Pattern and predictors of medical care received by hepatitis B carriers during pregnancy and after delivery.

OBJECTIVE: The objective of the study is to evaluate the pattern and predictors of medical care received by hepatitis B virus (HBV) carriers during pregnancy and after delivery in Hong Kong. STUDY DESIGN: The study is a retrospective analysis. METHODS: Pregnant HBV carriers and their infants were followed up for 9-12 months after delivery. Face-to-face interviews were conducted to investigate what medical care they received for HBV before, during and after pregnancy. RESULTS: Data were available for 412 HBV carriers. A total of 375 (91.0%) women were known HBV carriers before pregnancy. Routine antenatal screening picked out the remaining 37 (9.0%) HBV carriers; these women were younger, more likely to be smokers and had a lower level of education (P < 0.05) than known HBV carriers. In total, 356 of 412 (86.4%) HBV carriers did not receive any medical care for HBV during pregnancy. Known HBV carrier status, history of medical check-up and the use of antiviral treatment before pregnancy were significant predictors for HBV medical care during pregnancy (P < 0.05). The results show that 217 of 412 (52.6%) HBV carriers did not receive medical care for HBV after delivery. HBV medical care before pregnancy, use of antiviral treatment before pregnancy and a higher level of education were significant predictors for postpartum HBV medical care (P < 0.05). Multivariate analysis showed that HBV medical care before pregnancy (odds ratio [OR], 7.73; 95% confidence interval [CI], 3.21-18.65; P < 0.001) and the use of antiviral treatment (OR, 5.02; 95% CI, 1.41-17.81; P = 0.013) were associated with medical care during pregnancy. Medical care before pregnancy was also associated with postpartum HBV medical care (OR, 5.05; 95% CI, 3.29-7.51; P < 0.001). CONCLUSIONS: A significant proportion of HBV carriers did not receive HBV-related medical check-ups during and after pregnancy in Hong Kong despite the majority being aware of their carrier status. Medical care before pregnancy predicted antenatal and postpartum HBV medical care.

ß-catenin downregulates Dicer to promote ovarian cancer metastasis.

Ovarian cancer is a nearly uniform lethal disease and its highly aggressive metastatic phenotype portends a poor prognosis. Lack of a well-controlled, relevant experimental model has been a major obstacle to identifying key molecules causing metastasis. Here we describe the creation of a new isogenic model of spontaneous human ovarian cancer metastasis exhibiting opposite phenotypes-highly metastatic (HM) and non-metastatic (NM)-both in vitro and in vivo. HM was unique in its ability to metastasize consistently to the peritoneum, mimicking the major dissemination route of human ovarian cancer. In contrast, NM failed to form detectable metastases, although it was equally tumorigenic. Using comparative label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified ß-catenin, which we demonstrated for the first time as having a direct role in the pathogenesis of ovarian cancer metastasis. Our studies also revealed a previously unrecognized role of ß-catenin in the downregulation of multiple microRNAs (miRNAs) through attenuating miRNA biogenesis by targeting Dicer, a key component of the miRNA-processing machinery. One such downregulated miRNAs was miR-29s involved in epithelial-to-mesenchymal transition and subsequent stem cell traits. Silencing ß-catenin or overexpressing Dicer or miR-29 mimics in HM significantly reduced the ability of these cells to migrate. ß-catenin-knockdown cells also failed to metastasize in an orthotopic model of ovarian cancer. Meta-analysis revealed an increase in CTNNB1 and a decrease in DICER1 expression levels in the high-risk group. These results uncover ß-catenin as a critical factor in promoting ovarian cancer aggressiveness and a new mechanism linking between ß-catenin and miRNA downregulation underlying this process.

c-Kit mediates chemoresistance and tumor-initiating capacity of ovarian cancer cells through activation of Wnt/ß-catenin-ATP-binding cassette G2 signaling.

Cisplatin and paclitaxel are standard chemotherapy for metastatic ovarian cancer, but with limited efficacy. Cancer stem/progenitor cells (or tumor-initiating cells, TICs) are hypothesized to be chemoresistant, and the existence of TICs in ovarian cancer has been previously demonstrated. However, the key signals and molecular events regulating the formation and expansion of ovarian tumor-initiating cells (OTICs) remain elusive. Here, we show that c-Kit is not just a marker of OTICs, but also a critical mediator of the phenotype that can be a viable target for the treatment of ovarian cancer. In contrast to non-OICs, c-Kit was overexpressed in OTICs. Moreover, the use of small interfering RNA to inhibit c-Kit expression markedly attenuated the number and size of OTIC subpopulations, inhibited the expression of stem cell markers and decreased the tumorigenic capabilities of OTICs. Imatinib (Gleevec), a clinical drug that blocks c-Kit kinase activity, also demonstrated its inhibition potency on OTICs. In addition, cisplatin/paclitaxel, which killed non-OTICs, with c-Kit knockdown or imatinib revealed that this was critically required for intervening ovarian cancer progression and recurrence in vitro and in xenograft tumors in vivo. Similar results were obtained with OTICs derived from ovarian carcinoma patients. Studies into the mechanisms suggest an important role for the activation of Wnt/ß-catenin and ATP-binding cassette G2 downstream of c-Kit. The tumor-promoting microenvironment, such as hypoxia, could promote OTICs via upregulation of c-Kit expression. These results unravel an integral role for c-Kit in ovarian neoplastic processes and shed light on its mechanisms of action.

P-cadherin cooperates with insulin-like growth factor-1 receptor to promote metastatic signaling of gonadotropin-releasing hormone in ovarian cancer via p120 catenin.

Gonadotropin-releasing hormone (GnRH) is a potent prometastatic factor in ovarian cancer, but the intracellular signaling events are not well understood. The classical Gα(q)-phospholipase C signal transduction pathway known to operate in the pituitary is not involved in GnRH actions at non-pituitary targets. Here we showed that GnRH treatment of ovarian cancer cells led to a rapid and remarkable tyrosine phosphorylation of p120 catenin (p120(ctn)), which was mediated by P-cadherin. The use of P-cadherin small interfering RNA or neutralizing antibodies to inhibit P-cadherin expression and function resulted in diminished p120(ctn) activation, confirming that the effect was P-cadherin specific. On exploring how P-cadherin, which lacks intrinsic kinase activity, might regulate the activation of p120(ctn), we found that P-cadherin could induce the ligand-independent activation of insulin-like growth factor-1 receptor (IGF-1R). Inhibition of IGF-1R expression or its activity significantly inhibited GnRH-induced p120(ctn) activation, and the subsequent cell migration and invasion. In addition, we showed that IGF-1R regulation by P-cadherin was associated with complex formation between IGF-1R and P-cadherin, and this regulation was also observed to be in vivo correlated with metastasis. Furthermore, using a mouse model of ovarian cancer metastasis, GnRH receptor knockdown was shown to diminish peritoneal dissemination of tumors and ascites formation. These findings suggest for the first time that GnRH can initiate an outside-in p120(ctn) signal transduction through the cross-talk between P-cadherin and IGF-1R, thus providing a novel molecular mechanism by which GnRH may control the high level of aggressiveness and invasion and metastasis potential that are characteristic of ovarian cancer.

MAPK and PKC activity are not required for H(2)O(2)-induced arterial muscle contraction.

H(2)O(2)-induced pulmonary arterial smooth muscle (PASM) contractions are independent of Ca(2+) and myosin light chain phosphorylation. The purpose of this study was to determine whether mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1 and ERK2, or protein kinase C (PKC) activation is required for H(2)O(2)-induced contraction. Porcine PASM strips were stimulated with 1 mM H(2)O(2), 120 mM KCl, or 10 microM phorbol myristic acetate and freeze clamped at various times during the contractions. Changes in relative amounts of tyrosine/threonine phosphorylated MAPK compared with total MAPK were measured. MAPK tyrosine phosphorylation levels increased in correlation with tension development. However, 50 microM PD-98059, a MAPK/ERK kinase-MAPK kinase blocker, reduced MAPK phosphorylation below resting levels, even though the magnitude of the isometric tension development was unaltered. Freeze-clamped PASM strips were placed in a PKC activity assay buffer containing (32)P and CaCl(2) to measure the total myelin basic protein phosphorylation. The data show that: 1) the time courses of PKC activity and force produced in response to H(2)O(2) do not correlate, and 2) MAPK activation may be a concurrent event with, or a consequence of, tension development in response to a variety of agonists but is not responsible for contractions to H(2)O(2), high K(+), or phorbol esters.

Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction.

We have previously shown that p21-activated kinase, PAK, induces Ca(2+)-independent contraction of Triton-skinned smooth muscle with concomitant increase in phosphorylation of caldesmon and desmin but not myosin-regulatory light chain (Van Eyk, J. E., Arrell, D. K., Foster, D. B., Strauss, J. D., Heinonen, T. Y., Furmaniak-Kazmierczak, E., Cote, G. P., and Mak, A. S. (1998) J. Biol. Chem. 273, 23433-23439). In this study, we provide biochemical evidence implicating a role for PAK in Ca(2+)-independent contraction of smooth muscle via phosphorylation of caldesmon. Mass spectroscopy data show that stoichiometric phosphorylation occurs at Ser(657) and Ser(687) abutting the calmodulin-binding sites A and B of chicken gizzard caldesmon, respectively. Phosphorylation of Ser(657) and Ser(687) has an important functional impact on caldesmon. PAK-phosphorylation reduces binding of caldesmon to calmodulin by about 10-fold whereas binding of calmodulin to caldesmon partially inhibits PAK phosphorylation. Phosphorylated caldesmon displays a modest reduction in affinity for actin-tropomyosin but is significantly less effective in inhibiting actin-activated S1 ATPase activity in the presence of tropomyosin. We conclude that PAK-phosphorylation of caldesmon at the calmodulin-binding sites modulates caldesmon inhibition of actin-myosin ATPase activity and may, in concert with the actions of Rho-kinase, contribute to the regulation of Ca(2+) sensitivity of smooth muscle contraction.

Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle.

Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca2+ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160(ROCK)) can induce Ca2+-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 +/- 12% (n = 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca2+-independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.

Autoradiographic localization of 2[125I]iodomelatonin binding sites in the gastrointestinal tract of mammals including humans and birds.

In-vitro autoradiography was utilized to compare the distribution of 2[125I]iodomelatonin binding sites or putative melatonin receptors in the gastrointestinal tracts of humans, guinea pigs, mice, rats, hamsters, rabbits, ducks, chickens, pigeons, and quail. In humans, binding was detected in the mucosa of the colon, caecum, appendix, and on their blood vessels but not in the ileum. In the other mammals, significant binding was only demonstrated in the mucosa of the rabbit rectum, mouse colon, mouse rectum, and guinea pig ileum. The distribution of 2[125I]iodomelatonin binding in the avian gut varied with species. In the esophagus, binding was present in the lamina propria and blood vessels of all four birds. However, only the lamina propria of the chicken and quail proventriculus and ventriculus showed positive binding. For the duodenum and ileum, binding was very strong in the duck lamina propria, weak in the chicken lamina propria, and absent in the quail. In contrast, the pigeon muscle layer was weakly positive. The most striking species difference was found in the caecum where the duck lamina propria showed very strong binding, while the chicken lamina propria was only weakly positive. Conversely, the caecal muscle layer was strongly positive in chicken and quail but negative in duck and pigeon. In the rectum, a similar but less intense pattern of distribution was observed. The tremendous diversity in the distribution of 2[125I]iodomelatonin binding sites in the gastrointestinal tract is in accord with the hypothesis that melatonin may serve different functions in the gut of different species.

NMR studies of caldesmon-calmodulin interactions.

The binding of the calcium-regulatory protein calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. Two regions of caldesmon have been identified as putative calmodulin-binding domains. We have earlier reported on the binding of one of these domains to calmodulin (Zhang & Vogel (1994) Biochemistry 33, 1163-1171). Here we have studied the binding of CaM to synthetic peptides of CaD which contain: (1) both the first and second CaM-binding domains; (2) the second CaM-binding domain; and (3) the sequence between the first and second CaM-binding domains. Two-dimensional transferred nuclear Overhauser enhancement proton NMR measurements as well as circular dichroism studies of a 22-residue peptide NKETAGLKVGVSSRINEWLTK, which contains the second CaM-binding domain, show that only the C-terminal half of the peptide becomes alpha-helical upon binding to CaM. Somewhat surprisingly, the shorter 9-residue peptide SRINEWLTK was sufficient to form a 1:1 complex with CaM; this peptide appears to bind as a 3(10)-helix. Proton-carbon-13 correlation NMR titration studies with specifically labeled [methyl-13C]methionine CaM were used to study the participation of the hydrophobic regions in both domains of the dumbbell shaped CaM in peptide binding. Binding of a 54-residue CaD peptide containing both CaM-binding domains affects all the 8 Met residues in the two hydrophobic domains of CaM (only Met 76 in the linker region of CaM is not involved), while binding of the second CaM-binding domain of CaD influences principally Met 51, 71, and Met 124, 144. Simultaneous binding to CaM of two peptides comprising the first and the second CaM-binding domains also caused changes to all Met residues except Met 76. Taken together, these data demonstrate that both CaM-binding domains of CaD can bind simultaneously to the two hydrophobic regions of CaM.

Tryptophan residues in caldesmon are major determinants for calmodulin binding.

Calmodulin has been shown to interact with the COOH-terminal domain of gizzard h-caldesmon at three sites, A (residues 658-666), B (residues 687-695), and B' (residues 717-725), each of which contains a Trp residue [Zhan et al. (1991) J. Biol. Chem. 266, 21810-21814; Marston et al. (1994) J. Biol. Chem. 296, 8134-8139; Mezgueldi et al. (1994) J. Biol. Chem. 269, 12824-12832]. To determine the contribution of each of the three Trp residues in the calmodulin-caldesmon interaction, we have mutated the Trp residues to Ala in the COOH-terminal domain of fibroblast caldesmon (CaD39) and studied the effects on calmodulin binding by fluorescence measurements and using immobilized calmodulin. Wild-type CaD39 binds with a Kd of 0.13 x 10(-6) M and a stoichiometry of 1 mol of calmodulin per mol of caldesmon. Replacing Trp 659 at site A or Trp 692 at site B to Ala reduces binding by 22- and 31-fold (Kd = 2.9 x 10(-6) and 4.0 x 10(-6) M), respectively, and destabilizes the CaD39-calmodulin complex by 1.75 and 1.94 kcal mol-1, respectively. Mutation of both Trp 659 and Trp 692 to Ala further reduces binding with a Kd of 6.1 x 10(-6) M and destabilizes the complex by 2.17 kcal mol-1. On the other hand, mutation of Trp 722 at site B' to Ala causes a much smaller decrease in affinity (Kd = 0.6 x 10(-6) M) and results in a destabilization energy of 0.87 kcal mol-1. To investigate the relative importance of the amino acid residues near each Trp residue in the caldesmon-calmodulin interaction, deletion mutants were constructed lacking site A, site B, and site A + B. Although deletion of site A decreases binding of CaD39 to calmodulin by 13-fold (Kd = 1.7 x 10(-6) M), it results in tighter binding than mutation of Trp 659 to Ala at this site, suggesting that the residues neighboring Trp 659 may contribute negatively to the interaction. Deletion of site B causes a similar reduction in binding (Kd = 4.1 x 10(-6) M) as observed for replacing Trp 692 to Ala at this site, indicating that Trp 692 is the major, if not the only, binding determinant at site B. Deletion of both site A and site B drastically reduces binding by 62-fold. Taken together, these results suggest that Trp 659 and Trp 692 are the major determinants in the caldesmon-calmodulin interaction and that Trp 722 in site B' plays a minor role.

Skilled Hong Kong immigrants' intention to repatriate.

"An emphasis on skills in Australian immigration policy in the past decade has led to the increase of highly skilled Hong Kong immigrants. However, Australia has not been able to retain all of them.... This paper reports the results of an in-depth study on intention to repatriate and work in Hong Kong, conducted in Australia with 111 professional and managerial Hong Kong immigrants. Correlational and loglinear analyses on prediction of such an intention are presented. Research findings on the career-family dilemma experienced by a number of immigrants are likewise discussed."

Identification of C-type natriuretic peptide gene transcripts in glial cells.

C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, is found throughout the central nervous system (CNS), particularly in those regions involved in neuroendocrine regulation. Astrocytes, which have important physiological roles in normal neuronal functioning, express receptors of CNP. Using reverse transcription-polymerase chain reaction (RT-PCR), followed by hybridization with a digoxigenin-labelled cDNA probe, we have demonstrated the expression of CNP gene transcripts in both cultured mouse astrocytes and rat C6 glioma cells, with the former expressing the gene at a considerably higher level than the latter. Our data raise the possibility that CNP may act in autocrine and/or paracrine fashion in glial cell physiology and neuromodulate communication between glial cells and neurones.

Melatonin and 2[125I]iodomelatonin binding sites in the human colon.

2[125I]Iodomelatonin binding sites were identified in the mucosa of the human colon from Chinese patients with carcinoma of the rectum or colon using biochemical receptor assay and autoradiography. Melatonin in the colonic mucosa/submucosa and muscle layers were quantitated by radioimmunoassay. The binding of 2[125I]iodomelatonin to the membrane preparations of the human colonic mucosa/submucosa was stable, saturable, reversible and of high affinity. Rosenthal analysis from saturation studies performed at 21 degrees C yielded an equilibrium dissociation constant (Kd) of 61.7 +/- 4.48 pmol/L (n = 3) and maximum number of binding sites (B(max)) of 1.65 +/- 0.51 fmol/mg protein (n = 3). The linearity of the Rosenthal plots and unity of the Hill coefficient suggested that 2[125I]iodomelatonin was bound to a single class of binding sites. The radioligand binding was displaced by 2-iodomelatonin (Ki = 0.02 nmol/L), melatonin (0.65 nmol/L), 6-chloromelatonin (Ki = 5.33 nmol/L), 6-hydroxymelatonin (Ki = 33.8 nmol/L) and N-acetylserotonin (Ki = 122 nmol/L). The characteristic of the binding sites were similar to those reported in the jejunum of duck, chicken, and human but of higher affinity than those in the mouse colon. Autoradiography localizes the binding to the mucosa of the human colon. Radioimmunoassay revealed a melatonin concentration of 467 +/- 99 pg/g wet tissue of human colon (n = 6). Our findings suggest that melatonin may influence the human colonic functions through interaction with its receptors in the mucosa.

Regional myosin heavy chain expression in volume and pressure overload induced cardiac hypertrophy.

Although the overall shift towards the V3 myosin heavy chain (MHC) has been shown to be associated with cardiac hypertrophy, quantitative evidence describing regional expression is sparse. The aim of this study was to compare and contrast the regional ventricular myosin isoform expression in two distinct haemodynamic states: pressure and volume overload. Volume overload was achieved using an aortocaval fistula (ACF) model and pressure overload by two-kidney-one-clip (2K1C) hypertension. A separate group (UC-2K1C) had the clip removed 1 week prior to investigation. Sham operated rats (SHAM) served as controls. All groups were studied 4 weeks after surgery. Ventricular tissue samples (approximately 50 mg) were taken from the walls of the right ventricle (RV), septum and left ventricular (LV) free wall. Tissue samples (excluding RV) were divided into endocardium and epicardium, and myosin expression was determined using polyacrylamide gel electrophoresis. Cardiac hypertrophy was substantial in both LV (1.7-fold) and RV (1.9-fold) in ACF rats. The 2K1C rats had similar LV enlargement (1.6-fold) whereas RV hypertrophy was not as great (1.2-fold). Blood pressure (BP) was increased 65% in 2K1C rats, whereas there was no change in ACF rats with respect to SHAM animals. After unclipping (UC-2K1C), LV hypertrophy and BP had returned towards control levels. In general, V3 MHC expression was associated with increasing LV hypertrophy in both 2K1C and ACF models. However, there was a marked endo-epi differential (1.5:1) in the LV free wall and septum of 2K1C rats. In contrast, in ACF rats there was no differential V3 MHC expression in the LV or septal tissue, i.e. expression was similar in both endo- and epi-samples. Elevated expression of V3 MHC persisted despite normotension and regression of cardiac hypertrophy in UC-2K1C rats. Taken together with published results demonstrating that relative transmural myocyte hypertrophy in ACF rats (endo > epi) is in contrast to that seen in 2K1C rats (epi > endo), the present findings reveal that regional V3 myosin expression represents a distinct adaptational component of the overall cardiac hypertrophic response in both volume and pressure overload.

Alignment of caldesmon on the actin-tropomyosin filaments.

We have reported previously that each smooth-muscle caldesmon binds predominantly to a region within residues 142-227 of tropomyosin, but a weaker binding site also exists at the N-terminal region of tropomyosin [Watson, Kuhn, Novy, Lin and Mak (1990) J. Biol. Chem. 265, 18860-18866]. In view of recent evidence for the presence of tropomyosin-binding sites at both the N- and C-terminal domains of caldesmon, we have studied the binding of the N- and C-terminal fragments of human fibroblast caldesmon expressed in Escherichia coli to tropomyosin and its CNBr fragments. The N-terminal fragment, CaD40 (residues 1-152), binds tropomyosin, but the interaction is mostly abolished in the presence of actin. CaD40 binds strongly to Cn1B(142-281) of tropomyosin, but weakly to Cn1A(11-127). The C-terminal fragment, CaD39, which corresponds to residues 443-736 of gizzard caldesmon, binds tropomyosin, and the interaction is enhanced by actin. CaD39 binds to both Cn1A(11-127) and Cn1B(142-281) of tropomyosin. Our results suggest that the N-terminal domain of caldesmon interacts with the C-terminal half of one tropomyosin molecule, whereas the C-terminal domain binds to both N- and C-terminal regions of the adjacent tropomyosin molecule along the actin filament. In addition, the binding of the N-terminal domain of caldesmon to the actin-tropomyosin filament is weak, which may allow this domain to project off the thin filament to interact with myosin.

Audit of basal cell carcinoma in Princess Margaret Hospital, Hong Kong: usefulness of frozen section examination in surgical treatment.

A retrospective study was undertaken of 64 Chinese patients with primary (previously untreated) basal cell carcinoma (BCC) surgically treated by the plastic and reconstructive surgery team at the Princess Margaret Hospital, Hong Kong, from January 1988 to March 1994. Sixty-three (98%) were in the head and neck region, half on the nose. It was equally common in men (mean age 69 years) and women (mean age 67). Two women (3%) were younger than 35. The rate of complete excision increased after the introduction of frozen section examination. A complete excision rate of 89% (n = 57) was achieved by 1994. We conclude that frozen section examination should be done routinely in patients having BCC excised.

Use of preputial skin for the release of burn contractures in children.

Contractures are frequent sequelae of burn injuries. Progressive worsening of contractures with limitation of joint movement is common in children. The results of release procedures in 10 male Chinese children using their own preputial skin obtained from circumcision were reviewed. This technique yields reliable results and is well accepted by the parents.

Phosphorylation of the precursor sequence of rat B-type natriuretic peptide by p34cdc2 and MAP kinase.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), two distinct members of the natriuretic peptide family, share many features in common. However, differences in expression indicate that the processing mechanisms must be different. The leader sequence of rat BNP contains three potential phosphorylation sites for proline-directed kinases that are not present in the leader sequence of ANP. This study has examined how these sites are used by two somewhat different proline-directed kinases. A peptide containing these sites was phosphorylated in vitro by HeLa p34cdc2 kinase and by sea star p44mpk kinase at rates that were comparable to the rates with peptide substrates that are used to assay these enzymes. Sequence analysis of the phosphopeptide shows that both kinases phosphorylate only the two potential phosphorylation sites surrounding the cleavage site of the BNP precursor. The enzymatic potential for such a phosphorylation of BNP in cardiac tissue is demonstrated by immunoblots and kinase assays, showing that in fetal and in adult rat heart both the atria and the ventricles contain a mitogen-activated protein kinase homologue that can phosphorylate this preproBNP sequence.

Smooth-muscle mitogen-activated protein (MAP) kinase: purification and characterization, and the phosphorylation of caldesmon.

A single 42 kDa isoform of mitogen-activated protein (MAP) kinase is expressed in both embryonic and adult chicken gizzard. The gizzard MAP kinase, which cross-reacts with anti-p44mpk antibody, has been purified from adult chicken gizzard and partially characterized. The purification protocol employs phenyl-Sepharose, polylysine-agarose, hydroxyapatite, Mono-Q and phenyl-Superose column chromatography. The purified enzyme phosphorylates myelin basic protein and gizzard high-molecular-mass (h-)caldesmon. Sea-star p44mpk and gizzard MAP kinase phosphorylate h-caldesmon at identical sites at the C-terminal domain, as revealed by tryptic-peptide mapping of the phosphorylated protein. Phosphorylation of h-caldesmon by gizzard MAP kinase abolishes its interaction with polymerized tubulin. The specific activity of the purified gizzard kinase toward myelin basic protein is similar to that of brain tau kinase, but is only a fraction of that of activated sea-star p44mpk. This suggests that, although a large amount of MAP kinase is present in the gizzard, only a small percentage of the enzyme is activated normally. Autophosphorylation of the gizzard kinase, at least in part on tyrosine residues, activates its kinase activity.

MAP kinases from bovine brain: purification and characterization.

We have purified 42- and 44-kilodalton (kDa) isoforms of the mitogen-activated protein (MAP) kinase family from bovine brain. The kinases were assayed with myelin basic protein as the substrate and detected by anti-sea star p44mpk antibody. Purification was achieved using phenyl-Sepharose, polylysine-agarose, hydroxylapatite, and Mono-Q column chromatography. Both myelin basic protein and smooth muscle caldesmon, but not histone H1, served as good substrates. Based on chromatographic behaviors and specific activities toward myelin basic protein, it is likely that the 42-kDa brain isoform is similar to that of brain tau kinase. The 44-kDa enzyme, however, is a novel brain MAP kinase isoform not reported previously. Although it has been demonstrated that p44mpk can be activated in vitro through phosphorylation by the tyrosine kinase p56lck, neither of the brain kinases were significantly stimulated by the tyrosine kinases p56lck, p56lyn, or p59fyn. However, based on antibody cross-reactivity, a MAP kinase kinase is present in the crude brain extract. Both brain MAP kinases were capable of autophosphorylation which occurred, at least in part, on tyrosine residues. However, only the 44-kDa isoform showed a significant degree of coincident activation.