LIPIDAT: a database of lipid phase transition temperatures and enthalpy changes. DMPC data subset analysis. The systematic study of the mesomorphic phase properties of synthetic and biologically derived lipids began some 30 years ago. In the past decade, interest in this area has grown enormously. As a result, there exists a wealth of information on lipid phase behavior, but unfortunately these data have until now been scattered throughout the literature in a variety of books, proceedings and journals. The data have recently been compiled in a centralized database, LIPIDAT, with a view to providing ready access to the data and to the appropriate literature. LIPIDAT consists of a tabulation of all known mesomorphic and polymorphic phase transition temperatures and enthalpy changes for synthetic and biologically-derived lipids in the dry and in the partially and fully hydrated states. Also included is the effect of pH, and of salt and metal ion concentration and other additives such as proteins, drugs, etc., on the thermodynamic values. The methods used in making the measurements and the experimental conditions are reported. Bibliographic information includes comprehensive literature referencing and list of authors, but does not at the present time include article titles. As of this writing, the database is current through June, 1990 and is approaching 10,000 records in length. Each record contains 28 fields. In this paper we report the contents and present an analysis of LIPIDAT as it refers to fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). This database subset represents about 7% of all LIPIDAT records. It includes data collected over a 23-year period from 1967 to 1989 and consists of 702 records obtained from 336 articles in 55 different journals. The number of records per year rises steadily beginning in 1971, reaches a maximum of 89 records/year in 1977 and remains relatively constant at 60-70 records/year in the succeeding period. Journals making the greatest contribution to the DMPC subset include Biochimica et Biophysica Acta, Biochemistry, Chemistry and Physics of Lipids and the Biophysical Journal. These four journals account for 71% of the total records in the database subset. The analysis shows that differential scanning calorimetry, electron spin resonance, fluorescence, nuclear magnetic resonance and Raman spectroscopy are the methods most commonly used for DMPC transition temperature determination. An interesting pattern emerges as to the place in time the different methods assume or loose popularity.(ABSTRACT TRUNCATED AT 400 WORDS)


Effects of aspirin on 1,2-dimethylhydrazine-induced colonic carcinogenesis. The influence of aspirin (ASA) on 1,2-dimethylhydrazine (1,2-DMH)-induced colonic carcinogenesis was examined in weanling Sprague-Dawley rats. The incidence of adenocarcinomas in response to a single dose of 1,2-DMH was reduced 60% in rats receiving ASA for 1 week before and after the carcinogen. However, ASA had no effect on tumor incidence when initiated 4 weeks after a single dose of 1,2-DMH and continuing until the animals were killed at 36 weeks. The doses of ASA employed suppressed by 95% or more ex vivo colonic prostaglandin E2 (PGE2) production and reduced colonic mucosal cAMP levels in both rats exposed to 1,2-DMH and in age-matched controls. Proliferative activity of colonic mucosa as assessed from tritiated thymidine ([3H]dThd) incorporation into mucosal DNA was increased at 1 week but suppressed by 36 weeks after 1,2-DMH exposure. ASA significantly increased colonic mucosal DNA synthesis, suppressed colonic PGE2 production and reduced mucosal cAMP levels at both 1 and 36 weeks in rats given the 1,2-DMH vehicle. However, ASA failed to alter the enhanced mucosal DNA synthesis observed at 1 week or the suppressed DNA synthesis observed at 36 weeks after a single dose of 1,2-DMH, despite significant inhibition of colonic PGE2 production and reduction in mucosal cAMP levels by ASA. Treatment of rats for 1 week with ASA significantly inhibited basal and arachidonate stimulated decomposition of the 1,2-DMH intermediary metabolite methylazoxy-methanol, assessed ex vivo in colonic mucosal homogenates. Thus, while other mechanisms are not excluded, suppression of 1,2-DMH induced colonic carcinoma by concurrent administration of ASA may be linked in part to altered metabolic activation of this carcinogen via cyclooxygenase-dependent co-oxidation. By contrast, the previously reported suppression of the promotional phase of colonic carcinogenesis in rats by the delayed introduction of cyclooxygenase inhibitors may not be linked to inhibition of local colonic prostanoid production, since (i) inhibition of colonic prostanoid synthesis by ASA did not mimic this antipromotional effect, and (ii) the doses of non-steroidal anti-inflammatory drugs employed in some earlier studies may not significantly inhibit colonic prostanoid synthesis.


Activities of flavonoids for the cleavage of DNA in the presence of Cu(II): correlation with generation of active oxygen species. The activities of several flavonoids and the related nonflavonoid compound epicatechin were compared with respect to Cu(II)-induced strand scission of DNA by using two different assays. The same series of compounds was used to study the stoichiometry of Cu(II) reduction in the absence of DNA. The compounds were compared for their ability to generate superoxide, hydrogen peroxide and the Cu(II)-dependent production of hydroxyl radicals. Flavonoids were examined to assess the production of a charge-transfer complex with Cu and the rate of decay of the complexes were compared. All the compounds tested had some ability to cause DNA strand scission in the presence of Cu(II), with myricetin being the most active and galangin the least active. The ability to cause such scission correlated with the rate of decay of the charge-transfer complex, the ability to generate active oxygen species and with the stoichiometry of Cu(II) binding. Analysis of the data in the light of the structural differences between the flavonoids led to a discussion of alternative Cu(II)-sequestering mechanisms.


Induction of epidermal ornithine decarboxylase activity in mouse skin exposed to biogenic silica fibers. The present study demonstrates that biogenic silica fibers (BSF), previously shown to promote skin tumors in mice and more recently to promote the induction of mesotheliomas when injected into the pleural cavity of rats, rapidly induces epidermal ornithine decarboxylase (ODC) activity in SENCAR mice following topical application. The time course for induction of epidermal ODC by BSF was very similar to that observed following topical treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). Maximal ODC activity was observed 4-6 h following treatment with BSF. Cycloheximide (70 mg/kg i.p.) partially inhibited (61%) the induction of ODC by BSF at 5 h. In addition, retinoic acid (RA, 5 micrograms per mouse given 30 min before BSF) effectively inhibited BSF-induced ODC by 68%, while indomethacin (100 micrograms per mouse 2 h before BSF) had little or no effect. Copper(II) bis(diisopropylsalicylate) (2 mumol 30 min before BSF), an effective inhibitor of TPA-induced ODC activity and tumor promotion, also had little or no effect on BSF-induced ODC. The work described in this paper suggests that BSF induces epidermal ODC by a very specific mechanism that exhibits both similarities and differences with that of the phorbol ester, TPA. Nevertheless, this response strongly supports the conclusion that BSF is an effective tumor promoter in mouse skin and that ODC induction is an integral part of the mechanism of action of this environmental promoter.


Iron mobilization from crocidolite asbestos greatly enhances crocidolite-dependent formation of DNA single-strand breaks in phi X174 RFI DNA. The ability of the iron associated with asbestos to catalyze damage to phi X174 RFI DNA was determined and compared with iron mobilized from asbestos. Asbestos (1 mg/ml) suspended for 30 min in 50 mM NaCl containing 0.5 micrograms phi X174 RFI DNA, pH 7.5, did not catalyze detectable amounts of DNA single-strand breaks (SSB). However, addition of ascorbate (1 mM) resulted in 19, 26, 7 or 8% DNA with SSB for crocidolite, amosite, chrysotile or tremolite respectively. The percentage of DNA with SSB induced by each form of asbestos was directly related to its iron content. Inclusion of desferrioxamine B, which binds Fe(III) rendering it redox inactive, completely inhibited asbestos-dependent formation of DNA SSB, suggesting that iron was responsible for catalyzing the formation of DNA SSB. Mobilization of Fe(II) from crocidolite by citrate, EDTA or nitrilotriacetate (1 mM) in the absence of ascorbate resulted in 15, 33 or 63% DNA with SSB respectively. This activity was completely inhibited by compounds considered to be .OH scavengers, i.e. mannitol, 5,5-dimethyl-1-pyrroline N-oxide or salicylate (100 mM). Preincubation of crocidolite with citrate (1 mM) for 24 h resulted in mobilization of 52 microM iron and increased ascorbate-dependent induction of DNA SSB compared with crocidolite that was preincubated without citrate. Iron mobilized by citrate was entirely responsible for crocidolite-dependent formation of DNA SSB as evidenced by complete inhibition with desferrioxamine B. Therefore, the results of the present study strongly suggest that iron was responsible for asbestos-dependent generation of oxygen radicals, which resulted in the formation of DNA SSB. Mobilization of iron by chelators, followed by redox cycling, greatly enhanced crocidolite-dependent formation of DNA SSB. Thus, mobilization of iron in vivo by low mol. wt chelators may lead to the increased production of reactive oxygen species resulting in damage to biomolecules, such as DNA.


The stability of dioxin-receptor ligands influences cytochrome P450IA1 expression in human keratinocytes. Three dioxin-receptor ligands were analyzed for their effect on cytochrome P450IA1 mRNA expression in normal human keratinocytes. Although a 2 h pulsed treatment with the receptor agonists 2,3,7,8-tetrachlorodibenzofuran (TCDF) and beta-naphthoflavone (BNF) gave the same maximal induction response, the effect of BNF was transient compared to effect of TCDF. This was most likely due to metabolism of BNF as exemplified by the fact that a P450IA1 enzyme suicide-inhibitor, 1-ethynylpyrene, could prolong the induction response following a short BNF treatment. The TCDF induction of a reporter gene construct under the control of the -1140 to +2435 part of the CYPIA1 gene transiently transfected into HK was effectively inhibited by the dioxin-receptor antagonist alpha-naphthoflavone (ANF). In addition, ANF inhibited the accumulation of TCDF-activated nuclear receptors with capacity to bind to a xenobiotic response element. Interestingly, ANF could also suppress already maximally induced P450IA1 mRNA levels. The data demonstrate that the stability of the ligand influences the long-term effects on gene expression and that the effect of stable ligands may be masked due to receptor antagonist presence. In addition, the results support the hypothesis that a constant low level of activated nuclear receptors is required to maintain induced P450IA1 expression.


Novel detection by magnetic microcapsules in the human gastrointestinal tract of cross-linking agents and diet-dependent reactive oxygen species. Semi-permeable magnetic microcapsules previously shown able to trap gastrointestinal carcinogens and containing polyethyleneimine (PEI) were covalently labelled with [14CH3], and administered for the first time to humans (six healthy volunteers, 1.3 microCi/dose) in gelatin capsules together with radio-opaque gut transit markers (ROM), in order both to seek human endogenous cross-linking or bifunctional alkylating agents and assess gut transit features. No ill-effects were reported. Faecal ROM and 14C excretions were well correlated (r = 0.96), and net 14C recovery in faeces was 83-96%. Microcapsules were separated magnetically from faeces and 29-81% of specific labelling of microcapsules (nCi/10(6)) was found to have been removed during GI transit. Label cleavage out of these microcapsules was also found following in vitro anaerobic incubation with faecal slurries from two volunteers. On treatment with H2O2, label was removed selectively from the Fe-containing core in a dose-dependent manner. Therefore, label cleavage in vivo (not observed in rats consuming chow but found notably on consumption of low-fibre and/or high-beef human diets) is likely to arise from low mol. wt substances that give Fenton reaction producing hydroxyl radicals and oxidative demethylation. After GI transit, extensive core to membrane cross-linking in the microcapsules was found and was inversely related to faecal output. Cross-linking also was obtained to a greater extent during in vitro anaerobic incubation with faecal slurries. The GI mucosa would also be exposed to both types of agents, and several features of this microcapsule monitoring are in accord with putative risk-modulating effects. This first use of microcapsules for biomonitoring of the human GI tract thus seemed to be without hazard, and revealed extensive levels of agents likely to cause DNA damage.


DNA adducts detected by 32P-postlabelling, in the intestine of rats given bile from patients with familial adenomatous polyposis and from unaffected controls. Duodenal adenomas are common in patients with familial adenomatous polyposis (FAP) and cluster around the papilla, suggesting the involvement of bile in their development. Using 32P-postlabelling we determined levels of DNA adducts in the small bowel and colon of rats treated with bile from FAP and control patients. We found a significantly higher level of adducts in the small bowel of rats treated with FAP gallbladder bile compared with control gallbladder bile (P = 0.0034). This result supports the hypothesis that bile plays a role in the development of neoplasia in the foregut of FAP patients.


Balancing of energy-consuming processes of K 562 cells. A balance of ATP-consuming processes in human erythroleukemia (K 562) cells by use of the decreased 14CO2 formation from [1-14C]-glutamate following inhibition of energy-requiring processes is presented. This method was tested on Ehrlich mouse ascites tumour cells and was used in suspensions of K 562 cells with a low cell content. More than 90 percent of the ATP produced by oxidative phosphorylation could be accounted for in K 562 cells. Protein synthesis consumed about 35 percent, Na+/K(+)-ATPase about 20 percent and transcription processes 5-10 percent of the total ATP. The share of the Ca(2+)-dependent reactions was notably high at 25 percent in comparison with Ehrlich mouse ascites tumour cells, reticulocytes or hepatocytes. ATP consumption by DNA synthesis was assessed at 5-10 percent. Only less than 10 percent of the consumption of ATP produced oxidatively remained for other cellular reactions. The degree of coupling of K 562 cells was high in comparison with that of other eukaryotic cell types.


Alpha 1-adrenoreceptor blockade reduces the angiotensin II-induced vascular smooth muscle cell DNA synthesis in the rat thoracic aorta and carotid artery. We explored effects of alpha 1-adrenoreceptor blockade with prazosin on the increased vascular smooth muscle cell (SMC) DNA synthesis induced by angiotensin II (Ang II) in rats. Ang II was infused with or without prazosin or its solvent. Observations were compared with those in rats receiving saline or solvent. In group A, Ang II was infused for 2 weeks by subcutaneously implanted osmotic minipumps at a rate of 35 ng/100 g per minute. Group B received Ang II together with the alpha 1-adrenoreceptor antagonist prazosin (0.35 micrograms/100 g per minute). Group C received Ang II and 50% dimethyl sulfoxide (DMSO), the solvent of prazosin; group D received 50% DMSO; and group E received 0.9% NaCl (Ang II vehicle). All animals were infused with 5-bromo-2'-deoxyuridine for 2 weeks via separate minipumps to measure DNA synthesis. Ang II significantly increased the fraction of DNA synthesizing SMCs in the media of the thoracic aorta from 0.4 +/- 0.1% (mean +/- SD) in group E (n = 6) to 10.8 +/- 7.0% in group A (n = 8). Addition of prazosin to Ang II reduced the labeling fraction of SMCs to 3.0 +/- 2.2% (group B, n = 9). The remaining SMC DNA synthesis in the prazosin-treated group was probably due to the effects of the solvent of prazosin, i.e., 50% DMSO, since infusion of 50% DMSO alone increased the labeling fraction to 4.1 +/- 2.0% (group D, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)


Oxidized low density lipoproteins induce mRNA expression and release of endothelin from human and porcine endothelium. Experiments were designed to examine the effect of oxidized low density lipoproteins (Ox-LDLs) on the expression and the release of endothelin from cultured endothelial cells and intact blood vessels. Ox-LDLs (30-300 micrograms/ml), but not native low density lipoproteins (200 micrograms/ml), stimulated the expression of preproendothelin mRNA in porcine and human endothelial cells, leading to a time- and concentration-dependent release of the peptide into the culture medium. The Ox-LDL-stimulated release of endothelin was mimicked by acetylated low density lipoprotein and abolished by downregulation of protein kinase C by phorbol ester. In the intact porcine aorta, Ox-LDLs, but not native low density lipoproteins, also increased the release of peptide in an endothelium- and concentration-dependent manner. The maximal effect was observed at a concentration of 100 micrograms/ml. Incubation of the intact porcine aorta with the scavenger receptor antagonist dextran sulfate decreased the formation of endothelium evoked by Ox-LDLs. The Ox-LDL-stimulated production of the peptide was further augmented in the presence of thrombin (4 units/ml) and was unaffected by nitric oxide-generating compound 3-morpholinosydnonimine (10(-5) M). These results suggest that Ox-LDL may be an endogenous mediator of the augmented release of endothelin observed in hyperlipidemia and atherosclerosis. The increased production of the peptide could contribute to vasospastic events and may promote vascular smooth muscle proliferation and progression of atherosclerotic vascular disease.


Adrenergic blockade blunts adenosine concentration and coronary vasodilation during hypoxia. Myocardial hypoxia is thought to be an important stimulus for increasing interstitial adenosine concentration. The adenosine hypothesis of coronary control was investigated during steady-state hypoxia by making measurements of coronary venous and epicardial well adenosine concentrations in adrenergically intact dogs and in animals with alpha- and beta-receptor blockade. In the adrenergically intact group, hypoxia sufficient to lower coronary venous oxygen tension to 8 mm Hg increased coronary blood flow 243% from normoxic values. Both coronary venous and epicardial well adenosine concentrations were increased throughout the hypoxic period. In the adrenergically blocked group, hypoxia to a similar level of coronary venous oxygen tension produced an increase in coronary blood flow of only 75%, which was significantly less than in the adrenergically intact group (p less than 0.01). Coronary venous adenosine was only transiently elevated, and epicardial well adenosine was unchanged from control levels. In a separate group of alpha- and beta-receptor-blocked animals that received an infusion of L-homocysteine thiolactone during hypoxia, there was no difference in tissue S-adenosylhomocysteine levels compared with those of normoxic controls. It is concluded that much of the coronary vasodilation associated with systemic hypoxia is dependent on adrenergic activation and that adenosine may only play a role in sustained hypoxic vasodilation when adrenergic receptors are intact.


Depression of peak force without altering calcium sensitivity by the superoxide anion in chemically skinned cardiac muscle of rat. Among the mechanisms postulated to contribute to myocardial "stunning" is a depression of contractility by oxygen-derived free radicals. It has been suggested that these radicals might depress the calcium sensitivity of the contractile proteins. We have exposed the myofilaments (in chemically "skinned" rat cardiac muscle) to the superoxide anion and measured isometric force at controlled degrees of activation. Superoxide was generated by the xanthine/xanthine oxidase system: the effects to be described were shown to be specifically attributable to superoxide. Maximum calcium-activated force is reduced, or even completely abolished, in a dose-dependent fashion and without any alteration in calcium sensitivity. The myofilaments are highly sensitive to superoxide: significant force reduction has been shown to be caused by enzyme concentrations as low as 2 microunits/ml xanthine oxidase and with exposures of less than 1 minute to the generating system (at higher enzyme concentrations). Once force has been depressed, it cannot be recovered within the duration of the experiments described. When xanthine oxidase is applied during the calcium-induced contracture, tension falls steadily. However, a similar concentration is without immediate effect on the rigor contracture (evoked by applying ATP-free solutions). To account for the depression of maximum calcium-activated force, we conclude that some aspect of crossbridge behavior is particularly vulnerable to superoxide rather than that the radical has a nonspecific "proteolytic" effect. This action on the fundamental units of force production could contribute to myocardial stunning since the effects we report are consistent with many aspects of this phenomenon.


Immunolocalization and expression of functional and nonfunctional cell-to-cell channels from wild-type and mutant rat heart connexin43 cDNA. The carboxyl terminal cytoplasmic domain of distinct gap junction proteins may play an important role in assembly of functional channels as well as differential responsiveness to pH, voltage, and intracellular second messengers. Oligonucleotide-directed site-specific mutagenesis in a paired Xenopus laevis oocyte expression system was used to examine the expression of mRNAs encoding wild-type and carboxyl terminal mutant connexin43 (Cx43) proteins. Oocytes were stripped, injected with mRNA or distilled water (dH2O), preincubated for 16-20 hours, and then paired for 5-10 hours; this process was followed by electrophysiological recording using the dual voltage-clamp technique. Initial experiments compared the relative junctional conductances (Gjs) in oocyte pairs expressing Cx43 (382 amino acid residues) and two truncated mutants lacking most or a portion of the cytoplasmic carboxyl terminal. The shortest mutant (M241) contained 240 amino acid residues and was devoid of all phosphorylatable serine residues in the cytoplasmic tail; its length approximated the length of liver connexin26. The longest mutant (M257) tested contained 256 amino acid residues, including two serine residues. Oocyte pairs expressing M241 yielded a Gj similar to that of oocytes injected with dH2O, whereas M257 yielded a Gj similar to that of oocytes injected with Cx43. Immunoprecipitation studies showed that Cx43, M257, and M241 proteins were readily detectable in oocytes injected with their respective mRNAs, indicating that the lack of Gj observed with the M241 mRNA was not due to reduced translation. Immunocytochemical studies revealed that wild-type and both truncated mutants were localized to the area of cell-to-cell contact between the paired oocytes, indicating that protein targeting to the membrane was not inhibited in oocytes injected with M241 mRNA. Oocyte pairs expressing mutants in which serine residues were replaced with nonphosphorylatable amino acids (serine codon No. 255 AGC was converted to GCC, alanine, designated as M255S----A, and serine codon No. 244 AGC was converted to GGC, glycine, designated as M244S----G) showed Gjs similar to M257, indicating that these serine residues and, by inference, their phosphorylation state are not critical for expression of functional channels. The importance of the length of the carboxyl terminus was assessed by comparing the Gjs in a series of mutants that were intermediate in length between M257 and M241. Gradual shortening of the carboxyl terminus produced a gradual reduction of Gj relative to M257. However, simple deletion of amino acid residues 241-257 from the wild-type Cx43 did not affect Gj relative to M257.(ABSTRACT TRUNCATED AT 400 WORDS)


Kinetics of interaction of the lidocaine metabolite glycylxylidide with the cardiac sodium channel. Additive blockade with lidocaine. The recovery of the sodium channel from blockade by local anesthetic antiarrhythmic drugs is voltage dependent. Recovery from lidocaine-induced blockade is accelerated by hyperpolarization, whereas that from glycylxylidide (GX) blockade has been reported to be slowed by hyperpolarization. This striking difference occurs despite similarities in chemical structure. The fast recovery from GX block at depolarized potentials may lead to a partial reversal of lidocaine blockade when the two drugs are combined. We have examined the kinetics of interaction of GX with the cardiac sodium channel over a range of membrane potentials by measuring whole-cell currents in isolated rabbit myocytes under voltage clamp at 15 degrees C. In the absence of drug, slow inactivation developed with a time constant of 10.7 +/- 5.1 seconds (n = 6). During exposure to 74 mumol/l GX, block developed with a time constant of 7.0 +/- 3 seconds (n = 6). Because of the similar time course of slow inactivation and block, we used a high concentration of GX to induce a level of block sufficient for analysis. The onset of block was slower than that induced by lidocaine and was unaffected by variation of external sodium from 20 to 75 mmol/l. Use-dependent blockade of sodium channels was greater when pulse trains were applied from a holding potential of -100 than -140 mV. This suggested that recovery from GX block might be slower at -100 than -140 mV. Direct measurements gave time constants of recovery of 10.3 +/- 4.2 seconds at -100 mV (n = 6) and 4.1 +/- 0.4 seconds at -140 mV (n = 4). The combination of GX with lidocaine produced only additive blocking effects when pulse trains were applied from both holding potentials. Computer simulations of the requirements for the competitive displacement of a sodium channel blocker with slow kinetics by one with fast kinetics suggest that the recovery time constant of the fast drug must be 10-100-fold smaller than that of the slow drug. Rapid association kinetics effected by a large binding rate constant or a higher concentration of the fast blocking drug is also important. The simulations suggest that, for the interaction of GX and lidocaine, only additive blocking action should be observed over the range of stimulus frequencies used in these experiments.


Bovine angiotensin converting enzyme cDNA cloning and regulation. Increased expression during endothelial cell growth arrest. Angiotensin converting enzyme (ACE) is a zinc-containing dipeptidase that converts angiotensin I to angiotensin II, a powerful vasoconstrictor and smooth muscle growth factor. ACE activity has been shown to be dynamically regulated by hormones, ACE inhibitors, and endothelial cell growth state. To study how ACE expression is regulated, we isolated and sequenced the bovine ACE gene using both ACE-specific cDNA and genomic clones. Bovine ACE cDNA encodes a single polypeptide of 1,306 residues with a molecular mass of 150 kd. Bovine ACE is approximately 80% homologous to that of other species. It contains two homologous domains of equal size. Alignment of ACE sequences from bovine, human, mouse, and rabbit reveals that during evolution both domains have been highly conserved. We used the bovine ACE cDNA to study regulation of ACE gene expression during density-dependent growth arrest. As endothelial cells became growth-arrested (6 days after confluence), there was a 12-fold increase in ACE activity and a 90% decrease in DNA synthesis. Immunocytochemically detectable ACE markedly increased in growth-arrested cells. The increase in ACE was due to increased ACE gene expression, as assayed by RNase protection, which showed a 20-fold increase in ACE-specific mRNA. The present study shows that bovine ACE is highly regulated by endothelial cell growth state at the level of protein and mRNA expression. Such dynamic regulation may have important consequences for angiotensin II production during endothelial cell proliferation after arterial injury.


Alpha 1-receptor-independent activation of protein kinase C in acute myocardial ischemia. Mechanisms for sensitization of the adenylyl cyclase system. The activity of the adrenergic system plays an important role in the genesis of malignant arrhythmias and the spreading of the infarcted zone in acute myocardial ischemia. Acute myocardial ischemia induces an increased activity of adenylyl cyclase. This sensitization at the enzyme level as shown in the isolated perfused rat heart occurs rapidly after the onset of ischemia (5-15 minutes) and is rapidly reversible on reperfusion. With prolonged ischemia, it is only transient and is followed by a gradual loss of the adenylyl cyclase activity. The increased activity of adenylyl cyclase is even retained after partial purification, suggesting a covalent modification of the enzyme. Blockade of alpha 1-adrenergic receptors does not prevent this sensitization, demonstrating that it occurs independently of alpha 1-adrenergic receptor activation. Only blockade of protein kinase C by various inhibitors, such as polymyxin B or staurosporine, is able to completely prevent this sensitization process. Moreover, in acute myocardial ischemia an activation of protein kinase C could be identified using its translocation from the cytosol to the particulate fraction as an indicator. Blockade of alpha 1-adrenergic receptors using prazosin fails to prevent the activation of protein kinase C and consequently the sensitization of the adenylyl cyclase system, indicating that the ischemia-induced translocation of protein kinase C occurs independently of alpha 1-adrenergic receptors. These data characterize for the first time an important interaction of two effector enzymes of two distinct signal transduction pathways, i.e., the adenylyl cyclase system and the protein kinase C system in acute myocardial ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)


Subcellular distribution of beta 2-adrenergic receptors delineated with quantitative ultrastructural autoradiography of radioligand binding sites. beta-Adrenergic receptors play a critical role in signal transduction in the cardiovascular system. Regulation of beta-adrenergic receptor expression depends on multiple factors, including rates of synthesis and degradation of receptor protein and trafficking of receptors between the cell surface and putative intracellular compartments. To characterize the subcellular distribution of beta-adrenergic receptors and to delineate the ultrastructure of intracellular compartments in which adrenergic receptors reside in the steady state, we studied Chinese hamster fibroblast cells transfected with the human beta 2-adrenergic receptor gene. beta 2-Adrenergic receptors of these cells were covalently labeled with the lipophilic photoactivatable antagonist [125I]iodocyanopindolol diazarine, and specific radioligand binding sites were localized at the ultrastructural level of resolution with quantitative electron microscopic autoradiography. The density of beta 2-adrenergic receptors was approximately 100-fold greater in the plasmalemma than in any other compartment. Approximately 50% of total cellular receptors were identified on the cell surface in an apparently random, nonclustered distribution and without association with clathrin-coated pits or other cell surface structural specializations. The remaining receptors were distributed among intracellular membranous compartments, including smooth vesicles, rough endoplasmic reticulum, and the Golgi apparatus, organelles presumably involved in stages of receptor synthesis, degradation, or trafficking. In the basal state, there was no association of beta 2-adrenergic receptors with coated intracellular vesicles typical of the endocytotic pathway of selected cell surface receptors that function to internalize their extracellular ligands. These results are the first to rigorously quantify the subcellular distribution of beta-adrenergic receptors and unequivocally establish the presence of a substantial pool of intracellular receptors.


Exercise training reduces the sympathetic component of the blood pressure-heart rate baroreflex in man. 1. Exercise training reduces resting sympathetic activity, but the effects on sympathetic activation or withdrawal during baroreflex responses to blood pressure perturbations are controversial. The purpose of this study was to investigate the effects of training on both the vagal and sympathetic reflex heart rate responses to blood pressure changes. 2. Using 10 healthy males in a randomized cross-over design, we examined the effects of three 30 min cycling sessions at 70% of maximal capacity for 4 weeks on the steady-state reflex heart rate responses to perturbations in mean arterial pressure induced with injections of nitroprusside and phenylephrine. The method provides a sigmoidal relationship between changes in heart rate and blood pressure. The upper plateau (maximum tachycardia in response to blood pressure reduction) and lower plateau (maximum bradycardia in response to blood pressure elevation) are mainly mediated by the cardiac sympathetics and vagus, respectively. The slope of the relationship is a measure of reflex gain. 3. Training, which increased maximal oxygen consumption by 13 +/- 2% (mean +/- standard error of the difference), reduced supine and standing blood pressures by 3 +/- 1/3 +/- 1 mmHg (P less than 0.05) and 4 +/- 1/2 +/- 2 mmHg (P less than 0.05 for systolic), respectively, whereas resting heart rate was lowered by 6 +/- 1 beats/min (P less than 0.05). Reflex sensitivity in the presence of functioning vagus and sympathetics was not altered with training, but the vagal component of sensitivity, as assessed after sympathetic blockade with propranolol, was significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)


Renal, haemodynamic and hormonal interactions between atrial natriuretic factor and arginine vasopressin in patients with congestive heart failure. 1. Nine patients with compensated heart failure were infused with synthetic arginine vasopressin at a rate of 0.1 m-units min-1 kg-1 for 60 min to increase their plasma arginine vasopressin concentration. Synthetic human atrial natriuretic factor (3 pmol min-1 kg-1) or placebo was co-infused with the arginine vasopressin in random order in a single-blind cross-over design. 2. The resultant plasma concentrations of arginine vasopressin and atrial natriuretic factor fell to within the upper range observed in congestive heart failure. Compared with the infusion of arginine vasopressin alone, atrial natriuretic factor co-infusion enhanced both the urine flow rate and the sodium excretion rate (both P less than 0.05) without significant haemodynamic and hormonal effects. 3. Systematic blood pressure was elevated by arginine vasopressin infusion (P less than 0.05) without any change in heart rate. Co-infusion of atrial natriuretic factor did not affect these haemodynamic parameters. 4. These results suggest that an increased release of atrial natriuretic factor maintains water and sodium excretion in the presence of arginine vasopressin-induced renal modulations, and that the pressor effect of arginine vasopressin is not antagonized by the increased plasma level of atrial natriuretic factor in patients with congestive heart failure.


Alpha-adrenoceptor blockade by phentolamine inhibits adrenaline-induced platelet activation in vivo without affecting resting measurements. 1. The effects of phentolamine (500 micrograms/min) on platelet aggregability in vivo at rest and during adrenaline infusion were assessed by ex vivo filtragometry and measurements of plasma beta-thromboglobulin levels in 10 healthy male subjects. Plasma levels of von Willebrand factor antigen and free fatty acids were also measured. 2. Adrenaline induced marked and expected increases in heart rate and systolic blood pressure and decreased diastolic blood pressure when venous plasma adrenaline levels were elevated from 0.12 +/- 0.02 to 2.9 +/- 0.3 nmol/l (P less than 0.01). 3. Adrenaline caused platelet activation in vivo. Ex vivo filtragometry readings were shortened by 58 +/- 9% (P less than 0.01), plasma beta-thromboglobulin levels increased by 99 +/- 44% (P less than 0.01) and platelet counts increased by 26 +/- 6% (P less than 0.01). Plasma levels of von Willebrand factor antigen and free fatty acids increased by 53 +/- 5% and 475 +/- 113% (both P less than 0.01), respectively. 4. Phentolamine enhanced the beta-adrenergic vasodilator responses to adrenaline, as both the decrease in diastolic blood pressure and the reflexogenic increase in heart rate were enhanced (both P less than 0.01). Marked elevations in plasma noradrenaline levels were found during infusions of phentolamine and adrenaline (P less than 0.001). 5. Phentolamine did not alter platelet indices at rest, but abolished adrenaline-induced platelet activation, as filtragometry readings, plasma beta-thromboglobulin levels and platelet counts remained at, or below, resting levels. Responses of plasma levels of von Willebrand factor antigen and free fatty acids to adrenaline were not influenced by phentolamine and did not seem to influence platelet responses.(ABSTRACT TRUNCATED AT 250 WORDS)


Longitudinal study of platelet angiotensin II binding in human pregnancy. 1. Platelet angiotensin II binding, circulating angiotensin II levels, plasma renin substrate and plasma renin concentration were measured in a longitudinal study of 30 women during pregnancy and the puerperium. 2. There was a significant fall in platelet angiotensin II binding from 11 weeks gestation to 18 weeks gestation (P less than 0.01). There were no further significant changes in platelet angiotensin II binding until after delivery, a significant rise in platelet angiotensin II binding being found at 6 weeks post partum as compared with at 36 weeks gestation (P less than 0.01). There was no further significant change from 6 to 12 weeks post partum, and platelet angiotensin II binding at 6 and 12 weeks post partum in the pregnant cohort approximated to that in nonpregnant women. These changes parallel those known to occur in pressor responsiveness to angiotensin II in pregnancy. 3. Plasma angiotensin II concentration, plasma renin substrate and plasma renin concentration were all significantly higher during pregnancy than in the puerperium (P less than 0.001). There were no significant changes during pregnancy in plasma angiotensin II concentration or plasma renin concentration, although plasma renin substrate rose throughout. 4. Significant inverse correlations between platelet angiotensin II binding and plasma angiotensin II concentration (P less than 0.01), plasma renin substrate (P less than 0.01) and plasma renin concentration (P less than 0 greater than 001) were found during pregnancy. These data suggest that down-regulation of platelet angiotensin II binding by the components of the renin-angiotensin system pertains in pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelium-derived prostacyclin: effect of serum from women with normal and hypertensive pregnancy. 1. Pregnancy-induced hypertension (or pre-eclampsia) is characterized by vasoconstriction, platelet aggregation and altered capillary permeability, implying disordered endothelial function and/or structure. Serum from women with pregnancy-induced hypertension has been reported by others to be cytotoxic to endothelial cells in vitro. We hypothesized that such serum contains a factor that limits the ability of endothelial cells to produce and/or release prostacyclin. 2. Prostacyclin production by intact and damaged cultured human umbilical vein endothelial cells was measured after incubating these cells with serum from non-pregnant and normal pregnant women and women with pregnancy-induced hypertension. Confluent human umbilical vein endothelial cell monolayers (intact and damaged) were incubated with sera for 24 h at 37 degrees C followed by 1 h of incubation with added thrombin (stimulated production) or media (basal production). Supernatants were then collected for measurement of 6-keto-prostaglandin F1 alpha by radioimmunoassay. 3. Basal production of 6-keto-prostaglandin F1 alpha was greater in response to serum from non-pregnant women than to that from pregnant women. Within each group, sub-lethally damaged cells had a similar basal production of 6-keto-prostaglandin F1 alpha to that of intact cells. 4. Basal production of 6-keto-prostaglandin F1 alpha by intact or damaged cells incubated with sera from normal pregnant women and from women with pregnancy-induced hypertension was similar. 5. In all groups the addition of thrombin to intact endothelial cells increased 6-keto-prostaglandin F1 alpha production approximately 15-30-fold over basal levels, but only three- to five-fold in damaged endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Oestradiol-induced hypotension in spontaneously hypertensive rats: putative role for intracellular cations, sodium-potassium flux and prostanoids. 1. The effect of oestradiol alone and in combination with indomethacin on blood pressure, erythrocyte cation concentration and Na(+)-K+ flux has been studied in adult female normotensive and spontaneously hypertensive rats. 2. Oestradiol alone resulted in a significant decrease in blood pressure in spontaneously hypertensive rats (from 165.3 +/- 3.9 to 146.4 +/- 2.7 mmHg, P less than 0.001), whereas it induced a significant increase in normotensive rats (from 111.8 +/- 1.8 to 124.1 +/- 3.6 mmHg, P less than 0.001). When indomethacin and oestradiol were administered simultaneously or when indomethacin was given alone, no change in blood pressure occurred in spontaneously hypertensive rats (158.6 +/- 6.9 and 159.8 +/- 6.2 mmHg, respectively). 3. The fall in blood pressure induced by oestradiol in spontaneously hypertensive rats was associated with significant reductions in erythrocyte K+ concentration (from 127.4 +/- 1.2 to 116.9 +/- 1.7 mmol/l of cells, P less than 0.001), in erythrocyte Na+ concentration (from 14.3 +/- 0.8 to 13.0 +/- 0.6 mmol/l of cells, P less than 0.02), in ouabain-sensitive erythrocyte Na+ flux (from 17.8 +/- 0.3 to 16.0 +/- 0.4 mmol h-1 (l of cells)-1, P less than 0.01) and in ouabain-sensitive erythrocyte K+ flux (from 11.4 +/- 0.2 to 10.4 +/- 0.2 mmol h-1 (l of cells)-1, P less than 0.01). No change in blood pressure, erythrocyte cation concentration or Na(+)-K+ flux occurred when oestradiol and indomethacin were given together or when indomethacin was administered alone.(ABSTRACT TRUNCATED AT 250 WORDS)


Dose-response study of the redistribution of intravascular volume by angiotensin II in man. 1. The response of systemic and regional haemodynamic indices to increasing infusion rates of angiotensin II (1, 3 or 10 ng min-1 kg-1) or placebo [5% (w/v) D-glucose] was studied in eight normal male subjects. 2. As compared with placebo, angiotensin II infusion caused an incremental rise in the serum angiotensin II level [14.5 +/- 7.7 (placebo) to 187.2 +/- 36.1 (10 ng of angiotensin II min-1 kg-1) pmol/l; mean +/- 95% confidence interval] associated with a stepwise increase in total peripheral resistance [880 +/- 42 (placebo) to 1284 +/- 58 (10 ng of angiotensin II min-1 kg-1) dyn s cm-5] and a progressive reduction in cardiac output [8.3 +/- 0.4 (placebo) to 7.0 +/- 0.4 (10 ng of angiotensin II min-1 kg-1) litres/min]. 3. A stepwise fall in renal blood flow was observed with increasing angiotensin II infusion rate [1302 +/- 65 (placebo) to 913 +/- 64 (10 ng of angiotensin II min-1 kg-1) ml/min]. In contrast, calf blood flow was unaffected by 1 ng or 3 ng of angiotensin II min-1 kg-1 and was significantly increased by 10 ng of angiotensin II min-1 kg-1 (P less than 0.01). 4. Calf venous capacitance was uninfluenced by 1 ng of angiotensin II min-1 kg-1, but was significantly increased by both 3 ng (P less than 0.005) and 10 ng (P less than 0.001) of angiotensin II min-1 kg-1.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of oral lithium on bronchial reactivity in asthma. 1. Recognition that inositol phospholipid-derived second messengers are involved in the initiation and maintenance of airway smooth muscle contraction raises the possibility of new therapeutic approaches to the treatment of asthma. We anticipated that lithium, through its effects on cell signal transduction and ion-transport pathways, would be likely to protect the airways against constrictor stimuli. 2. We carried out a randomized, double-blind study of lithium carbonate in asthmatic patients. 3. After a 1 week run-in period, 27 patients were allocated lithium carbonate (800 mg) or placebo for 21 days with measurement of forced expiratory volume in 1 s, the dose of histamine causing a 20% fall in the forced expiratory volume in 1 s and serum lithium concentration on days 3, 7, 14 and 21. 4. Twenty-one patients completed the study (10 on lithium, 11 on placebo). Mean serum lithium levels for patients on active treatment were in the therapeutic range on all four occasions. 5. Lithium did not alter the forced expiratory volume in 1 s (P = 0.8) or the twice-daily peak expiratory flow (P = 0.15). It did, however, reduce histamine reactivity (the maximum difference between lithium and placebo was 1.2 doubling doses of histamine on day 21; 95% confidence interval 0.2-2.2 doubling doses), improve symptom scores (P less than 0.05) and reduce usage of beta-adrenoceptor agonist inhalers (P less than 0.05). 6. We conclude that lithium reduces bronchial reactivity in airway smooth muscle; this finding raises new therapeutic possibilities for the treatment of asthma.


Stimulation of human breast cancers by dietary L-arginine. 1. The amino acid L-arginine has been shown to enhance immune mechanisms and inhibit tumour growth in experimental animals, but although many of the immunological effects of arginine have been reproduced in man there have been few studies of its effects on human tumours. In this study the effects of arginine on human breast cancers were determined by measuring tumour protein synthesis and comparing this with immunohistochemical assessments of cell proliferation. 2. Patients with breast cancer were randomized to receive either a standard diet or arginine supplementation. At the time of surgery, the rate of tumour protein synthesis was measured by the incorporation of the stable isotope [1-13C]leucine into tumour protein. Tumours were also assessed histologically and by staining for the presence of the activation antigen Ki67. 3. The median rate of tumour protein synthesis was 10%/day (range 5.5-15.8%/day) in the control patients and 25.6%/day (range 9-37%/day) in the patients receiving arginine supplements (P less than 0.005, Wilcoxon rank sum test). The rates of protein synthesis correlated with Ki67 expression within these tumours (r = 0.78, P less than 0.001). A double-staining technique confirmed that tumour cells, rather than tumour-infiltrating lymphoreticular cells, expressed Ki67. 4. This study demonstrates that, in contrast to animal studies, L-arginine stimulates human tumours in vivo. This represents the first direct evidence that a single amino acid can modulate the behaviour of a human cancer.


Effect of diet-induced weight loss on total body bone mass. 1. Total body areal bone mineral density was measured by dual-energy X-ray absorptiometry in eight women before and 10 weeks after a very-low-calorie diet [405 kcal (1701 kJ)/day]. 2. The mean weight loss of 15.6 kg was accompanied by a statistically significant reduction in total body bone mineral density from 1.205 +/- 0.056 to 1.175 +/- 0.058 g/cm2 (mean +/- SD, P less than 0.005). 3. After cessation of the diet, weight gradually increased and by 10 months was similar to baseline values. Total body bone mineral density also increased after stopping the diet and mean values obtained 10 months after the diet did not differ significantly from initial values. Throughout the study total body bone mineral density values in all subjects were well within the range reported for normal subjects. 4. These data indicate that diet-induced weight loss is associated with rapid bone loss, subsequent weight gain being accompanied by increases in bone mass. Further studies are required to establish the clinical significance of these findings and, in particular, the skeletal distribution of bone loss.


Effect of acute and chronic administration of ethanol on the pancreatic exocrine response to cholecystokinin in rats fed different diets. 1. The effects of a diet rich in protein and fat, compared with a control diet, with or without chronic ingestion of ethanol on the pancreatic response to cholecystokinin were studied in rats after a 7-month treatment period. The acute effects of intraduodenal administration of 20% (v/v) ethanol were also analysed under these experimental conditions. 2. Animals receiving a diet rich in protein and fat showed a greater percentage increase in pancreatic output in response to cholecystokinin. 3. Chronic ethanol consumption reduced the basal secretion of protein and amylase; and even though the response capacity to cholecystokinin (considered as the percentage secretion on cholecystokinin stimulation with respect to basal secretion) was maintained, this led to hormone-stimulated secretion being decreased in comparison with the animals receiving water. In contrast, a lack of inhibition of basal volume flow and flow after cholecystokinin stimulation was seen after long-term ingestion of ethanol. 4. Acute administration of ethanol generally depressed cholecystokinin-stimulated pancreatic secretion. 5. On stimulation with cholecystokinin, the diet rich in protein and fat combined with long-term ingestion of ethanol led to non-parallel changes in the release of pancreatic enzymes, since an increase in trypsin secretion and a decrease in amylase secretion occurred concomitantly.


Variations in the glycoforms of serum alpha 1-antichymotrypsin in liver diseases and after liver transplantation. 1. Using crossed immunoaffinity electrophoresis with free concanavalin A in the first dimension, we studied the microheterogeneity of alpha 1-antichymotrypsin due to various glycoforms in sera from patients with various liver diseases and after liver transplantation. 2. Studies by isoelectric focusing and immunoblotting and by crossed immunoelectrophoresis without concanavalin A in the first dimension allowed us to show that there is no dramatic variation in electrophoretic heterogeneity of alpha 1-antichymotrypsin in the serum of patients with liver diseases or after liver transplantation when compared with that of normal subjects. Therefore the heterogeneity observed in crossed immunoaffinity electrophoresis is due to various interactions with concanavalin A. 3. The results were expressed as the ratio of concanavalin A non-reactive glycoforms plus concanavalin A weakly reactive glycoforms to concanavalin A reactive glycoforms, called R alpha 1-ACT. R alpha 1-ACT was significantly higher in patients with cirrhosis (n = 53) when compared with normal subjects (n = 30). The median R alpha 1-ACT was 1 (range 0.72-1.25) in normal subjects. It was 1.6 (range 1.18-3.02), 1.45 (range 0.65-4.12) and 2.24 (range 1.03-19) in cirrhosis of Child's grade A, B and C, respectively. There was a dramatic decrease in glycoforms with mostly biantennary glycans in some patients with Child's grade C cirrhosis. Serum levels of alpha 1-antichymotrypsin were lower than normal only in some patients with Child's grade C cirrhosis. 4. Among the patients with acute viral hepatitis studied (n = 17), five were studied longitudinally.(ABSTRACT TRUNCATED AT 250 WORDS)


A novel mucin sulphatase from human faeces: its identification, purification and characterization. 1. Colonic mucus is heavily sulphated and it is likely that this contributes considerably to its resistance to degradation by bacterial enzymes. The presence of a mucin-desulphating enzyme in faeces could therefore be very important in determining the rate of degradation of secreted mucus and hence the level of protection of the mucosa. 2. A novel assay for mucin sulphatase has been developed using biologically labelled human colonic [35S]sulphomucin as a substrate and a mucin sulphatase has been purified from faeces by sequential high-performance gel filtration and ion-exchange chromatography. 3. The mucin sulphatase has been shown to have a pH optimum of 4.5 and activity over the pH range 3-7. It has a pI of 4.0 and is inhibited by inorganic sulphate and phosphate. The purified enzyme preparation gave a single band on electrophoresis with a molecular mass of 15,000 Da. It has a Km of 41.9 mmol/l and a Vmax. of 1.17 katal/kg for glucose 6-sulphate. The enzyme was also shown to enhance fivefold the deglycosylation of [3H]glucosamine-labelled mucin by a faecal mucin glycosidase preparation. 4. Two bacteroides spp. isolated from normal human faeces, Bacteroides fragilis and B. thetaiotaomicron, were found to be producers of mucin-desulphating enzymes. 5. Mucin sulphatase is likely to be critical in determining the rate of enzymic degradation of secreted colonic mucin.


Accumulation of calcium by normal and dystrophin-deficient mouse muscle during contractile activity in vitro. 1. Accumulation of calcium by extensor digitorum longus muscles from dystrophin-deficient mdx and control C57BL/10 mice has been studied in vitro by measurements of total muscle calcium and by following the retention of 45Ca resulting from the incubation of muscles with the isotope for up to 2 h. 2. The rate of influx of calcium, calculated from the retention of 45Ca, was linear over 2 h in muscles at rest with no significant difference between mdx and control muscles. 3. Repetitive tetanic stimuli caused a substantial increase in 45Ca flux into both mdx and control muscles. This elevated rate of influx was maintained by control muscle, but not by mdx muscle after stimulation resulting in a significantly smaller total calcium flux into mdx muscle compared with control muscle by 1 h after stimulation. Similar changes were also seen in the total muscle calcium content of mdx and control muscles. Comparison of these results with those for loss of cytosolic creatine kinase previously reported (McArdle, A., Edwards, R.H.T. & Jackson, M.J. Clin. Sci. 1991; 80, 367-71) [1] indicate that control and dystrophin-deficient muscles release equivalent amounts of intracellular creatine kinase in response to the same accumulation of intracellular calcium. 4. These results therefore do not support the hypotheses that dystrophin deficiency in muscle leads to increased calcium influx during contractile activity, or that dystrophin-deficient muscle shows any inherent increased permeability to cytosolic proteins.


Perforin gene expression in T lymphocytes correlates with disease activity in immunoglobulin A nephropathy. 1. We studied perforin gene expression in T lymphocytes obtained from 26 patients with IgA nephropathy and from 15 healthy age-matched control subjects. 2. The majority of patients with IgA nephropathy (96%) had elevated perforin mRNA expression, whereas no perforin mRNA expression was detected in the T lymphocytes of normal control subjects. 3. A positive correlation was noted between perforin mRNA expression and urinary protein excretion. 4. Perforin mRNA expression correlated also with the histopathology in the renal tissue of patients with IgA nephropathy. 5. Sixty per cent of patients with grade III or IV histopathology had high perforin mRNA expression in T lymphocytes [more than (++)]. 6. These studies suggest that disregulation of perforin gene expression in T lymphocytes may be associated with the progression of IgA nephropathy and could be used as an indicator of disease activity.


Identification and analysis of transforming growth factor beta receptors on primary osteoblast-enriched cultures derived from adult human bone. Primary human osteoblast-enriched (PHO) cultures derived from adult trabecular bone were analyzed to determine the presence or absence of transforming growth factor beta (TGF-beta) receptors. Saturation binding studies were performed with 125I-TGF-beta in the absence or presence of 200-fold excess cold TGF-beta. Cross-linking experiments utilizing 125-I-TGF-beta were performed to identify specific cell surface binding proteins for TGF-beta. The saturation binding studies demonstrated saturable binding for TGF-beta on PHO cells. TGF-beta was cross-linked to cell surface binding proteins of 50 to 110 KDa and a high molecular weight component. Thus, these receptors appear to be similar in affinity, number per cell, and molecular weight to those previously identified with other cell types. The potential biological effects of TGF-beta on the growth of PHO cultures were evaluated by both 3H-thymidine incorporation and cell number determination. Growth of PHO cells in the presence of TGF-beta resulted in an approximately two-fold stimulation in cell number as compared to control cells while the 3H-thymidine experiments demonstrated a two to four-fold increase in thymidine uptake in the presence of TGF-beta. Radiographic emulsion studies revealed that the alkaline phosphatase positive and negative cell populations were responsive to the TGF-beta mitogenic stimulation. The cumulative findings of saturable binding, specific cell surface binding proteins, and biological effects suggest that functional TGF-beta cell surface receptors are present on primary osteoblast-enriched cultures derived from adult human trabecular bone.


Suspended judgment. AIDS and the ethics of clinical trials: learning the right lessons. There is a tribe of thinkers who believe that new technological advances and scientific discoveries raise new ethical issues. And it is the case, of course, that scientific and technological change--change in knowledge and power--implies a new realm for choice and, therefore, moral reflection. In the case presently concerning us, they see the AIDS social and scientific crisis as requiring the adoption of new ethical principles. I believe to the contrary. AIDS--more broadly, the entire spectrum of HIV disease--does not in itself present radically new issues at the level of principle. For example, the questions associated with confidentiality and HIV disease that have to this point dominated public discourse about morality and AIDS simply resurrect old questions raised by potentially deadly infectious disease. Recent discussions of the ethical issues associated with HIV-infectious health care workers fail to recognize that HIV is simply a new garb for the old question of the impaired or unsafe practitioner. HIV disease does have this effect on ethical reflection, however: It rubs our noses in issues that may have received insufficient attention to date. The urgency and poignancy associated with moral conflict and HIV disease forces us to face up to questions hitherto ignored. But this may be a good or a bd thing. In the heat of crisis, being human, we are likely to call for change without adequate consideration of the need for and effects of that change.(ABSTRACT TRUNCATED AT 250 WORDS)


Studies of ocular complications of AIDS Foscarnet-Ganciclovir Cytomegalovirus Retinitis Trial: 1. Rationale, design, and methods. AIDS Clinical Trials Group (ACTG). Cytomegalovirus (CMV) retinitis is the most common intraocular infection in patients with AIDS and affects an estimated 20% of these patients. Two drugs, ganciclovir and foscarnet, are currently available for the treatment of CMV retinitis. The Foscarnet-Ganciclovir CMV Retinitis Trial was designed: (1) to evaluate the relative efficacy and safety of foscarnet and ganciclovir for the treatment of CMV retinitis in patients with AIDS; and (2) to compare the relative benefits of immediate treatment versus deferral of the treatment for patients with disease not involving the posterior pole. Prior to randomization, patients were assigned to one of two strata based upon the location and extent of retinitis in the more severely involved eye. Patients with retinitis in zone 1 (posterior pole) or extensive retinitis (extent greater than or equal to 25% retina) in zones 2 and/or 3 (peripheral retina) were randomly assigned to immediate treatment with either ganciclovir or foscarnet. Patients with CMV retinitis confined to less than or equal to 25% of the retina and in zones 2 and/or 3 were offered the option of participating in the comparison of immediate treatment versus deferral of treatment. Patients opting to participate in this comparison were randomly assigned to immediate treatment versus deferral. Patients preferring to choose either immediate treatment or deferral (treatment preference design) were randomized only to foscarnet or ganciclovir for treatment of CMV retinitis. Patients in the deferral group were started on drug treatment when the retinitis became more immediately sight-threatening either by virtue of location (involvement of zone 1) or size (extent greater than 25% of the retina). Outcome measures included: survival, retinitis progression, visual function (visual acuity and visual field), drug side effects, and morbidity. Enrollment in the trial began in March 1990 and was completed in October 1991.


Patient and clinic factors predictive of missed visits and inactive status in a multicenter clinical trial. The Macular Photocoagulation Study Group. Obtaining complete patient follow-up in clinical trials is important for the analysis of treatment results and for good patient care. Incomplete data can introduce bias into study results and can alter conclusions concerning treatment efficacy. The purpose of this study was to explore in a case-control design patient and clinic factors that may be associated with missed visits or inactive status within three clinical trials conducted as part of the Macular Photocoagulation Study. All inactive patients, all patients with any missed visits, and a random sample of patients with no missed visits were selected for interview. A total of 175 patients (60% of 292 selected) participated in the study. Two factors were found to be significantly associated (P less than .05) both with inactive status and with having any missed visits: health problems and travel costs. Several patient and clinic factors were found to be associated only with inactive status. These included change in marital status since joining the study, retirement since study entry, unsatisfactory interactions with the study physician or clinic coordinator, too little time spent with the study physician, and patient's belief that participants should be paid to participate in clinical trials. Distance from the clinic greater than 100 miles, in addition to health problems any time during follow-up and problems with travel costs, was found to be associated only with having any missed visits. This study emphasizes the importance of maintaining good patient-staff interactions and sensitivity to the events in a patient's life in order to obtain complete patient follow-up.


Impact of random assignment on study outcome: an empirical examination. Sixty research investigations published in the biomedical literature were analyzed to examine the effect of design attributes on outcome. All 60 studies included a controlled trial involving a pretest, a therapeutic intervention, and a posttest across at least two groups. Thirty of the trials used random assignment of participants to treatment or control conditions and 30 trials employed some nonrandom method of subject assignment. Trial results were aggregated and evaluated by comparing effect sizes for the primary statistical test of the hypothesis. Data analysis revealed that the trial results, as measured by effect size, did not vary across therapeutic trials using random assignment versus those using nonrandom allocation of subjects. The impact of design attributes in the interpretation of multiple clinical trials addressing a similar research question is examined. The argument is made that various design attributes frequently associated with methodological quality should be considered as important moderator variables and their influence on trial outcome should not be assumed a priori but rather examined empirically.


Aspects of statistical design for the Community Intervention Trial for Smoking Cessation (COMMIT). We present statistical considerations for the design of the Community Intervention Trial for Smoking Cessation (COMMIT). One outcome measurement, the quit rate in randomly selected cohorts of smokers, is compared with another outcome measurement, the decrease in smoking prevalence, in terms of statistical efficiency and interpretability. The COMMIT study uses both types of outcome measurements. The merits of pair-matching the communities are considered, and sample size calculations take into account heterogeneity among pair-matched communities. In addition to significance tests based on the permutational (randomization) distribution, we also describe approaches for covariate adjustment. The COMMIT design includes 11 pair-matched communities, which should provide good power to detect a 10% or greater difference in quit rates between the intervention and control communities in cohorts of heavy smokers and in cohorts of light or moderate smokers. The power is only moderate to detect intervention effects on the decreases in overall smoking prevalence or in the prevalence of heavy smoking.


Termination of a clinical trial with no treatment group difference: the Lupus Nephritis Collaborative Study. The Lupus Nephritis Collaborative Study (LNCS) was a multicenter randomized clinical trial designed to assess the effects of standard drug therapy alone versus drug therapy plus plasmapheresis (plasma exchange) on the incidence of fatal or nonfatal renal failure associated with lupus nephritis. After 86 patients had been entered, with a mean of 97 weeks of follow-up, the trial was terminated partly due to lack of a beneficial effect of plasmapheresis. Although there are numerous methods for the statistical analysis of emerging results in a clinical trial, there have been relatively few descriptions of the application of these methods to the termination of a clinical trial when no favorable difference exists between groups. This report presents a review of the statistical methods employed for the pivotal interim analyses of the LNCS that were performed in order to help reach the decision to terminate the trial. These included the assessment of unconditional power post-hoc and the assessment of conditional power using an exact method appropriate for small sample sizes. Conditional power was used to assess the likelihood of detecting a significant treatment effect in the future given the data thus far observed and given reasonable hypotheses regarding the nature of the possible differences between the treatment groups. In addition, weighted-likelihood ratios (Bayes odds ratios) were computed to assess the likelihood of various alternative hypotheses given the present data. We show how such analyses can be useful in reaching a decision to terminate a trial that fails to show a treatment effect.


Distributed data analysis in a multicenter study: the CARDIA Study. Unlike distributed data entry, which is used in many large epidemiologic studies and multicenter clinical trials, distributed data analysis is a relatively new concept. This paper reports on the usefulness of such a system in the Coronary Artery Risk Development in Young Adults (CARDIA) Study. CARDIA distributes the entire examination dataset to participating centers soon after completion of each round of data collection. The process was designed to encourage more numerous, diverse, and rapid publications, and to allow for more efficient use of the manpower and expertise in centers. Responsibilities of the coordinating center have changed from a conventional coordinating center but remain substantial due to the need for collating, monitoring, verifying, and documenting the distributed data analysis (DDA) system. DDA is successful from the standpoint of implementation and operation--21 manuscripts representing work analyzed at six participating centers had been submitted for publication within 3.5 years of the completion of the baseline examination.


Distribution and characteristics of placental ANP receptors in normal and hypertensive pregnancy. The ANP (atrial natriuretic peptide) receptor binding site was studied in human placentas of normal and hypertensive pregnancy. The results showed there were specific high affinity ANP receptors in the nonbrush border (fetal side), and their affinity to ANP was higher than that in the microvillous membrane (meternal side). The ANP receptor affinity in the nonbrush border and microvillous membrane of normal pregnancy was higher than that of hypertensive pregnancy. Though the weight of placentas of hypertensive pregnancy was lower than that of normal pregnancy, high ANP concentrations in the placental tissues, umbilical and maternal blood were found in hypertensive pregnancy. It is believed that the distribution of ANP receptors in the placentas is related to hemodynamics, maternal exchange and fluid and electrolyte balance. The decrease of ANP receptors and lowering of affinity in hypertensive pregnancy may influence the the target cell effect of ANP, especially in the fetal side. This may be related to the pathogenesis of hypertensive pregnancy.


Distribution of Drosophila melanogaster transposable element sequences in species of the obscura group. Fifteen species belonging to the obscura group of the genus Drosophila were screened for sequences homologous to Drosophila melanogaster transposable elements (TEs) as an initial step in the examination of the possible occurrence of TEs at chromosomal inversion breakpoints. Blots of genomic DNAs from species of the obscura group were hybridized at three different stringencies with 14 probes representing the major families of TEs described in D. melanogaster. The probe DNAs included copia, gypsy, 412, 297, mdg1, mdg3, 3S18, F, G, I, jockey, P, hobo, and FB3. D. melanogaster TEs were not well represented in the species of the obscura group analyzed. The TEs that were observed generally exhibited heterogeneous distributions, with the exception of F, gypsy and 412 which were ubiquitous, and 297, G, Sancho 2, hobo and FB which were not detected.


[Detection of estrogen receptors (ER) in gastric cancer]. Using Avidn-biotin-peroxidase complex (ABC) immunohistochemical method, 68 gastric cancer specimens and 20 non-cancerous stomach specimens were examined for ER. In 38 of 68 gastric cancer specimens, ER concentrations were also biochemically determined by Dextran-coated charcoal (DCC) assay. The results showed that ER was absent in noncancerous stomach tissues. ER was present in 21 (30.9%) of gastric cancers. The ER positive cases were chiefly poorly differentiated gastric cancers. There were no significant statistical differences in the patient's age or sex, size of primary tumor and the ER positive rates. The results indicate that endocrine therapy might be useful for the ER positive gastric cancer patients.


[Chest wall and mediastinum invasion by lung cancer--a CT and MRI evaluation]. CT and MRI manifestations of 52 peripheral lung cancers located close to the pleural surface or mediastinal structures were studied correlating with the surgical and pathologic findings. Rib destruction as shown by CT and T2 weighted MRI showing the encroachment of tumor upon the chest wall were crucial in demonstrating chest wall invasion. An obtuse chest wall intersecting angle and the length of the neighboring borders of the tumor and chest wall were of limited value. Local pleural thickening was observed near the tumor in 54% of cases, over half of them showed obtuse angle between the tumor and the chest wall but without tumor invasion. T2 weighted MRI was more reliable than CT by showing different signal intensities for pleural thickening, inflammation, localized pleural effusion or tumor invasion to chest wall soft tissue.


[High dose chemotherapy with autologous bone marrow rescue for small cell lung cancer]. The efficacy of high dose chemotherapy with autologous bone marrow rescue (ABMR) was given to 10 patients with small cell lung cancer (SCLC). Four patients received high dose methotrexate plus ABMR followed by chest irradiation. Six patients giving complete response after induction chemotherapy were given intensive chemotherapy plus ABMR only. The over-all survival of these 10 patients was 30.5 months with a range of 6-96 months. Five of them are surviving as of this writing, 3 survived for more than two years and 2 for more than 5 years. Toxic reactions were not severe. Systemic metastasis was the Leading cause of treatment failure. ABMR was effective and safe. When combined with radiotherapy ABMR could be expected to improve the result of intensive chemotherapy for limited SCLC.


[AgNOR in the pathologic differential diagnosis of tumors]. AgNOR staining technique was applied to facilitate the pathologic differential diagnosis of 74 breast tumors and 95 non-Hodgkin's lymphoma (NHL). The results showed that the mean AgNOR count per nucleus was significantly different between benign and malignant tumors (P less than 0.01). An AgNOR count per cell less than 3 indicated benign breast lesion and more than 5 implied malignancy. In NHL, AgNOR count that nucleolus less than 4 was indicative of low-grade while more than 6 of high-grade malignancy. In 45 NHL patients followed up, the AgNOR count/cell was high in 28 patients with short survivals than otherwise (17 patients). These observations suggest that the AgNOR could provide useful information about the nucleolus activity in hyperplastic and neoplastic conditions. Electron microscopically, argyrophilic protein was irregular in shape and highly stained with silver in the fibrillar centers and the dense fibrillar component in NHL tumor cells as compared to that seen in normal lymphocytes. This further implies that the NOR Proteins could be used as a marker of ribosomal DNA and its level of transcription is higher in the malignant tumors.


[Angioneurotic edema caused by angiotensin-converting enzyme inhibitors]. Inhibition of angiotensin converting enzyme (ACE) may cause angioneurotic oedema. In order to define the clinical spectrum of this important adverse effect, we analysed data on 60 patients with angioneurotic oedema notified to the Drug Commission of the German Medical Association, after taking captopril (n = 24), enalapril (n = 25) or lisinopril (n = 11). In 48 cases the oedema affected the face, tongue and pharynx, while swelling of the extremities (n = 4), the trunk (n = 2) or the genitalia (n = 1) was observed less frequently. While oedema appeared most often after 1 to 21 days, it started within an hour in one patient, and only after 6 months of therapy in five patients. After discontinuation of the ACE inhibitor, the angioneurotic oedema resolved within 72 hours; additional therapeutic measures (glucocorticoids, antihistamines, adrenaline, C1 inhibitors) did not shorten the recovery time. In view of the increasing use of ACE inhibitors, the features of this unusual adverse reaction need to be widely recognized, since angioneurotic oedema of the larynx is potentially life-threatening.


Characterization of serum lysosomal enzymatic activities. I. Effect of dietary urea in dairy cows. The experiment was carried out on ten Holstein dairy cattle fed on conventional concentrate as base diet for 15 days, then turned on unconventional concentrate diet supplemented with 3% urea (CM-U) for 45 days to study the effect of four lysosomal enzymes in both states. The N-acetyl-beta-glucosaminidase enzyme level reflected the highest value among other enzymes inspite of its significant drop in the serum after feeding cows on the unconventional ureated diet. Beta-galactosidase was the sole enzyme to rise significantly in the serum of cows during the unconventional ureated diet feeding. Acid phosphatase and alpha-galactosidase showed highly significant decreases due to the effect of the tested diet.


[The use of ELISA systems for control of epizootics: safety of test standards using batch assays]. One of the advantages of ELISA techniques is to achieve a higher degree of safety by standardization. Bases of these standardizations should be, if available, international or national standards in combination with cutoff definitions. The cutoff definitions should be expressed, separate for the main kinds of samples, defined as a dilution of the standard. Those definitions can replace the mostly awkward and involved test descriptions as usual in official regulations. To guarantee a high grade of reliability it is a matter of urgent necessary to test (if possible by the producer) each serial (lot) of ELISA and to describe their capacities with regard of sensitivity and specificity. Separate pretests may be done with the main kinds of samples with well known reactions (negative and weak positive). The results are to compare to those of the cutoff-related dilutions of the standard in the same matrix. These descriptions of the capacities of each serial (lot) are safe bases for use of the serial. Key concepts are the quotients of the minimum positive value divided by the maximum negative or alternatively the mean value of the positive minus the threefold standard deviation divided by the mean value of the negative plus the threefold standard deviation.


Spatial and temporal patterns of distribution of the gap junction protein connexin43 during mouse gastrulation and organogenesis. Connexin43 (Cx43) is a member of the family of channel-forming proteins that make up the gap junction and are believed to provide pathways for cell-cell exchange of developmental signals. We have used immunofluorescence and confocal microscopy to characterize the patterns of distribution of Cx43 in postimplantation mouse embryos representing stages of development extending through gastrulation and the major period of organogenesis [through 13.5 days post coitum (dpc)]. We find that Cx43 is expressed early after implantation by the undifferentiated, pluripotent cells of the primitive embryonic ectoderm from which all tissues of the fetus are believed to be derived. As cells become committed to particular developmental pathways, there is a progressive restriction of Cx43 to specific areas and organ systems. The patterns are complex and not limited by germ layer of origin, although there is a clear preference for expression in ectodermal and, to a lesser extent, mesodermal derivatives. Expression in lens, retina, kidney, brain, pineal and pituitary glands is initiated early in organogenesis. In heart, the first clear signal for Cx43 appears in the ventricle at about 10 dpc and is only subsequently detected in the atrium at about 13-13.5 dpc. Particularly intriguing with regard to functional implications is the high level expression observed at sites of inductive interaction; the eye lens and optic cup, the infundibulum and the apical ectodermal ridge of the limb bud.


Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Fibroblast growth factors (FGFs) can influence the growth and differentiation of cultured cells derived from neuroectoderm, ectoderm or mesenchyme. The FGFs interact with a family of at least four closely related receptor tyrosine kinases that are products of individual genes. To investigate the role of FGFs in the growth and differentiation of embryonic tissues and to determine whether the individual FGF receptor genes might have specific functions, we compared the localization of mRNA for two FGF receptor genes, FGFR1 (the flg gene product) and FGFR2 (the bek gene product), during limb formation and organogenesis in mouse embryos (E9.5-E16.5). Although the two genes were coexpressed in some tissues, the differential expression of FGFR1 and FGFR2 in most embryonic tissues was striking. FGFR1 was expressed diffusely in mesenchyme of limb buds, somites and organ rudiments. In contrast, FGFR2 was expressed predominantly in the epithelial cells of embryonic skin and of developing organs. The differential expression of FGFR1 and FGFR2 in mesenchyme and epithelium respectively, suggests the receptor genes are independently regulated and that they mediate different functions of FGFs during development.


Expression of XMyoD protein in early Xenopus laevis embryos. A monoclonal antibody specific for Xenopus MyoD (XMyoD) has been characterized and used to describe the pattern of expression of this myogenic factor in early frog development. The antibody recognizes an epitope close to the N terminus of the products of both XMyoD genes, but does not bind XMyf5 or XMRF4, the other two myogenic factors that have been described in Xenopus. It reacts in embryo extracts only with XMyoD, which is extensively phosphorylated in the embryo. The distribution of XMyoD protein, seen in sections and whole-mounts, and by immunoblotting, closely follows that of XMyoD mRNA. XMyoD protein accumulates in nuclei of the future somitic mesoderm from the middle of gastrulation. In neurulae and tailbud embryos it is expressed specifically in the myotomal cells of the somites. XMyoD is in the nucleus of apparently every cell in the myotomes. It accumulates first in the anterior somitic mesoderm, and its concentration then declines in anterior somites from the tailbud stage onwards.


Inhibition of TSH bio-activity by synthetic beta TSH peptides. The in vitro bioactivity of the human beta TSH subunit was investigated utilizing eleven overlapping synthetic peptides representing the entire 112 residue sequence. The peptides were tested for both stimulatory and inhibitory activity in two sensitive bioassay systems: the first based on cAMP production in FRTL-5 rat thyroid cells, and the second based on stimulation of iodine trapping by the same continuous cell line. Peptides from three distinct regions of the beta-subunit showed concentration dependent inhibition of TSH bio-activity, including beta 1-15, beta 11-25, beta 31-45, beta 81-95, and beta 91-105 with IC50 values ranging from 150 to 304 microM. An additional peptide representing the entire sequence of the "intercysteine loop" region of beta TSH, beta 31-52, also inhibited TSH activity with somewhat higher potency than its fragment peptide beta 31-45 (IC50 of 87.5 +/- 14.7 microM for beta 31-52 versus 207 +/- 92.4 microM for beta 31-45). Three of these, beta 1-15, beta 31-45, and beta 31-52, also inhibited binding of TSH to the receptor in a radio-receptor assay, as previously reported (1), supporting their importance in receptor interaction. None of the synthetic peptides stimulated either cAMP production or iodine trapping. Two other overlapping peptides, beta 81-95 and beta 91-105, possessed bio-inhibitory activity but did not inhibit binding of labeled TSH. Computer analysis of this sequence predicted an extended turn structure for this region. This region has been referred to as the "determinant loop" as it is bounded by cysteine residues at positions 88 and 95 that many believe form a disulfide bond in the native subunit. The current data suggests the beta 88-95 region may play a role in receptor activation after initial binding of hormone to receptor.


Cyclic guanosine monophosphate analogs do not reverse bacterial toxin modulation of lactogen-stimulated NB2 cell mitogenesis. Pertussis toxin (PT) and cholera toxin (CT) have been shown to modulate lactogenic hormone-stimulated Nb2 cell mitogenesis, a lactogen-dependent cell line. As both toxins have been shown to alter guanylate cyclase activity in other cell systems, cyclic guanosine monophosphate (cGMP) analogs, 8-bromo or dibutyryl cGMP, were added to determine if they could reverse the toxin-mediated effects. In the absence of bacterial toxins, both cGMP analogs enhanced lactogen-stimulated Nb2 cell mitogenesis in a multiphasic pattern. At maximal enhancement, the effect was statistically significant but not marked (113 +/- 5%; p less than 0.01). Neither cGMP analog increased lactogenic binding site number or affinity so cGMP must affect lactogen action following receptor binding. Neither analog could stimulation Nb2 cell mitogenesis in the absence of lactogens so cGMP is not a second messenger for lactogens in this cell system. Finally, neither cGMP analog reversed the inhibitory effects of either bacterial toxin on lactogen-stimulated Nb2 cell proliferation. In summary, although bacterial toxins may be capable of altering guanylate cyclase activity, as addition of cGMP analogs do not reverse toxin-mediated effects on lactogen-stimulated mitogenesis, these toxins' actions must be mediated predominantly through other mechanisms that may have significant importance to lactogen signal transduction.


ACTH immunoactivity in normal rat tissues: modulation by hypophysectomy and adrenalectomy. This study investigates the effect of pertubation of the normal pituitary-adrenal axis on concentrations of adrenocorticotropin (ACTH)-like immunoactivity in peripheral tissues. We used a polyclonal antibody (West antibody) to measure ACTH-like immunoactivity in glacial acetic acid extracts of five tissues in adult male rats at increasing times (1, 7, 14, and 28 days) after hypophysectomy or adrenalectomy, and in normal control rats. Concentrations of ACTH-like immunoactivity were similar to those previously reported in liver, colon, heart, and small intestine and were not significantly affected by either hypophysectomy or adrenalectomy. While hypophysectomy also had no effect in the kidney, adrenalectomy resulted in a four-fold increase in extractable immunoactivity, first noticeable at seven days (p less than 0.005), but increasing progressively to 28 days (p less than 0.0005). Gel filtration showed that most of the increase in activity in kidneys of adrenalectomized rats corresponded to the 4.5 kD form comprising most of the serum ACTH immunoactivity and suggesting that the activity increase in kidney was largely due to ACTH derived from blood.


Comparison of time-integrated measurement of salivary corticosteroids by oral diffusion sink technology to plasma cortisol. We report preliminary data on the standardization of a device used in the recently developed "Oral Diffusion Sink" (ODS) technology for the time-integrated measurement of salivary corticosteroids. The concentrations of corticosteroids collected with the ODS devices were compared to plasma cortisol and saliva corticosteroid levels measured simultaneously. Six volunteers installed the ODS devices into their mouths during unstimulated and ACTH (250 micrograms) stimulated periods. Blood and saliva samples were also collected during these periods. The integrated plasma cortisol response and saliva corticosteroid levels were strongly correlated with the time-integrated total corticosteroid measurement of the ODS devices as well as with the cortisol and cortisone fractions. This preliminary data suggests that the accuracy of assessing adrenocortical activity by the measurement of salivary corticosteroids collected with the ODS device is high in both normal and stimulated conditions in normal volunteers. Continued standardization and studies in the practical use of this technology could lead to an important tool in assessing adrenocortical abnormalities. The use of such technology would increase the convenience while reducing the cost and invasiveness of current provocative testing of adrenal functioning.


Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase. Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.


Carbohydrate structure of a thrombin-like serine protease from Agkistrodon rhodostoma. Structure elucidation of oligosaccharides by methylation analysis, liquid secondary-ion mass spectrometry and proton magnetic resonance. The carbohydrate side chains of the thrombin-like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. Glycans obtained were characterized by digestion with exoglycosidases, methylation analysis and, in part, by liquid secondary-ion mass spectrometry and 1H-NMR spectroscopy. The results reveal that this snake venom glycoprotein contains partially truncated di-, tri- and tetraantennary complex type N-glycans carrying Fuc(alpha 1-6) residues at the innermost N-acetylglucosamine and solely (alpha 2-3)-linked sialic acid substituents. As a characteristic feature, ancrod oligosaccharides comprise mainly sialylated Gal beta 3GlcNAc beta lactosamine antennae. Furthermore, a small proportion of the sugar chains were found to carry a NeuAc alpha 3GalNAc beta 4GlcNAc beta antenna exclusively linked to C-2 of Man(alpha 1-3) residues of the pentasaccharide core. Thus, many of the glycans found represent novel glycoprotein-N-glycan structures.


Pharmacokinetics of the active metabolite of the prodrug repirinast in healthy Caucasian volunteers after a single oral dose. The pharmacokinetics of BAY w 8199, the active metabolite of the prodrug repirinast (BAY u 2372), has been investigated after oral administration of 150, 300 and 450 mg repirinast to twelve healthy male Caucasians. Plasma BAY w 8199 concentrations were very variable between subjects. The mean peak level (geom.mean; 1s-range) was 0.14 (0.08-0.25), 0.19 (0.13-0.29) and 0.24 (0.14-0.42) mg/l after the 150, 300 and 450 mg doses, respectively. Peak levels were reached 0.5-2.5 h after drug intake. Terminal half-lives were calculated as 5.9 h (150 mg), 8.0 h (300 mg) and 9.8 h (450 mg). The dose proportionality of the plasma profiles of BAY w 8199 and of its excretion in urine was demonstrated by testing several parameters. About 7.4% of each dose (calculated as BAY w 8199) was excreted in urine over 36 h. The renal clearance of about 27 l/h suggests that BAY w8199 is excreted by tubular secretion in addition to glomerular filtration.


VLA-2 is the integrin used as a collagen receptor by leukocytes. Cultured T cells and freshly isolated mononuclear leukocytes are able to bind collagen specifically. These leukocytes express equivalent levels of the integrins VLA-1, VLA-2 and VLA-3 which are collagen-binding receptors on other cells. However, only solubilized VLA-2 is able to bind collagen and only monoclonal antibodies specific for alpha 2 or beta 1 subunits are able to block the binding of intact cells to collagen. This restriction provides another example of the dependence of integrin specificity on the cell type on which it is expressed. It was also speculated that the inserted or I domain on the alpha subunits of VLA-1, VLA-2 and the beta 2 integrin family might have a role in collagen binding on the basis of its sequence homology to other types of collagen binding proteins. However, LFA-1, CR3 and p150,95 showed no collagen binding activity, suggesting that the I domain has another function.


Interaction between Epstein-Barr virus and a T cell line (HSB-2) via a receptor phenotypically distinct from complement receptor type 2. Epstein-Barr virus (EBV), the causative agent of mononucleosis and several human cancers, infects cells via complement receptor type 2 (CR2, CD21) which also serves as the receptor for the third complement component, C3. Expression of this receptor is restricted to B lymphocytes, immature thymocytes, and certain epithelial cells. In the present investigation; we describe the presence of a seemingly novel EBV receptor which is phenotypically distinct from CR2. Among various leukemic T cells studied, one, HSB-2, demonstrates no reactivity to several anti-CR2 antibodies, yet it reacts strongly with EBV as detected by incubation with biotin-conjugated virus and streptavidin-phycoerythrin. The virus binding is specific as demonstrated by blocking with anti-EBV antibodies and with non-conjugated virus. Aggregated C3 also binds HSB-2 and is capable of partially inhibiting EBV binding. The absence of CR2 on HSB-2 is further supported by the lack of expression of specific mRNA, assessed by Northern blotting analysis and polymerase chain reaction. Viral internalization and infection is demonstrated with electron microscopy, with detection of EBV-DNA by Southern blotting, and with detection of EBNA-1 transcripts by the polymerase chain reaction. Even though HSB-2 does not express CR2, it nevertheless displays transcripts which have some homology to a CR2 cDNA probe under low stringency hybridization conditions. This probe encompasses approximately the N-terminal half of CR2 which includes the EBV-binding epitope(s). The HSB-2 message is 5.2 kb, a size distinct from the 4.7-kb message of B cell CR2s. In contrast, the 5.2-kb message in not seen, under similar hybridization conditions, with a probe comprising the C-terminal half of CR2. Collectively, the data indicate that a receptor molecule having distinct phenotypic characteristics from the known CR2 protein on B cells is utilized by EBV to target human T lymphocytes.


Interleukin-1 up-regulates transcription of its own receptor in a human fibroblast cell line TIG-1: role of endogenous PGE2 and cAMP. The regulation of interleukin-1 receptor (IL-1R) mRNA expression by IL-1 in a human lung fibroblast cell line (TIG-1) was investigated. After 2 h of stimulation with human recombinant IL-1 alpha or IL-1 beta, the levels of T cell/fibroblast-type IL-1R mRNA increased, and the elevation was sustained for at least 72 h. IL-1 also stimulated synthesis of prostaglandin E2 (PGE2) and secondary cAMP accumulation. Exogenously added PGE2 increased the levels of both IL-1R mRNA and intracellular cAMP. Forskolin, cholera toxin and 8-Bromo adenosine (8-Br-cAMP) all increased IL-1R mRNA levels. Indomethacin blocked IL-1 stimulation of IL-1R mRNA expression, PGE2 production and cAMP. 125I-labeled IL-1 alpha-binding studies showed that this cell line expresses 2.6 x 10(4) IL-1R per cell with a Kd of 5.1 x 10(-10) M. After treatment of the cells with IL-1, the level of IL-1R increased over that of control cells. PGE2 also increased IL-1R without alteration in its affinity. Cross-linking experiments indicate that this cell line expresses the 80-kDa receptor molecule before and after treatment with PGE2; the molecular mass corresponds to the T cell/fibroblast type I IL-1R. These results indicate that IL-1 does not directly stimulate expression of IL-1R mRNA or cell surface IL-1R, but only indirectly by stimulation of endogenous PGE2.


[Laboratory breeding of the red-backed vole (Clethrionomys rufocanus bedfordiae)]. Laboratory matings were attempted to establish breeding colonies of red-backed voles (Clethrionomys rufocanus bedformidae) as experimental animals. For these mating, 10 pairs of red-backed voles which were captured in the Tohbetsu region of Hokkaido, Japan and their litters were used. In the results for two years, 1987 to 1988, the rates of pregnancy, birth and weaning were 35.4%, 94.5% and 79.5%, respectively. The mean litter size was 5:1 +/- 1.6 with a range of 1 to 9. The mean gestation period was 20.0 +/- 0.7 days with a range of 18 to 22. These results suggest that planned production of red-backed voles in the laboratory is possible. To determine intraregional variations of red-backed voles with a view to the establishment of a strain by inbreeding, restriction patterns of mitochondrial DNAs using seven restriction endonucleases were compared. Four different patterns were obtained from wild red-backed voles used in the present study.