Paget's disease of bone--current thinking and management. The objective of this article was to critically review the current knowledge on Paget's disease of bone, focusing primarily on possible etiologies and trends in management. Current courses of treatment are described, including calcitonin, diphosphonate and mithramycin therapy and the efficacy of each drug regime is analyzed. Finally, newer, more experimental pharmaceutical agents, such as gallium nitrate, are described. Information was obtained from English language medical and scientific journals and medical/physiology text books. Index Medicus (1983-1989) and MEDLINE computerized data base were used. Paget's disease of bone, paramyxovirus, osteoclast, calcitonin and diphosphonate were the terms indexed directly. Key authors indexed included Rebel, Baste and Paget. Further information was obtained from the National Association for the Relief of Paget's Disease (England). Current research focuses primarily on histological and hybridization studies over the past 15 yr in favor of a slow virus as the causative agent in the disease, and the evidence supporting this is analyzed in some detail. In particular, the discovery of nuclear and cytoplasmic inclusion bodies within the osteoclasts of patients with Paget's disease and their similarity to respiratory syncytial virus and measles virus are discussed. Some form of environmental or physiological trigger may be involved, but the exact mechanism remains obscure. The current treatment of Paget's disease is based around the prevention of bone resorption using various diphosphonates and calcitonins, but many of these are associated with severe side effects. Newer pharmaceutical agents to control bone turnover, including gallium nitrate, have given promising results in preliminary medical trials.


The relationship between beta-adrenoceptors and adrenergic responsiveness in trout (Oncorhynchus mykiss) and eel (Anguilla rostrata) erythrocytes. Experiments were performed in vitro specifically to elucidate the underlying mechanism(s) of the attenuated adrenergic responses of eel (Anguilla rostrata) erythrocytes. This was achieved by comparing beta-adrenoceptor numbers and affinities in addition to (i) Na+/H+ exchange activity, (ii) cell swelling and (iii) cyclic AMP formation mediated by catecholamines in eel and trout (Oncorhynchus mykiss) erythrocytes under normoxic and hypoxic conditions. Under normoxic conditions, eel erythrocytes displayed a total absence of Na+/H+ exchange activity (as determined from measurements of extracellular pH) after addition of noradrenaline (50-1000 nmol l-1) in contrast to a pronounced dose-dependent response in trout. Incubation of the blood under hypoxic conditions, to achieve approximately 50% haemoglobin O2-saturation, further increased the extent of Na+/H+ exchange activation in trout and elicited a statistically significant, although physiologically small (10% of the response in trout), activation of H+ extrusion activity in eel. Catecholamine-mediated cell swelling, although obvious in trout, was absent in eel when estimated under hypoxic conditions. Eel erythrocytes possessed approximately 50% fewer surface beta-adrenoceptors than did trout erythrocytes, although the dissociation constants (KD) of these receptors did not differ between eel and trout. The numbers and affinities of the erythrocyte beta-adrenoceptors were not significantly affected by the hypoxic incubation. Both eel and trout erythrocytes displayed a dose-dependent elevation of cyclic AMP concentration in response to noradrenaline that was further increased by hypoxia. Surprisingly, eel erythrocytes produced larger quantities of cyclic AMP despite the lower numbers of surface beta-adrenoceptors. Thus, the absence of adrenergic swelling and the attenuated H+ extrusion response in eel erythrocytes cannot be attributed to insufficient numbers of beta-adrenoceptors or to functional uncoupling of these receptors from adenylate cyclase. Instead, the differences between trout and eel may reflect differing numbers of Na+/H+ exchangers or fundamental differences in the manner by which these exchangers are activated by cyclic AMP.


Sequence analysis of the large (L) protein of simian virus 5. The complete nucleotide sequence of the large (L) protein gene of simian virus 5 (SV5) was determined from cDNA of the genomic RNA and mRNA, and found to be 6804 bases in length, exclusive of a poly(A) tract. The sequence contained an open reading frame of 6765 nucleotides encoding 2255 amino acids. Results of dot matrix comparisons of the L protein of SV5 with those of human parainfluenza type 3 virus and Sendai virus indicated that there are five conserved domains, and that each domain contains characteristic sequence(s). The L protein of SV5 was detected in purified virions using antiserum directed against an oligopeptide corresponding to the N-terminal region.


Characterization of primary human fibroblasts transformed by human papilloma virus type 16 and herpes simplex virus type 2 DNA sequences. Human papilloma virus type 16 (HPV-16) and herpes simplex virus type 2 (HSV-2) are human viruses implicated in the development of cancer, in particular cervical cancer. The ability of HSV-2 and HPV-16 to transform early passage human cells was examined in this report. For these studies, gingival fibroblasts were utilized. One gingival cell strain was derived from a normal individual (N-16). The second cell strain was derived from hyperplastic gingival tissue of an epileptic individual (R-30) treated with phenytoin, an antiseizure drug. A common side effect of phenytoin is the induction of gingival overgrowth. R-30 cells contained a stable chromosomal translocation between chromosomes 8 and 18 and expressed higher steady state levels of c-myc. HPV-16 DNA efficiently immortalized R-30 cells but not N-16 cells. R-30 cells cotransfected with HPV-16, and HSV-2 viral DNAs were more aneuploid than R-30 cells transfected with HPV-16 DNA alone. Additionally, R-30 cells cotransfected with both viral DNAs grew better in soft agar than R-30 cells transfected with HPV-16 DNA alone. HSV-2 DNA was detected in transformed cells by polymerase chain reaction. These results suggested R-30 cells were immortalized more efficiently by HPV-16 and further imply that HPV-16 and HSV-2 DNA fragments can cooperate during multistep transformation.


Glycoprotein 60 of equine herpesvirus type 1 is a homologue of herpes simplex virus glycoprotein D and plays a major role in penetration of cells. Monoclonal antibodies (MAbs) specific for equine herpesvirus type 1 (EHV-1) glycoprotein 60 (gp60) and gp 17/18 (F3132 and 5H6 respectively) were found to react with the same protein, which was identified as a homologue of herpes simplex virus type 1 gD. MAb F3132 strongly neutralized virus infectivity and inhibited the penetration of the virus into the cell. The effects on penetration were shared with three other MAbs against this protein (P68, F3116 and F3129), but no effect on virus penetration was found with any other anti-EHV-1 MAb tested. The level of glycosylation of gp60 was analysed using glycanase enzymes and glycosylation inhibitors, and consisted of mainly N-linked carbohydrate. The M(r) of non-N-glycosylated gp60 was 50K.


Immunity in strain 2 guinea-pigs inoculated with vaccinia virus recombinants expressing varicella-zoster virus glycoproteins I, IV, V or the protein product of the immediate early gene 62. The immunogenicity of specific varicella-zoster virus (VZV) proteins, with emphasis upon cell-mediated immune responses, was evaluated by immunizing strain 2 guinea-pigs with vaccinia virus recombinants that express gpI (vac-gpI), gpIV (vac-gpIV) and gpV (vac-gpV) or the IE-62 protein (vac-IE-62). Vac-gpI elicited the highest initial mean T cell proliferation response [stimulation index (S.I.) 3.8 +/- 0.9 S.E.M.] whereas inoculation with vac-gpV produced the lowest primary T cell response (S.I. 2.5 +/- 1.1 S.E.M.). T cell proliferation was detected for a shorter period after immunization with vac-gpV compared to vac-gpI, vac-gpIV or vac-IE-62. A comparison of the immunogenicity of vac-gpI and vac-IE-62 with the same proteins prepared by immunoaffinity purification showed that immunization with these proteins in either form elicited virus-specific IgG antibodies and T cell recognition. The presence or absence of IgG antibodies to the IE-62 protein was used to assess protection against challenge with guinea-pig cell-adapted infectious VZV in animals that had been inoculated with vac-gpI, vac-gpIV or vac-gpV. Immunization with vac-gpI and vac-gpIV restricted VZV replication but all animals given vac-gpV developed antibodies to IE-62 after challenge with infectious VZV. Priming of the T lymphocyte response was observed in all animals immunized with VZV-vaccinia virus recombinants after subsequent exposure to infectious VZV. These experiments with VZV vac-gpI, vac-gpIV and vac-gpV in guinea-pigs suggest variability in the capacity of herpesviral glycoproteins to elicit cell-mediated immunity in vivo. Induction of virus-specific immunity using IE-62 means that this major tegument protein of VZV could be a useful component for vaccine development.


The different interactions of a gIII mutant of pseudorabies virus with several different cell types. Glycoprotein gIII of pseudorabies virus (PrV) is multifunctional. It plays a role in the stable adsorption of the virus to its host cells by interacting with a cellular heparin-like substance. It also affects both release of mature virus from infected cell types and virulence. Thus, although non-essential for growth in vitro, gIII plays a central role in the biology of the virus. The primary attachment of a mutant, PrV2, which has an in-frame internal deletion and expresses a shortened version of gIII, and of wild-type (wt) virus, to MDBK cells has been shown to occur similarly. To ascertain whether different domains of gIII control the expression of the different biological functions of the gIII protein, we have compared several aspects of virus-host cell interactions of PrV2, of a gIII-null virus, and of wt virus. Our results showed that the deletion of the internal segment of the gIII glycoprotein affects adsorption and virus release differently, i.e. that these two functions of gIII appear to be independent of each other. Furthermore, we observed that although the primary adsorption of PrV2 and wt virus to MDBK cells is similar, PrV2 behaved like a gIII-null mutant with respect to virulence. The apparent contradiction between these two findings was resolved when it was found that although PrV2 binds as well as does wt to some cell types, it binds poorly to other cell types. The functional importance of different domains of gIII in virus adsorption thus differs, depending on the cell type with which the virus interacts.


Bovine coronavirus uses N-acetyl-9-O-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells. The importance of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant for bovine coronavirus (BCV) on cultured cells was analysed. Pretreatment of MDCK I (Madin Darby canine kidney) cells with neuraminidase or acetylesterase rendered the cells resistant to infection by BCV. The receptors on a human (CaCo-2) and a porcine (LLC-PK1) epithelial cell line were also found to be sensitive to neuraminidase treatment. The susceptibility to infection by BCV was restored after resialylation of asialo-MDCK I cells with Neu5,9Ac2. Transfer of sialic acid lacking a 9-O-acetyl group was ineffective in this respect. These results demonstrate that 9-O-acetylated sialic acid is used as a receptor determinant by BCV to infect cultured cells. The possibility is discussed that the initiation of a BCV infection involves the recognition of different types of receptors, a first receptor for primary attachment and a second receptor to mediate the fusion between the viral envelope and the cellular membrane.


Characterization of the genes encoding two of the major capsid proteins of epizootic haemorrhagic disease virus indicates a close genetic relationship to bluetongue virus. The sequences of the genes of two of the major capsid proteins of epizootic haemorrhagic disease virus serotype 1 (EHDV-1, Orbivirus genus, Reoviridae) have been determined by analyses of cDNA clones representing the L2 and S7 RNA segments. The EHDV-1 S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and has the capacity to encode 349 amino acids (M(r) 38,243). The EHDV-1 L2 RNA segment, which encodes the outer capsid VP2 protein (M(r) 113,249) is 2968 nucleotides in length and has an open reading frame of 971 codons. The potential secondary structure of the EHDV-1 S7 mRNA species, in particular that of the terminal regions, is comparable to those of the corresponding segments of bluetongue virus (BTV) and African horse sickness virus (AHSV); the EHDV-1 L2 mRNA species has a secondary structure similar to that of the L2 mRNA of BTV. The EHDV-1 VP2 and VP7 proteins, as well as those of the other two major structural proteins of EHDV published previously (the inner core VP3 protein and the second outer capsid, VP5), are closely related to the corresponding proteins of BTV. The EHDV and BTV VP7 sequences are more distantly related to the sequence of the AHSV VP7 protein published recently.


Detection of adeno-associated virus type 2 in human peripheral blood cells. The non-pathogenic human parvovirus, adeno-associated virus (AAV) is helper virus-dependent. However, it integrates into the cellular genome in the absence of its helper viruses. Therefore it could become a useful vector for gene therapy. Previous studies and our own results have shown that 40 to 80% of adults are seropositive for AAV and that seroconversion occurs during the first few years of life, but little is known about the route of natural infection with the virus. We used the polymerase chain reaction to detect the AAV-2 genome and identify AAV sequences within peripheral blood leukocytes (PBLs). We could detect AAV in PBLs of two of 55 healthy blood donors, and two of 16 haemophilic patients. AAV DNA replication and viral protein production in PBLs propagated in tissue culture were also examined. AAV DNA replicated very efficiently in the presence of helper adenovirus, but capsid proteins were produced at a lower level and the yield of infectious virus was very low. Our findings prove that in vivo infection of PBLs occurs, and that PBLs could mediate the spread of AAV infection to different body tissues.


Peripheral replication and latency reactivation kinetics of the non-neurovirulent herpes simplex virus type 1 variant 1716. The terminal portion of the herpes simplex virus (HSV) genome long repeat region has been shown to contain a neurovirulence gene. Both HSV-1 and HSV-2 mutants deleted in this gene fail to cause central nervous system (CNS) disease in mice. The HSV-1 strain 17 variant 1716, which has a 759 bp deletion encompassing the gene, grows normally in tissue culture but fails to grow following intracerebral inoculation of mice. This paper demonstrates that 1716 is capable of peripheral replication in the footpads of mice. However, no acute replication of virus is detectable in dorsal root ganglia up to 10 days after footpad inoculation. These results imply that the replication defect in 1716 is not host-specific, but is tissue- and/or cell type-specific. Latency reactivation kinetics demonstrate that 1716 is capable of establishing a latent infection, but the kinetics of reactivation are significantly impaired compared to wild-type virus and are dose-dependent. Lack of acute ganglionic replication combined with impaired reactivation kinetics support the conclusion that a proportion of 1716 genomes initiate a lytic infection which then aborts, and a proportion enter the latent state. The results with 1716 imply that its inability to replicate in CNS and peripheral nervous system neurons is specific, and that the block in replication is beyond the stage of adsorption and entry. A prerequisite for any live attenuated HSV vaccine is an inability to initiate CNS involvement following peripheral inoculation. In this respect, 1716 has prototype vaccine potential with the proviso that a direct extrapolation is being made from mouse to man.


Status of the ICP34.5 gene in herpes simplex virus type 1 strain 17. In the published DNA sequence of herpes simplex virus type 1 (HSV-1) strain 17 the coding region for the neurovirulence factor ICP34.5 is disrupted by a 2 bp insertion relative to the corresponding sequence in HSV-1 strain F; this difference would render 60% of the ICP34.5 coding sequence out of frame in strain 17. Re-investigation of the strain 17 sequence showed that the plasmid clone used as the major sequencing substrate for the region was atypical in that other clones did not possess the 2 bp insertion. It was concluded that the coding sequence for ICP34.5 is intact in HSV-1 strain 17 and that the HSV-1 DNA sequence should be revised at this locus.


Vaccinia virus encodes four putative DNA and/or RNA helicases distantly related to each other. Computer-assisted analysis of the amino acid sequences of vaccinia virus proteins containing the purine NTP-binding pattern revealed the seven motifs typical of the DNA (RNA) helicase superfamily II in the proteins I8R and A18R. Together with the previously described putative helicases D6R and D11L, the number of putative helicases of this superfamily encoded by the genome of vaccinia virus now reaches four. Aside from the helicase motifs, the sequences of I8R and A18R showed no strong similarity to each other, nor to D6R and D11L. Statistically significant similarity was demonstrated between I8R and the putative RNA helicases involved in pre-mRNA splicing in yeast, PRP2, PRP16 and PRP22, whereas A18R appeared to be related to the putative helicases encoded by the human DNA repair gene ERCC3 and the D10 gene of bacteriophage T5. These findings suggest that I8R may be an RNA helicase. Based on the known properties of the virion NTPases of vaccinia virus, it is possible that the I8R protein may be identical to the previously characterized virion NTPase II. A18R is likely to possess DNA and/or RNA helicase activity. Circumstantial evidence suggests that this activity might be involved in melting duplex structures in late mRNAs. The possibility of independent acquisition of the putative helicases I8R, A18R and a common progenitor to D6R and D11L by an ancestral poxvirus is discussed.


Modulation of sprouting in organ culture after axotomy of an identified molluscan neuron. We examined a variety of factors that might modulate the initiation of neurite outgrowth in an attempt to identify means by which its initiation might be accelerated. We examined this initiation from an identified molluscan neuron, Helisoma trivolvis buccal neuron B5 after axotomy, and determined whether the site of injury, temperature, ion channel blockers, pH, the second messenger cAMP, and protein synthesis affect the initiation of neurite outgrowth. Neurite outgrowth was assayed from axotomized neurons by filling the neurons intracellularly with Lucifer Yellow and examining the percentage of axons that extended (sprouted) new process after 9 or 24 h in organ culture. About one-third (31%) of axotomized neurons sprouted from the site of injury after 9 h (n = 22), and 88% (n = 20) sprouted after 24 h in saline at 22 degrees-24 degrees C when the injury was located 800 microns from the soma. Elevating the temperature to 32 degrees C or moving the lesion site to 400 or 1500 microns from the soma did not significantly alter the incidence of sprouting. Blocking sodium channels with tetrodotoxin [TTX (2 x 10(-5) M)] did not significantly reduce the incidence of sprouting, whereas the sodium channel agonist, veratridine (10(-5) M) did. The calcium channel blocker lanthanum (10(-6)-10(-4) M), stimulated neurite outgrowth; however, the organic calcium channel blocker verapamil (10(-3)-10(-5) M), and the calcium ionophore A23187 (10(-5) M), had no effect on sprouting. Exposure of neurons to the potassium channel blocker tetraethylammonium [TEA (20 mM)], elevation of intracellular pH with NH4Cl (5 mM), or treatment with the adenylate cyclase activator forskolin (10(-5) M) reduced the incidence of sprouting, whereas dideoxy-forskolin (10(-5) M) had no effect. Inhibition of protein synthesis with anisomycin (2 x 10(-4) to 2 x 10(-6) M) did not significantly suppress sprouting 24 h after axotomy. Both D and L isomers of glutamate (300 microM) stimulated sprouting. The present results suggest that the initiation of sprouting is regulated locally at or near the site of injury, and that blocking specific ion channels may either inhibit or enhance the initiation of neurite outgrowth.


Effect of zinc ion on the interaction of some amino acid compounds of copper(II) with hydrogen peroxide. Reaction of elemental copper and zinc powder mixtures with glycine (NH2.CH2COOH; HA) or aspartic acid (NH2CHCOOHCH2COOH; H2B) (in 1:1:2 ratio, respectively) in the presence of excess hydrogen peroxide (H2O2) at 50 degrees C, results in the formation of a new mixed metal peroxy carbonate compound corresponding to formula [Cu(Zn)2(O2(2-) (CO3)2(H2O)4], while the same reaction with elemental copper powder alone yields merely peroxy amino acid compounds having the formula [Cu(O2(2-)) (HA)2(H2O)] and [Cu(O2(2-)) (H2B) (H2O)2] for glycine and aspartic acid, respectively. These compounds have been characterized by elemental analysis, ESR, and electronic and IR spectra. It is interesting to note that both amino acids are converted to carbonate in the presence of zinc alone. A method analogous to that described above, for the reaction of elemental copper, zinc powder mixtures with succinic acid [(CH2COOH)2] or acetic acid (CH3COOH) in excess H2O2, on the other hand, gave a product essentially comprising copper succinate or acetate, respectively. These observations suggest an interesting and perhaps important phenomenon by which only the simple amino acids such as glycine and aspartic acid are converted to carbonates while their corresponding carboxylic acids form only their respective salts.


Oxovanadium(IV) complex formation by simple sugars in aqueous solution. The binding of oxovanadium(IV) to simple sugars in neutral or basic aqueous solution, as studied by EPR and electronic absorption spectroscopy, is reported. The complexation is favored in basic media and involves the coordination of the metal ion to couples of adjacent deprotonated hydroxyls of the sugar molecule. However, only the ligands provided with cis couples can adopt this chelating ligand behavior. The ability of the cis hydroxyl couples to yield chelated complexes has been related to the structural rearrangement (decrease of the O-C-C-O torsion angle in the five-membered chelated ring) needed to permit the oxovanadium(IV) coordination by the sugar molecule.


Potentiometric and spectroscopic studies of the Cu(II) complexes of Ala-Arg8-vasopressin and oxytocin: two vasopressin-like peptides. The results are reported of a potentiometric and spectroscopic study of the H+ and Cu2+ complexes of Ala-Arg8-vasopressin (Ala-AVP) and oxytocin at 25 degrees C and an ionic strength of 0.10 mol dm-3 (KNO3). The coordination chemistry of oxytocin and Cu(II) has been shown to be virtually identical to that of Arg8-vasotocin, forming unusually stable complexes with four nitrogen coordination (4N complexes) below pH 7. Spectroscopic evidence suggests weak interaction between Cu(II) and the sulphur atom of the -Cys6- residue in the 2N species (pH congruent to 6) but this is absent in the 4N complex. Evidence is also presented for perturbation of electronic transitions within the aromatic ring of the Tyr residue by Cu(II). While the physiological potency of Ala-AVP is very high, its coordination chemistry differs significantly from that of Arg8-vasopressin. With Cu(II) it forms complexes of similar stability to those with tetraglycine, demonstrating that the addition of an alanyl residue to the amino-terminal of the peptide destroys the conformation which is particularly favorable for rapid 4N coordination.


The coordination of copper(II) to 1-hydroxy-4-(glycyl-histidyl-lysine)-anthraquinone; a synthetic model of anthraquinone anti-cancer drugs. Results are reported of a pH-metric and spectroscopic (CD and ESR) study of the complexes formed between the pseudo-peptide 1-hydroxy-4-(Gly-His-Lys)-anthraquinone (Q-GHK) since, when complexed to copper ions, Q-GHK has been shown to be very effective in promoting the formation of free radicals and inducing DNA cleavage. Q-GHK forms very stable complexes with copper, the major species being bonded to three nitrogen donors in the coordination plane: an imidazole-N of the His residue and the peptide nitrogens of the Gly and His residues. This species is probably stabilized through bonding of the fourth planar coordination site of Cu(II) to the 9-anthraquinone oxygen. At high Q-GHK:copper ratios a second Q-GHK molecule is coordinated through its imidazole-N donor.


Thiamin mono- and pyrophosphatase activities from brain homogenate of Guamanian amyotrophic lateral sclerosis and parkinsonism-dementia patients. Thiamin-pyrophosphatase (TPPase) and thiamin-monophosphatase (TMPase) were determined using a spectrophotometric method at various pH values (5.5, 7.5, and 9.0) in brain tissue obtained at autopsy from amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD) patients from Guam and from Guamanian patients who died from other diseases (controls). TPPase separation by thin-layer polyacrylamide gel isoelectric focusing (IEF) was also performed using both gray and white matter. TPPase content, chemically determined at pH 9.0, was found to be significantly reduced in the frontal cortex of ALS and PD patients compared to controls. TMPase content, on the contrary, was unchanged. IEF analysis showed 9 clear-cut bands with TPPase activity in the pH range 5.4-7.2 and a broad band at pH 4.7-5.2. The enzymatic activity was higher in gray than in white matter. In one patient the pattern was clearly different, with two additional bands observed at pH 7.1 and 6.7, and thought to be due to genetic microheterogeneity.


Calcium, calmodulin and 3',5'-cyclic nucleotide phosphodiesterase activity in human muscular disorders. 3',5'-Cyclic nucleotide phosphodiesterase (PDE) is known to play an important role in the regulation of cyclic nucleotide levels in various tissues including the muscle. Previous studies have estimated the level of this enzyme in several neuromuscular disorders but the results have been variable. Moreover, there was no attempt made to correlate the enzyme levels with the levels of calcium and calmodulin, both of which regulate diverse biological processes including muscle contraction. In the present study we have estimated phosphodiesterase in the muscle of normal controls as well as patients with myotonic (MyD) and Duchenne muscular dystrophy (DMD) and amyotrophic lateral sclerosis (ALS). PDE was found to be increased significantly in all of the diseased muscles as compared to controls (P less than 0.01). But the increase could be coupled with an increase in calcium and calmodulin only in Duchenne dystrophic muscle.


CPT-11: a new derivative of camptothecin for the treatment of refractory or relapsed small-cell lung cancer. To evaluate the activity of CPT-11, which is a new derivative of camptothecin, against refractory or relapsed small-cell lung cancer (SCLC). Sixteen patients with refractory or relapsed SCLC were entered onto a prospective, non-randomized, single-institution phase II trial. All 16 patients had been pretreated heavily with some form of cisplatin-based combination chemotherapy. Five patients had received previous chemotherapy with cisplatin, vincristine, doxorubicin, and etoposide (CODE) as an induction therapy. Six patients had been treated with concurrent cisplatin and etoposide plus chest x-ray. The median time off chemotherapy was 7.3 months (range, 1.9 to 15.1 months). Patients were treated with a CPT-11 starting dose of 100 mg/m2 body surface given as a 90-minute intravenous (IV) infusion every week with subsequent doses based on toxicity. Fifteen patients were assessable for toxicity, response, and survival. Seven patients (47%; 95% confidence limits for an overall response rate, 21.4% to 71.9%) responded to CPT-11 with a median duration of response of 58 days. The major toxicities were myelosuppression (predominantly leukopenia), diarrhea, and pulmonary toxicity. CPT-11 is an active agent against refractory or relapsed SCLC and deserves to be studied more closely as both a single agent and in combination with other drugs to treat patients with SCLC.


Intensive combination chemotherapy, concurrent chest irradiation, and warfarin for the treatment of limited-disease small-cell lung cancer: a Cancer and Leukemia Group B pilot study. In prior Cancer and Leukemia Group B (CALGB) studies, combined chemotherapy and thoracic irradiation was superior to chemotherapy alone in limited-disease (LD) small-cell lung cancer (SCLC). A combined modality pilot study was performed to test the feasibility of adding warfarin to aggressive chemoradiotherapy for LD SCLC. Combination chemotherapy with doxorubicin 45 mg/m2 intravenously (IV) on day 1, cyclophosphamide 800 mg/m2 IV on day 1, and etoposide (ACE) 80 mg/m2 on days 1 to 3 was given every 21 days for the first three courses. The fourth and fifth courses substituted cisplatin 33 mg/m2 IV on days 1 to 3 for the doxorubicin, with concurrent chest irradiation to a total of 4,000 cGy given in 20 fractions during a 4-week period followed by a boost of 1,000 cGy in five fractions during a 1-week period. Prophylactic cranial irradiation, 3,000 cGy was given concurrently in 10 fractions during a 2-week period. Courses 6 to 8 again used ACE chemotherapy, but courses 4 to 8 were given on a 28-day schedule with dose adjustment for hematologic or renal toxicity. Warfarin was given throughout the treatment period titrated to achieve a prothrombin time (PT) of 1.5 to 2 times the control. Patients with histologically proven limited-stage SCLC, good performance status, and normal renal, hematologic, and hepatic functions were eligible. Sixty-one of 66 patients entered onto the study were eligible and assessable. Fifty-four (89%) (95%) confidence interval [CI], 78% to 95%) experienced an objective response, 35 (57%) achieved a complete response (CR) (95% CI, 44% to 70%), and 17 (28%) achieved a partial response (95% CI, 16% to 39%). Median durations were CR, 26.3 months; failure-free survival, 11.8 months; and survival, 18 months. Forty-one percent of the patients were alive at 2 years, 33% were alive at 3 years, and 25% were alive at 4 or more years. Median follow-up for survivors is 5 years (range, 3.5 to 5.9 years). Severe or life-threatening myelosuppression occurred in 90%, infection occurred in 34%, fever without documented infection occurred in 26%, and pulmonary toxicity occurred in 6%. Another 6% of patients experienced severe or life-threatening hemorrhages. There were four treatment-related fatalities. The pulmonary toxicities have been associated with the resumption of ACE chemotherapy after chest irradiation. These highly encouraging response and survival results compare favorably with any prior CALGB group study. Although they are somewhat more toxic, they are comparable to the best published results. A randomized study that examines the role of warfarin is underway.


Neoadjuvant chemotherapy and radiotherapy followed by surgery in stage IIIA non-small-cell carcinoma of the lung: report of a Cancer and Leukemia Group B phase II study. This phase II trial was designed to evaluate the feasibility, toxicity, response rates, and survival for neoadjuvant chemotherapy and radiotherapy (RT) followed by surgical resection in newly diagnosed patients with surgically staged IIIA non-small-cell lung carcinoma (NSCLC). Previously untreated patients with NSCLC underwent bronchoscopy, chest and abdominal computed tomography (CT), bone scan, and surgical staging of the mediastinum. Neoadjuvant treatment consisted of concurrent chemotherapy and RT. Patients then underwent surgical resection, which was followed in turn by additional chemotherapy and RT. Chemotherapy included cisplatin 100 mg/m2 on days 1 and 29, vinblastine 3 mg/m2 on days 1 and 3 and 29 and 31, and fluorouracil (5-FU) 30 mg/kg/d by infusion on days 1 to 3 and 29 to 31 (FVP). RT began on day 1 and included 3,000 cGy in 15 fractions. Surgery took place on day 55, and one more cycle of chemotherapy and an additional 3,000 cGy of RT began on day 85. Forty-one eligible patients (median follow-up, 53 months) were studied. N2 disease was present in 80%, whereas 20% had T3N0 or T3N1 lesions. Response to neoadjuvant chemotherapy and RT included no complete responses (CR), 21 (51%) partial responses (PR) or regressions, 19 (46%) stable disease (SD), and one (2%) progressive disease (PD). Thirty-one patients underwent surgery, and 25 were resected. In four of the 25 resection specimens, no viable tumor was present, whereas in three of the six unresectable patients, extensive biopsy results demonstrated only necrotic tumor. The maximum response achieved using all protocol treatment was 27 (66%) CRs, seven (17%) PRs or regression, six (15%) SDs, and one (2%) PD. Toxicity was substantial and primarily hematologic. There were six (15%) treatment-related deaths, which included three perioperative deaths and three chemotherapy-related toxicity deaths. The Kaplan-Meier curve indicated a 1-year survival of 58% and a median survival of 15.5 months. Nine patients (22%) remain disease-free. There was a reasonably high rate of PR associated with concurrent neoadjuvant chemotherapy and RT, and a high percentage of patients who ultimately were rendered completely disease-free. However, treatment-related morbidity and mortality was common. Median survival seemed to be only modestly improved beyond that achieved with less intensive means of treatment. However, a group has emerged of patients who enjoy prolonged disease-free survival and possible cure.


Outcomes in low-risk babies treated with half-dose chemotherapy according to the Third National Wilms' Tumor Study. To determine whether the 50% dose reduction of all chemotherapeutic agents recommended for babies (less than or equal to 12 months of age) by the Third National Wilms' Tumor Study (NWTS-3) produced acceptable toxicity without sacrificing any survival benefit. The 365 babies enrolled in NWTS-3 had tumors of varying histologies and stages. The present analysis was restricted to the 256 infants who had tumors of favorable histology, were free of metastasis at diagnosis, and received treatment according to NWTS-3 guidelines. Despite the recommended attenuation of drug doses observed in 75% of the chemotherapy courses received, outcomes for these babies were comparable to those obtained in older children given full doses of chemotherapy. Four-year survival rates for 256 babies with stages I (n = 199), II (n = 38), and III (n = 19) favorable-histology tumors were 96%, 95%, and 90%, respectively. The figures for 498 stage I, 342 stage II, and 373 stage III older children with favorable-histology lesions were 92%, 94%, and 91% in that order. There were no deaths from hematologic toxicity or infection among babies who received half-dose chemotherapy. The death rates for their older NWTS-3 counterparts was 1%. Less aggressive therapies advocated for babies in NWTS-3 provide acceptable levels of morbidity without compromising the excellent results previously reported for low-risk patients of all ages.


Chemical, physical, and histologic studies on four commercial apatites used for alveolar ridge augmentation. The purpose of this study was to evaluate four commercial apatite products. Subperiosteal alveolar ridge augmentation was performed on the maxilla of rats by implantation of granules of two dense products and of two porous products, and the tissue response was compared with the material characteristics obtained by chemical analysis and infrared spectrometry. None of the apatites caused osteoinduction or osteoconduction; fibrous encapsulation with multinuclear giant cells was observed around all four types. One of the apatites was fluorapatite and not hydroxylapatite, as claimed by the manufacturer. The tissue response to this implant material was dominated by multinuclear giant cells.


[Detection of Epstein-Barr virus DNA in salivary gland tumors]. To evaluate the relations between salivary gland tumors and Epstein-Barr virus (EBV), the levels of EBV-related antibodies were examined, and detection of EBV nuclear antigen (EBNA) and EBVDNA in tumor tissue was attempted by the anti-complement immunofluorescence technique and polymerase chain reaction, respectively. The mean VCA-IgG antibody level was increased to 925 (80-2560) in Warthin's tumor, 496 (40-2560) in mucoepidermoid tumor, and 206 (40-640) in pleomorphic adenoma. The positive rate of EA-IgG was high in Warthin's and mucoepidermoid tumors. VCA-IgA antibody was positive in 2 of the 7 cases of Warthin's tumor. EA-IgA antibody was negative in all cases. EBVDNA was detected in 7 of the 7 cases of Warthin's tumor, 3 of the 5 cases of mucoepidermoid tumor, and 2 of the 26 cases of pleomorphic adenoma. A relationship between Warthin's tumor and EBV was suggested by the 100% detection rate of the viral DNA.


The ultrastructure of oesophageal carcinomas: multidirectional differentiation. A transmission electron microscopic study of 43 cases. Forty-three oesophageal carcinomas, comprising 15 squamous cell carcinomas, 22 adenocarcinomas, 5 small cell carcinomas, and 1 adenosquamous carcinoma, were examined by transmission electron microscopy. The ultrastructural features of each tumour type are detailed. Multi-directional differentiation (heterogeneity) was observed in 11 cases: 5 squamous cell carcinomas with focal glandular features; 4 adenocarcinomas with focal squamous features; and 2 small cell carcinomas, one with glandular and the other with squamous features. Abnormal distributions of desmosomes in squamous cell carcinomas and of polarity of tumour cells in adenocarcinomas are described.


Ubiquitin: its potential significance in murine AA amyloidogenesis. Amyloid enhancing factor (AEF), which has recently been shown to have identity with ubiquitin (Ub), is believed to play a causative role in experimentally induced AA amyloidosis in mice. We have examined the profile of Ub in activated leukocytes and splenic reticulo-endothelial (RE) cells and its relationship with serum amyloid A protein (SAA) and AA amyloid deposits in an alveolar hydatid cyst (AHC)-infected mouse model of AA amyloidosis. Two monospecific antibodies, anti-ubiquitin (RABU) and anti-mouse AA amyloid, were used as immunological probes to localize Ub, SAA, and AA amyloid. In response to AHC infection, the dull and diffuse Ub immunoreactivity in normal mouse leukocytes and RE cells promptly changed to a discrete granular pattern suggesting an increase in the intracellular concentration of Ub and the formation of Ub-protein conjugates. This corresponded to an elevation in SAA levels, SAA uptake by RABU-positive phagocytic cells, co-localization of Ub-SAA immunoreactive splenocytes in the perifollicular areas, and deposition of Ub-bound AA amyloid in the splenic and hepatic tissues. These results suggest that Ub-loaded monocytoid cells may play an important role in the physiological processing of the sequestered SAA into AA amyloid. Aspects of AA amyloidogenesis are discussed in relation to other experimental models in which stress-induced Ub-protein conjugate formation and its transport to lysosomal vesicles have been studied.


Effects of visible and near-infrared lasers on cell cultures. The effect of 360, 632 and 780 nm light on NIH fibroblast cells was examined. Mitosis counts of irradiated cells at various energy doses were taken. Scanning electron micrographs of these cells were studied. It is suggested that low-level laser therapy in the visible and in the near-infrared region is due to cell respiration stimulation by either the endogenous porphyrins in the cell, or by the cytochromes.


Inhibition of phosphoinositide turnover by praziquantel in Schistosoma mansoni. The effect of praziquantel on phosphoinositide turnover was examined in Schistosoma mansoni to determine if this anthelminthic modulates signal transduction pathways in parasites. Adult worms were radiolabeled with [3H]myoinositol for 24 hr and total inositol phosphate levels determined in the presence of praziquantel. Praziquantel inhibited inositol phosphate turnover when activated with NaF plus AlCl3 or with the nonhydrolyzable guanine nucleotide-binding protein analogue GTP gamma S. Furthermore, praziquantel decreased basal turnover of inositol phosphates. Inhibition was seen in both male and female worms as well as in schistosomula. These data indicate that inhibition of phosphoinositide turnover may contribute to the effect of praziquantel on parasite survival within the definitive host.


Highly potent and selective heptapeptide antagonists of the neurokinin NK-2 receptor. Incorporation of D-Pro9 into substance P related peptides is known to enhance neurokinin NK-2 receptor agonist potency and selectivity with respect to other neurokinin receptors. We now report that replacement of D-Trp9 by D-Pro9 in the nonselective neurokinin antagonist [Arg5,D-Trp7,9, Nle11]-SP(5-11) gave a partial agonist with NK-2 receptor selectivity. Further incorporation of Pro10 provided the weak but selective NK-2 antagonist Arg-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (compound 4; NK-2 pKB = 5.9; NK-1 pKB = 4.7; NK-3 pKB less than 4.6). Addition of a suitable lipophilic N-terminal substituent (e.g. Boc, PhCO, cyclohexylcarbonyl) to this compound greatly enhanced NK-2 antagonist activity (compound 10, GR 83074; NK-2 pKB = 8.2), and combined with further optimization of the N-terminal amino acids, provided the extremely potent and selective NK-2 antagonist PhCO-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (compound 34, GR 94800; NK-2 pKB = 9.6; NK-1 pKB = 6.4; NK-3 pKB = 6.0). Compounds of this class produced a potent inhibition of NK-2 agonist-induced bronchoconstriction in the anaesthetized guinea-pig.


Methoxytetrahydropyrans. A new series of selective and orally potent 5-lipoxygenase inhibitors. Investigation of the SAR of the lead (methoxyalkyl)thiazole 1-[3-(naphth-2-ylmethoxy)phenyl]-1-thiazol-2-ylprop yl methyl ether (1, ICI 211965) led to the methoxytetrahydropyrans, a new series of 5-lipoxygenase (5-LPO) inhibitors exemplified by the parent compound 4-[3-(naphth-2-ylmethoxy)phenyl]-4- methoxy-3,4,5,6-tetrahydro-2H-pyran (4f). In vitro 4f inhibited leukotriene C4 (LTC4) synthesis in zymosan-stimulated plasma-free mouse macrophages and LTB4 synthesis in A-23187-stimulated human whole blood (IC50s 0.5 nM and 0.07 microM, respectively). In the rat 4f inhibited LTB4 synthesis in blood ex vivo and in zymosan-inflamed air pouch exudate with an ED50 3 h after oral dosing of 10 mg/kg in each system. In seeking more potent orally active compounds, strategies were explored in congeners of 4f for reducing lipophilicity without sacrificing potency. For example, replacement of 2-naphthyl of 4f by various aza- and oxoheterocycles afforded compounds in which log P is reduced by 1.7-2.3 units while potency in human whole blood in vitro was maintained or enhanced relative to 4f. In addition, the oxoheterocyclic replacements provided compounds with improved oral potency and the preferred compound from this group is 6-[[3-fluoro-5-(4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4- yl)phenoxy]methyl]-1-methylquinol-2-one (4y). In the in vitro systems, 4y inhibited LT formation with IC50s in mouse macrophages and human whole blood of 3 nM and 0.02 microM, respectively. 4y did not inhibit the synthesis of cyclooxygenase (CO) products at concentrations up to 500 microM in human blood, a selectivity for 5-LPO over CO of greater than 20,000-fold. In the rat 4y inhibited the formation of LTB4 in blood ex vivo and in inflammatory exudate with ED50s 3 h after oral dosing of 0.9 and 0.3 mg/kg, respectively. 4y was more potent in vitro in human whole blood and in rat blood ex vivo at 3 h than either the 5-LPO inhibitor A-64077 or the FLAP antagonist MK-886. Based on these data 4y (ICI D2138) has been entered into development as an orally active, selective 5-LPO inhibitor for clinical evaluation in inflammatory conditions in which LTs are believed to play a role.


Novel inhibitors of prolyl 4-hydroxylase. 3. Inhibition by the substrate analogue N-oxaloglycine and its derivatives. N-Oxaloglycine (3) is an alpha-ketoglutarate (1) analogue that is a competitive inhibitor of prolyl 4-hydroxylase (EC 1.14.11.2). A study of the structure-activity relationships of some other oxalo derivatives shows that substitution on the glycine moiety modulates activity stereoselectively and that if the omega-carboxylate is homologated or replaced by either acylsulfonamides or anilide, then activity is sharply reduced. This sensitivity to these changes is contrasted with the relative insensitivity of another putative alpha-ketoglutarate analogue, pyridine-2,5-dicarboxylic acid (2), and the implication is discussed that compounds of both series are unlikely to bind to prolyl hydroxylase in the same way even though both inhibit the enzyme competitively.


Inhibitors of blood platelet cAMP phosphodiesterase. 2. Structure-activity relationships associated with 1,3-dihydro-2H-imidazo[4,5-b]quinolin-2-ones substituted with functionalized side chains. A series of 1,3-dihydro-2H-imidazo[4,5-b]quinolin-2-one derivatives, substituted at the 7-position with functionalized side chains, was synthesized and evaluated as inhibitors of human blood platelet cAMP phosphodiesterase (PDE) as well as ADP- and collagen-induced platelet aggregation, in vitro. Structural modifications focused on variation of the side-chain terminus, side-chain length, and side-chain connecting atom. Functionality incorporated at the side-chain terminus included carboxylic acid, ester and amide, alcohol, acetate, nitrile, tetrazole, and phenyl sulfone moieties. cAMP PDE inhibitory potency varied and was dependent upon the side-chain terminus and its relationship with the heterocyclic nucleus. Methylation at N-1 or N-3 of the heterocycle diminished cAMP PDE inhibitory potency. Several representatives of this structural class demonstrated potent inhibition of ADP- and collagen-induced blood platelet aggregation and were half-maximally effective at low nanomolar concentrations. Amides 13d, 13f, 13h, 13k, 13m, and 13w are substantially more potent than relatively simply substituted compounds. However, platelet inhibitory properties did not always correlate with cAMP PDE inhibition across the series, probably due to variations in membrane permeability. Several compounds inhibited platelet aggregation measured ex vivo following oral administration to rats. Ester 11b, acid 12b, amide 13d, and sulfone 29c protected against thrombus formation in two different animal models following oral dosing and were found to be superior to anagrelide (2) and BMY 20844 (5). However, ester 11b and acid 12b demonstrated a unique pharmacological profile since they did not significantly affect hemodynamic parameters in dogs at doses 100-fold higher than that required for complete prevention of experimentally induced vessel occlusion in a dog model of thrombosis.


Inhibitors of blood platelet cAMP phosphodiesterase. 3. 1,3-Dihydro-2H-imidazo[4,5-b]quinolin-2-one derivatives with enhanced aqueous solubility. Two series of 1,3-dihydro-2H-imidazo[4,5-b]quinolin-2-one derivatives incorporating an additional site for acid salt formation were synthesized and evaluated as inhibitors of human blood platelet cAMP phosphodiesterase (PDE) and ADP-induced platelet aggregation. The objective of this study was to identify compounds that blended potent biological activity with a satisfactory level of aqueous solubility. From a series of 7-aminoimidazo[4,5-b]quinolin-2-ones, biological and physical properties were optimally combined in the 1-piperidinyl derivative 11c. However, this compound offered no significant advantage over earlier studied compounds as an antithrombotic agent in an animal model of small vessel thrombosis. A series of 7-alkoxy alkanoic piperazinamide derivatives, in which the additional basic nitrogen atom was remote from the heterocyclic nucleus and accommodated in a secondary binding region of the cAMP PDE enzyme, demonstrated greater intrinsic cAMP PDE inhibitory activity. Structural modifications of this series focused on variation of the piperazine substituent and side-chain length. The lipophilicity of the N-substituent influenced biological potency and aqueous solubility, with substituents of seven carbon atoms or less generally providing acceptable solubility properties. The N-(cyclohexylmethyl)piperazinamide 21h was identified from this series of compounds as a potent inhibitor of platelet cAMP PDE, IC50 = 0.4 nM, and ADP-induced platelet aggregation, IC50 = 0.51 microM after a 3-min exposure and 0.1 microM after a 15-min exposure of platelet-rich plasma to the drug. Evaluation of 21h and representative analogues in vivo using a rabbit model of small vessel thrombosis revealed significantly greater antithrombotic efficacy compared to that of previously studied compounds with similar intrinsic biological activity measured in vitro but inferior aqueous solubility.


Singlet oxygen-induced inhibition of cardiac sarcolemmal Na+K(+)-ATPase. We investigated the susceptibility of sarcolemmal Na+K(+)-ATPase to singlet oxygen. The role of this enzyme is regulation of Na+ concentration and thereby membrane potential. Inhibition of Na+ pump would lead to intracellular Ca2+ overload therefore further aggravating the injury caused by free radicals. Incubation of isolated sarcolemmal vesicles with irradiated rose bengal (150 nM) resulted in 86 +/- 1% inhibition of Na+K(+)-ATPase activity and histidine (25-100 mM) protected the enzyme in a dose-dependent fashion whereas SOD, catalase or mannitol (.OH radical scavenger) did not have any effect. Also, the inhibition of Na+K(+)-ATPase activity was dependent on rose bengal concentration, intensity of irradiation, duration of light exposure, showing that inhibition was directly related to amount of singlet oxygen generated. These results show that singlet oxygen may have significant disruptive effects on sarcolemmal function and may represent an important mechanism by which the oxidative injury to the myocardium induces arrhythmogenesis.


Lack of oscillations in cyclic AMP, cAMP-protein kinase and glycogen phosphorylase during the cardiac cycle in perfused rat hearts. It is unclear whether reported fluctuations in the level of adenosine 3',5'-cyclic monophosphate (cAMP) during a single cardiac cycle in ventricular muscle are associated with distal changes in cAMP-dependent processes. The degree of cAMP variation and its effect, if any, on biochemical sequelae during the cardiac cycle, were investigated by determining the level of cAMP and the activity ratios of cAMP-dependent protein kinase and glycogen phosphorylase in the rat ventricular myocardium. Isolated perfused hearts contracting at 240 beats/min and free of exogenously administered catecholamines were freeze-clamped, utilizing an automated clamping device capable of freezing the entire heart in less than 50 ms. The cardiac cycle was segmented into phases utilizing three different segmentation schemes. No significant difference was detected between phases regardless of the method of segmentation for cAMP, cAMP-dependent protein kinase, or glycogen phosphorylase levels. These results suggest that the levels of cAMP and the activities of cAMP-dependent protein kinase and glycogen phosphorylase do not vary significantly during a single cardiac cycle in the mammalian myocardium.


Isolated ventricular myocytes from failing and non-failing human heart; the relation of age and clinical status of patients to isoproterenol response. Single cardiac myocytes were isolated from the ventricles of failing and non-failing human hearts. The contraction amplitude, time-to-peak shortening and time to 50% and 90% relaxation were measured in cells stimulated at 0.2 Hz at 32 degrees C. The effects of increasing extracellular calcium and isoproterenol were investigated using cumulative concentration/response curves. Maximum contraction amplitude in high calcium or velocities of contraction or relaxation were not impaired in cells from failing hearts. Beta-adrenoceptor function in a single cell was assessed by the maximum contraction amplitude in the presence of isoproterenol relative to that with high calcium in the same cell (isoproterenol/calcium ratio). A decrease in the isoproterenol/calcium ratio correlated positively with an increase in the isoproterenol EC50 (concentration for half-maximal effect) for a cell (P less than 0.02, n = 39). The isoproterenol/calcium ratio in left ventricular myocytes decreased with increasing severity of disease, correlating with failure as defined by New York Heart Association class (P less than 0.001, n = 26 patients), left ventricular ejection fraction (P less than 0.001, n = 24), left ventricular end diastolic pressure (P less than 0.05, n = 21) and amount of diuretics prescribed (P less than 0.001, n = 26). In right ventricular myocytes, only increasing NYHA class correlated with decreasing isoproterenol/calcium ratios. There was a correlation of the isoproterenol/calcium ratio between right and left ventricular cells from patients with ischemic heart disease (P less than 0.05), n = 11). Beta-adrenoceptor subsensitivity occurred in mitral valve disease, ischemic heart disease, congenital abnormalities and congestive cardiomyopathy, but not in the right ventricle of patients with myocarditis. The isoproterenol/calcium ratio correlated negatively with the age of the patient (P less than 0.001, n = 26, left ventricle). Multiple regression indicated that the maximum contraction amplitudes in either high isoproterenol or high calcium declined significantly with age only, but that both age and severity of disease contributed to the decrease in isoproterenol/calcium ratio. Time-to-peak tension in isoproterenol, as well as relaxation times in high calcium also decreased with the age of the patient. Analysis of variance showed that between-patient variation was significantly greater than between-cell for most of the parameters measured. Beta-adrenoceptor desensitisation may be detected in individual myocytes from failing hearts, and this relates more to the severity of disease and the age of the patient rather than the etiology of heart failure. A decline in absolute contractility of muscle cells with age was detected.


The prevalence of hepatitis C in a regional level I trauma center population. Several studies have examined the prevalence of hepatitis B (HBV) and human immunodeficiency virus (HIV) in a trauma population. To our knowledge, no one has reported on the prevalence of hepatitis C (HCV). We prospectively studied the prevalence of HCV, as well as HBV, HIV, and syphilis in our adult regional level I trauma center population. Two hundred eighty-six consecutive trauma patients were tested for previous exposure to HCV using an anti-HCV mAb ELISA. Patients were also tested for exposure to HBV, HIV, and syphilis, and for illicit drug use. All rho values were calculated using Yates' corrected chi 2 or Student's t test. Twenty-two patients (7.7%) were found to have anti-HCV antibodies, five patients (1.7%) had active HBV, nine patients (3.2%) had HIV, and 16 patients (6%) were positive by RPR. Four (18%) of the patients seropositive for HCV tested positive for HBV, HIV, or syphilis as well. The HIV-positive patients were more likely than the HIV-negative patients to be HCV positive (rho = 0.018). Nine of the HCV seropositive patients (41%) tested positive for cocaine use. Cocaine users were more likely than nonusers to be HCV positive (rho = 0.0007). We have demonstrated the prevalence of HCV in our trauma population to be high (7.7%). It is well known that HCV has a high rate of chronicity, thus up to 90% of these patients are carriers and represent a substantial risk to health care workers. The two significant risk factors, HIV status and cocaine use, are difficult to elicit in the acute setting, reinforcing the need for adhering to universal precautions.


[A clinical and ultrastructural study of Fechtner syndrome in two Japanese families]. This is a report of Fechtner syndrome in two Japanese families. Six members of family I and three members of family II were studied. All but one had macrothrombocytopenia and leukocyte inclusion bodies, four had deafness, four had persistent proteinuria and none had cataracts. Under a diagnosis of ITP, two of them had splenectomy which resulted in no response. History revealed, other family members with deafness and/or nephritis were confirmed in both families. Ultrastructural studies of leukocytes showed oval inclusion bodies with unclear borders containing many fine ribosome like granules and randomly scattered filaments. Ultrastructural studies of macrothrombocytes were unremarkable except for a well-developed open canalicular system. More than half of megakaryocytes had uneven basophilic speckles in the cytoplasm, which were positive for Unna-Pappenheim staining. Ultrastructurally, widening of demarcating systems and remaining ribosomes were noted in the cytoplasma of mature megakaryocytes.


Localization of collagen (I) and collagenase mRNA by in situ hybridization during corneal wound healing after epikeratophakia or alkali-burn. The expression and localization of type I collagen and collagenase gene were studied by in situ hybridization using rabbit cornea during wound healing following epikeratophakia or alkali-burn. In corneas 24 days after epikeratophakia, alpha 1(I) collagen mRNA was detected in keratocytes which had migrated from the host cornea into the keratolens. In contrast, collagenase mRNA was detected in cells which seemed to be inflammatory cells around the suture between the host stroma and the keratolens. The increase of alpha 1(I) collagen mRNA in keratocytes was observed in corneas 94 days after epikeratophakia and in alkali-burned corneas 1-2 months after the burn. These results provide evidence that keratocytes synthesize collagen and that this synthesizing activity lasts for a long period during corneal wound healing.


Oxidation of 1,1-diphenylhydrazine to N-nitrosodiphenylamine by superoxide radical in the eye. By microsomes obtained from bovine ciliary body, 1,1-diphenylhydrazine was oxidized to N-nitrosodiphenylamine in the presence of NADPH. This reaction was stimulated by riboflavin which was recognized to be an electron carrier. The oxidizing activity by microsomes was markedly inhibited by superoxide dismutase, but not by SKF 525-A or carbon monoxide. Similarly, the oxidation of 1,1-diphenylhydrazine to its corresponding nitrosamine occurred in varying degrees when the hydrazine derivative was exposed to visible light in the presence of photosensitizers such as riboflavin, flavin adenine dinucleotide, flavin mononucleotide, lumiflavin, lumichrome, NAD+, NADH, NADP+, or NADPH. The photochemical oxidation was inhibited by active oxygen-scavengers such as superoxide dismutase, L-ascorbic acid or alpha-tocopherol. The superoxide radical involved in the photochemical reaction was determined by measuring the oxidation of epinephrine to adrenochrome. The oxidation of epinephrine was well correlated to that of 1,1-diphenylhydrazine. Thus, the present study provided evidence that the superoxide radical is responsible for the oxidation of a hydrazine derivative to a corresponding nitrosamine by ocular tissue microsomes and by photosensitizers.


Effect of forced-running stress on beta-adrenergic receptors in rat brain regions and liver. Rats were exposed to forced-running stress for 1 day, 3 days or a long term (approximately 2 weeks), and beta-adrenergic receptor binding was then assayed using [3H]dihydroalprenolol (DHA) in six brain regions and the liver. In the pons + med.obl., hypothalamus and midbrain, a reduction in beta-adrenergic receptor density was first evident on day 1. In contrast, a decrease in beta-adrenergic receptor density in the cerebral cortex and hippocampus was first evident on day 3. Decreased [3H]DHA binding in the pons + med.obl., cerebral cortex and hippocampus subsequently plateaued for the duration of the forced-running stress. In the midbrain and hypothalamus, however, decreased [3H]DHA binding subsequently returned to control levels despite the exposure to the forced-running stress. In the cerebellum and the liver, [3H]DHA binding did not change significantly throughout the stress. These results indicate that the forced-running stress induces both the time- and region-specific changes in beta-adrenergic receptors. Moreover, the rats showed either a behavioral depression or a spontaneous recovery of running activity during the 2 weeks following the end of the long-term stress. Thus, we also examined the relationship of beta-adrenergic receptors to these behavioral differences. [3H]DHA binding for the behavioral depression group was lower in the hippocampus and higher in the liver than for the spontaneous recovery group.


G-protein stimulation inhibits amiloride-sensitive Na/H exchange independently of cyclic AMP. G-proteins are heterotrimeric proteins involved in many transmembrane signaling events. Both the renal basolateral membrane and the renal brush border membrane contain large quantities of these proteins. G-proteins appear related to hormonal signaling in the basolateral membrane and presumably affect ion gating in the brush border. We investigated the influence of G-proteins on the amiloride-sensitive Na/H exchanger, the activity of which is regulated at least in part by cAMP-dependent protein kinase, by measuring the amiloride-sensitive component of [22Na+] uptake in rat renal brush border membrane vesicles (BBMV) in the presence of a pH gradient. Incubation of vesicles with AlF4- (10 microM Al3+, 10 mM F-) resulted in significant inhibition of amiloride-sensitive [22Na+] uptake at both 20 seconds and 5 minutes of incubation. Incorporation of GTP gamma S into BBMV by transient hypotonic lysis also resulted in significantly reduced amiloride-sensitive [22Na+] uptake compared to controls at both time points. This inhibition could be reversed by GDP beta S. Similar lysis in the presence of 10 microM GDP beta S alone had no significant effect. When Na(+)-dependent [14C]-D-glucose uptake into BBMV was studied no significant effect of these G-protein modulating agents was observed. Adenylate cyclase activity could not be stimulated in these BBMV preparations using standard techniques. Furthermore, cAMP-dependent protein kinase activity, strongly stimulated in these BBMV by exogenously added cAMP, was not stimulated by 10 microM GTP gamma S alone. These findings suggest that the amiloride-sensitive Na/H exchanger can be regulated by G-proteins independently of adenylate cyclase and cAMP-dependent protein kinase.


Endothelin-1 stimulates the Na+/H+ and Na+/HCO3- transporters in rabbit renal cortex. Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor identified to date, raising the strong possibility of its involvement in the pathogenesis of systemic hypertension. Whether ET-1 exerts a direct stimulating effect on sodium reabsorption in the renal proximal convoluted tubule, the dominant locus of sodium reabsorption in the nephron, is currently unknown. Such an effect would suggest yet another mechanism by which ET-1 might mediate systemic hypertension. In studies on membrane vesicles prepared from rabbit renal cortex, we show that ET-1 (10(-8) to 10(-11) M) exerts dose-dependent stimulation of the apical Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter; preincubation of vesicles with 10(-10) M ET-1 for five minutes enhanced the activity of each transporter by approximately 25%. This stimulation reflected an increase in the Vmax of each transporter but no change in the Km for sodium. The stimulatory effect of ET-1 was blocked in the presence of an ET-1 antiserum. Moreover, the stimulation of the apical Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter by ET-1 displayed specificity as indicated by the lack of effects on the activities of the apical Na(+)-glucose transporter and the basolateral Na(+)-succinate transporter. The data implicate ET-1 as a novel, direct and specific modulator of sodium reabsorption in the proximal tubule. As such, ET-1 might be a direct determinant of extracellular fluid volume under normal and pathophysiologic circumstances, including hypertensive disorders.


Mechanism of glycine protection in hypoxic injury: analogies with glycine receptor. Addition of glycine to the recirculating perfusate of isolated perfused rat kidneys protects against hypoxic injury to the medullary thick ascending limb and slows functional deterioration in the course of perfusion. This effect is dependent on dose; the earliest significant protection is seen at 0.25 mM, and the protective effects increase as glycine concentration is increased to 2 mM, the highest level tested. Two specific agonists of the strychnine-insensitive (NMDA) glycine receptor in neural membranes, 1-aminocyclopropane carboxylic acid (ACC) and d-serine, also exerted a cytoprotective effect at a concentration of 2 mM. On the other hand, 1-serine and taurine, ineffective agonists of the NMDA-glycine receptor but effective agonists of the strychnine-sensitive glycine receptor, had no protective effect in this system. Two antagonists to glycine at its binding site on the N-methyl-D-Aspartate (NMDA) receptor, 7-chlorokynurenic acid (2 mM) and indole-2-carboxylic acid (12.5 mM), did not reverse the cytoprotective action of 0.25 mM glycine. The data are consistent with a ligand-acceptor type of interaction to account for cytoprotection. The configuration of the glycine acceptor may resemble, but is not identical with, that of certain glycine receptors in the nervous system.


Multiple patterns of 11 beta-hydroxysteroid dehydrogenase catalytic activity along the mammalian nephron. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is thought to be a protective enzyme of the mineralocorticoid receptor (MR). We have previously demonstrated (Bonvalet et al, J Clin Invest 86:832-837, 1990) that 11 beta-OHSD is colocalized with MR along the rabbit nephron. In the present study, we examined whether 11 beta-OHSD is similarly located along the nephron of other mammals. Various tubular segments were microdissected from the mouse, rat, and rabbit nephron, and incubated for two hours at 37 degrees C in the presence of 11 nM [3H]-corticosterone (B). Thereafter, the respective amounts of B and [3H]-11dehydrocorticosterone (A) in the incubation solution were measured by HPLC. In the rabbit, the mouse and the rat, about 520 pmol/10 mm of B were transformed into A in tubular segments possessing MR, that is, the distal parts of the nephron (distal and collecting tubule). Differences appeared in the aldosterone-insensitive proximal tubule; in both the initial and final parts of this segment, 11 beta-OHSD activity was low (26 pmol/10 mm) in the rabbit and the mouse, and relatively high in the rat (328 pmol/10 mm). In the cortical part of the loop of Henle, where the presence of MR is still under discussion, 11 beta-OHSD activity was low in the mouse (70 pmol/10 mm), high in the rat (533 pmol/10 mm) and intermediate in the rabbit (227 pmol/10 mm). The comparison of these results with previous data obtained with immunohistochemical methods suggests that the proximal and distal nephron might express different isoforms of 11 beta-OHSD.


Renal receptors and effects of atrial natriuretic factor in compensatory renal hypertrophy. In the present study we investigated the in vivo and in vitro renal responsiveness to ANF, and the adaptation of ANF receptors in compensatory renal hypertrophy in the rat. One week after left nephrectomy (UNx), plasma levels of immunoreactive ANF, blood pressure (MAP), hematocrit (Hct), and urine flow rate (V) were unaltered compared to control (C) rats. Baseline GFR and potassium excretion (UKV) were significantly higher, and sodium excretion (UNaV) tended to be elevated in UNx rats. Administered ANF led to similar dose-related decreases in MAP and increases in Hct in UNx and C rats. However, at each dose of infused ANF, absolute values and the increase in GFR and UNaV were higher in UNx than in C rats. Hypertrophied (H) kidneys were removed from UNx and perfused in vitro to determine distribution and density of ANF receptors, responsiveness to ANF, and receptor-mediated organ clearance of 125I-ANF1-28. The density of ANF receptors in cortex, outer medulla, and papilla of H kidneys was not significantly different from that in C kidneys. In H isolated kidneys, ANF led to dose-related increases in GFR, V, UNaV, and UKV that were indistinguishable (P greater than 0.05) from those in C kidneys. Receptor-mediated organ clearance of 125I-ANF1-28 in isolated H kidneys was 2.8 +/- .02 ml/min, a value not significantly different (P greater than 0.05) from that in C kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)


[Proteolytic and collagenolytic activities of the bovine vitreous body]. The vitreous contains collagen and soluble non-collagen proteins. The degeneration of collagen takes place in a different manner than in the other proteins. It has been detected that the cattle vitreous does not show any collagenolytic activity against the basic types of collagen (I, II, IX, and XI). The vitreous digests haemoglobin only in the acidic section of pH (optimum 3.5-4.5). No proteolytic activity towards Hb in physiological pH has been found.


Inhibition of adenylate cyclase by delta 9-tetrahydrocannabinol in mouse spleen cells: a potential mechanism for cannabinoid-mediated immunosuppression. The ability of delta 9-Tetrahydrocannabinol (delta 9-THC) to modulate adenylate cyclase activity in mouse spleen cells was investigated. These studies were prompted by the recent identification and cloning of a G-protein coupled cannabinoid receptor localized in certain regions of the brain and the potential for a common mechanism between cannabinoid-mediated CNS effects and immunosuppression. Temporal addition studies were initially performed to identify the period of time when spleen cells in culture were most susceptible to the inhibitory effects of delta 9-THC, as measured by the day 5 IgM antibody forming cell response. delta 9-THC was only inhibitory when added to spleen cell cultures during the first 2 hr following antigen sensitization. In light of this time course, adenylate cyclase activity was measured in spleen cells incubated in the presence of 22 microM delta 9-THC for 5 min and subsequently stimulated with forskolin. delta 9-THC treated spleen cells demonstrated a 33% inhibition and a 66% inhibition in intracellular cAMP after a 5 or 15 min stimulation with forskolin, respectively. These studies suggest that inhibition of immune function by delta 9-THC may be mediated through the inhibition of intracellular cAMP early after antigen stimulation.


Effects of human interleukin 3, macrophage and granulocyte-macrophage colony-stimulating factor on monocyte function following autologous bone marrow transplantation. The effects of human interleukin 3 (IL-3), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied on the functional activity of human peripheral blood monocytes from healthy individuals and from eight patients at 4, 8 and 12 weeks following autologous bone marrow transplantation (ABMT). Functions studied included superoxide production, phagocytosis of Candida albicans and reduction of 3-[4,5-dimethylthiazol-2-yl]-2.5-diphenyl tetrazolium bromide (MTT). IL-3 and GM-CSF significantly enhanced the oxidative metabolism of monocytes from healthy individuals, while the effect of M-CSF was moderate. A considerable variability between healthy individuals was found in both resting and cytokine-stimulated monocytes with regard to superoxide production. All three investigated CSFs, i.e. IL-3, M-CSF and GM-CSF did not affect phagocytosis of C. albicans by the cells or their metabolic activity (reduction of MTT). In ABMT patients no deficit in the functional activity of monocytes was found at any time after transplantation and all three CSFs investigated did not modulate the functional activity of the cells. These results suggest that monocytes do not have a major role in infectious complications post-ABMT.


Effects of amphetamine on the development of MTV-induced mammary tumors in female mice. Female C3H/He mice carrying the mammary tumor virus (MTV) were monitored for mammary tumor incidence and latent periods while submitted to a daily subcutaneous injection with amphetamine (0,4 mg/kg/day). Results show that amphetamine caused an increase in incidence and a decrease in latency of tumors compared with placebo. There was also appreciated a correlation with the lethality of mice.


A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors. A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.


Identification of apamin binding sites in rat intestinal mucosa. Apamin, a specific blocker of one class of Ca(2+)-activated K+ channes, was used to detect the apamin receptors associated with K+ channels in the mucosa of the rat jejunum and colon. Two receptor sites for 125I-apamin have been identified. These sites differed in their affinity for apamin (jejunum: KD1 = 1.1 nM and KD2 = 170 nM; colon: KD1 = 0.5 nM and KD2 = 1.1 nM and KD2 = 140 nM) and the maximum number of sites (jejunum: B(max1) = 111 and B(max2) = 4030; colon: B(max1) = 187 and B(max2) = 7550 fmol/mg of protein). 125I-apamin binding was stimulated by K+ ions with K0.5 = 1.0 mM and inhibited by the neuromuscular blocker tubocurarine (KI = 50 microM). We interpret these data to demonstrate that the high-affinity, low-capacity binding sites reflect the existence of apamin-sensitive K+ channels in the intestinal mucosa.


[Comparative analysis of biological aggressiveness of dusts from different coal mines for determining the risk of development of pneumoconiosis among miners]. The aim of the study was to determine the changes in the respiratory system of animals administered intratracheally settled dusts sampled from the following coal mines: "Debieńsko" in Leszczyny, "Zabrze" in Zabrze, "Gliwice" in Gliwice, "Janina" in Libiaz and "Victoria" in Wałbrzych. Total dustiness in those mines in 1985-1988 was found to be as follows: "Debieńsko"--1.39 mg/m3-22.3 mg/m3, "Zabrze"--7.6 mg/m3-16.8 mg/m3, "Janina"--16.0 mg/m3-34.0 mg/m3, "Victoria"--3.7 mg/m3-15.6 mg/m3. In the examined dusts the content of crystalline silica determined using the Polezajev's method amounted to 3.5%-9.4% in "Debieńsko", 2%-10% in "Zabrze", 3.8% in "Gliwice", 3.6%-8.4% in "Janina" and 3.7%-11% in "Victoria". The biological aggressiveness of the mine dusts was determined using intraperitoneal, lung and hemolitic tests. The biochemical determinations of hydroxyproline level were made using the Stegemann's method modified by Hurych and Chvapil. The obtained results of biochemical analyses were examined statistically using the t-Student's test. The lung sections for histopathological examination were stained with hematoxylin and eosin. Collagen fibres were stained according to the Van Giesan's method. Certain discrepancies were found between epidemiological analysis concerning the incidence of pneumoconiosis and animal experiments focusing on the fibrogenic activity of the dusts from particular mines. The measurements of fibrogenic activity of dusts based on animal experiments and the determination of SiO2 content in dusts cannot be used for estimating the risk of pneumoconiosis. Therefore, biological exposure should be assessed on the basis of the monitoring of occupational environment.


[Evaluation of long-term occupational exposure to dust and its effect on health during production of ceramic tiles]. A medical examination has been carried out of 500 workers (290 men and 210 women) of a ceramic plates plant. Also, the measurements of dust concentrations were made at some standard work places . In the materials used for manufacturing the plates crystalline phases and the content of free crystalline silica were determined using the X-ray diffraction method. In the animal experiments the fibrogenic activity of all materials used in the plant was examined and compared to the fibrogenic activity of standard quartz. As a result of the medical examination 64 cases of pneumoconiosis were diagnosed (13% of the subjects). The incidence rate of pneumoconiosis was similar for men and women. The radiological changes characteristic of pneumoconiosis took approximately 24 years of the workers tenure to develop. Type q changes were most frequent (69%), types p and r were observed in 14% of workers (mostly women). In 31% of workers tuberous changes of size B were observed. In 43.8% of the subjects restrictive disorders of ventilation were found. In 30% of workers chronic bronchitis was diagnosed. Dust concentrations at 11 work places were measured using the individual dosimetry method. Total dust concentrations ranged from 0.6 mg/m3 at the electricians posts to 60.1 mg/m3 at the workposts where the furnace truck restorers worked. Dust concentrations exceeded the MACs at 7 workposts. The respirable fraction concentrations ranged from 0.1 mg/m3 to 8.4 mg/m3. During the replacement of asbestos ropes and asbestos board used for insulating the furnace trucks mineral fibres (0.1-0.5 fibre/cm3) were found in the air. The following crystalline phases were determined in the materials: kaolinite, illite, quartz, orthoclase and microline.(ABSTRACT TRUNCATED AT 250 WORDS)


[Interpretation of early radiological changes in the diagnosis of pneumoconiosis among coal miners]. Apart from certain changes which are typical for pneumoconiosis, the radiological picture of the lungs of sigma coal miners does frequently show some irregular small opacities of s, t and u types. The role and specificity of these changes in the early diagnosis of pneumoconiosis has not been too well defined by now. A 10-year study (conducted at 2 or 3 year intervals) was carried out among 150 miners from 2 mines characterized by different dust loading. Some irregular changes in the miners' lungs were observed. The control group derived from the same mines comprised 115 miners with no radiological changes found in their lungs. The evolution of radiological changes took place in 55.3% of the miners and was more intensive in the heavily dusted mine. Radiological changes were revealed in 38.3% of the controls. It was indicated that pneumoconiosis results much more frequently (38.6%) from the evolution of the irregular changes rather than directly from the proper radiological picture of the lungs (5.3%). In 44.7% of the subjects the changes of s, t and u type did not undergo any evolution, which may be due to their non-specific characteristics. The evolution of irregular opacities is dominating in the patients with bronchitis and emphysema. No significant correlation between smoking and the progress of irregular opacities was found. The observation of the further exposure to the dusts did not produce any clear results. The progress of the changes of s, t and u type was observed more frequently in those still working under ground, but more cases of pneumoconiosis were found in the miners who stopped working. This fact indicates that the further exposure affects the s, t and u type changes and confirms the observations by other authors concerning the manifestation of pneumoconiosis after the break of exposure. The results of the 10-year study prove that the miners with this sort of changes are exposed to a higher risk of pneumoconiosis, although the answer concerning specificity of irregular changes in the radiological picture has not yet been found.


The effect of endothelin 1 on the retinal microvascular pericyte. The effect of the highly vasoactive peptide endothelin 1 (ET1) was tested on bovine retinal microvascular pericytes propagated in vitro. Specific binding of 125I-ET1 to retinal pericytes was documented by autoradiography. ET1 caused contraction of pericytes at a concentration of 0.1 nM which was accompanied by increases in inositol phosphates. Exposure of pericytes to 10 nM ET1 resulted in the aggregation and realignment of muscle-specific actins into bundles which were oriented parallel to the long axis of the cell, and ET1 was also mitogenic to pericytes in the presence of low levels of fetal calf serum. These observations suggest that ET1 may play an important role in endothelial cell-pericyte interactions within the microvasculature of the retina and that it may be involved in the autoregulation of retinal blood flow.


Oestrogen and cyclical progestogen in postmenopausal hormone replacement therapy. To ascertain the effectiveness, safety and incidence of side effects of a postmenopausal hormone replacement regimen consisting of continuous conjugated equine oestrogens and cyclical medroxyprogesterone acetate given for the first 14 days of each calendar month. A six month, prospective, open label, two centre, outpatient study of continuous Premarin (0.625 mg; Wyeth-Ayerst) and cyclical Provera (10 mg; Upjohn). Dosage adjustment was allowed in one centre. Seventy-six postmenopausal women in Adelaide and Brisbane. Menopausal symptom score, serum lipid levels, routine biochemical and haematological indices, endometrial histology and clinical bleeding pattern, blood pressure, weight changes, side effects, withdrawal from the study, compliance and necessary dose adjustment. Eight women withdrew from the study and nearly 50% experienced some minor side effect. Where dosage adjustment was allowed, almost all side effects were eliminated. Most patients had acceptable regular withdrawal bleeds although some were deemed heavy. There was a statistically highly significant 54% reduction in the menopausal symptoms score at three months and a 62% reduction at six months. Endometrial biopsy at six months showed atrophic or secretory endometrium with no inappropriate proliferation or hyperplasia. Total cholesterol and low density lipoprotein (LDL) cholesterol levels were significantly decreased. The high density lipoprotein (HDL) cholesterol level remained unchanged and triglyceride levels were raised within the normal range. There were no other clinically relevant biochemical, haematological or clinical changes. Continuous conjugated equine oestrogens (0.625 mg) and cyclical medroxyprogesterone acetate (10 mg) for the first 14 days of each calendar month proved to be a safe and effective postmenopausal therapy regimen. Initial minor side effects were common but could be readily ameliorated with early follow-up and dose titration.


Isolation of Kunjin virus from a patient with a naturally acquired infection. To describe the first isolation of Kunjin virus from a patient with a natural infection. A 48-year-old female egg collector presented with muscle weakness, fatigue and extreme lethargy three weeks after developing rigors, headache, photophobia and nausea. Kunjin virus was isolated from an acute phase serum sample. The patient made a partial recovery after treatment for 10 days with Catovit (Boehringer Ingelheim), one tablet twice a day, and then declined further medical contact. The isolation of Kunjin virus from this patient confirms previous serological observations which suggested that this mosquito-borne virus caused febrile episodes in humans accompanied, on occasion, by polyarthralgia or mild central nervous system signs and symptoms.


Arboviral disease--United States, 1991.

Arboviral disease--United States, 1991. During 1991, state and local health departments reported 122 cases of human arboviral encephalitis to CDC. More than half (69) of the cases resulted from outbreaks of St. Louis encephalitis (SLE) in Arkansas and Texas. In addition, an epizootic of eastern equine encephalitis (EEE) extending from the Atlantic and Gulf coasts into the upper midwest caused sporadic human cases and a substantial loss of livestock. This report summarizes the reported cases of arboviral encephalitis in the United States during 1991 and underscores the continuing need for arbovirus surveillance and control.


Laboratory-based surveillance for rotavirus United States, January 1989-May 1991. Geographic and temporal trends of rotavirus detections in the United States for the period January 1989-May 1991 were determined by analyzing data reported monthly by 47 virology laboratories participating in the North American Rotavirus Surveillance System. Reports included complete information on the number of specimens tested, the number of test results positive for rotavirus, and the method used to detect rotavirus. Consistent trends in regional and geographic area were identified, with distinctly different peaks of rotavirus activity in the western and eastern states. Each year in the western states, rotavirus activity began in November and peaked in December-January, whereas in the eastern states activity began in January and peaked in February-March. These differences do not correlate with obvious trends in strain variation of rotavirus and remain unexplained. Unexpected reporting of summer rotavirus activity by some laboratories in 1989 was traced to the use of a single diagnostic kit and to two questionable laboratory practices: having more than six medical technologists perform the test and failure to use controls with the test. Laboratory-based surveillance of rotavirus activity has proven to be useful in identifying and correcting problems in laboratory methods for detecting rotavirus and will be a sensitive means for monitoring coverage of the rotavirus vaccine now being developed.


Molecular structure and pharmacological characterization of humEAA2, a novel human kainate receptor subunit. A cDNA encoding a novel human glutamate receptor subunit protein was isolated from a human hippocampal library. This cDNA, termed humEAA2, is most closely related to rat cDNAs for kainate receptor proteins and, when expressed in COS cells, is associated with high affinity kainate receptor binding. The relative potency of compounds in displacing [3H]kainate binding was kainate greater than quisqualate greater than domoate greater than L-glutamate much greater than 6,7-dinitroquinoxaline-2,3-dione greater than dihydrokainate greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. Homomeric expression of humEAA2 does not appear to elicit ligand-gated channel activity. Nevertheless, the molecular structure and pharmacology of high affinity kainate binding suggest that humEAA2 is a novel subunit protein of a human kainate receptor complex.


Relaxation of pig coronary arteries by new and potent cGMP analogs that selectively activate type I alpha, compared with type I beta, cGMP-dependent protein kinase. Smooth muscle preparations of human aorta or pig coronary arteries contain nearly equal amounts of cGMP-dependent protein kinase isozymes (cGMP kinase I alpha and I beta). In order to understand the roles of these isozymes in relaxing vascular smooth muscle, several new cGMP analogs were synthesized and tested for potencies in activating each enzyme and in relaxing pig coronary arteries. Analogs modified with a derivatized phenylthio group at the 8-position were as much as 72-fold more potent in activating purified cGMP kinase I alpha than cGMP kinase I beta. Electron-donating substituents, such as hydroxy, amino, and methoxy, on the phenyl ring enhanced the potencies of these analogs in activating cGMP kinase I alpha. The most potent of these cGMP analogs [8-(4-hydroxyphenylthio)-cGMP] was 17 times more potent (EC50 = 1.1 microM) as a muscle relaxant than the most efficacious analog tested previously. Among derivatives with an 8-halo group, 8-iodo-cGMP was the most potent compound (Ka = 9 nM for I alpha and 122 nM for I beta) for both I alpha and I beta. Analogs modified at the 1,N2-position or at both the 1,N2-and 8-positions of cGMP were highly potent for activating both isozymes. Within this group, 8-I-beta-phenyl-1,N2-etheno-cGMP had Ka values of 22 nM and 17 nM for cGMP kinase I alpha and I beta, respectively, whereas the Ka values of cGMP were 110 nM and 250 nM for the two isozymes. 8-I-beta-phenyl-1,N2-etheno-cGMP was the most potent muscle relaxant tested, with EC50 of 0.4 microM. For all cGMP analogs tested, there was a positive correlation between potency for activation of cGMP kinase I alpha and that for relaxation of pig coronary arteries. Assuming that the kinase assay conditions yielded a cyclic nucleotide specificity similar to that which would exist in intact cells, it was concluded that the cGMP kinase I alpha isozyme mediates the relaxation of pig coronary artery smooth muscle caused by cGMP elevation. However, an additional role for cGMP kinase I beta in the relaxation process could not be ruled out.


Study design considerations in developmental neurotoxicology. It is widely accepted that exposure to environmental factors during development can result in effects other than death, gross structural abnormality, or altered growth. One area of concern is the developing nervous system, which may be especially vulnerable to environmental perturbation. Testing chemicals for potential developmental neurotoxicity has received a high priority and testing guidelines have been published and recently revised by the U.S. Environmental Protection Agency. These guidelines are based on several principles of developmental neurotoxicity that have been developed during several years of research. In general, manifestation of neurotoxicity following developmental exposure can depend on the time at which exposure occurs and for the purposes of hazard detection, experiments should be designed to optimize the detection of neurotoxicity. In addition, maternal health and interaction with the offspring, as well as postnatal development are important design issues in developmental neurotoxicology. It is also widely accepted that several doses be used and multiple measures of neurotoxicity assessed in both genders at several points during the life span of the animal. Finally, the litter is usually regarded as the most appropriate statistical unit to control for genetic and maternal factors.


Characterization of opioid receptors mediating stimulation of adenylate cyclase activity in rat olfactory bulb. We have investigated the pharmacological profile of the opioid stimulation of adenylate cyclase activity in rat olfactory bulb, in order to identify the opioid receptor subtype(s) involved in this response. The synthetic delta-selective agonists (D-Ala2)deltorphin I, (2-D-penicillamine,5-D-penicillamine)-enkephalin, and (D-Ser-Leu5-enkephalyl)-threonine were effective stimulators of the enzyme activity, with EC50 values of 6.7, 420, and 63 nM, respectively. A significant increase was also observed with the mu-selective agonists (N-methyl-Phe3,D-Pro4)-morphiceptin, dermorphin, and (D-Ala2-N-methyl-Phe4-Gly-ol5)-enkephalin (DAGO). The latter two agonists displayed biphasic concentration-response curves, with high affinity components accounting for 75-80% of the maximal responses. The kappa-selective agonists U-50,488 and U-69,593 were ineffective, whereas (D-Ala2)dynorphin A-1-11, dynorphin A, dynorphin A-1-13, and dynorphin A-1-6 acted with a rank order of potency consistent with their affinity for delta receptors. The stimulatory responses of Leu-enkephalin, beta-endorphin, dynorphin A, and delta-selective agonists were counteracted by naltrindole with pA2 values of 9.39-8.93, whereas naloxone was less potent (pA2 = 8.17-7.59). The kappa-selective antagonist norbinaltorphimine was the least potent. The inhibition by naltrindole and naloxone of DAGO stimulation showed biphasic curves, with 90% of the response being antagonized more potently by naloxone than by naltrindole. These results demonstrate that delta- and mu- but not kappa-opioid receptor subtypes stimulate basal adenylate cyclase activity in rat olfactory bulb.


Beta-adrenergic receptor levels and function after growth of S49 lymphoma cells in low concentrations of epinephrine. Growth of S49 wild-type (WT) lymphoma cells for 24 hr with 3 nM epinephrine produces a very pronounced attenuation of cAMP accumulation in response to subsequent challenges with much higher concentrations of the catecholamine [Mol. Pharmacol. 36:459-464 (1989)]. We report here the effects of this treatment, in S49 WT, cyc-, and kin- cells, on the responsiveness of adenylate cyclase in partially purified membranes. The desensitization of adenylate cyclase in the S49 WT cells after 24-hr treatment was homologous, in that only responses to epinephrine were attenuated. The EC50 for epinephrine stimulation of adenylate cyclase was 54 +/- 8% (mean +/- standard error) higher in treated cells than in controls, and there was a 32 +/- 3% reduction in Vmax at supramaximal epinephrine concentrations. The treatment also caused a 34 +/- 9% reduction in the levels of the beta-adrenergic receptor (beta AR), which was of a sufficient magnitude to account for the homologous desensitization seen. The 24-hr treatment had similar effects in S49 kin- cells, where we observed a 28 +/- 4% decrease in Vmax, a 35 +/- 6% increase in EC50 for epinephrine stimulation of adenylate cyclase, and a 25 +/- 3% decrease in beta AR. In contrast, the 24-hr treatment had no measurable effect on adenylate cyclase activity in S49 cyc- cells. That is, the responsivity of adenylate cyclase reconstituted with Gs from S49 WT cells was not attenuated, although beta AR levels were significantly decreased. The desensitization of S49 cells with the 24-hr treatment was additive with that mediated by the cAMP-dependent protein kinase (cAPK). Further, unlike the cAPK-mediated attenuation, it was relatively insensitive to the levels of free Mg2+ in the adenylate cyclase reaction mixture. The characteristics of the desensitization produced by 24-hr treatment with 3 nM epinephrine, together with the observation that it is similar in S49 WT and kin- cells, demonstrates that the process in WT cells is, at least in part, independent of the rapid cAPK-mediated desensitization. It is also most likely that it is unrelated to the rapid cAMP-independent processes involving sequestration/internalization or the beta AR kinase, because those mechanisms require much higher receptor occupancies than the 0.2% occurring with 3 nM epinephrine. Thus, 24-hr treatment appears to produce attenuation of adenylate cyclase by causing down-regulation of beta AR, without involving any other known form of desensitization.


Existence and alpha 1-adrenergic stimulation of inositol polyphosphates in mammalian heart. The concentration-response curves and the time course of the effects of phenylephrine (0.01-100 microM) on force of contraction and on inositol polyphosphates in isolated electrically stimulated perfused rat hearts (Langendorff technique) were studied. A nonradiometric high performance liquid chromatography metal dye detection technique was used to determine absolute concentration masses/changes of inositol polyphosphates in heart. Products measured after separation with high performance liquid chromatography were inositol 1,4,5-trisphosphate (1,4,5-IP3), inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4) and its isomer 1,3,4,6-IP4, inositol 1,3,4,5,6-pentakisphosphate (1,3,4,5,6-IP5), and inositol hexakisphosphate (IP6). 1,4,5-IP3 (significant at 10 microM) and both IP4 isomers (significant at 1 microM) increased after alpha-adrenoceptor stimulation, whereas 1,3,4,5,6-IP5 and IP6 remained unaffected. Phenylephrine had a concentration-dependent positive inotropic effect (significant at 1 microM). All effects were antagonized by the alpha 1-adrenoceptor antagonist prazosin (0.1 microM), indicating receptor-mediated effects. In a time course study 1,4,5-IP3 was the first compound to increase significantly, within 1 min after stimulation; this rise was followed by an increase in 1,3,4,5-IP4 beginning within 2 min. The increase in all other inositol polyphosphates was slower (5-10 min). The increase in the force of contraction started at 2 min. For comparison, the effects of the beta-adrenoceptor agonist isoprenaline were studied. Isoprenaline produced a positive inotropic effect similar to that of phenylephrine, but all inositol polyphosphates remained unaffected. In conclusion, for the first time the existence of 1,3,4,5,6-IP5 and IP6 was observed in the heart. However, the physiological role of these inositol polyphosphate isomers in the heart remains to be elucidated, because, from the time course, they appear to have no acute intracellular second messenger function. Increased inositol polyphosphate turnover may be involved in the mechanism(s) whereby alpha 1-adrenoceptor stimulation produces an increase in myocardial force of contraction. Because the increase in 1,4,5-IP3 precedes and that in 1,3,4,5-IP4 coincides with the increase in the force of contraction, 1,4,5-IP3 may initiate and 1,3,4,5-IP4 may maintain the positive inotropic effect of alpha 1-adrenoceptor agonists.