The clinicopathological implication of myocytes with contraction bands in autopsy hearts. Quantitative analysis of myocytes with contraction bands in autopsy hearts. To define the normal extent of myocytes with contraction bands, 51 adult autopsied hearts including 30 normal and 21 common hypertrophic hearts were studied. Based on the histologic features, contraction bands were divided into mild and severe types. They were found in 48 and 32 of the total 51 cases, respectively. However, in five hearts, myocytes with contraction bands were more diffused or localized in one part of the ventricular wall. There were no special different treatments before death among the five cases or others. They seemed however to correlate with sudden death or acute cerebral damages. Therefore, reperfusion injury and metabolic cell injuries were considered. If these 5 cases were excluded, the normal extend of myocytes with contraction bands was considered as less than a 1.7% area in the mild type and a 0.5% area in the severe type found in the ventricular wall. These mild and severe contraction bands were considered related to autolysis or events occurring before death.


Desmoid tumours treated with triphenylethylenes. Desmoids are uncommon mesenchymal tumours that occur at single or multiple anatomical sites, occasionally in association with polyposis coli. This paper describes the use of the triphenylethylene tamoxifen, and a new chlorinated analogue, toremifene, in 20 patients with progressive desmoid disease. Clinical responses ranging from stabilisation of disease to complete resolution were observed in 65% of cases. The antitumour activity of this group of drugs has been attributed to their anti-oestrogenic behaviour. However, desmoids provide a clinical model of a purely mesenchymal tumour which appears to respond despite having generally low levels of hormone receptor. This emphasises the significance of stroma within breast (and other) tumours, in particular how the stroma may regulate the response to these drugs regardless of receptor status.


Epidemiology of vertebral osteoporosis.

Epidemiology of vertebral osteoporosis. Whereas the extent, morbidity and costs of hip and Colles' fracture are well recognised from epidemiological studies, those arising from vertebral fracture are less secure. Reasons relate to the uncertain definition of vertebral fracture and its variable clinical expression, and hence its incidence is not known. Utilising radiological criteria 50% or more of vertebral fractures may be asymptomatic. In the remainder morbidity is significant, particularly in the presence of multiple vertebral fractures. Although the true incidence of vertebral fracture is unknown there is evidence that it increases exponentially with age in much the same way as for hip fracture. Between the ages of 60 and 90 years the apparent incidence rises approximately 20-fold in women compared to a 50-fold increase in risk of hip fracture. The incidence of vertebral osteoporosis is two-fold lower in men than in women at all ages, comparable to the pattern observed with other osteoporotic fractures. However, there is a relatively high incidence of vertebral fracture in men during middle adult-life (probably due to trauma), so that the prevalence of vertebral fracture in the male community is not two-fold less than in women.


Quality of life in clinical trials of adjuvant therapies. International Breast Cancer Study Group (formerly Ludwig Group). Clinical trials of adjuvant therapies usually measure the effectiveness of treatments by comparing disease-free survival or overall survival. These take into consideration only indirectly the quality of life experienced by the patients. We present some approaches that were developed to assess the impact of adjuvant therapy on the quality of life of breast cancer patients, as well as new methods created to compare treatments based on time spent without symptoms and toxicity (TWiST). The integration of these two methods (measuring quality and comparing duration of time) will provide a new tool for evaluating benefits from treatments given in the adjuvant setting.


Reporting and interpreting adjuvant therapy clinical trials. International Breast Cancer Study Group (formerly Ludwig Group). Identifying effective adjuvant treatments for patients with node-negative breast cancer is made difficult by the heterogeneity of the disease, the relatively low event rate and long follow-up time required, and the small magnitude of effects of current therapies. Several aspects of clinical trials that influence the appropriate reporting and interpretation of statistical results are discussed. We point out that the P value is a measure of the statistical uncertainty of an observed outcome and depends on the number of events available for analysis; it is not a measure of the magnitude of a treatment effect. We recommend that the relative reduction in the risk of an event and its 95% confidence interval be presented as an estimate of the treatment effect size, and that absolute improvements be used to judge whether treatment benefits outweigh the costs for the patient population. We suggest that subgroup analyses are important to define treatment effects within groups with different prognoses, and should be used with the understanding that multiple comparisons increase the chance of a false-positive result. Subgroup analysis should rely on the estimates of relative treatment effect and should avoid the use of the P value to declare incorrectly that "treatment is effective for one subgroup but not for another." We present the meta-analysis (overview) as a powerful method to demonstrate the statistical significance of a modest treatment effect by increasing the number of events available for analysis. The interpretation of overviews should consider the potential for treatment interactions and the validity of indirect comparisons that are not protected by a randomized design.(ABSTRACT TRUNCATED AT 250 WORDS)


Advances in pediatric interventional radiology. The past year has seen important papers on the risk of acquired immunodeficiency syndrome in the invasive radiology laboratory with emphasis on awareness and prevention. Interventional catheterization techniques in congenital heart disease are reviewed. Valvoplasty of pulmonic stenosis has the greatest success and least complication rate. The majority of complications appear to be at the vascular access site and are more common in neonates and in procedures that are ultimately unsuccessful. Intrauterine valvoplasty of the aortic valve is reviewed as is angioplasty for a recurrent coarctation of the aorta. Closure of a persistent ductus arteriosus with a percutaneously introduced prosthesis and occlusion of congenital coronary artery arteriovenous fistulae with a variety of embolic agents is reviewed. The use of balloon-expandable stents in congenital heart disease is discussed. Traumatic rupture of the aorta, aortic and great vessel involvement in Takayasu's disease, and mucopolysaccharidosis are reviewed along with imaging of congenital anomalies of the aortic arch. The diagnosis of vascular complications of renal transplants is summarized.


Peripheral tolerance through clonal deletion of mature CD4-CD8+ T cells. Transgenic mice bearing the alpha beta transgenes encoding a defined T cell receptor specific for the male (H-Y) antigen presented by the H-2Db class I MHC molecule were used to study mechanisms of peripheral tolerance. Female transgenic mice produce large numbers of functionally homogeneous CD8+ male antigen-reactive T cells in the thymus that subsequently accumulate in the peripheral lymphoid organs. We have used three experimental approaches to show that male reactive CD8+ T cells can be eliminated from peripheral lymphoid organs after exposure to male antigen. (i) In female transgenic mice that were neonatally tolerized with male spleen cells, male reactive CD8+ T cells continued to be produced in large numbers in the thymus but were virtually absent in the lymph nodes. (ii) Injection of thymocytes from female transgenic mice into female mice neonatally tolerized with the male antigen, or into normal male mice, led to the specific elimination of male-reactive CD8+ T cells in the lymph nodes. (iii) Four days after male lymphoid cells were injected intravenously into female transgenic mice, male antigen-reactive CD8+ T cells recovered from the lymph nodes of recipient mice were highly apoptotic when compared to CD4+ (non-male reactive) T cells. These data indicate that tolerance to extrathymic antigen can be achieved through elimination of mature T cells in the peripheral lymphoid organs.


Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes. The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It was shown that a single cysteine residue (Cys-344) was labeled on the ATPase, with a 25% reduction in steady-state ATPase activity and no reduction in the steady-state rate of hydrolysis of p-nitrophenyl phosphate. The fluorescence intensity of the labeled ATPase was sensitive to pH, consistent with an effect of protonation of a residue of pK 6.8. Fluorescence changes were observed on binding Mg2+, consistent with binding to a single site of Kd 4 mM. Comparable changes in fluorescence intensity were observed on binding ADP in the presence of Ca2+. Binding of AMP-PCP produced larger fluorescence changes, comparable to those observed on phosphorylation with ATP or acetyl phosphate. Phosphorylation with P(i) also resulted in fluorescence changes; the effect of pH on the fluorescence changes was greater than that on the level of phosphorylation measured directly using [32P]P(i). It is suggested that different conformational states of the phosphorylated ATPase are obtained at steady state in the presence of Ca2+ and ATP and at equilibrium in the presence of P(i) and absence of Ca2+.


Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine. The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)


1H, 13C, and 15N NMR assignments and global folding pattern of the RNA-binding domain of the human hnRNP C proteins. The hnRNP C1 and C2 proteins are abundant nuclear proteins that bind avidly to heterogeneous nuclear RNAs (hnRNAs) and appear to be involved with pre-mRNA processing. The RNA-binding activity of the hnRNP C proteins is contained in the amino-terminal 94 amino acid RNA-binding domain (RBD) that is identical for these two proteins. We have obtained the 1H, 13C, and 15N NMR assignments for the RBD of the human hnRNP C proteins. The assignment process was facilitated by extensive utilization of three- and four-dimensional heteronuclear-edited spectra. Sequential assignments of the backbone resonances were made using a combination of 15N-edited 3D NOESY-HMQC, 3D TOCSY-HMQC, and 3D TOCSY-NOESY-HSQC as well as 3D HNCA, HNCO, and HCACO spectra. Side-chain resonances were assigned using 3D HCCH-COSY and 3D HCH-TOCSY spectra. Four-dimensional 13C/13C-edited NOESY and 13C/15N-edited NOESY experiments were used to unambigously resolve NOEs. The overall global folding pattern was established by calculating a set of preliminary structures using constraints derived from the sequential NOEs and a small number of long-range NOEs. The beta alpha beta-beta alpha beta domain structure exhibits an antiparallel beta-sheet with the conserved RNP 1 and RNP 2 sequences [Dreyfuss et al. (1988) Trends Biochem. Sci. 13, 86-91] located adjacent to one another as the two inner strands of the beta-sheet.


Heteronuclear 1H-15N nuclear magnetic resonance studies of the c subunit of the Escherichia coli F1F0 ATP synthase: assignment and secondary structure. Nuclear magnetic resonance (NMR) studies of the c subunit of F1F0 ATP synthase from Escherichia coli are presented. A combination of homonuclear (1H-1H) and heteronuclear (1H-15N) 2D and 3D methods was applied to the 79-residue protein, dissolved in trifluoroethanol. Resonance assignment for all the backbone amide groups and many C alpha H side-chain protons was achieved. Analysis of inter- and intraresidue 1H-1H nuclear Overhauser effect (NOE) data and scalar coupling constant information indicates that this protein contains two extended regions of predominant alpha-helical character (residues 10-40 and 48-77) separated by an eight-residue segment which displays little evidence of ordered secondary structure. This model is consistent with information about the molecular motion of the protein deduced from 15N-1H heteronuclear NOE data and observed pKa values of carboxylic acid groups.


Fragment E-2 from fibrin substantially enhances pro-urokinase-induced Glu-plasminogen activation. A kinetic study using the plasmin-resistant mutant pro-urokinase Ala-158-rpro-UK. In a previous study, it was shown that fibrin fragment E-2 selectively promotes the activation of plasminogen by pro-urokinase (pro-UK) [Liu, J., & Gurewich, V. (1991) J. Clin. Invest. 88, 2012-2017]. In this study, the kinetics of this promotion by fragment E-2 was studied. Alanine-158-rpro-UK (A-pro-UK), a recombinant plasmin-resistant mutant, was used in order to avoid interference by UK generation during the reaction. In some experiments, pro-UK was substituted in order to validate the mutant as a surrogate. In the presence of a range of concentrations (0-20 microM) of fragment E-2, a linear promotion of the catalytic efficiency of A-pro-UK against native Glu-plasminogen was seen which was 245.5-fold at the highest concentration of fragment E-2 and 450-fold at the highest ratio of E-2/plasminogen used. The promotion was largely a function of an increase in kcat, since fragment E-2 induced a less than 10-fold reduction in KM (8.50-1.40 microM). In contrast to this ligand, epsilon-aminocaproic acid (EACA) induced a biphasic promotion of the activation of Glu-plasminogen which was only 18-fold at maximum. Fragment E-2 did not promote the activation of Lys-plasminogen, but the catalytic efficiency of A-pro-UK was 19.7-fold greater against the open Lys-form than against the closed Glu- form of plasminogen. Fragment E-2 had no effect on the amidolytic activity of A-pro-UK or pro-UK, suggesting that the promotion of their activities was indirect and related to a fragment E-2-induced conformational change in Glu-plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)


Proteins derived from platelet alpha granules modulate the uptake of oxidized low density lipoprotein by macrophages. Activated platelets secrete from their alpha granules a protein-like factor which stimulates the uptake of oxidized low-density lipoprotein (Ox-LDL) by macrophages. The aim of the present study was to evaluate the effect of three purified proteins obtained from platelet alpha granules: platelet-derived growth factor (PDGF), platelet factor-4 (PF-4), and beta-thromboglobulin (B-TG), on the uptake of Ox-LDL by macrophages. Cellular degradation of Ox-LDL by the J-774 A.1 macrophage-like cell line, that was preincubated for 18 h at 37 degrees C, with increasing concentrations of partially purified PDGF, (designated PDGF-CMS-III) was increased by up to 36% in comparison to control cells preincubated without PDGF. This effect was due to PDGF-mediated increase in the number of macrophage receptors for Ox-LDL. The enhanced uptake of Ox-LDL by PDGF resulted in an increase in cellular cholesterol content. Preincubation of macrophages with two types of recombinant PDGF dimers (10 ng/ml), revealed that PDGF-BB stimulated Ox-LDL cellular degradation by 64%, whereas PDGF-AB demonstrated only 34% stimulation, in comparison to control cells that were not treated with PDGF. The stimulatory effect of PDGF-CMS-III and PDGF-AB were reduced by 20% and 28%, respectively, when incubated in the presence of H-7, a specific protein kinase C inhibitor. When macrophages were preincubated with B-TG, cellular uptake of Ox-LDL was reduced by up to 30% at 100 ng B-TG/ml. This effect, however, was obtained only when B-TG was present in the incubation medium. Cellular degradation of Ox-LDL was not affected by preincubation of the cells with PF-4. Pretreatment of PCM with anti-PDGF or anti-B-TG antibodies abolished the effects of PCM on Ox-LDL degradation by macrophages. PDGF, thus, may represent the protein-like factor present in PCM which stimulates Ox-LDL degradation by macrophages, whereas B-TG may have a role in the recognition of PCM particles by the macrophage scavenger receptor. Modulation of macrophage cholesterol content by proteins secreted from activated platelets may have an important role in foam cell formation and atherosclerosis.


Modulation of complement activation on hemodialysis membranes by immobilized heparin. To determine the effects of surface-associated heparin on the capacity of hemodialysis membranes to activate complement, cellulose acetate (CA) membranes that were untreated and CA membranes that had been coated with heparin (HCA) were incubated with C3-depleted serum repleted with radio-labeled C3. Next, the proteins in the supernatant and those eluted from the membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C3 activation was quantified by determining the radioactivity of the C3a-containing band in the gel. Total C3a generation (fluid phase C3a plus membrane-associated C3a) was three times greater in the presence of HCA compared with CA. Most (88%) of the C3a generated in the presence of HCA, however, was adsorbed onto the membrane surface. Consequently, there was more C3a in the CA supernatant than in the HCA supernatant. To determine the mechanism by which heparin enhanced alternative pathway activity, binding studies with radiolabeled factor B and factor H were performed. HCA bound 3.4 times more factor B and 20 times more factor H than did CA. The binding of these proteins, however, was not dependent on complement activation. Studies designed to test the functional activity of isolated factor H and factor B that had been adsorbed to the membrane showed that factor H was active on both CA and HCA, whereas factor B was active only on HCA. These data demonstrate that heparin immobilized onto CA hemodialysis membrane enhances C3 activation but produces low levels of C3a in the fluid phase because of high surface adsorption of the anaphylatoxin. Heparin appears to augment alternative pathway activity by favoring the interactions of factor B with other constituents of the amplification C3 convertase of the alternative pathway of complement.


Thromboxane receptor blockade attenuates chronic cyclosporine nephrotoxicity and improves survival in rats with renal isograft. The question of whether pharmacological inhibition of the thromboxane A2 activity prevents cyclosporine-induced chronic renal dysfunction in a Lewis rat model of renal isograft was addressed. Transplanted animals were given a daily oral dose of cyclosporine (20 mg/kg; N = 15), cyclosporine (20 mg/kg) and the thromboxane A2 receptor antagonist GR32191 (3 mg/kg twice daily, by gavage; N = 15), or the vehicle alone (N = 12). Treatments were started the day of kidney transplant, and animals were monitored for 1 year. Cyclosporine-treated animals developed renal insufficiency, as documented by serum creatinine levels of 0.49 +/- 0.09, 0.95 +/- 0.12, and 1.38 +/- 0.15 mg/dL before and after 6 and 12 months of observation, respectively. Cyclosporine and GR32191 used in combination partially but significantly prevented the deterioration of renal function (serum creatinine, basal, 0.52 +/- 0.06; month 6, 0.68 +/- 0.04; month 12, 0.93 +/- 0.10 mg/dL). At the end of the study, GFR, as insulin clearance, was significantly lower in rats given cyclosporine (0.28 +/- 0.09 mL/min/100 g) than in rats given cyclosporine plus GR32191 (0.45 +/- 0.05 mL/min/100 g) or than in vehicle-treated animals (0.56 +/- 0.07 mL/min/100 g). Similar results were obtained for the effective RPF, measured as p-aminohippurate clearance. At the same time points, comparable to whole-blood cyclosporine levels were found in rats receiving cyclosporine alone and in those given cyclosporine plus GR32191. More than 50% of the animals on cyclosporine alone died from uremia before the end of the observation period. By contrast, rats receiving cyclosporine in combination with GR32191 had a prolonged survival.(ABSTRACT TRUNCATED AT 250 WORDS)


Collateral sensitivity to azidothymidine in methotrexate resistant human leukemia cells. Resistance to methotrexate (MTX) is a well established clinical problem and strategies to circumvent this would obviously be beneficial. The human leukemia cell line, CCRF-CEM, was grown in folinic acid to study MTX resistance-reflecting more in vivo conditions. A 15.8-fold resistant subline, CEM/MTX, was evolved by stepwise increases in MTX exposure. The MTX resistant cell line exhibited both increased dihydrofolate reductase (DHFR) activity due to gene amplification as well as impaired MTX uptake. An additional mechanism of resistance to MTX was a 2-fold increase in thymidine kinase (TK). As a result of this increased TK activity, the CEM/MTX cells were collaterally sensitive to the nucleoside analogue, azidothymidine (AZT). CEM cells resistant to MTX possess properties that can be exploited by AZT and these studies may have clinical implications.


The growth hormone-insulin-like growth factor I axis and renal glomerular function. This study examined whether maneuvers that chronically raise or lower serum insulin-like growth factor I (IGF-I), within physiological and pathophysiological ranges, will affect glomerular hemodynamics. Pair-fed Munich Wistar rats received, for 6 to 7 days, continuous s.c. infusions of human recombinant IGF-I (rhIGF-I; 125 micrograms/day), vehicle, or s.c. injection of a synthetic growth hormone-releasing hormone antagonist (GHRH-ANT) (N = 7 in each group). Infusion of rhIGF-I raised serum IGF-I to about 180% of control values, and GHRH-ANT injections lowered serum IGF-I to about 33% of control. The IGF-I infusion induced an increase in left kidney weight when expressed in absolute units but not when expressed as a percentage of body weight; there was also an increase in glomerular volume in the IGF-I treated rats. GFR, single nephron GFR, and single nephron plasma flow also rose with IGF-I infusion, and these changes were associated with decreased afferent and efferent arteriolar resistance and increased glomerular ultrafiltration coefficient. GHRH-ANT injection did not affect kidney weight or glomerular volume; however, GFR, single nephron GFR, and single nephron plasma flow were reduced in association with an increase in efferent arteriolar resistance. There also was a tendency, not significant, for the glomerular ultrafiltration coefficient to decrease. The findings that a low dose of rhIGF-I, which raised the serum IGF-I only modestly, increased glomerular ultrafiltration and that reducing serum IGF-I below control values decreased glomerular dynamics suggest that physiological or pathophysiological changes in IGF-I may affect and possible help to regulate glomerular function.(ABSTRACT TRUNCATED AT 250 WORDS)


Responses of experimental rat tumours and a mouse colon tumour to flavone acetic acid. The toxicity in rats and mice and the responses of 5 rat lung tumours, a rat rhabdomyosarcoma and a mouse colon tumour to flavone acetic acid, FAA, were studied. The LD50 values of FAA in WAG/Rij and BN rats were about 350 and 525 mg/kg respectively, independent of whether the animals were tumour bearing or not. In contrast, the LD50 value of tumour bearing BCBA mice was 50 percent of that of non-tumour bearing mice. Neither tumour volume regression nor growth delay were observed in the rat tumours growing in syngeneic rats or in nude mice, in contrast to the response of colon 38 tumours in mice. Light microscopy revealed massive tumour necrosis in the mouse tumour while no significant changes were observed in the rat tumours.


Analysis of structure and transcriptional activation of an osmotin gene. A Nicotiana tabacum gene encoding the basic PR-like protein osmotin was isolated and characterized. The gene is derived from the N. sylvestris parent of N. tabacum. In cell suspension cultures of tobacco, the osmotin gene was shown to be transcriptionally activated by treatment with ABA. Transcriptional activation of the osmotin promoter was further investigated in transformed plants carrying copies of a fusion of the cloned promoter to the beta-glucuronidase reporter gene. In these plants, the osmotin promoter is transcriptionally activated by the hormones ABA and ethylene. The sensitivity of the osmotin promoter to ABA applied exogenously decreased with age in both roots and shoots of young seedlings. NaCl shock also activated the promoter in plant tissues. The osmotin promoter is much more active in root tissues than in shoot tissues.


Oral acitretin induces alterations in mouse liver phospholipid composition. 3 and 10 mg/kg/day acitretin, a new synthetic retinoid, were orally administered to two groups of Ng57Bln mice (12 animals each) respectively, over a period of six weeks. Control animals (N = 10) received corresponding quantities of arachis oil. Chromatographic analysis and quantitative determination of the isolated phospholipid classes revealed statistically significant alterations in the liver phospholipid composition of the treated animals under both acitretin dosages, which may be associated with changes in the metabolic activity, ionic transport and cell-cell interaction of the liver cellular components.


Asynchronous synthesis of membrane skeletal proteins during terminal maturation of murine erythroblasts. To study the changes in the synthesis of the major membrane skeletal proteins, their assembly on the membrane, and their turnover during terminal red blood cell maturation in vivo, we have compared early proerythroblasts and late erythroblasts obtained from the spleens of mice at different times after infection with the anemia-inducing strain of Friend virus (FVA). Metabolic labeling of these cells indicates striking differences between early and late erythroblasts. In early erythroblasts, spectrin and ankyrin are synthesized in large amounts in the cytosol with proportionately high levels of spectrin and ankyrin messenger RNA (mRNA). In contrast, only small amounts of these polypeptides are incorporated into the skeleton, which is markedly unstable. In late erythroblasts, however, the synthesis of spectrin and ankyrin and their mRNA levels are substantially reduced, yet the net amounts of these polypeptides assembled in the membrane skeleton are markedly increased, and the membrane skeleton becomes stable with no detectable protein turnover. The mRNA levels and the synthesis of the band 3 and 4.1 proteins are increased considerably in terminally differentiated normoblasts with a concomitant increase in the net amount and the half-life of the newly assembled spectrin and ankyrin. Thus, the increased accumulation of spectrin and ankyrin at the late erythroblast stage is a consequence of an increased recruitment of these proteins on the membrane and an increase in their stability rather than a transcriptional upregulation. This is in contrast to band 3 and 4.1 proteins, which accumulate in direct proportion to their mRNA levels and rates of synthesis. These results suggest a key role for the band 3 and 4.1 proteins in conferring a long-term stability to the membrane skeleton during terminal red blood cell differentiation.


Primary structure and in vitro expression of the N. crassa phosphoglycerate kinase. The primary structure of 3-phosphoglycerate kinase (PGK) from Neurospora crassa was determined by sequencing a full-length cDNA. The deduced 418 amino acids protein shows a considerable identity to PGKs of other organisms with all the residues thought to be important for the function of the yeast enzyme conserved. The cloned PGK cDNA could be efficiently expressed in vitro resulting in a product with the expected molecular weight.


Effect of antineoplastic agents on smooth muscle cell proliferation in vitro: implications for prevention of restenosis after transluminal angioplasty. The effect of several antineoplastic agents on vascular smooth muscle cell proliferation was studied in vitro. Both fluorouracil and cytarabine produced significant concentration-dependent inhibition of smooth muscle cell proliferation in cultured porcine pulmonary artery in vitro, while cyclophosphamide stimulated growth. For fluorouracil, inhibition was near maximal at a concentration of 13.0 microgram/mL and was seen with both coincubation and 2-hour preincubation of fluorouracil with quiescent cells. Fluorouracil is a promising agent for inhibition of intimal proliferation. Further work is warranted to determine its effect in vivo.


Design and testing of a high-flow autoperfusion catheter: an experimental study. An autoperfusion catheter is similar to an angioplasty balloon catheter with side holes in the guide-wire lumen proximal to the balloon. When the balloon of the autoperfusion catheter is deployed and inflated in an artery, the guide wire is removed, and the hub of the guide-wire lumen is capped. The catheter then allows passive distal perfusion by using ambient pressure to drive blood into the guide-wire lumen, through the balloon, and out the end hole. This article discusses the requirements and constraints of a high-flow autoperfusion catheter, summarizes attempts to modify standard angioplasty catheters for use as an autoperfusion catheter, and describes the design and testing of a custom autoperfusion catheter capable of delivering approximately 3 mL/sec at physiologic pressures. In a model of canine acute renal artery occlusion lasting 90 minutes, the custom autoperfusion catheter provided marked protection from acute tubular necrosis compared with conventional percutaneous transluminal angioplasty catheters. The authors conclude that the high-flow autoperfusion catheter may be useful as a temporary stent in cases of rupture, dissection, or penetrating wounds involving large arteries.


Budd-Chiari syndrome with long segmental inferior vena cava obstruction: treatment with thrombolysis, angioplasty, and intravascular stents. The authors describe a patient with Budd-Chiari syndrome caused by long segmental thrombotic obstruction of the inferior vena cava associated with paroxysmal nocturnal hemoglobinuria. The patient was successfully treated with a combination of local thrombolytic therapy, balloon angioplasty, and placement of Gianturco expandable metallic stents.


Oral contraception affects osteocalcin serum profiles in young women. The serum concentrations of osteocalcin (bone Gla protein) were followed by continuous blood sampling for 24 h in 9 healthy young women before and during treatment with oestrogen/progestogen combinations for oral contraception. There were marked fluctuations during the 24 h sampling period, values ranging from 0.5 to 10.0 ng/ml. Values displayed an apparent circadian rhythm. Daytime values were on average lower than nocturnal concentrations. During treatment with oral contraceptives there was a significant decrease in osteocalcin levels but fluctuations during the 24 h sampling period were still observed. Almost all individual values obtained at 30 min intervals were lower during treatment. For the whole group the mean osteocalcin concentration decreased by 1.4 ng/ml (p less than 0.01) during treatment. In postmenopausal women high serum levels of osteocalcin are supposed to reflect increased bone turnover secondary to enhanced bone resorption. Oestrogens are known to reduce osteocalcin levels and may reduce bone resorption. In healthy young women alternative mechanisms should be considered but the reduced osteocalcin serum levels in this short-term study indicate that oral contraceptive use may influence bone metabolism.


New rapid test for prenatal detection of trisomy 21 (Down's syndrome): preliminary report. To devise and evaluate a rapid screening method for detecting trisomy 21 (Down's syndrome) in samples of uncultured amniotic fluid cells. Non-radioactive in situ hybridisation with HY128, a 500,000 base pair yeast artificial chromosome probe specific for chromosome 21. Blinded study of 12 karyotypically normal amniotic fluid samples and eight samples trisomic for chromosome 21. Cytogenetic and obstetric services at a tertiary referral centre, Copenhagen. Time necessary to complete the test. Proportion of cell nuclei containing two and three hybridisation signals in karyotypically normal and abnormal amniotic fluid samples. The test could be completed within three to four days after amniocentesis. In the normal samples a mean of 73% (range 61-82%) of the amniotic cell nuclei showed two hybridisation signals and 6% (0-18%) showed three signals. By contrast, among the trisomic samples 29% (19-38%) of the nuclei exhibited two signals and 48% (31-60%) showed three signals. The technique clearly distinguished between normal and trisomic samples. Prenatal diagnosis with in situ hybridisation with chromosome specific probes was fast and may make it possible to screen for selected, aneuploidies. However, the technique is still at a preliminary stage and needs further evaluation and refinement.


Presymptomatic testing for Huntington's disease in the United Kingdom. The United Kingdom Huntington's Disease Prediction Consortium. To evaluate the United Kingdom Huntington's disease presymptomatic testing programme. Postal questionnaire survey to collect data on all tests performed by clinical genetics centres between 1987 and 1990. Genetic centres providing presymptomatic testing in the United Kingdom. 248 subjects at risk of Huntington's disease who had presymptomatic testing at their request. Sex, age, prior risk, and risk after testing. The risk of carrying the Huntington disease gene was reduced for 151 (61%) of the applicants and raised for 97 (39%). 158 (64%) of the subjects were female and 90 (36%) male. The median age at which the results were given was 32.5 years. The demand for testing was lower than expected and may have reached its peak in 1990. The excess of low risk results was not fully explained by the age effect. All the genetics centres concerned have agreed a common service protocol which requires extensive pre-test counselling and post-test follow up. The worth of the procedure remains to be decided. The availability of a large body of pooled data from all the United Kingdom testing centres, which individually are likely to have only a few results, will form a valuable resource for monitoring the long term psychosocial impact of testing.


Impaired endothelium-dependent relaxation of dog coronary arteries after myocardial ischaemia and reperfusion: prevention by amlodipine, propranolol and allopurinol. 1. Anaesthetized, open-chest dogs were subjected to 60 min of left circumflex coronary artery occlusion followed by 90 min of reperfusion. Endothelium-dependent and -independent relaxant responses of the isolated coronary arterial rings were then investigated. 2. The endothelium-dependent, acetylcholine-induced relaxation of ischaemic/reperfused arterial rings was significantly attenuated in comparison to control rings (1.9 fold rightward shift, ischaemic/reperfused maximum relaxation = 57 +/- 13% of control maximum relaxation; P less than 0.05). In contrast, glyceryl trinitrate produced similar relaxant responses in control and ischaemic rings. 3. Pretreatment of dogs with either amlodipine (3 micrograms kg-1 min-1, i.v.) or propranolol (1 mg kg-1, i.v.) completely prevented the postischaemic impairment of endothelium-dependent relaxant responses (100 +/- 3% and 90 +/- 5% of control maximum relaxation, respectively). 4. Allopurinol pretreatment (25 mg kg-1, p.o. 24 h previously, plus 50 mg kg-1 i.v. 5 min before arterial occlusion) partially protected against endothelial dysfunction by preventing the ischaemia-induced rightward shift of the acetylcholine relaxation curve and increasing the maximum relaxation response (83 +/- 7% of control rings). 5. These results confirm that endothelium-dependent coronary vascular relaxation is impaired by ischaemia and reperfusion, and that the ischaemia-induced impairment is reduced by pretreatment with amlodipine, propranolol or allopurinol.


Inhibitory effect of morphine on yawning induced by cholinoceptor and dopamine D2 receptor activation in rats. 1. Bromocriptine (2, 4 and 8 mg kg-1, i.p.), physostigmine (0.05, 0.1 and 0.2 mg kg-1, i.p.) and pilocarpine (1, 3 and 5 mg kg-1, i.p.) induced dose-dependent yawning in rats. 2. These responses were reduced in a dose-dependent manner by pretreatment with morphine. 3. The inhibitory effect of morphine was reversed by naloxone. 4. Naloxone alone induced slight but significant yawning. 5. The present results suggest that morphine inhibits yawning in rats at an opiate receptor downstream from the sites at which cholinoceptor and dopamine D2 activation induce yawning. The anatomical location of these sites remains to be established.


8-Hydroxy-2-(di-n-propylamino)tetralin impairs spatial learning in a water maze: role of postsynaptic 5-HT1A receptors. 1. The effects of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, on place navigation was studied by use of two spatial tasks in a water maze. 2. In the first experiment, rats treated subcutaneously with 100 and 300 (but not 30) micrograms kg-1 8-OH-DPAT were impaired in their ability to locate a hidden platform. The probe test confirmed the impairment of spatial navigation but the effect (time spent in the training quadrant) was quantitatively different, depending on whether 8-OH-DPAT was administered only before each training session, only before the probe test or in both conditions. 3. In the second experiment, rats received 150 micrograms 5,7-dihydroxytryptamine (5,7-DHT) intracerebroventricularly to destroy 5-hydroxytryptamine (5-HT)-containing neurones and 24 days later were examined for choice accuracy in a two-platform spatial discrimination task. 4. At 100 (but not 30) micrograms kg-1 8-OH-DPAT impaired rats' accuracy with no effect on latency and no errors of omission. In 5,7-DHT-treated rats, this dose had a greater effect, including errors of omission. Sham-operated rats injected with 300 micrograms kg-1 8-OH-DPAT were markedly impaired in accuracy but they had longer latencies and made more errors than controls. All the effects were increased in 5,7-DHT treated rats. 5. The results suggest that, at doses causing no apparent changes in motor behaviour or motivation, 8-OH-DPAT impairs spatial navigation by stimulating postsynaptic 5-HT1A receptors in the rat brain.


A new paradiscal injection technique for the relief of back spasm after chemonucleolysis. Back spasm, or spasm of the back muscles, is the commonest adverse reaction encountered after chemonucleolysis. In order to overcome this troublesome complication, the authors present a new 'paradiscal injection technique'. After the injection of chymopapain into the affected disc, the needle is withdrawn to just outside the annulus. Bupivacaine is injected into the paradiscal 'space' which acts upon the paravertebral muscles. Eighty consecutive patients have been treated by chemonucleolysis with paradiscal injection for pain relief. All patients were discharged the same day or the following day and no immediate complications occurred. When reviewed 3 weeks later, only three (3.8%) patients complained of back pain (which was different in character to that present before the injection or was exacerbated by the injection). Pain persisted in the same patients until 6 months after the injection but was negligible. None of the remaining patients had developed back pain as a result of chymopapain. The authors suggest that the addition of paradiscal injection of bupivicaine after cymopapain injection can reduce the incidence of spasm of the back muscles. This technique is a major contribution to increasing the efficacy of chemonucleolysis for the treatment of herniated lumbar disc.


Reemergence of spontaneous hypertension in hypoxia-protected rats returned to normoxia as adults. Five-week-old male spontaneously hypertensive rats (SHR) were either exposed to hypoxia or maintained in normoxia. Groups of rats were returned to normoxia after 8 or 12 weeks exposure to hypoxia while others remained in hypoxia or normoxia throughout the study. Subdivisions of the groups were sacrificed 2 or 6 weeks after return to normoxia at the same time as were rats continuously exposed to either normoxia or hypoxia. Hypoxia attenuated the development of systemic hypertension (P less than 0.05); however, this protection dissipated partially when rats were returned to normoxia. Norepinephrine concentration was significantly elevated and serotonin turnover (5-hydroxyindoleacetic acid/serotonin 5HIAA/5HT) was significantly decreased in caudal brainstem of hypoxic SHR and both were gradually normalized upon return to normoxia. Similarly, left ventricular hypertrophy was attenuated and adrenal catecholamine contents were increased with hypoxic exposure. Both gradually normalized upon return to normoxia. Mechanisms associated with the development of spontaneous hypertension reemerge when adult, previously hypoxic SHR are returned to a normoxic environment. These findings implicate long-term changes in central noradrenergic and serotonergic function as components of the cardiovascular adaptation to hypoxia which includes hypoxic moderation of spontaneous hypertension.


Identification of a 5-HT1A receptor positively coupled to planarian adenylate cyclase. The adenylate cyclase system present in particulate fractions prepared from two planarian species was tested for sensitivity to various neurotransmitters. While dopamine and other catecholamines were ineffective, serotonin was capable of stimulating the enzyme. Among the various serotonin agonists tested, only 8-OH-DPAT resulted effective on the adenylate cyclase activity, thus suggesting the presence in planarians of a serotonin receptor of the type 5-HT1A.


[Laparoscopic treatment of tubal pregnancy using local administration of prostaglandin F2 alpha]. The present study reports on new treatment of tubal pregnancies by laparoscope-guided intratubal injection of prostaglandin F2 alpha into the affected tube. In 9 cases treated with PG, the method proved to be successful in 89%, as indicated by reduction of human chorionic gonadotropin (hCG) serum values to less than 20 IU/l. Preoperative serum hCG levels were 137 to 2520 IU/l (mean 1170 IU/l). In 8 women the serum concentration of beta hCG decreased to less than 20 IU/l in a mean time of 12.3 days. One woman needed surgical intervention 8 days after treatment as she continued to have abdominal pain. Hysterosalpingography 3 to 6 months after treatment showed tubal patency on the side of the pregnancy in two of three women who were examined. It is concluded that local prostaglandin F2 alpha treatment is a suitable method for termination of some tubal pregnancies.


Phase I clinical and pharmacokinetics study of high-dose toremifene in postmenopausal patients with advanced breast cancer. Toremifene is an antiestrogen that binds strongly to estrogen receptors (ER). A total of 19 previously treated postmenopausal women with metastatic breast cancer whose performance status was good and whose ER status was positive or unknown were studied to determine the maximum tolerated dose of toremifene. Cohorts of patients received 200, 300, or 400 mg/m2 p.o. daily until relapse or unacceptable toxicity had occurred. Nausea, vomiting, and dizziness were dose-related. Three of five patients receiving 400 mg/m2 experienced moderate or severe vomiting and another developed reversible disorientation and hallucinations. Mild sweating, peripheral edema, vaginal discharge, and hot flushes were encountered at all doses. Reversible corneal pigmentation was identified in seven cases but was not of clinical importance. The pharmacokinetics of toremifene was studied weekly and in detail on day 42 using a high-performance liquid chromatographic (HPLC) assay that identified the parent compound and three active metabolites, N-desmethyltoremifene, (deaminohydroxy)toremifene, and didemethyltoremifene. Steady state was achieved at 1-3 weeks. The toremifene area under the curve and the maximal concentration were dose-dependent at high doses. The recommended phase II dose is 300 mg/m2 p.o. daily.


Management of the limitations in low-speed rotational transluminal angioplasty: technical note. The low-speed rotational transluminal angioplasty catheter system (ROTACS) is a recently available device for recanalization of occlusions prior to balloon angioplasty. The presence of large collaterals originating just proximal to the occlusion with an acute angle, and insufficient space between the puncture site and the occlusion for placing an introducer sheath are reported to be limitations for ROTACS. To avoid the rotating catheter entering the collateral, we propose a technique to create a pilot inlet on the proximal aspect of the occlusion. This technique was applied successfully in 4 patients. A contralateral approach was used in 3 patients, in whom an ipsilateral access was not possible because of the insufficient space to place an introducer sheath.


Myocardial mechanical, biochemical, and structural alterations induced by chronic ethanol ingestion in rats. To determine the effects of moderate ethanol consumption on the mechanical, biochemical, and structural characteristics of the heart, myocardial mechanical performance, contractile protein enzyme activity, and the number and size of myocytes were measured in male Fischer 344 rats after the ingestion of 30% oral ethanol. Papillary muscles removed from the left ventricle were greater in length, weight, and cross-sectional area than the corresponding muscles from the right side. However, no differences were found between control and ethanol-treated myocardium when either the left or right side was compared separately. Chronic ethanol ingestion resulted in an increase in resting tension in left ventricular muscles, with no alteration in peak developed tension. Moreover, time to peak tension was significantly prolonged, whereas a depression was observed in the peak rate of isometric tension development. Isotonically, left muscles from ethanol-treated rats revealed a prolongation of time to peak shortening and a marked depression in the velocity of shortening at physiological loads. No changes were noted in muscles from the right ventricle. Contractile protein enzyme activity revealed no differences in myofibrillar Mg(2+)-ATPase activity in right and left ventricular myocardium between control and ethanol-treated rats in the presence of EGTA. However, at physiological activating levels of calcium, an upward shift of the myofibrillar Mg(2+)-ATPase activity-calcium curve occurred in left myocardium, whereas a depression in this relation was seen in the right ventricle. As a result of chronic ethanol intake, a decrease was noted in the volume percent of myocardium occupied by myocytes, and that myocyte cell volume per nucleus was found to remain essentially constant throughout the various layers of the ventricular wall. Importantly, a 14% significant decrease in the total number of myocyte nuclei was demonstrated in the left ventricular myocardium of rats on chronic ethanol consumption. Thus, chronic but moderate alcohol ingestion resulted in depressed contractile performance, alterations in myofibrillar Mg(2+)-ATPase activity, and myocyte loss. These events may serve to function as preliminary indicators of the onset of heart failure of alcoholic origin in this animal model.


Antidiuretic hormone and atrial natriuretic peptide during lower body negative or positive pressure in hypertensive patients with and without left ventricular hypertrophy. Aim of the study was to evaluate the effect of cardiopulmonary receptors activation and deactivation on antidiuretic hormone (ADH) and atrial natriuretic peptide (ANP) incretion in hypertensive and normotensive subjects. Twenty-one male subjects, 7 normotensives and 14 mild hypertensives, 7 without and 7 with left ventricular hypertrophy (LVH) were admitted to the study. Each subject underwent selective loading and unloading of cardiopulmonary receptors, by application of a positive (LBPP) or negative (LBNP) pressure to the lower body. Blood samples were taken for measurement of ANP, ADH, PRA, immunoreactive renin, aldosterone, noradrenaline and adrenaline. ADH plasma concentration increased during cardiopulmonary receptors inhibition, but this increase became statistically significant (p less than 0.05) at a step of LBNP (-40 mm Hg), in which an involvement of the sinoaortic receptors cannot be excluded. ANP plasma levels increased progressively during LBPP (p less than 0.05 at least). These changes were significantly reduced in hypertensive patients with LVH.


Effect of fatty acid anilides on immune responses of Swiss mice. The possible relationship between fatty acid anilides and the toxic oil syndrome (TOS) which appeared in Spain in 1981 has been debated during recent years. These anilides have been detected as anomalous compound in toxic oils analysed. After treatment with one daily dose of 50 mg/kg of oleilanilide (88.86% pure) for 5 days, animals showed a tendency towards progressive loss of body weight and a significant increase in serum concentration of immunoglobulins. The percentage of suppressor T cells in spleen diminished significantly compared with the control group. Consequently, an increase in the helper T cells/suppressor T cells was also observed. The production of IgM and IgG in culture was significantly higher than in controls and no differences were seen in IgA synthesis. The functional studies of generation of specific IgM, IgA and IgG suppressor cells at variable doses of concanavalin A (Con A) showed paradoxical behaviour of suppressor T cells generated by low doses of Con A. A similar change occurred at higher doses of Con A. These results suggest that low-dose treatment with oleilanilides induces an alteration in the immune response in Swiss mice.


IL-6 enhances the generation of cytolytic T lymphocytes in the allogeneic mixed leucocyte reaction. Cytolytic T lymphocytes (CTL) require soluble proteins termed lymphokines to develop lytic activity. In this report we have studied two of the lymphokines involved in the development of CTL during the allogeneic mixed leucocyte reaction (MLR). High doses of dendritic cells induced lytic activity from purified CD8+ cells in both the murine and human MLR. Under these conditions, IL-2 and IL-6 were endogenously produced and secreted. Antibodies to IL-2 or the IL-2 receptor blocked CTL formation; however, anti-IL-6 receptor antibodies only partially inhibited the response while anti-IL-6 antibodies were largely ineffective. When limiting numbers of antigen-presenting cells were used CTL failed to develop, and neither IL-2 nor IL-6 was secreted into the culture supernatant. Although the addition of IL-6 to such cultures was ineffective in generating CTL, the combination of IL-2 and IL-6 resulted in a 4-5-fold increase in lytic activity over that of IL-2 alone. We conclude that in the allogeneic MLR, IL-2 and IL-6 contribute to the generation of lytically active CD8+ cells, and the effect of IL-6 is evident when the dose of antigen-presenting cell is limited.


Intracellular free Ca2+ fluxes and responses to phorbol ester in T lymphocytes from healthy elderly subjects. A group of healthy elderly subjects (greater than or equal to 75 years) was selected by the strict criteria of the SENIEUR protocol, and compared with healthy young (less than or equal to 35 years) volunteers. Mitogenic responses of peripheral blood mononuclear cells to phytohaemagglutinin and anti-CD3 were significantly reduced in the elderly (P less than 0.0002), thereby confirming that even though in perfect health, elderly individuals show impaired cell-mediated immunity. However, no abnormality of intracellular free Ca2+ fluxes could be detected in purified T cells from the elderly subjects when stimulated with anti-CD3 antibody. Nevertheless, both the proliferative responses of purified T cells to phorbol ester and calcium ionophore (Ionomycin) and the phorbol ester-induced inhibition of the Ca2+ response were defective in the elderly subjects (P less than 0.003 and P less than 0.0002, respectively). These data suggest that signal transduction and the generation of second messengers proceed normally in T cells from the elderly, but downstream events mediated by activation of protein kinase C are dysfunctional.


Kinetics of cytokine secretion by mononuclear cells of the blood from rheumatoid arthritis patients are different from those of healthy controls. Mononuclear cells from peripheral blood (PBMC) of rheumatoid arthritis (RA) patients and healthy controls were incubated with alpha-CD3. Cytokine secretion from 2 h to 72 h of incubation was measured by ELISA. There were no significant differences in secretion of T cell derived IL-2 and IL-4 in cultures from RA patients and controls. The macrophage-derived cytokines, IL-1 beta and tumour-necrosis factor-alpha (TNF-alpha) were secreted with a steep increase of concentration during the first 16 h of incubation by PBMC from RA patients. PBMC from healthy controls secreted both cytokines at a constantly rising rate with a maximum for TNF-alpha at 48 h and for IL-1 beta at 72 h. Interferon-gamma (IFN-gamma) is secreted in significantly reduced concentrations by PBMC from untreated RA patients compared with controls. Gold-salt treatment led to a slightly delayed and enhanced secretion of TNF-alpha and IL-1 beta, an enhanced secretion of IL-2 and a restored secretion of IFN-gamma.


Elevation of activated gamma delta T cell receptor bearing T lymphocytes in patients with autoimmune chronic liver disease. To study the possible role of T cells bearing the gamma delta T cell receptor (TCR) heterodimer in the pathogenesis of autoimmune chronic active hepatitis (AI-CAH) and primary sclerosing cholangitis (PSC) in children, we measured levels of gamma delta+ T cells in the peripheral blood, assessed the proportion of cells bearing the disulphide-linked (BB3+) and non-disulphide-linked (A13+) subtypes of the receptor, and studied the co-expression of TCR-gamma delta and the activation markers HLA-DR and IL-2 receptor (IL-2R), and the memory cell marker CD45RO. Percentage levels and absolute numbers of gamma delta +T cells were higher in both groups of patients than in controls (P less than 0.01), mainly as a result of an increase in both percentage levels and absolute numbers of the A13+ subtype (P less than 0.001). Co-expression of IL-2R and TCR-gamma delta was not found in controls but was present in some patients with AI-CAH (four out of 17) and PSC (six out of 12) at low levels (median 2.3%, range 1.7-5.0%). Expression of HLA-DR on gamma delta+ T cells was similar in both groups of patients and controls. The majority of gamma delta+ T cells in children with AI-CAH and PSC also expressed CD45RO (74.7 +/- 18.4% and 79.8 +/- 24.3%, respectively) at levels significantly higher than in controls (53.3 +/- 17.2%, P less than 0.01). These results suggest that autoimmune liver diseases in children are associated with an expansion and activation of gamma delta+ T cells in the peripheral blood, which may be important in the pathogenesis of these disorders.


Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes. Complement receptor 1 (CR1) on primate red blood cells (RBC) binds most complement-fixing immune complexes in the circulation. It has been postulated that by binding them, RBC keep immune complexes in the intravascular space and deliver them to the tissue macrophages of the mononuclear phagocyte system. We have developed an in vitro model to study the transfer of RBC-bound immune complexes (heat-aggregated IgG and DNA-anti-DNA) to phagocytic cells (human monocytes). Transfer of immune complexes from RBC to monocytes occurred significantly more rapidly than monocyte uptake of the same immune complexes from solution. In the transfer process, complex-bearing RBC were not bound or sequestered by the monocytes. To define the monocyte receptors involved in binding immune complexes from the RBC surface, monocyte receptors were blocked with MoAbs (anti-CR1, anti-FcRII) or EDTA (to block CR3). Monocyte binding of immune complexes primarily used CR1 with a small contribution from FcRII, and with little or no contribution from CR3 and FcRI. Uptake of immune complexes from solution employed the same monocyte receptors as binding of complexes from the RBC surface. Immune complexes in solution bound to RBC and to monocytes with equally high avidity (approximately 1 x 10(11) l/M), but monocytes expressed a 15-20-fold greater number of immune complex binding sites. We propose that immune complexes distribute between RBC and monocytes according to the binding capacity of these cells, such that at equal or high RBC/monocyte ratios as would be seen in the circulation immune complexes bind to RBC, but at low RBC/monocyte ratios (as would be seen in the sinusoidal circulation of the liver and spleen), most immune complexes bind to monocytes. To define the pathway by which immune complexes move from RBC to monocytes, their release from RBC CR1 was examined. Under various conditions, the dissociation rate was extremely slow, and did not increase with the addition of monocyte supernatants. To examine whether factor I-mediated processing of immune complexes enhances binding of immune complexes to monocytes, RBC-bound complexes were released with factor I, and binding of these 'processed' immune complexes to monocytes was examined. Monocyte binding of these processed immune complexes was slower than of control ones; furthermore, performance of transfer experiments at 4 degrees C, which significantly shows enzymatic processes, did not decrease the rate of immune complex transfer from RBC to monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)


[A case of adult onset phosphoglucomutase deficiency]. A case of 38-year-old male with adult onset phosphoglucomutase (PGM) deficiency was reported. The patient was admitted at Kawamura Hospital (Gifu City) for evaluation of easy fatiguability and exercise-induced weakness of the extremities since he was 20 years old. Physical examination revealed moderate muscle weakness, wasting of extremities, bilateral clubbed fingers and hypoesthesia of distal portion of extremities. Fasting plasma glucose was low (58 mg/dl). Venous concentration of lactate failed to rise after an ischemic forearm exercise test. An epinephrine tolerance test revealed hyperglycemic response. Studies of anaerobic glycolysis in vitro using muscle homogenates with the substrate between glucose-1-phosphate and glucose-6-phosphate showed decreased lactate production. Direct assay of individual muscle glycolytic enzymes demonstrated reduction of PGM activity (15% of normal, n = 12). Biopsy study with PAS staining of quadriceps femoris muscle demonstrated small amount of deeply staining glycogen in subsarcolemmal area. Electron microscopic examination revealed muscle destruction with small amount of glycogen in subsarcolemmal and intermyofibrillar spaces. Sural nerve biopsy showed degeneration of myelinated and unmyelinated fibers but there was no apparent accumulation of glycogen. From the clinical, biochemical and histopathological evidences, the patient might be a rare case of adult onset PGM deficiency.


Dietary practices, physical activity, and body-mass index in a selected population of Down syndrome children and their siblings. Thirty sibling pairs, each with one Down syndrome child between the ages of 2 and 14, were selected from families who had participated in an infant-stimulation program. The maternal and paternal educational levels were 14.9 and 16.9 years, respectively. The Down syndrome patients and their siblings were compared in terms of body-mass index, that is, weight/stature2 (w/s2); activity as measured on a questionnaire; and weekly caloric intake. There were no significant differences between the Down syndrome children and their siblings in terms of w/s2. The Down syndrome patients were less active than their siblings and spent significantly more time indoors, showing a preference for indoor activities. Caloric intake calculated as percentage of recommended allowance for height was somewhat less in the Down syndrome children--88.7%, compared with 95% in the siblings--but not significantly so. It is postulated that even though Down syndrome patients have been shown to be at risk for obesity, familial and other environmental factors, such as dietary control and involvement in physical activity, have an influence.


Differential occurrence of CSF-like activity and transforming activity of Mos during the cell cycle in fibroblasts. The Xenopus c-mos proto-oncogene product, Mosxe, possesses cytostatic factor (CSF) activity to arrest maturing oocytes in metaphase II and has weak transforming activity in mouse NIH3T3 cells. We show that Mosxe mutants bearing 'stabilizing' penultimate N-terminal amino acids are strongly transforming and can retard progression through the G2-M phases in Mosxe-transformed cells, probably via their CSF activity. On the other hand, a cyclin-Mosxe fusion protein, which undergoes abrupt degradation at the end of mitosis and is restored to its normal levels only after the G1 phase, transforms cells much less efficiently than a mutated cyclin-Mosxe fusion protein that is stable during M-G1 transition. Moreover, in low-serum medium, cells transformed by the unstable cyclin-Mosxe require a long period to enter the S phase, in contrast with the rapid entry into the S phase of cells transformed by the stable cyclin-Mosxe. These results provide strong evidence that unlike the physiological CSF activity, the transforming activity of Mos is exerted in the G1 phase of the cell cycle.


Adenovirus E1a prevents the retinoblastoma gene product from repressing the activity of a cellular transcription factor. The retinoblastoma (Rb) gene product forms a complex with the cellular transcription factor DRTF1, a property assumed to be important for mediating negative growth control because certain viral oncogenes, such as adenovirus E1a, prevent this interaction and mutant Rb alleles, which have lost the capacity to regulate growth, encode proteins that fail to associate with DRTF1. In this study, we show that the wild-type Rb protein can specifically repress transcription from promoters driven by DRTF1 whereas a naturally occurring mutant Rb protein cannot. Furthermore, Rb-mediated transcriptional repression can be overridden by adenovirus E1a; this requires regions in E1a necessary for cellular transformation. The Rb protein therefore acts in trans to repress the transcriptional activity of DRTF1 whereas adenovirus E1a prevents this interaction and thus maintains DRTF1 in a constitutively active state. The Rb protein and adenovirus E1a therefore have opposite effects on the activity of a common molecular target. Transcriptional repression mediated by the Rb protein and inactivation of repression by the E1a protein are likely to play an important role in mediating their biological effects.


T cell receptor (TCR) beta chain homodimers on the surface of immature but not mature alpha, gamma, delta chain deficient T cell lines. Transfected T cell receptor (TCR) beta chain genes are expressed as homodimers on the surface of immature (Sci/ET27F) but not on mature (58 alpha-beta-) T cell lines which lack TCR alpha, gamma and delta chains. The homodimer on Sci/ET27F cells is tightly bound to CD3 delta and CD3 epsilon while the association with CD3 gamma and CD3 zeta proteins is rather weak. Crosslinking of the TCR beta homodimers resulted in a strong and rapid calcium flux. In 58 alpha-beta- T cells the beta TCR chain could be easily visualized intracellularly but was not transported to the cell surface. The Scid cell lines considerably facilitate the molecular analysis of early differentiation events in the thymus which are likely to be regulated by the beta TCR homodimer.


Isradipine for the treatment of hypertension following coronary artery bypass graft surgery: a randomized trial versus sodium nitroprusside. In a randomized trial, a calcium antagonist, isradipine (ISR) and sodium nitroprusside (SNP) were compared in the management of hypertension in the early period following coronary artery bypass grafting (CABG). Patients with a mean arterial pressure (MAP) of greater than 100 mmHg were treated with a 6 h i.v. infusion of ISR (n = 98) or SNP (n = 100). Mean MAP at baseline was 113 (ISR) and 112 mmHg (SNP). Blood pressure control (MAP less than or equal to 90 mmHg within 25 min) was achieved in 92% (ISR) and 84% (SNP), within a mean of 12 and 15 min, respectively (P less than 0.01 between groups). At 25 min, mean percentage changes from baseline for ISR and SNP were: MAP -24.3% vs. -21.4% (P less than 0.05), heart rate +4.1% vs. +8.4% (P less than 0.01), rate-pressure-product -16.9% vs. -10.6% (P less than 0.001), cardiac index +19.2% vs. +4.6% (P less than 0.001), stroke volume index +16.1% vs. -1.9% (P less than 0.001), and peripheral vascular resistance -35.4% vs. -22.0%, (P less than 0.001). Treatment was discontinued before 6 h in 24 patients in each group because of low blood pressure. Hypotension (MAP less than 70 mmHg) and tachycardia were less frequent with ISR than with SNP. In conclusion, ISR is effective and well tolerated in the treatment of hypertension following CABG, and has a haemodynamic profile which may be more favourable than that seen after treatment with SNP.


Important roles of N-methyl-D-aspartate receptors in expression of amygdaloid-kindled seizure demonstrated by intraperitoneal administration of L-aspartate in dimethyl sulfoxide. Intraperitoneally (i.p.) administered L-aspartate (Asp) (20 mmol/kg) produced no behavioral or EEG change in nonkindled rats. Nonkindled rats that received 18, 19, or 20 mmol/kg Asp, dissolved in 10 or 15% dimethylsulfoxide (DMSO), i.p. developed masticatory movement, head nodding, and myoclonic jerks of the limbs, followed by wild running and subsequent tonic extension of the whole body. In contrast to the effects in nonkindled rats, i.p. injection of Asp 20 mmol/kg in 15% DMSO in amygdala-kindled rats precipitated electroclinical generalized seizures identical to kindled ones. When the kindled amygdala was pretreated with 2-amino-7-phosphonoheptanoic acid (2-APH), a potent and specific antagonist of N-methyl-D-aspartate (NMDA) receptors, the Asp/DMSO-induced generalized convulsion identical to kindled amygdala seizure was suppressed. 2-APH treatment of the contralateral amygdala was without such suppression. The results suggest that (a) Asp is ineffective when given alone (when given with DMSO, however, Asp evokes generalized seizures identical to kindled ones in amygdala-kindled rats, while it induces a qualitatively different generalized seizure in nonkindled rats; (b) NMDA receptors of the kindled amygdala play an important role in activation of the transsynaptic neurocircuit underlying the expression of kindled amygdala seizure; and (c) DMSO is useful in assessing potential central effects of compounds that do not readily penetrate the blood-brain barrier (BBB).


Characterization of yeast-expressed beta-actins, site-specifically mutated at the tumor-related residue Gly245. The tumorigenic cell line HUT14 expresses a beta-actin carrying a mutation at position 245. In this study, two mutant beta-actins with amino acid changes at position 245 replacing the wild-type glycine by an aspartic acid and a lysine residue, respectively, were produced in the yeast Saccharomyces cerevisiae, purified to homogeneity and characterized with respect to polymerization behaviour and interaction with myosin. The major functional effect of these mutations appears to be an impaired polymerization, while the interaction with myosin seems less influenced. In addition, the results also suggest the presence of a Ca(2+)-binding site in the region of residue 245 in actin.


Multiple binding sites in fibronectin and the staphylococcal fibronectin receptor. The binding of fibronectin to Staphylococci exhibits the properties of a ligand-receptor interaction and has been proposed to mediate bacterial adherence to host tissues. To localize staphylococcal-binding sites in fibronectin, the protein was subjected to limited proteolysis and, of the generated fragments, Staphylococci appeared to preferentially bind to the N-terminal fragment. Different fibronectin fragments were isolated and tested for their ability to inhibit 125I-fibronectin binding to Staphylococci. The results indicate that only the N-terminal region effectively competed for fibronectin binding. However, when isolated fragments were adsorbed to microtiter wells, we found that two distinct domains, corresponding to the N-terminal fragment and to the heparin-binding peptide mapping close to the C-terminal end of fibronectin, promoted the attachment of both Staphylococcus aureus Newman and coagulase-negative strain of Staphylococcus capitis 651. These same domains were recognized by purified 125I-labeled staphylococcal receptor, either when immobilized on microtiter wells or probed after adsorption onto nitrocellulose membrane. The heparin-binding domain is comprised of type-III-homology repeats 14, 15 and 16. To determine which repeats participate in this interaction, we isolated and tested repeats type III14 and type III16. We found that the major staphylococcal binding site is located in repeat type III14. The staphylococcal receptor bound the N-terminal domain of fibronectin with a KD of 1.8 nM, whereas the dissociation constant of the receptor molecule for the internal heparin-binding domain was 10 nM. Since the fusion protein ZZ-FR, which contains the active sequences of fibronectin receptor (D1-D3) bound only to the N-terminus, it is reasonable to assume that the bacterial receptor may have additional binding sites outside the D domains, capable of interacting with the internal heparin-binding domain of fibronectin.


The catalytic site is located on subunit I of the ATPase from Halobacterium saccharovorum. A direct photoaffinity labeling study. Nucleotide-binding sites of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum were labeled by ultraviolet irradiation in the presence of [alpha-32P]ATP. A high-affinity site, located on subunit I (98 kDa), was identified as catalytic by the following criteria: ATP bound to subunit I was hydrolyzed and the cross-linked nucleotide was ADP; the specificity for ATP or ADP compared to that of other nucleotides was high; the tightly bound radionucleotide was exchangeable in the presence of excess unlabeled ATP and Mg2+; photolabeling of this site and enzyme inhibition due to tightly bound ADP were both dependent on the presence of Mg2+ and showed identical Kd values; treatment that restored the activity of the ADP-inhibited enzyme also led to the release of the tightly bound nucleotide from subunit I. In addition, a non-catalytic nucleotide-binding site was found, located on subunit II (71 kDa). This site did not hydrolyze ATP, its occupation was Mg2+ independent and the affinity for ATP and the nucleotide specificity were much lower than that of subunit I. We suspect that this site is nonspecific. These results indicate that H. saccharovorum ATPase is different from F1-ATPases which contain the catalytic site on the second largest subunit, but may be similar to other archaebacterial and vacuolar ATPases.


Synthesis and spectroscopy of membrane receptor proteins. The gamma subunit of the IgE receptor. The high-affinity receptor for IgE is a tetrameric complex of subunits of the type alpha beta gamma 2. We report here conformational studies of the intact gamma subunit in trifluoroethanol and water/liposomes by circular dichroism and Fourier-transform infrared (FTIR) spectroscopy. In trifluoroethanol, the FTIR amide I' frequencies were consistent with two predominant conformational components, the beta-turn and alpha-helix, whilst in liposomes consisting of D2O and dimyristoylglycerophosphocholine (Myr2GroPCho), three components were observed. The third component present may contain some left-handed extended helix. Spectral simulation was carried out to demonstrate that the CD spectra were consistent with the component conformations identified from FTIR spectroscopy. The stimulated CD spectra were in excellent agreement with the experimental spectra. The intact gamma subunit conformation in trifluoroethanol was shown to possess 72% alpha-helical and 28% beta-turn conformations. In water/Myr2GroPCho liposomes the percentage of each conformational component present is 37%, 38% and 25% for the alpha-helix, beta-turn and extended structures, respectively. Assuming that the transmembrane fragment was alpha-helical, an excellent correlation was found between this derived alpha-helical content in water/liposomes (37%) and from hydrophobicity plots where the percentage of amino acids in the transmembrane domain is predicted by others to be 34%. It is suggested that the beta-turn detected by CD and FTIR was attributable to a 3(10) helix rather than a type I or type III reverse turn.


Iso-immune neonatal neutropenia due to an anti-Fc receptor III (CD16) antibody. We report a case of transient neonatal neutropenia due to a maternal iso-immunization against a non polymorphic region of the glycosylphosphatidylinositol-linked Fc receptor type III (CD16) on granulocytes. The mother's granulocytes were typed NA1-negative, NA2-negative and CD16-negative with human and monoclonal antibodies whereas her lymphocytes express the CD16 molecule. Expression of other markers were comparable to the controls. Flow cytometric analysis showed that maternal antibody recognized the granulocytes but not the lymphocytes from blood bank donors and that its binding was decreased on normal, phospholipase C-treated, granulocytes. The binding of commercial CD16 monoclonal antibodies was also dramatically decreased on normal granulocytes pre-incubated with maternal serum. The CD16 specificity of the antibody was confirmed by negative reactions with another CD16-deficient granulocytes. This observation leads us to conclude that cell-lineage specific differences of CD16 molecules are recognized by the patient's antibody. Moreover, we confirm that the absence of the FcRIII (CD16) on granulocytes is not associated with any pathology or susceptibility to infections and that, in the children, the blockade of this receptor by the maternal antibody only led to moderate neutropenia.


Glove use by orthodontists: results of a survey in England and Wales. Attitudes to glove wearing during treatment of patients were tested by distribution of a questionnaire to 2000 dentists known to be practising under the National Health Service regulations in England and Wales. Of the dentists who replied, 41 specialist orthodontists, representing approximately one-sixth of all orthodontists working in the general dental services in England and Wales, were identified. Results indicate that 39 per cent of these orthodontic respondents wore gloves routinely for all patients and procedures, while 49 per cent wore gloves for some patients or procedures, with 12 per cent never wearing gloves. Reasons given by the occasional glove wearers for not wearing gloves routinely included loss of tactile sensation, perceived small risk, lack of comfort, and restriction of movement. Six per cent of those who replied had experienced skin irritation considered to be associated with glove wearing, while latex gloves were preferred by 78 per cent of respondents who wore gloves.


Effect of diabetes on the cholinergic enzyme activities of the urinary bladder and the seminal vesicles of the rat. Choline acetyltransferase (ChAT) and acetyl-cholinesterase (AChE) activities were determined in the seminal vesicles and in two regions of the urinary bladder, the detrusor muscle and sphincter-trigon in control and streptozotocin(STZ)-induced diabetic male Sprague-Dawley rats. In this study, STZ was administered (65 mg/kg, i.p.) to induce diabetes 14 days prior to sacrifice and enzyme analysis. Diabetic rats exhibited significant increase in both ChAT and AChE activities in the detrusor compared to the control animals. Significant increases in ChAT activity, however, were observed only in the seminal vesicles of diabetic animals compared to the control group. AChE activity in the seminal vesicles and sphincter-trigon region of the diabetic rats was not altered significantly. These findings suggest that urogenital complications associated with diabetes may be related to the dysfunction of the peripheral cholinergic system.


Reduced ability of neutrophils to produce active oxygen species in streptozotocin-induced diabetic rats. We investigated a possible alteration in the ability of neutrophils to produce active oxygen species in streptozotocin (STZ)-induced diabetic rats. The production of superoxide of the neutrophils was assessed by luminol-dependent chemiluminescence (LDCL) after the stimulation by opsonized zymosan. Four days after STZ (60 mg/kg, ip) injection, blood glucose level increased by 399 +/- 9 mg/dl and the LDCL activity was significantly reduced in diabetic rats (control group: 5.12 +/- 1.53 KC/min2/10(6) cells, STZ group: 1.10 +/- 0.07 KC/min2/10(6) cells, p less than 0.01). At Day 17, blood glucose level was maintained high (598 +/- 8 mg/dl) and the LDCL activity (1.01 +/- 0.39 KC/min2/10(6) cells) was almost at the same level as that of diabetic rats at Day 4. Subcutaneous injection of insulin for 10 consecutive days increased the LDCL activity of diabetic rats in a dose-dependent manner (vehicle: 0.82 +/- 0.27 KC/min2/10(6) cells, 0.04 U/day: 1.61 +/- 0.09 KC/min2/10(6) cells, 0.40 U/day: 1.99 +/- 0.47 KC/min2/10(6) cells, 4.00 U/day: 3.33 +/- 0.43 KC/min2/10(6) cells). The data obtained herein indicate that an increased susceptibility to bacterial infection in diabetic rats results from impaired neutrophil function to produce active oxygen species.


The nucleotide sequence of the S RNA of Impatiens necrotic spot virus, a novel tospovirus. Impatiens necrotic spot virus (INSV) shares a number of properties with tomato spotted wilt virus (TSWV), the type species of the genus tospovirus within the family Bunyaviridae. INSV, however, differs from TSWV in plant host range and serology. In order to define the genomic structure and the taxonomic status of this TSWV-like virus, the nucleotide sequence of its genomic S RNA segment has been determined. The molecular data obtained demonstrate that, like TSWV, INSV has an ambisense S RNA molecule, encoding a non-structural protein in viral sense and the nucleocapsid protein in viral complementary sense. The level of nucleotide sequence homology between their S RNAs, as well as the divergence in amino acid sequence homology of their gene products, confirm previous conclusions from serological studies that INSV and TSWV represent distinct virus species within the newly created genus, tospovirus.


A model of the structure of human annexin VI bound to lipid monolayers. Annexin VI is an eight repeat member of the annexin family of proteins which are both water soluble and bind to negatively charged phospholipids in a calcium-dependent manner. Here we present a model for annexin VI based on fitting the three-dimensional structure of two annexin V molecules (Huber (1990) EMBO J. 9, 3867-3874) to the two-dimensional stain-excluding density of lipid-bound annexin VI (Newman (1989) J. Mol. Biol. 206, 213-219). Both annexin VI lobes could only be fitted with their convex faces closest to the lipid monolayer. This supports the hypothesis that annexin-lipid binding is mediated by the interaction between calcium bound to the loops protruding from the convex protein surface and phospholipid headgroups.


Detection of VAC-beta (annexin-8) in human placenta. A 36 kDa calcium/phospholipid binding protein in human placenta was identified as VAC-beta (annexin-8) by a combination of immunological and peptide mapping analyses. The protein is a minor product in placenta, accounting for less than 1% of extracted annexins. From 150 g of tissue, only 100 micrograms of the protein was isolated. By anion-exchange chromatography on diethylaminoethyl-cellulose annexin-8 coeluted with annexin-3. By gel filtration, the protein chromatographed as a broad peak, where half the product eluted as a monomer and half eluted as a heterodimer that was associated with a 10 kDa subunit. The combination of annexin-8 being a minor component in standard annexin preparations and it co-eluting with annexin-3 by ion exchange chromatography are likely to account for the failure of other labs to characterize the product.


Ectopic induction of dorsal mesoderm by overexpression of Xwnt-8 elevates the neural competence of Xenopus ectoderm. The ectoderm of early Xenopus gastrula is competent to become induced to neural tissue, but dorsal ectoderm is more neural competent than ventral ectoderm. It is a tenable, but as yet untested possibility that the higher neural competence of dorsal gastrula ectoderm is dependent on the presence of the dorsal mesoderm. To test this hypothesis we overexpressed Xwnt-8 in order to ectopically induce dorsal mesoderm in the ventral side of the embryo. We found that this elevated the level of neural competence of ventral ectoderm to that of dorsal ectoderm. The effect of Xwnt-8 on neural competence of ventral ectoderm was strictly correlated with its ability to enhance the amount of dorsal structures. The data indicate that the presence of dorsal mesoderm is a prerequisite for establishing the differences in neural competence between gastrula dorsal and ventral ectoderm.


Role of atrial natriuretic peptide in the pathogenesis of sodium retention in IDDM with and without glomerular hyperfiltration. The pathogenetic determinants of sodium retention in IDDM are not fully understood. The aim of this study was to elucidate the action of ANP in 11 IDDM patients with high GFR (greater than or equal to 135 ml.min-1 x 1.73 m-2), referred to here as HF patients; in 10 IDDM patients with normal GFR (greater than 90 and less than 135 ml.min-1 x 1.73 m-2), referred to here as NF patients; and 12 control subjects, here called C subjects, at baseline and during saline infusion administered on the basis of either body weight (2 mmol.kg-1 x 60 min-1; Saline 1) or of ECV (12 mM.ECVL-1 x 90 min-1; Saline 2) during euglycemic insulin-glucose clamp. C subjects and both HF and NF IDDM patients received a second Saline 1 infusion accompanied by ANP infusion (0.02 microgram.kg-1.min-1) at euglycemic levels. HF and NF patients were studied again after 3 mo of treatment with (10 mg/day). Quinapril (CI 906, Malesci, Florence, Italy), an ACE inhibitor without sulfhydryl group. At baseline, both HF and NF IDDM patients had higher plasma ANP concentrations than C subjects (HF, 36 +/- 4, P less than 0.01 and NF, 34 +/- 3, P less than 0.01 vs. C, 19 +/- 3 pg/ml). Plasma ANP and natriuretic response to isotonic volume expansion was impaired both in HF (44 +/- 8 pg/ml, NS vs. base) and NF (40 +/- 7 pg/ml, NS vs. base) compared with C (41 +/- 4 pg/ml, P less than 0.01 vs. base) during Saline 1. On the contrary, plasma ANP response to Saline 2 was similar in HF and NF patients and C subjects, but IDDM patients had still lower urinary sodium excretion rates. The simultaneous administration of ANP and Saline 1 resulted in comparable plasma ANP plateaus in C subjects and HF and NF patients. However, urinary sodium excretion rate was significantly lower in HF and NF patients than in C subjects: HF, 267 +/- 64, P less than 0.01 and NF, 281 +/- 42, P less than 0.01 vs. C, 424 +/- 39 mumol.min-1 x 1.73 m-2. During simultaneous administration of ANP and Saline 1, GFR and FF increased in C subjects, but not in HF and NF patients. HF and NF patients had higher urinary vasodilatory prostanoid excretion rates than C subjects at baseline. Saline infusion did not change urinary excretion rate of prostanoids either in C subjects or IDDM patients (both NF and HF).(ABSTRACT TRUNCATED AT 400 WORDS)


Meiotic recombination on artificial chromosomes in yeast. We have examined the meiotic recombination characteristics of artificial chromosomes in Saccharomyces cerevisiae. Our experiments were carried out using minichromosome derivatives of yeast chromosome III and yeast artificial chromosomes composed primarily of bacteriophage lambda DNA. Tetrad analysis revealed that the artificial chromosomes exhibit very low levels of meiotic recombination. However, when a 12.5-kbp fragment from yeast chromosome VIII was inserted into the right arm of the artificial chromosome, recombination within that arm mimicked the recombination characteristics of the fragment in its natural context including the ability of crossovers to ensure meiotic disjunction. Both crossing over and gene conversion (within the ARG4 gene contained within the fragment) were measured in the experiments. Similarly, a 55-kbp region from chromosome III carried on a minichromosome showed crossover behavior indistinguishable from that seen when it is carried on chromosome III. We discuss the notion that, in yeast, meiotic recombination behavior is determined locally by small chromosomal regions that function free of the influence of the chromosome as a whole.


Ribosomal DNA is a site of chromosome breakage in aneuploid strains of Neurospora. In wild-type strains of Neurospora crassa, the rDNA is located at a single site in the genome called the nucleolus organizer region (NOR), which forms a terminal segment on linkage group (LG) V. In the quasiterminal translocation strain T(I;V)AR190, most of the right arm of LG I moved to the distal tip of the NOR, and one or a few rDNA repeat units are moved to the truncated right arm of LG I. I report here that, in partial diploid strains derived from T(I;V)AR190, large terminal deletions result from chromosome breakage in the NOR. In most of these partial diploids, chromosome breakage is apparently frequent and the breakpoints occur in many parts of the NOR. The rDNA ends resulting from chromosome breakage are "healed" by the addition of new telomeres. Significantly, the presence of ectopic rDNA creates a new site of chromosome breakage in the genome of partial diploids. These results raise the possibility that, under certain conditions, rDNA is a region of fragility in eukaryotic chromosomes.


Construction and expression of diphtheria toxin-encoding gene derivatives in Escherichia coli. We reported earlier that in the periplasmic space of Escherichia coli, truncated derivatives of diphtheria toxin undergo limited proteolysis [Zdanovsky et al., Mol. Biol. 22 (1988) 1037-1293]. Here, we present data indicating that this proteolysis is reduced in cells bearing a mutation in the degP gene. We have also constructed hybrid genes whose products are not secreted into the periplasm. These hybrid genes were expressed in E. coli from both the pR promoter, controlled by the heat-inducible CI857 repressor, and from the P(lac) promoter, controlled by the IPTG-inducible LacI repressor. The latter system proved to be more productive.


T-cell repopulation following neonatal injection of non-obese diabetic (NOD) mice with anti-T-cell antibodies. Non-obese diabetic (NOD) mice injected with CD3 antibody as newborns have a reduced incidence of diabetes, raising the possibility that the neonatal injection caused a long-lasting change in circulating T cells. The present study shows that NOD and BALB/c mice injected with soluble CD3 antibody in the first 2 days of life sustained an 80-95% reduction in the number of circulating T cells lasting for 2-3 weeks, with T cells returning after 4 weeks, and reaching control values after 6 weeks. The T cells which appeared in intact mice 4-6 weeks after injection showed no excess of T-cell receptor (TcR) delta expressing cells. They had a similar distribution into CD4 and CD8 subsets as uninjected controls, and a similar usage and cell surface expression of four T-cell receptor V beta families. Labelled CD3 antibody was detected in the serum for up to 2 weeks after injection into neonates and was enriched in the thymus. Adoptively transferred T cells continued to be cleared from the circulation for 4 weeks following antibody injection. The properties of T cells which had been exposed to CD3 neonatally were investigated in animals who were first injected with CD3 antibody and then thymectomized. These animals had reduced numbers of T cells at 12 weeks of age. The surviving T cells showed a Ca2+ flux when stimulated but their proliferation in response to concanavalin A (Con A) was reduced, even in the presence of irradiated accessory cells or T-cell supernatant co-stimulator factors. Although the representation of four different V beta families was the same as in the uninjected controls, the density of expression of the T-cell receptor was reduced. The data indicate that the limited number of T cells which survive the injection are functionally deficient and that an intact thymus is required for full T-cell repopulation following neonatal CD3 injection into NOD mice.


Differential effects of pentoxifylline on the production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by monocytes and T cells. Pentoxifylline (PTX) is a methylxanthine compound known to inhibit the production of tumour necrosis factor-alpha (TNF-alpha) by monocytic cells. In this study, we found that PTX differentially regulates the production of TNF-alpha and interleukin-6 (IL-6). Indeed, PTX at high concentrations triggers the production of IL-6 but not of TNF-alpha by peripheral blood mononuclear cells (PBMC). Further experiments indicated that monocytes are responsible for this PTX-induced IL-6 production. When PBMC were stimulated with LPS, PTX was found to inhibit the secretion of TNF-alpha as well as the accumulation of TNF-alpha messenger RNA (mRNA). In contrast, no inhibitory effect was observed on the induction of IL-6. Similar results were obtained when PBMC were stimulated with OKT3 monoclonal antibody (mAb). In addition, the in vivo administration of PTX in transplant patients receiving the first dose of OKT3 allowed to decrease the systemic release of TNF-alpha but not of IL-6. Since monocytes represent a major source of TNF-alpha and IL-6 in these settings, additional experiments were performed in vitro on purified T cells stimulated with the CLB-T3/3, an anti-CD3 mAb which does not require the presence of accessory cells to activate T cells. In this system, PTX was found to inhibit the secretion of both TNF-alpha and IL-6 by T cells. We suggest that cAMP could be involved in these differential effects of PTX on production of TNF-alpha and of IL-6.


Immunosuppressive activity induced by nitric oxide in culture supernatant of activated rat alveolar macrophages. Alveolar macrophages (AM) from normal rats had immunosuppressive activity to mitogen-induced proliferative responses of splenic lymphocytes. We studied the mechanism and the implication of the nitric oxide synthetase pathway in AM-mediated suppression of concanavalin A (Con A)-induced lymphocyte proliferation. The culture supernatant from AM cultures alone did not have immunosuppressive activity to Con A-induced proliferative responses of non-adherent spleen cells (n-ad SC), but the culture supernatant from co-culture of AM and autologous n-ad SC had this activity. Con A-pulsed AM also liberated the immunosuppressive factor. When AM and autologous n-ad SC were cultured separately under the condition that medium could freely communicate, the culture supernatant did not suppress the Con A-induced proliferative response of n-ad SC. This indicated that the immunosuppressive factor was liberated when AM was activated by cell-to-cell contact with n-ad SC. Further, we examined the immunosuppressive activity of the culture supernatant of co-culture of AM and autologous n-ad SC to Con A-induced responses of allogeneic n-ad SC and xenogeneic murine n-ad SC, and allogeneic mixed leucocyte reaction, and found that this culture supernatant could suppress all these proliferative responses. Nitrate (NO2-) synthesis was markedly augmented in the culture supernatants of Con A-pulsed AM and co-culture of AM and n-ad SC. NG-monomethyl-L-arginine (MMA), a specific competitive inhibitor of the nitric oxide synthetase pathway (NOSP), extinguished both NO2- synthesis by AM and AM-mediated immunosuppressive activity. These data suggest that NOSP was important in AM-mediated suppression of Con A-induced lymphocyte proliferation.


Lower back pain. Laminectomies, spinal fusions, demographics, and socioeconomics. The models and analyses used in this study represent an important step in the continued search for the optimum use of surgery for the treatment of lower back pain. The likelihood of patients who are hospitalized with lower back pain in Massachusetts receiving either laminectomies or spinal fusions or both was increased when any of the following demographic, socioeconomic, or medical characteristics were present: white, male, well insured, young, routine admission, admitted to a medium-sized hospital, admitted to a teaching hospital, admitted to a hospital with a high occupancy rate, and discharged home.


Evaluation of 99mTc-mercaptoacetyltripeptides in mice and a baboon. Different derivatives of MAG, carrying amino acids such as D- or L-alanine, D-serine, D-2-aminobutyric acid, D-valine or D-phenylglycine were synthesized and their 99mTc-complexes were evaluated in mice and a baboon. The efficiency of renal handling of the examined 99mTc-complexes is influenced not only by their lipophilicity but also to a great extent by their configuration and the site of substitution. The renal excretion characteristics of 99mTc-MAGAG-DA are superior to those of 99mTc-MAG3 and the studied 99mTc-complexes in both animal species. In an attempt to improve the renal handling of 99mTc-MAG3 and to evaluate the effect of derivatization we have synthesized different derivatives of MAG3 in which one or more glycyl groups are replaced by other amino acids such as D- or L-alanine, D-serine, D-2-aminobutyric acid, D-valine or D-phenylglycine. Due to the presence of a chiral centre in the ligand core, exchange labelling of each of the MAG3 derivatives results in the formation of two diastereomeric technetium complexes. These isomers were separated by HPLC and evaluated in mice. Biodistribution in mice indicates that the efficiency of renal handling of the examined 99mTc-complexes is not only influenced by their lipophilicity but also to a great extent by their configuration. The renal excretion characteristics of isomer DA of 99mTc-MAGAG in mice are superior to those of all other studied 99mTc-complexes and also of the reference compound [131I]Hippuran. The isomers LB of several alanyl derivatives of 99mTc-MAG3 exhibit a pronounced renal retention in both mice and baboon. The results of the evaluation in a baboon confirm the superiority of 99mTc-MAGAG-DA over 99mTc-MAG3 and the other studied 99mTc-complexes.


Reproductive hormones and bone mineral density in women runners. We examined the relationships among reproductive hormone concentrations and bone mineral density (BMD) in 43 women runners classified as eumenorrheic (n = 24), oligomenorrheic (n = 8), or amenorrheic (n = 11). Results were compared with a eumenorrheic nonrunner control group (n = 11). Serum 17 beta-estradiol, progesterone, and dehydroepiandrosterone sulfate concentrations were determined in daily blood samples for 21 days, and integrated concentrations (areas under the curve) were calculated. BMD was assessed at the lumbar spine and proximal femur by dual-photon absorptiometry. As expected, 17 beta-estradiol, progesterone, and lumbar spine BMD were higher in the control and eumenorrheic runner groups than in the oligomenorrheic and amenorrheic runner groups (P less than 0.05). Progesterone concentration was significantly correlated with lumbar spine BMD in the eumenorrheic runners (r = 0.61). None of the steroid hormones was significantly related to BMD in the oligomenorrheic/amenorrheic group. The present data suggest that circulating levels of gonadal steroid hormones affect axial BMD in eumenorrheic runners.