Effects of (R)-8-OH-DPAT and the enantiomers of UH-301 on motor activities in the rat: antagonism of (R)-8-OH-DPAT-induced effects. The effects of the enantiomers of 5-fluoro-8-hydroxy-2-(dipropylamino)tetralin, UH-301 and the potent 5-HT1A-receptor agonist (R)-8-hydroxy-2-(dipropylamino)tetralin, (R)-8-OH-DPAT, on locomotion, rearing and total activity were studied in rats. The experiments were performed as tests either of exploratory activity in non-habituated rats or of motor activity of rats habituated to the environment for 2 h before drug injection. (R)-8-OH-DPAT increased locomotion and total activity and decreased rearing in both conditions. (R)-UH-301 increased locomotion and slightly also total activity in habituated rats and decreased rearing in both conditions. (S)-UH-301 decreased locomotion and rearing in both conditions but only in doses of 10 mumol/kg and above. Lower doses of (S)-UH-301 (10 mumol/kg) antagonized (R)-8-OH-DPAT-induced increases of locomotion and total activity. As (S)-UH-301 decreased rearing, per se, it was not able to antagonize the (R)-8-OH-DPAT induced decrease. These results further support previous data that (S)-UH-301 is a 5-HT1A antagonist while (R)-UH-301 is a 5-HT1A agonist.


A gibberellin response complex in cereal alpha-amylase gene promoters. The Amy32b gene is a representative member of a closely related family of alpha-amylase genes expressed under hormonal control in aleurone layers of barley grains. Transcription of this gene is induced by gibberellin (GA) and suppressed by abscisic acid. In this study, we functionally defined the promoter elements of the Amy32b gene that govern the developmental and hormonal control of its expression in aleurone. Two functionally distinct yet physically associated elements are essential: a gibberellin response element mediates regulation by GA and abscisic acid, and an Opaque-2 binding sequence (O2S) is thought to interact with a barley homolog of the maize endosperm-specific transcriptional regulator Opaque-2. An additional element CCTTTT, which with the O2S forms part of a canonical "endosperm box," is important in modulating the absolute level of expression of the Amy32b promoter, as is another separate, highly conserved element TATCCATGCAGTG.


Passive tumor targeting of soluble macromolecules and drug conjugates. The biodistribution of soluble macromolecules is governed extensively by their ability to penetrate endothelial layers. Many solid tumors possess vasculature that is hyperpermeable to macromolecules, not always correlating with the presence of interendothelial cell fenestrations. The exact physiological mechanisms responsible for this nonspecific leakiness are not yet fully understood. Together with enhanced vascular permeability, however, tumors usually lack effective lymphatic drainage; consequently, they selectively accumulate circulating macromolecules (up to 10% of an i.v. dose per gram in mice). This "enhanced permeability and retention effect" (EPR effect) has been studied extensively, and it is thought to constitute the mechanism of action of SMANCS (styrene-maleic/anhydride-neocarzinostatin), now in regular clinical use in Japan for the treatment of hepatoma. It seems likely that EPR also contributes to the anticancer activity of the N-(2-hydroxypropyl)methacrylamide copolymer-anthracycline conjugates which are shortly to undergo clinical evaluation in the U.K.


Contact dermatitis in hairdressers: the Italian experience. Gruppo Italiano Ricerca Dermatiti da Contatto e Ambientali. A multicenter study was performed in 9 Italian centers by members of the GIRDCA, to evaluate the frequency and source of contact sensitization in a group of 302 hairdressers with dermatitis. Occupational habits and use of preventive measures were specifically investigated both in these 302 hairdressers and in a further group of 240 hairdressers who answered a questionnaire. The results showed the presence of an occupationally relevant sensitization in 60.9% of the 302 hairdressers. This proportion included 52 hairdressers who had negative patch tests to the hairdressers' series but showed positive reactions to other allergens, such as nickel, rubber additives, preservatives and fragrances, which were judged relevant to their occupation. Among hair dyes, PPD caused 73 reactions (24.2%), PAP 32 reactions (10.6%), ONPPD 24 reactions (7.9%), and PTD 40 reactions (13.2%). A low incidence of sensitization was detected in our hairdressers to resorcinol and pyrogallol (1.3% for each substance). Among permanent wave allergens, positive reactions to GMTG were found in 11.3% of patients, while ATG gave a lower rate of positive reactions (5.0%). Allergic contact dermatitis due to APS was also relatively common (11.3%). 4 hairdressers in this study gave a positive reaction 30 min after a provocative test with latex gloves, patch testing to the rubber series being negative. Enquiry regarding preventive measures revealed that the majority of hairdressers use gloves when doing hair dyeing, but rarely use them for washing dyed hair or for doing permanent waving. The infrequent use of preventive measures by Italian hairdressers was confirmed by the results of the questionnaire, and possibly explains the high frequency of skin problems (12.5%) in the hairdressing population that was specifically interviewed.


How satisfied are parents of pre-school children who have special needs with the services they have received? A consumer survey. The parents of 41 pre-school children with special needs volunteered to participate in a consumer survey to ascertain their satisfaction with the services they had received and how these could be improved. The survey produced an overall high level of satisfaction, although parents felt they had not received as much information as they wanted on their child's condition (29%), available help for their family (44%), financial benefits (61%), or information about their child's future (61%). Families also felt that they had not received enough family support (43%), and that professionals regularly did not understand their concerns (32%). Ways in which these issues could be addressed are discussed, along with more general issues of such consumer satisfaction surveys.


Early social-emotional development in blind infants. In order to study the impact of blindness on social and emotional development during the first year of life, the level of social-emotional development was compared in blind and sighted 9- and 12-month-old infants. The five 9-month-old and the 17 12-month-old blind infants were completely blind from birth and exhibited no further serious disabilities. Social-emotional development was assessed with a scale from the Bielefeld Developmental Test for Blind Infants and Preschoolers containing three subscales on emotions, social interaction and impulse control. Compared to non-disabled infants, blind infants exhibited a more limited repertoire of facial expressions and less responsiveness. They less frequently attempted to initiate contact with their mothers (self-initiated interactions) or comply with simple requests and prohibitions than sighted infants. These differences in the social-emotional development of blind and sighted infants are traced back to the effects of blindness on the mother-child interaction. The lack of visual perception appears to impede particularly the acquisition of a dialogue concept.


Allergic contact dermatitis from resin hardeners during the manufacture of thermosetting coating paints. 5 production operators from 2 factories manufacturing thermosetting coating paint developed work-related skin disorders within 12 months of the introduction of a new powdered paint product. All 5 workers were found to have allergic contact dermatitis from 2 epoxy resin hardeners, both of which were commercial preparations of triglycidyl isocyanurate (TGIC). 2 of the workers had concomitant sensitization to epoxy resin in the standard series and several of the epoxy resin preparations at the workplace. TGIC has been reported as a contact sensitizer both in persons producing the chemical and among end-users of TGIC-containing products. These 5 reported cases document allergic contact dermatitis from commercial TGIC among exposed workers during an intermediate process of powdered paint manufacture. The possibility of substituting this epoxy resin hardener with less sensitizing alternatives should be explored.


Hand dermatitis in the pottery industry. Irritant hand dermatitis has long been recognized in the pottery industry. In our series, among workers handling glaze, sensitization to chromate was common and allergy to other metals and to biocides also occurred. Allergy to oil additives was found in mould makers. Whilst irritant hand dermatitis does occur, allergy to metals and biocides should be looked for in workers handling glazes, and allergy to oil additives in mould makers.


Failure of ondansetron to block the discriminative or reinforcing stimulus effects of cocaine in the rat. Ondansetron (GR38032F), a serotonin 5HT3 antagonist, is active in numerous behavioral paradigms and neurochemical systems. Since 5HT3 antagonists have been suggested as therapeutic agents for the treatment of drug abuse, the action of ondansetron on cocaine drug discrimination and self-administration paradigms in rats was investigated. Doses of ondansetron (0.001 - 1.0 mg/kg) had no effect on the discriminative stimulus properties of 10 mg/kg cocaine. In contrast SCH23390, a dopamine D1 antagonist known to block cocaine discrimination, acted as previously reported. Ondansetron did not augment the effects of SCH23390, but at higher doses, combinations of ondansetron and SCH23390 produced disruption of lever pressing in the presence of cocaine. Ondansetron (0.001-1.0 mg/kg) had no effect on the self-administration of various doses of cocaine, nor did it have any effect on reacquisition of cocaine self-administration in animals with a history of active administration followed by a period of abstinence. As before, SCH23390, known to block cocaine self-administration, acted as previously reported. Although other 5HT antagonists may prove to be efficacious in cocaine abuse, ondansetron appears unlikely to alter the subjective or rewarding stimulus properties of cocaine.


[Subacute progressive polyneuropathy syndrome in HIV infection. The efficacy of immunosuppressive treatment?]. A subacute advanced severe sensorimotor polyneuropathy developed over 6 months in a 47-year-old patient in stage 5 of an HIV infection (Walter Reed Hospital classification). Clinical examination, cranial computed tomography and spinal nuclear magnetic imaging failed to demonstrate any central nervous system complication. Cerebrospinal fluid showed a lymphocytic pleocytosis of 57/3 cells and total protein raised to 132 mg/dl as sign of an abnormal blood-brain barrier. Circulating immune complex in blood was raised to 30%. Assuming an immune-complex mediated neuropathy treatment with oral steroids was started, initially 150 mg daily. The signs of polyneuropathy regressed almost completely, even after prednisolone was discontinued. The proportion of circulating immune complexes in blood fell within 7 weeks to 10% during this treatment. It is suggested that in HIV-infected patients severe polyneuropathies may develop as part of a humoral immune reaction in which immunosuppressive treatment can be effective. Even in advanced HIV infection high-dosage and prolonged steroid treatment can be undertaken, under strictest indications, and may have impressive results.


An 88-kDa protein of Plasmodium falciparum is related to the band-3-binding domain of human erythrocyte ankyrin. Three tryptic-peptide sequences of an 88-kDa pair of phosphoproteins of the malaria parasite Plasmodium falciparum were determined. They exhibit a striking similarity to corresponding sequences of the 89-kDa domain of human erythrocyte ankyrin. [35S]Methionine labeling of the two proteins demonstrated their parasitic origin. Using an appropriate oligonucleotide probe, Southern-blot analysis of genomic malaria DNA and Northern-blot analysis of malaria RNA suggest the existence of ankyrin-related sequences in the parasite genome and the presence of an ankyrin-related transcript of about 3.2 kb. Our studies provide further evidence of malaria-specific analogues of host-cell proteins, implying an unusual kind of parasite/host interaction.


Cloning, sequencing and in vivo expression of genes encoding the F0 part of the sodium-ion-dependent ATP synthase of Propionigenium modestum in Escherichia coli. A DNA fragment containing the genes encoding subunits of the F0 part of the sodium-translocating ATPase of Propionigenium modestum was cloned in Escherichia coli and sequenced. The predicted amino acid sequences of subunits a, b and c of the P. modestum ATPase were compared with those of the corresponding subunits of proton-translocating ATPases from other bacteria and chloroplasts. Deletion mutants of E. coli, lacking different genes for ATPase subunits, were transformed with a recombinant plasmid, containing the genes for the subunits a, c, b, delta and part of alpha of the ATPase of P. modestum. Functionally reconstituted ATPase activity could be demonstrated for the transformants. The identity of the vector containing P. modestum genes was verified by restriction analysis of plasmid DNA.


Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181. A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.


Determination of the sequence coding for the beta subunit of the human high-affinity IgE receptor. The cDNA encoding the beta subunit of the human high-affinity IgE receptor was cloned by a combination of various polymerase chain reactions (PCR). A major portion of the beta cDNA was amplified using primers homologous within the sequences of rat and mouse. The 3' unknown sequence was preferentially amplified using the RNA template-specific PCR and the improved two-step PCR. The 5' unknown sequence was specifically amplified by our newly developed PCR walking. Random heptanucleotides tagged with a unique sequence at the 5' end were used as the walking primer. Finally, the entire coding region was amplified and sequenced. The two extracellular loops of the human beta subunit were the least homologous to those of rat and mouse.


Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein. The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S-modulin, the CAR (cancer-associated retinopathy) protein and recoverin, the Ca(2+)-dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.


Combinatorial RNA splicing alters the surface charge on the NMDA receptor. Transcripts encoding four NMDA receptor subunits, generated from the NMDAR1 gene by alternative RNA splicing, have been demonstrated in adult rat brain. RNA transcripts derived from cDNAs encoding each form direct the formation of functional NMDA receptors in Xenopus oocytes. The two amino acid cassettes of 21 and 37 amino acids found in the splice variants increase the positive extracellular surface charge on the subunits and may thereby modulate the functional properties of the receptor.


Deduced amino acid sequence and E1-E2 equilibrium of the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle. Comparison with the Ca(2+)-ATPase of rabbit fast twitch muscle. The cDNA encoding a Ca(2+)-transport ATPase of frog (Rana esculenta) skeletal muscle was isolated and characterized. The deduced amino acid sequence, consisting of 994 residues, showed 89% identity to the fast twitch muscle sarcoplasmic reticulum Ca(2+)-ATPases of chicken and rabbit. Northern blot analysis using a fragment of this cDNA as probe detected a 5.0 kb message in frog skeletal muscle but did not detect any mRNA encoding sarcoplasmic reticulum Ca(2+)-ATPase in frog cardiac muscle. The enzymatic properties of the amphibian skeletal muscle Ca(2+)-ATPase were compared with those of the rabbit fast twitch muscle Ca(2+)-ATPase by functional expression of the cDNAs in COS-1 cells. The amphibian Ca(2+)-ATPase displayed a reduced apparent affinity for Ca2+ and an increased apparent affinity for the inhibitors, vanadate and thapsigargin, relative to the mammalian enzyme. This may be explained by a mechanism in which relatively more of the E2 conformation accumulated in the frog Ca(2+)-ATPase than in the mammalian enzyme.


Mutational analysis of the role of Glu309 in the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle. Site-specific mutagenesis was used to analyse the role of the residue, Glu309, in the function of the Ca(2+)-ATPase of frog skeletal muscle sarcoplasmic reticulum by substitution with Ala or Lys. At pH 6.0, 100 microM Ca2+ was unable to prevent phosphorylation from Pi, consistent with previous observations on the Ca(2+)-ATPase of rabbit fast twitch muscle [Clarke, D.M., Loo, T.W, Inesi, G. and MacLennan, D.H. (1989) Nature 339, 476-478]. At neutral pH, however, micromolar concentrations of Ca2+ were sufficient to inhibit phosphorylation of the Glu309----Lys mutant from inorganic phosphate, suggesting that at least one high-affinity Ca2+ site was relatively intact in this mutant. The Glu309----Lys mutant was unable to form a phosphoenzyme from ATP at all Ca2+ concentrations studied (up to 12.5 mM), whereas phosphorylation of the Glu309----Ala mutant occurred at 12.5 mM Ca2+, but not at Ca2+ concentrations in the submillimolar range. Kinetic studies demonstrated a reduced rate of dephosphorylation of the E2P intermediate in the Glu309----Lys mutant. A less pronounced stabilization of E2P was observed with the Glu309----Ala mutant, suggesting a possible role of the charge at the position of Glu309 in phosphoenzyme hydrolysis.


An open randomized comparative study of the effect of goserelin depot and danazol in the treatment of endometriosis. Zoladex Endometriosis Study Team. To compare the efficacy and safety of goserelin depot and danazol for endometriosis. Open, randomized comparative trial. Multicenter European academic clinical institutions. A total of 307 patients with laparoscopically diagnosed endometriosis were randomized to goserelin (n = 204) or danazol (n = 103); 249 patients underwent second look laparoscopy (175 received goserelin and 74 danazol) and were analyzed for efficacy. A 3.6-mg depot of goserelin monthly subcutaneously or oral danazol 200 mg three times a day administered for 24 weeks. Efficacy assessments were based on changes in visible deposits at laparoscopy before and after treatment and subjective symptom scores at 4-week intervals during treatment and 8-week intervals after treatment for up to 24 weeks. Safety was assessed by adverse event reporting and clinical laboratory measures. There were similar proportions of symptomatic (73%) and asymptomatic (but infertile) (27%) and comparable distribution of different severity of endometriosis randomized to each treatment. Significantly fewer patients randomized to goserelin (6.4%) withdrew during treatment compared with 20.4% randomized to danazol (P less than 0.05). There were significantly reduced visible deposits of endometriosis found post-treatment (P less than 0.0001) within each group but no differences between the treatments. The mean total subjective symptoms scores remained significantly less than entry at 24 weeks post-treatment (P less than 0.05). Hypoestrogenic side effects were more common in those receiving goserelin, particularly hot flushes, but anabolic/androgenic side effects of weight gain and muscle cramps were more common in those receiving danazol. The monthly administered 3.6-mg depot preparation of goserelin was highly effective at inducing resolution of endometriotic implants and relieving the symptoms of endometriosis with prevention of their return during 24 weeks follow-up in the majority of patients. However, results were not significantly different from those achieved with danazol 600 mg/d.


Direct effects of medroxyprogesterone acetate, danazol, and leuprolide acetate on endometrial stromal cell proliferation in vitro. To assess the direct effects of three endometriosis chemotherapeutic agents (medroxyprogesterone acetate [MPA], danazol, and leuprolide acetate [LA]) on endometrial cell proliferation. Analysis of cell proliferation in vitro. Proliferative phase endometrial stromal cells isolated from biopsy specimens and grown in short-term culture served as a model for stromal components of endometriotic implants. Biopsies obtained from volunteers with regular cycles and without endometriosis or endometrial pathology. Medroxyprogesterone acetate, danazol, and LA were added to nutrient media with the following supplements: 2.5% calf serum (CS) only; 2.5% CS+estradiol (E2) 110 pmol/L; 2.5% CS+E2 550 pmol/L; 10% preovulatory serum (net E2 624 pmol/L). Cumulative [3H]-thymidine incorporation as a reflection of cell proliferation. Medroxyprogesterone acetate and danazol exerted significant antiproliferative effects on stromal cell proliferation (P less than 0.05), effects that were enhanced by an absence of exogenous E2. Leuprolide acetate exerted no consistent effects. These data imply that MPA and danazol but not LA exert direct effects, suppressing growth of endometriotic implants.


Evolution of spontaneous endometriosis in the baboon (Papio anubis, Papio cynocephalus) over a 12-month period. To document the spontaneous evolution of endometriosis, a repeat laparoscopy was performed in 11 baboons after 10 and/or 12 months. The mean number of endometriotic lesions had increased significantly after 10 months (P less than 0.02) because of a high proportion of new lesions (82%). These implants were mainly subtle (67%) and localized on the uterine peritoneum (58%). Progression of endometriosis did not go beyond revised AFS stage I. Additionally, repeat laparoscopy in 10 baboons with an initially normal pelvis showed an endometriosis incidence of 70% after 10 to 12 months. Remodeling of the lesions was apparent in both groups after 12 months. These results suggest that endometriosis is moderately progressive in the baboon. It is possible that multiple laparoscopies could favor the development of endometriosis.


Laparoscopic CO2 laser uterine nerve ablation for treatment of drug resistant primary dysmenorrhea. Twenty women with drug-resistant primary dysmenorrhea were subjected to laparoscopic CO2 laser uterine nerve ablation. Menstrual pain assessed by a linear analog pain score showed a reduction of 33%, decreasing from 7.5 +/- 0.5 preoperatively to 5.0 +/- 1.7 postoperatively. The procedure was free of major complications and should be considered as a second-line therapeutic option in women who have failed medical treatment using nonsteroidal antiinflammatory agents or OCs.


Application of the cavitron ultrasonic surgical aspirator (CUSA) for gynecological laparoscopic surgery using the rabbit as an animal model. To study the potential application of the cavitron ultrasonic surgical aspirator (CUSA) in gynecological laparoscopic surgery using a rabbit animal model. Twenty-six rabbits were prospectively randomized into two groups. Laparoscopically directed standard injuries were made on the randomly assigned horn and sidewall in all animals with the CUSA. Contralateral injuries were made with a contact neodymium-yttrium aluminum garnet (Nd:YAG) laser in group 1 and with bipolar cautery in group 2. Adhesion and inflammation scores were assessed for two animals in each group at 24, 48, and 72 hours, and seven animals in each group at 14 days. University animal research facility. Adhesion and inflammation scores were compared between animals in the CUSA versus Nd:YAG study and the CUSA versus bipolar cautery at 14 days. No significant difference in uterine or sidewall adhesion scores was noted between the CUSA versus Nd:YAG or the CUSA versus bipolar cautery. Bipolar cautery produced significantly less inflammation on the uterine horn compared with the CUSA (3.0 +/- 0.2 versus 5.3 +/- 0.7, P = 0.0001), but no difference in sidewall inflammation was noted between the CUSA compared with bipolar cautery. No difference in inflammation was observed between the CUSA and the Nd:YAG laser. The bipolar cautery appears to be preferable to the CUSA for coagulation of uterine lesions, although dissection of the uterus is not possible with bipolar cautery. The CUSA and the Nd:YAG appear to be comparable for uterine horn dissection. Because the CUSA causes similar adhesion formation and tissue inflammation at the sidewall when compared with the Nd:YAG laser and bipolar cautery and may be less likely to damage blood vessels, ureters, or other collagen-rich tissues, the CUSA may represent a promising new surgical tool for laparoscopically directed peritoneal dissection.


Presymptomatic testing for late-onset genetic disorders: lessons from Huntington's disease. Huntington's disease is an inherited, neurodegenerative disorder, usually of adult onset. Since the identification of linked markers, more than 1000 presymptomatic tests have been performed worldwide and multiple ethical issues have been encountered in relation to informed consent, testing of children, exclusion testing during pregnancy, and confidentiality. Further ethical problems are anticipated after identification of the causal mutation (or mutations). As Huntington's disease is a model for other disorders of adult onset for which testing is becoming possible, the successful resolution of these ethical issues is of great importance. A failure to do so might discredit genetic testing as a whole.


The Tiresias complex: Huntington's disease as a paradigm of testing for late-onset disorders. Huntington's disease represents the first disorder for which positional cloning techniques successfully localized an autosomal gene--in 1983. Events since that time have proved the gene recalcitrant to identification and characterization. Since 1986, presymptomatic and prenatal testing for Huntington's disease has been available internationally, although on a limited basis. Testing for Huntington's disease provides an excellent model for designing service programs for genetic testing for late-onset, fatal disorders, particularly when the gene is not yet in hand and no therapeutic intervention is possible. Special training and precautions must be in place before presymptomatic genetic testing should be offered.


Response to hepatitis B vaccine of persons positive for antibody to hepatitis B core antigen. The significance of antibody to hepatitis B core antigen (anti-HBc) present in a person's serum without hepatitis B surface antigen (HBsAg) or its antibody (anti-HBs) is unknown. Serum specimens from 281 persons initially positive only for anti-HBc by enzyme immunoassay (EIA) were retested by radioimmunoassay (RIA), and of these, 177 (63%) remained positive for anti-HBc by both assays. Of these 177 persons, 3 were positive for HBsAg, and 72 possessed low levels of anti-HBs [less than 10 sample ratio units; (SRU's)]. When persons positive for anti-HBc by EIA and RIA were given one 20-micrograms dose of plasma-derived hepatitis B vaccine and tested for anti-HBs 1 month later, a booster response was observed in 14 of 41 (34%) persons with low level anti-HBs and 3 of 50 (6%) persons negative for anti-HBs. Of those positive only for anti-HBc by EIA but negative by RIA, only 3 of 37 (8.1%) showed a booster response. Of those who completed the three-dose immunization series and did not show a booster response, 63 of 80 (78.8%) developed anti-HBs levels greater than 10 standard ratio unit. The majority of persons with isolated anti-HBc will have a primary rather than a booster response to hepatitis B vaccine.


Hemodynamic, neurohumoral, and metabolic responses to amino acid infusion in patients with cirrhosis. In patients with cirrhosis the renal response to amino acid infusion is controversial. In addition, the renal and systemic metabolic effects of amino acids are unknown. Therefore, the present study examined the effects of amino acids on renal hemodynamics, renal and systemic oxygen (O2) consumption, and hormones in patients with cirrhosis. Twelve patients received an 8% amino acid solution for 30 minutes at a rate providing 250 mg of amino acids/kg body wt. Renal blood flow increased by 45% (P less than 0.05) and the glomerular filtration rate by only 9% (P greater than 0.05). Renal vascular resistance decreased by 23% (P less than 0.05), and renal perfusion pressure did not change significantly. Renal and systemic O2 consumption and pulmonary artery plasma glucagon level significantly increased. There were no significant changes in plasma osmolality, plasma volume, and plasma atrial natriuretic peptide concentrations. In conclusion, the results show that amino acid-induced renal vasodilation caused hyperperfusion but not renal hyperfiltration in patients with cirrhosis. In addition, renal hyperemia was associated with renal and systemic hypermetabolism.


Immunosuppressive properties of chenodeoxycholic and ursodeoxycholic acids in the mouse. Cell-mediated immunity is impaired during cholestasis, and there is evidence that bile acids play a role in this immune defect. Ursodeoxycholic acid (UDCA), which corrects the immunological abnormalities observed in primary biliary cirrhosis, could counter the detrimental effects of the endogenous bile acids. Accordingly, we assessed the respective effects of cholestasis, chenodeoxycholic acid (CDCA), and UDCA, using mixed lymphocyte culture as a model of allogeneic immune response. CDCA induced a dose-dependent inhibition of the proliferative response (0-150 mumol/L). Mononuclear cells obtained from bile duct-ligated mice had a normal immunostimulatory effect, whereas responder cells obtained from such animals showed a profoundly impaired proliferative response, suggesting that responder T cels are the main target of the cholestasis-induced immune defect. Supplementation of cultures with exogenous interleukins partially compensated for the inhibitory effect of 25 mumol/L CDCA, but not for that of 50 mumol/L CDCA, suggesting that impaired secretion of interleukins is not the only factor involved in the effect of bile acids. In contrast to CDCA, UDCA had no inhibitory effect on the allogenic immune response at concentrations of up to 50 mumol/L.


Evidence for a storage pool defect in platelets from cirrhotic patients with defective aggregation. The mechanisms underlying the defective platelet function in cirrhotic patients were investigated. Eleven cirrhotic patients with mild disease (group 1), 20 patients with severe cirrhosis (group 2), and 31 controls were studied. Platelet aggregation was significantly reduced in cirrhotics compared with controls. Compared with controls, cirrhotic patients in group 2 showed a significant reduction in the total content of adenosine triphosphate (57.8 +/- 7.8 vs. 26.1 +/- 6.3 mumol/10(11) platelets; P less than 0.05), 5-hydroxytryptamine (285 +/- 26 vs. 104 +/- 38 nmol/10(11) platelets; P less than 0.05), beta-thromboglobulin (2129 +/- 120 vs. 1223 +/- 161 ng/10(8) platelets; P less than 0.01), and platelet factor 4 (1389 +/- 108 vs. 805 +/- 176 ng/10(8) platelets; P less than 0.05). In patients with severe disease, an increase in plasma beta-thromboglobulin-platelet factor 4 ratio, an index of in vivo platelet activation, was observed (controls, 3.50 +/- 0.50; group 1, 4.02 +/- 0.80; and group 2, 6.59 +/- 1.15). Our data indicate the existence of a platelet storage pool defect, which may favor the bleeding tendency of cirrhotic patients.


Selective accumulation of endogenously produced porphyrins in a liver metastasis model in rats. The possibility of using the porphyrin precursor 5-aminolevulinic acid to cause selective porphyrin accumulation in tumors was examined. Syngeneic colon carcinomas CC531 were implanted in the livers of Wag/Rij rats. Groups of three to six animals each were given 2 mg/mL of 5-aminolevulinic acid in drinking water from the 8th, 14th, or 17th day after tumor implantation. Two other groups received either 2.5 or 5 mg/kg of Photofrin II (Photomedica Inc., Raritan, NJ) intravenously on day 17. On day 19 the livers were removed and porphyrin concentrations were measured in normal livers and tumors by solvent extraction and high-performance liquid chromatography. Protoporphyrin accumulated progressively in tumors with increasing duration of 5-aminolevulinic acid administration (P = 0.0001), whereas no increase was found in normal livers. After 11 days of 5-aminolevulinic acid administration the porphyrin concentration ratio between tumors and livers was 4:1. In contrast, after Photofrin II administration the concentration was higher in normal livers than in tumors (1:3 ratio, tumor to liver). Enzyme measurements showed a threefold decrease in ferrochelatase activity in tumors compared with livers (P less than 0.001). In conclusion, oral administration of 5-aminolevulinic acid results in progressive accumulation of protoporphyrin in a transplantable colon carcinoma without accumulation in the surrounding liver tissue. This selective accumulation of porphyrins appears to be caused by a relative ferrochelatase deficiency in malignant tissue. 5-Aminolevulinic acid administration may be a suitable approach to photosensitizing liver tumors for photodynamic therapy or to early detection of tumors by fluorescence in ultraviolet light.


[Pelviscopic salpingectomy in ruptured extrauterine pregnancy using an automatic stapler-incision instrument]. The case report presented describes pelviscopic salpingectomy with ruptured tubal pregnancy carried out by an automatic stapling and cutting instrument. This instrument (EndoGIA 30) was developed for endoscopic intestinal surgery by the Autosuture company. It can be inserted via a 12 mm trocar and allows threefold clip suture of 3.3 cm length on each side of the automatically cut tissue. The clip suture is absolutely blood dry and does not pull anatomic structures out of shape. Unfortunately, the instrument is available for single use only and thus more expensive than the traditional "Semm's three-ligature method" (1979). Nevertheless, it will add an improvement to minimal invasive surgery. One of the indication for its use is to avoid laparotomy in individual cases.


Rat mitochondrial coupling factor 6: molecular cloning of a cDNA encoding the imported precursor. A cDNA clone encoding the precursor to the rat mitochondrial protein coupling factor 6 (F6) has been isolated and sequenced. The deduced amino acid sequence of the rat precursor protein shows 78% and 74% identity with the human and bovine F6 pre-proteins, respectively.


Determination of hormonal response in uterine cancer cell lines by the ATP bioluminescence assay and flow cytometry. Although progesterone receptor status has been shown to correlate with response to hormonal therapy, not all progesterone receptor-positive patients respond to this treatment and additional biologic assays are needed to help better predict clinical response to hormonal therapy. This study explored the potential of the ATP bioluminescence assay and flow cytometry as biological assays of hormonal response. Five uterine cancer cell lines were used: AE7, ECC-1, HEC1A, AN3, and SKUT1B. Cells were exposed to Provera or tamoxifen at 0.1, 0.2, 0.5, 1, 2, and 5X (X equal to peak plasma concentrations: 1.0 micrograms/ml Provera and 0.1 micrograms/ml tamoxifen). For correlation, estrogen and progesterone receptors were determined by the standard dextran-coated charcoal method. Only AE7 and ECC-1 were positive for progesterone receptors (501 fmol/mg AE7, 194 fmol/mg ECC-1) and the rest were negative (less than 8 fmol/mg). Tamoxifen exerted no inhibition to the above cell lines. Meanwhile, Provera exerted dose-response inhibition on both AE7 and ECC-1 cell lines. The effects of accumulation of G0-G1 phase and reduction of S, G2 cells (P less than 0.05), but not on the HEC1A cell line (P = 0.4). These changes confirmed the antiproliferative property of Provera. Further studies are needed to establish the role of the ATP bioluminescence assay and flow cytometry as biological assays of hormonal response.


[Extragenital endometriosis with multiple stenoses of the small intestine]. This paper reports on the case of a 38-year-old woman with multiple stenoses in the proximal and middle section of the jejunum, as well as in the terminal ileum, who had been symptomatic for nine years, with symptoms progressively reflecting obstruction due to mechanical subileus. The cause was found to be extragenital endometriosis which, with the mucosa remaining intact, was associated with hormone-dependent bleeding with subsequent fibrosis leading to segmental thickening of the bowel wall and narrowing of the lumen. The diagnostic work-up included a radiological study of the small bowel as described by Sellingk, abdominal sonography and a CT exam; the X-ray study and the CT scan revealed stenosing of the bowel, while sonography and CT showed wall thickening. The treatment of this condition comprises resection of the affected bowel segment together with anti-estrogen treatment or ovariectomy, and in this respect differs notable from the more common Crohn's disease of the small bowel, from which it must be diagnostically differentiated.


Low-pressure balloon angioplasty of an occluded portacaval shunt. Safe angioplasty of a portacaval shunt requires particular knowledge of the tissue characteristics of an anastomosis and the behavior of a balloon during inflation. The nature and true diameter of a portacaval shunt anastomosis are more difficult to evaluate than those of a peripheral arterial lesion, and complications are potentially more hazardous than those related to peripheral arterial angioplasty. We suggest that in some instances low pressure and incomplete balloon inflation are all that is necessary to yield safe and satisfactory results.


Increased expression of 5q31 fragile site in a Bloom syndrome family. In this work, we report spontaneous chromosomal breakpoints and fragile site expression induced by 5-fluorodeoxyuridine (FdUrd) and FdUrd plus caffeine in a family with Bloom's syndrome (BS) and 2 healthy donors. Standard and G-banded metaphases from each individual and each treatment were analyzed. Among the 59 common fragile sites (c-fra) identified in this work, only the frequency of 5q31 was significantly increased in the BS family with respect to healthy donors (P less than 0.005). A remarkable coincidence between the breakpoints involved in spontaneous chromosome aberrations and induced c-fra was found in BS homozygote patients. The importance of the interaction between fragile sites and chromosome rearrangements in cancer is discussed.


Human T cells expressing V beta 8 do not predominantly recognize DR2 alloantigen. A panel of seven monoclonal antibodies recognizing human T-cell antigen receptor (TcR) V alpha or V beta subsets has been used to measure TcR gene expression in peripheral blood lymphocytes and mixed lymphocyte culture responses (MLR) between DR2- and DR2+ (DRw15+) donors. There were no significant differences between DR2- and DR2+ donors in per cent T cells in fresh peripheral blood labelled with any of these antibodies, which included an antibody recognizing V beta 8. This indicates strongly that increased negative selection of V beta 8+ T cells does not occur in DR2+ compared with DR2- individuals. In MLR between DR2- and DR2+ donors the only significant change compared with fresh peripheral lymphocytes was that T cells expressing V beta 5.1 were decreased in DR2- lymphocyte populations responding to DR2 alloantigen. No changes in levels of V beta 8+ T cells were detected in MLR between DR2- and DR2+ donors. This suggests that V beta 8+ T cells are not predominantly reactive against DR2 (DRw15). The data support the concept that alloreactivity against a single class II major histocompatibility complex (MHC) mismatch is mediated by T cells expressing a range of different TcR V beta molecules.


Characterization of the progeny of precursor-T (pre-T) cells maintained in vitro by interleukin-3 (IL-3). Development of T-cell function in vivo. We have recently described a bone marrow culture system which is able to maintain a portion of the precursor-T (pre-T) cell compartment of adult murine marrow in vitro, in the presence of interleukin-3 (IL-3), for at least 2 weeks. However, because growth in IL-3 might also induce the differentiation of the pre-T cells, it is necessary to determine the extent to which the developmental potential of the pre-T cells is altered during their residency in vitro. Previously, we analysed the progeny of cultured pre-T cells and compared their intrathymic development, their appearance in the periphery, and their V beta gene utilization to that of the progeny of fresh pre-T cells. Within these parameters, the cells derived from cultured marrow cells did not differ significantly from cells derived from fresh marrow cells. However, these studies did not allow us to determine the functional status of the T-cell progeny of cultured marrow. In the work presented here, we analysed the functional potential of T cells which were derived either from fresh pre-T cells or pre-T cells which had been maintained for 1 week in vitro. The T-cell mediated functions analysed included mitogen- and alloantigen-induced proliferation, IL-2 production, and generation of cytotoxic T cells. We found that the cultured pre-T cells were capable of giving rise to mature, immunocompetent T cells which did not differ significantly from the progeny of fresh pre-T cells in their functional potential.


An efficacy trial of a mammalian cell-derived recombinant DNA hepatitis B vaccine in infants born to mothers positive for HBsAg, in Shanghai, China. We conducted a randomized, double-blind clinical trial of an experimental mammalian cell-derived DNA hepatitis B vaccine (Betagen, Connaught Laboratories Ltd, Toronto, Canada) to determine its efficacy in infants born to mothers who were carriers of hepatitis B surface antigen (HBsAg). Four groups of 55 infants received injections as follows: (1) a licensed plasma-derived vaccine (Lanzhou, Lanzhou Institute for Biological Products, Lanzhou, People's Republic of China), 20 micrograms; (2) Betagen, 20 micrograms; (3) Betagen, 20 micrograms+hepatitis B immune globulin (HBIG); and (4) Betagen, 10 micrograms+HBIG. Vaccine injections were given at birth and at 1 and 6 months and HBIG was given at birth. The vaccines were compared to a historical placebo control group. The efficacy of Betagen alone was 82.6% compared to 51.0% for the Lanzhou. Efficacy of Betagen increased with the concomitant use of HBIG. No infants who were HBsAg negative at birth and/or were born to hepatitis B e antigen (HBeAg) negative mothers became carriers. The rate of HBsAg in infants receiving Betagen alone, and born to mothers who were HBeAg positive, decreased from 60% at birth to 20% by the ninth month, compared to 62.5% and 50% (respectively) for Lanzhou. The percentage of infants with protective levels of antiHBs was significantly higher for Betagen alone than for Lanzhou, but the geometric mean titre of antiHBs for responders was not significantly different. We have shown that Betagen alone is highly efficacious in preventing the development of hepatitis B in infants born to mothers who are carriers of HBsAg and is also highly effective in reducing the carriage of HBsAg in infants who are HBsAg positive at birth and/or born to HBeAg positive mothers.


Reliability and validity of self and proxy reporting of morbidity data: a case study from Beirut, Lebanon. We compared the self-reported illnesses (heart disease, back pain, rheumatoid arthritis, hypertension, and pulmonary disease) and smoking histories of 100 cases and 100 controls matched for age and sex with reports of this information from proxy informants from the same household in two areas in the city of Beirut. In addition, both cases and controls were given physical examinations to evaluate the accuracy of the responses. The level of agreement between the responses of subjects and of their informants varied from one condition to the other. Heart disease had the highest level of agreement, with the proportion of agreement greater than 93% for the cases and the controls and having chi values of 0.79 and 1.0, respectively. The report of back pain exhibited the lowest level of agreement, with responses showing a proportion of agreement of 74% for the cases and 90% for the controls, with chi values of 0.49 and 0.50, respectively. In comparing the responses of subjects and proxy informants with the results of physical examinations, heart disease had the highest level of agreement (J index ranged from 0.69 to 0.84), and back pain had the lowest level of agreement (J index ranged 0.42 to 0.48). These results show that proxy informants are good respondents for members of the same household and that health interview surveys are accurate for data collection of well defined chronic conditions.


Retinal development in very-low-birth-weight infants fed diets differing in omega-3 fatty acids. Full-field electroretinograms (ERGs) were obtained from very-low-birth-weight (VLBW) neonates to determine whether omega-3 (omega-3) fatty acids are essential for normal human retinal development. Eighty-one infants born at 30.4 (standard deviation, +/- 1.5) wk gestation were, within 10 d of birth, either enrolled to receive mother's milk (naturally containing both omega-6 and omega-3 essential fatty acids) or randomized to receive one of the infant formulas. Corn oil-based Formula A contained mainly linoleic acid (18:2 omega-6) and was low in all omega-3 fatty acids. Soy oil-based Formula B contained ample alpha-linolenic acid (18:3 omega-3) but no long-chain omega-3. Formula C, supplemented with both alpha-linolenic acid and marine oils, was comparable to human milk in long-chain omega-3. Full-field ERGs were obtained in the special care nursery from infants aged 36 and 57 wk postconception. Ten healthy preterm infants born at 35 wk gestation were tested at 36 wk postconception. Significant differences were found among groups in rod ERG function. Post hoc comparisons showed that infants fed Formula A had significantly higher rod thresholds than infants receiving long-chain omega-3 (human milk, Formula C, and intrauterine). Infants receiving Formula B had intermediate thresholds that were significantly higher than those of infants receiving intrauterine nutrition. Analysis of the leading edge of the a-wave showed that b-wave differences originated at the photoreceptor level. Differences were not present in infants at 57 wk postconception. No significant differences among groups were found in cone b-waves at 36 or 57 wk postconception. Oscillatory potentials had significantly longer implicit times at 57 wk postconception in infants fed Formula A than in infants receiving human milk. These findings suggest that retinal function varies with the dietary supply of omega-3 fatty acids in VLBW infants.


Epidermal growth factor stimulates corneal epithelial cell attachment to fibronectin through a fibronectin receptor system. Epidermal growth factor (EGF) stimulates corneal epithelial migration in vivo and in vitro. Antibody against fibronectin inhibits this effect in vitro, suggesting that a fibronectin-dependent mechanism in involved. To elucidate the action of EGF, we placed rabbit corneal epithelial cells, preincubated in the absence or presence of EGF (10 ng/ml), into wells coated with fibronectin. After 45 minutes of incubation, the numbers of cells attached to the wells were counted. Preincubation with EGF for 6 hr was not effective, but preincubation for 9 hr significantly increased the numbers of cells attached to the wells. These numbers were not increased further by additional preincubation. When concentrations of EGF were reduced, numbers of attached cells decreased proportionally, but remained significantly higher than the numbers obtained with cells not exposed to EGF. The EGF-stimulated attachment to the fibronectin matrix was inhibited in a dose-dependent fashion by antifibronectin IgG and by GRGDSP, a synthetic peptide that mimics the amino acid sequence of the cell-binding domain of fibronectin. The authors conclude that a fibronectin/fibronectin receptor system mediates EGF-induced stimulation of cellular attachment. These findings suggest that EGF may increase the expression of fibronectin receptors.


Tertiary myotubes in postnatal growing pig muscle detected by their myosin isoform composition. The postnatal development of skeletal muscles was studied in growing pigs from 8 to 210 d of age. Indirect immunoperoxidase staining of frozen sections of porcine semimembranosus muscle and longissimus muscle revealed a distinct population of small fibers (tertiary myotubes) that were stained specifically by an antibody (anti-NE) selective for the developmental (embryonic and neonatal) isoforms of muscle myosin. At 8 d of age the other larger fibers were already anti-NE negative and differentiated into Types I and II. A gradual decrease in the number of anti-NE positive fibers together with a gradual increase in area of the remaining positive fibers was observed throughout the pigs' growth. These results may indicate that hyperplastic growth does not cease at birth. Possible mechanisms to explain the origin of these tertiary myotubes containing developmental isoforms of myosin are suggested.


Promoting interaction during sociodramatic play: teaching scripts to typical preschoolers and classmates with disabilities. We investigated the effects of teaching sociodramatic scripts on subsequent interaction among three triads, each containing 2 typical children and 1 child with autistic characteristics. The same type and rate of teacher prompts were implemented throughout structured play observations to avoid the confounding effects of script training and teacher prompting. After learning the scripts, all children demonstrated more frequent theme-related social behavior. These improvements in social-communicative interaction were replicated with the training of three sociodramatic scripts (i.e., pet shop, carnival, magic show) according to a multiple baseline design. These effects were maintained during the training of successive scripts and when the triads were reconstituted to include new but similarly trained partners. Results provided support for the inclusion of systematic training of scripts to enhance interaction among children with and without disabilities during sociodramatic play.


Fading teacher prompts from peer-initiation interventions for young children with disabilities. This study examined a system for fading teacher prompts to children who served as peers in peer-initiation interventions for young children with disabilities. A teacher taught peers to direct social initiations to children with disabilities, provided verbal prompts for those initiations, and introduced a system that provided peers with visual feedback about the social interactions of the children with disabilities. She then systematically withdrew the verbal prompts to peers, and subsequently faded the visual feedback system. Peer initiations increased when the intervention began and resulted in increases in social interaction for the children with disabilities. As the teacher systematically faded the prompts and visual feedback to the peers, social interaction continued at the levels found during intervention and was maintained during a short maintenance period.


Training supervisors in a collaborative team approach to promote peer interaction of children with disabilities in integrated preschools. Three supervisors of integrated preschools were trained in a collaborative team approach to encourage resource and classroom teachers to develop strategies that promote peer interaction of all children, including children with disabilities. The focus of classroom teachers' behaviors and the interactive play of children with disabilities were measured daily in both a training (indoor play period) and a generalization (outdoor play period) setting. In a multiple baseline design, supervisors were individually trained in a collaborative team approach using a manual, modeling, and role playing; then they implemented the approach with classroom and resource teachers. We found that after supervisor training, classroom teachers increased their behaviors directed towards children with disabilities and decreased their behaviors directed towards nondisabled children. Moreover, we found a doubling of the interactive play of children with disabilities and, for two of the three classes, an increase in the interactive play of comparison children, randomly selected by the classroom teachers. Changes in both teachers' and children's behaviors were also found in the generalization setting. The implications of the results for interventions in community settings are discussed.


Treatment of acute wheezing and dyspnea attacks in children under 2 years old: inhalation of fenoterol plus ipratropium bromide versus fenoterol. Two treatment regimens for the initial treatment of acute wheezing were evaluated in 61 wheezing infants. Thirty-one patients received fenoterol (F) (0.1 mg/kg) and placebo (P) and 30 patients received fenoterol (F) (0.1 mg/kg) plus a fixed dose of ipratropium bromide (IB) (50 micrograms). Both groups received the drugs by inhalation using an ultrasonic nebulizer and face mask. A clinical score system based on wheezing and rib cage retraction was established and evaluations were performed before and at 15, 30, and 45 minutes after treatment. After the last evaluation based on the clinical score, it was decided whether to repeat or not to repeat the treatment. Our results showed that a combination of a beta agonist and ipratropium bromide (FB) was more effective than a beta agonist alone (F) in reducing wheezing and dyspnea during an acute attack (63.4 versus 25.8%; p less than 0.05).


Brief report: therapeutic manipulations in severe nocturnal asthma. A nonconventional approach in a severe high-risk asthmatic. A patient with severe nocturnal asthma of multifactorial pathogenesis with high-risk features leading to several episodes of nocturnal respiratory arrests is described. Despite aggressive conventional therapy with bronchodilators and glucocorticoid agents, the patient had progressive worsening within the year prior to admission. After a nonconventional approach consisting of: high-dose inhaled steroids, afternoon dose of prednisone, addition of troleandomycin therapy, high-dose inhaled ipratropium at bedtime, maximizing serum theophylline concentrations in the early morning, and nasal CPAP through the night; the patient's pulmonary functions were optimized with minimal or no reduction in morning FEV1, and decreased airways hyperresponsiveness to methacholine.


Left ventricular mass by M-mode echocardiography. A comparison of various M-mode echocardiographic methods for assessment of left ventricular mass (LVM) was done in 21 subjects. The anatomical LVM was taken as Standard; it varied from 64.55 to 341.82 g. Of the six different M-mode echo methods compared, the method of Devereux and Reichek (1977) was found to correlate best with anatomical LVM (r = 0.99; SD = 49.54). By this method LVM = 1.4 [(LVIDd + LVPWTd + IVSTd)3 - (LVIDd)3] - 14 g.


Left ventricular muscle mass by echocardiography: methodology. The limitations of electrocardiography for diagnosing left ventricular hypertrophy (LVH), due to unacceptable accuracy and lack of serial quantifications, are well known. The use of angiocardiography for LVH assessment is invasive, hazardous and costly. Echocardiography provides an excellent method of estimation of left ventricular muscle mass, which is simple, non-hazardous, accurate and reproducible.


Evidence for GTP-binding protein involvement in the tyrosine phosphorylation of the T cell receptor zeta chain. The zeta subunit of the T cell receptor (TCR) is a prominent substrate for a TCR-activated tyrosine kinase. Tyrosine phosphorylation of the zeta subunit in response to antibody-mediated receptor cross-linking was synergized in permeabilized T cells by either of two non-hydrolyzable GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) or guanosine 5'-[beta, gamma-imido]triphosphate Gpp(NH)p. ATP analogues did not significantly affect antibody-induced tyrosine phosphorylation. Unlike the GTP analogues, the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP beta S) did not enhance phosphorylation of zeta. The effect induced by the GTP analogues required TCR occupancy and was independent of protein kinase C. Taken together these observations implicate a GTP-binding protein in the modulation of TCR-induced tyrosine phosphorylation.


A cytosolic inhibitor of vacuolar H(+)-ATPases from mammalian kidney. Regulation of the vacuolar H(+)-ATPase in organellar and transepithelial acidification has been attributed to the effects of the proton electrochemical gradient across the membrane or to changes in the number of proton pumps. We now report the identification and purification of a protein from bovine kidney cytosol that inhibits both ATPase activity and proton translocating activity of vacuolar H(+)-ATPases. Its relative molecular weight (M(r)) is 6300, similar to that for protein inhibitors of the mitochondrial F0F1-ATPase. The newly identified cytosolic inhibitor protein may participate in the physiologic regulation of the vacuolar H(+)-ATPase by suppressing activity directly.


Regulation of vascular smooth muscle tone by caldesmon. Caldesmon is an actin-binding protein present in smooth muscle cells that also inhibits actin-activated myosin ATPase activity. To assess the possible role of caldesmon in the regulation of smooth contraction, we investigated the effects of synthetic peptides on force directly recorded from single hyperpermeable smooth muscle cells of ferret aorta and portal vein. GS17C, a peptide that contains the residues from Gly651 to Ser667 of the caldesmon sequence plus an added cysteine at the C terminus, binds calmodulin in a Ca(2+)-dependent manner and also binds to F-actin but does not inhibit actomyosin ATPase activity (Zhan, Q., Wong, S.S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814). In cells in which Ca2+ was clamped at pCa 7.0, GS17C induced a dose-dependent contraction (EC50 = 0.92 microM) in aorta cells, whereas it evoked little or no contraction in portal vein cells. The GS17C-induced contraction in aorta cells was inhibited at higher Ca2+ concentrations (above pCa 6.6) and by pretreatment with calmodulin. Another peptide, C16AA, which contains the residues from Ala594 to Ala609 and does not bind actin or calmodulin, did not induce contraction. Our results strongly suggest that GS17C induces contraction by the displacement of the inhibitory region of endogenous caldesmon and, furthermore, that caldesmon present in these smooth muscle cells regulates contraction by providing a basal resting inhibition of vascular tone.


A tissue-specific small nuclear ribonucleoprotein and the regulated splicing of the calcitonin/calcitonin gene-related protein transcript. Based on a correlation between expression patterns of an abundant tissue-specific small nuclear ribonucleoprotein (N protein) and the calcitonin gene-related protein (CGRP) splicing choice, a small nuclear ribonucleoprotein (the N factor) has been hypothesized to potentially function as a trans-acting factor involved in the regulation of the alternative splicing of the calcitonin/CGRP transcript. RNA analysis indicated that most rat, human, and simian cell lines and tissues making the CGRP mRNA splicing choice expressed the N factor mRNA. These data led us to address the effect of ectopic expression of the N factor in HeLa cells, which exhibit a calcitonin splicing choice when expressing the calcitonin/CGRP gene. Expression of the N factor exerts no effect on the calcitonin/CGRP splicing choice in HeLa cells. Furthermore, several cell lines such as the human 293 cell line make the CGRP mRNA splicing choice in the absence of any detectable level of the mRNA encoding the N factor. Together, these data reveal that the N protein is neither sufficient nor required for the tissue-specific CGRP splicing decision and that the N protein is not the trans-acting factor regulating the alternative splicing of the calcitonin/CGRP gene.


The mitochondrial F1ATPase alpha-subunit is necessary for efficient import of mitochondrial precursors. The mitochondrial import and assembly of the F1ATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA1 and hsp70SSC1 and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the F1ATPase alpha-subunit (ATP1p) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the F1ATPase alpha-subunit (delta ATP1) versus mitochondria that lack the other major catalytic subunit, the F1ATPase beta-subunit (delta ATP2). Yeast mutants lacking the alpha-subunit but not the beta-subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the F1 beta subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1 beta-subunit precursor accumulates as a translocation intermediate in absence of the F1 alpha-subunit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the beta-subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the F1 alpha-subunit likely functions as an "assembly partner" to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import and assembly of proteins in mitochondria.


Energy-dependent efflux of methotrexate in L1210 leukemia cells. Evidence for the role of an ATPase obtained with inside-out plasma membrane vesicles. Earlier studies from our laboratory (Dembo, M., Sirotnak F. M., and Moccio, D. M. (1984) J. Membr. Biol. 78, 9-17) suggested that methotrexate (MTX) efflux from L1210 cells was mediated predominantly by an ATP-dependent, outwardly directed, mechanism. To examine this process further, we utilized predominantly (74%) inside-out plasma membrane vesicle preparations derived from an L1210 cell variant (L1210/R24) with 15-fold reduced Vmax for [3H]MTX influx. Efflux of [3H]MTX, under nonionic buffer conditions, in these inside-out membrane vesicles was temperature and ATP dependent (apparent Km = 0.40 +/- 0.06 mM), osmotically sensitive, and unaffected by protonophores. The presence of K+, Na+, Cl-, and HCO3- at their physiological concentrations had no effect on [3H]MTX efflux. Other triphosphonucleotides (GTP and CTP), but not a nonhydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), could also stimulate efflux, but to a lesser extent. Also, ATP gamma S and orthovanadate were potent inhibitors of ATP-dependent efflux of [3H]MTX. Other experiments revealed a system with low saturability for [3H]MTX during efflux (apparent Km = 46 +/- 7 microM), but extremely high capacity (106 +/- 15 pmol/min/mg protein), and a pH optimum in the range of 5.5-6. However, appreciable efflux was measured in the physiological range of pH 6.7-6.9. A number of inhibitors or copermeants for ATP-dependent [3H]MTX efflux in intact L1210 cells were inhibitors of ATP-dependent efflux in inside-out plasma membrane vesicles, including, cholate, bromosulfophthalein, verapamil, quinidine, and reserpine. These findings and other results showing that bromosulfophthalein will completely inhibit efflux are consistent with a role for an ATPase in [3H]MTX efflux, and suggest that the process under study is the bromosulfophthalein-sensitive, ATP-dependent route responsible for the majority of [3H]MTX efflux in intact L1210 cells.


Movement of Cys-697 in myosin ATPase associated with ATP hydrolysis. To detect movement of Cys-697 (SH2) in myosin subfragment-1 (S-1) associated with ATP hydrolysis, SH2 was labeled with the environmentally sensitive fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). Complex formation of S-1 labeled at Cys-697 with MIANS (MIANS-S-1) with adenyl-5'-yl imidodiphosphate and ADP resulted in a significant decrease in the fluorescence intensity of approximately 40 and 30%, respectively. When ATP was added to MIANS-S-1, the fluorescence intensity decreased rapidly by approximately 40%, and this fluorescence level was maintained during the steady state of ATP hydrolysis. As the substrate was used up, the fluorescence intensity increased to approximately 70% of the original value. These results together with model experiments with MIANS-N-acetylcysteine indicate that in the presence of ATP, the MIANS fluorophore attached to SH2 is located in a less hydrophobic environment than is the fluorophore in the absence of ligand and that the hydrolysis of ATP enhances hydrophobicity around the fluorophore. Acrylamide fluorescence quenching studies of MIANS-S-1 confirmed these results, indicating that addition of ATP and ADP to MIANS-S-1 results in an increase in the Stern-Volmer quenching constant of the fluorophore by factors of approximately 3 and 2.5, respectively. The present observations suggest that binding of ATP causes a movement of SH2 toward the protein surface, whereas it goes back into the protein interior after ATP hydrolysis. The results also confirmed previous observations by a chemical cross-linking approach (Hiratsuka, T. (1987) Biochemistry 26, 3168-3173).


Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase. The reactive thiol Cys-697 (SH2) in myosin ATPase was labeled with a fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) (Hiratsuka, T. (1992) J. Biol. Chem. 267, 14941-14948). Although the tryptophan fluorescence of myosin subfragment-1 (S-1) was slightly affected by incorporation of the MIANS fluorophore, the tryptophan fluorescence of the resultant S-1 derivative (MIANS-S-1) was enhanced by ATP in a manner similar to that of unlabeled S-1. The quenching of tryptophan fluorescence of MIANS-S-1 was shown to result from a transfer of the excitation energy from tryptophanyl residue(s) to the MIANS fluorophore attached to SH2, which absorbed and fluoresced maximally at 325 and 418 nm, respectively. The energy transfer measurements were performed in the presence of acrylamide and compared to those performed in the absence of the quencher. The energy transfer efficiencies were found to be unaltered by acrylamide, indicating that the observed fluorescence energy transfer is originated exclusively from the tryptophanyl residue(s) that are not affected by acrylamide, i.e. the ATP-sensitive tryptophanyl residue(s) of S-1 (Torgerson, P. M. (1984) Biochemistry 23, 3002-3007). The distance between the tryptophanyl residue(s) and Cys-697 was calculated to be 27 A assuming a single donor-acceptor pair. Trp-510 is proposed to be one of the ATP-sensitive tryptophanyl residues.


Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids and by partial unfolding. SecA protein, a principal component of the protein export machinery of Escherichia coli, is found both in the cytoplasm and inner membrane of cells. Previous in vitro and in vivo studies demonstrated that the interaction of SecA with the inner membrane requires the presence of physiological levels of anionic (acidic) phospholipids. In this report the degree of SecA insertion into model membranes and the conformational changes associated with this event have been examined. The extent of association of SecA with model membranes was determined by photolabeling with a hydrophobic reagent, and the depth of insertion of the protein into the phospholipid bilayer was determined by the amount of quenching of SecA fluorescence by both brominated and spin-labeled phospholipids. These methods demonstrated that SecA penetrates deep within the acyl chain region of the phospholipid bilayer. It was also found that SecA penetration into vesicles was associated with a major conformational change in the protein. This change can be induced by higher temperatures and involves a partial unfolding event as judged by differential scanning calorimetry, SecA fluorescence and increased sensitivity to proteolysis. These properties suggest the induction of a molten-globule-like conformation in a portion of the SecA polypeptide. This change was also induced at lower temperatures by the presence of membranes containing a physiological amount of the anionic phospholipid, phosphatidylglycerol. The partial unfolding and concomitant deep insertion of SecA into membranes may aid in the insertion of precursor proteins into the inner membrane and may influence possible interactions between SecA and the integral membrane export machinery components.


Glomerular actions of a free radical-generated novel prostaglandin, 8-epi-prostaglandin F2 alpha, in the rat. Evidence for interaction with thromboxane A2 receptors. 8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha) and related compounds are novel prostanoid produced by a noncyclooxygenase mechanism involving lipid peroxidation. Renal ischemia-reperfusion injury increased urinary excretion of these compounds by 300% over baseline level. Intrarenal arterial infusion at 0.5, 1, and 2 micrograms/kg per min induced dose-dependent reductions in glomerular filtration rate (GFR) and renal plasma flow, with renal function ceasing at the highest dose. Micropuncture measurements (0.5 microgram/kg per min) revealed a predominant increase in afferent resistance, resulting in a decrease in transcapillary hydraulic pressure difference, and leading to reductions in single nephron GFR and plasma flow. These changes were completely abolished or reversed by a TxA2 receptor antagonist, SQ 29,548. Competitive radioligand binding studies demonstrated that 8-epi-PGF2 alpha is a potent competitor for [3H]SQ 29,548 binding to rat renal arterial smooth muscle cells (RASM) in culture. Furthermore, addition of 8-epi-PGF2 alpha to RASM or isolated glomeruli was not associated with stimulation of arachidonate cyclooxygenase products. Therefore, 8-epi-PGF2 alpha is a potent preglomerular vasoconstrictor acting principally through TxA2 receptor activation. These findings may explain, in part, the beneficial effects of antioxidant therapy and TxA2 antagonism observed in numerous models of renal injury induced by lipid peroxidation.


Predominant activation and expansion of V gamma 9-bearing gamma delta T cells in vivo as well as in vitro in Salmonella infection. Gamma delta T cell receptor-positive cells (gamma delta T cells) have recently been implicated to play a role in the protection against infectious pathogens. Serial studies on gamma delta T cells in 14 patients with salmonella infection have revealed that the proportions of gamma delta T cells (mean +/- SD: 17.9 +/- 13.2%) in salmonella infection were significantly increased (P less than 0.01) compared with 35 normal controls (5.0 +/- 2.6%) and 13 patients with other bacterial infections (4.0 +/- 1.4%). Expansion of gamma delta T cells was more prominent in the systemic form (28.9 +/- 10.8%) than in the gastroenteritis form (10.5 +/- 7.9%) of salmonella infection (P less than 0.01). Most in vivo-expanded gamma delta T cells expressed V gamma 9 gene product. Increased activated (HLA-DR+) T cells were observed in all the six patients with the systemic form and four of the seven with gastroenteritis form. Especially in the six with systemic form, gamma delta T cell activation was significantly higher than alpha beta T cell activation at the early stage of illness (P less than 0.01). When peripheral blood lymphocytes from normal individuals were cultured with live salmonella, gamma delta T cells were preferentially activated and expanded and most of them expressed V gamma 9. Purified gamma delta T cells also responded to live salmonella in vitro. The present study suggests that human gamma delta T cells play a role in the protection against salmonella infection in vivo.


Alterations of phasic coronary artery flow velocity in humans during percutaneous coronary angioplasty. Studies using Doppler catheters to assess blood flow velocity and vasodilator reserve in proximal coronary arteries have failed to demonstrate significant improvement immediately after coronary angioplasty. Measurement of blood flow velocity, flow reserve and phasic diastolic/systolic velocity ratio performed distal to a coronary stenosis may provide important information concerning the physiologic significance of coronary artery stenosis. This study was designed to measure these blood flow velocity variables both proximal and distal to a significant coronary artery stenosis in patients undergoing coronary angioplasty. A low profile (0.018-in.) (0.046-cm) Doppler angioplasty guide wire capable of providing spectral flow velocity data was used to measure blood flow velocity, flow reserve and diastolic/systolic velocity ratio both proximal and distal to left anterior descending or left circumflex coronary artery stenosis. These measurements were made in 38 patients undergoing coronary angioplasty and in 12 patients without significant coronary artery disease. Significant improvement in mean time average peak velocity was noted in distal coronary arteries after angioplasty (before 19 +/- 12 cm/s; after 35 +/- 16 cm/s; p less than 0.01). Increases in proximal average peak velocity after angioplasty were less remarkable (before 34 +/- 18 cm/s; after 41 +/- 14 cm/s; p = 0.04). Mean flow reserve remained unchanged after angioplasty both proximal (1.5 +/- 0.5 vs. 1.6 +/- 1; p greater than 0.10) and distal (1.6 +/- 1 vs. 1.5 +/- 0.8; p greater than 0.10) to a coronary stenosis. Before angioplasty, mean diastolic/systolic velocity ratio measured distal to a significant stenosis was decreased compared with that in normal vessels (1.3 +/- 0.5 vs. 1.8 +/- 0.5; p less than 0.01). After angioplasty, distal abnormal phasic velocity patterns generally returned to normal, with a significant increase in mean diastolic/systolic velocity ratio (1.3 +/- 0.5 vs. 1.9 +/- 0.6; p less than 0.01). Phasic velocity patterns and mean diastolic/systolic velocity ratio measured proximal to a coronary stenosis were not statistically different from values in normal vessels (1.8 +/- 0.8 vs. 1.8 +/- 0.5; p greater than 0.10) and did not change significantly after angioplasty (1.8 +/- 0.8 vs. 2.13 +/- 0.9; p greater than 0.10). Flow velocity measurements may be performed distal to a coronary stenosis with the Doppler guide wire. Phasic velocity measurements made proximal to a coronary stenosis differed from those in the distal coronary artery. Both proximal and distal flow reserve measurements made immediately after angioplasty were of limited utility. Changes in distal flow velocity patterns and diastolic/systolic velocity ratio appeared to be more relevant than the hyperemic response in assessing the immediate physiologic outcome of coronary angioplasty.


Apolipoprotein(a) gene accounts for greater than 90% of the variation in plasma lipoprotein(a) concentrations. Plasma lipoprotein(a) [Lp(a)], a low density lipoprotein particle with an attached apolipoprotein(a) [apo(a)], varies widely in concentration between individuals. These concentration differences are heritable and inversely related to the number of kringle 4 repeats in the apo(a) gene. To define the genetic determinants of plasma Lp(a) levels, plasma Lp(a) concentrations and apo(a) genotypes were examined in 48 nuclear Caucasian families. Apo(a) genotypes were determined using a newly developed pulsed-field gel electrophoresis method which distinguished 19 different genotypes at the apo(a) locus. The apo(a) gene itself was found to account for virtually all the genetic variability in plasma Lp(a) levels. This conclusion was reached by analyzing plasma Lp(a) levels in siblings who shared zero, one, or two apo(a) genes that were identical by descent (ibd). Siblings with both apo(a) alleles ibd (n = 72) have strikingly similar plasma Lp(a) levels (r = 0.95), whereas those who shared no apo(a) alleles (n = 52), had dissimilar concentrations (r = -0.23). The apo(a) gene was estimated to be responsible for 91% of the variance of plasma Lp(a) concentration. The number of kringle 4 repeats in the apo(a) gene accounted for 69% of the variation, and yet to be defined cis-acting sequences at the apo(a) locus accounted for the remaining 22% of the inter-individual variation in plasma Lp(a) levels. During the course of these studies we observed the de novo generation of a new apo(a) allele, an event that occurred once in 376 meioses.


Intramural methotrexate therapy for the prevention of neointimal thickening after balloon angioplasty. The study was performed to test the hypothesis that high local, intramural concentrations of antineoplastic agents at the site of balloon injury inhibit vascular smooth muscle cell proliferation without systemic toxicity. The predominant mechanism for recurrent stenosis after coronary balloon angioplasty is neointimal thickening due to medial smooth muscle cell proliferation. The clinical use of potent antiproliferative agents to prevent restenosis has been limited by the potential for severe systemic side effects. Local therapy with these agents may be effective and free of systemic complications. After bilateral balloon angioplasty of the carotid arteries of 14 juvenile farm pigs, the dilated arterial segments were treated locally with methotrexate (6.25 mg/ml, total dose 25 mg) or 0.9% saline solution through a perforated balloon catheter. The animals were then killed 30 days after balloon injury to determine the effects of this therapy on neointimal thickness. In an additional six animals, tritium-labeled methotrexate was used to determine the concentration and duration of detectability of methotrexate in the wall of the treated arteries and in the systemic circulation. Two hours after drug instillation the concentration of labeled drug was greater than 1,000-fold greater in the wall of the treated artery than in circulating blood, and this ratio remained between 50 and 100 for at least 7 days. Despite this difference, the mean intimal thickness 30 days after the procedure was similar in the 10 methotrexate-treated arteries and the 18 saline-treated arteries (59 +/- 30 vs. 56 +/- 25 microns, p = 0.6). The morphologic appearance of the neointima was similar in each group and suggested an important role for mural thrombus in the genesis of the intimal thickening. Treatment with intramural methotrexate, delivered through a perforated balloon catheter at the selected concentration and total dose, failed to prevent intimal thickening after balloon injury. Nonetheless, the perforated balloon catheter appears to be a promising means of delivering a high local concentration of drugs with potentially life-threatening systemic side effects. The optimal concentrations and combinations of candidate drug therapies warrant further evaluation.


The dynamics of disability and functional change in an elderly cohort: results from the Alameda County Study. To examine changes in functional status over time by age, gender, and ethnicity in a representative sample of older persons. Six-year prospective cohort study. Alameda County, California. 508 persons 65 years old and older at baseline in 1984. Activities of daily living (ADL) dependence, mobility impairment, and functioning on an 18-item scale. The prevalence of ADL dependence and mobility impairment at baseline increased with age, while function decreased. Particularly striking differences occurred for those 80 and older. Changes in function over the 6-year follow-up showed a similar pattern. While death rates for males were higher, females had poorer initial functioning, and surviving females declined more than surviving males. The incidence of ADL dependence and mobility impairment during follow-up was similar for males and females, although females survived longer with incident disability than did males. Blacks had poorer baseline functioning, more ADL dependence and mobility impairment, and declined more than non-Blacks during follow-up. Some of the baseline difference in function between Blacks and non-Blacks was due to higher rates of chronic illness and co-morbidity. In spite of the general downward trend in functioning over the 6 years, 13% of the males and 20% of the females improved. Age-related changes in function for older persons are complex and result in much heterogeneity. Clarifying the reasons for such heterogeneity is an important and challenging area of research.


The physician and Down syndrome: are attitudes changing? A survey of attitudes of pediatricians, child neurologists, and pediatric surgeons toward Down syndrome is compared with a similar study reported in 1975. A contemporary physician would be much more aggressive in treating a child with Down syndrome who has associated anomalies such as duodenal atresia or congenital heart disease. Few would recommend institutionalization of a person with Down syndrome; the majority expect that such individuals could productively spend their adult lives in group homes. Positive changes in physicians' attitudes during the past 15 years have been influenced by parent advocacy groups, court decisions, and studies showing that the ultimate intellectual and social skills of Down syndrome children are greater than was previously believed. The most prominent variable associated with attitudes was the physician's age: the older the physician, the more likely he or she would be nonsupportive of active treatment on behalf of the Down syndrome individual. These findings suggest that ongoing education of medical students and pediatric residents in the field of developmental disabilities and bioethics is required in order to promote well-informed advocacy for the mentally handicapped.


IL-1 receptor antagonist protein production and gene expression in rheumatoid arthritis and osteoarthritis synovium. IL-1 can participate in the perpetuation of arthritis through direct stimulation of synoviocytes and augmentation of matrix degradation. Hence, local production of the IL-1R antagonist protein (IRAP) might be an important negative feedback signal that regulates synovitis. We assessed synovial IRAP production in synovia from 30 individuals, by using a specific mAb and the immunoperoxidase staining method. IRAP was detected in 11 of 12 rheumatoid arthritis (RA) synovial tissues (ST) and was located primarily in the sublining, particularly in perivascular regions enriched for macrophages. Some staining was observed in the intimal lining of the synovium, although this was significantly less than in the sublining (p less than 0.05). Nine of 12 osteoarthritis (OA) tissues were positive for IRAP. In contrast to RA, the staining was observed primarily in the synovial lining in OA, with only minimal sublining IRAP being detected. Synovia from four patients without arthritis were negative (three autopsy specimens and one post-traumatic sample). Of the other two patients with miscellaneous diagnoses, one sample was negative (tenosynovitis) and one was positive (seronegative inflammatory arthritis) (sublining). Studies of serial sections and double-immunostaining experiments indicated that macrophages are the major cells containing immunoreactive IRAP. IRAP gene expression in vivo was determined by performing in situ hybridization on ST from 17 arthritis patients. RNA sense IRAP probes did not hybridize to any tissues. Anti-sense IRAP probes bound to two of nine RA tissues, two of six OA tissues, one of one seronegative inflammatory arthropathy tissue, and none of one flexor tenosynovitis tissue. As with immunoreactive protein, IRAP mRNA was primarily localized to cells in the synovial lining in OA but was more prominent in perivascular lymphoid aggregates in RA and seronegative inflammatory arthropathy. Northern blot analysis was performed on RNA isolated from nine ST. The appropriately sized IRAP band was identified in six of nine samples (five of six RA and one of three OA). Supernatants from cultured RA and OA ST cells contained immunoreactive and biologically active IRAP. Hence, IRAP gene expression and protein production occur in RA and OA synovium, albeit in different distributions.


Expression, molecular association, and functions of C3 complement receptors CR1 (CD35) and CR2 (CD21) on the human T cell line HPB-ALL. We have investigated the expression, molecular association, ligand binding properties, and ability to transduce intracellular signals of CR1 and CR2 C3 receptors on cells of the human HPB-ALL T cell line. CR1 and CR2 on HPB-ALL cells bound polymeric C3b and C3dg and several anti-CR1 and anti-CR2 mAb recognizing different epitopes of the receptors on normal peripheral blood cells. Immunoprecipitated CR1 and CR2 exhibited similar m.w. to those of the receptors on normal peripheral blood T and B lymphocytes. CR1 and CR2 were partially associated in the form of CR1/CR2 complexes in the cell membrane as assessed by the ability of the receptors to cocap and cointernalize and to form a detergent-sensitive complex upon immunoprecipitation analysis. Triggering of CR2 with mAb OKB7 that recognizes an epitope associated with the ligand binding site of the receptor induced an increase in intracellular free calcium concentration in HPB-ALL cells. The signal provided by mAb OKB7 did not synergize with that triggered by anti-CD3 mAb UCHT1. Triggering of CR1 did not result in changes in intracellular free calcium concentration. Our observations have significance for the biology of normal human T cells because the majority of peripheral blood T cells that express CR1 also expressed CR2 and because a change in (Ca2+)i was induced by mAb OKB7 in purified normal T cells. These functions may be relevant for the regulatory role of C3 fragments on the immune response to T-dependent Ag and for the penetration into T cells of lymphocytotropic viruses.


Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease. Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated spleen cells, all fractions protected the BALB/c recipients. Unfractionated bone marrow cells showed modest protection. Evaluation of the dose response characteristics of the suppressive activity of the low and middle density (1.060-1.068 g/ml) bone marrow cell fraction showed that reproducible protection could be achieved at a 5:1 ratio of inducing to suppressing cells. The low density fractions of both bone marrow and spleen cells had a marked depletion of typical TCR(+)-alpha beta CD4+ or CD8+ T cells, and a predominant population of TCR(+)-alpha beta CD4- CD8- T cells. Purified populations of the latter cells suppressed GVHD. Recipients given unfractionated C57BL/Ka spleen cells and protected with low-density bone marrow or spleen cells were chimeras.


Structure of the gene for the alpha-chain of the mouse high affinity receptor for IgE (Fc epsilon RI). Full-length genomic clones for the alpha-chain of mouse Fc epsilon RI were isolated and the exon/intron structure of the gene determined. The gene consisted of 5 exons, of which the first and second comprised the 5' untranslated region and the leader sequence; the third and fourth, the extracellular domain; and the fifth, the transmembrane and cytosolic domains, plus the 3' untranslated region. The upstream region was highly homologous to that of the rat counterpart. Primer extension and RNase protection analyses revealed multiple transcription initiation sites, between 30 and 120 nucleotides 3' of the putative TATA box. Comparison with other Fc receptor genes (rat Fc epsilon RI, mouse Fc gamma RIII alpha, human Fc gamma RIIIB and human Fc gamma RIIIA alpha) revealed a high degree of gene organization conservation.


IL-4 up-regulates IL-1 receptor antagonist gene expression and its production in human blood monocytes. IL-4 down-regulates the productions of IL-1 and TNF-alpha in human monocytes. We examined whether the productions of IL-1 and a specific receptor antagonist of IL-1 (IL-1Ra) in human blood monocytes were regulated differently. Highly purified blood monocytes, isolated by centrifugal elutriation from healthy donors, were stimulated with LPS in the presence or absence of IL-4, and their productions of IL-1 and IL-1Ra were measured by Northern blot and immunoblot analyses. IL-1 and IL-1Ra were produced by monocytes stimulated with LPS, but not with IL-4 alone. Marked up-regulation by IL-4 of IL-1Ra production in LPS-stimulated monocytes was observed at both the mRNA and protein levels. Maximal expressions of IL-1 beta and IL-1Ra mRNA in LPS-stimulated monocytes were observed 2 h and 8 h, respectively, after stimulation. The enhancement of IL-1Ra production by IL-4 was concluded to be due to enhanced gene transcription, because there was no difference in the half-lives of IL-1Ra mRNA in monocytes cultured with and without IL-4. Up-regulation of IL-1Ra production by IL-4 was also observed in monocytes stimulated with adherent IgG at both the mRNA and protein levels. This unique property of IL-4 may be important in down-regulation of the IL-1-initiated immune and/or inflammatory response, not only directly through inhibition of IL-1 production, but also indirectly through up-regulation of IL-1Ra production.


Efficacy of hepatitis B vaccination in primary school children from a village endemic for Schistosoma mansoni. To determine whether chronic Schistosoma mansoni infection interferes with hepatitis B virus (HBV) immunization, 308 schoolchildren aged 6-12 years with no evidence of prior HBV infection (156 with active schistosomiasis) were vaccinated with three 5-micrograms injections of recombinant DNA-derived HBV vaccine. The vaccine was given in the deltoid muscle at time 0 and 1 and 7 months later. All vaccinees were examined 1 and 3 years after vaccination for quantitative antibody to hepatitis B surface antigen (anti-HBs). Seroconversion was detected in 284 vaccinated children (92%), of whom 271 had a good (51-300 mIU/mL) or excellent (greater than 300 mIU/mL) anti-HBs response. Sixteen other children (5%) had evidence of natural HBV infection (antibody to hepatitis B core antigen). Of those with good or excellent response, 99% retained high antibody titers for 3 years. Response was not influenced by S. mansoni infection. Hepatomegaly and splenomegaly were associated with reduced vaccine response.


Skin, soft tissue, and bone infections due to Mycobacterium chelonae chelonae: importance of prior corticosteroid therapy, frequency of disseminated infections, and resistance to oral antimicrobials other than clarithromycin. Little is known of clinical disease due to Mycobacterium chelonae chelonae. One hundred skin, soft tissue, or bone isolates of this rapidly growing mycobacterium were identified over 10 years. Clinical disease included disseminated cutaneous infection (53%); localized cellulitis, abscess, or osteomyelitis (35%); and catheter infections (12%). Underlying conditions with disseminated infection included organ transplantation, rheumatoid arthritis, and autoimmune disorders; 92% involved corticosteroid use. Trauma and medical procedures were risk factors for localized infections. Corticosteroids and chronic renal failure were risk factors for catheter infections. Overall, 62% of patients were receiving corticosteroids and 72% were immunosuppressed. MICs of six oral antimicrobials were obtained for 180 isolates by broth microdilution. Up to 20% of isolates were susceptible to doxycycline, ciprofloxacin, ofloxacin, and sulfamethoxazole. In contrast, 100% were susceptible to clarithromycin (MICs less than or equal to 1 microgram/mL). Disease due to M. chelonae chelonae usually occurs in the setting of corticosteroid therapy and is often disseminated; the organisms require high MICs of oral antimicrobials other than clarithromycin.


Streptokinase-producing streptococci grown in human plasma acquire unregulated cell-associated plasmin activity. Group A streptococci grown in the presence of human plasma generated plasmin from plasminogen and captured the functional enzyme to a specific cell-surface receptor. Bacteria-bound plasmin was not regulated by alpha 2-antiplasmin present in the medium. The ability of the bacteria to acquire cell-associated plasmin activity was dependent on both the presence of plasminogen in the culture medium and the production of a bacterial plasminogen activator, streptokinase. The ability of group A streptococci to produce a plasminogen activator and capture resulting plasmin in an unregulatable form could provide the organism with a mechanism for invasion of normal tissue barriers.


Lumbar motion trends and correlation with low back pain. Part II. A roentgenological evaluation of quantitative segmental motion in lateral bending. A radiographic study was undertaken to describe the relationship between the magnitude of coupled lumbar motion in lateral bending and the presence of low back pain: correlation between pain and motion, relationship between motion category and motion and symmetry of lumbar motion. Survey. Chiropractic college student health center and private chiropractic clinic. 249 subjects: 114 with low back pain, 29 asymptomatic with no history and 106 asymptomatic with history. Of these, 194 were freshman volunteers and 55 were new private clinic low back pain patients. None. Net lumbar segmental tilt and rotation in lateral bending: corrected and uncorrected for segmental malposition with the patient standing in the upright neutral position. Statistical analysis demonstrated no significant relationship between coupled lumbar motion and low back pain (p greater than .01). The presence of type II motion could account for, on average, less than 5% loss of segmental tilt in the lumbar spine. Asymmetries between left and right side motion averaged 45 to 100% of unilateral range of motion. This study suggests that back pain is not an indication for the routine use of lateral bending films for the identification of alterations in the magnitude of lumbar segmental motion in lateral bending. It further indicates that type II motion cannot be ruled out as a normal variant. The paucity of symmetrical lumbar motion suggests that segmental tilt or coupled rotation asymmetry, in and of itself, should not be considered an indication for spinal manipulation.


Lumbar apophyseal ring fractures in adolescents. Fracture of the vertebral ring apophysis in the lumbar spine is an uncommon condition that has been reported in adolescent patients presenting with low back pain. The pathophysiology is considered to be a fracture of the posterior ring apophysis in association with a herniated disc. The nature and the extension of the lesion is best established by computed tomography scan with sagittal reconstruction. We present three cases to illustrate the classic clinical and radiologic findings. Management of the condition is also discussed.