Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein in chick embryonic muscle cells in culture. The pattern of protein phosphorylation was found to change in differentiating chick embryonic myoblasts in culture. The extent of phosphorylation of 42-, 50-, and 100-kDa proteins increased while that of a 63-kDa protein declined in extracts of myoblasts that had been cultured for increasing periods. Of these, the increase in phosphorylation of the 100-kDa protein occurred most dramatically in extracts of myoblasts in an early stage of differentiation and was specifically inhibited by trifluoperazine (TFP) and other calmodulin (CaM) antagonists including chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). Treatment of increasing concentrations of TFP to culture medium also decreased the phosphorylation state of the 100-kDa protein and the degree of myoblast fusion in parallel. In addition, levels of both the kinase activity and the 100-kDa protein but not of CaM appeared to rise in the cells cultured for longer periods. These results suggest that (1) a Ca2+/CaM-dependent protein kinase is responsible for phosphorylation of the 100-kDa protein, (2) the TFP-mediated myoblast fusion block may be associated with the inhibitory effect of the drug against the kinase activity, and (3) the increase in phosphorylation state of the 100-kDa protein during myogenic differentiation is due to the rise in levels of the kinase and its substrate.


V4, a gene required for the transition from growth to development in Dictyostelium discoideum. The V4 gene of Dictyostelium discoideum is regulated in a nutrient-dependent manner and is deactivated immediately upon the onset of development. V4 is expressed only during growth, but its expression is not required for growth. We propose that the V4 gene product plays a role in the transition from growth to development. We have tested this hypothesis by antisense mutagenesis. Cells transformed with a V4 antisense construct contained no detectable endogenous V4 mRNA. These cells grew normally, but they failed to aggregate. Under conditions which normally promote development, V4 antisense transformants failed to deactivate vegetative-specific genes. These cells also were unable to induce the expression of the cAMP cell surface receptor, the cyclic nucleic phosphodiesterase, and contact sites A, all of which are normally induced under such conditions. Surprisingly, cells transformed with a V4 sense construct displayed a similar morphological and biochemical phenotype as the antisense cells, whereas cells transformed with the parental vector exhibited a normal biochemical and morphological phenotype. These results demonstrate that expression of the V4 gene during growth is required for the proper initiation of development.


Effect of free fatty acids on insulin receptor binding and tyrosine kinase activity in hepatocytes isolated from lean and obese rats. We demonstrated previously that high physiological concentrations of free fatty acids (FFA) rapidly decrease insulin binding, degradation, and action in isolated rat hepatocytes. In this study, hepatocytes from lean and obese Sprague-Dawley rats (Alab, Stockholm) were preincubated with or without 0.4 mM oleic acid, and the effect on insulin binding and tyrosine kinase activity was measured. In the absence of exogenous FFA, insulin binding was reduced in hepatocytes from obese compared with lean rats (mean +/- SE reduction 44 +/- 7%, n = 8, P less than 0.01). Furthermore, the inhibitory effect of oleic acid added to hepatocytes from lean rats (n = 8; 40 +/- 9%, P less than 0.01) was not seen in cells from obese rats. Treating obese rats with Etomoxir, a carnitine palmitoyl transferase I inhibitor, increased insulin binding to isolated hepatocytes by 41 +/- 13% (n = 5, P less than 0.05). There was no difference in total binding to partially purified insulin receptors from solubilized hepatocytes from lean and obese rats, whether cells were or were not preincubated with oleic acid. Tyrosine kinase activity of partially purified receptors from basal or insulin-stimulated cells was not affected by either obesity, treatment with Etomoxir, or preincubating the cells with oleic acid. Thus, both obesity and elevated ambient FFA levels are associated with impaired insulin cell surface binding to isolated hepatocytes, possibly through an effect of lipid oxidation on the internalization/recycling of the insulin-receptor complex without any perturbation of the receptor tyrosine kinase activity. The data suggest that the reduced insulin binding to hepatocytes from obese rats is due to elevated ambient FFA levels.


Tolbutamide inhibits cAMP-dependent phosphorylation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The activity of a bifunctional enzyme, liver 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (F-2,6-Pase), which regulates the level of liver fructose-2,6-bisphosphate (F-2,6-P2), the most potent activator of PFK, is modulated by its phosphorylation rate mainly catalyzed by cAMP-dependent protein kinase A (PKA). To elucidate the action mechanism of sulfonylurea on liver F-2,6-P2 production, effects of tolbutamide on PKA-dependent phosphorylation of purified liver PFK-2/F-2,6-Phase protein and on kinase and phosphatase activities of the purified enzyme were examined in vitro. The purified enzyme was phosphorylated in the presence of the catalytic subunit of PKA, and tolbutamide inhibited the enzyme phosphorylation catalyzed by PKA in a dose-dependent manner. By adding the same dosages of tolbutamide used in the phosphorylation experiment, reduced activity of PFK-2 and increased activity of F-2,6-Pase in the presence of PKA were restored to the levels observed in the absence of PKA. On the other hand, carboxytolbutamide, an inactive metabolite of tolbutamide, had little effect on enzyme phosphorylation and activity. Our results indicate that tolbutamide inhibits a phosphorylation of the liver PFK-2/F-2,6-Pase catalyzed by PKA along with an activation of PFK-2 and an inactivation of F-2,6-Pase, leading to liver F-2,6-P2 production.


Lymphadenopathy in sclerosing cholangitis: pitfall in the diagnosis of malignant biliary obstruction. We retrospectively reviewed abdominal computed tomographic (CT) studies from 20 patients with sclerosing cholangitis and found evidence of abdominal lymphadenopathy in 13 patients. Enlargement occurred primarily in areas draining the liver, such as the gastrohepatic ligament or celiac axis (N = 8), the porta hepatis (N = 7), and the pancreaticoduodenal region (N = 2). One patient had reactive adenopathy and retroperitoneal fibrosis. The presence of benign reactive lymphadenopathy in at least one intraabdominal location was confirmed by pathological examination of excised lymph nodes in seven patients. Malignancy was excluded by surgical exploration or clinical follow-up. We conclude that enlarged lymph nodes are a common finding by CT in patients with sclerosing cholangitis. Enlarged reactive lymph nodes in this condition should not be mistaken for evidence of periportal metastasis or cholangiocarcinoma.


Parenchymal changes of the liver in cholangiocarcinoma: CT evaluation. We evaluated parenchymal changes of the liver in 92 patients (41 peripheral types and 51 hilar types) with cholangiocarcinomas studied by bolus-enhanced computed tomography (CT). In 39% of patients with the peripheral type, a wedge-shaped increased enhancement of the liver was observed peripheral to the tumor on bolus-enhanced CT. Tumor was observed in all cases. In 58.8% of patients with the hilar type, a segmental or lobar increased degree of enhancement of the liver was observed, but the tumor was demonstrated in only 58.8%. Atrophy was accompanied by areas of increased enhancement in 80% of hilar type and 25% of peripheral type. Areas of increased degree of enhancement corresponded to a wedged-shaped perfusion defect on CT during arterial portography. On magnetic resonance imaging (MRI), those lesions showed hyperintensity on T2-weighted images. Most of these changes were considered to be due to reversible hepatic parenchymal ischemia secondary to portal vein invasion by the tumor.


Hepatic vascular and bile duct injury after ethanol injection therapy for hepatocellular carcinoma. Three patients with serious hepatic vascular injury after percutaneous ethanol injection therapy for hepatocellular carcinoma (HCC) are described. In two cases, immediately after ethanol injection, liver function tests deteriorated and computed tomography (CT) showed a wedge-shaped low-density area due to liver infarction. In the other case, 1 year after ethanol injection, the intrahepatic bile duct in the right lobe was dilated and the right hepatic lobe atrophied. Endoscopic retrograde cholangiography showed stenosis of the main right hepatic duct. Since complications include both acute and delayed vascular injury after ethanol injection, patients should be followed for a long period after treatment.


Ruptured hepatoma: detection with lipiodol angiography. Hemorrhage from ruptured hepatoma is a serious complication. Transcatheter arterial embolization (TAE) is a choice of the treatment for control of hemorrhage. But conventional angiography may be useless for detection of bleeding site from ruptured hepatoma. We report two cases of ruptured hepatoma with detection of bleeding site by Lipiodol angiography.


Characterization of insulinlike growth factor I receptors in human colon cancer. A possible role of insulinlike growth factor I (IGF-I) and IGF-I receptors in the proliferation of human colon cancer has, to the best of the authors' knowledge, not been examined as yet. To determine a role of IGF-I in colon cancer, several human colon cancer cell lines and colon cancers were screened for specific binding to 125I-IGF-I. Almost all the human colon cancer cells examined were variably positive for specifically binding 125I-IGF-I. Almost half the colon cancer cell lines examined showed significant growth response to 6.6 nmol/L IGF-I. Dose-dependent growth effects of exogenously added IGF-I (0.05-3.3 nmol/L) were shown in a representative human colon cancer cell line (Colo-205). To determine if IGF-I binding sites on colon cancer cells were similar or different from that described on other cells, the binding affinity, binding specificity, and molecular size of the IGF-I binding sites were characterized in representative human colon cancer cell lines (HCT-116 and Colo-205). The optimal binding assay conditions for measuring maximum number of 125I-IGF-I binding sites on the cells, in vitro, were determined and found to be similar to that described on other cells. Scatchard analysis of the specific binding data showed the presence of a single class of high-affinity binding sites [disassociation constant (Kd) = approximately 1.0-2.0 nmol/L], with a binding capacity of 1.0-2.0 x 10(5) sites per cell. The molecular weight of IGF-I receptors on cell membranes was determined by gel electrophoresis of the affinity cross-linked proteins, followed by autoradiography. A single band of binding proteins with a molecular mass of approximately 300 kilodaltons was evident under nonreducing conditions and separated out into two bands of approximately 240 and approximately 130 kilodaltons under reducing conditions. The 130-kilodalton band represents the alpha subunit of IGF-I receptors, and the 240-kilodalton band may represent aggregates of the receptor subunits that were not reduced completely. The widespread existence of high-affinity binding sites for IGF-I in established human colon cancer cell lines and freshly resected human colon cancers and the proliferative effect of IGF-I in several colon cancer cell lines, in vitro, reflect that IGF-I may be an important endocrine/paracrine/autocrine factor in the growth regulation of colon cancers in situ.


Calcitonin gene-related peptide mediates the gastric hyperemic response to acid back-diffusion. Disruption of the gastric mucosal barrier with resultant increased acid back-diffusion leads to a marked increase in gastric mucosal blood flow (GMBF). This increase in GMBF is blocked by ablation of capsaicin-sensitive sensory neurons. The gastric arterioles are densely innervated by afferent neurons containing vasodilator peptides, calcitonin gene-related peptide (CGRP) being the most potent of these. We investigated (a) whether CGRP is the vasodilator mediator released by acid stimulation of capsaicin-sensitive sensory neurons and (b) whether the resultant hyperemia protects against the acid-induced mucosal injury. When the stomach was perfused with 0.15N HCl plus 15% ethanol, GMBF significantly increased by 70%. This hyperemic response was completely blocked by intra-arterial infusion of human CGRP8-37 (500 pmol/min), a CGRP-receptor antagonist, close to the stomach. With the blockade of the hyperemic response to acid back-diffusion, gross and histological mucosal damage were significantly aggravated. It is concluded that CGRP mediates the gastric hyperemic response to acid back-diffusion and that this gastric hyperemic response is an important protective factor against acid-induced injury.


Atriopeptins and Escherichia coli enterotoxin STa have different sites of action in mammalian intestine. Studies with Escherichia coli-induced heat-stable enterotoxin (STa), an activator of intestinal particulate guanylate cyclase, have established an independent role for cyclic guanosine monophosphate (cGMP) as an intracellular mediator of intestinal salt and water secretion. The present study addressed whether atriopeptins (APs), known activators of particulate guanylate cyclase in other tissues, function as physiological agonists for cGMP-linked Cl- secretion in intestine. APs, in contrast to STa, caused no or only minor changes in cGMP levels in freshly isolated rat intestinal villus and crypt cells and in cultured human colon carcinoma cell lines (HT29glc-, CaCo-2, and T84). Conversely, APs, but not STa, induced a large increase in intracellular cGMP levels in the undifferentiated small intestinal cell lines IEC-6, IEC-18, and INT407. Addition of AP II (atrial natriuretic peptide fragment 5-27) to stripped mucosa of rat proximal colon in Ussing chambers caused a transient increase in the transepithelial potential difference (PD), which most likely represents an increase in Cl- secretion. In contrast, a sustained increase in PD was observed in response to STa or 8Br-cGMP. The AP II-provoked increase in PD was blocked by the neurotoxin tetrodotoxin. Immunohistochemical detection of cGMP in this tissue provided evidence for a different localization pattern of cells responding with an increase in cGMP levels to STa (colonocytes and goblet cells) or AP (specific cells in the submucosa) in rat proximal colon. This indicates that APs, unlike STa, do not directly stimulate the colonic epithelial cells but possibly provoke Cl- secretion by release of a neurotransmitter in the submucosa.


Effect of colonic inflammation on mucin inhibition of Escherichia coli RDEC-1 binding in vitro. Intestinal mucus protects the underlying epithelium against adhesion and invasion by microbial pathogens and their products. In inflamed colonic mucosa there are both histochemical and biochemical changes in the major organic constituent of mucus, goblet cell-derived mucin. To determine if these changes result in differences in functional properties of mucin, inhibition of adherence of piliated Escherichia coli, strain RDEC-1 (serotype O15:H-), by mucin purified from distal colons of normal rabbits was compared with inhibition by mucin from colons of rabbits with dinitrochlorobenzene-induced colitis. Histology from inflamed colons revealed evidence of chronicity with a chronic inflammatory cell infiltrate, depletion of mucous glands, and branching of crypts. Mucin was purified from crude mucus of distal colonic mucosa by sequential ultracentrifugation and characterized by gel electrophoresis and amino acid analysis. The rabbit enteropathogen RDEC-1 was grown to promote expression of adherence pili. A nonpiliated mutant, strain M34, was used as a negative control. A concentration-dependent inhibition of piliated RDEC-1 binding was shown using mucins derived from both inflamed and noninflamed colons. However, equivalent dry weights of mucin purified from inflamed colons showed less inhibition of bacterial binding (10.3% inhibition +/- 5.2%; mean +/- SD) compared with mucin from normal colons (47.6% +/- 10.8%; P less than 0.05). Mucin purified from additional animals with hapten-induced colitis but treated with the oral leukotriene B4-receptor antagonist SC-41930 showed intermediate inhibition of RDEC-1 binding (35.6% +/- 14.3%). It is concluded that mucin derived from inflamed distal colon of rabbits failed to inhibit in vitro binding of piliated RDEC-1, and by decreasing mucosal inflammation, this functional alteration of mucin was partially reversed.


Verapamil alters eicosanoid synthesis and accelerates healing during experimental colitis in rats. In inflammatory bowel disease, prostaglandins are mucosal protective whereas leukotrienes are proinflammatory. Recent evidence suggests that the formation and action of leukotrienes are calcium-dependent, whereas the formation and action of prostaglandins are not. To examine the possibility that, because of differential regulation of arachidonic acid metabolism, calcium channel blockade might alter mucosal eicosanoid synthesis and accelerate healing during inflammatory bowel disease, we treated a 4% acetic acid-induced colitis model with verapamil and/or misoprostol and determined the effects on colonic macroscopic injury, mucosal inflammation as measured by myeloperoxidase activity, in vivo intestinal fluid absorption, and mucosal prostaglandin E2 and leukotriene B4 (LTB4) levels as measured by in vivo rectal dialysis. In colitic animals, verapamil treatment significantly improved colonic fluid absorption and macroscopic ulceration. This mucosal-protective effect of verapamil occurred in the presence of a twofold reduction in mucosal LTB4 synthesis. In noncolitic animals, verapamil alone had no effect on in vivo fluid absorption, macroscopic ulceration, or myeloperoxidase activity but did induce a threefold reduction in LTB4 synthesis in addition to shifting arachidonic acid metabolism towards a sixfold stimulation of prostaglandin E2 synthesis. Our results show that, when administered before the experimental induction of colitis, the calcium channel blocker, verapamil, has a mucosal-protective effect that occurs as a consequence of reduced mucosal leukotriene synthesis and increased prostaglandin synthesis. This differential regulation of arachidonic acid metabolism may play an important role in the development of novel therapeutic agents for inflammatory bowel disease.


Fecal and stomal bile acid composition after ileostomy or ileoanal anastomosis in patients with chronic ulcerative colitis and adenomatosis coli. Fecal bile acids (FBA) were analyzed by gas-liquid chromatography in 29 patients [17 with ulcerative colitis (UC) and 12 with adenomatosis coli (AC)] and 5 healthy volunteers. Seven UC and 9 AC patients had undergone total colectomy and J-shaped ileal pouch-anal anastomosis (JAA). The mean daily FBA output was similar for JAA and terminal ileostomy patients (approximately 370 mg/day), and was about 1.5 times that of healthy Japanese volunteers (approximately 235 mg/day). The output of unconjugated bile acids, secondary bile acids, and 7-dehydroxylated bile acid was higher in JAA patients with UC than in terminal ileostomy patients, but there were no significant differences between the groups. The total fecal bacterial count in JAA patients was 10 times that in terminal ileostomy patients but was 1/10 of that in healthy volunteers. In patients with defunctioning high ileostomy, FBA output increased markedly (maximum, 3054 mg/day), and serum cholesterol levels were also significantly lower (P less than 0.05). These results suggest that after JAA the bile acid metabolism and fecal bacterial flora undergo more normalization than after terminal ileostomy.


Absence of hepatitis C viral RNA from saliva and semen of patients with chronic hepatitis C. Body fluids from 14 patients with chronic hepatitis C were analyzed for the presence of hepatitis C viral RNA using the polymerase chain reaction. Nucleic acids were extracted from serum, saliva, and semen in guanidinium thiocyanate, subjected to reverse transcription, and then amplified by a double polymerase chain reaction technique using "nested" primers from the highly conserved 5' non-coding region of the hepatitis C virus genome. Hepatitis C viral RNA was found in the serum of all patients in titers ranging between 10(-1) and 10(-3). The hepatitis C viral genome was not detected in any saliva or semen sample. These findings suggest that body fluids of patients with chronic hepatitis C are rarely, if ever, contaminated with the hepatitis C virus. This may help to explain the infrequent transmission of this disease by sexual or close physical contact.


Atrial natriuretic factor in experimental cirrhosis in rats. Atrial natriuretic factor (ANF) is a cardiac hormone with potent natriuretic, diuretic, and vasorelaxant properties. Although abnormalities in ANF release, plasma level, and renal receptors have been described in humans and/or animals with cirrhosis and ascites, little is known about the ANF hormonal system in cirrhosis without ascites. The aim of this study was to examine the ANF hormonal system in an animal model of cirrhosis to determine whether compensated cirrhosis is associated with changes in the ANF hormonal system. Pair-fed rats were studied 5-7 weeks after either sham surgery or bile duct ligation. Bile duct-ligated (BDL) rats had elevated portal pressure and cirrhosis but did not have ascites. ANF messenger RNA levels were increased onefold in the atria of BDL rats. Sham-operated and BDL rats had similar plasma ANF levels. Competitive binding inhibition studies of isolated glomeruli showed a single class of receptors in sham-operated and BDL rats. The equilibrium dissociation constant was similar in sham-operated (0.51 nmol/L) and BDL (0.63 nmol/L) rats. Glomerular ANF receptor density increased significantly in BDL rats. Cyclic guanosine monophosphate generation in isolated glomeruli in response to 100 nmol/L ANF decreased slightly but not significantly in BDL rats. It was concluded that the ANF hormonal system is altered in cirrhosis without ascites; atrial messenger RNA level and glomerular ANF receptor density are increased.


Sequence analysis of active mariner elements in natural populations of Drosophila simulans. Active and inactive mariner elements from natural and laboratory populations of Drosophila simulans were isolated and sequenced in order to assess their nucleotide variability and to compare them with previously isolated mariner elements from the sibling species Drosophila mauritiana and Drosophila sechellia. The active elements of D. simulans are very similar among themselves (average 99.7% nucleotide identity), suggesting that the level of mariner expression in different natural populations is largely determined by position effects, dosage effects and perhaps other factors. Furthermore, the D. simulans elements exhibit nucleotide identities of 98% or greater when compared with mariner elements from the sibling species. Parsimony analysis of mariner elements places active elements from the three species into separate groups and suggests that D. simulans is the species from which mariner elements in D. mauritiana and D. sechellia are most likely derived. This result strongly suggests that the ancestral form of mariner among these species was an active element. The two inactive mariner elements sequenced from D. simulans are very similar to the inactive peach element from D. mauritiana. The similarity may result from introgression between D. simulans and D. mauritiana or from selective constraints imposed by regulatory effects of inactive elements.


Dosage compensation of the copia retrotransposon in Drosophila melanogaster. Dosage compensation in Drosophila has been studied at the steady state RNA level for several single-copy genes; however, an important point is addressed by analyzing a repetitive, transposable element for dosage compensation. The two issues of gene-specific cis control and genomic position can be studied by determining the extent of dosage compensation of a transposable element at different chromosomal locations. To determine whether the multicopy copia transposable element can dosage compensate, we used the X-linked white-apricot (wa) mutation in which a copia element is present. The extent of dosage compensation was determined for the white and copia promoters in larvae and adults in two different genomic locations of the wa allele. We conclude that copia is able to dosage compensate, and that the white promoter and the copia promoter are not coordinate in their dosage compensation abilities when assayed under these various conditions. Thus, two transcriptional units, one within the other, both of which are able to dosage compensate, do so differently in response to developmental stage and genomic position.


Molecular analysis of Ac transposition and DNA replication. Molecular events associated with transposition of the mobile element Activator (Ac) from the P locus of maize have been examined in daughter lineages of twinned sectors. Genetic and molecular analyses indicate that the donor Ac has excised from only one of the two daughter chromosomes in these lineages. Cloning and sequence analyses of target sites on daughter chromosomes indicate that Ac insertion can occur either before or after the completion of DNA replication. Transpositions from a replicated donor site to both unreplicated and replicated target sites imply that most transpositions of Ac occur during or shortly after the S phase of the cell cycle.


The yeast SFL2 gene may be necessary for mating-type control. We previously reported the isolation of the yeast suppressor gene for flocculation, SFL2 (TUP1). SFL2 gene disruption results in pleiotropic phenotypes; the sfl2 null mutation also causes a morphological change similar to shmoo in both the MAT alpha and MATa/alpha cells. The MAT alpha and MATa/alpha sfl2 null mutant cells incorporate chitin into the new growth zone in the same way as the alpha-factor-treated MATa cells. In order to clarify the molecular basis of this morphological change, we examined the effect of the sfl2 null mutation on the mRNA production of various genes involved in mating-type control. The transcripts of both the STE2 (an a-specific gene) and STE3 (an alpha specific gene) genes are detected in the MAT alpha and MATa/alpha cells carrying the sfl2 null mutation. In addition, mRNA of the GPA1 gene (haploid-cell-specific gene) is also detected in the MATa/alpha sfl2 cells. However, there is no significant difference in the levels of the MAT alpha 2 and MATa1 transcripts. These results suggest that the SFL2 gene product may be necessary for alpha 2 and a1-alpha 2 repression.


Purification and N-terminal amino acid sequences of two polypeptides encoded by the mcrB gene from Escherichia coli K-12. This report provides a purification method for the two proteins, 51 kDa and 33 kDa, both encoded by the same mcrB gene of the McrBC restriction system in Escherichia coli K-12. The two proteins were produced in large quantity using a T7 expression system and copurified to near homogeneity by DEAE-Sepharose and Affi-Gel blue column chromatography. The N-terminal amino acid sequences of these purified McrB proteins were the same as those predicted from the mcrB DNA sequence by Ross et al. [J. Bacteriol. 171 (1989b) 1974-1981]. The 33-kDa protein totally overlaps the C-terminal part of the 51-kDa protein.


Hydroxypyridinium collagen cross-links in human liver fibrosis: study of alveolar echinococcosis. Liver samples from patients with three different types of liver diseases, alveolar echinococcosis (a dense and irreversible fibrosis), hepatocellular carcinoma and alcoholic cirrhosis, were analyzed for their content in hydroxypyridinium cross-links found in mature collagen. We demonstrated the presence of small amounts of pyridinoline in control livers (0.27 +/- 0.06 pmol/pmol of collagen). Pyridinoline content was increased in fibrotic livers, with the highest values found in patients with alveolar echinococcosis (up to 1.33 pmol/pmol of collagen). The deoxy analogue of pyridinoline was not detected in either normal or fibrotic livers. Pyridinoline levels, expressed as picomoles per picomole of collagen, were similar in all patients with carcinoma (0.7 +/- 0.05 pmol/pmol of collagen). They were heterogeneous in patients with alveolar echinococcosis and were particularly high in patients with alcoholic cirrhosis (1.04 +/- 0.11 pmol/pmol of collagen). These results demonstrate for the first time the presence of an hydroxypyridinium cross-link in liver fibrosis and suggest that pyridinoline measurement might be an important criterion in assessing the irreversibility of human liver fibrosis.


Survival and prognostic factors in 212 Italian patients with genetic hemochromatosis. Two hundred twelve Italian patients with genetic hemochromatosis (181 men, mean age 50 +/- 11 yr; and 31 women, mean age 49 +/- 10 yr) were followed for a median period of 44 mo (range = 3 to 218 mo). Alcohol abuse was present in 31 subjects (15%), and chronic HBV and HCV infection were seen in 19 (9%) and 35 (24%) of 145 cases tested, respectively. Twenty-four patients (11%) had concomitant beta-thalassemia trait. Liver biopsy revealed cirrhosis in 146 and a noncirrhotic pattern in the other 66. Perls' stain was degree III in 37 patients and IV in 171 patients. One hundred eighty-five patients underwent weekly venesection, and iron depletion was achieved in 122 cases after total iron removal of 3 to 41 gm. Death occurred in 44 patients after 3 to 198 mo and was due to hepatocellular carcinoma in 20 cases, liver failure in 10, extrahepatic cancer in six, heart failure in three and hemochromatosis unrelated causes in five. Cancer has developed in seven other patients still alive (hepatocellular in five and extrahepatic in two). No deaths were observed among noncirrhotic patients; cumulative survival rates in cirrhotic patients were 85%, 75%, 60% and 47% at 3, 5, 8 and 10 yr, respectively. Univariate analysis in the 146 cirrhotic patients showed that age greater than 60 yr, alcohol abuse, cardiomyopathy, skin pigmentation, portal hypertension, hypoalbuminemia, hypergammaglobulinemia and Child class B or C had significant negative prognostic value. At multivariate analysis, only alcohol abuse, gamma-globulins greater than 2.0 gm/dl and Child class B or C maintained their negative prognostic values (p less than 0.01, hazard ratio 2.7; p less than 0.001, hazard ratio 2.8; and p less than 0.001, hazard ratio 4.3, respectively).


A chromosome 17:7 translocation is associated with a hepatitis B virus DNA integration in human hepatocellular carcinoma DNA. Chronic hepatitis B virus infection is often associated with major structural rearrangements of both the integrated viral DNA and the associated cellular sequences. We present here the structure of a single-copy hepatitis B virus insert cloned from human hepatocellular carcinoma DNA recently reported to encode a novel transcriptional trans-activator function. The hepatitis B virus portion of the clone consists of two colinear fragments covering the X gene with its promoter and enhancer (nucleotides 717 to 1796) and a 3' truncated pre-S/S gene (nucleotides 2703 to 423). The lack of the entire pre-C/C gene caused a fusion of the 3' end of the X gene with sequences upstream from the pre-S gene. The structure of the integrated viral DNA fragments suggests insertion of hepatitis B virus replication intermediates into cellular DNA and subsequent recombination between these primary integrations to generate the final structure of the clone. The 5' and 3' cellular flanking sequences mapped to the centromeric alpha-satellite DNA of chromosome 17 and to the short arm of chromosome 7 (p14-pter), respectively, indicating that chromosomal translocation was associated with the hepatitis B virus DNA integration. Because this is the fourth case reported in which hepatitis B virus-associated rearrangements have affected chromosome 17, it is conceivable that a loss of important cellular genes (such as the p53 antioncogene on chromosome 17) may be a crucial step in hepatitis B virus-related hepatocarcinogenesis.


Three different patterns of hepatitis C virus infection in chimpanzees. The relationship between hepatitis C virus RNA and hepatitis C virus-associated antibodies (antibody against the putative capsid protein and C-100 antibody) was determined by nested polymerase chain reaction and enzyme-linked immunosorbent assay in serial serum samples obtained from eight chimpanzees experimentally infected with hepatitis C virus. Three different patterns emerged from the polymerase chain reaction data: the first (group 1) was acute resolving hepatitis with transient appearance of HCV RNA (two cases). The second (group 2) had chronic hepatitis with persistent hepatitis C virus RNA positivity (four cases) and the third (group 3) had chronic hepatitis with intermittent appearance of hepatitis C virus RNA (two cases). In four of eight animals, hepatitis C virus RNA was first detectable in serum 1 wk after inoculation. Although serum HCV RNA was detected in all infected chimpanzees, two were positive only for antibody against the putative capsid protein, whereas two were positive only for antibody to C-100 antigen. In four of eight cases, antibody against the putative capsid protein appeared earlier than did antibody to C-100 antigen, was detected just before or coincident with rising glutamate pyruvate transaminase values and remained positive for a long time even after recovery. Six of eight animals (75%) were still hepatitis C virus RNA positive 1 yr after inoculation, suggesting that the risk of development of the chronic carrier state is high in hepatitis C virus infection. Furthermore, there did not appear to be a good correlation between antibody titer in serum and hepatitis C virus infectivity titer.


Atrial natriuretic peptide in portal vein-ligated rats: alterations in cardiac production, plasma level and glomerular receptor density and affinity. The atrial natriuretic peptide hormonal system is altered to a variable degree in patients with cirrhosis. Portal pressure and portal-systemic shunting are also varied in cirrhosis. We used a portal vein-ligated rat model with predictable portal hypertension to study the effects of portal hypertension alone on the atrial natriuretic peptide hormonal system. Sham-operated rats were used as controls. Mean portal pressure was significantly increased in portal vein-ligated rats (portal vein-ligated rats, 21.7 +/- 0.74 cm H2O; sham-operated rats, 13.7 +/- 0.47 cm H2O; p less than 0.0001). Plasma atrial natriuretic peptide decreased 50% in the portal vein-ligated rats (p less than 0.0001). Atrial natriuretic peptide messenger RNA level was decreased by 40% to 60% in the left and right atria and in the ventricles of portal vein-ligated rats (p less than 0.05 for each chamber). Only one class of glomerular binding site was identified by competitive binding studies. The atrial natriuretic peptide glomerular receptor density increased in the portal vein-ligated rats (portal vein-ligated rats, 1,660 +/- 393; sham-operated 725 +/- 147 fmol/mg protein, p less than 0.02), whereas affinity decreased (portal vein-ligated, 1.69 +/- 0.49; sham-operated, 0.55 +/- 0.12 nmol/L, p less than 0.02). No difference was seen in the amount of cyclic GMP generated by atrial natriuretic peptide stimulation in isolated glomeruli from portal vein-ligated and sham-operated rats.(ABSTRACT TRUNCATED AT 250 WORDS)


PCR-assisted localization of the human SRPR gene. A polymerase chain reaction (PCR)-based assay has been designed that detects the presence of the human signal recognition particle receptor (SRPR) gene in inter-species somatic cell hybrids. By using hybrids containing various fragments of chromosome 11q, SRPR has been mapped to a chromosomal region flanked by the 11q23 and 11q24 breakpoints associated with the constitutional and neuroepithelioma (11;22) translocations, respectively.


Complementation of a DNA repair deficiency in six human tumor cell lines by chromosome 11. Human tumor cells, after x-irradiation during the G2 phase of the cell cycle, show an abnormally high frequency of persistent chromatid breaks and gaps resulting from deficient DNA repair. Addition of a single human chromosome 11 from normal fibroblasts by micro-cell fusion to cell lines from six different tumors resulted in efficient repair of the radiation-induced damage to the level in normal cells. For one of the cell lines, addition of the long arm of chromosome 11 was sufficient to restore repair efficiency. In four of the six tumor lines, restoration of efficient DNA repair by chromosome 11 was associated with tumor suppression in nude mice. These results suggest that chromosome 11 carries a DNA repair gene or genes that complement the repair deficiency of tumor cells and that this gene for at least one tumor is localized to the long arm.


PKU mutations R408Q and F299C in Norway: haplotype associations, geographic distributions and phenotype characteristics. Details are given concerning the phenylketonuria (PKU) mutations R408Q and F299C. Both mutations were identified among 47 PKU patients, derived from the Norwegian PKU registry. A novel PKU mutation (R408Q) was identified, by single-strand conformation polymorphism analysis, on six out of eight mutant haplotype 12 chromosomes and on none of the other PKU chromosomes. The F299C mutation occurred exclusively on mutant haplotype 8, and was the only mutation associated with this haplotype (on six chromosomes). One patient homozygous for each mutation was found. The patient homozygous for F299C manifested severe PKU, whereas the R408Q homozygote exhibited a mild PKU variant. Pedigree analysis of these families has not, so far, revealed consanguinity. Information on the place of birth of the relevant grandparents of the PKU patients with these mutations suggests that each of these mutations in Norway has originated from a common gene source.


Detection by DGGE of a new polymorphism closely linked to the adenomatous polyposis coli region. The EF5.44 locus is in close proximity to the chromosome 5 region to which the genetic defect responsible for familial adenomatous polyposis has been mapped. We have devised two oligonucleotides that promote the specific polymerase chain reaction (PCR) amplification of a 365-bp sequence in this region. Analysis by denaturing gradient gel electrophoresis of the resulting fragment has unravelled individual differences that could be identified as a single base pair change in a MnlI restriction site. This PCR assayable polymorphism increases the informativeness at this locus, and should be useful in the presymptomatic diagnosis of familial adenomatous polyposis.


Protein kinase C activation modulates tumour necrosis factor-alpha priming of human neutrophils for zymosan-induced leukotriene B4 release. Neutrophil (PMN) activation by the yeast component zymosan involves the complement receptor type 3 (CD11b/CD18). Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) augmented the zymosan-stimulated leukotriene B4 (LTB4) release from PMN, reaching a fourfold increase at 10(-9) M. Co-incubation of PMN with 10(-9) M rhTNF-alpha and staurosporine resulted in a further dose-dependent increase, which became significantly greater than a purely additive effect at a staurosporine concentration of 10 nM. This synergy was maintained at all doses of staurosporine tested. In addition, doses of phorbol 12-myristate 13-acetate (PMA) that do not activate protein kinase C (PKC) (below 10(-9) M) also augmented the zymosan-stimulated release of LTB4. However, doses of PMA above 10(-9) M progressively inhibited the response to levels below that of zymosan alone. Staurosporine at 50 nM completely prevented, and 10(-9) M rhTNF-alpha partially but significantly (P less than 0.02 at 10(-8) M PMA, P less than 0.01 at 10(-7) M PMA) reversed, this high-dose PMA inhibition. PKC activation thus opposes the priming effect of rhTNF-alpha on neutrophils, while PKC inhibition may enhance the ability of rhTNF-alpha to prime PMN for zymosan activation. The combined effect of rhTNF-alpha and staurosporine suggests an intracellular synergy rather than simply a direct action due to increased zymosan receptor expression. Thus there appear to be mechanisms whereby the responses of neutrophils may be augmented without activating PKC. Indeed, kinase activation may even exert a degree of feedback control that is antagonized by rhTNF-alpha treatment.


Cytokine-induced (interleukins-3, -6 and -8 and tumour necrosis factor-beta) activation and deactivation of human neutrophils. The effect of various cytokines [interleukin-3(IL-3), IL-6, IL-8, tumour necrosis factor-beta (TNF-beta)] on human neutrophils (PMN) was analysed with regard to the generation of leukotrienes and the involvement of guanosine triphosphate (GTP)-binding proteins (G proteins). Incubation of cytochalasin B-pretreated PMN with cytokines alone did not lead to a generation of leukotrienes. However, the cytokines affected the formyl-methionyl-leucyl-phenylalanine-(FMLP)-induced formation of leukotrienes in a time-dependent manner. Preincubation of the cells with the different cytokines for short periods (15 seconds at 37 degrees) enhanced the subsequent FMLP-induced leukotriene generation, whereas preincubation for prolonged times resulted in a reduced formation of leukotrienes. These results correlated with the respective G protein-associated guanosine triphosphatase (GTPase) activities within isolated membrane fractions. The present study indicates a modulation of the FMLP-induced leukotriene formation by diverse cytokines via interaction with the GTP-binding proteins.


Genetic analysis of the Tn21 mer operator-promoter. The mercury resistance operon, mer, of the transposon Tn21 is transcribed from two overlapping divergent promoters: PR for the regulatory gene, merR, and PTPCAD for the structural genes, merTPCAD. Transcription of merTPCAD is repressed in the absence of Hg(II) and activated in the presence of Hg(II) by the regulatory protein, MerR. In addition, MerR represses its own expression regardless of the presence of Hg(II). MerR binds as a dimer to a single region of dyad symmetry lying between the -35 and -10 hexamers of PTPCAD. Analysis of the expression of transcriptional fusions to hydroxylamine- and oligonucleotide-generated mutants of this divergent operator-promoter region identified key bases involved in MerR-dependent repression of PTPCAD and of PR and in activation of PTPCAD. Six of the seven mutants affecting the palindromic region were altered in their ability to bind the MerR protein in vitro as measured by fragment retardation assays. These differences in in vitro MerR binding correlated well with the in vivo measurements of repression or of activation. Bases identified as functionally relevant by this genetic analysis coincide extensively with those previously identified as relevant via in vivo footprinting. Four major points emerge from this analysis: (i) transition and transversion mutations within the spacer between the -10 and -35 hexamers of PTPCAD generally have little effect on the MerR-independent (i.e., unregulated) expression of either promoter; (ii) alteration of certain bases in the MerR-binding dyad affects repression of PTPCAD differently than repression of PR; (iii) certain dyad changes can impair activation of PTPCAD more severely than repression of this promoter; and (iv) mutations in the -10 hexamer of PTPCAD which also effect PR expression define one of two potential -10 hexamers in PR as actually functional in vivo.


Rhizobium leguminosarum CFN42 lipopolysaccharide antigenic changes induced by environmental conditions. Four monoclonal antibodies were raised against the lipopolysaccharide of Rhizobium leguminosarum bv. phaseoli CFN42 grown in tryptone and yeast extract. Two of these antibodies reacted relatively weakly with the lipopolysaccharide of bacteroids of this strain isolated from bean nodules. Growth ex planta of strain CFN42 at low pH, high temperature, low phosphate, or low oxygen concentration also eliminated binding of one or both of these antibodies. Lipopolysaccharide mobility on gel electrophoresis and reaction with other monoclonal antibodies and polyclonal antiserum indicated that the antigenic changes detected by these two antibodies did not represent major changes in lipopolysaccharide structure. The antigenic changes at low pH were dependent on growth of the bacteria but were independent of nitrogen and carbon sources and the rich or minimal quality of the medium. The Sym plasmid of this strain was not required for the changes induced ex planta. Analysis of bacterial mutants inferred to have truncated O-polysaccharides indicated that part, but not all, of the lipopolysaccharide O-polysaccharide portion was required for binding of these two antibodies. In addition, this analysis suggested that O-polysaccharide structures more distal to lipid A than the epitopes themselves were required for the modifications at low pH that prevented antibody binding. Two mutants were antigenically abnormal, even though they had abundant lipopolysaccharides of apparently normal size. One of these two mutants was constitutively unreactive toward three of the antibodies but indistinguishable from the wild type in symbiotic behavior. The other, whose bacteroids retained an epitope normally greatly diminished in bacteroids, was somewhat impaired in nodulation frequency and nodule development.


A single regulatory gene integrates control of vitamin B12 synthesis and propanediol degradation. The cob operon of Salmonella typhimurium encodes enzymes required for synthesis of adenosyl-cobalamin (vitamin B12). The pdu operon encodes enzymes needed for use of propanediol as a carbon source, including an adenosyl-cobalamin-dependent enzyme, propanediol dehydratase. These two operons both map near min 41 of the S. typhimurium linkage map and are transcribed divergently. Here we report that the cob and pdu operons form a single regulon. Transcription of this regulon is induced by either glycerol or propanediol. The metabolism of these compounds is not required for induction. Propanediol induces the regulon either aerobically or anaerobically during growth on poor carbon sources. Aerobically glycerol induces only if its metabolism is prevented by a mutational block such as a glpK mutation. Under anaerobic conditions, glycerol induces in both glpK+ and glpK mutant strains during growth on poor carbon sources. A new class of mutations, pocR, prevents induction of the cob/pdu regulon by either propanediol or glycerol and causes a Cob- Pdu- phenotype. The pocR gene is located between the cob and pdu operons and appears to encode a trans-acting protein that acts as a positive regulator of both operons. Transcription of the pocR regulatory gene is induced, even without the PocR protein, during aerobic growth on poor carbon sources and during anaerobic respiration. With the functional PocR protein, transcription of the pocR gene is autoinduced by propanediol but not by glycerol. The growth conditions that increase pocR gene expression correlate with growth conditions that allow high induction of the cob/pdu regulon. A model for control of this regulon suggests that the PocR protein is a transcriptional activator of both the cob and pdu operons and that both glycerol and propanediol can individually serve as effectors of the PocR protein. We suggest that global control mechanisms cause variation in the level of the PocR protein; an increased level of the PocR protein permits higher induction by propanediol or glycerol.


The poc locus is required for 1,2-propanediol-dependent transcription of the cobalamin biosynthetic (cob) and propanediol utilization (pdu) genes of Salmonella typhimurium. In this communication we present evidence that indicates that 1,2-propanediol (1,2-PDL) is a positive effector of the transcription of the cobalamin (vitamin B12) biosynthetic (cob) operon and of the 1,2-PDL utilization (pdu) genes. The stimulatory effects of 1,2-PDL were demonstrated using Mu d-lac transcriptional fusions to cob and pdu. Significantly increased levels of transcription of the cob and pdu operon fusions were measured in cultures grown under both anoxic and highly aerated conditions when 1,2-PDL was present in the culture medium. We have isolated mutants that carry lesions that render both pdu and cob transcription unresponsive to 1,2-PDL. These mutations were mapped to the region between cob and pdu (41 min), and they define the poc locus PDL and cobalamin). The poc locus is required for the positive regulatory effects of 1,2-PDL to be exerted. Complementation of Poc function was achieved in trans by an F' plasmid carrying the poc+ locus, suggesting that at least one diffusible product regulates the expression of cob and pdu in S. typhimurium.


Interaction between DNA and alpha/beta-type small, acid-soluble spore proteins: a new class of DNA-binding protein. DNA in spores of Bacillus and Clostridium species is associated with small, acid-soluble proteins (SASP) of the alpha/beta type; the presence of these proteins is a major factor in causing spore resistance to UV light, alpha/beta-type SASP did not bind to single-stranded DNA, single- or double-stranded RNA, or DNA-RNA hybrids in vitro. However, these proteins bound a variety of double-stranded DNAs and conferred protection against DNase cleavage. The binding of alpha/beta-type SASP to DNA saturated at a protein/DNA ratio (wt/wt) of 4:1 to 5:1, which is approximately 1 SASP per 4 bp. alpha/beta-type SASP-DNA interaction did not require divalent cations, was independent of pH between 6 and 8, and, for some SASP-DNA pairs, was relatively insensitive to salt up to 0.3 M. The relative affinity of alpha/beta-type SASP for different DNAs was poly(dG).poly(dC) greater than poly(dG-dC).poly(dG-dC) greater than plasmid pUC19 greater than poly(dA-dT).poly(dA-dT), with poly(dA).poly(dT) giving no detectable binding. This order in alpha/beta-type SASP-DNA affinities parallels the facility with which the DNAs adopt an A-like conformation, the conformation in alpha/beta-type SASP-DNA complexes. An oligo(dG).oligo(dC) of 12 bp was bound by alpha/beta-type SASP. While a 26-bp oligo(dG).oligo(dC) bound more tightly than the 12-mer, there was no significant increase in affinity for alpha/beta-type SASP with further increase in size of oligo(dG).oligo(dC). In contrast, binding of alpha/beta-type SASP to oligo(dA-dT).oligo(dA-dT) was minimal up to at least a 70-mer, and binding to poly(dA-dT).poly(dA-dT) was very cooperative. In addition to blocking DNase digestion, binding of alpha/beta-type SASP to DNA blocked (i) cleavage of the DNA backbone by hydroxyl radicals and orthophenanthroline-Cu2+, (ii) DNA cleavage by restriction enzymes, in particular those with specificity for GC-rich sequences; and (iii) in vitro transcription of some but not all genes. However, methylation of dG residues by dimethyl sulfate was not affected by alpha/beta-type SASP binding.


Genome maps of Campylobacter jejuni and Campylobacter coli. Little information concerning the genome of either Campylobacter jejuni or Campylobacter coli is available. Therefore, we constructed genomic maps of C. jejuni UA580 and C. coli UA417 by using pulsed-field gel electrophoresis. The genome sizes of C. jejuni and C. coli strains are approximately 1.7 Mb, as determined by SalI and SmaI digestion (N. Chang and D. E. Taylor, J. Bacteriol. 172:5211-5217, 1990). The genomes of both species are represented by single circular DNA molecules, and maps were constructed by partial restriction digestion and hybridization of DNA fragments extracted from low-melting-point agarose gels. Homologous DNA probes, encoding the flaAB and 16S rRNA genes, as well as heterologous DNA probes from Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, were used to identify the locations of particular genes. C. jejuni and C. coli contain three copies of the 16S and 23S rRNA genes. However, they are not located together within an operon but show a distinct split in at least two of their three copies. The positions of various housekeeping genes in both C. jejuni UA580 and C. coli UA417 have been determined, and there appears to be some conservation of gene arrangement between the two species.


Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides. It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase. These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center. Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga. This technique monitors the stretching frequency of CO bound at the oxygen binding site and can be used to characterize the oxidases in situ with membrane preparations. Oxidases that have a heme-copper binuclear center are recognizable by FTIR spectroscopy because the bound CO moves from the heme iron to the nearby copper upon photolysis at low temperature, where it exhibits a diagnostic spectrum. The FTIR spectra indicate that the binuclear center of the R. sphaeroides aa3-type cytochrome c oxidase is remarkably similar to that of the bovine mitochondrial oxidase. Upon deletion of the ctaD gene, encoding subunit I of the aa3-type oxidase, substantial cytochrome c oxidase remains in the membranes of aerobically grown R. sphaeroides. This correlates with a second wild-type R. sphaeroides is grown photosynthetically, the chromatophore membranes lack the aa3-type oxidase but have this second heme-copper oxidase. Subunit I of the heme-copper oxidase superfamily contains the binuclear center. Amino acid sequence alignments show that this subunit is structurally very highly conserved among both eukaryotic and prokaryotic species. The polymerase chain reaction was used to show that the chromosome of R. sphaeroides contains at least one other gene that is a homolog of ctaD, the gene encoding subunit I of the aa3-type cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)


Identification of a missing link in glycosylphosphatidylinositol anchor biosynthesis in mammalian cells. A large number of mammalian proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthetic intermediates of the GPI anchor have been identified in mammalian cells. The early GPI precursors are sensitive to phosphatidylinositol (PI)-specific phospholipase C (PLC). However, all of the later GPI precursors, which contain 1 or more mannose residues, are PI-PLC-resistant, suggesting that there is another unidentified precursor. Here, we report the identification of this missing link. This GPI precursor can only be labeled with glucosamine and inositol, and is resistant to PI-PLC but sensitive to GPI-phospholipase D. It accumulates in large quantity only in mutants which are defective in the addition of the first mannose residue to the elongating GPI core. Thus, fatty acylation of glucosaminylphosphatidylinositol, to render it PI-PLC-resistant, is an obligatory step in the biosynthesis of mammalian GPI anchor precursors.


Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin. We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2, of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was 67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells.


Enhancement of phospholipase C delta 1 activity in the aortas of spontaneously hypertensive rats. Spontaneously hypertensive rats (SHR), which develop hypertension approximately 10 weeks after birth, are considered to provide a good animal model for human essential hypertension. We report here that the abnormal activation of phospholipase C delta 1 (PLC-delta 1) may be one of the main causes of hypertension. Levels of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol are found to be higher in the aortas of 12-week-old SHR than in age-matched normotensive Wistar-Kyoto rats (WKY), although the levels in the aortas of 7-week-old SHR, which have normal blood pressure, are the same as in WKY. Moreover, PLC activity is also higher in the aortas of 12-week-old SHR. Judging from Western blot analysis and immunoabsorption of PLCs, this activation is found to be due to that of PLC-delta 1. PLC-delta 1 from rat aorta is expressed significantly from 7 to 12 weeks, which correlates with the development of hypertension in SHR. The activity of PLC-delta 1 in the aortas of 12-week-old SHR is more markedly activated at low Ca2+ concentration than that of age-matched WKY. These results suggest that the abnormal enhancement of PLC-delta 1 activity is responsible for accumulation of inositol 1,4,5-trisphosphate and diacylglycerol, leading to continuous hypertonicity of vascular smooth muscle in SHR. The activity of PLC-delta 1 in the aortas of 12-week-old SHR is significantly higher at low Ca2+ concentration than that of normotensive WKY.


Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes. The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.


A rabbit reticulocyte ubiquitin carrier protein that supports ubiquitin-dependent proteolysis (E214k) is homologous to the yeast DNA repair gene RAD6. The two isoforms of the 14-kDa ubiquitin carrier protein (E2(14k)) are unique among rabbit E2s in efficiently supporting ubiquitin-protein ligase (E3)-mediated ubiquitination of proteins destined for degradation. To begin determining the structural basis for this property, we have isolated a cDNA encoding the predominant reticulocyte isoform of the E2 from a rabbit skeletal muscle library. The sequence predicts a protein of 152 amino acids with a molecular weight of 17,293. Expression of the cDNA in Escherichia coli and purification of the recombinant protein revealed an E2 with high affinity for E3 and ubiquitin activating enzyme (E1). The latter high affinity interaction appears to be between the ubiquitin charged form of E1 and the uncharged form of E2 and does not result in a stable complex between these two enzymes. The predicted sequence shows regions of strong homology with other sequenced E2s, suggesting that these regions may be involved in binding to E1 and/or in ubiquitin transfer from E1, functions common to all E2s. Surprisingly, the E2(14k)) sequence is markedly more similar to Saccharomyces cerevisiae RAD6 (69% identity) than to its proposed homologs UBC4/UBC5 (38% identity). The sequence is identical to that recently reported for a human 17-kDa E2 which can complement rad6 mutants thereby identifying rabbit E2(14k) as a RAD6 homologue. The biochemical properties of this previously uncharacterized human 17-kDa E2 are now defined and its misassignment as a homologue of rabbit E2(17k) is corrected. Our findings resolve current confusion regarding relationships among E2s and define yeast RAD6, rabbit E2(14k), and the human 17-kDa E2 as a subclass of E2s which biochemically support E3-mediated conjugation and ubiquitin-dependent proteolysis and physiologically play a role in DNA repair.


Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol. In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.


Lysine 274 is essential for fructose 2,6-bisphosphate inhibition of fructose-1,6-bisphosphatase. Lysine 274 is conserved in all known fructose-1,6-bisphosphatase sequences. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray crystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphatase was mutated to alanine by the polymerase chain reaction, and the T7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of the enzyme were purified to homogeneity, and their specific activity, substrate dependence, and inhibition by fructose 2,6-bisphosphate and AMP were compared. While the mutant exhibited no change in maximal velocity, its Km for fructose 1,6-bisphosphate was 20-fold higher than that of the wild-type, and its Ki for fructose 2,6-bisphosphate was increased 1000-fold. Consistent with the unaltered maximal velocity, there were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The Ki for the allosteric inhibitor AMP was only slightly increased, indicating that Lys274 is not directly involved in AMP inhibition. Fructose 2,6-bisphosphate potentiated AMP inhibition of both forms, but 500-fold higher concentrations of fructose 2,6-bisphosphate were needed to reduce the Ki for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274----Ala mutant was evident at fructose 2,6-bisphosphate concentrations (approximately 100 microM) well below those that inhibited the enzyme, which suggests that fructose 2,6-bisphosphate interacts either with the AMP site directly or with other residues involved in the active site-AMP synergy. The results also demonstrate that although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2,6-bisphosphate than fructose 1,6-bisphosphate, probably because it binds the 2-phospho group of the former while other residues bind the 1-phospho group of the substrate. It is concluded that the enzyme utilizes Lys274 to discriminate between its substrate and fructose 2,6-bisphosphate.


Isolation, characterization, and amino acid sequences of auracyanins, blue copper proteins from the green photosynthetic bacterium Chloroflexus aurantiacus. Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. Auracyanin A was described previously (Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988) Biochemistry 27, 7858-7863) and is not glycosylated. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa. The amino acid sequences of the B forms are presented. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different. Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins. Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein. All three auracyanins exhibit a midpoint redox potential of +240 mV. Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer. Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center.


Glu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase. The fructose-2,6-bisphosphatase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to be structurally and functionally homologous to phosphoglycerate mutase. Both enzymes catalyze their reactions via phosphoenzyme intermediates which utilize an active site histidine as a nucleophilic phosphoacceptor and another histidine as a proton donor to the leaving group. Glu327 in the bisphosphatase domain of the rat liver bifunctional enzyme is conserved in all phosphoglycerate mutase structures and is postulated, by modelling studies, to be located in the active site. Glu327 was mutated to Ala, Gln, or Asp. The mutant and wild-type enzymes were expressed in Escherichia coli with a T-7 RNA polymerase-based expression system and purified to homogeneity by substrate elution from phosphocellulose. The Glu327 mutants had apparent molecular weights of 110,000 by gel filtration and had unaltered 6-phosphofructo-2-kinase activity. Circular dichroism showed that the secondary structure of the Glu327 mutant enzyme forms was the same as the wild-type enzyme. The maximal velocity of the fructose-2,6-bisphosphatase of the Glu327----Ala, Glu327----Gln, and Glu327----Asp mutants was 4, 2, and 20%, respectively, that of the wild-type enzyme, but the rate of phosphoenzyme formation of the mutants was reduced by at least a factor of 1000. In addition, the rate constants of phosphoenzyme hydrolysis for the Glu372----Ala and Glu327----Gln mutants were 2.7 and 1.3%, respectively, of the wild type, whereas the rate constant for the Glu327----Asp mutant was 60% of the wild-type value. Glu327 was not a substrate or product binding site determinant since the Km for fructose-2,6-bisphosphate and Ki for fructose-6-phosphate of the mutants were not appreciably changed. The results implicate Glu327 as part of a catalytic triad in fructose-2,6-bisphosphatase and suggest that it influences the protonation state of the active site histidine residues during phosphoenzyme formation and/or acts as a base catalyst to enhance the nucleophilic attack of water on the phosphoenzyme intermediate.


Permissible amino acid substitutions within the putative nucleoside binding site of herpes simplex virus type 1 encoded thymidine kinase established by random sequence mutagenesis [corrected]. We determined the essentiality of all amino acid replacements within an 11-codon sequence in the putative nucleoside-binding site of thymidine kinase encoded by herpes simplex virus type 1. This involved partial randomization of 11 codons in the gene to create a degenerate library, followed by genetic complementation using a tk- Escherichia coli strain and selection of unnatural active enzymes. We produced and tested 53,000 variants; of which 190 were found to be biologically active. Sequence analyses of functional variants revealed a high degree of flexibility in accommodating different types of amino acid substitutions in this region. However, no replacement was tolerated at proline-173, whereas tyrosine-172 could be replaced by only phenylalanine. To further define permissible substitutions at specified positions, we constructed a library with randomization at only four test codons. We produced and tested 600,000 variants; of which only 5 were active. Again proline-173 was conserved, and only tyrosine and phenylalanine were found at position 172. The identification of these conserved amino acids should provide important insights into the understanding of the structural basis of catalysis by this enzyme.


The energy transduction mechanism is different among P-type ion-transporting ATPases. Acetyl phosphate causes uncoupling between hydrolysis and ion transport in H+,K(+)-ATPase. H+,K(+)-ATPase, Na+,K(+)-ATPase, and Ca(2+)-ATPase belong to the P-type ATPase group. Their molecular mechanisms of energy transduction have been thought to be similar until now. Ca(2+)-ATPase and Na+,K(+)-ATPase are phosphorylated from both ATP and acetyl phosphate (ACP) and dephosphorylated, resulting in active ion transport. However, we found that H+,K(+)-ATPase did not transport proton nor K+ when ACP was used as a substrate, resulting in uncoupling between energy and ion transport. ACP bound competitively to the ATP-binding site of H+,K(+)-ATPase. The hydrolysis of ACP by H+,K(+)-ATPase was stimulated by cytosolic K+, the half-maximal stimulating K+ concentration (K0.5) being 2.5 mM, whereas the hydrolysis of ATP was stimulated by luminal K+, the K0.5 being 0.2 mM. Furthermore, during the phosphorylation from ACP in the absence of K+, the fluorescence intensity of H+,K(+)-ATPase labeled with fluorescein isothiocyanate increased, but those of Na+,K(+)-ATPase and Ca(2+)-ATPase decreased. These results indicate that phosphorylated intermediates of H+,K(+)-ATPase formed from ACP are not rich in energy and that there is a striking difference(s) in the mechanism of energy transduction between H+,K(+)-ATPase and other cation-transporting ATPases.


Low affinity superphosphorylation of the Na,K-ATPase by ATP. Pre-steady-state phosphorylation of purified Na,K-ATPase from red outer medulla of pig kidney was studied at 25 degrees C and an ample range of [tau-32P]ATP concentrations. At 10 microM ATP phosphorylation followed simple exponential kinetics reaching after 40 ms a steady level of 0.76 +/- 0.04 nmol of P/mg of protein with kapp = 73.0 +/- 6.5 s-1. At 500 microM ATP the time course of phosphorylation changed drastically, since the phosphoenzyme reached a level two to four times higher at a much higher rate (kapp greater than or equal to 370 s-1) and in about 40 ms dropped to the same steady level as with 10 microM ATP. This superphosphorylation was not observed in Na,K-ATPase undergoing turnover in a medium with Mg2+, Na+, and ATP, suggesting that it required the enzyme to be at rest. Superphosphorylation depended on Mg2+ and Na+ and was fully inhibited by ouabain and FITC. After denaturation the phosphoenzyme made by superphosphorylation had the electrophoretic mobility of the alpha-subunit of the Na,K-ATPase, and its hydrolysis was accelerated by hydroxylamine. On a molar basis, the stoichiometry of phosphate per ouabain bound was 2.40 +/- 0.60 after phosphorylation with 1000 microM ATP. The results are consistent with the idea that under proper conditions every functional Na,K-ATPase unit can accept two, or more, phosphates of rapid turnover from ATP.


Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells. ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.


Activated cGMP phosphodiesterase of retinal rods. A complex with transducin alpha subunit. Purified G-protein (transducin) activated with the nonhydrolyzable analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and cGMP phosphodiesterase (PDE) from retinal rods are added to protein-stripped disc membranes. Specific binding of the mainly soluble alpha subunit of G-protein with GTP gamma S bound (G alpha GTP gamma S, activator of the PDE) to the disc membrane in the presence of PDE is measured from gel scans or experiments with labeled G-protein alpha subunit (G alpha). Its variation as a function of G concentration matches the theoretical variation of G alpha involved in the activation of PDE calculated with previously estimated dissociation constants (Bennett, N., and Clerc, A. (1989) Biochemistry 28, 7418-7424), and the G alpha bound/PDE ratio at saturation is close to 2. No increase of G alpha binding to the membrane is observed when purified inhibitory subunit of PDE (PDE gamma) is added together with or instead of total PDE, and excess PDE gamma remains soluble. These results suggest that activated PDE is a complex with the activator G alpha GTP rather than PDE from which the inhibitory subunits have been removed. A method for purifying PDE gamma with a high yield of recovery and activity is described.


Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction. Nitrogenase binds and hydrolyzes 2MgATP yielding 2MgADP and 2Pi for each electron that is transferred from the iron protein to the MoFe protein. The iron protein alone binds but does not hydrolyze 2MgATP or 2MgADP and the binding of these nucleotides is competitive. Iron protein amino acid sequences all contain a putatitive mononucleotide-binding region similar to a region found in other mononucleotide-binding proteins. To examine the role of this region in MgATP interaction, we have substituted glutamine and proline for conserved lysine 15. The amino acid substitutions, K15Q and K15P, both yielded a non-N2-fixing phenotype when the genes coding for them were substituted into the Azotobacter vinelandii chromosome in place of the wild-type gene. The iron protein from the K15Q mutant was purified to homogeneity, whereas the protein from the K15P mutant could not be purified in its native form. Unlike wild-type iron protein, the purified K15Q iron protein showed no acetylene reduction, H2 evolution, or ATP hydrolysis activities when complemented with wild-type MoFe protein. The K15Q iron protein and the normal iron protein had a similar total iron content and both proteins showed the characteristic rhombic EPR signal resulting from the reduced state of the single 4Fe-4S cluster bridging the two subunits. Unlike the wild-type iron protein, addition of MgATP to the K15Q iron protein did not result in the perturbation necessary to change the EPR signal of its 4Fe-4S center from a rhombic to an axial line shape. Also unlike the wild-type iron protein, addition of MgATP to K15Q iron protein in the presence of the iron chelator, alpha,alpha'-dipyridyl, did not result in a time-dependent transfer of iron to the chelator. Thus, even though the K15Q iron protein contains a normal 4Fe-4S center, it does not respond to MgATP like the wild-type protein. Examination of the ability of the K15Q iron protein to bind MgADP showed no change from the wild-type iron protein, but its ability to bind MgATP decreased to 35% of the wild-type protein. Thus, in A. vinelandii iron protein, lysine 15 is not needed for interaction with MgADP but is involved in the binding of ATP, presumably through charge-charge interaction with the gamma-phosphate. Based on the above data, this lysine appears to be essential for the MgATP induced conformational change of wild-type iron protein that is required for activity.(ABSTRACT TRUNCATED AT 400 WORDS)


[3H]ryanodine as a probe of changes in the functional state of the Ca(2+)-release channel in malignant hyperthermia. The defect in malignant hyperthermia (MH) alters the binding of [3H]ryanodine to the Ca(2+)-release channel by increasing its apparent affinity for the binding site. In sarcoplasmic reticulum (SR) membranes from both normal and mutant pigs the apparent Kd is dependent on a number of parameters. Adenosine 5'-(beta,gamma-methylene)triphosphate, ionic strength, and Ca2+ each increase the apparent affinity of the binding site for [3H]ryanodine. Equilibrium and kinetic evaluation of the binding of [3H]ryanodine to these membranes demonstrates that the MH defect in pigs increases the apparent affinity of the membranes for [3H]ryanodine by increasing the amount of high affinity relative to low affinity binding sites. Both the association and dissociation of [3H]ryanodine with all three types of membranes (normal, heterozygous MH, homozygous MH) are characterized by two or more components, with the relative ratios of these components altered by the MH defect. These findings suggest that the observed Kd is the weighted average of the binding of ryanodine to two or more interconvertible states of the channel. Dilution of [3H]ryanodine bound to normal membranes at high Ca2+ into low Ca2+ solutions enhances the rate of dissociation. This conversion occurs to a much lesser extent with MH membranes, suggesting that the MH defect may alter the rate at which the high affinity form of the protein converts to the low affinity form.


Hexadecylphosphocholine inhibits translocation of CTP:choline-phosphate cytidylyltransferase in Madin-Darby canine kidney cells. The mechanism of the inhibition of phosphatidylcholine biosynthesis by the phospholipid analogue, hexadecylphosphocholine, was investigated in Madin-Darby canine kidney cells. In the presence of 50 mumol/liter hexadecylphosphocholine, there was a translocation of CTP:choline-phosphate cytidylyltransferase (EC 22.7.7.15) activity from the membranes to the cytosol of the cells. Since we recently demonstrated that hexadecylphosphocholine also inhibits protein kinase C in vitro, [methyl-3H]choline labeling experiments were repeated with phorbol ester-desensitized cells. In these cells the same inhibitory effect of hexadecylphosphocholine was measured. As a consequence of inhibition, the [methyl-3H]choline incorporation into the phosphocholine pool was increased time-dependently. In addition, there was no evidence for a difference between the choline uptake of control and hexadecylphosphocholine-treated cells. Likewise, the amount of diacylglycerol, a known activator of the translocation process, was not reduced. Finally, we showed that the inhibitory effect of hexadecylphosphocholine on CTP:choline-phosphate cytidylyltransferase translocation cannot be explained by the detergent properties of this phospholipid analogue. Therefore, we suggest a direct inhibitory effect of hexadecylphosphocholine on the translocation of CTP:choline-phosphate cytidylyltransferase.


Alanine scanning mutagenesis and functional analysis of the fibronectin-like collagen-binding domain from human 92-kDa type IV collagenase. The human 72-kDa (CLG4A) and 92-kDa (CLG4B) type IV collagenases contain a domain consisting of three contiguous copies of the fibronectin (FN)-derived type II homology unit (T2HU), T2HU-1, T2HU-2, and T2HU-3. To investigate the functional role of this domain, we have constructed plasmids expressing beta-galactosidase fusion proteins with one or more of the CLG4B-derived T2HU. The gelatin binding assays demonstrate that a single copy of T2HU-2 renders beta-galactosidase capable of binding gelatin. The three repeats, however, differ dramatically in their capacity to bind gelatin, with T2HU-1 and T2HU-3 having significantly less binding activity than T2HU-2. Using alanine scanning mutagenesis we have defined the amino acid residues (Arg307, Asp309, Asn319, Tyr320, Asp323) that are critical for gelatin binding of T2HU-2. The low gelatin binding of T2HU-1 compared to T2HU-2 was traced to the non-conserved residues Ala228-Ala and Leu253-Pro. The results suggest that the gelatin binding of the type IV collagenase proenzyme is mediated by the FN-like domain, although the presence of another gelatin-binding site cannot be excluded. The FN domain-mediated binding, however, is not a rate-limiting step in the hydrolysis of gelatin by the enzyme.


Synergistic interaction between the probucol phenoxyl radical and ascorbic acid in inhibiting the oxidation of low density lipoprotein. Chain-breaking antioxidants such as butylated hydroxytoluene, alpha-tocopherol, and probucol have been shown to decrease markedly the oxidative modification of low density lipoprotein (LDL). Their mechanism of action appears to involve scavenging of LDL-lipid peroxyl radicals. The purpose of this study was to investigate the occurrence of radical reactions produced during oxidation of LDL and LDL-containing probucol initiated by lipoxygenase or copper. In addition, we have investigated the possibility of a synergistic interaction between ascorbate and probucol in inhibiting the oxidation of LDL. Incubation of LDL-containing probucol and lipoxygenase produced a composite electron spin resonance (ESR) spectrum due to the endogenous alpha-tocopheroxyl radical and probucol-derived phenoxyl radical. The spectral assignment was further verified by chemical oxidation of alpha-tocopherol and probucol. In the presence of ascorbic acid, these radicals in the LDL particle were reduced to their parent compounds with concomitant formation of the ascorbate radical. In both the peroxidation of linoleic acid and the copper-initiated peroxidation of LDL, the antioxidant activity of probucol was significantly increased by low (3-6 microM) concentrations of ascorbate. The probucol-dependent inhibition of LDL oxidation was enhanced in the presence of ascorbic acid. We conclude that the reaction between the phenoxyl radical of probucol and ascorbate results in a synergistic enhancement of the antioxidant capacity of these two compounds and speculate that such reactions could play a role in maintaining the antioxidant status of LDL during oxidative stress in vivo.


Protein-DNA interactions at a dioxin-responsive enhancer. Mutational analysis of the DNA-binding site for the liganded Ah receptor. The liganded Ah receptor activates transcription by binding to a specific DNA-recognition motif within a dioxin-responsive enhancer upstream of the CYP1A1 gene. Analyses of mutant enhancers by gel retardation reveal that each base pair within the domain 5'CGTG(GCAC)3' is essential to the receptor-enhancer interaction. The three base pairs immediately flanking each end of the essential domain contribute less strongly to receptor binding. Analyses of enhancer function by transfection reveal that a mutation in the essential domain, which abolishes receptor-DNA binding, obliterates enhancer function. Mutations outside the essential domain, which diminish, but do not abolish, receptor-DNA binding, also obliterate enhancer function. Additionally, one mutation adjacent to the essential binding motif does not affect receptor-DNA binding, but destroys enhancer activity. These findings imply that transcriptional enhancement by the dioxin-responsive system cannot be predicted solely by the strength of the receptor-enhancer interaction.


Hydroxylated metabolites of the antimalarial drug primaquine. Oxidation and redox cycling. Oxidation and redox cycling of the hydroxylated metabolites of the antimalarial drug primaquine (i.e. 5-hydroxyprimaquine, 5-hydroxydemethylprimaquine, and 5,6-dihydroxy-8-aminoquinoline) were studied. The three metabolites readily oxidized under physiological conditions, forming hydrogen peroxide and the corresponding quinone-imine derivatives as the main products. The latter compounds were characterized by visible, NMR, and infrared spectroscopy. Concomitant formation of drug-derived radicals and hydroxyl radicals was attested by direct and spin-trapping EPR experiments, respectively. The use of the spin stabilization method indicated that the radicals derived from 5-hydroxydemethylprimaquine and 5,6-dihydroxy-8-aminoquinoline are of the o-semiquinone type. Tentative structures are proposed for the radicals based on product identification and computer simulation of the experimental EPR spectra. The quinone-imines obtained from the reduced metabolites did not react at appreciable rates with NADPH but underwent redox cycling upon addition of ferredoxin:NADP+ oxidoreductase, forming hydrogen peroxide and hydroxyl radicals. The effect of antioxidant enzymes on hydroxyl radical yield obtained during oxidation and redox cycling indicates that the main route for hydroxyl radical formation is the metal ion-catalyzed reaction between the drug-derived radicals and hydrogen peroxide. Taken together, the results indicate that hydrogen peroxide is the potential toxic product formed from the primaquine metabolites.


Dioxin- and Ah receptor-dependent protein binding to xenobiotic responsive elements and G-rich DNA studied by in vivo footprinting. DNA-protein interactions before and after transcriptional activation of the carcinogen- and dioxin-inducible enhancer of the murine CYP1A1 gene were detected in vivo by treatment with dimethyl sulfate followed by ligation-mediated, polymerase chain reaction-aided genomic sequencing. Following 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment of mouse Hepa-1 hepatoma cells, evidence of protein binding was detected at the sequence 5' CACGCNA/T 3' within two previously defined xenobiotic response elements (XREs). The observed XRE footprint was similar to that previously identified by in vitro methylation protection footprints and attributed to the binding protein for 2,3,7,8-tetrachlorodibenzo-p-dioxin the Ah receptor. No XRE footprinting was observed in Hepa-1 mutant cells possessing a defective Ah receptor. Unexpectedly, evidence of protein binding was also detected at a G-rich DNA sequence immediately adjacent to one of the XREs. Footprinting of the G-rich sequence element, like that of XRE1 and XRE2, was dependent on the presence of a functional Ah receptor. The Ah receptor is therefore able to bind to its own DNA target sites in vivo and is also required for the binding of a second factor to the G-rich element.


ATPase-deficient mutants of the Escherichia coli DNA replication protein PriA are capable of catalyzing the assembly of active primosomes. The PriA replication protein of Escherichia coli (formerly replication factor Y or protein n') is multifunctional. It is a site-specific, single-stranded DNA-dependent ATPase (dATPase), a 3'----5' DNA helicase, and guides the ordered assembly of the primosome, a mobile, multiprotein DNA replication priming/helicase complex. Although PriA is not absolutely required for viability, priA null mutant cells grow very slowly, have poor viability, and form extensive filaments. In order to assess which of the multiple activities of PriA are required for normal replication and growth, site-directed mutagenesis was employed to introduce single amino acid substitutions for the invariant lysine within the consensus nucleotide-binding motif found in PriA. Biochemical characterization of the representative purified mutant PriA proteins revealed them to be completely deficient in nucleotide hydrolysis, incapable of translocation along a single-stranded DNA binding protein-coated single-stranded DNA template, and unable to manifest the 3'----5' DNA helicase activity of wild-type PriA. These mutant proteins were, however, capable of catalyzing the assembly of active primosomes in vitro. Furthermore, when supplied in trans to insertionally inactivated priA cells, plasmids containing a copy of these mutant priA genes restored the wild-type growth rate, viability, and cell morphology. Based on these results, a model for PriA function in vivo is discussed.


Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin. The carbohydrate binding specificity of Mr = 30,000 lectin (CBP30) from baby hamster kidney (BHK) cells has been studied by inhibition of binding of the radiolabeled lectin to asialofetuin-Sepharose using model oligosaccharides and glycopeptides. CBP30 binds type I or II Gal beta(1----3(4))GlcNAc chains but not Gal(beta 1----3)GalNAc. The inhibitory potency of straight chain polylactosamine structures or complex-type branched glycans is increased in proportion to the number of Gal(beta 1----3(4)) units present. Fucosylation or sialylation of terminal galactose residues or further substitution by (alpha 1----3)-linked galactose or N-acetylgalactosamine does not affect binding whereas substitution of the penultimate N-acetylglucosamine residue drastically reduces binding. Thus, blood group A, H type I or H type II structures, shows high affinity whereas Lex, Lea, and Leb structures bind poorly. CBP30 binds to murine Engelbreth-Holm-Swarm (EHS) tumor laminin and human amniotic fluid fibronectin but not human plasma fibronectin. Binding involves polylactosamine glycans as well as tri- and tetraantennary complex-type glycans present in EHS laminin and amniotic fluid fibronectin but absent in plasma fibronectin. Proteolytic fragments of EHS laminin (E1X/Nd, P1, E8, and E3) bind CBP30, but only fragment E8 supports attachment and spreading of BHK cells. BHK cell adhesion to EHS laminin or fragment E8 was not disturbed by CBP30-specific antibodies, but at relatively high concentrations (45 micrograms/ml) CBP30 inhibited spreading and partially attachment of cells on laminin.


Phorbol esters inhibit the dioxin receptor-mediated transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Tetradecanoyl phorbol acetate (TPA) has been shown to inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced mouse P450IA1 benzo[a]pyrene hydroxylase activity (Raunio, H., and Pelkonen, O. (1983) Cancer Res. 43, 782-786). When we co-administered TPA and TCDD to C57BL/6 mice, the accumulation of TCDD-inducible liver P450IA1 and P450IA2 mRNA, as well as kidney P450IA1 mRNA, was greatly inhibited. When nuclear run-on assays were conducted, maximal levels of transcriptional activation were achieved for both liver Cyp1a-1 and Cyp1a-2 with 1 micrograms/kg (approximately equal to 3.0 nmol/kg) TCDD. TCDD elicited a dose-dependent increase in the rates of gene transcription, which paralleled the induction of P450IA1 and P450IA2 mRNA. Only Cyp1a-1 gene transcription was elevated in kidney. When these experiments were repeated following the co-administration of TPA with TCDD, the levels of TCDD-mediated transcriptional increases in liver Cyp1a-1 and Cyp1a-2 and P450IA1 and P450IA2 mRNAs were dramatically inhibited. The reduction in Cyp1a gene transcription by TPA could be accounted for by reduced DNA binding of the dioxin receptor to the xenobiotic-responsive element (XRE) sequences, as measured by gel-retardation analysis. Analysis of nuclear [3H]TCDD dioxin receptor by sucrose density gradients demonstrated that the inhibition of Cyp1a gene transcription and DNA binding by TPA resulted from a reduction in nuclear dioxin receptor concentration.


Cyclic AMP acutely stimulates translocation of the major insulin-regulatable glucose transporter GLUT4. Facilitated glucose transport across plasma membranes is mediated by a family of transporters (GLUT1-GLUT5) that have different tissue distributions and Km values for transport. It has been shown that insulin stimulates glucose transport in fat and muscle tissues by causing the redistribution of one of these proteins (GLUT4) from inside the cell to the plasma membrane. Previous studies have shown that agents that change cAMP levels are able to modulate glucose transport in fat cells. The aim of this study was to investigate the mechanisms responsible for modulation of glucose transport by cAMP. 2-Deoxyglucose transport and insulin-regulatable glucose transporter (GLUT4) immunoreactivity in plasma and low density microsomal membranes were measured in adipocytes incubated for 30 min with insulin or dibutyryl-cAMP (Bt2cAMP). Low concentrations of Bt2cAMP (10 microM) increased 2-deoxyglucose uptake by translocating GLUT4 from low density microsomal membranes to the plasma membranes. Bt2cAMP at 1000 microM inhibited glucose transport below basal but further increased translocation of GLUT4. The effect of Bt2cAMP on translocation was additive to that of 7 nM insulin. We conclude that in rat adipocytes, Bt2cAMP acutely translocates GLUT4 but inhibits its activity to transport glucose.


Autophosphorylation of inositol 1,4,5-trisphosphate receptors. Inositol 1,4,5-trisphosphate (IP3) releases internal stores of calcium by binding to a specific membrane receptor which includes both the IP3 recognition site as well as the associated calcium channel. The IP3 receptor is regulated by ATP, calcium, and phosphorylation by protein kinase A, protein kinase C, and calcium/calmodulin-dependent protein kinase II. Its cDNA sequence predicts at least two consensus sequences where nucleotides might bind, and direct binding of ATP to the IP3 receptor has been demonstrated. In the present study, we demonstrate autophosphorylation of the purified and reconstituted IP3 receptor on serine and find serine protein kinase activity of the IP3 receptor toward a specific peptide substrate. Several independent purification procedures do not separate the IP3 receptor protein from the phosphorylating activity, and many different protein kinase activators and inhibitors do not identify protein kinases as contaminants. Also, renaturation experiments reveal autophosphorylation of the monomeric receptor on polyvinylidene difluoride membranes.