On the supramolecular organization of gramicidin channels. The elementary conducting unit is a dimer. The question, whether the conducting channels formed by the linear gramicidins are dimers (as is generally believed) or tetramers (as has been recently proposed [Stark G., M. Strässle, and Z. Takacz. 1986. J. Membr. Biol. 89:23-37; Strässle, M., G. Stark, M. Wilhelm, P. Daumas, F. Heitz, and R. Lazaro. 1989. Biochim. Biophys. Acta. 980:305-314]) has been addressed in single-channel experiments. The experimental approach was based on the ability of electrophysiological (single-channel) experiments to resolve the number of hybrid channel types that could form between gramicidin A or C and O-pyromellityl-gramicidin A or C (in which a pyromellitic acid residue has been esterified to the ethanolamine-OH group [Apell, H.-J., E. Bamberg, H. Alpes, and P. Läuger. 1977. J. Membr. Biol. 31:171-188]). The presence of the bulky, negatively charged pyromellityl group at the channel entrances endows the hybrid channels with characteristically different features and thus facilitates the resolution of the different hybrid channel types. Only two hybrid channel types were detected, indicating that the conducting channels are membrane-spanning dimers. There was likewise no evidence for lateral association between conducting channels and nonconducting monomers. These results can be reconciled with those of Stark et al. (op. cit.) if gramicidin channel formation involves a (slow) folding into beta 6.3-helical monomers followed by the dimerization step.


Multichannel recordings from membranes which contain gap junctions. We have studied multichannel patch-clamp recordings in earthworm axon septal membranes that contain gap junctions. Though all channels have the same conductance and selectivity, the probabilities of the conductance levels in the majority of the recordings could not be fit by assuming independent and identical channels; in these cases, we found that at least two different open probabilities were required to explain the data. The data thus suggest that, within one junctional membrane complex, there exists a heterogenous channel population of similar but not identical channel types. The analysis also revealed cases where cooperativity between individual channels was the only explanation for the amplitude histograms of the observed multichannel activity. The conclusions drawn are based on a theoretical analysis of multichannel current-amplitude histograms. We derive two tests for independent and identical channels. We analyze the effects of mode shifting. These results are based on the ratio of peaks in the histograms; they are independent of the number of channels in the patch and the model of channel gating. In some cases failure to fulfill the criteria of these tests implied an interdependence or cooperativity between channels. Lastly, we have devised statistical tests for stability of the recording in the presence of variance due to finite sample size.


Electrostatic coupling of ion pumps.

Electrostatic coupling of ion pumps. In this paper the electrostatic interactions between membrane-embedded ion-pumps and their consequences for the kinetics of pump-mediated transport processes have been examined. We show that the time course of an intrinsically monomolecular transport reaction can become distinctly nonexponential, if the reaction is associated with charge translocation and takes place in an aggregate of pump molecules. First we consider the electrostatic coupling of a single dimer of ion-pumps embedded in the membrane. Then we apply the treatment to the kinetic analysis of light-driven proton transport by bacteriorhodopsin which forms two-dimensional hexagonal lattices. Finally, for the case of nonordered molecules, we also consider a model in which the pumps are randomly distributed over the nodes of a lattice. Here the average distance is equal to that deduced experimentally and the elemental size of the lattice is the effective diameter of one single pump. This latter model is applied to an aggregate of membrane-embedded Na, K- and Ca-pumps. In all these cases the electrostatic potential considered is the exact solution calculated from the method of electrical images for a plane membrane of finite thickness immersed in an infinite aqueous solution environment. The distributions of charges (ions or charged binding sites) are considered homogeneous or discrete in the membrane and/or in the external solution. In the case of discrete distributions we compare the results from a mean field approximation and a stochastic simulation.


Re-irradiation of soft-tissue sarcoma.

Re-irradiation of soft-tissue sarcoma. Re-irradiation for local recurrence of malignancy after radical radiotherapy is of proven benefit at head and neck sites but has seldom been used elsewhere. This paper reports a series of 10 patients re-irradiated with external-beam techniques for local recurrence of soft-tissue sarcoma of the limb and limb girdle following initial limb conserving management with surgery and radiotherapy (dose range 33-60 Gy). Median survival was 14 months following re-treatment. Two cases received treatment with high-energy electrons and the rest with megavoltage photons. Five patients re-treated with radical intent (dose range 40-60 Gy) had a median survival of 36 months and median recurrence-free survival of 16 months. All five patients treated palliatively (dose range 12-50 Gy) have died, although two demonstrated local control until death. Acute reactions were not severe. Radionecrosis was seen in one patient who was re-irradiated twice (total dose 145 Gy) and subsequently required amputation. One other case required amputation for persistent local disease, but in the remaining eight, limb conservation was achieved. Re-irradiation of soft-tissue offers good local control and may avoid amputation.


Subchronic methamphetamine treatment enhances ouabain-induced striatal dopamine efflux in vivo. The effect of manipulation of the Na+ gradient between the intracellular and extracellular media on striatal dopamine (DA) efflux under steady-state conditions after subchronic methamphetamine (MAP) treatment was investigated. Rats were injected with 4 mg/kg MAP or saline (i.p.), once daily for 14 days. Seven days after the last injection, ouabain (10(-4) M), a selective inhibitor of the Na+,K(+)-ATPase, was infused locally through a semi-permeable probe in the striatum. Ouabain induced a significantly greater (P less than 0.01) increase of the DA concentrations in the striatal perfusate in the subchronic MAP than the control group. The levels of 3,4-dihydroxyphenylacetic acid (DOPAC) (P less than 0.05) and 5-hydroxyindoleacetic acid (5-HIAA) (P less than 0.05) were significantly higher in the subchronic MAP than in the control group. Reserpine pretreatment (5 mg/kg, i.p.) did not affect the enhanced ouabain-induced DA efflux (P less than 0.01) in the subchronic MAP group, and the levels of DOPAC (P less than 0.01), 5-HIAA (P less than 0.01) and HVA (P less than 0.01) were also significantly higher in the subchronic MAP than in the control group. In contrast, alpha-methyl-p-tyrosine (250 mg/kg, i.p.) pretreatment abolished the ouabain-induced efflux of DA, DOPAC and HVA, but not 5-HIAA, in both groups. Specific striatal [3H]ouabain binding and striatal Na+,K(+)-ATPase activity in the subchronic MAP and control groups did not differ significantly. These results suggest that subchronic MAP treatment facilitates the efflux of newly synthesized DA, which is induced by the ouabain-induced decrease of the Na+ gradient between intracellular and extracellular media.(ABSTRACT TRUNCATED AT 250 WORDS)


Cholecystokinin induces c-fos expression in hypothalamic oxytocinergic neurons projecting to the dorsal vagal complex. Systemic administration of cholecystokinin (CCK) decreases gastric motility and stimulates pituitary secretion of oxytocin (OT). Although peripheral OT does not affect gastric function, increasing evidence suggests that central OT secretion acting within the dorsal vagal complex (DVC) can alter gastric motility. To evaluate whether systemically administered CCK is capable of activating oxytocinergic neurons projecting to the DVC, we utilized fluorogold retrograde labeling from the DVC in combination with c-fos and OT immunocytochemical staining to quantitatively analyze paraventricular nucleus (PVN) neurons of rats following injection of CCK at a dose known to cause maximal pituitary OT secretion (100 micrograms/kg i.p.). Our results showed that 2320 +/- 63 PVN neurons were retrogradely labeled from the DVC; 146 +/- 21 (6.3%) of these contained OT, and these cells were predominantly located in the medial parvocellular subdivision of the PVN. Of all retrogradely labeled cells, 671 +/- 112 (28.9%) expressed c-fos after CCK stimulation, and 68 +/- 14 of these (10.1%) contained OT. Approximately 50% of the OT-containing neurons retrogradely labeled from the DVC stained positively for c-fos. Many magnocellular OT neurons in the PVN that were not retrogradely labeled from the DVC also expressed c-fos after CCK stimulation. These results demonstrate that parvocellular OT neurons projecting to the DVC are co-activated along with magnocellular OT neurons projecting to the pituitary following administration of a large dose of CCK, and lend support to a possible functional role for OT as a central neurotransmitter that modulates vagal efferent traffic to the gastrointestinal tract.


Effect of neonatal capsaicin treatment on cholecystokinin-(CCK8) satiety and axonal transport of CCK binding sites in the rat vagus nerve. Cholecystokinin (CCK) binding sites which accumulate at ligatures placed on the rat vagus nerve may mediate the satiety actions of CCK. Treatment of neonatal rats with capsaicin attenuated the satiety effect of injected CCK in adult life. Capsaicin pretreatment also reduced, but did not eliminate, the accumulation of CCK binding sites proximal and distal to ligatures on either cervical trunk. A similar effect was observed following ligation of subdiaphragmatic vagal trunks. The CCK receptor antagonists, MK-329 and L-365,260, inhibited binding to capsaicin- and vehicle-treated nerves to a similar degree. Densities of CCK binding sites in the nucleus tractus solitarius and area postrema were also markedly affected by neonatal capsaicin treatment.


Convergence of subthalamic and striatal efferents at pallidal level in primates: an anterograde double-labeling study with biocytin and PHA-L. Small injections of two highly sensitive anterograde tracers, Phaseolus vulgaris-leucoagglutinin (PHA-L) and biocytin, into the striatum and the subthalamic nucleus of squirrel monkeys (Saimiri sciureus) have revealed a high degree of convergence of striatal and subthalamic fibers upon single pallidal cells. Both afferent systems formed highly complex band-like patterns that were largely in register with one another. At single cell level, the somata of pallidal neurons were closely surrounded by subthalamic terminal varicosities, whereas the dendrites were entwined mostly by striatal fibers. Typically, a subthalamopallidal fiber coursed in a caudorostral direction and arborized according to a uniform pattern along its trajectory in the pallidum. Numerous thin and markedly varicose axon collaterals detached themselves at right angle from the main subthalamopallidal fiber. These highly branched collaterals were mostly oriented along the mediolateral plane and entwined rather loosely the dendrites but surrounded very closely the somata of pallidal neurons. In contrast, a striatopallidal fiber travelled in a rostrocaudal direction. Its initial segment made only en passant contacts with pallidal cell bodies, whereas its distal end closely entwined the dendrites of pallidal neurons, forming arrangements similar to 'woolly' type fibers. These results suggest that a single subthalamic fiber may influence a rather large collection of pallidal neurons in a similar fashion, compared to the striatal input which appears to exert a more specific control upon selected sets of the same pallidal neurons.


The fibroma-like variant of epithelioid sarcoma. A fibrohistiocytic/myoid cell lesion often confused with benign and malignant spindle cell tumors. Five cases of a previously undescribed variant of epithelioid sarcoma are presented. This variant differs from the usual lesion in its absence of the typical necrobiotic nodular epithelioid pattern. It is instead composed of deceptively bland fibrohistiocytic and myoid cells arranged in a fibroma-like or dermatofibroma-like pattern with an affinity for osseous involvement. The clinical presentation, ultrastructural features, and presence of vimentin and low molecular weight keratin within the tumor cells justifies their designation as an epithelioid sarcoma variant.


Combined assessment of vascular and myometrial invasion as a model to predict prognosis in stage I endometrioid adenocarcinoma of the uterine corpus. The prognostic significance of vascular invasion as compared with other pathologic features was evaluated in 102 cases of endometrioid adenocarcinoma confined to the uterus (Stage I) treated by hysterectomy. By univariate analysis, survival most closely correlated with patient age, architectural grade, depth of myometrial invasion, vascular invasion, and the presence of perivascular lymphocytic infiltrates. Among these, vascular invasion and the presence of perivascular lymphocytic infiltrates were the best indicators of prognosis. In contrast to perivascular lymphocytic infiltrates, the presence of a lymphocytic infiltrate at the tumor-myometrial junction was not related to outcome. The presence of vascular invasion was found to be associated closely with perivascular lymphocytic infiltrates. These two features may be related biologically and were designated "vascular invasion-associated changes." By multivariate analysis with the Cox proportional hazards model, the depth of myometrial invasion and the presence of vascular invasion-associated changes were found to provide a highly reliable model for predicting outcome. The highly predictive value of vascular invasion as a prognostic factor in Stage I endometrial carcinoma suggests that it is the mechanism by which occult metastasis develops in patients whose disease progresses after hysterectomy. It is likely that other variables correlating with recurrence, such as the presence of deep myometrial invasion and high tumor grade, may act by increasing the probability of vascular invasion and subsequent metastasis.


Tumor cell dissemination triggers an intrathecal immune response in neoplastic meningitis. The intrathecal immune response in neoplastic meningitis (NM) was studied by quantitation of immune parameters such as immunoglobulin G (IgG); IgM; interleukins (IL) 1, 2, 4, and 6; soluble IL-2 receptors (sIL-2R); interferon gamma (IFNy); tumor necrosis factor-alpha (TNF alpha); and three tumor markers, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and fibronectin (FN), in 47 paired cerebrospinal fluid (CSF) and serum samples from patients with NM from different carcinomas, malignant melanoma, and lymphoma. Elevated IgG and IgM indices, CSF oligoclonal Ig bands, and CSF IL-6 indicated an intrathecal immune activation in most patients with NM. Results for IL-1, IL-2, and IL-4 were always negative. sIL-2R and IFNy were detected occasionally but not associated with specific malignant neoplasms. CSF TNF alpha was detected only in NM from cases of malignant melanoma. None of the immune parameters proved useful for the differentiation of NM from autoimmune or inflammatory conditions. Immune parameters were not correlated with tumor markers CEA, AFP, or FN. Results for AFP were positive only in a case of glioblastoma. CEA was a useful and specific diagnostic parameter in carcinomatous NM. CSF FN levels frequently were elevated but are not specific for NM.


Angiogenic effects of macrophages isolated from ascitic fluid aspirated from women with advanced ovarian cancer. The cellular components of ascitic fluid aspirated from the peritoneal cavity of women with advanced ovarian cancer were separated on a Ficoll gradient. Isolated macrophages, which were further purified, elaborated a growth factor which was mitogenic for human endothelial cells isolated from umbilical veins, arteries and the omental microvasculature in vitro, and was angiogenic in vivo. It is postulated that the macrophage-derived factor enhances tumor neovascularization of the widespread ovarian-derived peritoneal malignant lesions appearing in these patients, thus contributing to their rapid growth and metastasis, and the poor prognosis of the disease.


Mechanisms of natural resistance to antifolates in human soft tissue sarcomas. Efforts to use fresh human sarcoma cells for evaluating antifolate resistance with an in situ thymidylate synthesis assay using 5-[3H] deoxyuridine were unsuccessful because of low thymidylate synthesis activity in enzymatically disaggregated tumors. By incubating tumor cell suspensions in supplemented RPMI-1640 medium with 10% fetal bovine serum for 3 days, activity of the in situ thymidylate synthesis assay markedly increased (1.42 versus 0.03 pmol/h/10(7) cells), thus allowing 75% of samples to be evaluated for antifolate sensitivity. By criteria developed with a methotrexate-resistant and -sensitive cell line, this assay indicated that most sarcomas are naturally resistant to methotrexate (12 of 15). Natural resistance to 10-ethyl-10-deazaaminopterin and trimetrexate was also observed in 60% of the samples (nine of 15, respectively). The results from the 3-day in situ assay were confirmed by specific tests for resistance mechanisms in most sarcoma samples. The resistance mechanisms detected were impaired polyglutamylation, an increased level of dihydrofolate reductase, and amplification of this gene. These results encourage further exploration of this assay to predict response to antifolates in individual patients and to evaluate efficacy of new antifolates as candidates for clinical trial.


Elevated expression of pp60c-src in low grade human bladder carcinoma. The results presented in this report demonstrate that the tyrosine-specific protein kinase activity of pp60c-src is elevated over that recorded in normal bladder mucosa in a subset of human bladder carcinomas. Increased kinase activity was observed mainly in low grade bladder lesions and was associated, at least in part, with elevated levels of pp60c-src expression. Extension of this analysis to a panel of human bladder carcinoma cell lines confirmed previous observations of low pp60c-src kinase activity in three lines established from high grade (GIII) bladder tumors and revealed increased kinase activity in three alternative bladder lines derived from GI or GII tumors. Use of an anti-phosphotyrosine antibody in Western blot analysis revealed the presence of novel phosphotyrosyl cellular substrates in human bladder cell lines and tumors displaying elevated pp60c-src kinase activity. These observations suggest an association for the src protooncogene in urothelial cell differentiation events.


Tumor-promoting phorbol ester down-regulates the androgen induction of prostate-specific antigen in a human prostatic adenocarcinoma cell line. Prostate-specific antigen (PSA) is the most sensitive marker available for monitoring the progression of prostate cancer and response to therapy. In a previous study, we demonstrated tissue-specific expression of PSA glycoprotein and mRNA and its regulation through the androgen receptor. In this study, we examine the effects of protein kinase A (PKA) and protein kinase C (PKC) on the androgen regulation of PSA in a human adenocarcinoma cell line, LNCaP. Northern blot analysis demonstrated that forskolin, an activator of PKA, had no effect on the androgen regulation of PSA. However, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a direct activator of PKC, showed a time- and dose-dependent repression of the androgen regulation of PSA glycoprotein and mRNA. The biologically inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, had no effect. Staurosporine, a PKC inhibitor, blocked the TPA-mediated repression of the androgenic stimulation of PSA glycoprotein. In addition, the calcium ionophore, A23187, was able to simulate the actions of TPA, presumably through activation of PKC via calcium mobilization. In summary, the androgenic regulation of PSA protein and mRNA is repressed by tumor-promoting phorbol esters through the PKC pathway. This indicates that the effects of TPA may be secondary to repressed gene transcription or altered mRNA stability. In addition, this study emphasizes that the androgenic regulation of PSA is complex and may involve other extracellular transduction signals.


Serum prostate specific antigen levels in mice bearing human prostate LNCaP tumors are determined by tumor volume and endocrine and growth factors. The ability of prostate-specific antigen (PSA) to predict tumor volume and stage in patients with prostate cancer would be improved if factors regulating its production and clearance were better defined. A thorough understanding of the pharmacokinetics (regulation of production, metabolism, and excretion) of PSA has been precluded, however, by the absence of an in vivo animal model. The purposes of this study are to develop a murine model for evaluating PSA pharmacokinetics in vivo and to assess factors that influence PSA production in vitro. The human prostate cancer cell line, LNCaP, was chosen because it is androgen sensitive and PSA positive. Although LNCaP cells are usually nontumorigenic when inoculated s.c. in athymic mice, coinoculation of 1 x 10(6) LNCaP cells with 1 x 10(6) human bone fibroblasts reliably produces PSA-secreting carcinomas. This LNCaP model provides accurate correlation between tumor volume and serum PSA levels (r = 0.94) and demonstrates that tumor volume and androgens are codeterminants of circulating PSA levels. Following castration, serum PSA levels decrease rapidly up to 8-fold and increase up to 20-fold following androgen supplementation, without detectable castration-induced tumor cell death or concomitant changes in tumor volume. Serum PSA levels increase 0.24 ng/ml/mm3 of tumor, which is approximately 5-fold less than that estimated for humans. Most likely this reduced PSA index (PSA:tumor volume ratio) results from a 7-fold faster clearance of PSA in athymic mice than in humans; other than this shorter half-life, PSA elimination in the murine model appears similar to that in humans, with both following first-order kinetics characteristic of a two-compartment model. Interestingly, following prolonged growth (greater than 21 days) in castrate hosts, LNCaP tumors are capable of adapting to an androgen-deprived environment whereby LNCaP tumors regain the ability to secrete PSA in amounts similar to the precastrate state. In LNCaP cells, androgens increase PSA mRNA levels 4-fold in vivo and in vitro. PSA mRNA expression is also altered by various growth factors. Changes in PSA production induced by androgens and growth factors do not always parallel changes in LNCaP cell growth rate induced by these factors, suggesting that PSA production occurs independently of cell growth rate and may be influenced by various interrelated factors, including hormonal and stromal milieu. Observations from this murine model suggest that androgens and tumor volume are independent determinants of serum PSA levels and imply that decreases in circulating PSA following antiandrogen therapy may not always reflect a corresponding reduction in tumor volume.


Induction of T-cell-mediated immunity against MethA fibrosarcoma by intratumoral injections of a bacillus Calmette-Guérin nucleic acid fraction. MY-1, which consists of DNA and RNA extracted and purified from bacillus Calmette-Guérin (BCG), has been shown to have strong antitumor activity against various experimental tumors. To examine the role of T cells in the antitumor mechanism of MY-1, the effect of MY-1 injection on the development of tumor-specific immunity against MethA fibrosarcoma was investigated. MY-1 injections inhibited tumor growth less effectively in T-cell-deficient nude mice than in normal BALB/c mice. MethA tumor growth was suppressed after inoculation with L3T4-positive lymphocytes from tumor-bearing mice treated with MY-1. MethA-specific delayed-type hypersensitivity was also detected in tumor-bearing mice treated with MY-1. Immunohistochemical analyses showed that many L3T4-positive and a few Lyt2-positive cells infiltrated the regressing tumors. These results indicate that intratumoral MY-1 injections induce a MethA-specific, L3T4-positive cell-mediated, delayed-type hypersensitivity, which is necessary for the tumor regression.


Allogeneic tumor-specific cytotoxic T lymphocytes. We report the development of cytotoxic T lymphocytes specific for an allogeneic brain tumor in a rat model. DA strain cytotoxic T cell precursors stimulated by an allogeneic tumor (9L gliosarcoma) from the Fischer rat could generate a population of cytotoxic T lymphocytes that lysed the allogeneic 9L tumor but failed to lyse other targets, including Fischer concanavalin-A(ConA)-stimulated lymphoid blast targets. DA T cells depleted of reactivity to the Fischer haplotype (DA-F) retained reactivity to the 9L tumor, demonstrating that T cell precursors with specificity for normal Fischer alloantigens were not required for the generation of a response to the 9L Fischer tumor. The preferential lysis of the tumor target did not simply reflect a higher density of Fischer target antigens on the tumor than that found on normal Fischer ConA blast targets. First, the relative densities of class I antigen on the 9L tumor and normal Fischer ConA blasts were comparable. Second, cytotoxic T cells could not be generated from DA-F precursors when Fischer ConA blasts were used as stimulators. If DA-F T cells were simply responding to the higher density of Fischer antigen found on 9L tumor, it would have been expected that the ConA blasts expressing comparable levels of antigen to that found on the tumor would have generated cytotoxicity for both the 9L and ConA targets. We conclude that the cytotoxic T cells are specific for a determinant expressed only by the tumor. Such tumor-specific cytotoxic T cells could be useful in vivo for adoptive immunotherapy of brain tumors.


Mechanisms of activated Ca2+ entry in the rat pancreatoma cell line, AR4-2J. The characteristics of Ca2+ entry activated by surface receptor agonists and membrane depolarization were studied in the rat pancreatoma cell line, AR4-2J. Ca2+ mobilization activated by substance P, bombesin, or muscarinic receptor stimulation was found to involve both Ca2+ release and entry. In addition, depolarization of the surface membrane of AR4-2J cells with elevated concentrations of K+ activated Ca2+ entry. Ca2+ entry induced by membrane depolarization was inhibited by the L-channel antagonist, nimodipine, while that due to surface receptor agonists was not inhibited by this agent. The microsomal Ca(2+)-ATPase inhibitor, thapsigargin, caused both depletion of the agonist-sensitive intracellular Ca2+ pool and sustained Ca2+ influx indistinguishable from that produced by bombesin or methacholine. These results confirm that, unlike the pancreatic acinar cells from which they are presumably derived, AR4-2J cells express voltage-sensitive, dihydropyridine-inhibitable Ca2+ channels. However, in contrast to previous reports with this cell line, in the AR4-2J cells in use in our laboratory, and under our experimental conditions, surface receptor agonists (including substance P) do not cause Ca2+ influx through voltage-sensitive Ca2+ channels. Instead, we conclude that agonist-activated Ca2+ mobilization is initiated by (1,4,5)IP3-mediated intracellular Ca2+ release and that Ca2+ influx is regulated primarily, if not exclusively, by the state of depletion of the (1,4,5)IP3-sensitive intracellular Ca2+ pool.


Discordant results in human chorionic gonadotropin assays. Discordance has been reported in human chorionic gonadotropin (hCG) concentrations measured by different immunoassay kits. We examined the results for 40 serum samples assayed with 10 different hCG immunoassay kits. Results varied considerably. Individual sample results varied by as much as 58-fold. Average results for different kits varied by as much as 1.4-fold for pregnancy (20 samples) and 2.2-fold for trophoblast disease (20 samples) serum. We investigated the causes of this discordance. hCG or hCG beta are general names for mixtures of hCG, hCG alpha, or hCG beta immunoreactive molecules in serum. These mixtures include regular hCG, nicked hCG (missing peptide linkages at beta 44-45 or beta 47-48), carbohydrate variants of hCG, hCG missing the beta-subunit C-terminal segment, free beta-subunit, beta-core fragment, and free alpha-subunit. We prepared standards for each of these major variants and measured their reactivities in the 10 hCG immunoassay kits. Free beta-subunit reactivity varied from nonrecognition (anti-beta:anti-alpha type kits; Hybritech Tandem-R and others) to overrecognition (one kit had five-fold greater affinity for free beta than for hCG). Kits with antibodies to beta-subunit C-terminal segment (Organon NML and others) failed to recognize hCG missing this segment, a component of serum hCG in trophoblast disease. Kits with anti-hCG antibodies (Serono MAIA-clone and others) had minimal recognition of nicked hCG (12%), a component of all serum hCG samples, and consistently gave the lowest values with all serum samples. We conclude that differences in recognition of nicked hCG, free beta, and these other hCG variants cause discordance in hCG immunoassay results.


Ratio of serum tartrate-inhibitable acid phosphatase to total serum protein in benign prostatic hypertrophy and prostatic carcinoma. The activity concentration and the specific activity (the ratio of enzyme activity to total serum protein) of the tartrate-inhibitable fraction of acid phosphatase [orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2; TIAP] were related to benign prostatic hypertrophy and to prostatic carcinoma. As expected, the TIAP activity concentrations assayed in the sera of patients with benign prostatic hypertrophy were within the range of those assayed in normal human sera. In contrast, the specific activities of TIAP determined in the sera of patients with benign prostatic hypertrophy were significantly higher than those determined in the control group. In the sera of prostatic carcinoma patients, both the TIAP activity concentrations and the TIAP specific activities differed significantly (F = 730) from the normal values.


Modified immunoradiometric assay of parathyroid hormone-related protein: clinical application in the differential diagnosis of hypercalcemia. We have developed a sensitive, specific solid-phase immunoradiometric assay (IRMA) of parathyroid hormone-related protein (PTH-RP) with use of affinity-purified polyclonal immunoglobulins. Antibodies recognizing PTH-RP(37-74) are immobilized to a polystyrene bead to "capture" analytes from the sample; antibodies to epitopes within the 1-36 amino acid region of PTH-RP are labeled with 125I. This IRMA recognizes PTH-RP(1-74) and PTH-RP(1-86) equivalently, but does not detect N-terminal or C-terminal fragments of PTH-RP, intact human parathyrin (PTH), or fragments of PTH. PTH-RP is not stable in plasma at 3-5 degrees C or room temperature, but a mixture of aprotinin (500 kallikrein units/L) and leupeptin (2.5 mg/L) improves PTH-RP stability in blood samples. In plasma collected in the presence of these protease inhibitors from normal volunteers and patients with various disorders of calcium metabolism, PTH-RP concentrations were above normal (greater than 1.5 pmol/L) in 91% (42 of 46) of patients with hypercalcemia associated with nonhematological malignancy. In plasma from patients with other hypercalcemic conditions (e.g., primary hyperparathyroidism, sarcoidosis, and vitamin D excess), PTH-RP was undetectable. Above-normal concentrations of PTH-RP and total calcium decreased to normal in a patient with an ovarian cyst adenocarcinoma after surgical removal of the tumor. We conclude that PTH-RP is related to and probably the causative agent of hypercalcemia in most patients with cancer, and that measurements of PTH-RP are useful in the diagnosis and management of patients with tumor-associated hypercalcemia.


Increased plasma amylase in the family of a patient with 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency. A patient with 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase, EC 4.1.3.4) deficiency presented consistently above-normal values of plasma amylase (EC 3.2.1.1). Activities measured were in the lower normal range in family members not proven heterozygotes and in the upper normal range in the proven heterozygotes. Heterozygosity was proven by intermediate HMG-CoA lyase activities determined in cultured fibroblasts and in lymphocytes in the parents and the paternal grandmother. Because all of the family members had diseases of the pancreas, colon, and liver, we question whether the heterozygote state contributes to the impaired function of these organs. Our findings of significantly increased amylase activities in the heterozygotes and the patient, in comparison with the other family members, support this hypothesis.


Cross-reactivity of the B/B' subunit of the Sm ribonucleoprotein autoantigen with proline-rich polypeptides. Using recombinant fusion proteins representing different regions of the human Sm B/B' polypeptide, the 4B4 monoclonal anti-Sm antibody was found to bind a C-terminus epitope that is proline-rich. 4B4 cross-reacted with the p24 gag protein of HIV-1 and with other polypeptides rich in proline residues, including collagen. BALB/c mice immunized with human collagen not only produced antibodies to the immunizing antigen but also antibodies to Sm. This immune mouse serum also recognized C-terminus B/B' fusion proteins. These data suggest that the Sm B/B' antigen contains a poly-Pro epitope that is shared by several autoantigens and retroviral proteins. These sites may be important in the induction of autoantibodies through molecular mimicry.


In situ hybridization of interleukin-1 in CD14-positive cells in rheumatoid arthritis. Interleukin 1 (IL-1) has been implicated as an inflammatory mediator in rheumatoid arthritis (RA). Many cell types, including macrophages, lymphocytes, fibroblasts, and endothelial cells, can produce IL-1 and it is known that IL-1 production is under transcriptional control. It has, however, been difficult to define in vivo the predominant cellular source of this mediator in RA. Here, we have used the combination of in situ hybridization of mRNA and cellular immunophenotyping with monoclonal antibodies to show that the IL-1 beta gene is expressed predominantly by CD14-positive macrophages in synovial tissue from patients with RA. Synovial macrophages were also associated with the immunoreactive IL-1 peptide. These cells appear to be the major source of IL-1 beta within the rheumatoid synovium in vivo and must be regarded as playing a central role in the chronic inflammation and joint destruction of RA.


An immunosuppressant compound, FK-506, prevents the progression of autoimmune myocarditis in rats. A new immunosuppressive compound, FK-506, is a macrolide produced by Streptomyces tsukubaensis. It is reported that FK-506 prolongs the viability of allogenic grafts of the heart and kidney in vivo and inhibits the development of autoimmune diseases. Furthermore, immunosuppressive therapy of myocarditis in humans has been given special attention by various observers; however, it is controversial. This study investigates the effects of FK-506 on experimental autoimmune myocarditis in rats. We performed two experiments. In Experiment 1, FK-506 was given intramuscularly on Days 11-20 after the first immunization. The rats were immunized twice (on Day 0 and Day 7). They were injected subcutaneously in the footpads with 1.0 mg of human cardiac myosin in equal volumes of complete Freund's adjuvant supplemented with Mycobacterium tuberculosis. They were divided into four groups: Control (six rats, saline), group 1 (six rats, FK-506: 0.1 mg/kg/day), group 2 (seven rats, FK-506: 0.32 mg/kg/day), and group 3 (six rats, FK-506: 1.0 mg/kg/day). To investigate the histologic extent of myocarditis, we formulated a histologic score (0-3). Histologic scores were: Control, 1.90 +/- 0.14; group 1, 0.97 +/- 0.46; group 2, 0.03 +/- 0.05; and group 3, 0 +/- 0. The indices of heart weight/body weight were: Control, 0.74 +/- 0.10%; group 1, 0.45 +/- 0.05%; group 2, 0.35 +/- 0.03%; and group 3, 0.35 +/- 0.03%. In Experiment 2, FK-506 was given on Days 1-10 after the first immunization, earlier than in Experiment 1. The rats were similarly divided into four groups. Each group was given the same dose of FK-506 as in Experiment 1. Histologic scores were: Control 1.49 +/- 0.24; group 1, 1.60 +/- 0.22; group 2, 0.29 +/- 0.41; and group 3, 0.03 +/- 0.03. The indices of heart weight/body weight were: Control, 0.69 +/- 0.15%; group 1, 0.76 +/- 0.09%; group 2, 0.42 +/- 0.08%; and group 3, 0.37 +/- 0.03%. Accordingly, in Experiments 1 and 2, the effects of FK-506 on autoimmune myocarditis were dose-dependent. On the other hand, in Experiments 1 and 2, not only in the control group but also in all treated groups, the titers of anti-myosin IgG were high. In conclusion, even if it is administered just before the onset of myocarditis, FK-506 is extremely effective at suppressing autoimmune myocarditis, despite a high titer of anti-myosin IgG.


Essential fatty acids and iron are involved at distinct stages of the proliferative cycle but not in the activation of human T cells. In the absence of serum, optimal lymphocyte proliferation is obtained when cultures are supplemented with transferrin and an essential fatty acid (EFA). In order to study the effects of iron in conjunction with EFA on T-cell proliferation, we have utilized a chemically defined serum-free culture system to achieve better control of the variables involved. This system includes three different serum-free media (SFM) that differ in total iron content and source of iron: (i) transferrin-free medium containing a high concentration (500 microns) of a soluble iron salt in the form of ferric citrate (Fe-SFM); (ii) iron-saturated human transferrin (5 micrograms/ml) (T-SFM); and (iii) iron-free medium (SFM(-Fe)) without any apparent source of iron. None of these SFM supported proliferation of T cells stimulated by the combination of phorbol 12,13-dibutyrate/ionomycin or phytohemagglutinin. Restoration of the proliferative response was only observed following supplementation of the iron-containing media with linoleic acid (complexed to bovine serum albumin (LA/BSA)). In cultures containing LA/BSA, the addition of iron alone in the absence of transferrin (Fe-SFM) resulted in similar responses to the transferrin-containing medium (T-SFM). Low levels of RNA synthesis in mitogen-stimulated T cells could be demonstrated in the presence or absence of iron and the addition of LA/BSA resulted in marked enhancement of RNA synthesis, regardless of the availability of iron. Cell cycle analysis showed that 91-94% of the cells cultured in SFM were arrested in G0/G1. These cells could progress through the cell cycle following the addition of LA/BSA, but only in the iron-containing media. Unlike DNA or RNA synthesis, activation of T cells could be demonstrated in SFM with or without iron as shown by the normal induction of c-fos and early growth response gene mRNA, normal expression of IL2 and transferrin receptors, and normal IL2 production, despite the arrest of cells in G0/G1. These results suggest that although human T-cell growth is iron and EFA dependent, the early events of T-cell activation are both iron and EFA independent.


Effects of methotrexate on rat liver regeneration after partial hepatectomy. 1. Methotrexate was administered immediately after partial (70%) hepatectomy, resulting in complete inhibition of dihydrofolate reductase in 24 h-regenerating liver. 2. At 48 h and 72 h after partial hepatectomy, thymidylate synthase activity was increased, whereas thymidine kinase was inhibited, by the injection of methotrexate. The DNA and RNA contents and the liver weight were also reduced in methotrexate-treated rats. 3. The immunoblotting assay showed that methotrexate stimulated the synthesis of thymidylate synthase protein in 48 h-regenerating liver. At the same time, thymidylate synthase activity was directly inhibited by methotrexate. The mechanisms of inhibition of these enzymes by methotrexate appeared to be different.


Molecular biology of islet amyloid polypeptide. We investigated the relationship between non-insulin-dependent diabetes mellitus (NIDDM) and islet amyloid polypeptide (IAPP) gene by restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-direct sequencing analysis. Endonuclease BglII and/or PvuII RFLP analysis revealed no positive correlation of IAPP gene with NIDDM. In PCR-direct sequencing of 25 NIDDM patients, no nucleotide sequence differences were found. These data do not support the view that IAPP plays an important role in the pathogenesis of NIDDM. cDNAs encoding cat, rat, mouse, guinea pig and degu IAPP precursors were also cloned, and comparison of these predicted amino acid sequences clarified the species difference, especially between amyloid-forming and non-amyloid-forming species. Amino acid residues 25-28 of mature IAPP might be responsible for their amyloidogeneity. The alternative splicing transcripts of guinea pig IAPP gene were identified by using PCR. If these types of transcripts are translated, N-terminal mutated IAPP might be produced and act as an antagonist. The signal peptide cleavage site of rat IAPP precursor was also identified by an in vitro translation and processing system.


Noninvasive mapping of muscle representations in human motor cortex. We used transcranial magnetic stimulation to map the cortical representations of 4 upper extremity muscles (abductor pollicis brevis, flexor carpi radialis, biceps, and deltoid) of 10 normal subjects. Three stimuli were delivered to scalp positions 1 cm apart, and the amplitude and latency of the motor evoked potentials (MEPs) were averaged for each position. Maps were described in terms of number of excitable scalp positions, amplitude of MEPs, scalp positions for evoking largest amplitude MEPs, and threshold for producing MEPs. We compared different muscles across subjects and the same muscles on the left and right sides in individual subjects. Distal muscles had larger representations with higher amplitude MEPs and lower thresholds. Biceps and deltoid on the left had larger representations and higher MEP amplitudes than on the right. Maps showed a somatotopic progression on the scalp of proximal to distal muscles along a posteromedial to anterolateral axis.


Magnetic brain stimulation with a double coil: the importance of coil orientation. The human motor cortex can be excited by currents induced by a transient magnetic field generated in a coil over the scalp. A 9 cm mean diameter circular coil centered at the vertex is optimally placed for exciting the hand area. Anticlockwise current flow in the coil preferentially excites the left hemisphere and vice versa. A double coil has been used to investigate the orientation of inducing currents at which activation of cortical neural elements is maximal. The inducing current flowed in the same direction in the central segment of the coil and followed a monophasic wave form. The coil was rotated through 360 degrees over the motor area in increments of 45 degrees and compound muscle action potentials from the first dorsal interosseous muscle were recorded. The largest responses were obtained with the coil at about 50 degrees to the parasagittal plane with a backward flowing inducing current. The optimal angle did not depend on stimulus intensity or background voluntary contraction. This orientation corresponds to an maximal induced current flowing forwards approximately at right angles to the central sulcus. It is postulated that horizontal neural elements are aligned in this direction and are preferentially excited by these monophasic magnetic stimuli. The results have important implications for mapping the motor areas with magnetic stimulators.


Relevance of stimulus duration for activation of motor and sensory fibers: implications for the study of H-reflexes and magnetic stimulation. Electric stimuli with durations of 0.5-1.0 msec are optimal for studies of H-reflexes. It is more difficult to obtain H-reflexes with shorter duration stimuli or with magnetic stimulation. In order to understand this behavior, we studied the excitation thresholds for motor and sensory fibers in the ulnar, median and tibial nerves using both electric and magnetic stimulation. For short duration electrical stimuli (0.1 msec) the threshold for motor fibers is lower than for sensory fibers. For longer duration electric stimuli (1.0 msec) the threshold for sensory fibers is lower. For magnetic stimulation the threshold for motor fibers is much lower than for sensory fibers. Thus, stimulus duration is a critical parameter for sensory fiber excitation, and current magnetic stimulators are not optimal.


Jitter measurement by axonal micro-stimulation. Guidelines and technical notes. Single fiber EMG (SFEMG) with axonal micro-stimulation is a convenient method to study the neuromuscular jitter at the individual motor end-plates. Compared to the original method of jitter measurement in voluntarily activated muscle, it has the advantage of perfect control of the discharge rate, including pauses in activity, useful in quantitative estimation of the neuromuscular transmission defect. It obviates the need to search for muscle fiber pairs. It can be used in young children and in uncooperative patients, as well as those with impaired voluntary motor control. It is useful in animal experiments as well as veterinary medicine. The technique eliminates the possibility of overestimating the jitter due to unrecognized interdischarge interval dependent jitter, as well as that of underestimating it due to unrecognized low jitter in split muscle fibers. The technique has certain pitfalls causing under- or overestimation. The paper gives practical guidelines and hints as to how to avoid some of these, particularly errors due to overlooked threshold stimulation and to unrecognized direct muscle fiber stimulation.


Motor evoked potentials: a new method of controlled facilitation using quantitative surface EMG. A new method of assessing MEP facilitation using surface EMG under computer control is described. Transcranial magnetic stimulation with voluntary contraction at a value between 2 and 6% of maximal surface EMG activity produced responses with shortest latencies and largest amplitudes and power. No significant changes occurred when facilitation increased beyond this level. These results are similar to previously published studies using a force transducer, confirming the reliability of the system as an alternative method of monitoring voluntary contraction. The controlling system may potentially provide an easy, flexible, quantitative method of ensuring adequate, reproducible facilitation with minimum EMG interference for testing in a clinical setting.


Normal conduction in pathways traversing an asymptomatic multiple sclerosis plaque. A 31-year-old woman developed right facial myokymia as the initial manifestation of multiple sclerosis (MS). An MRI scan revealed a focal signal abnormality confined to the left dorsolateral pontomedullary region. Brain-stem auditory evoked potentials (BAEPs), somatosensory evoked potentials (SEPs), and blink reflex (BR) failed to show a conduction abnormality through the left brain-stem lesion. Instead, BAEP and BR indicated a conduction defect in the right pons and EMG showed myokymic discharges in right facial muscles. Our findings provide rare documentation of normal conduction through a presumably asymptomatic MS plaque. The abnormal MRI signal likely represents tissue edema, rather than demyelination. This case demonstrates that physical findings in MS patients may correlate better with electrophysiological abnormalities than with MRI abnormalities.


Effects of fatigue on the stretch reflex in a human muscle. The effects of fatigue on the electromyographic (EMG) reflex activities were compared during sustained voluntary contractions and contractions evoked by electrical stimulation (30 Hz) in the human first dorsal interosseus (FDI). Short latency (SL), medium latency (ML) and long latency (LL) reflex responses to a ramp-and-hold stretch of the muscle were recorded and analysed in 27 healthy subjects of both sexes. The amplitude of the reflex components was normalized as function of the amplitude of the surface action potential (SAP) recorded in response to the supramaximal stimulation of the motor nerve. The results indicate that for a similar reduction of force, SL and ML are significantly reduced after fatigue induced by voluntary contractions but they are not when the fatigue test is performed by electrical stimulation at the motor point. In voluntary fatigue experiments, the LL component showed no significant decrease below control values, but an enhancement was observed during electrically evoked contraction. This enhancement remained above control values for at least 15 min during the recovery period, whereas SL and ML decreases returned to control within 5 min after the fatigue tests. The electrical stimulation applied to the skin overlying the FDI at an intensity lower than the motor threshold did not affect SL and ML, but enhanced LL for about 15 min. On the contrary, the anaesthesia of the skin overlying the FDI induced a decrease in LL without significant change of SL and ML. It is concluded that muscle reflex fatigue is present during sustained voluntary contractions and decreases SL and ML responses to quick stretches.(ABSTRACT TRUNCATED AT 250 WORDS)


Physiological analysis of motor reorganization following lower limb amputation. It is now known that amputation results in reorganization of central motor pathways, but the mechanism for the changes is unclear. One possibility is alteration of the excitability of the alpha motoneurons. We studied motor reorganization and excitability of alpha motoneurons to Ia input in 6 subjects with unilateral lower limb amputation. A Cadwell MES-10 stimulator was used to deliver transcranial magnetic stimuli through a circular coil centered on the sagittal axis 4 cm anterior to Cz and through an 8-shaped coil positioned over scalp locations 1 cm apart along the coronal axis. Surface EMG was recorded bilaterally from quadriceps femoris, the first muscle immediately proximal to the site of amputation. Excitability of the spinal alpha motoneuron pool to Ia afferents was assessed by determining the ratio of the maximal H reflex to the maximal M response (H/M ratio) elicited in the quadriceps femoris. Stimuli of equal intensity delivered to optimal scalp positions recruited a larger percentage of the alpha motoneuron pool in muscles ipsilateral to the stump than in those contralateral to the stump (P less than 0.01). Mean onset latencies of motor evoked potentials were shorter in ipsilateral muscles than in contralateral muscles (P less than 0.01). Muscles ipsilateral to the stump showed a trend toward activation from a larger number of scalp positions than those contralateral to the stump (P = 0.06). There was no difference in the quadriceps H/M ratios (7.2% ipsilateral vs. 10.9% contralateral). The absence of changes in the excitability of the alpha motoneuron pool in the presence of motor reorganization targeting muscles proximal to the stump suggests that reorganization occurs proximal to the alpha motoneuron level.


The effects of cooling supplementary motor area and midline cerebral cortex on neuronal responses in area 4 of monkeys. It has been postulated that the supplementary motor area (SMA) is involved in the initiation of movement and in gating of afferent input to motor cortex (MI) from peripheral receptors. We studied the responses of 119 neurons in MI to imposed disturbances of wrist-movement performance generated by the introduction of torque pulses before, during and after localized cooling of the SMA in conscious monkeys. The cooling of SMA did not prevent monkeys from making these simple movements. Eighty-two neurons responded to the wrist perturbations. Only 7 of these neurons changed their responsiveness with unilateral or bilateral cooling of SMA. From the data we have obtained on MI neuronal firing patterns, the SMA does not appear to modulate the long-latency trans-cortical stretch reflex during the periods in a task that we have investigated. Nor does it prevent animals from performing these simple movements to a visual target.


Slow positive dorsal cord potentials activated by heterosegmental stimuli. Heterosegmental slow positive waves (HSPs) and segmental spinal cord potentials were recorded from the cord dorsum in ketamine-anesthetized rats. Forepaw stimulation produced HSPs in the lumbo-sacral enlargement (lumbar HSPs), whereas hind paw stimulation evoked HSPs in the cervical cord (cervical HSP). Both the HSP and the secondary component of the slow positive wave (P2s) in the segmental spinal cord potential were highly vulnerable to anesthetics and completely disappeared after spinal cord transection at the C1/2 level, indicating that both the HSP and P2s are produced by a long feedback loop via supraspinal structures. The lumbar HSP evoked by forepaw stimulation was maximal in amplitude at the L5 level and more dominant in the ipsilateral cord dorsum than in the contralateral one, but widely distributed in the lumbo-sacral cord. A variability of onset (7-18 msec for cervical and 5-17 msec for lumbar HSPs) and peak (22-35 msec for cervical and 12-50 msec for lumbar HSPs) suggests the existence of several nuclei to form the feedback loops for descending impulses to produce the HSPs. There were no peak latency differences between the HSPs and P2s. Since there were several similar characteristics between the P2s and HSP such as a high vulnerability to anesthetic, a complete disappearance after high spinal transection and similar response curves to graded intensities of stimulation, there may be a close relationship between their feedback nuclei and the pathways mediating them. All wide dynamic range (WDR) neurons (12/12) in lamina V of Rexed responded to heterosegmental stimulation with inhibition of firing.(ABSTRACT TRUNCATED AT 250 WORDS)


Topographic mapping of the human motor cortex with magnetic stimulation: factors affecting accuracy and reproducibility. We recorded motor evoked potentials (MEPs) from deltoid, biceps brachii, abductor pollicis brevis and flexor carpi radialis muscles of 5 normal volunteers during transcranial magnetic stimulation. With the subjects at rest, an 8-shaped magnetic coil was used to deliver 30 stimuli to different scalp positions 0.5 or 1.0 cm apart. The variability in amplitude and latency of MEPs was studied as a function of the scalp position stimulated, the number of stimuli at each position, and the percentage of maximal peripheral M responses (%M) elicited. The results were used to estimate the optimal number of stimuli at each position and the optimal spacing of scalp positions for topographic mapping of the human motor cortex. The amplitude and latency variability of MEPs were higher when suboptimal scalp positions were stimulated. Consequently, a larger number of stimuli were required to determine representative MEP amplitudes at suboptimal positions. In addition, there was an inverse relationship between %M recruited by transcranial magnetic stimuli in different subjects and the variability in MEP amplitude and latency. Latency variability was less pronounced than amplitude variability. Optimal sampling conditions are required to produce the best topographic maps, particularly to show subtle reorganization patterns in the human motor cortex.


Cloning and expression of a human ATP-citrate lyase cDNA. A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.


The nucleotide and partial amino acid sequences of rat fetuin. Identity with the natural tyrosine kinase inhibitor of the rat insulin receptor. Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full-length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin receptor tyrosine kinase in vitro. Therefore, we conclude that RF619 and pp63 cDNA encode the same protein, i.e. authentic rat fetuin which is a functional tyrosine kinase inhibitor.


A membrane-bound metallo-endopeptidase from rat kidney. Characteristics of its hydrolysis of peptide hormones and neuropeptides. A membrane-bound metallo-endopeptidase that hydrolyzes human parathyroid hormone (1-84) and reduced hen egg lysozyme between hydrophilic amino acid residues was isolated from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563-571]. In this study, the hydrolyses of various peptide hormones and neuropeptides by the metallo-endopeptidase were examined using an automated gas-phase protein sequencer. The purified enzyme hydrolyzed the oxidized insulin B chain and substance P most rapidly, followed by big endothelin 1, neurotensin, angiotensin 1, endothelin 1, rat alpha-atrial natriuretic peptide and bradykinin, in this order. The enzyme mainly cleaved these peptides at bonds involving a hydrophilic amino acid residue. However, it cleaved bonds between less hydrophilic amino acid pairs in several short peptides, e.g. at the His5-Leu6 bond in oxidized insulin B chain, the Ile28-Val29 bond in big endothelin-1 and the Ile5-His6 and Phe8-His9 bonds in angiotensin 1. The enzyme cleavage sites of oxidized insulin B chain and angiotensin 1 were different from the reported sites cleaved by meprin and by endopeptidase 2, respectively. Kinetic determination of bradykinin hydrolysis by the purified enzyme yielded values of Km = 18.1 microM and kcat = 0.473 s-1, giving a ratio of kcat/Km = 2.62 x 10(4) s-1.M-1. The Km value was about 20-fold lower than that reported for meprin and endopeptidase 2. These results indicate that the membrane-bound metallo-endopeptidase from rat kidney is distinguished from meprin and endopeptidase 2 in its substrate specificity and is not parathyroid hormone specific, but has potential capacities to inactivate various biologically active peptide hormones and neuropeptides in vivo.


Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis. Lactococcus lactis strains produce an extracellular subtilisin-related serine proteinase in which immunologically different components can be distinguished. Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified. By Western-blot analysis it was found that all monoclonal antibodies recognize all denatured proteinase components. The distinction between the different components could be made under native conditions only, indicating that binding regions are masked in the native molecule. In a L. lactis proteinase which was inactivated by the substitution Asp30----Asn under native conditions, only one epitope could be detected. This demonstrates that autoproteolytic activity is required to make specific binding regions accessible for (monoclonal) antibodies.


Molecular cloning and characterization of a constitutively expressed heat-shock-cognate hsc71 gene from rainbow trout. A rainbow trout major heat-shock-protein-like gene (hsp 70) and corresponding cDNA clones were isolated by hybridization to heterologous hsp70 probes. DNA sequencing revealed that this gene is structurally similar to a mammalian heat-shock-cognate hsc70 gene and consists of eight introns. Northern blot and primer extension analyses showed that the corresponding mRNA is constitutively abundant in different trout tissues and salmonid cell lines. Fragments of the isolated gene containing the -900 - +30 and -217 - +58 sequence were linked to a bacterial chloramphenicol acetyltransferase reporter gene and transiently transfected into salmonid cells. The expression pattern of these constructs supports our conclusion that the isolated genomic and cDNA clones correspond to a trout heat-shock-cognate hsc70 gene.


Effects of brain-gut related peptides on cAMP levels in myenteric ganglia of guinea-pig small intestine. This study was designed to test the hypothesis that stimulation of adenylate cyclase and elevation of cAMP is involved in the signal transduction process for substance P, calcitonin gene-related peptide, vasoactive intestinal peptide, cholecystokinin or gastrin releasing peptide in myenteric ganglia. Enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine were used to study changes in levels of cAMP in response to application of the brain-gut peptides in the presence and absence of forskolin. Application of substance P and calcitonin gene-related peptide were found to increase intraganglionic cAMP in a dose-dependent fashion when a phosphodiesterase inhibitor was present. The ED50 values for substance P and calcitonin gene-related peptide were 5 microM and 0.75 microM, respectively. The presence of forskolin in the incubation medium resulted in significant upward shifts of the dose-response curves for both peptides. Neither vasoactive intestinal peptide, cholecystokinin nor gastrin releasing peptide stimulated increases in intraganglionic cAMP under the same experimental conditions used for substance P and calcitonin gene-related peptide.


Synthesis and secretion of alpha 2-macroglobulin by human glioma established cell lines. Human alpha 2-macroglobulin (alpha 2M) is a high molecular weight plasma proteinase inhibitor exhibiting a broad specificity; in fact it is capable of binding endopeptidases from all known classes of proteases (Barret 1981). Two human glioma cell lines, namely an astrocytoma and a glioblastoma, were found to synthesize and secrete in the culture medium a protein which resembles the serum alpha 2M for immunological, biochemical and biological features. Using polyclonal antibodies to serum alpha 2M, an alpha 2M-like factor could be detected in the cytoplasm and in the culture medium of the tumor cells. Furthermore this factor accumulated in cytoplasmic granules if cells were incubated with monensin and its production was dramatically reduced following a treatment with cycloheximide. This protein behaved like the serum alpha 2M in immunoblotting analysis and exhibited the same antiproteolytic activity. Its role in human brain is unknown at present. Since interactions of proteinases and proteinase-inhibitors appear to influence the host-tumor immune response and to play a crucial role during the migration of metastasizing tumor cells, alpha 2M expression observed in these glioma cells could be involved in tumor cell proliferation and invasion.


Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death. The ultrastructural changes in the pyramidal neurons of the CA1 region of the hippocampus were studied 6 h, 24 h, 48 h, and 72 h following a transient 10 min period of cerebral ischemia induced by common carotid occlusion combined with hypotension. The pyramidal neurons showed delayed neuronal death (DND), i.e. at 24 h and 48 h postischemia few structural alterations were noted in the light microscope, while at 72 h extensive neuronal degeneration was apparent. The most prominent early ultrastructural changes were polysome disaggregation, and the appearance of electron-dense fluffy dark material associated with tubular saccules. Mitochondria and nuclear elements appeared intact until frank neuronal degeneration. The dark material accumulated with extended periods of recirculation in soma and in the main trunks of proximal dendrites, often beneath the plasma membrane, less frequently in the distal dendrites and seldom in spines. Protein synthesis inhibitors (anisomycin, cycloheximide) and an RNA synthesis inhibitor (actinomycin D), administered by intrahippocampal injections or subcutaneously, did not mitigate neuronal damage. Therefore, DND is probably not apoptosis or a form of programmed cell death. We propose that the dark material accumulating in the postischemic period represents protein complexes, possibly aggregates of proteins or internalized plasma membrane fragments, which may disrupt vital cellular structure and functions, leading to cell death.


Angiotensin converting enzyme inhibition prevents development of muscle and nerve dysfunction and stimulates angiogenesis in streptozotocin-diabetic rats. The effects of the angiotensin converting enzyme inhibitor lisinopril on slow and fast twitch muscle contractile properties, nerve conduction and hypoxic resistance, and muscle and nerve capillary density were examined in streptozotocin-diabetic rats. Prolongation of soleus contraction and relaxation were partially prevented by treatment (p less than 0.01). A 22% deficit in fast twitch extensor digitorum longus tetanic tension production was also ameliorated (p less than 0.01). Sciatic motor and sensory conduction velocity, 25% and 12% reduced by diabetes respectively, were 75% normalized by lisinopril (p less than 0.01). There was a 47% increase in resistance to hypoxic conduction block with diabetes (p less than 0.01). Lisinopril treatment resulted in normal hypoxic resistance. Capillarization of nerve and muscle was little affected by diabetes; however, there was a 17% increase in capillary density in sciatic nerve, and a 40% increase in extensor digitorum longus muscle with lisinopril (p less than 0.01). For soleus, a smaller treatment-induced increase in capillary density led to an elevated capillary/muscle fibre ratio (p less than 0.01). These results suggest that lisinopril promoted angiogenesis. It was concluded that the beneficial effect of preventive lisinopril treatment is likely to depend upon a reduction of peripheral vascular resistance and improvement of tissue blood flow, which implicates relative hypoxia as an important factor in the development of myopathy and neuropathy in experimental diabetes.


Molecular genetic analysis of the mldr mouse: a spontaneous revertant at the mld locus containing a recombinant myelin basic protein gene. The mld mutation is a complex genetic lesion affecting the myelin basic protein (MBP) locus in the mouse. The mutation consists of a variety of DNA rearrangements including: tandem duplication of the MBP structural gene, partial inversion of the 3' end of the upstream gene copy, duplication of a region flanking the rearrangement junction in the upstream copy and insertion between the two gene copies of a segment of extraneous DNA not associated with the wild-type MBP locus. The net result of the mutation is a dysfunctional MBP locus. Homozygous mld/mld mice produce very little MBP and consequently very little myelin. They exhibit a clinical phenotype characteristic of hypomyelination (shaking, convulsions). We have discovered a revertant mld mouse which does not exhibit clinical symptoms of hypomyelination. Genetic analysis indicates that the reversion is allelic to mld. We have designated the revertant locus mldr. Restriction analysis of mldr genomic DNA indicates that there is a single intact MBP gene. Analysis of various junction regions using the polymerase chain reaction indicates that the single MBP gene in mldr is derived by recombination from the 5' end of the upstream gene and the 3' end of the downstream gene. Studies on MBP expression in mldr mice indicate that the developmental regulation, level of expression and pattern of post-transcriptional processing of MBP gene products in mldr are similar to wild type. These results indicate that the recombinant MBP gene in mldr is fully functional. From this we infer that the MBP-deficient phenotype of the original mld mutant is attributable to the complex rearrangements in the upstream gene copy which render the locus dysfunctional.(ABSTRACT TRUNCATED AT 250 WORDS)


Synthetic human tRNA(UUULys3) and natural bovine tRNA(UUULys3) interact with HIV-1 reverse transcriptase and serve as specific primers for retroviral cDNA synthesis. Full-length and 5'-truncated variants of human (h) tRNA(UUULys3) were synthesized by in vitro transcription using SP6 RNA polymerase. Bovine(b) tRNA(SUULys3) was purified from calf liver. Both full-length tRNA species were shown to be biologically active in an aminoacylation assay. Gel retardation assays revealed that both full-length tRNA species, as well as a 5'-truncated h-tRNA(UUULys3) molecule containing 24 nucleotides (nt) at the 3' end (Lys24), interact with human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Competition studies with these three tRNA species demonstrate that the 3' end of h-tRNA(UUULys3) contributes to the interaction with HIV-1 RT. Escherichia coli tRNA(UUULys) and tRNA(UUCGlu2) were also able to interact with the enzyme, whereas unrelated RNA molecules such as E. coli 5S rRNA did not bind to RT. Both b-tRNA(SUULys3) and h-tRNA(UUULys3) molecules, as well as the 5'-truncated variants, could be demonstrated to prime cDNA synthesis specifically using a HIV-1 RNA template, prepared by in vitro transcription, indicating that other viral or cellular proteins are not essential for this process. E. coli tRNA(UUULys) and tRNA(UUCGlu2), although able to interact with HIV-1 RT, failed to prime retroviral transcription. Products of cDNA synthesis were characterized by polymerase chain reaction, demonstrating that at least 18 nt at the 3' ends of h-tRNA(UUULys3) and b-tRNA(SUULys3) are still present in the cDNA product, whereas the 5' ends of both primer molecules were removed by the RNase H activity of HIV-1 RT.


Studies of gut mucosal protein synthesis in a non-steroidal anti-inflammatory drug (NSAID) model of inflammatory bowel disease. The extent to which defects in protein synthesis occurred in an experimental indomethacin induced rat model of nonsteroidal enteropathy has been examined. Male rats (nine) were fed indomethacin (8 mg/kg/day) for three days mixed with a powdered form of chow. The control group of rats (nine) were fed the same diet for three days without indomethacin. After the feeding period, both groups were fed a normal solid diet for four days. At the end of this period, the fractional rates of intestinal protein synthesis was determined by the 'flooding dose' technique. The mucosal protein, RNA and DNA contents in the proximal ileum of animals with enteropathy were not significantly different from controls (p greater than 0.05). Experimental enteropathy induced selective increases in the fractional rates of protein synthesis (26% increase, p less than 0.03) and RNA activities (23% increase, p less than 0.04). There were no significant changes in any of these variables in the duodenum (p greater than 0.05 in all instances). These changes may partly reflect the activity of those processes responsible for the pathogenic changes in NSAID enteropathy.


Aggressive extracorporeal shock wave lithotripsy of gall bladder stones within wider treatment criteria: fragmentation rate and early results. Two hundred and twenty patients with a total of 412 gall bladder stones of between 8 and 38 mm in size were treated with extracorporeal shock wave lithotripsy, using the overhead module Lithostrar Plus. Fifty six per cent of stones were solitary (mean (SD) diameter 23 (5) mm) and 9.5% of the patients had more than three stones. Stones were successfully disintegrated in 218 patients (fragmentation size less than 5 mm in 80%, less than 10 mm in 19%). Some 65% of patients required one treatment and the rest two or three. A mean (SD) of 4100 (1800) shock waves with a pressure of 700 bar were applied. Twenty four to 48 hours after lithotripsy a transient but significant increase in serum transaminase activities (31%) and in bilirubin (29%), urinary amylase (27%), and blood leukocyte (62%) values was observed. In 29% of patients there was a transient microhaematuria, in 2% transient macrohaematuria, and in 25% painless petechiae of the skin. Ultrasound showed temporary gall bladder wall oedema in 13%, temporary distension of the gall bladder in 11%, and transient common bile duct distension in 8% after treatment. After discharge from hospital, 31% of patients complained of recurrent colic that responded to simple analgesics. Four to eight weeks after therapy, four patients developed biliary pancreatitis and 11 biliary obstruction that was managed by endoscopy. To date, 105 patients have been followed for over 12 months. Sixty one of these had a solitary stone, 17 had two, and 27 had three or more stones. A total of 59 patients, including 44 with a primary solitary stone, eight with two stones, and seven with three or more stones are completely stone free.


High conductance anion channel in Schwann cell vesicles from rat spinal roots. Potassium uptake, possibly together with chloride, is one of the presumed functions of Schwann cells in the peripheral nervous system. However, the presence of chloride channels has not been demonstrated in adult Schwann cells. We present here a new method which allows single channel recordings to be made from Schwann cells in situ without enzymatic treatment. Isolated rat spinal roots were split mechanically into several bundles. Within about 30 min after this procedure small bleb-like vesicles (approximately 20-30 microns in diameter) with a clean surface appeared at the edges of the fibre bundles. Immunofluorescence microscopy with a surface marker for Schwann cell membranes (monoclonal antibody O4) revealed that the vesicles originate from Schwann cells. In standard patch clamp recordings with symmetrical bath and pipette solutions (excised inside-out configuration) an anion channel with the following characteristics was mainly observed: 1) single channel slope conductance of 337 +/- 5 pS in 125 mM KCl and 209 +/- 6 pS in 125 mM K+ methylsulphate; 2) ion permeability ratio: PCl/PK/Pgluconate = 1/0.12/0.06; 3) linear current-voltage relationship (range +/- 60 mV); and 4) voltage- and time-dependent inactivation (the channel was most active at potentials +/- 20 mV). Pharmacologically, the channel was completely blocked with zinc (1 mM) and barium (10 mM). A similar anion channel, showing characteristics 1-4), has been described in cultured Schwann cells of newborn rats (Gray et al., 1984). We now demonstrate that this channel is also present in adult Schwann cells in situ.


Binding of monoclonal antibody specific for domain Ia/II of Pseudomonas aeruginosa exotoxin A at pH 4 strongly neutralizes exotoxin A-induced cytotoxicity in cell culture and in vivo. Mouse monoclonal antibodies (MAbs) against Pseudomonas aeruginosa exotoxin A (Ex-A) were established, and 4 of 20 MAbs were extensively studied for analysis of the structure-function relationship of Ex-A. IN vivo experiments demonstrated that MAb Ex-3C7 protected mice either injected with Ex-A or infected with Ex-A-producing P. aeruginosa from death caused by Ex-A at the highest rate, followed by MAbs Ex-4F2 and Ex-8H5, in that order. MAb Ex-2A10 failed to rescue the mice. MAb Ex-3C7 (immunoglobulin G1 [IgG1]) inhibited incorporation of Ex-A into target cells and strongly neutralized cytotoxicity in cell culture but did not inhibit an enzymatic activity of Ex-A, ADP-ribosyltransferase, at all. The MAb also bound Ex-A, even at a low pH of 4, and recognized amino acid residues 241 to 297 (domain Ia/II), suggesting that MAb Ex-3C7 can interfere with the conformational change and/or processing of Ex-A by keeping a complex of Ex-A and antibody stable at low pH in the phagolysosome. MAb Ex-4F2 (IgG1), which recognizes residues 550 to 590 (domain III), strongly inhibited Ex-A incorporation and neutralized cytotoxicity in cell culture but only weakly inhibited ADP-ribosyltransferase. MAb Ex-8H5 (IgG1), which recognizes residues 591 to 613 (domain III), also inhibited cytotoxicity in cell culture, but weakly. In contrast to the above three MAbs, MAb Ex-2A10 (IgG2b) greatly inhibited ADP-ribosyltransferase but showed no inhibition of Ex-A incorporation and no neutralizing activity against cell toxicity. A line of evidence indicates that (i) domain Ia/II plays an important role in the pathogenesis of Ex-A and (ii) MAbs that inhibit an intracellular postbinding process, such as conformational change, processing, and translocation of Ex-A in target cells, can display potent inhibitory activity against cytotoxicity in vivo, as well as in cell culture, and would be a good candidate for therapy of pseudomonal infections.


Longitudinal study of Plasmodium falciparum polymorphic antigens in a malaria-endemic population. Plasmodium falciparum merozoite surface antigens MSP1 and MSP2 and an exported antigen, Exp-1, exhibit allelic polymorphism in natural populations. To explain this, one hypothesis is that antigen polymorphisms are maintained by frequency-dependent immune selection. An expectation of the hypothesis is that rare variants have an advantage over common variants because of a lower level of acquired immunity against them and thus increase in frequency until an equilibrium is attained. To test this hypothesis, the frequencies of polymorphic epitopes of MSP1, MSP2, and Exp-1 were determined among isolates from malaria patients in an urban area of The Gambia, during different periods of one transmission season (1988) and in different years (1982, 1983, 1988, and 1989). The frequencies remained very stable throughout the period of study, alternative epitope variants remaining either rare or common, without shifts in relative frequencies. These results are discussed with reference to the immune-selection hypothesis, with the conclusion that frequencies of the major dimorphic serological classes of MSP1 are probably not maintained by immune selection.


Cloning and expression of the gene for the Avi-3 antigen of Mycobacterium avium and mapping of its epitopes. The Avi-3 antigen, which is found only in Mycobacterium avium culture sonic extracts, is species specific and results in strong skin test activity in guinea pigs sensitized with heat-killed M. avium. Its gene was cloned by using a previously developed single-probe method and was sequenced. The gene encoded a 194-amino-acid polypeptide with a molecular weight of 21,500. A recombinant Avi-3 antigen expressed in Escherichia coli reacted with monoclonal and polyclonal antibodies raised against the native Avi-3 antigen. To identify epitopes on this protein for immunodiagnostic purposes, various parts of the Avi-3 antigen were expressed as beta-galactosidase fusion proteins, using pUR and pURS expression vectors. The clones screened by both antibody reactivity and T-cell proliferative activity defined fragments with coexisting B- and T-cell epitopes. A B-cell epitope (Asn-176 to Ala-186) and two T-cell epitopes (Glu-75 to Ile-86 and Arg-155 to Leu-164) were thus defined. The synthetic polymerized peptides of the T-cell epitopes were proven to elicit a delayed cutaneous hypersensitivity reaction in guinea pigs. This mapping method would be useful in the development of a subunit vaccine consisting of an immunodominant B-cell epitope linked to a T-cell epitope in the vicinity.


Colonization factor antigen CFA/IV (PCF8775) of human enterotoxigenic Escherichia coli: nucleotide sequence of the CS5 determinant. Human enterotoxigenic Escherichia coli isolates expressing the colonization factor antigen CFA/IV (previously designated PCF8775) produce plasmid-encoded CS5 fimbriae. The nucleotide sequence of the region encoding the major CS5 fimbrial subunit was determined. The subunit is synthesized as a precursor of 203 amino acids (20.85 kDa) with a mature protein of 181 amino acids corresponding to a size of 18.6 kDa. The CS5 subunit shows homology to the corresponding component of porcine enterotoxigenic E. coli F41, particularly within the signal sequence and at the carboxy terminus.


Role of mycoplasma infection in the cytopathic effect induced by human immunodeficiency virus type 1 in infected cell lines. In addition to previously reported tetracycline analogs, other antibiotics known for antimycoplasmal activities inhibited the cytopathic effect in CEM cl13 cells infected with human immunodeficiency virus type 1 (HIV-1) or HIV-2 but were unable to block virus replication. A contaminating mycoplasma was isolated from our CEM cl13 cells and identified as a strain of Mycoplasma fermentans. Following infection of lymphoblastoid (CEM) or promonocytic (U937 and THP1) cell lines with HIV-1, cytopathic effect was observed only in association with mycoplasmal contamination. Moreover, HIV-1 infection of U937 cells after experimental inoculation with a human isolate of M. fermentans led to pronounced cell killing. We have verified that this effect is not merely an artifact caused by arginine and/or glucose depletion in the cell culture medium. These results confirm that mollicutes, in particular M. fermentans, are able to act synergistically with HIV-1 to kill infected cells in some in vitro systems.


Conserved outer membrane protein of Neisseria meningitidis involved in capsule expression. In Neisseria meningitidis, translocation of capsular polysaccharides to the cell surface is mediated by a transport system that fits the characteristics of ABC (ATP-binding cassette) transporters. One protein of this transport system, termed CtrA, is located in the outer membrane. By use of a CtrA-specific monoclonal antibody, we could demonstrate that CtrA occurs exclusively in N. meningitidis and not in other pathogenic or nonpathogenic Neisseria species. Nucleotide sequence comparison of the ctrA gene from different meningococcal serogroups indicated that CtrA is strongly conserved in all meningococcal serogroups, independent of the chemical composition of the capsular polysaccharide. Secondary structure analysis revealed that CtrA is anchored in the outer membrane by eight membrane-spanning amphipathic beta strands, a structure of proteins that function as porins.


Characterization of an antigenically conserved heat-modifiable major outer membrane protein of Branhamella catarrhalis. Branhamella catarrhalis is a common cause of otitis media in children and of respiratory infections in adults with chronic bronchitis. Little is known about the antigenic structure of the outer membrane proteins (OMPs). In this study, two murine monoclonal antibodies, 7D6 and 5E8, were developed and used to characterize the major heat-modifiable OMP (OMP C/D) of B. catarrhalis. Immunoblot assays indicated that OMP C/D is heat modifiable, having a molecular mass of 55 kDa at room temperature and a mass of 60 kDa when heated under reducing conditions. Expression of the epitopes is independent of growth phase and growth media. Both epitopes are present in 51 of 51 strains of B. catarrhalis tested and are highly specific for Branhamella strains, being absent from a variety of other gram-negative species. Antibody 5E8 recognizes an epitope which is expressed on the surface of the intact bacterium. We conclude that OMP C/D is a major, heat-modifiable OMP antigen that expresses at least one stable, conserved epitope on the surface of B. catarrhalis. Future studies should focus on the role of OMP C/D in pathogenesis and on its potential role as a vaccine antigen.


Identification and characterization of lipopolysaccharide-binding proteins on human peripheral blood cell populations. Previous research in this laboratory, using photoactivatable radioiodinated lipopolysaccharide derivatized with sulfosuccinimidyl-2-(p-azidosalicylamide)-1,3'-dithiopropionate (125I-ASD-LPS), has resulted in the identification of a specific LPS receptor with a molecular mass of approximately 73 kDa on murine lymphocytes and splenic macrophages. The experiments presented in this report investigated whether a similar LPS-binding protein was also expressed on human peripheral blood populations, including monocytes, lymphocytes, neutrophils, platelets, and erythrocytes. Each cell population was incubated with 125I-ASD-LPS, UV irradiated, washed, reduced, and solubilized, and the cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. On all of the cell populations, except erythrocytes, a similar 73-kDa LPS-binding protein was present. In addition, each population also expressed lower-molecular-weight secondary LPS-binding proteins, some of which were conserved among the populations. Binding of the photoactivatable LPS probe was found to be both time and temperature dependent. These data support the concept that the 73-kDa LPS-binding protein is conserved on multiple cell types from a variety of species.


Plasmodium falciparum-infected erythrocytes do not adhere well to C32 melanoma cells or CD36 unless rosettes with uninfected erythrocytes are first disrupted. Plasmodium falciparum malaria parasites modify the human erythrocytes in which they grow so that some parasitized erythrocytes (PE) can cytoadhere (C+) to host vascular endothelial cells or adhere in rosettes (R+) to uninfected erythrocytes. These C+ and R+ adherence properties of PE appear to mediate much of the pathogenesis of severe malaria infections, in part by blocking blood flow in microvessels. From one parasite strain, PE were selected in vitro for C+ R+ or C+ R- adherence properties and examined in model adherence assays. The C+ R+ PE cytoadhered poorly to C32 melanoma cells or to immobilized CD36 in a settled-cell assay when uninfected human erythrocytes were present and formed rosettes with PE. C+ R- PE adhered well in the same assays. However, C+ R+ PE adhered very well, even better than C+ R- PE, when the rosettes were disrupted and the C+ R+ PE were purified. Adding back rabbit erythrocytes, which do not form rosettes with C+ R+ PE, had simply a dilutional effect. The ability of rosettes to interfere with the detection of adherence must be dealt with in all future assays of malarial PE adherence. Individual PE were observed attached simultaneously to C32 cells and to a few erythrocytes, suggesting that C+ and R+ adherence properties are coexpressed on the same PE. Coexpression of these adherence properties on the same PE may have pathological importance in vivo, where passage of rosettes through capillaries may shear uninfected erythrocytes from rosetted PE and allow direct PE attachment to postcapillary venule walls before rosettes reform.


Efficacy of copper and silver ions with iodine in the inactivation of Pseudomonas cepacia. Alternatives to chlorination of water have been sought for reasons which include trihalomethane formation, possible bacterial regrowth, the high concentrations of chlorine required in certain circumstances, and the taste, odour and bodily irritation in chlorine-treated water. Electrolytically generated Cu and Ag ions at low levels, in addition to very low chlorine concentrations, have been suggested as an alternative to routine chlorination. We have examined the combination of Cu and Ag ions with low levels of iodine. Pseudomonas cepacia was grown either in rich medium or under nutrient restriction prior to disinfection. Survival of the organism and its ability to regrow after treatment as well as the effects of varying buffers, metal ion and iodine concentrations were determined. Low concentrations of metal ions (100 ppb Cu and 11 ppb Ag) and iodine (200 ppb) were more effective than either metal ions or iodine alone against Ps. cepacia grown on rich agar or in low nutrient buffer. After iodination, buffer-grown suspensions recovered to their original cell concentrations within 7 d. When Cu and Ag ions were used with or without iodine, regrowth was prevented. The results show that low concentrations of Cu and Ag in combination with iodine permit effective disinfection of bacteria after cultivation on either rich media or under nutrient restriction. These results, along with published data, suggest that the combination of these metals with halogenation may have applications in the disinfection of both recreational and potable water.


Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats. The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats.


Preserved endothelium-dependent vasodilation at the vasospastic site in patients with variant angina. Endothelial dysfunction has been implicated as a cause of coronary vasospasm in patients with variant angina. This study aimed to determine if endothelium-dependent vasodilation evoked with substance P (SP) was altered at the spastic site where vasospasm was induced by acetylcholine (ACH) in patients with variant angina. It has been shown that SP evokes endothelium-dependent vasodilation with no direct effect on vascular smooth muscle in excised human coronary arteries. SP and ACH were infused into the coronary arteries in nine patients with variant angina in whom coronary arteriograms showed normal or mild atherosclerotic lesions. The vasomotor responses of coronary arteries were assessed by quantitative arteriography. ACH at a high dose (100 micrograms/min) provoked coronary vasospasm associated with anginal attack in all patients. In contrast, SP at graded doses (13.5, 40, and 135 ng/min) caused the dose-dependent and comparable increases in the coronary diameter at the spastic and control sites. ACH at a low dose (10 micrograms/min) also caused comparable vasodilation at the spastic and control sites in patients with normal coronary arteries. Coronary vasodilating responses to SP were comparable in patients with variant angina and those with atypical chest pain. The results indicate that endothelium-dependent vasodilation evoked with SP and ACH at the low dose was present at the vasospastic site in patients with variant angina. These findings suggest that the ACH-induced coronary vasospasm in patients with variant angina results from hyperreactivity of vascular smooth muscle to ACH but not from endothelial dysfunction.


Monocyte chemotactic and activating factor is a potent histamine-releasing factor for basophils. Monocyte chemotactic and activating factor (MCAF) is a recently cloned cytokine that causes chemotaxis of basophils. In our pursuit of cytokines affecting basophil function, we studied the effect of MCAF on histamine secretion from basophils. Leukocytes from 20 donors, 10 allergic and 10 normal subjects, were studied. MCAF caused dose-dependent release of histamine at concentrations of 10(-8) and 10(-7) M, and the mean release was 31.25 +/- 2.9% at the highest concentration. In the same experiments the mean histamine release by anti-IgE and histamine releasing factor (HRF) was 27.05 +/- 4% and 32.70 +/- 2.7%, respectively. All 20 subjects responded to MCAF with significant histamine release. Allergic subjects released significantly more histamine than normals in response to anti-IgE (P less than 0.01) but not to MCAF (P = 0.2) and HRF (P = 0.1). The histamine release was significantly correlated between MCAF and HRF (P less than 0.01), but not between MCAF and anti-IgE (P greater than 0.05). The histamine release by MCAF was complete within the first 3 min. MCAF-induced degranulation was a calcium-dependent process. Leukocytes depleted of monocytes responded equally well to MCAF. Using an anti-MCAF affinity column we determined that greater than 50% of HRF activity of crude PBMC supernatant could be attributed to MCAF. Thus, we established that MCAF is a potent secretagogue for basophils.


Oestrogen receptor analysis: correlation between enzyme immunoassay and immunohistochemical methods. To compare oestrogen receptor measurements on fresh tissue done by an enzyme immunoassay (ER-EIA, Abbott) and an immunohistochemical technique (ER-ICA, Abbott) using formalin fixed, paraffin was embedded material. The ER-EIA is based on a sandwich immunoassay using fresh frozen tissue, and the absorbance values were read by a Quantum Analyzer. Sections were cut from the corresponding paraffin wax blocks, and after pretreatment with pronase and DNase, the slides were incubated with monoclonal oestrogen antibody. The immunoperoxidase staining was scored semiquantitatively, incorporating both the intensity and percentage of positive staining (HSCORE). HSCORE rating of the ER-ICA slides gave a significant correlation with the quantitative ER-EIA values (r = 0.76; p less than 0.001). The overall sensitivity and specificity of the immunohistochemical method was 88% and 93%, respectively. ER-ICA on routinely processed material can be a valuable alternative when biochemical analysis of oestrogen receptor content is not available. Furthermore, immunohistochemical staining identifies oestrogen receptor positivity at a cellular level, which ensures that the analysed material is representative. This technique could therefore be particularly valuable in small tumours and in situ lesions.


Heterogeneity of vascular endothelial cells with relevance to diagnosis of vascular tumours. To determine the distribution of factor VIII related antigen, CD31, CD34 and CD36 in normal and malignant human vascular tissues using a panel of well characterised monoclonal antibodies. Frozen and fixed material from a wide range of normal tissues and routinely processed material from 43 benign and malignant vascular tumours were examined. Single immunocytochemical labelling was performed using the APAAP technique. Double staining involved the sequential use of APAAP with the peroxidase method. Human vascular endothelium was antigenically heterogeneous. One of the most restricted markers was factor VIII related antigen, despite its having been widely used in diagnostic pathology as a marker of vascular endothelium and of the tumours which arise from it. Three antibodies against factor VIII related antigen, CD31 (JC70) and CD34 (QBend 10) were identified as immunostaining routinely processed, formalin fixed, paraffin wax sections. Each antibody gave different staining when tested on a range of vascular tumours, both benign and malignant. A small panel of three reagents (factor VIII related antigen, CD31 (JC70) and CD34 (QBend 10)) should be used by diagnostic pathologists who want to show the presence of cells of endothelial origin in routine material.