Cooperative effects of bombesin, substance P and methacholine on the release of intestinal neurotensin in rats.

The secretion of ileal neurotensin (NT) results from events occurring at the apical and basal side of the N-cells. The hypothesis of a functional relationship between cholinergic and peptidergic neurones with the N-cell was investigated in the present study utilizing the isolated vascularly perfused rat ileum. Intraarterial methacholine (MC, 10(-4) M) evoked a prompt and well sustained release of NT in the portal effluent (plateau value at 500% of basal). This effect was dose-dependent over the range of 10(-6) M to 10(-4) M. Bombesin (B) provoked a dose-dependent peak secretion of NT (800% of basal at 10(-7) M) followed by a rapid return to almost basal levels. The B-induced NT release remained unaltered upon 10(-6) M tetrodotoxin (TTX) or 10(-5) M atropine infusion. Substance P (SP) potently stimulated the release of NT. The maximal response, consisting of a sustained secretion, was observed at a concentration of 10(-7) M (350% of basal) while 10(-6) M SP induced a transient release. TTX or atropine did not reduce significantly the SP-induced secretion of NT. Neurokinin A and B did not increase NT concentrations in the portal effluent. B synergistically increased the secretion of NT induced by SP. Atropine or TTX did not modify the effect of combined SP and B infusion. MC potentiated the release of NT induced by B but not that evoked by SP. Combined infusion of SP, B and MC produced the largest output of NT. In conclusion, B, SP and MC are strong stimulants of NT release in rats. In addition, the cooperative effects of these transmitters argue in favor of a complex functional relationship between the intramural nervous network and the intestinal N-cells in rats.


Biochemical basis for mouse resistance to hyaline droplet nephropathy: lack of relevance of the alpha 2u-globulin protein superfamily in this male rat-specific syndrome.

It is well-established that binding of a chemical to alpha 2u-globulin is the rate-limiting step in the development of male rat-specific hyaline droplet nephropathy. Mice synthesize mouse urinary protein (MUP), a protein which is very similar to alpha 2u-globulin, but this protein does not render the mouse sensitive to a similar renal toxicity. Therefore, the purpose of the present study was to determine the biochemical basis for mouse resistance to hyaline droplet nephropathy. Male Fischer 344 rats and B6C3F1 mice excreted 12.24 +/- 0.60 and 14.88 +/- 0.99 mg of alpha 2u-globulin and MUP daily, indicating that quantitative differences in protein excretion were not involved in the species specificity of the nephropathy. With d-limonene as a model hyaline droplet inducing agent, both rat and mouse liver microsomes oxidized the terpene to its 1,2-epoxide (the metabolite that binds reversibly to alpha 2u-globulin in vivo), demonstrating that metabolic differences do not determine the mouse resistance to this lesion. In spite of the formation of the epoxide intermediate, no binding of [14C]d-limonene equivalents to mouse kidney proteins was observed. In contrast, about 40% of the d-limonene equivalents in male rat kidney was reversibly bound to renal proteins. The renal reabsorption of alpha 2u-globulin and MUP was markedly different, as rats reabsorbed about 60% of the total filtered load of alpha 2u-globulin, but MUP was not reabsorbed by the mouse kidney. Given the absence of MUP in mouse kidney, in vitro equilibrium saturation binding studies were also conducted to determine whether MUP could bind the epoxide metabolite. alpha 2u-Globulin bound [14C]d-limonene-1,2-oxide with an apparent Kd of 4 x 10(-7) M. However, under identical experimental conditions, MUP failed to bind the epoxide. These data indicate that two major biochemical differences between alpha 2u-globulin and MUP contribute to mouse resistance to hyaline droplet nephropathy. Under both in vivo and in vitro conditions, MUP does not bind d-limonene-1,2-oxide, the rate-limiting step in the development of the nephropathy. However, even if MUP did bind the epoxide, the fact that it is not reabsorbed into the mouse kidney precludes its involvement in a syndrome involving renal protein overload. Finally, the absence of an interaction between d-limonene, a model hyaline droplet inducer, and the protein most similar to alpha 2u-globulin suggests that no other protein in the alpha 2u-globulin superfamily is likely to cause hyaline droplet nephropathy in other species.


Induction of quinone reductase and glutathione in bone marrow cells by 1,2-dithiole-3-thione: effect on hydroquinone-induced cytotoxicity.

Stromal cells from bone marrow are susceptible to toxicity induced by several redox-active metabolites of benzene, including hydroquinone (HQ). We have previously shown that tert-butyl-hydroquinone (tBHQ) can induce quinone reductase (QR) in bone marrow stroma as well as protect stromal cells against HQ-induced toxicity. Current studies investigate the underlining mechanisms of chemoprotection against HQ in DBA/2- and C57Bl/6-derived bone marrow stromal cells. The chemoprotector 1,2-dithiole-3-thione (DTT) has been used in these studies due to tBHQ toxicity to stromal cells at higher concentrations. Pretreatment of cells with DTT prior to HQ administration protected cells against HQ-induced toxicity. DTT induced QR activity in a dose-dependent manner in stromal cells from both strains of mice. However, there were no corresponding changes in glutathione transferase activity. DTT also increased cytosolic glutathione (GSH) concentrations by approximately 85% in both strains. Since bone marrow stroma consists primarily of fibroblasts and macrophages, we also evaluated QR activity in the separate cell types from the two strains of mice. There were differences in basal and DTT-induced QR activity between fibroblasts and macrophage cells derived from the same strain of mice, as well as the expected differences between strains. Additionally, dicoumarol, an inhibitor of QR activity, potentiated HQ-induced toxicity in both strains of bone marrow stromal cells. Thus, cellular glutathione, QR activity, and their inducibility by chemoprotective agents such as DTT may prove to be important factors in chemically induced bone marrow toxicity and carcinogenicity.


[Chronic congenital neutropenia treated with granulocyte colony-stimulating factor (G-CSF)].

Kostmann's syndrome is a congenital disorder characterized by persistent severe neutropenia. It has a high morbidity and mortality on account of serious bacterial infection. A case which was successfully treated with G-CSF is reported.


Percutaneous transrenal ureteral occlusion: indication and technique.

Several techniques for achieving palliative ureteral occlusion in cases of underlying malignant diseases are known to exist. We performed nine ureteral occlusions on seven patients, using two different techniques (occlusion by detachable balloon and by "Harzmann Olive"). Initially, complete occlusion of all ureters was attained; in two cases a second occluding intervention had to be carried out after a period of 6 and 14 weeks. Six of seven patients enjoyed a marked improvement of their quality of life after occlusion. Complications were down to a minimum. In comparison with other techniques described in the literature, Harzmann's method seems to be the simplest, as well as the most fully developed one. It may also be recommended for patients in an advanced tumor stage.


Biophysical mechanism of the scavenger site near T cell-presented epitopes.

We seek to identify consensus sequences in digested fragments of antigenic proteins regulating selection and major histocompatibility complex (MHC)-restricted presentation to T cells of epitopes within those fragments. One such pattern, of recurrent, hydrophobic sidechains forming a longitudinal hydrophobic strip when a sequence is coiled as an alpha-helix, is found in or near most T cell-presented epitopes. Such recurrent hydrophobicity may lead to protease-protected coiling of the fragment against endosomal membranes and transfer to MHC molecules. This concept leads to better identification of T cell-presented sequences and possible to engineering of T cell-presented vaccines to affect their potency and MHC restriction.


Diazepam and atropine as premedicants: no discrimination by monoamine metabolite and catecholamine measurements in cerebrospinal fluid and plasma.

The relationships between self-reported assessments of the quality of the preoperative night's sleep, preoperative anxiety, and several biochemical and physiological indicators of stress reaction were investigated in pregnant women at term receiving no premedication (n = 15), a placebo tablet (n = 15), diazepam 5 mg p.o. (n = 15), or atropine 0.01 mg/kg i.m. (n = 15), in connection with spinal analgesia for elective caesarean section. In the patients receiving no premedication, the subjective estimate of the quality of the preoperative night's sleep was negatively associated with concentrations of noradrenaline (NA) and its metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG) in CSF, and with plasma adrenaline. The anxiolytic effect of diazepam was reflected as significantly lower plasma levels of another metabolite of NA, 3,4-dihydroxyphenylglycol (DHPG). Placebo and diazepam, and to a lesser extent atropine, confounded the statistical relationships between the clinical and biochemical responses found in the patients with no premedication. On the whole, the biochemical monoamine measurements were of little use in determining the clinical effects of different kinds of premedicants.


Treatment of terminal cancer pain in Finland: a second look.

A questionnaire concerning the treatment of cancer pain was sent to 10% and 5% random samples of Finnish physicians in 1985 and in 1990, respectively. The physicians were asked about their current practice in the treatment of pain in their cancer patients, and about their main clinical problems when treating pain. Three simulated patient cases were presented, and the adequacy of the suggestions for therapy was evaluated. The results indicated that Finnish physicians had adopted a more rational and effective analgesic therapy during the 5-year period. Treatment suggestions for the simulated patient cases had improved both in terms of daily doses of analgesics and of dose intervals, but the doses of opioids were still below those commonly used in chronic cancer pain. The clinical difficulties experienced by the physicians had changed: instead of being frustrated by the inefficacy of their treatment as in 1985, physicians were now working on the problem of finding a suitable preparation and dosage. The results suggest that the voluntary activity of patient organizations, a few pain clinics, and a few clinicians interested in pain treatment have been able to improve the practising physicians' theoretical knowledge in 5 years. In contrast to the improvement in the knowledge and skills, changing attitudes takes much longer. As many as 39% of physicians who see cancer patients at least occasionally, reported that they still had not acquired the prescription sheets necessary to prescribe opioids to outpatients.


Comparison between the clinicopathologic features of AFP-positive and AFP-negative gastric cancers.

We compared 27 cases of alpha-fetoprotein (AFP)-positive gastric cancer with 478 cases of AFP-negative gastric cancer in our department. The incidences of AFP-positive gastric cancer were 5.4% (27/505), 7.2% (23/218), and 2.1% (4/187) for overall, advanced, and early cases of gastric cancer, respectively. Sex, age distribution, pathologic type, and serum carcinoembryonic antigen levels were similar between these two groups. Borrmann III type cancer, lymph node metastasis, lymphatic and venous microinvasion of the gastric wall, and incidence of synchronous and metachronous liver metastasis occurred more often in the AFP-positive group. It was suspected that the character of early venous invasion contributed to the high incidence of liver metastasis. Liver metastasis occurred in 72% of AFP-positive patients, all of whom died within 2 yr. Long-term survival of the AFP-positive group was worse than that of the negative group. The 1-, 3-, 5-, and 7-yr survival rates of both groups were 38.7%, 11.6%, 11.6%, 11.6%, and 71.3%, 57.8%, 52.8%, 49.6%, respectively (p less than 0.001).


Hyperamylasemia in patients with the acquired immunodeficiency syndrome.

Marked elevations of serum amylase, unexplained despite extensive evaluation in patients with acquired immunodeficiency syndrome (AIDS), prompted this retrospective review of 85 patients to determine the prevalence of hyperamylasemia and identify any associated demographic and etiologic factors. Of 39 patients who had amylase determinations, 54% had hyperamylasemia (2/3 pancreatic, 1/3 salivary) and 31% had pancreatitis. Biliary tract disease, alcohol intake, and opportunistic infections were similar in hyperamylasemic and normoamylasemic subjects. Non-Caucasian race, intravenous drug abuse, renal dysfunction, alkaline phosphatase elevation, and pentamidine use were more prevalent in patients with hyperamylasemia (p less than 0.001, p less than 0.001, p less than 0.01, p less than 0.05, and p less than 0.05, respectively). However, by stepwise deletion multiple regression analysis, only non-Caucasian race, pentamidine use, and Mycobacterium avium-intracellulare infection were significant, independent predictors of hyperamylasemia (R2 = 0.65). Followed over time, in a historical prospective manner, case fatality rates (66.6% and 61.1%) and median survival times (101 and 84 days) were similar in the hyperamylasemic and normoamylasemic groups. We conclude that, although pancreatitis occurs frequently in AIDS, hyperamylasemia is often of salivary origin and clinical outcome is unaffected. Certain demographic factors are strongly associated with hyperamylasemia in AIDS patients, but multiple, concurrent, etiologic factors are probably operative in these patients.


Ethanol and human saliva: effect of chronic alcoholism on flow rate, composition, and epidermal growth factor.

Parotid saliva samples from 24 alcoholic subjects without evidence of cirrhosis were analyzed for changes in flow rate, composition, and epidermal growth factor (EGF) secretion. Mean (+/- SE) stimulated parotid saliva flow rate (ml/min/gland) was significantly (p less than 0.01) lower in alcoholic subjects than in matched control subjects. Reduction in parotid saliva flow rate was associated with significant (p less than 0.05) decrease in total protein and amylase secretion in this group of patients. In addition, secretion of immunoreactive EGF, a specific salivary protein, was also markedly reduced (p less than 0.05) in alcoholic patients. None of the parotid saliva samples from the alcoholic subjects had detectable bioactivity of EGF in saliva. These data suggest that chronic alcohol ingestion is associated with significant changes in parotid saliva secretion and its composition, which may perpetuate and compound ethanol-induced injury to the upper gastrointestinal tract.


Case report: distinctive immune abnormalities in a patient with procainamide-induced lupus and serositis.

To gain insight into the immunopathogenesis of drug-induced autoimmune disorders, lymphocyte and immunoglobulin distributions and cytokine levels were monitored in the peripheral blood and pleural fluid of a patient with procainamide-induced lupus and pleural effusion. Approximately 80% of the B cells in both compartments were CD5+ compared to 10% to 25% in normal adults. CD4/CD8 ratio and percentage CD4 were normal in peripheral blood. Serum levels of IgG (particularly IgG2), IL-6, and soluble IL-2R were slightly elevated, and those of IgA were significantly elevated compared to normal controls. Analysis of the pleural effusion revealed an increased CD4/CD8 ratio because of an increased percentage of CD4+CD29+ helper memory T cells, lack of expression of the resting B-cell marker CD21, immune complex deposition and complement consumption, increased relative levels of ANA, abnormally high levels of IL-6 and soluble IL-2R, and detectable levels of IL-1b, IFN-g and TNF-a. These observations provide evidence for the involvement of CD5+ B cells and differential helper T-cell activity in procainamide-induced lupus and for an association between local lymphocyte activation and organ pathology.


Antibodies against the cystic fibrosis transmembrane regulator.

Rabbit polyclonal antibodies have been raised against high-performance liquid chromatography purified synthetic peptides corresponding to two discrete regions of the cystic fibrosis transmembrane regulator (CFTR) protein: the R-domain (residues 785-796) and the extreme COOH-terminus (residues 1467-1480). Antibodies (Ab) to each of these peptides were affinity purified either by passage over a peptide-derivatized agarose matrix (Ab 785) to produce monospecific polyclonal antibodies or by protein A affinity chromatography to purify the immunoglobulin G1 fraction free of other serum proteins (Ab 1467). These antibodies recognize a candidate CFTR protein in the colonic cell line T84, as determined by Western blot analysis and by immunoprecipitation and labeling of the precipitate with [gamma-32P]ATP in the presence of protein kinase A. Both antibodies precipitated CFTR-related polypeptides from four mammalian cell types (HeLa, Bsc-40, HEp-2, and Chinese hamster ovary cells) transfected with the full-length CFTR cDNA clone using a vaccinia T7 protein expression system. Similar results were observed using a yeast CFTR expression system. In each case the Mr values of the bands observed were consistent with that expected for the CFTR protein. These antibodies should be useful probes for the immunocytochemical localization, immunoaffinity purification, and ultimately the functional characterization of the CFTR protein.


Characterization of myocardial Na(+)-Ca2+ exchange in rainbow trout.

This study compared Na(+)-Ca2+ exchange from the hearts of rainbow trout with that from canines. In several respects, trout cardiac Na(+)-Ca2+ exchange is functionally similar to that from dogs and other mammals. Trout cardiac Na(+)-Ca2+ exchange is stimulated approximately 200% after 30-min incubation with 10 micrograms/ml chymotrypsin at 21 degrees C, similar to mammals. On the other hand, both the temperature and pH dependencies are strikingly different between the trout and canine myocardial Na(+)-Ca2+ exchange. While canine heart Na(+)-Ca2+ exchange exhibits a Q10 of greater than 2 (similar to values observed in other mammals), that from trout is relatively insensitive to temperature with a Q10 of approximately 1.2. The absolute rates of Na(+)-Ca2+ exchange in trout heart are four- to sixfold higher than that in mammals when measured at 7 degrees C. Furthermore, the temperature insensitivity of trout myocardial Na(+)-Ca2+ exchange is retained when the exchanger is reconstituted into an asolectin bilayer, suggesting that this property is intrinsic to the protein and not dependent on species differences in lipid bilayer composition. Trout Na(+)-Ca2+ exchange is not markedly stimulated by alkaline pH, in contrast to mammals, and this characteristic is also maintained after reconstitution. Western blots of trout cardiac sarcolemma run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis react with antibodies raised against the canine Na(+)-Ca2+ exchanger with a similar pattern of bands (70, 120, and 160 kDa). Furthermore, a cDNA probe from canine Na(+)-Ca2+ exchanger hybridizes on Northern blots of trout heart mRNA to a 7-kb band, similar to that in mammals. Thus, while important functional differences in Na(+)-Ca2+ exchange exist between trout and mammalian hearts, the molecular basis is not yet known.


Regulation of hepatic protein synthesis in chronic inflammation and sepsis.

The regulation of protein synthesis was determined in livers from control, sterile inflammatory, and septic animals. Total liver protein was increased in both sterile inflammation and sepsis. The rate of protein synthesis in vivo was measured by the incorporation of [3H]phenylalanine into liver proteins in a chronic (5 day) intra-abdominal abscess model. Both sterile inflammation and sepsis increased total hepatic protein synthesis approximately twofold. Perfused liver studies demonstrated that the increased protein synthesis rate in vivo resulted from a stimulation in the synthesis of both secreted and nonsecreted proteins. The total hepatic RNA content was increased 40% only in sterile inflammation, whereas the translational efficiency was increased twofold only in sepsis. The increase in translational efficiency was accompanied by decreases in the amount of free 40S and 60S ribosomal subunits in sepsis. Rates of peptide-chain elongation in vivo were increased 40% in both sterile inflammation and sepsis. These results demonstrate that sepsis induces changes in the regulation of hepatic protein synthesis that are independent of the general inflammatory response. In sterile inflammation, the increase in protein synthesis occurs by a combination of increased capacity and translational efficiency, while in sepsis, the mechanism responsible for accelerated protein synthesis is an increased translational efficiency.


NaCl-dependent expression of amiloride-blockable Na+ channel in Xenopus oocytes.

RNA was isolated from chicken lower intestine (both colon and coprodeum) and injected into Xenopus oocytes. 22Na+ fluxes measured after 1-4 days demonstrated the induction of an amiloride-blockable pathway. The Na+ transporter expressed by the exogenous RNA had a high affinity to amiloride (inhibitory constant less than 0.1 microM), but was insensitive to ethylisopropyl amiloride, i.e., it is likely to be the apical Na+ channel. Functional channels were readily expressed in oocytes injected with RNA derived from chickens fed a low-NaCl diet. On the other hand, no channel activity was detected in oocytes injected with RNA isolated from chickens fed a high-NaCl diet. Thus the previously reported regulation of transport by the dietary NaCl intake involves modulations in the level of mRNA that codes either for the Na+ channel or a posttranscriptional regulator of the channel.


Perturbation of regulated secretion in the pancreatic acinar cell line, AR42J.

The regulated secretory pathway comprises accelerated discharge of proteins in response to hormonal stimuli, their presence in secretory granules (SG), and a long intracellular residence time. Dexamethasone induction of AR42J results in an increase in granule content and responsiveness to cholecystokinin (CCK). We studied the effects of conditions implicated in sorting of secretory proteins into the regulated pathway using [35S]methionine pulse-chase protocols that examine transport of secretory proteins from the rough endoplasmic reticulum (RER)----SG and specifically from the Golgi complex (GC)----SG. The latter uses a chase at 20 degrees C to allow accumulation of labeled proteins in the trans-Golgi, followed by a shift to 37 degrees C that initiates their transport to SG under test conditions. Quantitation of CCK-8-stimulated discharge of prestored amylase and of newly synthesized labeled proteins that have entered SG during the chase enables us to examine the effect of perturbants over selected parts of the pathway. The effects of acidic intracellular compartments, the cytoskeleton, protein synthesis, ATP, and temperature on pre- and post-Golgi entry of proteins into the regulated pathway were studied. NH4Cl, monensin, Na azide, incubation at 20 degrees C, and pertussis toxin retarded RER----SG transport without affecting amylase discharge. Only incubation with 20 mM NH4Cl or 1 microM monensin inhibited transfer of newly synthesized proteins from the late GC----SG. RER----Golgi or intra-Golgi transport thus appears to require ATP and possibly guanosine 5'-triphosphate (GTP)-binding proteins. Acidic compartments appear to be essential for sorting of secretory proteins from the GC----SG.


Role of polyamines in glucocorticoid effects on pancreatic acinar AR42J cell growth and differentiation.

We previously found that glucocorticoids inhibit growth and increase differentiation in rat pancreatic acinar AR42J cells. In the current study, we examined the role of polyamines in these effects. Treatment of AR42J cells with the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) inhibited DNA synthesis. Thus polyamines are required for AR42J cell growth. However, we have previously shown that dexamethasone (Dex) increased AR42J cell ODC activity and mRNA levels. In the current study, we found that Dex treatment increased cellular putrescine levels. These increases in ODC and putrescine occurred during Dex-induced inhibition of DNA synthesis. Therefore, in AR42J cells, ODC activity and polyamine levels are not strictly growth related. To examine the requirement for glucocorticoid induction of ODC activity in glucocorticoid stimulation of differentiation, we examined the effects of DFMO on amylase gene expression and cholecystokinin binding. DFMO reduced cell amylase content while having little effect on mRNA levels in both Dex-treated and untreated cells. In contrast, DFMO had little effect on control CCK binding but inhibited the Dex-induced increase. Thus polyamines are necessary for growth and glucocorticoid-induced differentiation of AR42J cells; however, effects of glucocorticoids on AR42J cell growth and differentiation are not mediated by effects on ODC.


Postnatal changes in the substance P receptor on rabbit gastric smooth muscle.

We used radioligand binding to tissue homogenates and isometric contraction of muscle strips to characterize the substance P (SP) receptor on gastric smooth muscle from 1- (newborn) and 7-day-old and 4- and 11-wk-old (weanling) rabbits. Scatchard analysis for newborns was curvilinear, suggesting the presence of multiple binding sites. In newborns the dissociation constant (Kd) of high-affinity binding site was 2.2 +/- 0.3 nM, and the maximum binding (Bmax) was 0.57 +/- 0.06 pmol/mg DNA. The number of high-affinity binding sites decreased with age, disappearing by 11 wk. The Kd for the low-affinity site was more than two orders of magnitude greater than that of the high-affinity site. In competitive binding studies with [3H]SP, the order of potency for the neurokinins was SP much greater than neurokinin A (NKA) greater than neurokinin B (NKB), suggesting that the high-affinity binding sites were NK-1 receptors. [125I]NKA is also bound to newborn tissue homogenate with high affinity. With [125I]NKA the order was NKA greater than SP greater than NKB, suggesting that NK-2 receptors were also present. In contraction studies, atropine and tetrodotoxin had no effect on tachykinin-stimulated contraction, suggesting solely myogenic tachykinin effects on this tissue. In newborn rabbits, the potency and efficacy of SP and NKA were similar. The half-maximal effective dose (ED50) of SP was nearly two orders of magnitude less in newborn rabbits than in weanlings; the potency of NKA did not change.(ABSTRACT TRUNCATED AT 250 WORDS)


Electrical and mechanical effects of acetylcholine and substance P in subregions of canine colon.

Effects of acetylcholine (ACh) and substance P on the electrical and mechanical activities of the circular muscle layer of the canine proximal colon were studied. Because this muscle layer is bordered by two different pacemaker regions, responses from segments containing either a single pacemaker region or no pacemaker region were compared with responses of the complete muscle layer. Concentration-response relationships for ACh and substance P were similar between the various segments, suggesting that receptors for these agonists are expressed throughout the layer. The dominant contractile pattern induced by ACh and substance P in each segment was a 1- to 3-cycle/min rhythm. In a like manner, these agonists also elicited an electrical pattern in which a long-duration slow wave occurred one to three times per minute between short-duration slow waves. Low concentrations of nifedipine (0.01 microM) selectively antagonized the 1- to 3-cycle/min rhythm. In circular muscles with no pacemaker region, ACh (1 microM) caused depolarization, induced oscillations in membrane potential averaging 24 +/- 5 mV in amplitude and 2.9 +/- 0.9 cycles/min in frequency, and generated rhythmic contractions at the same frequency. This "interior" circular muscle was functionally innervated by cholinergic excitatory nerves. Exposure to ACh (1 microM) did not alter the conduction of slow waves through the thickness of the circular layer. In summary, the excitatory neurotransmitters, ACh and substance P, induce a dominant electrical and contractile rhythm throughout the circular muscle layer that is different from the spontaneous rhythms produced at either the myenteric or submucosal border.


Selected polyclonal antibodies and ADH challenge in frog urinary bladder: a label-fracture study.

It is clearly established that the changes induced by antidiuretic hormone (ADH) in its target epithelial cells result from the insertion in the apical membrane of new components that contain channels for water. We have already undertaken an initial study of these channels by raising polyclonal antibodies against Triton X-100 apical extracts from ADH-treated bladders and approached their purification by different adsorption steps. In the present study, we used the label-fracture technique to investigate the localization of the binding sites of the obtained polyclonal antibodies on the apical membrane of ADH-stimulated frog urinary bladder. The results obtained clearly demonstrated a preferential labeling by the selected antibodies of morphological structures such as the groove arrays and the fusion images that are generally accepted as being involved in ADH-induced changes in water permeability of the apical membrane.


Comparison of the effects of BAY K 8644 on cardiac Ca2+ current and Ca2+ channel gating current.

Effects of (-)-BAY K 8644 on Ca2+ channel function were studied in guinea pig ventricular myocytes. It was found that the compound has both voltage-dependent stimulatory and inhibitory effects on the Ca2+ current (ICa), in agreement with prior studies. The basis for these effects was studied by evaluating the effects of (-)-BAY K 8644 on the Ca2+ channel gating current. It was found that the voltage-dependent inhibitory effects of the drug on ICa could be well explained by similar reductions in the amount of gating charge moved. However, the stimulatory effect of (-)-BAY K 8644 on ICa could not be simply correlated with changes in the amount of gating charge moved. Although the drug produced a shift of the charge-voltage relationship to more negative potentials, the drug actually reduced the total amount of movable gating charge. Thus it could be demonstrated that there are membrane potentials where (-)-BAY K 8644 reduced the Ca2+ channel gating current while enhancing ICa. In addition, the drug was found to slow the decay of the gating current during repolarization. It seems likely that (-)-BAY K 8644 has a dual effect on Ca2+ channels: affecting both the voltage dependence of gating charge and the relationship between open probability and charge movement.


Disseminated histoplasmosis with unusual cutaneous lesions in a patient from the Philippines.

The incidence and prevalence of histoplasmosis in Southeast Asia has not been extensively described. The first microbiologically documented case of disseminated histoplasmosis with cutaneous papulonodules in a 56-year-old woman from the Philippines is reported. She presented with fever and generalized papulonodular lesions in various stages, which evolved into vesicles with central necrosis that resembled molluscum contagiosum with an indurated erythematous halo. Biopsies revealed a granulomatous mass of lymphohistiocytic and epithelioid cells with intracellular budding yeast cells and dark nuclei. Cultures were positive for Histoplasma capsulatum. The patient was treated with amphotericin B (3 g) and 5-fluorocytosine (50 mg/kg/day), followed by ketoconazole (400 mg/day). Her clinical course was complicated by intractable hemolytic anemia that was initially treated with corticosteroids. A splenectomy was subsequently performed. Pneumonia and a brain abscess caused by Nocardia asteroides were secondary complications. Nine months after her admission, repeat testing was diagnostic for systemic lupus erythematosus. This patient serves to re-emphasize that cutaneous lesions in an immunocompromised patient must be evaluated by biopsy and culture analysis. Disseminated histoplasmosis in the immunocompromised host may present with unusual cutaneous lesions, and must be considered even in a nonendemic area.


Serial serum C-reactive protein levels as an aid to the management of melioidosis.

Of 46 patients with clinical melioidosis, 35 (22 culture-positive and 13 culture-negative) had relatively uneventful disease courses, with elevated serum C-reactive protein (CRP) concentrations (greater than 5 mg/dl) that decreased with the commencement of appropriate antibiotic therapy, and continued to show an uninterrupted decrease (mean 29.4 days, range 12-52 days) to the normal range (less than 1 mg/dl), with resolution of their infections. In five culture-positive patients with complicated disease courses, CRP concentrations remained elevated (greater than 5 mg/dl) until the underlying disorders were successfully managed, or until the antibiotic regimen was changed, and CRP values then decreased to the normal range. During surveillance, elevated CRP concentrations (greater than 10 mg/dl) led to the diagnosis of reactivation of infection in three afebrile patients, while the serum CRP values in other patients remained within the normal range in the absence of intercurrent complications. The CRP estimations may be helpful in ascertaining active infection in patients with low serum levels of specific IgM antibody, and serial measurements of serum CRP in patients with clinical melioidosis may be useful in determining the optimal duration of treatment and for detecting occult or unresolved infection with Pseudomonas pseudomallei.


Mechanism of action of atracurium on airways.

Histamine-releasing drugs may produce significant effects on airways in high-risk populations. To determine if clinically relevant doses of atracurium produce adverse effects on airways, we measured changes in airway resistance in the lung periphery of anesthetized Basenji-Greyhound dogs before and after intravenous (iv) administration of atracurium. A wedged bronchoscope technique was used to measure collateral system resistance (Rcs). After a stable baseline was obtained, atracurium (1.2 or 0.5 mg/kg) or histamine (200 micrograms) were administered as an iv bolus, and percent increase in Rcs was calculated. On separate days dogs were pretreated with the histamine 1 receptor antagonist, chlorpheniramine (0.2 mg/kg iv), with or without atropine (0.2 mg/kg iv) and ranitidine (0.75 mg/kg iv) and the experiment repeated. Histamine (200 micrograms) increased Rcs 97 +/- 24% at 30 s (8 sublobar segments), whereas a second dose increased Rcs 77 +/- 15%. Pretreatment with chlorpheniramine (0.2 mg/kg iv) totally prevented increases in Rcs (9 sublobar segments). Atracurium (1.2 mg/kg) increased Rcs to 174 +/- 35% at 3 min (14 sublobar segments), whereas 0.5 mg/kg had little effect (10 sublobar segments). A second bolus of atracurium (1.2 mg/kg) increased Rcs to only 54 +/- 14% (P less than 0.01). Chlorpheniramine pretreatment (0.2 mg/kg iv) reduced the response to the initial dose of atracurium to only 26 +/- 14% (10 sublobar segments). Pretreatment with a combination of atropine and chlorpheniramine (4 sublobar segments), or ranitidine and chlorpheniramine (5 sublobar segments), did not attenuate the increase in Rcs significantly more than chlorpheniramine pretreatment alone.(ABSTRACT TRUNCATED AT 250 WORDS)


Exercise-induced pulmonary hemorrhage in horses with experimentally induced allergic lung disease.

The lungs of sensitized horses were exposed to aerosolized ovalbumin. Some horses (n = 4) were given ovalbumin in 1 lung only, whereas in others (n = 7), ovalbumin or vehicle were inoculated in the cranial, ventral, and caudal regions of the caudal lung lobe. Horses were exercised 5 hours after ovalbumin exposure. Immediately before exercise, endoscopy failed to reveal any abnormality. After exercise, endoscopic examination of horses subjected to unilateral ovalbumin exposure revealed extensive blood in airways leading to the exposed lung in all horses. Blood was not observed in the airways leading to the control lung. Mean (+/- SEM) minimum volume of the exposed and control lungs was 9.5 +/- 1.5 and 5.5 +/- 1.6 L, respectively; this difference was statistically significant (P less than 0.05). Bronchoscopy of horses subjected to regional ovalbumin or vehicle exposure and exercise revealed a small amount of blood-tinged fluid in the bronchi serving the regions of the lung inoculated with ovalbumin. Minimum volumes of such regions were not significantly different from one another. However, their minimum volume was significantly (P less than 0.05) larger than that of vehicle-inoculated regions. Gross and histologic examination confirmed inflammation and hemorrhage in the ovalbumin-exposed, but not the control lungs or lung regions. Thus, exercise can cause blood from an injured region of lung to appear in the larger airways. Regional differences in lung structure and function do not influence the appearance of blood in the airways.


Development of monoclonal antibody ELISA for simultaneous detection of bovine coronavirus, rotavirus serogroup A, and Escherichia coli K99 antigen in feces of calves.

A rapid ELISA was developed for simultaneous detection of bovine coronavirus (BCV), rotavirus (RV) serogroup A, and Escherichia coli K99 antigen in feces of calves. A mixture of 3 monoclonal antibodies specific for BCV, RV, or K99 was used successfully to capture the antigens; the same antibodies labeled with peroxidase were used to detect BCV, RV, or K99. The triple ELISA was compared with standard reference diagnostic methods by examining feces from experimentally and naturally infected and healthy calves. All the components of the test were highly specific (greater than 90%) and sensitive (BCV, 77%; K99, 93%; RV, 100%) when used in a format requiring short incubation steps at 20 C and visual recording of results.


Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2.

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.


Variations in rRNA content of marine Vibrio spp. during starvation-survival and recovery.

The degree and temporal context of variations in ribosome content during nutrient starvation of two copiotrophic marine bacteria, Vibrio alginolyticus and Vibrio furnissii, have been examined. The organisms were starved either by nutritional shift-down or by consumption of limiting nutrients resulting from growth into stationary phase. Measurements of the amount of hybridization to 16S rRNA-specific probes revealed that the cells retained between 10 and 26% of their original rRNA content after 15 days of starvation. In V. alginolyticus, losses in stationary-phase cells occurred rapidly (1 to 2 days), whereas cells shifted into starvation remained larger and retained considerably more rRNA. The ability of V. alginolyticus to recover from starvation was assessed after cells were maintained for 2, 8, and 15 days in nutrient-depleted medium. The pattern of recovery at the level of rRNA accumulation depended upon the duration of nutrient deprivation and the manner in which it was imposed. Stationary-phase cells starved for 2 days had only slight relative increases in rRNA levels after excess nutrients were added. As the duration of starvation lengthened to 8 and 15 days, increasingly greater amounts of rRNA (30 and 70 times preenrichment values, respectively) were transcribed after nutrient enrichment. Shift-down cells recovered from 2 and 8 days of starvation without extensive rRNA production. After 15 days, nutrient enrichment caused 16S rRNA levels to increase 30-fold. The results indicate that the mechanisms controlling starvation-survival in these marine bacterial species are linked to the physiological state at the onset of starvation and that the subsequent pattern of recovery will depend upon how starvation was initiated.


Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A.

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.


Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.


HLA-D region genes and rheumatoid arthritis (RA): importance of DR and DQ genes in conferring susceptibility to RA.

The distribution of HLA-D region antigens was studied in three groups (I, IIa, and IIb) of patients with rheumatoid arthritis (RA): group I comprised 43 patients with mild, non-progressive RA, controlled by non-steroidal anti-inflammatory drugs without progression or erosions; group II comprised 94 patients with severe disease, who had earlier been treated with non-steroidal anti-inflammatory drugs and all had incomplete response requiring treatment with gold (sodium aurothiomalate). Of these, 46 patients (group IIa) responded to gold and the disease was well controlled, and the remaining 48 patients (group IIb) did not respond to gold and developed gold induced toxic reactions, including thrombocytopenia or proteinuria, or both. HLA-D region antigens were defined by serological and molecular (Southern blot analysis and oligonucleotide typing) techniques. The results show that DR4 was significantly increased in all three groups of patients. The prevalence of DR1, or DR1 in DR4 negative patients, and DR3 and DR4 associated DQw7 specificities, however, showed differences in these three groups of patients. The prevalence of DR1 and of DR1 in DR4 negative patients was increased only in patients with mild (group I) RA, but not in patients with severe (groups IIa and IIb) disease. On the other hand, the prevalence of DR4 associated DQw7 was significantly increased in patients with severe disease, but not in patients with mild RA. In addition, DR3 was significantly increased only in patients with severe disease who developed gold induced toxic reactions (group IIb). These data suggest that the HLA-D region genes which cause susceptibility to mild RA may be different from those causing susceptibility to severe RA. The results suggest that both DR and DQ (A, B) genes may be important in conferring susceptibility to RA: DR in mild disease and DQ in severe RA.


Different capabilities of monocytes from patients with systemic lupus erythematosus and rheumatoid arthritis to induce glycosylation alterations of acute phase proteins in vitro.

The effect of conditioned medium on the biosynthesis and glycosylation profile of acute phase proteins secreted by the human hepatoma cell line Hep G2 was studied. Conditioned medium was prepared from nonactivated [CM-LPS(-)] and ex vivo lipopolysaccharide activated [CM-LPS(+)] monocytes from eight patients with active rheumatoid arthritis (RA), five patients with active systemic lupus erythematosus (SLE), and seven healthy subjects. The biosynthesis of albumin, alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor and the profile of glycosylation of proteinase inhibitor were analysed. CM-LPS(-) from patients with SLE had a similar effect to CM-LPS(-) from healthy subjects. In contrast, CM-LPS(-) from patients with RA had the same effect as CM-LPS(+) from healthy donors. A similar effect to that of CM-LPS(+) of healthy subjects was seen with CM-LPS(+) from patients with SLE and with CM-LPS(+) from patients with RA. The treatment of CM-LPS(+) with antibodies against interleukin 6 neutralised most of its ability to induce changes in the biosynthesis and glycosylation of acute phase proteins. Antibodies to interleukin 1 and tumour necrosis factor alpha had only a limited effect on the ability of CM-LPS(+) to induce changes of albumin and alpha 1-antichymotrypsin syntheses, whereas they had no effect on the biosynthesis and glycosylation of proteinase inhibitor. These results indicate that: (a) monocytes isolated from patients with active SLE and active RA have different capabilities of inducing alterations of acute phase proteins in vitro; (b) ex vivo activation of monocytes from patients with SLE leads to the full induction of its capabilities to change acute phase proteins, whereas the activation of monocytes from patients with RA has no additive effects; and (c) interleukin 6 seems to be a major cytokine involved in the regulation of the glycosylation pattern of acute phase proteins.


Palliative intubation for dysphagia in patients with carcinoma of the esophagus.

One hundred seven consecutive patients seen over a 6-year period with dysphagia secondary to advanced primary carcinoma of the esophagus underwent intubation. One hundred five patients underwent pulsion intubation. In 2 patients pulsion intubation was not possible, and laparotomy and traction intubation was performed. For the intubated group there were 65 men and 40 women (ratio, 1.6:1), with a mean age of 71.3 +/- 10.5 years (range, 36 to 92 years). Of the 105 patients who had pulsion intubation, a perforation developed in 11 (10.5%), which was responsible for the death of 4 patients (3.8%). A further 3 patients died of malignant cachexia, which resulted in an overall mortality of 6.7%. Late complications included tube displacement (4 patients; 3.8%) and tube obstruction (32 patients; 30.5%). Tube obstruction was due to advancement of malignant disease in 26 patients (81.2%) and food bolus impaction in the remaining 6 patients (18.8%). Pulsion intubation for advanced carcinoma of the esophagus can be performed with a low morbidity and early mortality. However, there is a substantial long-term morbidity of tube obstruction in almost a third of survivors.


Platelet protection by aprotinin in cardiopulmonary bypass: electron microscopic study.

To evaluate the functional integrity of platelets in patients administered the proteinase inhibitor aprotinin during cardiopulmonary bypass, 20 patients undergoing a complicated and prolonged open heart operation were studied. They were randomized to receive either a high dose of aprotinin (total dose, 6 to 7 x 10(6) KIU) before and during cardiopulmonary bypass (10 patients) or a placebo (10 patients). Blood samples were collected preoperatively, at the termination of bypass, and 90 minutes thereafter to assess platelet count and aggregation on extracellular matrix, which was studied by scanning electron microscopy. On a scale of 1 to 4, mean preoperative platelet aggregation grades were similar in both groups (3.5 +/- 0.5). Postoperatively, at the termination of cardiopulmonary bypass and 90 minutes thereafter, all 10 patients treated with aprotinin revealed normal, unchanged platelet aggregation (grade, 3.5 +/- 0.5), whereas all placebo-treated patients showed severely disturbed aggregation (grade, 1.4 +/- 0.5) (p less than 0.001). The platelet count was similar in both groups before and after operation (preoperatively, 182 +/- 75 x 10(9)/L and 146 +/- 30 x 10(9)/L, and postoperatively, 87 +/- 13 x 10(9)/L and 80 +/- 27 x 10(9)/L for the aprotinin and placebo groups, respectively). Total 24-hour postoperative bleeding and blood requirement were significantly lower in the aprotinin group (371 +/- 84 mL and 2 +/- 0.7 units, respectively) compared with the placebo group (608 +/- 28 mL and 3.4 +/- 1.3 units, respectively) (p less than 0.01). These results demonstrate that improved postoperative hemostasis is directly related to the complete preservation of platelet function achieved by the protective properties of aprotinin.


Retention of epidermal growth factor receptors in the endoplasmic reticulum of adenovirus-infected cells.

The epidermal growth factor (EGF) receptor is down-regulated during early infection with adenovirus, and this has been attributed to accelerated internalization and degradation of the receptor in the absence of ligand (Carlin, Tollefson, Brady, Hoffman & Wold (1989) Cell 57, 135-144]. Using pulse-chase analysis, we show that loss of functional EGF receptors after infection of human KB and A431 cells with adenovirus type 5 is accompanied by accumulation of a receptor precursor that remains fully sensitive to endoglycosidase H, indicative of retention in the endoplasmic reticulum. A truncated receptor, normally secreted by A431 cells, also accumulates intracellularly as an endoglycosidase H-sensitive precursor. In no case is the block in intracellular transport of EGF receptors complete. We conclude that both stimulation of EGF receptor internalization and degradation and inhibition of intracellular transport of newly synthesized EGF receptors from the endoplasmic reticulum towards the cell surface contribute to EGF receptor down-regulation in adenovirus-infected cells.


Glucose transporter expression and glucose utilization in skeletal muscle and brown adipose tissue during starvation and re-feeding.

Starvation (48 h) decreased the concentration of mRNA of the insulin-responsive glucose transporter isoform (GLUT 4) in interscapular brown adipose tissue (IBAT) (56%) and tibialis anterior (10%). Despite dramatic [7-fold (tibialis anterior) and 40-fold (IBAT)] increases in glucose utilization after 2 and 4 h of chow re-feeding, no significant changes in GLUT 4 mRNA concentration were observed in these tissues over this re-feeding period. The results exclude changes in GLUT 4 mRNA concentration in mediating the responses of glucose transport in these tissues to acute re-feeding after prolonged starvation.


Type II intermediate-filament proteins from wool. The amino acid sequence of component 5 and comparison with component 7c.

Component 5 is one of the four type II intermediate-filament proteins found in the hard keratin wool. It was isolated as the S-carboxymethyl derivative from Merino wool and its amino acid sequence was determined by manual and automatic sequencing of peptides produced by chemical and enzymic cleavage. Component 5 is an N-terminally blocked molecule of 503 residues and Mr (not including the blocking group) of 56,600. The blocking group has not been identified. The amino acid sequence of component 5 shows 77% sequence identity with that of component 7c, another type II wool intermediate-filament protein [Sparrow, Robinson, McMahon & Rubira (1989) Biochem. J. 261, 1015-1022]. The sequence similarity extends from the N-termini of the two molecules to residue 459 (component 5 sequence); however, there is no recognizable sequence similarity in the remaining C-terminal 43 amino acid residues. Details of procedures used in determining the sequence of component 5 have been deposited as a Supplementary Publication SUP 50168 (80 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire, LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1992) 281, 5. The information comprises: (1) details of chemical and enzymic methods used for cleavage of component 5, peptide CN1, the peptide mixture CN2/3 and various other peptides, (2) details of the procedures used for the fractionation and purification of peptides from (1), including Figures showing the elution profiles from the chromatographic steps used, and (3) details of the method used to determine the C-terminal sequence of component 5.


Insulin-like growth factor-I (IGF-I) and especially IGF-I variants are anabolic in dexamethasone-treated rats.

The administration of insulin-like growth factor-I (IGF-I) via subcutaneously implanted osmotic pumps partially reversed a catabolic state produced by the co-administration of 20 micrograms of dexamethasone/day to 150 g male rats. Marked dose-dependent effects on body weight and nitrogen retention were produced, with the highest IGF-I dose, 695 micrograms/day, giving a 6 g increase in body weight over 7 days, compared with a 19 g loss in the dexamethasone-only group and an 18 g gain in pair-fed controls. Two IGF-I analogues that bind poorly to IGF-binding proteins, the truncated form, des(1-3)IGF-I, and a variant with an N-terminal extension as well as arginine at residue 3, LR3IGF-I, were approx. 2.5-fold more potent than IGF-I. The response with LR3IGF-I was particularly striking because this peptide binds 3-fold less well than IGF-I to the type 1 IGF receptor. The increased potencies of the IGF-I variants may relate to the substantially increased plasma levels of IGF-binding proteins, particularly IGFBP-3, produced by the combined treatment of dexamethasone with IGF-I or the variants. These binding proteins would be expected to decrease the transfer of IGF-I, but not that of the variants, from blood to tissue sites of action. Measurements of muscle protein synthesis at the end of the treatment period and muscle protein breakdown by 3-methylhistidine (3MH) excretion throughout the experiment indicated coordinate anabolic effects of the IGF peptides on both processes. Thus 3MH excretion was decreased at the highest IGF-I dose from 83.5 +/- 4.2 (S.E.M.) mumol/kg per 7 days to 65.1 +/- 2.2, compared with 54.9 +/- 1.2 in the pair-fed controls. Part of this response in 3MH excretion may have reflected a decrease in gut protein breakdown, because IGF-I and especially the IGF analogues increased the gut weight by up to 45%. Notwithstanding the effects on protein synthesis and breakdown, the fractional carcass weights remained low in the IGF-treated groups, although the increase in total carcass weight reflected nitrogen rather than fat gain. The dexamethasone-induced changes in liver, spleen and heart weight were restored towards normal by the IGF treatment. The experiment demonstrates the potential of IGF-I treatment of catabolic states and especially the value of modified forms of growth factors that bind weakly to IGF-binding proteins.


Cellular mechanism of the insulin-like effect of growth hormone in adipocytes. Rapid translocation of the HepG2-type and adipocyte/muscle glucose transporters.

The cellular mechanism whereby growth hormone (GH) acutely stimulates adipocyte glucose uptake was studied in cultures of primary rat adipocytes differentiated in vitro. Preadipocytes were isolated by collagenase digestion of inguinal fat-pads from young rats and were differentiated in the presence of 3-isobutyl-1-methylxanthine, insulin and dexamethasone. The development of an adipocyte morphology (i.e. lipid inclusions) was observed over 6 days after initiation of differentiation. Coincident with this phenotypic change was an increase in glyceraldehyde-3-phosphate dehydrogenase (GPDH) activity and in cellular content of the HepG2-type (Glut1) and adipocyte/muscle (Glut4) glucose transporter isoforms as determined by Western immunoblotting of total cellular protein. Age-matched undifferentiated cells expressed the Glut1 transporter and low levels of GPDH, but neither accumulated lipid nor exhibited measurable expression of the Glut4 protein. On day 6 after the initiation of differentiation, GH and insulin stimulated 2-deoxy[14C]glucose uptake in a dose- and time-dependent fashion in adipocytes cultured under serum-free conditions for at least 15 h. Western-blot analysis of subcellular fractions revealed that both GH and insulin rapidly (within 20 min) stimulated translocation of the Glut1 and Glut4 proteins from a low-density microsomal fraction to the plasma membrane. Confirmatory evidence was provided in immunocytochemical experiments utilizing antisera directed against the C-terminal region of the Glut4 protein and a fluorescein isothiocyanate-labelled second antibody. Observation of the cells via confocal laser microscopic imaging was consistent with glucose transporter redistribution from an intracellular region to the plasma membrane after treatment with GH or insulin. On the basis of these data, we suggest that the insulin-like effect of GH on adipocyte glucose transport involves translocation of the Glut1 and Glut4 proteins to the plasma membrane. Furthermore, stimulation of glucose-transporter translocation by both GH and insulin may indicate a common cell signalling element between the adipocyte GH and insulin receptors or, alternatively, the existence of multiple cellular mechanisms for stimulating glucose-transporter translocation.


Cyclic AMP-dependent modulation of cardiac Ca channels expressed in Xenopus laevis oocytes.

Cyclic AMP-dependent modulation of cardiac L-type voltage-dependent Ca channel (VDCC) has been probed in Xenopus laevis oocytes injected with poly(A+) RNA from rat heart. A 2 to 3 fold increase of the Ba current amplitude was routinely obtained upon microinjection of cAMP (50-500 microM). Inhibition of protein kinase A (PKA) dramatically reduced the Ba current amplitude, indicating that cAMP-dependent modulation plays an important role in maintaining the basal activity of expressed Ca channels. Moreover, the effects of the DHP agonist Bay K 8644 on kinetic properties of expressed Ba current (IBa,C) were dependent on PKA activation. The results suggest that most expressed cardiac L-type VDCCs are phosphorylated and demonstrate that reconstitution in Xenopus oocytes is a suitable approach to address how phosphorylation regulates VDCC activity.


In the GluR1 glutamate receptor subunit a glutamine to histidine point mutation suppresses inward rectification but not calcium permeability.

Recent papers have described glutamine to arginine point mutations of the cloned AMPA/Kainate receptor subunits that alter current-voltage relationship and suppress Ca2+ permeability, thus linking these two characteristics. We describe a glutamine to histidine mutation at the same position, which alters current-voltage relationship but retains Ca2+ permeability, thus dissociating the two properties.


Concomitant expression of receptor subtype and isopeptide of endothelin by human adrenal gland.

We studied whether specific receptors for endothelin (ET) isopeptide exist in human aldosterone-producing adenoma and normal adrenal cortex, and whether ET isopeptides are produced by human adrenal gland. Competitive binding studies using [125I]ET-1 as a radioligand revealed the presence of a single class of high-affinity binding sites for ET-1 with the apparent KD of 70 +/- 31 pM and Bmax of 226 +/- 139 fmol/mg protein in adenoma membranes almost comparable to those in adjacent normal cortex. The apparent Ki for ET-2 and ET-3 were 89 +/- 33 pM and 82 +/- 16 pM, respectively. Northern blot analysis of poly(A)+ RNA of adenoma and adjacent normal cortex using cDNAs for ET receptor subtype (ETA, ETB) and ET isopeptide (ET-1, ET-3) as probes revealed that ETA and ETB receptors as well as ET isopeptides (preproET-1, preproET-3) are concomitantly expressed in both tissues. Our data demonstrate for the first time that ET receptor subtype (ETA and ETB) and ET isopeptide (ET-1 and ET-3) are concomitantly expressed by human adrenal cortex, suggesting the potential role of ETs as a local mediator in human adrenal gland.


Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages.

A murine macrophage cell line, J774, expresses high levels of the enzyme nitric oxide synthase (NOS) and produces large amounts of nitric oxide (NO) when activated with recombinant interferon (IFN)-gamma and a low concentration of LPS (10 ng/ml). Both the expression of NOS and the production of NO were inhibited by recombinant IL-10 in a dose-dependent manner. The inhibition was effective only when the cells were pretreated with IL-10; addition of IL-10 at the same time or after IFN-gamma activation was without effect. These results demonstrate that IL-10, a product of Th2 (helper T lymphocyte 2) cells, can antagonise the function of IFN-gamma, a product of Th1 cells, by modulating the mechanism of synthesis of nitric oxide in the macrophages.


Evidence for polyamine channels in protoplasts and vacuoles of Arabidopsis thaliana cells.

The presence of ion channels permeable to polyamines in the plasma membrane and tonoplast of Arabidopsis thaliana cultured cells was investigated by means of the patch-clamp technique in the whole-cell configuration. Evidence is shown for channels, activated by depolarizations in protoplasts and by hyperpolarizations in vacuoles, with slow time course of activation, permeable to putrescine, spermidine and spermine.


Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding.

To clarify the role of CD36 as a TSP receptor and to investigate the mechanisms of the TSP-CD36 interaction, transfection studies were performed using CD36-cDNA in a CDM8 plasmid. Jurkat cells transfected with CD36 cDNA express an 88kD membrane surface protein and acquire the ability to bind thrombospondin. The TSP amino acid sequence, CSVTCG, mediates the interaction of thrombospondin with CD36. CD36 transfectants but not control transfectants bind radiolabeled tyrosinated peptide (YCSVTCG). The hexapeptide inhibits thrombospondin expression on activated human platelets and results in diminished platelet aggregation. CSVTCG-albumin conjugates support CD36-dependent adhesion of tumor cells. We conclude that the CSVTCG repeat sequence is a crucial determinant of CD36 thrombospondin binding.


FK-506 binding protein from Tolypocladium inflatum: resistance of FKBP/FK-506 complex against proteolysis.

A 12-kDa peptidyl-prolyl-cis/trans-isomerase was purified 225-fold to homogeneity from the cyclosporin producing fungus Tolypocladium inflatum. The enzyme is highly sensitive to the immunosuppressant FK-506 but not to cyclosporin and thus belongs to the class of FK-506 binding proteins (FKBP). Interestingly the FKBP/FK-506 complex is resistant against proteolytic digestion by the endoproteases GluC and LysC, in contrast to the free FKBP, which is readily cleaved by these proteases. This protection may play a role in the effects of FK-506 in the living cell.


Inhibition of selectin-dependent tumor cell adhesion to endothelial cells and platelets by blocking O-glycosylation of these cells.

Expression of sialosyl-Le(x) (SLe(x)) and sialosyl-Le(a) (SLe(a)) on tumor cell lines HL60, Colo205, and U937 was greatly suppressed by application of benzyl-alpha-GalNAc for inhibition of O-linked carbohydrate chain extension, which resulted in reduced adhesion of tumor cells to activated endothelial cells or platelets mediated by ELAM-1 (E-selectin) or GMP-140 (P-selectin). Inhibitors or modifiers of N-glycosylation had no effect on expression of SLe(x) or SLe(a) in these tumor cells. These findings suggest the possibility that targeting of O-glycosylation inhibitors or modifiers to tumor cells may effectively suppress metastatic potential.


Involvement of the "tethered-ligand" receptor in thrombin inhibition of platelet adenylate cyclase.

Thrombin is thought to activate platelets through multiple signaling pathways. Recently a new thrombin receptor was identified (Vu et al., Cell 64:1057-1068, 1991) that recognizes alpha-thrombin's anion-binding exosite. Thrombin cleaves this receptor generating a new N-terminal ("tethered-ligand") that activates the receptor. We report here that this receptor is involved in alpha-thrombin inhibition of platelet adenylate cyclase, a process thought mediated by thrombin's high-affinity pathway. In gel-filtered human platelets, iloprost-stimulated cAMP levels were lowered by alpha- and zeta-thrombin addition and, to a much lesser extent, by gamma-thrombin. The alpha- and zeta-thrombin mediated decreases in cAMP were prevented by the thrombin anion-binding exosite inhibitor, BMS 180742, implying that binding to thrombin's anion-binding exosite was required. The iloprost-stimulated increase in cAMP was also reversed (in a concentration-dependent fashion) by a peptide mimicking the new N-terminal of the "tethered-ligand" thrombin receptor (SFLLRNPNDKYEPF). In broken cell preparations, platelet adenylate cyclase activity was also inhibited by SFLLRNPNDKYEPF (but not by a similar peptide used as a control, FSLLRNPNDKYEPF). These results support the hypothesis that thrombin inhibition of platelet adenylate cyclase activity is mediated, at least in part, via the "tethered-ligand" receptor. Moreover, this data is consistent with the "tethered-ligand" receptor mediating the high affinity actions of alpha-thrombin.


Different receptors mediate stimulation of nitric oxide-dependent cyclic GMP formation in neurons and astrocytes in culture.

The ability of various compounds to stimulate cyclic GMP accumulation was studied in neuronal and astrocyte-enriched primary cultures from rat cerebrum. Glutamate was the only agonist eliciting a response in neurons whereas several agonists had an effect in astrocytes but only those due to norepinephrine and glutamate were of considerable magnitude. The responses were markedly inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The effect of glutamate appears to be mediated predominantly by NMDA receptors in neurons and by quisqualate AMPA-insensitive receptors in astrocytes.


Recombinant E-selectin-protein mediates tumor cell adhesion via sialyl-Le(a) and sialyl-Le(x).

E-selectin (previously known as ELAM-1) is one of the adhesion molecules expressed on activated endothelium. Here we show that HL-60 cells express sialyl-Le(x), but not Sialyl-Le(a) on their surface, a colon carcinoma cell line COLO 205 express both these epitopes and another colon carcinoma COLO 320 does not express either one of them. HL-60 and COLO 205 cell adhere strongly to E-selectin coated microwells, whereas COLO 320 does not adhere at all to E-selectin. Finally we provide evidence that monoclonal anti-sialyl-Le(x) can abolish part of the adherence of HL-60 cells to recombinant E-selectin. The adherence of COLO 205 cells can be decreased by either monoclonal anti-sialyl-Le(a) or anti-sialyl-Le(x) antibodies. These results indicate that cell-associated sialylated carbohydrate moieties can act as ligands for recombinant E-selectin.


Occurrence of differentiated keratin peptide(K1) in cultured human squamous cell carcinomas.

To date, the largest keratin peptide(K1, 68 KD) has been absent in cultured human squamous cell carcinomas. Using a low salt aqueous solution, not containing high salt and Triton X-100, as a washing buffer for keratin extraction, followed by two dimensional polyacrylamide gel electrophoresis, immunological techniques and Northern blot analysis, we demonstrated K1 peptide in two kinds of cultured human squamous cell carcinomas. Until now keratin extraction has been done using high salt/Triton X-100 solution during which K1 peptide may be removed together developed an affinity with the buffer. Many investigators may have therefore overlooked K1.


Cortisol secretion induced by substance P from bovine adrenocortical cells and its inhibition by calmodulin inhibitors.

When primary cultured bovine adrenocortical cells were treated with substance P (SP) at concentrations higher than 10 pM, cortisol output increased in a dose-dependent fashion. Although other neurokinins, such as neurokinin A (NKA) and neurokinin B (NKB), were also effective in secreting cortisol, SP was the most potent among the tested neurokinins, the potency order being SP greater than NKA much greater than NKB. This suggests that the NK-1 type receptor on adrenocortical cells may be the site of action of SP on cortisol secretion. The maximal response in SP-induced cortisol secretion was comparable to that elicited by adrenocorticotropic hormone (ACTH). SP-induced cortisol secretion was dependent upon extracellular Ca2+ concentrations, and 45Ca2+ uptake into adrenocortical cells treated with SP was long-lasting. While, in the case of ACTH, 45Ca2+ uptake proceeded transiently, the increase in intracellular cAMP content was much greater compared with that of SP. Although KT-5720, an inhibitor of protein kinase A, inhibited potently ACTH-induced cortisol secretion, SP-induced secretin was not affected by this inhibitor at all. On the other hand, calmodulin inhibitors, such as calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, were not more effective in inhibiting SP-induced cortisol secretion than secretion induced by ACTH. The present study indicates that SP may be one of the physiological stimulants of cortisol secretion and that an increase in intracellular Ca2+ concentration and the subsequent activation of calmodulin may precede SP-induced cortisol secretion.


Nitric oxide but not prostacyclin is an autocrine endothelial mediator.

Using porcine aortic endothelial cells, the present study investigates whether stimulation of prostacyclin (PGI2) and nitric oxide also causes elevation of the respective second messengers cAMP and cGMP in the endothelial generator cells. The calcium ionophore A23187 at 0.3-3 microM increased endothelial cGMP levels up to 27-fold in an L-arginine-dependent manner as assessed through complete inhibition by NG-monomethyl-L-arginine (100 microM). The 36-fold PGI2 stimulation by 3 microM A23187 was not accompanied by an intracellular increase in cAMP or an enhanced cAMP efflux. Correspondingly, the PGI2 mimetic iloprost (10 pM-100 microM) did not change endothelial cAMP levels. However, forskolin (1-100 microM) and prostaglandin E2 (PGE2) (0.1-10 microM) produced concentration-dependent increases in cAMP with a 9-fold and 8-fold stimulation at 100 microM forskolin and 10 microM PGE2, respectively. These results demonstrate that in contrast to NO, PGI2 acts as a strictly paracrine hormone without affecting the respective second messenger cAMP in the endothelial generator cells.


Properties of redox-inactivated bleomycins. In vitro DNA damage and inhibition of Ehrlich cell proliferation.

Blenoxane, bleomycin A2, bleomycin B2, and demethyl bleomycin A2 and products of their reactions with Fe2+ and oxygen were used to explore the relationship between their capacity to carry out in vitro DNA strand scission and their growth inhibitory activity against Ehrlich cells. Reaction of Fe2+, bleomycin and O2 in the absence of DNA decreased the subsequent effectiveness of various bleomycin congeners to degrade DNA in the presence of Fe2+ and oxygen. In comparison with controls, this loss of strand scission activity was not paralleled by equivalent decreases in growth inhibition. Demethyl bleomycin A2 retained full biological activity relative to bleomycin A2, despite being only 30% as effective as bleomycin A2 in its ability to cleave DNA in vitro. Prior reaction of bleomycins with Fe2+ did not alter their capacity to reduce oxygen or affect their ability to generate the activated intermediate which, for native bleomycin structures, is competent to cleave DNA in vitro.


Covalent binding of 14C- and 35S-labeled thiocarbamides in rat hepatic microsomes.

The covalent binding of a series of 14C- or 35S-labeled benzimidazole-2-thione (MBI) derivatives to rat liver microsomal proteins was studied to determine the mechanisms of hepatic monooxygenase oxidation of model anti-hyperthyroid compounds. All thiocarbamides tested (including methimazole) produced an NADPH-dependent loss of cytochrome P450 (P450) chromophore which could be prevented by the addition of glutathione (GSH). The covalent binding of MBI to liver microsomal proteins from dexamethasone (DEX)-pretreated rats was enhanced 10-fold with NADPH, unaffected by P450 inactivation with 1-aminobenzotriazole (ABT) and attenuated by GSH addition. Heat treatment of microsomes to inactivate the flavin-containing monooxygenase (FMO) decreased the observed binding. Equivalent amounts of [35S]- and [14C]MBI were covalently bound to hepatic microsomal proteins, suggesting retention of both the carbon and sulfur portions of the molecule in the MBI/protein adduct. Thiophilic reagents effected release of covalently bound [14C]- and [35S]MBI in equal amounts suggesting the presence of disulfide bonds between an MBI-derived sulfenic acid and microsomal protein thiols. Coincubation with bovine serum albumin (BSA) resulted in NADPH-dependent binding of [14C]-MBI to BSA sulfhydryls which was blocked by prior treatment of BSA with iodoacetamide. 1-Methyl-benzimidazole-2-thione (MMBI) also covalently bound to microsomal proteins and BSA but at levels lower than with MBI. P450, however, appeared to be more important than FMO in the metabolism of MMBI based on the effects of microsome heat pretreatment or ABT addition. In addition, ca. 1.5-fold more 35S- than 14C-label became bound. The covalent binding of [35S]1,3-dimethyl-benzimidazole-2-thione (DMMBI) to microsomal proteins was ca. six times greater than that of [14C]DMMBI. ABT, catalase and superoxide dismutase had a minimal effect on [35S]DMMBI binding, while FMO inactivation decreased binding by ca. 30%. These findings suggest that both monooxygenases contribute significantly to the hepatic metabolism of thiocarbamides. However, FMO activates thiocarbamides primarily to sulfenic acids, whereas P450 appears to produce both sulfenic acid and other reactive sulfur-derived metabolites. Thiol groups of P450 and other proteins are the molecular targets for these reactive species formed during the hepatic metabolism of anti-hyperthyroid drugs.


Comparison of cyclosporine and FK506 effects on glutathione levels in rat cochlea, brain, liver and kidney.

Cyclosporine treatment (50 mg/kg/day, p.o.) caused increases in rat renal reduced glutathione (GSH) levels of 205 and 673%, respectively, after 5 and 10 days. No changes were seen in liver GSH with either dose of cyclosporine. FK506 (2.5 mg/kg/day, p.o., for 7 days) caused an approximately 200% increase in kidney GSH, and an approximately 250% increase in hepatic GSH levels. Oxidized glutathione (GSSG) was never more than 1-2% of the level of the reduced form in any tissue from control animals. Small increases in the ratios of oxidized to reduced glutathione were seen in livers and kidneys from both cyclosporine- and FK506-treated animals. No changes in GSH or GSSG levels were seen in brains or cochleas from any animal.


Bleomycin regulation of transforming growth factor-beta mRNA in rat lung fibroblasts.

Pulmonary fibrosis is a well-known toxic response to bleomycin treatment. Here we demonstrate the direct effects of bleomycin on lung fibroblasts that resulted in a marked increase of collagen synthesis as compared with total noncollagen protein synthesis. Bleomycin treatment of rat lung fibroblast cultures resulted in an increase of total cellular transforming growth factor-beta (TGF-beta) mRNA and increased secretion of TGF-beta protein into the conditioned media. beta 2-Microglobulin was measured as an mRNA that did not increase with bleomycin treatment. The bleomycin-induced increase of TGF-beta mRNA was decreased by cells cultured in the presence of either cycloheximide, an inhibitor of protein synthesis, or 2-mercapto-1-(beta-4-pyridethyl) benzimidazole, an inhibitor of RNA synthesis. To assess the mechanism underlying increased steady-state mRNA levels, the nuclear fraction was isolated from bleomycin-treated cells and the TGF-beta transcripts were determined. Transcription of TGF-beta mRNA was increased 12 h after bleomycin treatment, whereas the transcription of type I procollagen, type III procollagen, and beta-actin mRNAs were increased after 48 h of bleomycin treatment. beta 2-Microglobulin mRNA synthesis was not increased within this time frame. These results suggest bleomycin regulation of TGF-beta at both the mRNA and protein levels. Rats lung fibroblasts were separated by cell sorting into two subpopulations. One population of fibroblasts demonstrated increased procollagen type I mRNAs, whereas fibroblasts in the other population had increased procollagen type III mRNA. Following bleomycin treatment, TGF-beta mRNA was shown to be located more prominently in those fibroblasts that contain primarily collagen type I mRNAs.


Alterations in DNA synthesis and cellular constituents in mouse lung following bleomycin injections.

Changes in the DNA synthesis and cellular constituents of mouse lung following repeated bleomycin (BLM) injections were studied. ICR mice were administered BLM subdermally for 10 days. Wet lung weight was increased 1.36 times on day 5 after the final administration compared with control mice receiving an identical volume of saline only for 10 days. The total number of cells in the bronchoalveolar lavage fluid of the BLM group reached a maximum on day 14, and histologic investigation of the lungs revealed marked cellular infiltrations. The labeling index obtained by the antibromodeoxyuridine monoclonal antibody method for cells was increased from days 5 to 14 in the BLM group. By day 28, these inflammatory changes had subsided and fibrotic remodeling had occurred. DNA polymerase activity in the lung tissue reached its maximal level on day 5 and remained unchanged until day 14. This phenomenon occurred in parallel with increases in DNA content and synthesis. During this period, an increase in DNA polymerase-beta activity and new induction of DNA polymerase-alpha activity were observed by phosphocellulose column chromatography. From these observations, it is concluded that: (1) repeated injections of BLM cause DNA injury in lung cells; (2) there is a subsequent increase in the DNA repair function as supported by the finding of an increase in DNA polymerase-beta activity; and (3) these lead further to cell proliferation as supported by the increase in both DNA polymerase-alpha activity and DNA content. Thus, a close relationship between morphologic changes and altered DNA synthesis was observed in the lungs of mice after BLM injections.


HIV inhibitors targeted at the reverse transcriptase.

HIV inhibitors targeted at the virus-associated reverse transcriptase (RT) can be divided into two groups, depending on whether they are targeted at the substrate or nonsubstrate binding site. To the first group belong the 2',3'-dideoxynucleosides (i.e., DDC, DDI), 3'-azido-2',3'-dideoxynucleosides (i.e., AZT), 3'-fluoro-2',3'-dideoxynucleosides (i.e., FLT), 2',3'-didehydro-2',3'-dideoxynucleosides (i.e., D4C, D4T) and carbocyclic derivatives thereof (i.e., carbovir), 2'-fluoro-ara-2',3'-dideoxynucleosides, 1,3-dioxolane derivatives (i.e., 2',3'-dideoxyl-3'-thiacytidine), oxetanocin analogues and carbocyclic derivatives thereof (i.e., cyclobut-G) and the 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) derivatives. These compounds need to be phosphorylated intracellularly to their triphosphate forms before they act as competitive inhibitors or alternate substrates (chain terminators) of HIV RT. The second group includes the tetrahydro-imidazo[4,5,l-jk][1,4]-benzodiazepin-2(1H)one (TIBO), 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine (HEPT), dipyrido[3,2-b:2',3'-e]-[1,4]diazepin-6-one (nevirapine) and pyridin-2(1H)one derivatives, which interact as such, noncompetitively, with a specific allosteric binding site of HIV-1 RT. Compounds belonging to the two different groups may give rise to synergism which combined, and, likewise, viral resistance to the compounds may arise through different mutations, depending on the nature of the compounds and the group to which they belong.


Nonnucleoside inhibitors of HIV-1 reverse transcriptase: nevirapine as a prototype drug.

Nevirapine, a dipyridodiazepinone, is a highly specific inhibitor of HIV-1 reverse transcriptase (RT) which exhibits an IC50 = 84nM in enzyme assays and IC50 = 40nM against HIV-1 replication in cell culture. This nonnucleoside inhibitor acts noncompetitively with respect to nucleoside triphosphates, template and primer suggesting that nevirapine does not bind to the active site of RT. Studies employing an azido analogue of nevirapine as a photoaffinity probe indicated that one molecule of inhibitor is sufficient to inactivate one molecule of heterodimeric enzyme and demonstrated that only the p66 subunit of p66/p51 heterodimeric RT is covalently labeled by this probe. When subjected to trypic mapping, Tyr 181 and Tyr 188 were labeled with probe and consequently these aromatic residues are apparently near or actually within the RT binding site for nevirapine. The extent to which Tyr 181 and Tyr 188 participate/contribute to nevirapine binding was determined by making amino acid substitutions at these positions using the corresponding residues from HIV-2 RT which is not sensitive to nevirapine. A change at either position dramatically decreased the enzymes' sensitivity to nevirapine, as well as to TIBO derivative and Merck L-693,593, indicating that both Tyr 181 and 188 are crucial for inhibitor-enzyme interaction. Cell culture selection in the continued presence of nevirapine results in the appearance of resistant HIV-1, Tyr 181 to Cys, raising the concern that combination drug therapy will be required in the clinic.


Interferons in the persistence, pathogenesis, and treatment of HIV infection.

Interferon (IFN) plays an important role in the treatment and pathogenesis of HIV disease. Recent studies show beneficial effects of IFN alpha in the treatment of HIV-associated Kaposi's Sarcoma and early HIV-infection. Moreover, cell culture studies support these beneficial effects. HIV infection of monocytes is blocked by IFN alpha administered at the time of viral challenge. The IFN alpha-treated cells show no evidence of HIV infection. Viral gene products produced in monocytes infected with HIV then treated with IFN alpha gradually decrease to baseline. Large quantities of proviral DNA are seen in the HIV-infected IFN alpha-treated cells with little evidence for viral transcription suggesting true microbiological latency. While most viral infections of cells result in IFN production, HIV is a notable exception. Indeed, HIV does not induce monocytes to produce IFN alpha and blocks its production following poly(I).poly(c) stimulation. This allows HIV yet another mechanism to evade an important host antiviral response. Paradoxically, the appearance of IFN activity in sera of HIV-infected patients is associated with disease progression, not resolution. Recent observations showing that the interaction between HIV-infected monocytes and PBMC results in the production of IFN alpha s with reduced anti-HIV activity may help explain this paradox. Thus, IFN alpha plays an important but complex role in HIV disease. The elucidation of cellular factors that regulate the antiretroviral effects of IFN alpha may lead to the development of novel therapeutic strategies for HIV infection.


An antigenic structure of the trans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), as defined by a panel of monoclonal antibodies.

In order to study an antigenic structure of the trans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), tax1 antigen, we generated and characterized a panel of rat anti-tax1 monoclonal antibodies (MAbs) designated WATM-1, WATM-2, WATM-3, and WATM-4. These MAbs were derived from WKA rats immunized with HTLV-I-transformed (HTLV-I+) syngeneic T cells. Immunoblot assays showed that: (1) All the MAbs reacted with the tax1 antigen in HTLV-I+ cell lines and a recombinant tax1 antigen, PX141 (containing entire tax1 polypeptide); (2) WATM-3 and WATM-4, but not WATM-1 or WATM-2, reacted with a truncated tax1 antigen, XD59 (tax1 amino acids 180-338); (3) None of them reacted with another truncated tax1 antigen, XD128 (tax1 amino acids 1-47 and 286-353); and (4) each of the four MAbs had different reactivity with tax1-related antigens in the range 38-41 kDa expressed in simian cell lines infected with various HTLV-I-related simian retroviruses (STLV-I). None of the MAbs reacted with HTLV-II tax antigen. Human sera containing anti-tax1 antibodies interfered specifically with the antigen-specific binding of all the MAbs. These results suggest that the present rat MAbs are directed against various epitopes on the tax1 antigen. An antigenic structure of the tax1 antigen deduced from reactivity of a panel of anti-tax1 MAbs including the present rat MAbs is discussed.


Identification of distinct epitopes of endoglin, an RGD-containing glycoprotein of endothelial cells, leukemic cells, and syncytiotrophoblasts.

Endoglin is a glycoprotein expressed predominantly on human endothelial cells. It was first identified with mAb 44G4, produced against the pre-B acute lymphoblastic HOON cell line. We now report that four mAbs independently produced against human umbilical vein endothelial cells (HUVECs), chronic myelogenous leukemia in blast crisis, or U-937 pro-monocytic cells stimulated with phorbol myristate acetate also react with endoglin. High levels of reactivity of all mAbs were observed with HUVEC, while intermediate levels were seen with HOON and U-937 cells. By sequential immunoprecipitation from HUVEC and U-937 cell extracts, it was established that RMAC8, HEC-19, 8E11, and 1G2 mAbs react with the same protein as 44G4. Three distinct epitopes recognized by 44G4, RMAC8, and 1G2 mAbs were identified by competitive radioimmunoassay and flow cytometry. The HEC-19 epitope is spatially related to the 44G4 epitope, whereas the 8E11 epitope is most closely related to the 1G2 epitope. Western blot analysis showed that all antibodies react with the endoglin dimer (Mr = 170,000) purified from placenta. Immunostaining of sections of full-term placenta revealed reactivity not only with fetal vessels but also with the syncytiotrophoblast, the fetal cell layer which interfaces with maternal blood. When HUVEC monolayers were treated with the different mAbs to endoglin, prior to incubation with U-937 cells, a 5- to 10-fold stimulation of adhesion was observed. A fibronectin hexapeptide containing RGD, but not the corresponding RGE peptide, was capable of inhibiting the increased adhesion, when tested with mAb 44G4 and RMAC8. However, the same peptides had no effect on the binding of any of the five anti-endoglin mAbs to cells. Since 44G4 and RMAC8 recognize two distinct epitopes of endoglin, and since all five mAbs stimulated adhesion, the results suggest that a signal has been triggered through endoglin on HUVECs. Endoglin might be implicated either directly, by binding to a specific integrin-like ligand, or indirectly, by regulating the level of adhesion between certain integrins and their receptors.


Purification and patch clamp analysis of a 40-pS channel from rat liver mitochondria.

Patch clamp analysis of membranes reconstituted with a fraction isolated from detergent-solubilized mitochondrial membranes by affinity chromatography on immobilized quinine earlier indicated the presence of two classes of ion channels, of about 40- and 140-pS conductance in medium including 150 mM KCl. Now a 57-kDa constituent of the quinine-affinity column eluate has been identified as the 40-pS channel. Protein fractions derived from the quinine-affinity column eluate by preparative isoelectric focusing with a Rotofor cell have been reconstituted into phospholipid vesicle membranes by detergent dialysis, and vesicles have been enlarged for patch clamping by dehydration and rehydration. Voltage clamp analysis has been carried out on excised patches bathed symmetrically in buffered medium containing 150 mM KCl and 100 microM CaCl2. Patches of membrane incorporating the 57-kDa protein exhibit 40-pS conductance transitions. The magnitude of conductance transitions is similar when Na+ replaces K+ in the bathing medium, indicating little selectivity of the 40-pS channel for K+ relative to Na+. Another fraction derived from the quinine-affinity column eluate is found to contain the larger channel, now estimated to have an average conductance of about 130 pS. Patches of control membrane prepared in the same way but without protein exhibit no channel activity.


cDNA cloning and sequencing of rat alpha 1-macroglobulin.

cDNA clones coding for the plasma protease inhibitor alpha 1-macroglobulin were isolated from a rat liver library. The obtained cDNA sequence contained 4701 nucleotides and had an open reading frame coding for a 1,500 amino acid long protein, including a 24 amino acid signal peptide. The identity of the deduced protein sequence as alpha 1-macroglobulin was established by comparison with published peptide sequences of the protein. The mature protein shares 53% and 57% overall amino acid identity with the two other identified members of the rat alpha-macroglobulin family, alpha 1-inhibitor 3 and alpha 2-macroglobulin. A sequence typical for an internal thiol ester was identified. Of the 24 cysteines, 23 are conserved with alpha 2-macroglobulin. However, instead of the two most C-terminal cysteines in alpha 2-macroglobulin, which forms a disulfide bridge in the receptor binding domain, alpha 1-macroglobulin contains phenylalanine. One mRNA species hybridizing with the alpha 1-macroglobulin probe was observed in rat and mouse liver RNA (approximately 6.2 kb), whereas no corresponding transcript was detected in RNA from human liver.


Solubilization and molecular characterization of active galanin receptors from rat brain.

Galanin receptors were solubilized from rat brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Binding of 125I-galanin to the soluble fraction was time- and temperature-dependent, saturable, and reversible. Scatchard analysis of binding data indicated that the soluble extract contained a single class of galanin binding sites with a Kd of 0.8 nM and a Bmax of 26 fmol/mg of protein. Unlabeled galanin and its fragments galanin(2-29) and galanin(1-15) antagonized the binding of 125I-galanin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane receptors. Galanin(3-29) was found inactive. Binding of 125I-galanin to CHAPS extracts was inhibited by guanine nucleotides with the following rank order of potency: GMP-P-(NH)P greater than GTP greater than GDP. Molecular analysis of the soluble galanin receptor by covalent cross-linking of 125I-galanin to CHAPS extracts using disuccinimidyl tartrate and further identification on SDS-PAGE indicated that the soluble galanin binding site behaves as a protein of Mr 54,000. After incubation of CHAPS extracts with 125I-galanin, gel filtration on Sephacryl S-300 followed by ultracentrifugation on sucrose density gradient revealed a binding component with the following hydrodynamic parameters: Stokes radius, 5 nm; s20,w, 4.5 S; Mr, 98,000; frictional ratio, 1.6. GMP-P(NH)P treatment of CHAPS extracts gave rise to a molecular form with the following characteristics: Stokes radius, 4 nm; s20,w, 3.3 S; Mr, 57,000; frictional ratio, 1.4.(ABSTRACT TRUNCATED AT 250 WORDS)


A rapamycin-selective 25-kDa immunophilin.

FKBP25, a previously uncharacterized 25-kDa FK506- and rapamycin-binding protein, was purified to homogeneity from calf thymus, brain, and spleen, and the sequence of a 215 amino acid (aa) 24-kDa C-terminal peptide was established. The N-terminal domain (101 aa) is unrelated to any known protein, is hydrophilic, and is predicted by circular dichroism spectroscopy to be largely alpha-helix. The C-terminal domain (114 aa) is homologous to FKBP12 and other FKBPs but has a potential nuclear targeting sequence and a unique insertion of seven amino acids in one of its loops. FKBP25 displays the rotamase activity characteristic of FKBPs; the activity is inhibited by the immunosuppressants rapamycin (Ki = 0.9 nM) and FK506 (Ki = 160 nM), but not cyclosporin A. The protein, its rapamycin selectivity, and the potential nuclear targeting sequence are discussed in terms of the structure of hFKBP12.


Density separation of human red blood cells on self forming Percoll gradients: correlation with cell age.

Human red blood cells were density separated on self-forming Percoll gradients. Redistribution of density fractionated red blood cells was studied by recentrifugation on self-forming Percoll gradients. A protocol that avoids centrifugation of red cells prior to removal of white cells and introduces EDTA before red cell pelleting completely avoided redistribution. Dense red cells separated according to this method were senescent on the basis of a biochemical and a physical criterion: the increase in the band 4.1a:4.1b ratio (Mueller, T., Jackson, C.W., Dockter, M.E. and Morrison, M. (1987) J. Clin. Invest. 79, 492-499) and the loss of maximum deformability. Characterization also included the relative content of two surface proteins (complement receptor 1, CR1 (Ripoche, J. and Sim, R.B. (1986) Biochem. J. 235, 815-821); decay accelerating factor, DAF) on density fractionated red cells. Unlike cytoplasmic proteins, these proteins face similar conditions, whether located on circulating reticulocytes or aging red cells. Both components were lost linearly within experimental errors with cell density and were lower by 60 and 40% in dense than light cells, respectively.