Correlation in the expression of type IV collagenase and the invasive and chemotactic abilities of myelomonocytic cells during differentiation into macrophages. Monomyelocytic phagocytes originate in the bone marrow and while differentiating into macrophages migrate to inflammatory foci and target tissues by egress from the capillary blood vessels. During such diapedesis, the cells must traverse tissue barriers such as basement membrane, which has type IV collagen as its principal structural element. We studied whether the expression of type IV collagenase activity, invasion through basement membrane, and the response to inflammatory chemoattractants are related to each other and to the process of differentiation of murine M1 myeloid leukemia cells into macrophages. M1 cells stimulated with mouse lung-conditioned medium (MLCM) or interleukin 6 (IL6) differentiate into macrophages by 72 h, as determined by expression of Fc receptors, induction of lysozyme, and morphological changes from blast cells to mature macrophages. During this process of differentiation the invasive ability of the cells and the amount of type IV collagenase in the supernatants from the invading cells continuously increased up to 72 h. Zymographic analysis of supernatants of the invading cells revealed a single 100-kd metalloproteinase with gelatinolytic activity. Chemotaxis towards arachidonic acid metabolites, which are present in inflamed tissues, was detected only in differentiated cells. Studies with thioglycolate (TG)-elicited peritoneal macrophages gave results similar to those obtained with differentiated M1 cells, showing that the ability to invade basement membrane, the expression of type IV collagenase, and the chemotactic response to inflammatory chemoattractants all increased with the differentiation of myeloid cells and reached their highest expression in fully differentiated cells.


Expansion of stimulus-evoked metabolic activity in monkey somatosensory cortex after peripheral denervation. The 2-deoxy-glucose (2DG) technique was used to study changes in stimulus-evoked metabolic activity in the somatosensory cortex of the squirrel monkey Saimiri sciureus after unilateral digit amputation. Two to 52 weeks after digit 2 on the left hand was removed, a somatic stimulus was applied to digit 3 bilaterally. In area 3b corresponding to the deafferented side of the brain, the area of stimulus-evoked metabolic activity was greater than that on the opposite, control side of the brain within the same animal. The extent of the topographic projection map of 2DG label in area 3b on the deafferented side of the brain was 1.92 to 4.75 times greater than that on the control side. There was no difference, however, in the topographical area of stimulus-evoked metabolic activity between the left and right somatosensory cortices in a normal, unoperated animal. These data suggest that the changes in functional organization observed using electrophysiological recordings in somatosensory cortex after peripheral denervation may have a metabolic substrate.


The effects of melanocortins and electrical fields on neuronal growth. The effects of the neuropeptide molecules, adrenocorticotrophic hormone or alpha-melanocyte stimulating hormone, on various parameters of in vitro nerve growth, have been studied in the presence and absence of a small applied electric field. Striking effects on rates of nerve growth occurred. Selected concentrations of either substance alone, produced a three- to fourfold increase in nerve growth rate; other concentrations were inhibitory. The applied electric field alone increased rates of growth threefold. Further augmented rates of growth were seen when either neuropeptide was present simultaneously with an applied field. Under these latter conditions, galvanotropic (cathodal) orientation persisted, while nerve branching was suppressed. Given the clinical interest in melanocortin-stimulated nerve regeneration, perhaps a combined electrical and neuropeptide approach would be warranted.


Expression of c-met proto-oncogene in COS cells induces the signal transducing high-affinity receptor for hepatocyte growth factor. By transfection of the expression plasmid containing a human c-met cDNA into COS-7 cells, high-affinity binding sites specific for HGF with a Kd value of 30 pM were newly detected. Furthermore, only in the c-met transfected COS-7 cells, but not in the control COS-7 cells, DNA synthesis was markedly induced in response to HGF. Thus, transient expression of exogenous c-met cDNA resulted in the appearance of high-affinity receptor for HGF and conversion of the normally non-responsive COS-7 cells into the HGF-responsive cells. These results provide evidence for identifying the c-met product as a signal transducing high-affinity receptor for HGF.


Modulation of activities and RNA level of the components of the plasminogen activation system during fusion of human myogenic satellite cells in vitro. Primary cultures of human myogenic stem cells (satellite cells) mimic myogenic differentiation. During this process, the expression of the components of the plasminogen activation system underwent modulation. Activities and mRNA levels of tissue-type and urokinase-type plasminogen activator were increased in a reproducible pattern during differentiation. A modulation of the mRNA level of PAI-2 was also observed. Human satellite cells expressed a urokinase receptor and also the mRNA level of this component underwent modulation. With the exception of PAI-1 mRNA, the level of all mRNAs increased from Day 4 to Day 8, i.e., just before myoblasts fusion, and then remained high at later stages. The modulation of the plasminogen activating activity indicates that this system is directly involved in the fusion process of myogenic differentiation.


N-cadherin localization in early heart development and polar expression of Na+,K(+)-ATPase, and integrin during pericardial coelom formation and epithelialization of the differentiating myocardium. N-cadherin, a Ca(2+)-dependent cell adhesion molecule, has been localized previously to the mesoderm during chick gastrulation and to adherens junctions in beating avian hearts. However, a systematic study of the dynamic nature of N-cadherin localization in the critical early stages of heart development is lacking. The presented work defines the changes in the spatial and temporal expression of N-cadherin during early stages of chick heart development, principally between Hamburger and Hamilton stages 5-8, 18-29 hr of development. During gastrulation N-cadherin appears evenly distributed in the heart forming region. As development proceeds to form the pericardial coelom (stages 6, 7, and 8, i.e., between 22 and 26 hr of development) N-cadherin localization becomes restricted to the more central areas of the mesoderm. The localization also shows a periodicity that correlates closely with the distance between foci of cavities that eventually coalesce to form the coelom. This distribution suggests that N-cadherin may have a function in the sorting out of somatic and splanchnic mesoderm cells to form the coelom. This separation of the mesoderm in the embryo for the first time physically delineates the precardiac mesoderm population. Concomitant with cell sorting during coelom formation, the precardiac cells change shape and show a distinct polarity as conveyed by (1) the apical expression of N-cadherin on precardiac cell surfaces lining the pericardial coelom, (2) the primarily lateral expression of Na+,K(+)-ATPase, and (3) an enrichment of integrin (beta 1 subunit) on basal cell surfaces. The somatic mesoderm cells apparently down-regulate N-cadherin expression. N-cadherin is also absent from the precardiac cells close to the endoderm. The latter cells eventually form the endocardium, i.e., the endothelial lining of the heart. By contrast, in the tubular, beating heart N-cadherin is found throughout the myocardium. In summary, immunolocalization patterns of N-cadherin during early cardiogenesis suggest that this cell adhesion molecule has a major role in the dynamics of pericardial coelom formation. Subsequently, its continued expression during cell differentiation of the cardiomyocyte to form the myocardium, but not endocardium, suggests N-cadherin is an essential morphoregulatory molecule in heart organogenesis.


Sequential activation of three myogenic regulatory genes during somite morphogenesis in quail embryos. We report the cloning of two new quail myogenic cDNAs, quail myogenic factor 2 (qmf2) and qmf3, which encode helix-loop-helix proteins homologous to mammalian myogenic factors myogenin and myf-5. In situ hybridization has been used to investigate the developmental expression of qmf2 and qmf3, as well as qmf1, the quail homologue to mammalian MyoD1, during the formation of the brachial somites. These studies show that qmf1 and qmf3 are activated sequentially in medially localized somite cells, immediately following somite formation but prior to myotome formation. qmf1, qmf2, and qmf3 are expressed in the myotome of compartmentalized somites. These findings suggest that determination of the myogenic cell lineage in quail somites is a progressive process controlled by influences of the neural tube on the expression of the qmf regulatory genes in newly forming somites.


A case of an undifferentiated small cell carcinoma of the esophagus with a primary abdominal mass. This paper reports a case with an undifferentiated carcinoma of the esophagus which primarily developed symptoms due to metastatic lesions. The case was a 59-year-old woman with a primary manifestation of an abdominal mass and with subsequent dysphagia. A protruding lesion with ulceration was found at the lower third of the thoracic esophagus by endoscopic examination and was histologically proved to be an undifferentiated carcinoma by biopsy. The abdominal mass was initially thought to be due to metastasis to an abdominal lymph node based on the diagnosis image finding at admission, but it was consequently found by autopsy to be a metastatic tumor in the liver. Therefore, undifferentiated carcinoma of the esophagus should be take into account for differential diagnosis of an abdominal mass.


Regulation of fat body mitochondrial respiration in Periplaneta americana by a novel factor from the corpus cardiacum. Respiration of fat body (Periplaneta americana) mitochondria is increased by pretreatment of the tissue with corpus cardiacum (CC) extract. The magnitude of the increase depends on the type of substrate supplied for oxidation. With 5 mM pyruvate the respiration increased 22%, decreasing to 0 with 1 mM pyruvate. In contrast, 50 microM and 0.2 mM palmitic acid supported an increase in CC-stimulated respiration of 14 and 44%, respectively. Unlike crude CC extract, the synthetic hyperglycemic peptides CCI and CCII failed to alter the respiratory activity of fat body mitochondria. In common with the action of CC extract pretreatment of the fat body in vitro with 10(-5) M cyclic AMP, 10(-5) M 8-bromo-cyclic AMP, or 10(-5) M forskolin increased mitochondrial respiration approximately 30%. Octopamine (10(-4) M) elicited a response similar to that obtained with CC extract. Neither 10(-5) M cyclic AMP nor 10(-5) M 8-bromo-cyclic AMP stimulated respiration when applied directly to the mitochondria. These results suggest that the factor in CC extract manifests its effect intracellularly through the activation of a cyclic AMP-dependent protein kinase. This interpretation is also based on the finding that diamide, an inhibitor of protein kinase, inhibits CC-dependent and cyclic AMP-dependent mitochondrial respiration. The physiological role of the CC factor responsible is not known.


Steroid production by ovarian follicles of the viviparous guppy (Poecilia reticulata) and its regulation by precursor substrates, dibutyryl cAMP and forskolin. Production in vitro of estradiol-17 beta, testosterone, 17 alpha-20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P), 17 alpha-hydroxyprogesterone, and progesterone by follicles of the guppy at various stages of oocyte growth and gestation was investigated. Basal production of estradiol-17 beta was highest in 0.8- and 1.2-mm follicles, whereas that of testosterone and 17 alpha,20 beta-P was highest in 1.6-mm (postvitellogenic) follicles. Levels of these steroids declined after fertilization and were undetectable in late gestation and postpartum follicles. 17 alpha-Hydroxyprogesterone and progesterone levels were low at all stages. Thus, none of these steroids appears to be involved in maintaining gestation. Regulation of estradiol-17 beta and 17 alpha,20 beta-P secretion by vitellogenic (1.0 mm) and postvitellogenic follicles by precursor substrates, dbcAMP (0.1 to 10 mM) and forskolin (1 to 100 microM), was also investigated. Vitellogenic follicles synthesized increased quantities of estradiol-17 beta in the presence of exogenous testosterone, whereas estradiol-17 beta production by postvitellogenic follicles was not altered by testosterone. These results suggest decreased aromatase activity in the postvitellogenic follicles. Dibutyryl cAMP and/or forskolin stimulated testosterone and estradiol-17 beta production by vitellogenic follicles but did not stimulate conversion of testosterone to estradiol-17 beta, suggesting that the adenylate cyclase system stimulates estradiol-17 beta production by stimulating testosterone production but does not mediate conversion of testosterone to estradiol-17 beta. Postvitellogenic follicles synthesized increased quantities of 17 alpha,20 beta-P in response to 17 alpha-hydroxyprogesterone in a dose-dependent manner. Although 1 microM of forskolin stimulated 17 alpha,20 beta-P production by postvitellogenic follicles in the absence of exogenous 17 alpha-hydroxyprogesterone, 100 microM of forskolin inhibited 17 alpha,20 beta-P production. Dibutyryl cAMP, however, did not affect 17 alpha,20 beta-P production. In the presence of 50 ng of 17 alpha-hydroxyprogesterone, dbcAMP (10 mM) and forskolin (1 to 100 microM) suppressed 17 alpha,20 beta-P production. It is suggested that cAMP mediates 17 alpha,20 beta-P production up to a certain threshold level, beyond which it inhibits 17 alpha,20 beta-P production.


Mechanism of action of a cytotonic enterotoxin produced by Aeromonas hydrophila. In this study, we describe the mechanism of action of a cytotonic enterotoxin produced by two isolates of Aeromonas hydrophila. Isolates SSU and Ah65 are of different origin and both are capable of producing either a cytotoxic enterotoxin or aerolysin. A cytotonic enterotoxin produced by diarrheal isolate SSU, which was purified and characterized in our laboratory, elevated intracellular cAMP and PgE2 levels in cultured Chinese hamster ovary (CHO) cells. Likewise, enterotoxic activity expressed by a cytotonic enterotoxin was detected in the culture filtrate of a fish isolate (Ah65) after cytotoxic activity was neutralized with homologous aerolysin monoclonal antibodies. This cytotonic enterotoxin also elevated intracellular cAMP and PgE2 levels in CHO cells, suggesting a cholera toxin-like mechanism of action for Aeromonas cytotonic enterotoxins.


Contribution of the fnr and arcA gene products in coordinate regulation of cytochrome o and d oxidase (cyoABCDE and cydAB) genes in Escherichia coli. The individual and the combined effect of the fnr and arcA regulatory gene products on cytochrome o oxidase and cytochrome d oxidase gene expression in Escherichia coli were evaluated using lacZ reporter fusions to the cyo-ABCDE and cydAB operons. Fnr repressed cyo-lacZ and cyd-lacZ expression during anaerobic growth but not during aerobic growth conditions. ArcA functioned as an anaerobic repressor of cyo-lacZ expression while, in contrast, it activated cydAB expression during both aerobic and anaerobic growth. ArcA and Fnr appear to function independently of each other to control cyo-ABCDE operon expression. In contrast, FNR repression of cydAB expression was dependent on arcA+, as indicated by the inability of fnr+ plasmids to repress cyd-lacZ expression in an arcA strain. Under no conditions tested did Fnr activate cydAB expression. Most, but not all, of the observed aerobic/anaerobic regulation of cyo and cyd was accounted for by the two transcriptional regulators. These data suggest the existence of additional levels of anaerobic gene control in E. coli. Additionally, the expression of the fnr regulatory gene, and regulation of the anaerobic respiratory genes, narGHJI, dmsABC and frdABCD, was found to be independent of ArcA.


A herpesvirus regulatory protein appears to act post-transcriptionally by affecting mRNA processing. Post-transcriptional control mechanisms play an important role in regulating gene expression for a number of viruses, especially in the regulation of late gene products. In this study we have investigated the mode of action of ICP27, an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) required for late gene expression. Transfection experiments have demonstrated that ICP27 can activate or repress expression depending on the target gene. Here, we show that the regulatory activity of ICP27 is independent of the target gene promoter sequences but, instead, depends on the presence of different mRNA processing signals. The activation function correlated with different polyadenylation sites, whereas the repressor function correlated with the presence of introns either 5' or 3' to the target gene-coding sequences. Poly(A)+ RNA levels were increased by ICP27 in transfections with a target gene having only an AATAAA recognition signal but no G/U box within the usual distance. In contrast, in the presence of ICP27, spliced target mRNAs were decreased 5- to 10-fold in transfections with target genes containing a 5' or 3' intron. These results suggest that this essential HSV-1 regulatory protein acts post-transcriptionally to affect mRNA processing and point to possible interactions between splicing and polyadenylation factors.


Inhibition of muscle differentiation by the adenovirus E1a protein: repression of the transcriptional activating function of the HLH protein Myf-5. Myogenic differentiation can be inhibited by the adenovirus E1a protein in the rat L6 muscle cell line. The present investigation provides evidence that E1a interferes with the expression of myogenin and the activity of Myf-5, the two myogenic helix-loop-helix (HLH) proteins that are expressed in L6 muscle cells. In nuclei of E1a-expressing L6 cells, Myf-5 protein accumulates to normal or even elevated levels and shows no alterations of its ability to bind to the DNA-binding site (CANNTG). However, trans-activation of muscle-specific reporter genes by Myf-5 is strongly inhibited. The same inhibition by E1a can be shown for the other myogenic HLH proteins, MyoD, myogenin, and MRF4/Myf-6, that have been expressed in 10T1/2 fibroblasts. In contrast to the normal level of Myf-5 expression, synthesis of myogenin is entirely abolished in the differentiation-defective L6-E1a cells. Here, we demonstrate that the carboxy-terminal trans-activator domain and probably the basic-HLH (bHLH) region of Myf-5 constitute targets for the inhibition by E1a. The effect of E1a depends on its intact transforming regions but not on the transcriptional activator domain. Our data suggest that activation of myogenin gene expression and the establishment of the differentiated phenotype may require functional Myf-5. Expression of the Myf-5 gene, however, is apparently independent of auto- or cross-regulation by the myogenic HLH proteins.


Intravitreous granulation tissue and retinal detachment following pars plana injection for cytomegalovirus retinopathy. Recently, the pars plana route of injection has been used to administer drugs for the treatment of severe intraocular infections. We observed a complication of this method in a patient with AIDS and cytomegalovirus (CMV) retinopathy.


Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA. Membrane lipid peroxidation processes yield products that may react with DNA to cause oxidative modifications. We have investigated this possibility and have found that calf thymus DNA exposed to autooxidized lipids causes the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG). 8-OH-dG formation in DNA was measured using high-pressure liquid chromatography with electrochemical detection. Methyl linolenate oxidized for different lengths of time was exposed to DNA. The amount of 8-OH-dG formed in DNA was proportional to the amount of lipid peroxidation as measured by the thiobarbituric reactive substances present. The formation of 8-OH-dG in DNA by autooxidized methyl linolenate was dependent on the presence of the transition metal ions Cu or Fe and was inhibited by various scavengers, including superoxide dismutase and catalase. This implicates the involvement of oxygen free radicals in the process. Liposomes formed from phosphatidylcholine (82%) and methyl arachidonate (18%) were peroxidized for different lengths of time and then exposed to DNA. 8-OH-dG was formed in DNA by exposure to Cu(II) and peroxidized liposomes. Under these conditions, Fe(III) was slightly less effective than Cu(II) in mediating 8-OH-dG formation. These observations clearly show that 8-OH-dG formation in DNA may result from processes that may occur during intracellular lipid peroxidation.


Antioxidant and anticataractogenic effects of topical captopril in diquat-induced cataract in rabbits. 1-[(2s)-3-Mercapto-2-methylpropionyl]-L-proline (captopril), an antihypertensive and free radical scavenger, protected the rabbit lens from peroxidative and oxidative damage induced by 1 mM diquat in vitro. To evaluate the anticataract efficacy of captopril, an experimental group of five rabbits was treated with topical captopril (1% in 0.15 M NaCl, w/v), and 50 microliters was instilled onto both eyes four times a day for a total of 8 weeks. Following the same procedure, the eyes of five rabbits were treated with topical 0.15 M NaCl as a control for captopril treatment. At the end of the first week of treatment, a single intravitreal dose of 120 nmole diquat in 30 microliters of 0.15 M NaCl was injected into the right eye of each rabbit of both the groups. As a control for intravitreal diquat injection, the left eye of all the rabbits were injected with the diluent, 30 microliters per eye. The intravitreal diquat or its diluent injection was only for one time. From slit-lamp biomicroscopic observation of the diquat-injected right eyes, the anticataract effect of captopril in the treatment group was indicated by the finding that in four of five rabbits the cataract did not advance; whereas in four of five rabbits treated with the diluent the cataract progressed to grade 3. The lenses in the diluent-injected control left eyes of the rabbits treated with the captopril or diluent were normal. However, since the number of animals used for the in vivo studies was few, further confirmation of the anticataract effect of captopril is necessary. In diquat-injected right eyes of animals treated with captopril, the integrated rate of O2- production was about 50% less (p less than .001) in the aqueous humor, vitreous humor, and lens, compared with O2-, 33.49 +/- 2.26 microM (mean +/- SEM) in the aqueous humor, 17.12 +/- 0.75 microM in the vitreous humor, and 31.44 +/- 1.29 nmole/g wet weight in the lens of the diquat-injected right eyes treated with the diluent. Similar significant (p less than .01) differences in the production of .OH and H2O2 in eye tissues were also observed.(ABSTRACT TRUNCATED AT 400 WORDS)


High levels of Mn-superoxide dismutase in serum of patients with neuroblastoma and in human neuroblastoma cell lines. Levels of serum manganese superoxide dismutase (Mn-SOD) in normal children aged from 1 to 14 years and children with various hematological and malignant diseases were determined by enzyme-linked immunosorbent assay (ELISA). In the normal children, the serum Mn-SOD levels gradually increased in proportion to age. By 8 years of age, the Mn-SOD level was nearly at the adult level. The normal values of serum Mn-SOD (mean +/- SD) of children below 4 and above 8 years old were 48 +/- 10.2 ng/ml and 84 +/- 22.5 ng/ml, respectively. Assuming the upper limit of normal Mn-SOD level in serum to be the mean value +/- 2 SD of children at each age, high serum levels of Mn-SOD were found for 8 of 12 patients with neuroblastoma, three of four patients with Wilms tumor, and four of five patients with acute myeloid leukemia. The patients with neuroblastoma exhibited a transient increase in Mn-SOD following chemotherapy, but after 1 week the levels decreased markedly to the control levels. The changes in serum Mn-SOD levels in the patients with neuroblastoma correlated well with the levels of neuron-specific enolase. Mn-SOD was intensely stained in bone marrow cells of patients whose cancer cells had moved into the bone marrow. High levels of Mn-SOD were also found in cultured human neuroblastoma cells. These data indicate that Mn-SOD is expressed in neuroblastoma cells, may serve as one of the diagnostic and prognostic markers for the neuroblastoma, and may be useful to predict the effectiveness of chemotherapy for neuroblastoma and the recurrence of this disease.


Free radical yields from the homolysis of peroxynitrous acid. A recent study reports discordant results for maximal free-radical yields from the decomposition of peroxynitrous acid using different assay reagents and a puzzling decrease (to zero) of the radical yields with increasing pH. An assay method using 2,2,'azino-bis(3-ethyl-benzthiazoline sulphonate) (ABTS) has been tested using stopped-flow kinetic studies, and the results imply that the assay is satisfactory when [HOONO] much greater than [-OONO] but that reactions involving peroxynitrite anion interfere at higher pH. Evidence is presented that peroxynitrite was an interfering species in the earlier studies.


[Cytomegalovirus associated diseases of the heart]. Latent cytomegalovirus (CMV) infection is not uncommon in the juvenile and adult population. The full blown disease is mostly restricted to immunosuppressed and immunodeficient patients, but may also occur in healthy individuals. The acute CMV-myocarditis often takes a mild course with only transient changes of left ventricular hemodynamics or a pericardial effusion as assessed by echocardiography. In our patient population it was characterized by the presence of anti-interfibrillary antibodies. In acute myocarditis the virus genome can be detected by in-situ hybridization in 42% (40% in the myocytes, 21% in the interstitial cells and 41% in endothelial cells). In patients with perimyocarditis CMV-DNA is found in 24% of patients in the myocytes, in 24% of patients in the interstitial cells and in 50% of patients in the endothelium. In healthy controls CMV-DNA could be assessed only in interstitial and endothelial cells (70% and of the infected 30% of positive cases) but not in the myocytes. In dilated cardiomyopathy (DC) CMV-DNA can be found in 48%. Particularly in myocytes in 45% of cases, in interstitial cells in 50% and in the endothelium of small vessels in 68%. An induction of the disease by a chronic local stimulation of the immune system is a likely pathogenetic explanation of the immuno phenomena observed in parallel to the viral persistence. Additive damage by chronic CMV infection can be caused by the infection of the endothelium and smooth muscle cells of the intima of coronary arteries. There are some reports of CMV-DNA detection in the arterial walls or atherosclerotic plaques of patients with atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)


The role of development and adrenal steroids in the regulation of the mineralocorticoid receptor messenger RNA. The ontogeny, adrenal-feedback regulation and regional distribution of the mineralocorticoid receptor (MR) mRNA were examined in the rat brain and kidney. In the kidney, MR mRNA levels in the adult were only 25-30% of the neonatal concentration. Adrenalectomy caused a 35% increase in total brain MR mRNA and a 94% increase in kidney MR mRNA levels. Examination of the regional distribution of the MR mRNA within the brain revealed that the hippocampus had the highest levels, and the mRNA abundance increased after adrenalectomy. The administration of dexamethasone to intact animals resulted in a significant reduction of MR mRNA in the kidney of neonatal rats but not in the brain. These data indicate that there are developmental changes in MR gene expression in kidney and that adrenal steroids can modulate MR gene expression in both the brain and kidney.


Gastrointestinal autonomic nerve tumours: a case report with ultrastructural and immunohistochemical studies. A case of gastrointestinal autonomic nerve tumour with light microscopic, immunohistochemical and ultrastructural examination is reported. The tumour was composed of spindle cells or large cells with clear cytoplasm and showed intense staining for vimentin and focal staining for neuron-specific enolase, chromogranin, synaptophysin, gastrin, P substance and S-100 protein. Ultrastructural examination showed long processes with dense core granules and the absence of features characteristic of other gastrointestinal stromal tumours. In addition we noted small traces of basal lamina and the absence of synaptic vesicles. It seems that the biological behaviour of gastrointestinal autonomic nerve tumours is aggressive but there are too few reports on which to conclude anything about their prognosis. Our findings suggest that tumour has a neuroectodermal differentiation.


Point mutations in the upstream region of the alpha-galactosidase A gene exon 6 in an atypical variant of Fabry disease. Single point mutations in the upstream region of exon 6 of the alpha-galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; 301Arg----Gln (902G----A) in a case that has already been published and 279Gln----Glu (835C----G) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; 328Gly----Arg (982G----A) in the downstream region of exon 6 in one case and two combined mutations, 66Glu----Gln (196G----C)/112Arg----Cys (334C----T), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the alpha-galactosidase A gene.


Detailed analysis of an amplified region at chromosome 11q13 in malignant tumors. We have examined the amplification unit at chromosome band 11q13 in 12 primary tumors and one cancer cell line (A431) with 15 DNA markers. The amplified region and size varied from one tumor to another; the smallest amplicon was estimated to be 700 kb long and the largest was 4.5 Mb long at maximum, on the basis of a physical map. Furthermore, the DNA amplified in tumors was not always continuous, because in three cases one locus within the amplicon was not amplified. The amplified region common to all 13 cancers consisted of 500 kb of DNA which incorporated eight defined loci (BCL-1, cCI11-524, cCI11-283, cCI11-234, HBI-1, cCI11-454, HSTF1, and INT2). As two of them, cCI11-524 and cCI11-454, were found to contain DNA sequences conserved in other species, one or both of these loci might encode the gene(s) that may be associated with progression of these tumors.


Structure of the human TNF receptor 1 (p60) gene (TNFR1) and localization to chromosome 12p13 [corrected]. Clones encoding the entire coding and 3' untranslated region of the human type I tumor necrosis factor receptor (p60) gene (TNFR1) were isolated by hybridization using probes derived from TNFR-1 cDNA. The gene was characterized by restriction mapping. DNA blot analysis and sequence analysis. The coding region and the 3' untranslated region are distributed over 10 exons. Each of the four repeats, comprising the extracellular ligand binding domain and characterizing a receptor superfamily, is interrupted by an intron. However, the intron-exon structure is not conserved in the nerve growth factor receptor gene, another member of this superfamily. By PCR analysis of human-mouse somatic cell hybrids and in situ hybridization using biotinylated genomic TNFR1 DNA, we localized the gene to human chromosomal band 12p13. This corresponds to the homologous murine gene localized at the distal region of mouse chromosome 6.


Regulation of carbachol- and histamine-induced inositol phospholipid hydrolysis in a human oligodendroglioma. A stable cell line derived from a human oligodendroglioma (HOG) was used to study the regulation of muscarinic- and histamine receptor-mediated phosphoinositide hydrolysis. Both carbachol and histamine increased inositol monophosphate (InsP) accumulation in a dose- and time-dependent manner in the presence of lithium and the effect of simultaneous addition of carbachol and histamine was additive, implying independent signal transduction pathways. Homologous desensitization of muscarinic, but not histamine receptors, could be demonstrated although neither receptor type appeared to be heterologously desensitized. [3H]InsP accumulation in HOG cells was also stimulated by fluoride, suggesting guanosine triphosphate (GTP)-binding protein involvement, but phosphoinositide (PtdIns) hydrolysis was not sensitive to pertussis toxin. Phorbol ester-activation of protein kinase C (PKC) inhibited both muscarinic and histamine receptor-stimulated InsP release but did not attenuate either the fluoride-induced release of InsP nor beta-adrenergic receptor-mediated stimulation of adenylate cyclase activity. Taken together, we conclude that muscarinic and histamine receptors are differentially regulated through both PKC-dependent and -independent mechanisms, and that feedback inhibition of PtdIns turnover occurs proximal to the GTP binding proteins.


[Epstein-Barr virus infection--a lympho- and epitheliotropic infection]. Epstein-Barr virus (EBV) has long been thought to be primarily a B-lymphotropic virus. This tropism becomes obvious in the association of the virus with diseases that become manifest in lymphoproliferative conditions, such as acute infectious mononucleosis or endemic Burkitt's lymphoma. In the course of mononucleosis, however, viraemia cannot be detected and B-lymphocytes infected with EBV in vitro produce only small amounts of the virus. In contrast, recent studies document that EBV replicates in the epithelial cells in the mouth, and pronounced secretion of virus can also be detected. Cells of the basal layer of the epithelium can be infected via the EBV-specific CR2 receptor. Upon mitosis of the cells of the basal layer, EBV genome in episomal form is partitioned to the daughter cell. On the other hand, differentiation and maturation of the epithelial cells is paralleled by active virus production. Thus, there is evidence that the epithelial EBV infection is the main factor in the persistence and production of EBV. Accordingly, the EBV infection of epithelial cells which can result in diseases, seems to be the primary process, leading to the infection of B-lymphocytes and then to other diseases. Diseases associated with infection of epithelial cells by EBV and diseases involving B-lymphocytes are discussed with reference to this idea.


[Condylomata acuminata in children--detection of HPV 6/11 and 2. Local therapy with interferon-beta hydrogel]. Four cases of genital warts in children (girls) are reported. HPV 6/11-DNA was identified in two cases, and HPV 2-DNA in one. In one case no virus identification was possible. The clinical features of the HPV 2-induced genital warts showed the typical morphology of condylomata acuminata. The mode of transmission of the virus, in absence of sexual contact, could not be explained. The HPV 2-associated genital warts might have been transmitted by autoinoculation from warts on the hands. Topical treatment with IFN-beta-hydrogel was applied over 8 weeks, either as single-agent therapy (1 case) or as adjuvant therapy after removal of the condylomata (3 cases). No remission was seen with the single-agent therapy. In one case the genital warts reappeared after adjuvant therapy, but in the other two cases no recurrence was seen.


Uptake of 2-fluoro-2-deoxy-D-[U-14C]-glucose during chemotherapy in murine Lewis lung tumor. Mice bearing intramuscular Lewis lung tumor were treated with BCNU and doxorubicin (ADM) to study chemotherapy-induced changes in the uptake of 2-fluoro-2-deoxy-[U-14C]glucose (FDG). A decreased FDG uptake, tumor regression and a diminished proportion of aneuploid versus diploid cells as evaluated by DNA flow cytometry were seen after treatment with BCNU but not with ADM; HPLC indicated that most of the 14C activity in tumors was from FDG6-phosphate. The results suggest that changes in FDG uptake reflect the effectiveness of antitumor therapy. FDG may be valuable in follow-up studies of cancer treatment.


Transitional mucosa at anastomosis. A cause of local tumor recurrence in patients with rectal cancer after anterior resection. Transitional mucosa adjacent to colorectal cancers is essentially characterized by an excess of sialomucins at the expense of the normally predominant sulphomucins in epithelial cells lining the intestinal crypts which presents the early stage of oncogenic transformation of colorectal epithelium. The presence or absence of sialomucins at the resection margins was studied histochemically using the high iron diamine-alcian blue(HID-AB) stain in 64 rectal cancer patients in Dukes' B stage who underwent curative anterior resection. The correlation was revealed between the presence of sialomucins at the resection margins and subsequent development of local tumour recurrence. Fourteen of 27 patients (51.9%) with sialomucins predominant pattern at either resection margin developed local recurrence compared with 4 of 37 patients (10.8%) with sulphomucins predominant pattern (P less than 0.001). It is suggested that determination of the transitional mucosa around anastomosis in patients treated for the rectal carcinoma by anterior resection appears to identify those with a higher risk of local recurrence.


Synthesis and biological studies of neutral technetium (V) complexes containing NNOS donor sets. Complexation of ligands containing an N3S donor set has been affected with [99mTc]. These are part of a ligand series of analogous structures which exhibit similar chemistry and potentially interesting biology. The complexes which have been characterized with [99Tc] as [TcOL] are neutral and lipophilic and their biological behaviour has been assessed in rats. After HPLC purification of the no-carrier added preparation, brain uptake of the tracers was greater than 1% at 15 min p.i. Muscle activity was significant with slow blood clearance.


Enteric Clostridium perfringens infection associated with parvoviral enteritis in dogs: 74 cases (1987-1990). Parvovirus infection was confirmed by fluorescent antibody staining of or viral isolation from specimens of small intestine in 181 (17%) of 1,110 dogs necropsied between July 1, 1987 and Dec 31, 1990. Clostridium perfringens was isolated from 74 (69%) of 108 dogs with parvovirus infection from which specimens of jejunum also had been obtained for culture of anaerobic bacteria. Gram-positive bacilli in association with focal to diffuse necrosis of the superficial portions of the villi were observed in histologic sections of specimens of small intestine from 56 (98%) of 57 dogs from which parvovirus and C perfringens had been identified. These findings indicate that C perfringens frequently proliferates in dogs with parvovirus infection.


6 alpha-hydroxypenicillanic acid-S(S)-oxide and analogues: synthesis and antimicrobial activity. The synthesis and in vitro antibacterial activity of a series of 6-oxygenated penicillanic acid sulfoxides is described. 6 alpha-Hydroxypenicillanic acid-S(S)-oxide (1a) exhibits weak Gram-negative antibacterial activity and appears to be similar to amdinocillin (5) in its mode of action. 6 alpha-Hydroxypenicillanic acid-S(R)-oxide (4a) has a broader spectrum of activity, but again is rather weak. The corresponding 6 beta-hydroxy series is essentially devoid of activity.


Biosynthesis of griseolic acids: incorporation of 13C-labeled compounds into griseolic acid A. Biosynthesis of griseolic acids, competitive inhibitors of cyclic nucleotide phosphodiesterase, was investigated with the culture of a producing strain of Streptomyces griseoaurantiacus. 13C-Labeled and 15N-labeled compounds were added into the culture, and 13C-enriched and 15N-enriched griseolic acid A was isolated from the culture medium and analyzed by 13C NMR and 15N NMR spectroscopy. The compounds added to growth medium were [2-13C]acetate, [1,2-13C]acetate, [1,4-13C]succinate, [1-13C]glucose, [6-13C]glucose, [2-13C]ribose, and [1-13C, 15N]glycine. The results suggest that adenosine, which is formed from amino acids and sugars contributes the adenine and ribose moieties to griseolic acid A. The data also suggest that a dicarboxylic acid from the Krebs tricarboxylic acid cycle contributes to the dicarboxylic part of the compound.


Conjugative transfer of Enterococcus faecalis plasmid pAD1: nucleotide sequence and transcriptional fusion analysis of a region involved in positive regulation. The Enterococcus faecalis plasmid pAD1 undergoes conjugative transfer in response to cAD1, a peptide sex pheromone emitted by potential bacterial recipients. Regulation of pAD1 transfer involves a number of plasmid-encoded determinants:iad, which determines a peptide-competitive inhibitor iAD1; signal sensing and transducing elements; and negative and positive regulators. The key positive regulator(s) of the pheromone response is believed to be encoded within a segment designated the E region of the plasmid. In this study, we analyzed the nucleotide sequence and transcription within the E region. An open reading frame designated traE1 was identified; its inferred protein consists of 118 amino acids. Insertional mutagenesis of traE1 resulted in a complete loss in plasmid transfer capability. Analysis of Tn917-lac insertions giving rise to transcriptional lacZ fusions showed that traE1 is transcribed only under cAD1 inducing conditions. Analysis of additional lacZ fusions within the region provided some insight into the roles of potential regulatory signals within and around the nucleotide sequences reported here. A regulatory role appearing to involve read-through of certain key transcription termination sequences seemed evident.


Interactions among mutations that cause altered timing of gene expression during sporulation in Bacillus subtilis. The ski4::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but that are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). ski4::Tn917lac caused a small defect in sporulation, but in combination with a null mutation in kinA, it caused a much more severe defect. The insertion mutation was in an 87-amino-acid open reading frame (orf87 bofA) that controls the activation of a sigma factor, sigma K, at intermediate times during sporulation. The ski4 mutation caused the premature expression of cotA, a gene controlled by sigma K. An independent mutation that causes the premature activation of sigma K also caused a synthetic (synergistic) sporulation phenotype in combination with a null mutation in kinA, indicating that the defect was due to altered timing of gene expression directed by sigma K. Expression of ski4 was shown to be controlled by the sporulation-specific sigma factor sigma E.


The lysP gene encodes the lysine-specific permease. Escherichia coli transports lysine by two distinct systems, one of which is specific for lysine (LysP) and the other of which is inhibited by arginine ornithine. The activity of the lysine-specific system increases with growth in acidic medium, anaerobiosis, and high concentrations of lysine. It is inhibited by the lysine analog S-(beta-aminoethyl)-L-cysteine (thiosine). Thiosine-resistant (Tsr) mutants were isolated by using transpositional mutagenesis with TnphoA. A Tsr mutant expressing alkaline phosphatase activity in intact cells was found to lack lysine-specific transport. This lysP mutation was mapped to about 46.5 min on the E. coli chromosome. The lysP-phoA fusion was cloned and used as a probe to clone the wild-type lysP gene. The nucleotide sequence of the 2.7-kb BamHI fragment was determined. An open reading frame from nucleotides 522 to 1989 was observed. The translation product of this open reading frame is predicted to be a hydrophobic protein of 489 residues. The lysP gene product exhibits sequence similarity to a family of amino acid transport proteins found in both prokaryotes and eukaryotes, including the aromatic amino acid permease of E. coli (aroP) and the arginine permease of Saccharomyces cerevisiae (CAN1). Cells carrying a plasmid with the lysP gene exhibited a 10- to 20-fold increase in the rate of lysine uptake above wild-type levels. These results demonstrate that the lysP gene encodes the lysine-specific permease.


Identification of the vibriobactin receptor of Vibrio cholerae. Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.


Purification and properties of the NADH reductase component of alkene monooxygenase from Mycobacterium strain E3. Alkene monooxygenase, a multicomponent enzyme system which catalyzes the epoxidation of short-chain alkenes, is induced in Mycobacterium strain E3 when it is grown on ethene. We purified the NADH reductase component of this enzyme system to homogeneity. Recovery of the enzyme was 19%, with a purification factor of 920-fold. The enzyme is a monomer with a molecular mass of 56 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is yellow-red with absorption maxima at 384, 410, and 460 nm. Flavin adenine dinucleotide (FAD) was identified as a prosthetic group at a FAD-protein ratio of 1:1. Tween 80 prevented irreversible dissociation of FAD from the enzyme during chromatographic purification steps. Colorimetric analysis revealed 2 mol each of iron and acid-labile sulfide, indicating the presence of a [2Fe-2S] cluster. The presence of this cluster was confirmed by electron paramagnetic resonance spectroscopy (g values at 2.011, 1.921, and 1.876). Anaerobic reduction of the reductase by NADH resulted in formation of a flavin semiquinone.


Early Caulobacter crescentus genes fliL and fliM are required for flagellar gene expression and normal cell division. The biogenesis of the Caulobacter crescentus polar flagellum requires the expression of more than 48 genes, which are organized in a regulatory hierarchy. The flbO locus is near the top of the hierarchy, and consequently strains with mutations in this locus are nonmotile and lack the flagellar basal body complex. In addition to the motility phenotype, mutations in this locus also cause abnormal cell division. Complementing clones restore both motility and normal cell division. Sequence analysis of a complementing subclone revealed that this locus encodes at least two proteins that are homologs of the Salmonella typhimurium and Escherichia coli flagellar proteins FliL and FliM. FliM is thought to be a switch protein and to interface with the flagellum motor. The C. crescentus fliL and fliM genes form an operon that is expressed early in the cell cycle. Tn5 insertions in the fliM gene prevent the transcription of class II and class III flagellar genes, which are lower in the regulatory hierarchy. The start site of the fliLM operon lies 166 bp from the divergently transcribed flaCBD operon that encodes several basal body genes. Sequence comparison of the fliL transcription start site with those of other class I genes, flaS and flaO, revealed a highly conserved 29-bp sequence in a potential promoter region that differs from sigma 70, sigma 54, sigma 32, and sigma 28 promoter sequences, suggesting that at least three class I genes share a unique 5' regulatory region.


Synthesis of a cytochrome c derivative with prolonged in vivo half-life and determination of ascorbyl radicals in the circulation of the rat. Since cytochrome c and acetylated cytochrome c disappear from the circulation with a half-life of 4 min, these proteins cannot be used for in vivo detection of superoxide radicals and related metabolites. To determine superoxide and other radicals in vivo, a cytochrome c derivative (SMAC) was synthesized by linking 1 mol of poly(styrene-co-maleic acid) butyl ester (SM) to cytochrome c, followed by acetylation of its lysyl amino groups. SMAC retained 8 and 80% of cytochrome c activity to react with ascorbyl and superoxide radicals, respectively. However, SMAC did not serve as a substrate for cytochrome c reductase and cytochrome c oxidase. When injected intravenously to the rat, SMAC circulated bound to albumin with a half-life of 130 min. SMAC was rapidly reduced in the circulation of intact animals. Treatment of animals with paraquat markedly enhanced the reduction of the circulating SMAC. We have synthesized an SM-conjugated superoxide dismutase (SOD) derivative (SM-SOD) that circulates bound to albumin with a half-life of 6 h. Kinetic analysis revealed that SM-SOD effectively inhibited the superoxide-dependent reduction of SMAC either in the presence or absence of 0.5 mM albumin. However, the reduction of the circulating SMAC was not inhibited by SM-SOD both in normal and paraquat-treated animals. Plasma samples from both animal groups also reduced cytochrome c and SMAC by an SOD-insensitive mechanism. However, after treatment with ascorbate oxidase, both plasma samples lost their activity to reduce cytochrome c and SMAC. These and other results suggest that ascorbyl radical might principally be responsible for the reduction of circulating SMAC and that plasma levels of ascorbyl radical might increase in paraquat-treated animals.


Inactivation-reactivation of aconitase in Escherichia coli. A sensitive measure of superoxide radical. The rapid inactivation of aconitase by O2-, previously seen to occur in vitro, was explored in vivo. A fraction of the aconitase in growing, aerobic, Escherichia coli is inactive at any instant but can be activated by imposition of anaerobic conditions. This reactivation occurred in the absence of protein synthesis and was inhibited by the ferrous chelator alpha,alpha'-dipyridyl. This fraction of inactive, but activatable, aconitase was increased by augmenting O2- production with paraquat, decreased by elevation of superoxide dismutase, and increased by inhibiting reactivation with alpha,alpha'-dipyridyl. The balance between inactive and active aconitase thus represented a pseudoequilibrium between inactivation by O2- and reactivation by restoration of Fe(II), and it provided, for the first time, a measure of the steady-state concentration of O2- within E. coli. On this basis, [O2-] was estimated to be approximately 20-40 pM in aerobic log phase E. coli containing wild type levels of superoxide dismutase and approximately 300 pM in a mutant strain lacking superoxide dismutase.


Heparin binding to protein C inhibitor.

Heparin binding to protein C inhibitor. Protein C inhibitor is a plasma protein whose ability to inhibit activated protein C, thrombin, and other enzymes is stimulated by heparin. These studies were undertaken to further understand how heparin binds to protein C inhibitor and how it accelerates proteinase inhibition. The region of protein C inhibitor from residues 264-283 was identified as the heparin-binding site. This differs from the putative heparin-binding site in the related proteins antithrombin and heparin cofactor. The glycosaminoglycan specificity of protein C inhibitor was relatively broad, including heparin and heparan sulfate, but not dermatan sulfate. Non-sulfated and non-carboxylated polyanions also enhanced proteinase inhibition by protein C inhibitor. Heparin accelerated inhibition of alpha-thrombin, gamma T-thrombin, activated protein C, factor Xa, urokinase, and chymotrypsin, but not plasma kallikrein. The ability of glycosaminoglycans to accelerate proteinase inhibition appeared to depend on the formation of a ternary complex of inhibitor, proteinase, and glycosaminoglycan. The optimum heparin concentration for maximal rate stimulation varied from 10 to 100 micrograms/ml and was related to the apparent affinity of the proteinase for heparin. There was no obvious relationship between heparin affinity and maximum inhibition rate or degree of rate enhancement. The affinity of the resultant protein C inhibitor-proteinase complex was also not related to inhibition rate enhancement, and the results showed that decreased heparin affinity of the complex is not an important part of the catalytic mechanism of heparin. The importance of protein C inhibitor as a regulator of the protein C system may depend on the relatively large increase in heparin-enhanced inhibition rate for activated protein C compared to other proteinases.


A comparison of three heparin-binding serine proteinase inhibitors. The purpose of this study was to compare three heparin-binding plasma proteinase inhibitors in order to identify common and unique features of heparin binding and heparin-enhanced proteinase inhibition. Experiments with antithrombin, heparin cofactor, and protein C inhibitor were performed under identical conditions in order to facilitate comparisons. Synthetic peptides corresponding to the putative heparin binding regions of antithrombin, heparin cofactor, and protein C inhibitor bound to heparin directly and interfered in heparin-enhanced proteinase inhibition assays. All three inhibitors obeyed a ternary complex mechanism for heparin-enhanced thrombin inhibition, and the optimum heparin concentration was related to the apparent heparin affinity of the inhibitor. The maximum inhibition rate and rate enhancement due to heparin appeared to be unique properties of each inhibitor. In assays with heparin oligosaccharides of known size, only the antithrombin-thrombin reaction exhibited a sharp threshold for rate enhancement at 14-16 saccharide units. Acceleration of antithrombin inhibition of factor Xa, heparin cofactor inhibition of thrombin, and protein C inhibitor inhibition of thrombin, activated protein C, and factor Xa did not require a minimum saccharide size. The differences in heparin size dependence and rate enhancement of proteinase inhibition by these inhibitors might reflect differences in the importance of the ternary complex mechanism and other mechanisms, alterations in inhibitor reactivity, and orientation effects in heparin-enhanced proteinase inhibition.


Expression of a ubiquitin derivative that conjugates to protein irreversibly produces phenotypes consistent with a ubiquitin deficiency. Ubiquitin (Ub) exists in a dynamic equilibrium between the free form and the conjugated form. This equilibrium is maintained and regulated through the antagonistic actions of the conjugation system and a class of enzymes referred to collectively as the Ub-protein hydrolases. Using a previously described epitope-tagged Ub approach (Ellison, M., and Hochstrasser, M. (1991) J. Biol. Chem. 266, 21150-21157) we show here that a single amino acid substitution at the carboxyl terminus of Ub (Gly-76 to Ala-76) results in a derivative of Ub (UbA-76) that becomes irreversibly conjugated to protein when expressed in the yeast Saccharomyces cerevisiae, producing a profound effect on the Ub-conjugate equilibrium. The major target of UbA-76 conjugation is itself (and presumably wild-type Ub) producing unanchored chains at the expense of the free form. Unsurprisingly, the expression of UbA-76 results in yeast phenotypes that would be expected in situations of Ub deprivation. Such cells show slow growth characteristics and sensitivity to various forms of environmental stress and to ultraviolet light. In view of these findings, the expression of UbA-76 in higher organisms may represent a convenient epigenetic strategy for examining the physiological consequences of Ub deprivation or Ub-protein hydrolase disfunction in living cells without the need for gene disruption or replacement. The observation that UbA-76 couples to itself irreversibly also provides an effective tool for elucidating the role of Ub as the proteolytic signal.


Sodium-dependent nucleoside transport in choroid plexus from rabbit. Evidence for a single transporter for purine and pyrimidine nucleosides. The overall goal of this study was to determine the mechanisms by which nucleosides are transported in choroid plexus. Choroid plexus tissue slices obtained from rabbit brain were depleted of ATP with 2,4-dinitrophenol. Uridine and thymidine accumulated in the slices against a concentration gradient in the presence of an inwardly directed Na+ gradient. The Na(+)-driven uptake of uridine and thymidine was saturable with Km values of 18.1 +/- 2.0 and 13.0 +/- 2.3 microM and Vmax values of 5.5 +/- 0.3 and 1.0 +/- 0.2 nmol/g/s, respectively. Na(+)-driven uridine uptake was inhibited by naturally occurring ribo- and deoxyribonucleosides (adenosine, cytidine, and thymidine) but not by synthetic nucleoside analogs (dideoxyadenosine, dideoxycytidine, cytidine arabinoside, and 3'-azidothymidine). Both purine (guanosine, inosine, formycin B) and pyrimidine nucleosides (uridine and cytidine) were potent inhibitors of Na(+)-thymidine transport with IC50 values ranging between 5 and 23 microM. Formycin B competitively inhibited Na(+)-thymidine uptake and thymidine trans-stimulated formycin B uptake. These data suggest that both purine and pyrimidine nucleosides are substrates of the same system. The stoichiometric coupling ratios between Na+ and the nucleosides, guanosine, uridine, and thymidine, were 1.87 +/- 0.10, 1.99 +/- 0.35, and 2.07 +/- 0.09, respectively. The system differs from Na(+)-nucleoside co-transport systems in other tissues which are generally selective for either purine or pyrimidine nucleosides and which have stoichiometric ratios of 1. This study represents the first direct demonstration of a unique Na(+)-nucleoside co-transport system in choroid plexus.


Intramolecular translocation of the protein radical formed in the reaction of recombinant sperm whale myoglobin with H2O2. A sperm whale myoglobin gene containing multiple unique restriction sites has been constructed in pUC 18 by sequential assembly of chemically synthesized oligonucleotide fragments. Expression of the gene in Escherichia coli DH5 alpha cells yields protein that is identical to native sperm whale myoglobin except that it retains the terminal methionine. Site-specific mutagenesis has been used to prepare all the possible tyrosine----phenylalanine mutants of the recombinant myoglobin, including the three single mutants at Tyr-103, -146, and -151, the three double mutants, and the triple mutant. All of the mutant proteins are stable except the Tyr-103 mutant. Introduction of a second mutation (Lys-102----Gln) stabilizes the Tyr-103 mutant. Absorption spectroscopy suggests that the active sites of the mutant proteins are intact. EPR and absorption spectroscopy show that all the proteins, including the triple mutant devoid of tyrosine residues, react with H2O2 to give a ferryl species and a protein radical. The presence of a protein radical in all the mutants suggests that the radical center is readily transferred from one amino acid to another. Cross-linking studies show, however, that protein dimers are only formed when Tyr-151 is present. Tyr-103, shown earlier to be the residue that primarily cross-links to Tyr-151 (Tew, D., and Ortiz de Montellano, P. R. (1988) J. Biol. Chem. 263, 17880-17886), is not essential for cross-linking. Electron transfer from Tyr-151 to the heme, which are 12 A apart, occurs in the absence of the intervening tyrosines at positions 103 and 146. The present studies show that the peroxide-generated myoglobin radical readily exchanges between remote loci, including non-tyrosine residues, but protein cross-linking only occurs when radical density is located on Tyr-151.


Structure-function analysis of casein kinase 2 with synthetic peptides and anti-peptide antibodies. Casein kinase 2 (CK2) is a ubiquitous, multifunctional protein-seryl/threonyl kinase that has been implicated in cellular regulation. Synthetic peptides were patterned after three highly conserved regions in CK2: the N terminus (CK2-NT); the lysine-rich, kinase subdomain III segment (CK2-III) (nomenclature of Hanks et al. (Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42-52)); and a 10-residue segment located near kinase subdomain X that is shared between CK2 and p34cdc2 (CK2/cdc2). The CK2-III and CK2/cdc2 peptides markedly stimulated the autophosphorylation of the alpha- and alpha'-subunits of purified CK2 from sea star oocytes, and they elicited up to 2-fold increases in its casein or phosvitin phosphotransferase activity. These peptides completely reversed nearly total inhibition of CK2 phosphotransferase activity toward itself, casein, and phosvitin by either heparin or poly(Glu,Tyr; 4:1), whereas CK2-NT was ineffective. Elution of CK2 from heparin-agarose with the CK2-III peptide indicated that this region of CK2 might mediate heparin binding to CK2. Affinity-purified rabbit polyclonal antibodies developed against both CK2-III and CK2/cdc2, but not CK2-NT, also produced up to 1.8-fold enhancements of the casein and phosvitin phosphotransferase activities of purified CK2. All three of the antipeptide antibody preparations immunoreacted with the alpha- and alpha'-subunits of CK2 on Western blots. These studies indicate that kinase subdomains III and X are involved in the modulation of CK2 phosphotransferase activity.


Evidence that the hamster tunicamycin resistance gene encodes UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase. A cDNA clone isolated from Chinese hamster ovary cells conferred elevated GlcNAc-1-P-transferase (GPT) activity and resistance to tunicamycin in transfected cells (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250-14255). It had been assumed that this cDNA, termed TRG for tunicamycin resistance gene, encoded GPT enzyme. However, other functions were not ruled out. Thus, by one of several mechanisms, the TRG protein could have instead functioned by activation of the transfected host's endogenous GPT enzyme. To analyze the biochemical function of the TRG protein, hamster TRG cDNA was stably expressed at high levels in Chinese hamster ovary cells. In addition, several antipeptide polyclonal antibodies directed against the predicted TRG protein were obtained. With these tools in hand, experiments were performed to test the hypothesis that the TRG encodes GPT enzyme, as well as to rule out other possible functions for the TRG protein. These experiments included examination of the effects of solubilization of membranes on TRG-dependent GPT activity, the apparent binding of tunicamycin to the TRG protein, and the immunoadsorption of GPT activity with TRG protein-specific antibodies. From these results, we conclude that the hamster TRG most likely encodes GPT enzyme.


Monooxygenase activity of cytochrome c peroxidase. Recombinant cytochrome c peroxidase (CcP) and a W51A mutant of CcP, in contrast to other classical peroxidases, react with phenylhydrazine to give sigma-bonded phenyl-iron complexes. The conclusion that the heme iron is accessible to substrates is supported by the observation that CcP and W51A CcP oxidize thioanisole to the racemic sulfoxide with quantitative incorporation of oxygen from H2O2. Definitive evidence for an open active site is provided by stereoselective epoxidation by both enzymes of styrene, cis-beta-methylstyrene, and trans-beta-methylstyrene. trans-beta-methylstyrene yields exclusively the trans-epoxide, but styrene yields the epoxide and phenylacetaldehyde, and cis-beta-methylstyrene yields both the cis- and trans-epoxides and 1-phenyl-2-propanone. The sulfoxide, stereoretentive epoxides, and 1-phenyl-2-propanone are formed by ferryl oxygen transfer mechanisms because their oxygen atom derives from H2O2. In contrast, the oxygen in the trans-epoxide from the cis-olefin derives primarily from molecular oxygen and is probably introduced by a protein cooxidation mechanism. cis-[1,2-2H]-1-Phenyl-1-propene is oxidized to [1,1-2H]-1-phenyl-2-propanone without a detectable isotope effect on the epoxide:ketone product ratio. The phenyl-iron complex is not formed and substrate oxidation is not observed when the prosthetic group is replaced by delta-meso-ethylheme. CcP thus has a sufficiently open active site to form a phenyl-iron complex, to oxidize thioanisole to the sulfoxide, and to epoxidize styrene and beta-methylstyrene. The results indicate that a ferryl (Fe(IV) = O)/protein radical pair can be coupled to achieve two-electron oxidations. The unique ability of CcP to catalyze monooxygenation reactions does not conflict with its peroxidase function because cytochrome c is oxidized at a distinct surface site (DePillis, G. D., Sishta, B. P., Mauk, A. G., and Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19334-19341).


The insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor mediates IGF-II-induced motility in human rhabdomyosarcoma cells. Insulin-like growth factor-II (IGF-II) is an autocrine growth and motility factor for human rhabdomyosarcoma. It interacts with three different receptors: the IGF-I, the IGF-II, and the insulin receptor. A specific function of the IGF-II receptor in mediating IGF-II responses has not been defined. In this report we investigate the mechanism of IGF-II-mediated motility in rhabdomyosarcoma cells. We demonstrate that IGF-II and [Leu27]IGF-II, an analog selective for the IGF-II receptor, stimulate motility at concentrations in which they interact only with their own receptor. An antibody that blocks the IGF-I receptor does not inhibit either peptide activity, while an antibody specific for the IGF-II receptor suppresses the IGF-II-induced motility. This antibody does not interfere with rhabdomyosarcoma cell proliferation. We conclude that in rhabdomyosarcoma cells IGF-II stimulates two different responses mediated by distinct receptors: 1) a mitogenic response through the type I receptor and 2) a motility response through the type II receptor.


Photoaffinity labeling and characterization of isolated inositol 1,3,4,5-tetrakisphosphate- and inositol hexakisphosphate-binding proteins. We have isolated high affinity inositol (1,3,4,5)-tetrakisphosphate (IP4)- and inositol hexakisphosphate (IP6)-binding proteins from detergent-solubilized rat brain membranes using a P1-tethered IP4 derivative linked to an Affi-Gel support. To determine the identity, binding characteristics, and distribution of the individual IP4 recognition sites, we have synthesized an IP4 photoaffinity label probe, 125I-(D,L)-1-O-[N-(4-azidosalicyloxy)-3-aminopropyl-1-phospho]- IP4 (125I-ASA-IP4). Two apparently distinct IP4-binding proteins (IP4BP), isolated with the IP4 affinity column, display high affinity and selectivity for IP4 over inositol trisphosphate (IP3), inositol pentakisphosphate (IP5), and IP6. The first IP4-binding protein (IP4BP1) which has a KD for IP4 of 4 nM, is comprised of a protein at 182 kDa which is specifically photolabeled with high affinity by 125I-ASA-IP4. The second, IP4BP2, has an affinity for IP4 of 1.5 nM and contains proteins at 84 and 174 kDa, both of which are specifically photoaffinity labeled. A putative IP6-binding protein (IP6BP), also isolated with the IP4 affinity column, binds IP6 with a KD of 14 nM and comprises three proteins of 115, 105, and 50 kDa. The 115- and 105-kDa subunits, but not the 50-kDa subunit, specifically incorporate the photolabel. The IP4BP (182, 174, and 84 kDa) and IP6BP (115 and 105 kDa) proteins are specifically photolabeled in the crude membrane, partially purified, and purified fractions. These receptor-binding proteins vary in inositol phosphate specificity and in the effects of pH, Ca2+, and heparin on IP4 photoaffinity labeling. In addition, IP4BP and IP6BP are enriched in the brain but differ in their regional localizations within the brain.


Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase receptor. The role of the urokinase receptor (uPAR) in the internalization of the urokinase-plasminogen activator inhibitor type-1 (uPA.PAI-1) complex has been investigated. First, exploiting the species specificity of uPA binding, we show that mouse LB6 cells (that express a mouse uPAR) were unable to bind or degrade the human uPA.PAI-1 complex. On the other hand, LB6 clone 19 cells, which express a transfected human uPAR, degraded uPA.PAI-1 complexes with kinetics identical to the human monocytic U937 cells. We also show by immunofluorescence experiments with anti-uPA antibodies that in LB6 clone 19 cells, the uPA.PAI-1 complex is indeed internalized. While at 4 degrees C uPA fluorescence was visible at the cell surface, shift of the temperature to 37 degrees C caused a displacement of the immunoreactivity to the cytoplasmic compartment, with a pattern indicating lysosomal localization. If uPA.PAI-1 internalization/degradation is mediated by uPAR, inhibition of uPA.PAI-1 binding to uPAR should block degradation. Three different treatments, competition with the agonist amino-terminal fragment of uPA, treatment with a monoclonal antibody directed toward the binding domain of uPAR or release of uPAR from the cell surface with phosphatidylinositol-specific phospholipase C completely prevented uPA.PAI-1 degradation. The possibility that a serpin-enzyme complex receptor might be primarily or secondarily involved in the internalization process was excluded since a serpin-enzyme complex peptide failed to inhibit uPA.PAI-1 binding and degradation. Similarly, complexes of PAI-1 with low molecular mass uPA (33 kDa uPA), which lacks the uPAR binding domain, were neither bound nor degraded. Finally we also show that treatment of cells with uPA.PAI-1 complex caused a specific but partial down-regulation of uPAR. A similar result was obtained when PAI-1 was allowed to complex to uPA that had been previously bound to the receptor. The possibility therefore exists that the entire complex uPA.PAI-1-uPAR is internalized. All these data allow us to conclude that internalization of the uPA.PAI-1 complex is mediated by uPAR.


Topology of the membrane-bound alkane hydroxylase of Pseudomonas oleovorans. The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that is expressed and active in both Escherichia coli and P. oleovorans. Its primary sequence contains eight hydrophobic stretches that could span the membrane as alpha-helices. The topology of alkane hydroxylase was studied in E. coli using protein fusions linking different amino-terminal fragments of the alkane hydroxylase (AlkB) to alkaline phosphatase (PhoA) and to beta-galactosidase (LacZ). Four AlkB-PhoA fusions were constructed using transposon TnphoA. Site-directed mutagenesis was used to create PstI sites at 12 positions in AlkB. These sites were used to create AlkB-PhoA and AlkB-LacZ fusions. With respect to alkaline phosphatase and beta-galactosidase activity each set of AlkB-PhoA and AlkB-LacZ fusions revealed the expected complementary activities. At three positions, PhoA fusions were highly active, whereas the corresponding LacZ fusions were the least active. At all other positions the PhoA fusions were almost completely inactive, but the corresponding LacZ fusions were highly active. These data predict a model for alkane hydroxylase containing six transmembrane segments. In this model the amino terminus, two hydrophilic loops, and a large carboxyl-terminal domain are located in the cytoplasm. Only three very short loops near amino acid positions 52, 112, and 251 are exposed to the periplasm.


Slow interaction of 5'-adenylyl-beta,gamma-imidodiphosphate with Escherichia coli DNA gyrase. Evidence for cooperativity in nucleotide binding. We have examined the kinetics of interaction between Escherichia coli DNA gyrase and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP) in the presence and absence of ATP. In the absence of ATP, [alpha-32P]ADPNP binds extremely slowly to gyrase, with an apparent second-order rate constant (k1) of 120 M-1 min-1. Similarly, the limited negative supercoiling of closed-circular DNA caused by ADPNP binding is slow, requiring at least 2 h to reach completion in the presence of 100 microM ADPNP. A very slow but detectable rate of dissociation of ADPNP from gyrase was measured, with a rate constant of 3.5 x 10(-4) min-1. The calculated dissociation constant for ADPNP is thus 2.9 microM. ADPNP is a potent competitive inhibitor of ATP-dependent DNA supercoiling. Inhibition is established much more rapidly than can be accounted for by the slow rate of ADPNP binding in the absence of ATP. We have found that ATP can accelerate the rate of [32P]ADPNP binding by more than 15-fold (k1 = 1,850 M-1 min-1). The ATP-promoted rate enhancement requires the presence of DNA; in the absence of DNA, ATP has no effect on the rate of binding. Relaxed closed-circular, nicked-circular, and linear pBR322 DNA are all equally effective cofactors for ATP-stimulated binding of ADPNP. After a short lag, the presence of ATP also greatly speeds up ADPNP dissociation from gyrase bound initially to closed-circular DNA, with the restoration of DNA supercoiling activity. This effect is not observed in the presence of nicked-circular or linear DNA, suggesting that ADPNP dissociates more rapidly from gyrase bound to supercoiled DNA. The results of ADPNP binding provide evidence for cooperative interactions between the nucleotide binding sites. To account for these data, a model is proposed for the interaction of nucleotides at the two ATP binding sites on DNA gyrase.


Phosphorylation by actin kinase of the pointed end domain on the actin molecule. Fragmin from plasmodium of Physarum polycephalum binds G-actin and severs F-actin in the presence of Ca2+ over 10(-6) M. The fragmin-actin complex consisting of fragmin and G-actin nucleates actin polymerization and caps the barbed (fast growing) end of F-actin, regardless of the concentrations of Ca2+, and the actin filaments are shortened. Actin kinase purified from plasmodium abolishes the nucleation and capping activities of the complex by phosphorylating actin of the fragmin-actin complex (Furuhashi, K., and Hatano, S. (1990) J. Cell. Biol. 111, 1081-1087). This inactivation of the complex leads to production of long actin filaments. We obtained evidence that Physarum actin is phosphorylated by actin kinase at Thr-201, and probably at Thr-202 and/or Thr-203, with 1 mol of phosphate distributed among them. This finding raises the possibility that the site of phosphorylation, Thr-201 to Thr-203, is positioned on the pointed (slow growing) end domain of the actin molecule, because growth of actin filaments from the fragmin-actin complex occurs only from the pointed end. These observations are consistent with a model of the three-dimensional structure of G-actin. Inactivation of the fragmen-actin complex may follow phosphorylation of the pointed end domain of actin.


Protein products of the rat kallikrein gene family. Substrate specificities of kallikrein rK2 (tonin) and kallikrein rK9. Two closely related kallikrein-like proteinases having little activity toward the standard synthetic amide substrates of tissue kallikreins were isolated from the rat submandibular gland. They were found to be the protein products of the rKlk2 (tonin) and the rKlk9 genes by amino acid sequence analysis (nomenclature of the genes and proteins of the kallikrein family is according to the proposal of the discussion panel from the participants of the KININ '91 meeting held Sept. 8-14, 1991, in Munich, Germany). These two proteinases of similar structure also had very similar physicochemical properties. They differed from other kallikrein-related proteinases in having high pHi values of 6.20 (rK2) and 6.85 (rK9). Kallikrein rK2 was purified as a single peptide chain, whereas rK9 appeared as a two-chain protein after reduction. Their enzymatic properties were also very similar and differed significantly from those of other rat kallikrein-related proteinases. Unlike the five other kallikrein-related proteinases we have purified so far, kallikrein rK9 was not inhibited by aprotinin. rK9 also differed from rK2 by its tissue localization. The prostate gland contained only rK9 where it was the major kallikrein-like component. The amino acids preferentially accommodated by the proteinase S3 to S2' subsites were identified using synthetic amide and protein substrates. Unlike other kallikrein-related proteinases, rK2 had a prevalent chymotrypsin-like specificity, whereas rK9 had both chymotrypsin-like and trypsin-like properties. Both rK2 and rK9 preferred a prolyl residue in position P2 of the substrate and did not accommodate bulky and hydrophobic residues at that position, as did most of the other kallikrein-related proteinases. This P2-proline-directed specificity is necessary for processing the precursors of several biologically active peptides. Subsites accommodating residues COOH-terminal to the scissile bond were also important in determining the overall substrate specificity of these proteinases. rK2 and rK9 both showed a preference for hydrophobic residues in P2'. Other subsites upstream of the S3 subsite were found to intervene in substrate binding and hydrolysis. The restricted specificity of rK2 and rK9 is consistent with the presence of an extended substrate binding site, and hence with a processing enzyme function. Their P1 specificities enabled both proteinases to release angiotensin II from angiotensinogen and from angiotensinogen I, but rK9 was at least 100 times less active than rK2 on both substrates. The substrate specificities of rK2 and rK9 were correlated with key amino acids defining their substrate binding site.(ABSTRACT TRUNCATED AT 400 WORDS)


Point mutations which drastically affect the polymerization activity of encephalomyocarditis virus RNA-dependent RNA polymerase correspond to the active site of Escherichia coli DNA polymerase I. The inhibitor sensitivity and functional domains of recombinant encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) have been extensively analyzed. The inhibitor profiles of EMC virus 3Dpol and Escherichia coli DNA-dependent RNA polymerase are distinct, and experiments with substrate analogs indicate that EMC virus 3Dpol lacks reverse transcriptase activity. Twenty amino acid substitutions were engineered in EMC virus 3Dpol based on sequence alignments of viral RNA-dependent RNA polymerases that identified conserved amino acid residues within motifs. Ten out of 17 conservative substitutions within the four most conserved motifs reduced the RNA polymerase activity of the mutants to 0-6% of the activity of the wild-type enzyme, demonstrating the importance of these amino acids in the structure and/or function of EMC virus 3Dpol. Remarkably, 5 of the 10 mutations in EMC virus 3Dpol which had the most drastic effect on its RNA polymerase activity (D240E, S293T, N302Q, G332A, and D333E) were found to correspond to active site residues in E. coli DNA-dependent DNA polymerase I (Klenow). Our results reveal that a basic structural and functional framework is conserved in the most distantly related classes of nucleic acid polymerases and demonstrate the validity of modeling the active site of an RNA-dependent RNA polymerase on the known structure of a DNA polymerase.


Expression, purification, and characterization of a recombinant ribonuclease H from Thermus thermophilus HB8. Thermus thermophilus ribonuclease H was overexpressed and purified from Escherichia coli. The determination of the complete amino acid sequence allowed modification of that predicted from the DNA sequence, and the enzyme was shown to be composed of 166 amino acid residues with a molecular weight of 18,279. The isoelectric point of the enzyme was 10.5, and the specific absorption coefficient A0.1%(280) was 1.69. The enzymatic and physicochemical properties as well as the thermal and conformational stabilities of the enzyme were compared with those of E. coli RNase HI, which shows 52% amino acid sequence identity. Comparison of the far and near UV circular dichroism spectra suggests that the two enzymes are similar in the main chain folding but different in the spatial environments of tyrosine and tryptophan residues. The enzymatic activities of T. thermophilus RNase H at 37 and 70 degrees C for the hydrolysis of either an M13 DNA/RNA hybrid or a nonanucleotide duplex were approximately 5-fold lower and 3-fold higher, respectively, as compared with E. coli RNase HI at 37 degrees C. The melting temperature, Tm, of T. thermophilus RNase H was 82.1 degrees C in the presence of 1.2 M guanidine hydrochloride, which was 33.9 degrees C higher than that observed for E. coli RNase HI. The free energy changes of unfolding in the absence of denaturant, delta G[H2O], of T. thermophilus RNase H increased by 11.79 kcal/mol at 25 degrees C and 14.07 kcal/mol at 50 degrees C, as compared with E. coli RNase HI.


Mutations that alter the activity of the Rous sarcoma virus protease. Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing "flaps," and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P,G106V, as well as the triple mutant, H65G,R105P,G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein.


Analysis of substrate interactions of the Rous sarcoma virus wild type and mutant proteases and human immunodeficiency virus-1 protease using a set of systematically altered peptide substrates. In the preceding study, mutant Rous sarcoma virus (RSV) proteases are described in which three amino acids found in the human immunodeficiency virus-1 (HIV-1) protease (PR) were substituted into structurally comparable positions (Grinde, B., Cameron, C.E., Leis, J., Weber, I., Wlodawer, A., Burstein, H., Bizub, D., and Skalka, A. M. (1992) J. Biol. Chem. 267, 9481-9490). In this report, the activity of the wild type and these mutant PRs are compared using a set of RSV NC-PR peptide substrates with single amino acid substitutions in each of the P4 to P3' positions. With most substrates, the relative activities of the two active mutants followed that of the RSV PR. Substitutions in the P1 and P1' positions were an exception; in this case, the mutants behaved more like the HIV-1 PR. These results confirm predictions from structural analyses which indicate that residues 105 and 106 of the RSV PR are important in forming the S1 and S1' binding subsites. These results, further analyzed with the aid of computer modeling of the RSV PR with different substrates, provide an explanation for why only partial HIV-1 PR-like behavior was introduced into the above RSV PR mutants.


mRNA capping enzyme. Isolation and characterization of the gene encoding mRNA guanylytransferase subunit from Saccharomyces cerevisiae. The highly purified yeast mRNA capping enzyme is composed of two separate chains of 52 (alpha) and 80 kDa (beta), responsible for the activities of mRNA guanylyltransferase and RNA 5'-triphosphatase, respectively (Itoh, N., Yamada, H., Kaziro, Y., and Mizumoto, K. (1987) J. Biol. Chem. 262, 1989-1995). The gene encoding the mRNA guanylyltransferase subunit (alpha subunit), CEG1, has been isolated by immunological screening of a yeast genomic expression library in lambda gt11 with polyclonal antibodies directed against purified yeast capping enzyme. The identity of CEG1 was confirmed by epitope selection and by expressing the gene in Escherichia coli to give a catalytically active mRNA guanylyltransferase. The gene is present in one copy per haploid genome, and encodes a polypeptide of 459 amino acid residues. From its primary structure as well as its mRNA size, it was concluded that the alpha and the beta subunits of yeast mRNA capping enzyme are encoded by two separate genes, not as a fused protein. CEG1 is located on the chromosome VII by a pulse-field gel electrophoresis. Gene disruption experiment indicated that CEG1 is essential for the growth of yeast. We have also found another open reading frame (ORF2) which lies in close proximity to CEG1 in our clones and encodes a 450 amino acid-polypeptide of yet unknown function.


Flanking host sequences can exert an inhibitory effect on the cleavage step of the in vitro mu DNA strand transfer reaction. The effect of flanking host sequences on the cleavage step of the in vitro Mu DNA strand transfer reaction was investigated. Insertion of a mini-Mu molecule into certain sites in pUC19 results in insertions that demonstrate a decreased ability to form Type 1 complexes in subsequent rounds of transposition. Similarly, changes in the flanking host sequences directly adjacent to the Mu ends by in vitro mutagenesis can also result in Type 1-deficient mini-Mu molecules. Further examination of the inhibition revealed that Type 1 deficient mini-Mu molecules are capable of forming uncut synaptic complexes at normal levels but are compromised in their ability to serve as substrates for phosphodiester bond hydrolysis at the Mu ends. This cleavage defect can be overcome by addition of the Mu B protein and ATP to the reaction. Our data suggest that one of the roles of the B protein may be to provide a mechanism whereby Mu prophages with inhibitory flanking sequences can overcome this obstacle and avoid being trapped at unproductive locations.


Nitroxide metabolites from alkylhydroxylamines and N-hydroxyurea derivatives resulting from reductive inhibition of soybean lipoxygenase. One proposed mechanism of the inactivation of lipoxygenase by inhibitors is the reduction of the catalytically active ferric form of the enzyme to its ferrous form. Recent studies have shown that compounds containing the hydroxamate moiety are potent inhibitors of lipoxygenase. The hydroxamate portion of the inhibitor is thought to bind to iron at the catalytic site of the enzyme. We now report evidence that the NOH of the hydroxamate group of N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, N-[(E)-3-(3-phenoxyphenyl)prop-2-enyl]acetohydroxamic acid (BW A4C), and N-(1-benzo(b)thien-2-ylethyl)-N-hydroxyurea (Zileuton) is oxidized by lipoxygenase to form their corresponding nitroxides, which are directly detected by electron paramagnetic resonance spectroscopy. It is consistently found that the selected NOH-containing compounds, e.g. alkylhydroxylamines or N-hydroxyureas, are also oxidized by lipoxygenase to form their corresponding nitroxides.


A phenylalanine in peptide substrates provides for selectivity between cGMP- and cAMP-dependent protein kinases. Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.


Slow-binding inhibition of NAD+ glycohydrolase by arabino analogues of beta-NAD. Modifications at the 2'-position of the nicotinamide-ribosyl moiety influence dramatically the nature of the interactions of the modified beta-NAD+ with calf spleen NAD+ glycohydrolase (EC 3.2.2.6), an enzyme that cleaves the nicotinamide-ribose bound in NAD(P)+. Nicotinamide arabinoside adenine dinucleotide (ara-NAD+) and nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide (araF-NAD+) are not hydrolyzed at measurable rates and are the first documented examples of reversible slow binding inhibitors of this class of enzyme. The kinetic data obtained are consistent with both slow kon and koff rate constants in the formation of an enzyme-inhibitor complex, i.e. the association rate constants are about 10(4) and 10(6) slower than diffusion rates, respectively, for araF-NAD+ and ara-NAD+, and the half-life of the complex is about 3-10 min for both analogues. The kinetic model does not account for a slow turnover of an ADP-ribosyl-enzyme intermediary complex. AraF-NAD+ is one of the most potent inhibitors described for NAD+ glycohydrolase.


The role of the C-terminal domain in collagenase and stromelysin specificity. Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.


Aldosterone does not alter apical cell-surface expression of epithelial Na+ channels in the amphibian cell line A6. The steroid hormone aldosterone regulates reabsorptive Na+ transport across specific high resistance epithelia. The increase in Na+ transport induced by aldosterone is dependent on protein synthesis and is due, in part, to an increase in Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. To examine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a mechanism by which aldosterone increases apical membrane Na+ conductance, apical cell-surface proteins from the epithelial cell line A6 were specifically labeled by an enzyme-catalyzed radioiodination procedure following exposure of cells to aldosterone. Labeled Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical cell surface. The activation of Na+ transport across A6 cells by aldosterone was not accompanied by alterations in the biochemical pool of Na+ channels at the apical plasma membrane, despite a 3.7-4.2-fold increase in transepithelial Na+ transport. Similarly, no change in the distribution of immunoreactive protein was resolved by immunofluorescence microscopy. The oligomeric subunit composition of the channel remained unaltered, with one exception. A 75,000-Da polypeptide and a broad 70,000-Da polypeptide were observed in controls. Following addition of aldosterone, the 75,000-Da polypeptide was not resolved, and the 70,000-Da polypeptide was the major polypeptide found in this molecular mass region. Aldosterone did not alter rates of Na+ channel biosynthesis. These data suggest that neither changes in rates of Na+ channel biosynthesis nor changes in its apical cell-surface expression are required for activation of transepithelial Na+ transport by aldosterone. Post-translational modification of the Na+ channel, possibly the 75,000 or 70,000-Da polypeptide, may be one of the cellular events required for Na+ channel activation by aldosterone.


The transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a chimera consisting, at the amino terminus, of a Ser/Thr protein kinase (PK) with features of a signal peptide and a transmembrane (TM) helical segment, and at the carboxy-terminus, of the ribonucleotide reductase (Chung et al., 1989, 1990). Membrane immunofluorescence of ICP10 transformed cells with antibodies to synthetic peptides located upstream or downstream of the TM indicates that ICP10 is a membrane-spanning protein. Site-directed and deletion mutants were used to further characterize ICP10-PK. Mutation of Gly106 in catalytic motif I or of the invariant Lys in catalytic motif II, and deletion of both motifs (amino acids 106-178) did not eliminate kinase activity. PK activity was retained by the invariant Lys mutant expressed in bacteria and following protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to membrane filters. Both ICP10 and the invariant Lys mutant bound 14C-labeled rho-fluorosulfonylbenzoyl 5'-adenosine, an ATP affinity analog. The deletion mutant had 4-fold lower kinase activity than ICP10-PK, and it was insensitive to Mn2+, suggesting that these motifs are involved in Mn2+ activation of kinase activity. PK activity was lost by deletion of the TM segment (amino acid residues 85-106).