transcription-polymerase chain reaction (RT-PCR) in endothelial cells from human aorta and hepatic artery and in the ECV304 cell line derived from human umbilical vein endothelial cells. CB1 receptor-binding sites were detected by the high-affinity antagonist radioligand [(125)I]AM-251. In ECV304 cells, both the highly potent synthetic cannabinoid agonist HU-210 and the endogenous ligand anandamide induce activation of mitogen-activated protein (MAP) kinase, and the effect of HU-210 was completely blocked, whereas the effect of anandamide was partially inhibited by SR141716A, a selective CB1 receptor antagonist. Transfection of ECV304 cells with CB1 receptor antisense, but not sense, oligonucleotides caused the same pattern of inhibition as SR141716A. This provides more definitive evidence for the involvement of CB1 receptors in MAP kinase activation and suggests that anandamide may also activate MAP kinase via an additional, CB1 receptor-independent, SR141716A-resistant mechanism. The MAP kinase activation by anandamide in ECV304 cells requires genistein-sensitive tyrosine kinases and protein kinase C (PKC), and anandamide also activates p38 kinase and c-Jun kinase. These findings indicate that CB1 receptors located in human vascular endothelium are functionally coupled to the MAP kinase cascade. Activation of protein kinase cascades by anandamide may be involved in the modulation of endothelial cell growth and proliferation.

The tumor suppressor protein p53 is a transcription factor that can positively regulate the expression of critical target genes involved in negative control of cell growth or induction of apoptosis; p53 is also able to suppress the transcription of other genes by virtue of its ability to bind components of the basal transcription machinery. Over 50% of human tumors are characterized by p53 mutations that result in a loss of wild-type p53 (wtp53) function in the transcriptional control of these target genes. We have exploited this loss of p53 function in the regulation of gene transcription to develop a novel gene therapy strategy that maximizes expression of the potential therapeutic gene in tumors while simultaneously down-regulating the same gene in normal cells. In one construct (unit I), the potential therapeutic gene (in this case represented by a luciferase reporter) is placed under the control of a promoter such as the heat shock protein 70 gene promoter, which is repressed by wtp53 but overexpressed in many tumor cells with defective p53 function. Residual expression of the reporter in normal cells is repressed by cotransfection of another construct (unit II) consisting of a repressor of unit I under the control of a promoter that is activated by wtp53 expression. Unit II contains a promoter with a consensus wtp53 binding site driving a transcriptional repressor or an antisense construct for the gene in unit I. Our results suggest that this dual control approach may represent a strategy with wide applications in the field of cancer gene therapy.

Tumor necrosis factor (alpha) (TNF-(alpha) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-(alpha) may influence cell-matrix interactions by altering the clustering as well as internalization of the (&agr;)5(beta)1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-(alpha) caused an increase in the intracellular staining of (alpha)5(alpha)1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37 degrees C after antibody-labeling of their surface (alpha)5(beta)1 integrins at 4 degrees C confirmed an increase in the rate of (alpha)5(beta)1 integrin internalization which was at least 3 times greater after TNF-(&agr;) exposure, based on the half-life for antibody-labeled surface integrins to reach equilibrium with non-labeled integrins within the intracellular pool. Interestingly, the total cell surface expression of (alpha)5(beta)1 integrins was relatively constant after TNF-(alpha) exposure despite the enhanced rate of internalization, suggesting an accelerated recycling of the internalized (alpha)5(beta)1 integrins back to the cell surface. This response was confirmed by the measurement of labeled integrin recycling, which showed a significant (P<0.01) increase in the rate of recycling of the internalized integrins in TNF-treated endothelial cells. Enhanced internalization and subsequent recycling of (alpha)5(beta)1 integrins by endothelial monolayers exposed to TNF-(alpha) may facilitate the redistribution of cell-surface integrins in response to this inflammatory cytokine and may also modify cell-matrix interactions leading to reduced integrity and increased protein permeability of the lung endothelial monolayers.

Treatment of primary rat hepatocytes or tranfected HepG2 cells with the alpha(1B)-adrenergic receptor (alpha(1B)AR) agonist phenylephrine (PE) significantly inhibited interleukin 6 (IL-6)-induced STAT3 binding, tyrosine phosphorylation, and IL-6-induced serum amyloid A mRNA expression. Western analyses and in vitro kinase assays indicate that this inhibition is not due to either down-regulation of STAT3 protein expression nor inactivation of upstream-located JAK1 and JAK2. Blocking the new RNA and protein syntheses antagonized the inhibitory effect of PE on IL-6-activated STAT3, suggesting synthesis of an inhibitory factor(s) is involved. The inhibitory effect of PE on IL-6 activation of STAT3 was also abolished by the tyrosine phosphatase inhibitor sodium vanadate, indicating involvement of protein tyrosine phosphatases. Furthermore, preincubation of the cells with the specific MEK1 inhibitor PD98059 or a dominant negative MEK1 reversed the inhibitory effect of PE, and expression of constitutively activated MEK1 alone abolished IL-6-activated STAT3. Taken together, these data indicate that PE inhibits IL-6 activation of STAT3 in hepatic cells by a p42/44 mitogen-activated protein kinase-dependent mechanism, and tyrosine phosphatases are involved. This inhibitory cross-talk between the alpha(1B)AR and IL-6 signaling pathways implicates the alpha(1B)AR involvement in regulating the IL-6-mediated inflammatory responses.

Objective: Immaturity of the endothelial-dependent relaxation is thought to be characteristic of the newborn pulmonary elastic arteries. In adulthood, the reactivity of different pulmonary arterial segments varies. Therefore, we investigated the presence of endothelial heterogeneity in perinatal porcine pulmonary arteries and compared it with the adult by studying the bradykinin-, substance P- and acetylcholine-induced relaxations in different arteries., Methods: Three types of pulmonary arteries (large conduit elastic, distal branching and resistance-sized; in situ diameters 0.7-1.7, 0.3-0.5 and 0.1-0.2 mm, respectively) were isolated from lungs of adult (nine months), young (60-84 h), newborn (4 min) and near-term foetal pigs. They were mounted for isometric force recording, contracted first with K+ = 125 mmol/l (reference contraction). Cumulative concentration-response curves to acetylcholine, substance P or bradykinin were obtained from prostaglandin F2 alpha (30 mumol/l) precontracted vessels. The effects of captopril and O2(95 or 8%) were also determined. Experiments were terminated by adding 100 mumol/l papaverine, obtaining maximal relaxation, which was used for normalising relaxations., Results: (i) Acetylcholine: In resistance arteries, relaxations were absent in the newborn and the adult. In conduit arteries, they were present from 60-84 h onward. (ii) Substance P: In resistance arteries, relaxations were only present in the adult. In the other two types of arteries, rudimentary relaxations were present from the mature foetal stage onward. (iii) Bradykinin: In resistance arteries, identical relaxations were present at all ages which, in the foetus and the adult, were insensitive to changes in O2 levels (95 to 8%). In conduit arteries, concentration-dependent relaxations were present from birth, increasing in amplitude with age and these were potentiated by captopril. Foetal conduit arteries relaxed to the single application of 0.1 mumol/l bradykinin, indicating age-dependent tachyphylaxis., Conclusions: (i) Bradykinin is unique among endothelium-dependent vasodilators in being able to relax all vascular segments, at all ages, subject to tachyphylaxis and bradykinin-breakdown but independent of the prevailing O2 concentration. (ii) Heterogeneity of the relaxations between conduit and resistance arteries is evident from the mature foetal stage onward. (iii) The type of agonist, the type of vessel and the age each independently determine the presence or absence of endothelial relaxations. Therefore, the perinatal pulmonary circulation is not immature with respect to endothelial-dependent relaxation; rather, the nature of this process changes within the perinatal period and between birth and adulthood.

Effects of acute ethanol (EtOH) exposure on motoneuron excitability and properties of synaptic transmission were examined in spinal cords of postnatal rats. Whole-cell patch clamp recordings and intracellular recordings with high-resistance electrodes were carried out in motoneurons of 1- to 4-day-old postnatal rats. To determine the effects of extracellular EtOH on action potential waveform, properties of current-evoked soma action potentials and motoneuron ability to generate repetitive action potential firing were examined. During a brief EtOH (70 mM) exposure, larger depolarizing current was required for action potential generation, the duration of the after hyperpolarizing potential increased, and fewer action potentials were produced during a prolonged intracellular current injection. These effects were reversed within 20 min of EtOH removal from the extracellular solution. To determine whether the reduced probability of action potential generation was associated with changes in synaptic transmission, properties of evoked synaptic potentials and spontaneous synaptic currents were investigated. In the presence of EtOH, the amplitude of dorsal root-evoked synaptic potentials was reduced, the frequency of spontaneous excitatory postsynaptic currents decreased, while the frequency of inhibitory postsynaptic currents increased. Our data suggested that acute EtOH exposure suppressed motoneuron electrical activity by decreasing motoneuron excitability and shifting the balance between excitatory and inhibitory synaptic transmission toward inhibition.

In this study we investigated the distribution of a recently cloned polyspecific organic anion transporting polypeptide (Oatp2) in rat brain by nonradioactive in situ hybridization histochemistry and immunofluorescence microscopy. The results demonstrate that Oatp2 is expressed in brain capillary and in plexus epithelial cells. At the blood-brain barrier (BBB), Oatp2 expression could be co-localized with the endothelial marker vWF (von Willebrand factor) but not with the astrocyte marker GFAP (glial fibrillary acidic protein). In choroid plexus epithelial cells, Oatp2 could be localized to the basolateral cell pole, whereas the first member of the Oatp gene family of membrane transporters to be cloned (Oatp1) co-localized with the alpha(1)-subunit of Na,K-ATPase at the apical plasma membrane domain. Because Oatp1 and Oatp2 have been previously shown to mediate transmembrane transport of a wide variety of amphipathic organic compounds, including many drugs and other xenobiotics, the histochemical localization of Oatp2 at the BBB and of Oatp1 and Oatp2 in the choroid plexus imply a role for these transporters in the active exchange of amphipathic solutes between the blood, brain, and cerebrospinal fluid compartments. (J Histochem Cytochem 47:1255-1263, 1999)

Background & Aims: Multispecific organic anion-transporting polypeptides (Oatps) are involved in the transcellular movement of amphipathic compounds in many tissues including the liver, kidney, and blood-brain barrier. Recently, a high-affinity digoxin transporter (Oatp2) was cloned from rat brain and shown to be also expressed in the liver., Methods: We investigated the cellular and subcellular distribution of Oatp2 in rat liver by in situ hybridization technology and immunofluorescence microscopy and compared its substrate specificity with that of Oatp1 in complementary RNA-injected Xenopus laevis oocytes., Results: The results show a selective basolateral (sinusoidal) expression of Oatp2 in midzonal to perivenous hepatocytes, but not in periportal or the innermost layer of perivenous hepatocytes. Common substrates of both Oatp1 and Oatp2 include bile salts, steroid conjugates, thyroid hormones (T3, T4), ouabain, and the endothelin receptor antagonist BQ-123 (Michaelis constants: Oatp1, approximately 600 micromol/L; Oatp2, approximately 30 micromol/L). Other organic anions including sulfolithotaurocholate, bilirubin monoglucuronide, and sulfobromophthalein were transported only by Oatp1., Conclusions: These results provide definite evidence for the partially overlapping and partially selective substrate specificities of Oatp1 and Oatp2. The unique acinar distribution of Oatp2 might indicate that it represents a high-affinity "backup" system for complete hepatocellular removal of certain cholephilic substances from portal blood plasma.

The ability of ethanol to inhibit regenerative processes in the liver is thought to play a key role in the development of alcoholic liver disease. To understand the underlying mechanisms, we investigated the effects of ethanol on the Janus kinasesignal transducer and activator transcription factor (JAK-STAT) signaling pathways in hepatocytes. Treatment of freshly isolated adult rat hepatocytes with 10-100 mM ethanol rapidly (< 3 min) inhibits interleukin-6 (IL-6)-induced STAT3 activation, tyrosine and serine phosphorylation and IL-6-induced CCAAT enhancer binding protein (C/EBP) alpha and beta mRNA expression. Western analyses, in vitro kinase assays and in vivo cell labelling assays indicate that this inhibitory effect is not due to blocking the upstream-located JAK1, JAK2 or Tyk2 activation. On the contrary, acute ethanol exposure significantly potentiates IL-6-induced JAK1 autophosphorylation in vitro and in vivo. Pretreatment with sodium vanadate, a non-selective tyrosine phosphatase inhibitor, or with MG132 and lactacystin, proteasome inhibitors, does not abolish the ethanol inhibition of IL-6-induced STAT3 activation, suggesting that activation of protein tyrosine phosphatases or the ubiquitin-proteasome pathway is not involved. In view of the critical role of IL-6 signaling in liver regeneration, these findings suggest that the ability of biologically relevant concentrations of ethanol to markedly inhibit IL-6-induced STAT3 phosphorylation is one of the cellular mechanisms involved in the pathogenesis and progression of alcoholic liver diseases.

Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway leading to prostacyclin (PGI2) production. Patients with severe pulmonary hypertension have a PGIS deficiency of their precapillary vessels, but the importance of this deficiency for lung vascular remodeling remains unclear. We hypothesized that selective pulmonary overexpression of PGIS may prevent the development of pulmonary hypertension. To study this hypothesis, transgenic mice were created with selective pulmonary PGIS overexpression using a construct of the 3.7-kb human surfactant protein-C (SP-C) promoter and the rat PGIS cDNA. Transgenic mice (Tg+) and nontransgenic littermates (Tg-) were subjected to a simulated altitude of 17,000 ft for 5 weeks, and right ventricular systolic pressure (RVSP) was measured. Histology was performed on the lungs. The Tg+ mice produced 2-fold more pulmonary 6-keto prostaglandin F1alpha (PGF1alpha) levels than did Tg- mice. After exposure to chronic hypobaric hypoxia, Tg+ mice have lower RVSP than do Tg- mice. Histologic examination of the lungs revealed nearly normal arteriolar vessels in the Tg+ mice in comparison with vessel wall hypertrophy in the Tg- mice. These studies demonstrate that Tg+ mice were protected from the development of pulmonary hypertension after exposure to chronic hypobaric hypoxia. We conclude that PGIS plays a major role in modifying the pulmonary vascular response to chronic hypoxia. This has important implications for the pathogenesis and treatment of severe pulmonary hypertension.

Objectives: To estimate the prevalence of lower female genital tract chlamydial (GTC) and gonococcal infection in 3 different ethnic groups in China, and to analyze the risk factors influencing these infections., Methods: A cross-sectional survey + physical and laboratory examinations. 1,800 married women aged 18-35 Han (600, from Dujiangyan city, Sichuan), Naxi (600, from Yanyuan county, Sichuan), and Dai (600, from Jinghong Prefecture, Yunnan) were investigated between March 1996 to March 1997. Information was obtained about their personal history, socioeconomic status, medical history, STDs, sexual history, contraceptive history, results of both examinations. Tabulation, t-test, Chi-square test, multiple-logistic models were used to analyze the data., Results: 3.2% of Han, 12.8% of Naxi and 4.5% of Dai women tested positive for gonococci; and 9.1% of Han, 16.7% of Naxi and 27.5% of Dai women tested positive for GTC infection. The differences of GTC and gonococcal infections between Han and Naxi, and Han and Dai women were significant., Conclusions: STDs are common in China today; both GTC and gonoccocal infections are related to non-use of condom, having sexual partners other than husband, number of abortions, and low education; and condom has a function in prevention of STDs.

Aim: To explore the in vitro sensitivity of artesunate-sensitive and -resistant Plasmodium falciparum to chloroquine and amodiaquine and to observe the effect of artesunate combined with chloroquine and artesunate combined with amodiaquine on artesunate-resistant P. falciparum., Methods: The sensitivity of the artesunate-sensitive and -resistant P. falciparum to chloroquine and amodiaquine and their combination with artesunate was compared by using Rieckmann's in vitro micro-technique., Results: The ID50 and ID95 values of chloroquine, amodiaquine and artesunete were 90.9, 50.9, 9.6 nmol/L and 320.0, 320.0, 40.0 nmol/L to the artesunate-sensitive P. falciparum respectively and were 112.0, 133.5, 85.1 nmol/L and 320.0, 320.0, 400.0 nmol/L to artesunate-resistant P. falciparum, respectively. When artesunate was combined with chloroquine, the ID50 values of the 2 drugs were 3.2 and 20.0 nmol/L to the artesunate-resistant P. falciparum. When artesunate was combined with amodiaquine, the ID95 values of the 2 drugs were 3.2 and 5.0 nmol/L to the artesunate-resistant P. falciparum, respectively., Conclusion: The artesunate-resistant P. falciparum has no cross resistance to chloroquine and amodiaquine, however, artesunate combined with chloroquine or amodiaquine exhibit an apparent synergistic effect in vitro.

Transcription of the rat alpha1B adrenergic receptor (alpha1B AR) gene is controlled by three promoters (P1, P2, and P3), which generate 2.3-, 2.7-, and 3.3-kb transcripts, respectively. The expression of the 2.3-kb mRNA species is tissue-specific. To explore the underlying mechanism, the P1 promoter was analyzed. DNase I footprinting of the P1 promoter yielded three protected regions: Plfl(-49 to -62); P1f2 (-73 to -90), and P1f3 (-95 to -115). Sequence analysis of P1f3 revealed the presence of an SV40 core C enhancer-like element. In gel mobility shift assays, P1f3 was found to bind a sequence specific protein, which was competed away by a SV40 core C enhancer consensus oligonucleotide. Mutations of this enhancer-like core sequence within P1f3 significantly reduced specific protein binding to P1f3 and inhibited P1 promoter activity. The distribution of the protein which binds to P1f3 is restricted. These findings suggest that the P1 promoter is controlled by a cell-type-specific transcription factor, which may account for the tissue-specific expression of 2.3-kb rat alpha1B AR mRNA species.

Nuclear factor 1 (NF1) has been reported to be a transcriptional activator for some genes and a transcriptional silencer for others. Here we report that in Hep3B cells, cotransfection of NF1/L, NF1/Red1, or NF1/X with the alpha1B adrenergic receptor (alpha1BAR) gene middle (P2) promoter increases P2 activity to more or less the same degree, whereas in DDT1 MF-2 cells cotransfection of NF1/L or NF1/Red1 causes a small but statistically significant decrease in the P2 promoter activity, and NF1/X causes a greater, 70% inhibition. Further experiments using truncated NF1/X mutants indicate that NF1/X contains both positive and negative regulatory domains. The positive domain, located between amino acids 416 and 505, is active in Hep3B cells, whereas the negative domain, located between amino acids 243 and 416, is active in DDT1 MF-2 cells. These functional domains are also capable of regulating transcription when isolated from their natural context and fused into the GAL4 binding domain. Furthermore, NF1 affinity purified from rat liver nuclear extracts copurified with a non-DNA binding protein, which can bind to the P2 promoter of the alpha1BAR gene via interacting with NF1. Taken together, these findings indicate that NF1/X contains both activation and suppression domains that may be recognized and modulated by cell type-specific cofactors. This may be one of the mechanisms whereby NF1 can activate or suppress the expression of different genes, and it may also underlie the tissue-specific regulation of the alpha1B AR gene.

We demonstrated previously that selective thrombosis of the blood vessels of solid tumors in mice can be achieved by targeting the extracellular domain of tissue factor by means of an antibody to an experimentally induced marker on tumor vascular endothelium. In the present study, we extend this finding to a naturally occurring marker of tumor vascular endothelium, vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 is expressed by vascular endothelial cells in Hodgkin's disease and various solid tumors in mice and humans. It is absent from vascular endothelial cells in normal tissues in mice, with the exception of the heart and lungs, where it is present on venules. A monoclonal antibody to murine VCAM-1 was covalently linked to the extracellular domain of human tissue factor to create a "coaguligand." After i.v. administration to severe combined immunodeficient mice bearing human Hodgkin's tumors, the coaguligand localized selectively to VCAM-1-expressing vessels, caused thrombosis of those vessels, and retarded tumor growth. The coaguligand also localized to VCAM-1-expressing vessels in the heart and lungs of the mice but did not induce thrombosis in these sites. An immunohistochemical evaluation of the distribution of a monoclonal anti-phosphatidylserine (PS) antibody in the mice showed that the VCAM-1-expressing vessels in the tumor expressed PS, whereas the VCAM-1-expressing vessels in the heart and lungs lacked PS. The lack of thrombotic effect of the coaguligand on heart and lung vessels may be because PS is needed to provide the procoagulant surface upon which coagulation complexes can assemble. The requirement for coincident expression of the targeted marker and PS on tumor endothelium probably contributes to the selectivity of thrombotic action and the safety of coaguligands.

Activation of alpha1B adrenergic receptors (alpha(1B)AR) promotes DNA synthesis in primary cultures of hepatocytes, yet expression of alpha(1B)AR in hepatocytes rapidly declines during proliferative events. HepG2 human hepatoma cells, which do not express alpha(1B)AR, were stably transfected with a rat alpha1B(AR) cDNA (TFG2 cells), in order to study the effects of maintained alpha(1B)AR expression on hepatoma cell proliferation. TFG2 cells had a decreased rate of growth compared to mock transfected HepG2 cells as revealed by a decrease in [3H]thymidine incorporation into DNA. Stimulation of alpha(1B)AR with phenylephrine caused a further large reduction in TFG2 cell growth, whereas no effect on growth was observed in mock transfected cells. Reduced cell growth correlated with increased percentages of cells found in G0/G1 and G2/M phases of the cell cycle. In TFG2 cells, phenylephrine increased p42MAPkinase activity by 1.5- to 2.0-fold for up to 24 h and increased expression of the cyclin dependent kinase inhibitor protein p21Cip1/WAF1. Treatment of TFG2 cells with the specific MEKI inhibitor PD98059, or infection with a -/- MEK1 recombinant adenovirus permitted phenylephrine to increase rather than decrease [3H]thymidine incorporation. In addition, inhibition of MAP kinase signaling by PD98059 or MEK1 -/- blunted the ability of phenylephrine to increase p21Cip1/WAF1 expression. In agreement with a role for increased p21Cip1/WAF1 expression in causing growth arrest, infection of TFG2 cells with a recombinant adenovirus to express antisense p21Cip1/WAF1 mRNA blocked the ability of phenylephrine to increase p21Cip1/WAF1 expression and to inhibit DNA synthesis. Antisense p21Cip1/WAF1 permitted phenylephrine to stimulate DNA synthesis in TFG2 cells, and abrogated growth arrest. These results suggest that transformed hepatocytes may turn off the expression of alpha1B(ARs) in order to prevent the activation of a growth inhibitory pathway. Activation of this inhibitory pathway via alpha1B(AR) appears to be p42MAPkinase and p21Cip1/WAF1 dependent.

To understand the mechanisms by which ethanol inhibits hepatocyte proliferation, we studied the effects of ethanol on p42/44 mitogen-activated protein kinase (MAPK), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) in normal and regenerating rat liver. Treatment of rat hepatocytes with 100 mM ethanol in vitro for 16 h prolonged the activation of p42/44 MAPK and p38 MAPK induced by various agonists. Such treatment also increased basal JNK activity, but did not potentiate or prolong agonist-induced JNK activation. Ethanol potentiation of the activation of p42/44 MAPK was abolished by pertussis toxin. In contrast, chronic ethanol consumption in vivo inhibited the activation of p42/44 MAPK, p38 MAPK and JNK induced either by partial hepatectomy or by various agonists. However, both acute and chronic ethanol inhibited hepatocyte proliferation induced by insulin and epidermal growth factor. A selective inhibitor of p42/44 MAPK partially prevented the inhibition of hepatocyte proliferation caused by acute, but not by chronic, ethanol exposure, whereas a selective inhibitor of p38 MAPK further inhibited hepatocyte proliferation under both conditions. These data suggest that acute and chronic ethanol inhibit hepatocyte proliferation by different mechanisms. The effect of acute ethanol may be related to the prolongation of p42/44 MAPK activation, whereas inhibition of hepatocyte proliferation by chronic ethanol may be due to inhibition of p38 MAPK activation.

Objective: The objective of this multicentre, randomized, prospective and comparative study was to evaluate and compare the efficacy and safety of 1 g intravenous ceftriaxone (active ingredient of Rocephin), 3 g intravenous cefoiaxime (active ingredient of clafron), and 2.25 g intavenous cefuroxime (active ingredient of Zinacef)., Method: In this multicentre, randomized, prospective and comparative study, patients received 1 g of ceftriaxone intravenously once a day (group A), or 1 g of cefotaxime intravenously three times a day (group B), or 0.75 g of cefuroxime intravenously three time a day (group C). 197 patients were enrolled in the study, and in 142 (48 in group A, 46 in group B and 48 in group C) we were able to make an evaluation., Result: The overall efficacy (bacteriological eradication plus clinical cure or clear improvement) of ceftriaxone, cefotaxime and cefuroxime were 81%, 83%, 79% respectively (P > 0.05). The eradication rate for three groups were 80%, 78%, 75% (P > 0.05). No adverse events occured., Conclusion: Data obtained in our study indicate that for the majority of patients with lower respiratory tract infections, 1 g ceftriaxone, 3 g cefotaxime and 2.25 g cefuroxime are effective and safe, and 7 days therapy is enough, but the use of 1 g ceftriaxone is more convenient.

Objective: To improve the diagnosis of retrocaval ureter., Method: 20 cases of retrocaval ureter with an average age of 33.4 years were reported. The diagnosis of this disease depends on urography and retrograde ureterography. Operation was the principal treatment. Ureter orthopedics and reduction were performed in 19 cases of type 1 (low loop) except one case of type II (high loop). After removing 3 - 4 cm retrocaval ureter with pathological changes, we anastomosed the ureter without tension and regained its normal anatomic position., Result: B-ultrasound and excretory urography showed no stenosis and improvement of hydronephrosis., Conclusion: Ureter orthopedics and reduction are ideal for the treatment of retrocaval ureter.

The modification of tyrosine residues in proteins to 3-nitrotyrosine by peroxynitrite or other potential nitrating agents has been detected in biological systems that are subject to oxidative stress. A convenient semi-quantitative method has been developed to assay nitrated proteins in biological fluids and homogenates using a competitive ELISA developed in our laboratory. This assay selectivity detected 3-nitro-l-tyrosine residues in a variety of peroxynitrite-treated proteins (BSA, human serum albumin (HSA), alpha1-antiprotease inhibitor, pepsinogen and fibrinogen) and also in a nitrated peptide, but had a low affinity for free 3-nitro-L-tyrosine and 3-chloro-L-tyrosine. The IC50 values for the inhibition of antibody binding by different nitrated proteins were in the range 5-100 nM, suggesting that the antibody discriminated between nitrotyrosine residues in different environments. The presence of nitrotyrosine in plasma proteins was detected by Western blot analysis and quantified by the ELISA. A concentration of 0. 12+/-0.01 microM nitro-BSA equivalents was measured in the proteins of normal plasma which was increased in peroxynitrite-treated plasma and was elevated in inflammatory conditions. HSA and low-density lipoprotein (LDL) isolated from plasma contained 0.085+/-0.04 and 0. 03+/-0.006 nmol nitro-BSA equivalents/mg protein, respectively. Comparison of the level of nitration in peroxynitrite-treated HSA and LDL in the presence and absence of plasma indicates that nitration and presumably oxidation is inhibited by plasma antioxidants. The presence of nitrotyrosine in LDL is consistent with previous reports implicating peroxynitrite in the oxidative modification of lipoproteins and the presence of a low concentration of oxidized LDL in the blood.

Objective: To elucidate the role of NGF in the regeneration of facial nerve., Methods: The superior buccal division of facial nerve of adult New Zealand rabbit was transected and a nerve growth chamber created. The chamber of the experimental side was filled with NGF/normal saline and that of the control side with normal saline alone. Four and eight weeks after operation, the regenerated nerves in the chambers were dissected for histological studies., Results: Four weeks after operation, the average thickness of myelin sheath and the average number of myelinated axons were 0.779 +/- 0.475 micron, 2.024 +/- 1.999 (n = 11) in experimental group and 0.413 +/- 0.132 micron, 368 +/- 171 (n = 8) in control sides respectively. There was significant difference between the experimental sides and control sides (P < 0.05). Eight weeks after operation, the regenerated nerve appeared more mature. There were significant difference in the average diameters, the thickness of myelin sheath and the number of myelinated axons between the experimental and control sides (P < 0.05)., Conclusion: NGF within a silicone chamber enhanced facial nerve regeneration in New Zealand rabbits.

Two polypeptides with molecular masses of 34 and 30 kDa were copurified from rat liver during DNA affinity purification of a sequence-specific transcription factor binding to the footprint II sequence within the P2 promoter of the rat alpha1B adrenergic receptor (alpha1B AR) gene, and were identified by microsequencing their endoproteinase Lys-C-derived peptides as histone H1d and histone H1c, respectively. Histone H1 was previously reported to bind to the nuclear factor 1 (NF1) recognition sequence, although the specificity of this binding has been controversial. Here, DNA mobility shift and supershift assays, DNase I footprinting and mutational analyses indicated that the binding of histone H1 to the NF1 sites located within footprint II of the alpha1B AR gene P2 promoter is nonspecific. Transient cotransfections into Hep3B cells of histone H1d cDNA with CAT constructs containing promoter regions of different genes resulted in generalized and non-specific suppression of CAT activity. The histone H1d-mediated repression of the activities of the alpha1B AR gene P2/CAT or beta2 AR gene P(-186/1307)/CAT constructs was reversed by the cotransfection of a cDNA encoding the sequence-specific transcription factor NF1/X, and the fold increase in CAT activities was similar to that obtained in the absence of histone H1d. These results suggest that sequence-specific transcription factors counteract the histone H1-mediated transcriptional repression in vivo by a true activation, which is different from the in vitro antirepression in histone H1-repressed chromatin templates (Laybourn and Kadonaga, (1991) Science 254: 238-245).

cAMP markedly increases alpha 1B adrenergic receptor (alpha 1B-AR) expression in FRTL-5 and PC C13 rat thyroid cells, DDT1MF-2 smooth muscle cells, primary rat hepatocytes, and K9 rat liver cells. Here, we used DDT1MF-2 cells to evaluate further the mechanisms by which cAMP stimulates alpha 1B-AR expression. Receptor binding assays, Northern blotting, and nuclear run-on analyses demonstrated that forskolin (1 microM) in the presence of isobutylmethylxanthine (0.25 mM) increased alpha 1B-AR numbers, mRNA level, and gene transcription rate by 2.3 +/- 0.2-, 2.5 +/- 0.3-, and 3.5 +/- 0.2-fold over control, respectively. Dibutyryl cAMP (1 mM) plus isobutylmethylxanthine (0.25 mM) also enhanced alpha 1B-AR density by 2.7 +/- 0.1-fold over control. Further experiments demonstrated that the induction of alpha 1B-AR by forskolin requires new protein synthesis and is protein kinase A dependent. In DDT1MF-2 cells transfected with alpha 1B-AR gene P2 promoter/CAT constructs, both forskolin and dibutyryl cAMP significantly increased P2 promoter activity. The P2 promoter region of the rat alpha 1B-AR gene (-813 to -432) contains a cAMP response element (CRE) (-444 to -437) and an AP2 binding site (-647 to -638). Mutations in either one of these elements alone led to a decrease in both basal and cAMP-induced P2 promoter activity. Mutations in both elements caused a further inhibition of basal transcription and a complete block of cAMP-induced P2 promoter activity. Direct binding of purified activator protein 2 (AP2) to the AP2 element in the P2 promoter was reported previously. Gel mobility shift and super-shift assays using liver nuclear extracts from either rat liver or DDT1MF-2 cells demonstrated that the CRE in the alpha 1B-AR gene bound CRE binding protein. These data indicate that both the CRE and the AP2 element in the P2 promoter contribute to basal as well as cAMP-induced transcription of the alpha 1B-AR gene in DDT1MF-2 cells.

Interleukin-6 (IL-6) induced activation of Signal Transducer and Activator Transcription Factor 3 (Stat3) is a critical step in liver regeneration. Chronic ethanol consumption is known to increase the plasma concentration of IL-6, yet the ability of the liver to regenerate and the regenerative induction of several IL-6 initiated events are impaired in chronic alcoholic liver disease. We hypothesized that chronic ethanol consumption inhibits IL-6 dependent signal transduction. To test this hypothesis, the effect of ethanol on the Stat3 signal transduction pathway was studied in the adult rat liver. In vitro treatment of freshly isolated normal adult rat hepatocytes with 50-100 mM ethanol for 30 min blocked IL-6-induced Stat3 activation. Long-term ethanol intake in vivo significantly attenuated the activation of Stat3 induced either in vivo by partial hepatectomy or in vitro by IL-6. In contrast, short-term ethanol consumption enhanced the regenerative induction of Stat3 but inhibited IL-6 induced Stat3 activation. These data suggest that the inhibition of liver regeneration by chronic ethanol consumption is, at least in part, mediated by modulating the activation of Stat3.

In the rat liver, NF1 and CP1 bind to the major P2 promoter of the alpha1B adrenergic receptor gene to generate footprint II. Here we show that, in DDT1MF-2 smooth muscle cells, the major protein bound to footprint II is not NF1 but Sp1, which binds to the 5'-portion of the footprint II sequence (footprint IIb). Mutational analyses demonstrate that the CCCGCG sequence in footprint IIb is critical for Sp1 binding and P2 promoter activity. A second GC box in the P2 promoter also binds the Sp1 protein and contributes to the P2 promoter activity. Gel shift assays indicate that footprint II can bind Sp1, NF1, and CP1, and that the binding of these 3 proteins is mutually exclusive. This is also indicated by the results of functional cotransfection experiments, where transient overexpression of NF1 and Sp1 together caused a similar increase in the activity of a P2/CAT reporter construct as overexpression of either Sp1 or NF1 alone, indicating lack of additivity. The preferential interaction of footprint II with Sp1 in DDT1MF-2 cells and NF1 in liver appears to be due to low levels of NF1 expression in DDT1MF-2 cells and low levels of Sp1 in liver. These observations suggest that NF1 and Sp1 are the major transcription factors involved in controlling the P2 promoter in liver versus DDT1MF-2 cells, respectively, which may be one of the mechanisms responsible for the complex tissue-specific regulation of the expression of the alpha1B adrenergic receptor gene.

The role of an ATP-dependent K+ channel (K(ATP)+) relaxation in the porcine pulmonary vasculature from birth to adulthood was investigated in vitro using levcromakalim on isolated, prostaglandin F2alpha (30 microM)-precontracted conduit arteries (CA), resistance arteries (RA), and veins (PV). Vessels from neonatal pulmonary hypertensive piglets exposed to chronic hypobaric hypoxia (CHH, 51 kPa) for 3 d, either from birth or from 3 d of age were also studied. Levcromakalim relaxed all vessels in a concentration- and glibenclamide-sensitive manner. In normal CA, the maximal extent of relaxation and sensitivity (EC50) increased between birth and 17 d. Endothelium-removal increased EC50 at 17 d only. Indomethacin (10 microM), but not N(G)-monomethyl-L-arginine (L-NMMA) (30 microM), inhibited relaxation in CA from newborn, 3-d-old, and adult animals. In RA, levcromakalim-induced relaxations did not change during development and endothelium-removal attenuated relaxations in 3-d-old but not in adult animals. At both ages in RA, L-NMMA attenuated relaxations and subsequent addition of L-arginine (1 mM) restored them. In PV, maximal relaxation increased between birth and 6 d with no change of EC50. At all ages, relaxation was partially endothelium-dependent and inhibited by L-NMMA (except in the newborn). Indomethacin only attenuated relaxations in veins from 6- and 17-d-old animals. CHH did not influence relaxant responses in CA and PV but decreased EC50 in RA. Thus K(ATP)+ channel activation caused relaxation from birth onward in all vascular segments with varying endothelium dependence. CHH did not affect relaxation in the large vessels and up-regulated those in RA. These findings indicate a possible role for K(ATP)+ channels during normal adaptation and a potential therapeutic role in the management of pulmonary hypertensive newborn infants.

To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX.

It is an important topic in medicine and genetics, which has not yet been studied in depth, that whether high-dose iodine, especially iodine excess in mother's body, can cause impediment of brain development in babies as iodine deficiency do. An animal model of goiter was reconstructed in mice with feeding them water containing high level of iodine for three months. Weight of brain, protein and nucleic acid concentrations were measured in one-, seven-, 14-, 21- and 30-day old young mice, and morphological changes in the brain and their abilities of learning and memory were observed in 30-day old young mice, born to mothers with high-iodine goiter. Results indicated that weight of brain, protein content, ratio of protein to DNA, RNA content, and ratio of RNA to DNA all decreased significantly, and DNA content increased in the brain of high-iodine mice, as compared with those in normal iodine mice. Abilities of learning and memory in 30-day old mice decreased. And, those changes began from the seventh to 14th days after their birth. It suggests that excessive intake of iodine, as iodine deficiency, can not only cause high-iodine goiter, but also damage nervous system leading to retardation of brain development and impediment of its function.

We measured venous blood concentrations of lidocaine in 15 patients undergoing lower abdominal or extremity surgery after epidural injection of lidocaine. Patients were divided into either an infusion group (Group I, n = 6) or a bolus injection group (Group II, n = 9). We administered 15 ml of 2% lidocaine into the epidural space at 1 mL/min in Group I and at 1 mL/3 in Group II. Venous blood was drawn at 3, 6, 9, 12, 15, 18, 21, 30, 60, and 90 min after injection for measurement of plasma lidocaine concentrations. The peak plasma concentration (Cmax) of lidocaine in Group I was significantly less than that in Group II (P < 0.0005). We conclude that slow epidural infusion can produce a lower Cmax of lidocaine compared with that of a bolus administration and thereby decrease the potential for systemic toxicity of the local anesthetic.

The sexually dimorphic nucleus of the preoptic area (SDN-POA) is the most striking structure displaying a morphological sex difference in the rat brain. A potentially homologous nucleus has been identified in the human hypothalamus. The objective of the present study was to pursue the putative homology of the rat and human SDN-POA by determining whether they express the same transmitter phenotype. We employed in situ hybridization histochemistry for GAD mRNA to show whether the neurons of the SDN-POA produce GABA. In both the rat and human, high levels of GAD65 and GAD67 mRNA are present in most, if not all, SDN-POA neurons. No sex difference is evident in the level of expression in either the rat or human. The data indicate that neurons of the SDN-POA in both the rat and human are GABA-producing and argue for the homology of these nuclei in the rat and human hypothalamus.

The 5' upstream region from --490 to --540 (footprint II) within the dominant P2 promoter of the rat alpha(1b) adrenergic receptor (alpha(1b)AR) gene is recognized by a sequence-specific DNA-binding protein (B. Gao, M. S. Spector, and G. Kunos, J. Biol. Chem. 270:5614-5619, 1995). This protein, detectable in Southwestern (DNA-protein) blots of crude nuclear extracts as 32- and 34-kDa bands, has been purified 6,000-fold from rat livers by DEAE-Sepharose, heparin-Sepharose, and DNA affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV cross-linking of the purified protein indicated the same molecular mass as that in crude extracts. Methylation interference analysis revealed strong contact with a TTGGCT hexamer and weak contact with a TGGCGT hexamer in the 3' and 5' portions of footprint II, respectively. Nucleotide substitutions within these hexamers significantly reduced protein binding to footprint II and the promoter activity of P2 in Hep3B cells. The purified protein also bound to the nuclear factor 1 (NF1)/CTF consensus sequence, albeit with lower affinity. Gel mobility supershift and Western blotting (immunoblotting) analyses using an antibody against the NF1/CTF protein identified the purified 32- and 34-kDa polypeptides as NF1 or a related protein. Cotransfection into Hep3B cells or primary rat hepatocytes of cDNAs of the NF1-like proteins NF1/L, NF1/X, and NF1/Redl resulted in a three- to fivefold increase in transcription directed by wild-type P2 but not by the mutated P2. Partial hepatectomy markedly decreased the levels of NF1 in the remnant liver and its binding to P2, which paralleled declines in the rate of transcription of the alpha(1b)AR gene and in the steady-state levels of its mRNA. These observations indicate that NF1 activates transcription of the rat alpha(1b)AR gene via interacting with its P2 promoter and that a decline in the expression of NF1 is one of the mechanisms responsible for the reduced expression of the alpha(1b)AR gene during liver regeneration.

By utilizing the oxygen-sensitive Escherichia coli Mn-superoxide dismutase (Mn-SOD) promoter, we have developed a vector system that expresses high levels of cloned foreign genes. The promoter for the bacterial Mn-SOD, as well as both 5'-untranslated and transcriptional termination sequences were ligated to a synthetic linker containing two restriction enzyme cloning sites. The vector also contained the gene for beta-lactamase, which confers ampicillin resistance to the host bacterium and provides a selectable marker. After screening and selection, high level of expression was achieved by exposure to the superoxide-generating agent paraquat (methyl viologen) as the inducer. To test the vector, both native and mutated human Mn-SOD cDNAs were cloned and expressed, respectively. To determine the optimal concentration of inducer necessary for maximal expression, recombinant bacteria were exposed to increasing concentrations of paraquat and subsequently assayed for superoxide dismutase (SOD) activity. The highest expression was induced by 20 microM paraquat, and approached 50% of total soluble protein.

Primer extension and RNase protection analyses of the rat beta 2-adrenergic receptor (beta 2AR) gene identify two transcription start points at -64 and -220 nt, respectively. Transient transfections of putative promoter/pCAT constructs into DDT1 MF-2 cells indicate that fragments -36 to -100 (PI) and -186 to -312 (P2) are sufficient to promote transcription, whereas -911 to -1122 contains a negative regulatory element(s). RNase protection analysis of the 3' untranslated region (3'-UTR) indicates the presence of two transcripts with 3'-UTR of 111 and 604 nt exclusive of the poly(A+) tails. Northern blots of beta 2AR mRNA using full-length and partial cDNA probes indicate that a major 2.2 kb and a minor 1.6 kb species arise from the use of alternative promoters as well as different polyadenylation signals. DNase I footprinting and DNA mobility shift assays (DMSA) using rat liver nuclear extracts identify a number of transcription factors binding to sequence elements within or upstream from P1 and P2, including Spl, CRE, CPl, AP-2, NF-1, NF-kappa B, and C/EBP. Supershift assays using antibodies against C/EBP alpha and C/EBP beta and mutational analyses indicate that the protein binding to the C/EBP consensus recognition site at -925 to -933 is C/EBP alpha. The activity of promoter/CAT constructs containing the C/EBP recognition site is significantly decreased by cotransfection of C/EBP alpha but not C/EBP alpha but not C/EBP beta into either DDT1 MF-2 cells or primary rat hepatocytes. Partial hepatectomy causes a transient decrease in C/EBP alpha, as measured by DMSA, and an increase in beta 2 AR mRNA levels and rate of transcription in the remnant liver. Thus, derepression via C/EBP alpha is likely involved in the up-regulation of beta 2AR in the regenerating rat liver.

Combinatorial peptide libraries have proved to be a valuable tool for the study of the interaction of a functional protein with its ligand. Here, the epitope for a monoclonal antibody 201/9, raised against beta-factor XIIa, has been identified with a two-step approach using peptide libraries attached to a polymer (polyvinylidene difluoride) membrane. First, the octapeptide libraries with two amino acids defined at position 2 and 4, represented by the formula X-O2-X-O4-X-X-X-X, were synthesized on a sheet of polymer membrane in which X represents a mixture of all the natural -amino acids except cysteine, while O2 and O4 each represent a single amino acid. The libraries were probed with the antibody 201/9, and the bound antibody was detected with a sensitive chemiluminescent method. In the first cycle, the peptide mixtures X-Phe-X-Gln-X-X-X-X showed the strongest signal development. In the second cycle Phe and Gln were incorporated into new libraries consisting of sequences O1-Phe-X-Gln-X-X-X-X, X-Phe-O3-Gln-X-X-X-X, X-Phe-X-Gln-O5-X-X-X, X-Phe-X-Gln-X-O6-X-X, X-Phe-X-Gln-X-X-O7-X, and X-Phe-X-Gln-X-X-X-O8. After probing these new peptides, the residues representing the core sequence of the epitope for monoclonal antibody 201/9 were elucidated. The sequence Ser-Phe-Leu-Gln-Glu-Asn, identified as the immunodominant epitope, correlates well with the sequence Ser-Phe-Leu-Gln-Glu-Ala previously identified (Gao, B., and Esnouf, M. P. (1996) J. Immunol. 157, 183-188) in a scan of overlapping peptides based on the sequence of human beta-factor XIIa.

Constitutive 70-kDa heat shock protein (hsc70) is a mixture of monomers and oligomers in ADP, while in ATP it is monomeric unless certain DnaJ homologs are present which induce hsc70 to form large polymers in an ATP-dependent reaction. A key question regarding polymerized hsc70 is whether it is able to bind protein substrates. Polymerized BiP, the hsc70 present in the endoplasmic reticulum, has been found to bind substrates in vitro although substrates appear to bind only to monomeric BiP in vivo. In this study, we investigated whether substrate binds to polymerized cytoplasmic hsc70 in vitro. Although both stoichiometric ATP and high concentrations of cytochrome c peptide monomerized hsc70, direct binding studies provided no evidence that cytochrome c peptide binds to polymerized hsc70. Furthermore, the time course of cytochrome c peptide and clathrin binding to hsc70 suggested that rather than binding to polymerized hsc70, they monomerized it by reducing free monomer, thereby shifting the monomer-polymer equilibrium toward monomer. We conclude that peptide and protein substrates bind at least an order of magnitude more weakly to polymerized hsc70 than to monomer, suggesting that polymerization of hsc70 in vivo, perhaps by DnaJ homologs, may store it in an inactive form.

The discontinuous epitopes of beta-factor XIIa for three mAbs were mapped by a linear peptide-based immunoblotting technique, referred to as multiple interactive residues of recognition. The Abs were incubated with a set of overlapping synthetic peptides, deduced from the cDNA sequences of beta-factor XIIa, on a polymer membrane, and the signal was amplified by an ECL assay. Several discrete sequences of the protein were recognized by each Ab. The recognized peptides were further characterized using alanine substitution analogues and peptides of different lengths. The discontinuous epitopes found for each Ab were formed by several peptides and were composed of 20 to 31 residues. The sequence FLQEA was recognized by all three Abs and was the immunodominant peptide. Abs 201/9 and 2/15 bound to very similar discontinuous sequences, but with subtle differences. The sequences 76RLHEAFSP83, 88HDLALLRLQE97, 178GFLEG182, 146FLQEA150, and 237IRE239 formed the epitope for mAb 201/9, whereas 76RLHEAFSP83, 90LALLRLQE97, 146FLQEA150, and 235AWIREHT241 formed the epitope for Ab 2/15. The third Ab 202/7 recognized the sequences 79EAFSP83, 93LRLQE97, 133WGHQF137, and 146FLQEA150. We suggest that these sequences represent the sites to which the Abs bind. This procedure provides a sensitive and convenient tool to elucidate discontinuous epitopes for the binding of Abs, receptor ligand-binding sites, or enzyme inhibitor binding sites.

We determined the Guanine Deaminase (GD) activity of 200 patients with different diseases. It was found that GD activity of hepatic patients is higher than that of health adults, while the GD activity of other patients is in the normal range. There is a linear correlation between GD activity and ALT in patients with chronic hepatitis, billiary obstruction, and between GD activity and total bilirubin in patients with chronic active hepatitis, biliary obstruction and liver cirrhosis. Moreover, the GD activity of patients positive for anti-HCV is significantly increased. So GD activity in serum is a specific and sensitive index to estimating hepatic functions and can be used in the diagnosis of acute and chronic hepatitis, cirrhosis of liver, and C virus hepatitis.

The rat suprachiasmatic nucleus (SCN) is comprised of neurons that contain gamma-amino butyric acid (GABA) colocalized with one or more peptides. In the present study, the authors employed in situ hybridization histochemistry to determine whether the human SCN also contains GABA neurons using synthetic oligonucleotide probes complementary to sequences of two isoforms of the GABA-forming enzyme, glutamic acid decarboxylase (GAD), GAD65, and GAD67. Most, if not all, SCN neurons appear to express both GAD65 mRNA and GAD67 mRNA with the content of GAD67 greater than GAD65. Both isoforms also are expressed in some neurons of the anterior hypothalamic area, in small neurons of the paraventricular nucleus but not in the supraoptic nucleus. These data indicate that neurons in the human SCN, like those in rodents, use GABA as a neurotransmitter.

Six flavonoids were isolated from the aerial part of Epimedium brevicornum and identified as baohuo side I, 2"' O-rhamnosyl icariside II, sagittatoside B, baohuoside II, ikarisoside F and ikarisoside C by means of UV, IR, 1H-NMR, 13C-NMR and FAB-MS spectral analysis, they were isolated from this plant for the first time.

Resistance tests, by conventional methods and by observing the time for knockdown, showed no evidence for any build up of resistance to deltamethrin in malaria vectors from areas where millions of bednets have been treated with this compound annually for up to 7 years. However, a strain of Culex quinquefasciatus which had been bred in a factory in which volatile pyrethroids are handled had developed unequivocal resistance to deltamethrin. Observation of the time for knockdown gave clearer discrimination between resistant and susceptible strains than did observation of percentage mortality after a standard exposure time.