Daltroban blocks thromboxane responses in the pulmonary vascular bed of the cat. The influence of daltroban (BM13.505; SK&F 96148), a thromboxane (Tx) A2-receptor-blocking agent, on responses to the TxA2 mimics U-46619 and U-44069 was investigated in the pulmonary vascular bed of the intact-chest cat under constant-flow conditions. Daltroban (5 mg/kg iv) had no significant effect on mean baseline vascular pressures but significantly decreased responses to the TxA2 mimics without altering responses to prostaglandin (PG) F2 alpha or PGD2 or the PGD2 metabolite 9 alpha, 11 beta-PGF2. Dose-response curves for U-46619 and U-44069 were shifted to the right in a parallel manner, and daltroban had no significant effect on responses to norepinephrine, serotonin, angiotensin II, BAY K 8644, endothelin-(ET) 1, ET-2, or platelet-activating factor (PAF). After administration of daltroban, responses to U-46619 returned to 50% of control in 90 min and responses to the PG and TxA2 precursor arachidonic acid were decreased significantly. These results suggest that daltroban selectively antagonizes TxA2-receptor-mediated responses in a competitive and reversible manner. These data provide support for the hypothesis that discrete TxA2 receptors unrelated to receptors stimulated by PGF2 alpha, PGD2, or 9 alpha, 11 beta-PGF2 are present in the pulmonary vascular bed of the cat. The present data suggest that pulmonary vasoconstrictor responses to PAF and ET peptides are not dependent on activation of TxA2 receptors in the cat.


A thromboxane mimetic, U-46619, produces plasma exudation in airways of the guinea pig. Thromboxane A2 (TxA2) has been implicated in airway responses to allergen and in the bronchial hyperresponsiveness observed in asthma. Furthermore a TxA2 receptor antagonist and a TxA2 synthase inhibitor inhibit plasma exudation in airways induced by inhaled platelet-activating factor. To evaluate whether TxA2 has any direct effect on plasma exudation in the airways, we studied the effect of a stable TxA2 mimetic (U-46619; 2, 20, and 200 nmol/kg iv) on lung resistance (RL) and Evans blue dye extravasation (marker of plasma albumin; 20 mg/kg iv) at the airway levels of trachea, main bronchi, and proximal and distal intrapulmonary airways in anesthetized, tracheostomized, and mechanically ventilated guinea pigs. Injection of U-46619 produced an immediate and marked dose-dependent increase in RL, which peaked at approximately 30 s. At the highest dose of U-46619, we also observed a later increase in RL, starting at approximately 3 min and reaching a second peak at approximately 8 min. Mean systemic blood pressure increased in a dose-dependent manner [maximum 82 +/- 8 (SE) mmHg]. U-46619 also produces dose-dependent plasma exudation, measured as Evans blue dye extravasation, at all airway levels as well as into the tracheal lumen. Airway responses to U-46619 (200 nmol/kg iv) were abolished in animals pretreated with the TxA2 receptor antagonist ICI-192605 (0.5 mg/kg iv). We conclude that U-46619, despite being a vasoconstrictor, is potent in inducing plasma exudation in airways and that this effect is mediated via a TxA2 receptor.


Exercise training prevents decline in stroke volume during exercise in young healthy subjects. Stroke volume (SV) increases above the resting level during exercise and then declines at higher intensities of exercise in sedentary subjects. The purpose of this study was to determine whether an attenuation of the decline in SV at higher exercise intensities contributes to the increase in maximal cardiac output (Qmax) that occurs in response to endurance training. We studied six men and six women, 25 +/- 1 (SE) yr old, before and after 12 wk of endurance training (3 days/wk running for 40 min, 3 days/wk interval training). Cardiac output was measured at rest and during exercise at 50 and 100% of maximal O2 uptake (Vo2max) by the C2H2-rebreathing method. VO2max was increased by 19% (from 2.7 +/- 0.2 to 3.2 +/- 0.3 l/min, P less than 0.001) in response to the training program. Qmax was increased by 12% (from 18.1 +/- 1 to 20.2 +/- 1 l/min, P less than 0.01), SV at maximal exercise was increased by 16% (from 97 +/- 6 to 113 +/- 8 ml/beat, P less than 0.001) and maximal heart rate was decreased by 3% (from 185 +/- 2 to 180 +/- 2 beats/min, P less than 0.01) after training. The calculated arteriovenous O2 content difference at maximal exercise was increased by 7% (14.4 +/- 0.4 to 15.4 +/- 0.4 ml O2/100 ml blood) after training. Before training, SV at VO2max was 9% lower than during exercise at 50% VO2max (P less than 0.05). In contrast, after training, the decline in SV between 50 and 100% VO2max was only 2% (P = NS). Furthermore, SV was significantly higher (P less than 0.01) at 50% VO2max after training than it was before. Left ventricular hypertrophy was evident, as determined by two-dimensional echocardiography at the completion of training. The results indicate that in young healthy subjects the training-induced increase in Qmax is due in part to attenuation of the decrease in SV as exercise intensity is increased.


Architecture of the vir regulons of group A streptococci parallels opacity factor phenotype and M protein class. Group A streptococci have traditionally been categorized into two broad groups based on the presence or absence of serum opacity factor (OF). Recent studies show that these two groups vary in a number of properties in addition to the OF phenotype, including sequence variations in the constant region of the antiphagocytic M protein genes, the presence or absence of immunoglobulin G Fc receptor proteins, and the presence or absence of multiple M protein-like genes situated in a tandem array. The M protein genes (emm) in OF- streptococcal strains are known to be part of a regulon of virulence-related genes controlled by the trans-acting positive regulatory gene, virR, situated just upstream of emm. In OF+ strains, however, the region adjacent to virR is occupied by an M protein-related, type IIa immunoglobulin G Fc receptor gene (fcrA), and the relative position of emm has not been determined. To further define the vir regulon in OF+ streptococci, we used the polymerase chain reaction to show that fcrA49 is situated immediately upstream of emm49 in the OF+ type 49 strain CS101. This result shows for the first time the separate identity and genetic linkage of these two genes in the vir regulon of an OF+ group A streptococcal strain and confirms our previous hypothesis that emm49 exists as the central gene in a trio of emm-like genes. Additionally, using DNA hybridizations, we found considerable sequence divergence between OF- and OF+ group A streptococci in virR and in the noncoding sequences between virR and the emm or fcrA expression site. We found, however, a high degree of sequence conservation in this region within each of the two groups of strains.


A nonapeptide to the putative F-actin binding site of annexin-II tetramer inhibits its calcium-dependent activation of actin filament bundling. A synthetic nonapeptide, Val-Leu-Ile-Arg-Ile-Met-Val-Ser-Arg, corresponding to residues 286-294 of annexin-II tetramer (A-IIt), was shown to completely inhibit the Ca(2+)-dependent bundling of F-actin by this protein. The inhibitory effect of the nonapeptide required preincubation with F-actin and was reversed by the addition of excess A-IIt. Kinetic analysis suggested that the nonapeptide reduced the K(0.5) but not the Vmax of F-actin bundling. In contrast, addition of excess nonapeptide to A-IIt-bundled F-actin did not reverse F-actin bundle formation. Although the nonapeptide produced a dose-dependent inhibition of A-IIt-dependent F-actin bundling, the binding of A-IIt to F-actin was not affected. These results identify a domain of A-IIt that is involved in the bundling activity of the protein and suggest that this domain binds transiently with F-actin, resulting in activation of the bundling activity of A-IIt.


Protein-protein interaction studied by site-directed mutagenesis. Characterization of the annexin II-binding site on p11, a member of the S100 protein family. p11, a member of the S100 protein family, forms a stable heterotetrameric complex with annexin II. The p11-binding site of annexin II resides in the N-terminal 14 residues, which form an amphiphatic alpha-helix with the hydrophobic face representing the contact site for p11 (Johnsson, N., Marriott, G., and Weber, K. (1988) EMBO J. 7, 2435-2442). We show that a corresponding peptide can be used to purify recombinant p11 by affinity chromatography. To map the annexin II-binding site on p11, we have produced progressively truncated p11 derivatives by site-directed mutagenesis. Our analysis reveals that a highly hydrophobic region between residues 85 and 91 is indispensable for annexin II-binding. It is located in the C-terminal extension, following the second distorted EF-hand. Using a series of single amino acid replacements, we have identified individual hydrophobic residues, which seem to represent contact points for annexin II. Most notably, substitution of tyrosine 85 or phenylalanine 86 by alanine drastically reduces the affinity of p11 for annexin II, whereas replacement of these residues by tryptophan has no or only a marginal effect. Thus, hydrophobic side chains on both annexin II and p11 are involved in complex formation.


Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein required for the proliferation and differentiation of granulocyte and macrophage precursors. Previous investigations have identified regions in human and murine GM-CSF that are required for bioactivity. In the present study, alanine substitution mutagenesis was undertaken to define more precisely specific amino-terminal residues in murine GM-CSF that are involved in bioactivity and receptor binding. Five double alanine mutants were identified that showed at least 10-fold reductions in bioactivity (K14AK20A, K14AE21A, H15AK20A, H15AE21A, K20AE21A). Each of these mutants maintained a normal N-linked glycosylation pattern when expressed in COS-1 cells, suggesting that native polypeptide backbone conformation was preserved. The purified prokaryotic expression products of two mutants (K14AE21A and H15AE21A) had a 100-fold decrease in bioactivity and a decrease in receptor binding, indicating that the side chains of K14, H15, and E21 are required for optimal receptor binding and maximal bioactivity.


The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase. Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.


Characterization of the transcription activator protein C1 of bacteriophage P22. We cloned, expressed, and purified the positive regulatory protein C1 of the temperate phage P22 of Salmonella typhimurium. The purified protein was characterized as to its amino acid composition, protein sequence, molecular weight, and antigenicity. P22 C1 was shown to be a tetrameric protein composed of four identical subunits with M(r) = 10,000. Moreover, we identified and characterized two P22 C1-dependent phage promoters, P(RE) and Pa23, whose function was completely dependent on C1 both in vitro and in vivo. These two promoters share a common TTGCN6TTGC/T motif in their -35 regions, the same motif recognized by the analogous phage lambda transcription activator protein cII. P22 C1 protein bound selectively to this region and centered on the TTGC repeat motif. Binding and transcription experiments demonstrated that the two promoters respond coordinately to C1 activation. Last, in contrast to lambda cII, the C1 protein exhibited little cooperativity with Escherichia coli RNA polymerase for DNA binding, but because of its stronger inherent binding ability, achieved an overall promoter affinity similar to that observed for cII at its cognate promoter signals.


Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps. ATP-dependent calcium pumps that reside in intracellular organelles are encoded by a family of structurally related enzymes, termed the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPases (SERCA), which each have a distinct pattern of tissue-specific and developmentally regulated expression. A COS-1 cell expression system was used to examine the biochemical properties of the isoforms: SERCA1 (fast-twitch skeletal muscle). SERCA2a (cardiac/slow-twitch skeletal muscle), SERCA2b (ubiquitous smooth- and non-muscle), and SERCA3 (non-muscle). Each isoform was expressed efficiently and appeared to be targeted to the endoplasmic reticulum. All isoforms displayed qualitatively similar enzymatic properties and were activated by calcium in a cooperative manner with a Hill coefficient of 2. The quantitative properties of SERCA1 and SERCA2a (the muscle isoforms) were identical in all respects. SERCA2b, however, appeared to have a lower turnover rate for both calcium transport and ATP hydrolysis. SERCA3 displayed a reduced apparent affinity for calcium, an increased apparent affinity for vanadate, and an altered pH dependence when compared with the other isoforms. These properties are consistent with an enzyme in which the equilibrium between the E1 and E2 conformations is shifted toward the E2 state.


The nucleotide binding/hinge domain plays a crucial role in determining isoform-specific Ca2+ dependence of organellar Ca(2+)-ATPases. Several isoforms of organellar Ca(2+)-ATPases have been identified, each of which is expressed in a tissue-specific manner. In order to examine the functional properties of fast-twitch (SERCA 1a), cardiac/slow-twitch (SERCA 2a), and non-muscle (SERCA 3) isoforms of the Ca(2+)-ATPase, cDNAs of each type were expressed transiently in COS-1 cells. A study of the Ca2+ dependence of Ca2+ uptake showed that SERCA 1 and SERCA 2 have identical Ca2+ dependences (K0.5 = pCa 6.87 +/- 0.03 and pCa 6.87 +/- 0.02, respectively), but SERCA 3 has a lower Ca2+ dependence (K0.5 = pCa 6.32 +/- 0.03). A study of the ATP dependence of Ca2+ uptake showed that SERCA 1, 2, and 3 have almost identical ATP dependences. Average Hill coefficients derived from Ca2+ uptake curves ranged from 1.7 to 1.8 for the three isoforms. In order to identify which regions of the linear sequence determine this difference in Ca2+ dependence, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed. Chimeric Ca(2+)-ATPases containing the nucleotide binding/hinge domain of SERCA 2 had SERCA 2 type Ca2+ dependence, but both nucleotide binding/hinge and COOH-terminal transmembrane domains of SERCA 3 were required for SERCA 3 type Ca2+ dependence. Accordingly, structural interactions between the nucleotide binding/hinge and COOH-terminal transmembrane domains appear to determine isoform-specific Ca2+ dependences.


Down-regulation of scatter factor in MRC 5 fibroblasts by epithelial-derived cells. A model for scatter factor modulation. Scatter factor/hepatocyte growth factor (SF/HGF) is a multifunctional cytokine produced by embryonic fibroblasts and other mesenchymal cells that affects the growth and/or the movement of certain epithelia. Here we report that expression of scatter factor activity by MRC 5 cells, a strain of normal human embryonic lung fibroblasts, is greatly reduced as a result of co-culture of these cells with SVK14, an SV40-transformed human keratinocyte cell line. Using a cDNA probe to the beta chain of human HGF, we have found that the fall in SF activity in MRC 5/SVK14 co-cultures is accompanied by the loss of SF/HGF transcripts. As the inhibition of SF activity coincides with the disappearance of SF/HGF transcript, we conclude that inhibition of the SF activity expressed by MRC 5 cells by co-culture with SVK14 involves transcriptional regulation.


The systemic vasculitides.

The systemic vasculitides. Taken together, the systemic vasculitides constitute a small but significant component in the practice of many primary care physicians. Like most diseases with autoimmune aspects, the vasculitides increase in prevalence with age. Of paramount importance is careful differentiation, to provide early appropriate treatment and to monitor adverse effects.


Determination of isradipine and its pyridine metabolite in serum by capillary column gas chromatography with nitrogen-selective detection. A relatively simple, sensitive and precise gas chromatographic method for the determination of isradipine, a calcium antagonist of the dihydropyridine type, and its main metabolite in serum is described. Using a one-step extraction procedure, a wide-bore column and a nitrogen-phosphorus detector, a limit of quantitation of 0.5 and 2.0 nM for isradipine and the metabolite was found. No interferences from several drugs were observed. The method was successfully used in a pharmacokinetic study in hypertensive women during pregnancy.


Effects of repeated botulinum toxin injections on orbicularis oculi muscle. Histologic evaluation was conducted on 12 orbicularis oculi specimens from 11 patients with essential blepharospasm and Meige's disease who had received an average of 11.3 injections of botulinum A toxin over 3.5 years. Denervation was demonstrated by the spread of acetylcholinesterase staining on muscle fibers when specimens were evaluated within 11 weeks of the last injection. When specimens were taken after 12 weeks, spread of acetylcholinesterase was confined to the neuromuscular junctions, with little fiber size variability resembling normal muscle. Fibrosis seen in three specimens could be correlated to prior surgery. Repeated injections of botulinum toxin into human muscle do not appear to cause irreversible muscle atrophy or other degenerative changes. Denervation changes (fiber size variability, acetylcholinesterase spread) appear to correlate to the time interval since the last injection.


The effect of the temperature and duration of sample storage on the measurement of lymphocyte subpopulations from HIV-1-positive and control subjects. EDTA-anticoagulated blood samples from 19 HIV-1-positive subjects and 13 healthy laboratory worker controls were analysed for three lymphocyte subpopulations (CD3, CD4, CD8 T cells) (lysed whole blood method, Becton Dickinson FACScan flow cytometer) at 0, 24, 48, 72 and 96 h after venesection, having been stored at either 4 degrees C, 12 degrees C, 16 degrees C or 21 degrees C. In samples stored at 4 degrees C and 12 degrees C there was a significant fall in both %CD3 and %CD 4, and a significant rise in %CD8. At 16 degrees C the %CD8 remained stable, while there were marginal rises in %CD3 and %CD4. At 21 degrees C, the %CD8 again remained stable, while %CD3 and %CD4 rose significantly with time. These trends were independent of HIV-1 status. At each temperature studied, the rates of change of lymphocyte subpopulations were independent of each other. These results suggest that a temperature range of 14-16 degrees C may be optimal for sample storage prior to measurement of T cell subsets. They emphasise the importance of strict control on conditions if samples are to be kept for any length of time before analysis.


Fc epsilon R11/CD23 receptor distribution in patch test reactions to aeroallergens in atopic dermatitis. There is increasing evidence that exposure to organic allergens may induce or exacerbate lesional skin in patients with atopic dermatitis. In this study, patients with atopic dermatitis were patch tested to 11 common organic allergens and to control chambers containing 0.4% phenol and 50% glycerin in 0.9% saline. In biopsies from positive patch test reactions, patch test control skin, lesional eczematous and non-lesional skin from atopic individuals, and normal skin from non-atopic volunteers, the presence and distribution of macrophages (RFD7+), dendritic cells (RFD1+), and Langerhans cells, and the expression of the low-affinity receptor for IgE (CD23) were investigated. In patch test reactions and lesional skin samples, inflammatory infiltrates of diffusely distributed macrophages (RFD7+), dendritic cells (RFD1+), T lymphocytes (RFTmix+), and Langerhans cells (CD1+) were seen, the latter being present in both the epidermis and the dermis. The numbers of Langerhans cells were reduced in the epidermis and increased in the dermis in patch test reactions and lesional skin compared to their controls. Double staining revealed a change in the distribution of CD23 antigen. In patch test control and non-lesional biopsies many macrophages and only a few Langerhans cells within the dermal infiltrates expressed this antigen. In patch test reaction and lesional skin samples, however, the proportion of CD23+ dermal Langerhans cells had increased compared to macrophages. Furthermore, in these latter samples an increased proportion of dermal CD1+ cells expressed the dendritic cell (RFD1+) marker. These results show that following antigen challenge there are marked similarities between the phenotype of the cellular infiltrate in patch test reaction and lesional skin biopsies, and also demonstrate a changing distribution of CD23 on antigen-presenting cells.


Relative importance of prior basal cell carcinomas, continuing sun exposure, and circulating T lymphocytes on the development of basal cell carcinoma. This 36-month prospective study of a group of 61 people at high risk to develop multiple basal cell carcinomas (BCC) examined the circulating lymphocyte subsets of the population, patterns of sun exposure, and the longitudinal development of basal cell carcinoma. Sun exposure status was highly correlated with immune status defined by the CD4/CD8 T-lymphocyte ratio. There were significantly more BCC at 18 and 36 months in the 35 patients with high sun exposure and low CD4/CD8 ratio than in the 20 patients with low sun exposure and high CD4/CD8 ratio. A multivariate analysis assessed the relative importance of prior basal cell carcinoma, sun exposure, and immune status on the development of the skin cancer. Basal cell carcinoma developing in the previous 18 months and sun exposure during those 18 months were the first and second most important variables in determining development of basal cell carcinoma during the next 18 months. CD4/CD8 ratio had no additional predictive ability once prior skin cancers and sun exposure were accounted for. A low ratio of CD4/CD8 cells correlated with high sun exposure during the preceding 18 months.


Dental management considerations in children with attention-deficit hyperactivity disorder. Children suffering from attention-deficit hyperactivity disorder frequently have numerous orofacial anomalies of concern to the dentist. Behavioral manifestations of the disorder frequently impair the patient's ability to perform home care adequately, make dental treatment arduous, and place the patient at risk of physical abuse from family and peers. Familiarity with the symptoms and treatment of the disorder will better prepare the dentist to meet the needs of this unique group of patients.


The small non-structural protein NS2 of the autonomous parvovirus minute virus of mice is required for virus growth in murine cells. Mutants of the autonomous parvovirus minute virus of mice (MVM) strains MVM(p) and MVM(i) that either fail to produce or produce a truncated NS2 protein, were deficient in the production of infectious virus and attained lower levels of viral DNA synthesis than wild-type virus following infection of a series of normal and transformed murine cell lines. Mutant virus growth and the levels of DNA replication were similar to those of wild-type virus in the rat, hamster and human lines tested. These results suggest that the requirement of NS2 for the growth of MVM is murine species-specific.


Dental management considerations in children who stutter. Stuttering is one of the most common speech disorders of childhood. It is characterized by involuntary pauses, reiteration and prolongation of sounds and syllables. Oral motor incoordination may make provision of dental treatment arduous. Familiarity with the verbal and nonverbal manifestations of the disorder and with the possible adverse psychosocial implications prepares the dentist best to meet the needs of this unique group of children.


Pediatric dental education and community service: a combined approach. Dental educators nationwide have expressed concern regarding the decreasing pediatric clinical experience available to undergraduate dental students. Some educators have suggested that dental programs should utilize extramural clinics and rotations to enhance current patient pools. This paper presents a successful clinical program that is designed to 1) augment the dental education of predoctoral dental students, and 2) provide dental care for an underserved pediatric dental population in an urban community.


Identification of a novel N-methyl-D-aspartate receptor population in the rat medial thalamus. To evaluate the possibility of pharmacologically distinct N-methyl-D-aspartate (NMDA) receptor subtypes, quantitative autoradiography was used to determine the potency of several compounds as inhibitors of L-[3H]glutamate or [3H]MK-801 binding to rat brain NMDA receptors in 10 brain regions. Competitive NMDA receptor antagonists displayed differing pharmacological profiles in the forebrain, cerebellum, and medial regions of the thalamus (midline nuclei). For example, compared with other competitive antagonists, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonate (CPP) and LY-233536 were especially weak displacers of L-[3H]glutamate binding in the cerebellum. In the the medial thalamus, CPP and D-2-amino-5-phosphonopentanoate displayed relatively low affinities, whereas LY-233536 was relatively potent. The noncompetitive NMDA receptor antagonists also displayed regional variations in their pharmacological profiles. Relative to other regions, [3H]MK-801 binding in the cerebellum was weakly displaced by MK-801 and potently displaced by dextromethorphan and SKF-10047. In the medial thalamus, 1-[1-(2-thienyl)-cyclohexyl]piperidine was relatively potent and SKF-10047 was relatively weak. These results confirm previous suggestions that the cerebellum contains a distinct NMDA receptor subtype and indicate that nuclei of the medial thalamus contain a novel NMDA receptor subtype that is distinct from both those found in the cerebellum and in the forebrain.


Distribution of preproatrial natriuretic peptide mRNA in rat brain detected by in situ hybridization of DNA oligonucleotides: enrichment in hypothalamic and limbic regions. The expression and distribution of mRNA encoding preproatrial natriuretic peptide (ppANP) in rat brain has been investigated by in situ hybridization of two 35S-labeled synthetic DNA oligonucleotides, based on a cDNA clone sequence that encodes rat ppANP. The highest relative concentrations of ppANP mRNA were detected in the medial preoptic hypothalamic nucleus ("anteroventral/third ventricle region") and the medial habenula. Moderate concentrations of ppANP mRNA were observed in the CA1 pyramidal cells of the hippocampus, the endopiriform nucleus, the arcuate nucleus, the zona incerta, and cells of the pontine tegmental and peduculopontine nuclei. Several of these regions, including the habenula and the hypothalamic areas, have previously been reported to contain atrial natriuretic peptide (ANP)-like immunoreactivity, but the expression of ppANP mRNA in CA1 pyramidal cells suggests the occurrence of differential translation of ppANP mRNA into protein product in different brain regions, or the existence of different immunological forms of the peptide. The abundance of ppANP mRNA in brain was relatively low in comparison with that previously reported for many other mRNA species encoding other brain neuropeptides. These results demonstrate that ANP gene expression occurs in discrete neuronal populations of the CNS and that studies of the regulation of this expression should now be possible using quantitative in situ hybridization.


Modification of Pseudomonas aeruginosa virulence factors by sub-inhibitory concentrations of antibiotics. This study's objectives were to evaluate the effects of subminimum inhibitory concentrations (MICs) of tobramycin, gentamicin, netilmicin, streptomycin, ciprofloxacin, cefotaxime and piperacillin on proteinase production, alginate and siderophore synthesis by two strains of Pseudomonas aeruginosa. One of these strains, of recent clinical isolation, was mucoid. In fact it is well known that mucoid strains are more resistant than non-mucoid; there is, moreover, evidence that in cystic fibrotic lungs the non-mucoid P. aeruginosa are invariably replaced by mucoid variants. Our results show that subinhibitory concentrations of beta-lactams and quinolones significantly reduced the amount of alginate. Protease production was affected by all antibiotics tested.


Renin-angiotensin system unresponsiveness in phaeochromocytoma. Water immersion to the neck is able to provoke a profound suppression of the renin-angiotensin system in several clinical conditions associated with hyper-reninaemia. Both hyper-reninaemia and secondary aldosteronism have sometimes been described in phaeochromocytoma. We report on two patients, with surgically proven phaeochromocytoma, in whom water immersion, performed before surgery, failed to induce any significant change in plasma renin activity.


A block in full-length transcript maturation in cells nonpermissive for B19 parvovirus. Vertebrate parvoviruses share a similar genomic organization, with the capsid proteins encoded by genes on the right side and nonstructural proteins encoded by genes on the left side. The temporal and cell-specific appearances of these two types of gene products are regulated by a variety of genetic mechanisms. Rodent parvovirus structural proteins, for example, are encoded by a separate promoter which is positively regulated by nonstructural-gene products. In contrast, for the human B19 parvovirus, the analogous structural-gene promoter is nonfunctional, and both left- and right-side transcripts originate from a single promoter and are highly processed. Using a combination of sensitive RNA analyses of wild-type and mutant templates, we have found that the relative abundance of these alternatively processed transcripts appears to be governed by unique postinitiation events. In permissive cells, the steady-state level of right-side structural-gene transcripts predominates over that of left-side nonstructural-gene transcripts. In nonpermissive cells transfected with the B19 virus genome, nonstructural-gene transcripts predominate. Removal of 3' processing signals located in the middle of the viral genome increases transcription of the far right side. Disruption of a polyadenylation signal in this region makes readthrough of full-length right-side transcripts possible. These results suggest that the abundance of B19 virus RNAs is determined by active 3' processing and is coupled to DNA template replication.


Role of the adenovirus E3-19k conserved region in binding major histocompatibility complex class I molecules. The adenovirus early region 3 glycoprotein E3-19k binds to and down regulates major histocompatibility complex (MHC) class I molecules in infected cells. We previously identified a 20-amino-acid conserved region in E3-19k by comparison of protein sequences from four different adenovirus serotypes. The roles of the E3-19k C-terminal and adjacent conserved regions in the interaction with MHC class I molecules have been examined. A functional class I-binding glycoprotein was expressed from the cloned E3 18.5-kDa open reading frame of adenovirus type 35. Truncations and single-amino-acid mutations in the adenovirus type 35 glycoprotein were created by site-directed in vitro mutagenesis and tested for the ability to associate with MHC class I molecules. Deletion of most of the transmembrane domain and cytoplasmic tail did not affect binding to class I molecules. However, removal of an additional 11 amino acids eliminated binding and changed the conformation of the adjacent conserved region. Separate mutations of residues Asp-107 and Met-110, within the conserved region, severely reduced or eliminated binding. These data indicate that the E3-19k conserved region plays a crucial role in binding to MHC class I molecules.


[Induction of tubular basement membrane (TBM) antigen specific suppressor T cells in BALB/c mice]. Transfer of tubular basement membrane (TBM)-primed thymocytes from BALB/c mice that had been immunized with allogeneic TBM antigen without adjuvant prevented the development of interstitial nephritis (IN) in recipient BALB/c mice that had been immunized with TBM antigen with complete Freund's adjuvant (CFA) to produce IN. TBM antigen was prepared from TBM of normal ddY mice (ddY TBM antigen). BALB/c mice were highly susceptible to IN and showed a high immune response to TBM antigen when they were immunized with TBM antigen in CFA. Development of IN in high responder BALB/c mice was clearly suppressed by transfer with ddY TBM-thymocytes. The anti-TBM antibody response and the proliferative response of splenic T cells to TBM antigen were also depressed by the transfer. The cell extract of ddY TBM-thymocytes had also suppressive activity on the development of IN and on the immune response to TBM antigen. This simple system without any adjuvant for inducing the thymocytes, which has strong suppressive activity on the development of IN and on the immune response to TBM antigen, may allow us to analyse the role of suppressor T cells in negative regulation of IN.


High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells. Sindbis virus is an alphavirus with a very wide host range, being able to infect many birds and mammals as well as mosquitoes. We have isolated a monoclonal antibody that largely blocks virus binding to mammalian cells. This antibody was found to be directed against the C-terminal domain of the high-affinity laminin receptor, a 67-kDa protein present on the cell surface that binds with high affinity to basement membrane laminin and that is known to be important in development and in tumor invasion. This receptor is believed to be formed from a 295-amino-acid polypeptide that is modified in some unknown way after translation. The primary sequence of this 295-amino-acid protein is highly conserved among mammals. We found the hamster amino acid sequence to be identical to a mouse sequence and to differ at only two amino acids from a human sequence and at two amino acids from a bovine sequence. To verify the importance of the laminin receptor for infection by Sindbis virus, hamster cells were stably transfected with the gene encoding the 295-amino-acid protein under the control of a high-efficiency promoter. Such transfected hamster cells overexpressed the laminin receptor at the cell surface, bound severalfold more Sindbis virions than did the parental cells, and became infected by Sindbis virus with a higher efficiency. In contrast, cells transfected with the antisense gene expressed less laminin receptor on the surface and were less susceptible to the virus. Binding of the virus varied linearly with the amount of laminin receptor on the cell surface, whereas infectivity measured with a plaque assay varied with the 1.4 power of the receptor concentration, suggesting that interaction with more than one receptor aids virus penetration. By these criteria, the laminin receptor functions as the major receptor for Sindbis virus entry into mammalian cells. We also found that the anti-laminin receptor antibody partially blocked Sindbis virus binding to mosquito cells, suggesting that the laminin receptor is conserved in mosquitoes and functions as a Sindbis virus receptor in this host. The wide distribution of this highly conserved receptor may be in part responsible for the broad host range exhibited by the virus, which infects a wide range of mammals and birds as well as its mosquito vector and can infect many different tissues within these hosts.(ABSTRACT TRUNCATED AT 400 WORDS)


[A comparison of bronchodilating drugs in the treatment of stable COPD]. The purpose of this study was to establish the optimal bronchodilating drug among therapies currently available for clinical treatment of the stable phase of chronic obstructive pulmonary disease (COPD). The efficacy of ipratropium bromide 40 micrograms, salbutamol 200 micrograms, and ipratropium bromide 40 micrograms plus salbutamol 200 micrograms was compared in 14 patients with COPD. Daily PEFR was obtained during the last seven days of a 2 week period incorporating drug inhalation four times daily. FEV1 and FVC were assessed on the final day of the treatment period. In the absence of bronchodilating medication, FEV1 was 1.27 +/- 0.13 l (52.9 +/- 5.1% pred). With ipratropium bromide 40 micrograms alone, FEV1 was 1.43 +/- 0.13 l (59.8 +/- 5.3% pred). A similar value was obtained for salbutamol 200 micrograms: 1.45 +/- 0.14 l (61.0 +/- 5.4% pred). However, FEV1 following the administration of ipratropium bromide 40 micrograms in combination with salbutamol 200 micrograms was 1.51 +/- 0.13 l (63.6 +/- 5.3% pred). The percent increase in FEV1 (compared to the value obtained without medication) was significantly higher with combined ipratropium bromide 40 micrograms plus salbutamol 200 micrograms (122.2 +/- 3.8%) than with either ipratropium bromide 40 micrograms (114.8 +/- 5.5%) or salbutamol 200 micrograms (116.5 +/- 4.4%) alone. Furthermore, the daily post-dilator PEFR improved significantly more with the combined therapy four times a day (311 +/- 29 l/min) than with either ipratropium bromide 40 micrograms (296 +/- 30 l/min) or salbutamol 200 micrograms (303 +/- 29 l/min) therapy alone. There was no discernible difference between results obtained with ipratropium bromide 40 micrograms versus salbutamol 200 micrograms.(ABSTRACT TRUNCATED AT 250 WORDS)


Are cardiac and vascular "amplifiers" both necessary for the development of hypertension? The vascular amplifier leads to enhancement of all resistance responses, from full dilatation to maximum constriction. The mechanism of resistance amplification is narrowing of the resistance vasculature, which is approximately constant at all levels of vasomotor tone. From the literature, the site of narrowing is localized to the small arteries and large arterioles. The narrowing at rest leads during constriction, to patchy reduction in blood flow in the microcirculation. The enhanced resistance responses of the vascular amplifier during constriction, increase blood pressure (BP) upstream, which minimizes the hemodynamic effects on the microcirculation and helps to maintain venous return. Concentric left ventricular (LV) hypertrophy is an amplifier of stroke volume and cardiac output. It reinforces the elevation of BP upstream from the site of vascular narrowing. This appears important for the initiation and maintenance of hypertension, in view of findings in SHR showing: (1) that in the course of normal development of hypertension the vascular amplifier properties develop before the onset of hypertension, which occurs in parallel with an increase in rate of LV hypertrophy; (2) after brief periods of enalapril treatment, hypertension redevelops in parallel with the redevelopment of LV hypertrophy, whilst the vascular amplifier properties remain suppressed; (3) treatment with immuno-sympathectomy plus prazosin prevents the development of both LV hypertrophy and hypertension but only produces gradual suppression of the vascular amplifier properties. The role of the sympathetic nervous system on LV-hypertrophy is mediated through alpha 1-adrenoceptors.


Remodeling of the heart (membrane proteins and collagen) in hypertensive cardiopathy. The basis for impaired left ventricular function of hearts in moderate to severe stages of hypertrophy and congestive heart failure remains uncertain. At the cellular level, the mechanisms governing the movements of calcium in the myocardium are actually depressed and might at least in part account for the slowing of the maximum shortening velocity and the impaired relaxation. These alterations of membrane proteins seem particularly important in species where the slowing of Vmax cannot be a consequence of the myosin heavy chain shift. They lead to an unstable equilibrium of calcium homeostasis and to calcium overload in heart failure. On the other hand, the enhanced density and remodeling of collagen in the hypertrophied heart, which would depend on elevation in circulating aldosterone, impair myocardial stiffness with diastolic dysfunction and lead to altered pumping capacity of the heart. Disturbances of calcium metabolism and matrix collagen remodeling enhance early afterdepolarizations and arrhythmias.


Angiotensin II, vascular structure and blood pressure. Angiotensin II (Ang II) in low dose raises blood pressure slowly by a mechanism which is not understood, but which is clearly different from the better known direct vasoconstrictor effect. Vascular hypertrophy develops during this slow pressor response, but is not wholly a consequence of the increase of pressure. We discuss non-pressor mechanisms by which Ang II may act as a growth factor to promote structural vascular change. Studies with cultured vascular smooth muscle cells suggest at least three possibilities, but none of these has been tested in vivo during slow pressor infusion of Ang II. The action of growth factors may be important in hypertension since increased arterial pressure causes vascular hypertrophy. Growth factors influence markedly the extent of this hypertrophic response and, however produced, vascular hypertrophy has an important influence on resistance and arterial pressure in hypertension.


[Characteristics of surgical interventions on account of uncomplicated duodenal ulcers]. The peculiarities of performance of the operative intervention for duodenal ulcer in 291 patients are presented. The technical difficulties encountered at the time of operative intervention in uncomplicated course of the disease can be conditioned by constitution of a patient, or peculiarities of the pathological process. It is expedient to conduct the operative treatment of the patients with an uncomplicated duodenal ulcer at a specialized therapeutic institution, where the choice of a type for forthcoming operation is made on the basis of the findings of preoperative examination, and operative intervention is performed by the highly qualified experts.


[Tactics of the surgical treatment of acute intestinal obstruction]. The results of treatment of 184 patients with acute ileus of non-tumour genesis operated on at the clinic within the period of from 1981 to 1990 are presented. In elimination of ileus without the surgical intervention, together with intensive therapy, intestinal decompression was used. Of 19 patients with acute ileus associated with peritonitis operated on, 12 underwent resection of the intestine. The use of enterosorption as well as extracorporeal hemosorption contributed to elimination of endogenous intoxication.


[Laparoscopy in penetrating wounds of the abdomen]. Within the recent 5 years, the authors observed 103 sufferers with penetrating injury to the abdomen. Operated on were 79 patients. Of them, 39 underwent explorative laparotomy. Laparoscopy was performed in obscure diagnosis in sufferers with associated trauma who were unconscious, in those with the signs of alcohol intoxication, in drug addicts etc. The advantages of the use of laparoscopy in the diagnosis, and in some patients--in the treatment of injuries were noted.


[The treatment of pneumothorax and hemothorax in multiple rib fractures and associated trauma]. The results of treatment of 480 sufferers with multiple costal fractures and associated trauma to the chest were analysed. Resulting from trauma, 55 (25.5%) patients developed pneumothorax, 71 (32.8%)--hemothorax, 90 (41.7%)--hemopneumothorax. Treatment of pneumo- and hemothorax in most cases was conservative (puncture of the pleural cavity was usually performed, rarely--its drainage). In 47 sufferers with associated trauma who were at a forced position (lying on their back), the aimed catheterization of the pleural cavity by means of the trocar stilette curved under the angle of 60 degrees was used. For the treatment of clotted hemothorax, the streptokinase was used with a positive effect noted in 6 of 7 patients. Indications for thoracotomy are restricted in patients with associated chest trauma in presence of shock and acute blood loss.


Abnormal lipid and fatty acid compositions of kidneys from mice with polycystic kidney disease. Renal cyst development in polycystic kidney disease (PKD) involves hyperplastic growth and extensive membrane alterations, suggesting abnormal membrane composition and function. Using thin-layer and gas-liquid chromatography, we analyzed the lipid components of the kidneys from 120-day-old DBA/2FG-pcy (pcy) having PKD as compared to normal DBA/2J (DBA) mice. At sacrifice, kidneys from pcy mice were four times larger than DBA controls, indicating that extensive renal cyst growth had occurred. The ratios of cholesterol/phospholipid, choline glycerophospholipid (GPC)/ethanolamine glycerophospholipid (GPE) and alkenylacyl GPE/diacyl GPE were higher (by 25%, 41% and 72%, respectively) in the cystic kidneys, while total phosphatidylinositol (PI), GPE and cardiolipin (DPG) were lower (by 13%, 23% and 27%, respectively). With respect to fatty acid compositions, there were significantly lower levels of docosahexaenoic acid (DHA, 22:6n-3) and higher levels of adrenic acid (AdA, 22:4n-6) in the phospholipids of pcy mouse kidneys. These changes were not present in serum, indicating that they were not generalized differences. Interestingly, the lower level of DHA in GPE was found to be associated with the alkenylacyl, but not the diacyl species. The fatty acids comprising the product/substrate ratio for the delta 4 desaturase activity were lower across all phospholipids, indicating a possible abnormality in polyunsaturated fatty acid metabolism in this model of PKD. These lipid abnormalities may influence membrane-mediated events such as receptor activation, signal transduction, ion transport and enzyme activities. The renal pathophysiologies associated with PKD may be related to the tissue lipid abnormalities described herein.


Transcriptional repression of the E2-containing promoters EIIaE, c-myc, and RB1 by the product of the RB1 gene. The protein product of the retinoblastoma susceptibility gene, p110RB1, is a nuclear phosphoprotein [W.H. Lee, J.Y. Shew, F.D. Hong, T.W. Sery, L.A. Donoso, L.J. Young, R. Bookstein, and E.Y. Lee, Nature (London) 329:642-645, 1987] with properties of a cell cycle regulator (K. Buchkovich, L.A. Duffy, and E. Harlow, Cell 58:1097-1105, 1989; P.L. Chen, P. Scully, J.Y. Shew, J.Y. Wang, and W.H. Lee, Cell 58:1193-1198, 1989; J.A. DeCaprio, J.W. Ludlow, D. Lynch, Y. Furukawa, J. Griffin, H. Piwnica-Worms, C.M. Huang, and D.M. Livingston, Cell 58:1085-1095, 1989; and K. Mihara, X.R. Cao, A. Yen, S. Chandler, B. Driscoll, A.L. Murphree, A. TAng, and Y.K. Fung, Science 246:1300-1303, 1989). Although the mechanism of action of p110RB1 remains unknown, several lines of evidence suggest that it plays a role in the regulation of transcription. We now show that overexpression of p110RB1 causes repression of the adenovirus early promoter EIIaE and the promoters of two cellular genes, c-myc and RB1, both of which contain E2F-binding motifs. Mutation of the E2 element in the c-myc promoter abolishes p110RB1 repression. We also demonstrate that a p110RB1 mutant, which is refractory to cell cycle phosphorylation but intact in E1a/large T antigen-binding properties, represses EIIaE with 50- to 80-fold greater efficiency than wild-type p110RB1. These data provide evidence that hypophosphorylated p110RB1 actively represses expression of genes with promoters containing the E2F-binding motif (E2 element).


[The prevalence of spondylopathies among the crane operators in the port of Venice]. A group of 78 crane operators were examined using the "EPM Research Unit" method for assessing spine function. Crane operators work in a fixed posture characterized by a flexed position of the cervical spine, isometric load of the extensor cervical muscles and increased dorsal kyphosis. The prevalence of spinal disorders was 39.7% for the cervical tract, 37.2% for the dorsal tract and 38.5% for the lumbar region. The results were compared with those concerning a control group. A statistically significant difference was found in the number of cervical and dorsal complaints (O.R. = 3.33 and 2.69), whereas the difference was nearly significant for the lumbar tract (O.R. = 1.65). The results suggest that this category of workers may be subject to an increased risk for the spine.


A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome. PRP16 is an RNA-dependent ATPase required for the second catalytic step of splicing in vitro. A dominant suppressor of a branchpoint mutation in Saccharomyces cerevisiae, the prp16-1 allele, contains a Tyr to Asp change in the nucleotide-binding site consensus sequence. We now find that cells harboring the prp16-1 allele have a general growth defect that is exacerbated at cold temperatures. The mutant is dominant over the wild-type gene when overexpressed. Purified Prp16-1 protein binds to the spliceosome with apparently wild-type affinity; however, it only weakly complements the second-step block in a PRP16-depleted extract. Analysis of purified Prp16-1 revealed that the rate of ATP hydrolysis is greatly reduced. These results can account for the dominant negative growth phenotype and argue that the ATPase activity of PRP16 is essential for its role in splicing. Moreover, since PRP16 is a member of the DEAD/H box families, these findings have important implications for a large class of proteins.


Gonadal-independent developmental changes in activation of N-methyl-D-aspartate receptors involved in gonadotropin-releasing hormone secretion. Using hypothalamic explants of male rats, we have shown that the N-methyl-D-aspartate (NMDA) receptors involved in a stimulatory control of gonadotropin-releasing hormone (GnRH) secretion were transiently activated at 25 days around the time of onset of puberty. This was evidenced by studying the dose-related inhibition of veratridine-induced GnRH secretion by MK-801, a use dependent antagonist of NMDA receptors. An increase in sensitivity of GnRH secretion to the inhibitory effect of MK-801 was used as a marker of increased activation of NMDA receptors involved in stimulation of GnRH secretion. Here, we report on data obtained in intact and castrated rats at different ages. The aim was to determine whether the absence of gonads would affect the developmental changes in activation of NMDA receptors that we described recently. In pubertal (50-day-old) rats, orchidectomy resulted in an activation of NMDA receptors which was nonsignificant after 4 days but significant after 13 days. In prepubertal rats orchidectomized at 5 or 10 days and studied 10 days later, the NMDA receptors involved in GnRH secretion were also more activated than in intact animals. Using explants of intact and castrated animals, a similar increase in activation of NMDA receptors was observed between 15 and 25 days of age, a period preceding onset of puberty. Subsequently, between 25 and 50 days, a reduction in NMDA receptor activation was seen. This decrease was observed in intact rats showing normal sexual development and in castrated rats as well.(ABSTRACT TRUNCATED AT 250 WORDS)


Antagonism of ceruletide, a cholecystokinin analog, to the neurochemical effects of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, phencyclidine and MK-801, on regional dopaminergic neurons in the rat brain. In the present study, we investigated the effects of ceruletide (CL), a cholecystokinin analog, on the neurochemical response to non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, phencyclidine (PCP) and MK-801, of the dopaminergic neuron systems in the discrete regions of the rat brain. Systemically administered PCP (7.5 mg/kg, i.p.) or MK-801 (1.0 mg/kg, i.p.) produced significant increases in the tissue contents of dopamine metabolite, homovanillic acid (HVA), in the prefrontal cortex, the nucleus accumbens and the olfactory tubercle but not in the nucleus caudatus putamen after 60 min. The effects of NMDA receptor antagonists in the nucleus accumbens and the prefrontal cortex were partially antagonized by pretreatment with CL (80 and 400 micrograms/kg, i.p., at 60 min prior to the drugs). While CL alone decreased the dopaminergic metabolism only in the nigrostriatal pathways in naive rats, the present results indicated that CL also attenuates the activities of the meso-limbic and meso-cortical dopaminergic neuron systems when these are enhanced by either PCP or MK-801.


[The surgical treatment of preperitoneal inguinal hernia: a comparison between the methods of Rives and Stoppa]. In groin hernia surgery pre-peritoneal prosthetic repair is a valid alternative to traditional inguinal repair in patients with a large area of transversalis fascia weakness: direct, inguinoscrotal, recurrent, bilateral hernias. Pre-peritoneal prosthetic surgical approach by Rives' technique (little unilateral prosthesis) has been used in 121 cases (24% bilateral and 67% recurrent hernias) and by Stoppa's technique (great bilateral prosthesis) in 95 cases (26% bilateral and 55% recurrent hernias). The results demonstrated 9.9% morbidity and 5.7% recurrences by Rives' technique vs 3.1 morbidity and complete absence of recurrences by Stoppa's technique. These results confirm the validity of large prosthetic pre-peritoneal repair in groin surgery.


[Congenital duodenal stenosis. A case treated in adulthood]. The authors report their experience the diagnosis and management of congenital duodenal malformation in adults. They emphasize problems and difficulties in this disease of pediatric age and the physiopathological adaptation in these years. They point out that surgery can modify important metabolic functions and sub-clinical pathological conditions that the malformation made up.


Analysis of proteins binding to the proximal promoter region of the human cytomegalovirus IE-1/2 enhancer/promoter reveals both consensus and aberrant recognition sequences for transcription factors Sp1 and CREB. Expression of the immediate early 1 and 2 gene (IE-1/2) of human cytomegalovirus, an important pathogen in immunosuppressed patients, is controlled by a strong enhancer/promoter. To define the promoter domain within this large cis-active region of about 550 nucleotides, DNA-protein interactions were studied. DNase I footprinting experiments using procaryotically expressed transcription factor Sp1 revealed an extensive interaction of this transcription factor with both consensus and aberrant recognition elements within the IE-1/2 promoter region. Protection of these Sp1 binding sites could also be observed when nuclear extracts prepared from HeLa cells and permissive human fibroblast cells were used. After in vitro mutagenesis of Sp1 targets and transient expression of mutagenized CAT-expression plasmids, however, no significant reduction in CAT activities was found. By analyzing a series of 5' deletion mutants of the IE-1/2 promoter region, a strong cis-acting element was localized between nucleotides -94 and -78, upstream of sites that interact with Sp1. Gel retardation experiments demonstrated binding of recombinant transcription factor CREB to this motif which reveals it as an aberrant CREB recognition sequence. Thus, this study identifies several previously unknown binding sites for transcription factors Sp1 and CREB within the proximal promoter region of the IE-1/2 gene, which differ markedly in their relevance for constitutive promoter function.


Characterization of the factors binding to a PEPCK gene upstream hypersensitive site with LCR activity. A previously described upstream hypersensitive site (HS) in the PEPCK gene at -4800 bp, termed HS A (1), has been characterized and determined to bind at least two factors. One of these is a member of the ubiquitous CREB/ATF family, and the second is a novel tissue specific protein, pep A. A construct carrying HS A and the PEPCK proximal promoter was tested in transgenic mice and its CAT activity compared to the proximal promoter alone. The HS A was shown to drive tissue-specific, position-independent transcription of the CAT reporter gene 2-3 fold more effectively than the proximal promoter alone, with a concommitant 4-5 fold higher expression of CAT. Protein binding activity has been localized to a 33 bp region. This region contains a CRE (2) which is shown to bind a member of the CREB/ATF family through competition assays with an oligo containing a CRE from the proximal promoter and by the appearance of a supershift when the factor/oligo complex was exposed to CREB polyclonal antibody. Through restriction enzyme digests and competition of protein binding with an oligonucleotide homologous to HS A with a mutated CRE we have characterized a putative binding site for a liver-specific factor. In vitro and 'in vivo' footprinting studies complement each other, as well as, mobility shift assay data in designating the binding site of the proteins. The CREB/ATF factor and Pep A bind independently of each other during short term incubations, however, both factors can be accomodated on the DNA substrate as a function of extended time of incubation. Preliminary biochemical analysis defines the subunit molecular mass of the CREB/ATF like proteins at 55, 42, and 35 kD, while the tissue specific material exists as a single homogeneous subunit polypeptide in SDS of molecular mass = 49 kD.


Cloning, expression, and crystallization of recoverin, a calcium sensor in vision. Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.


Changing patterns in cytoskeletal mRNA expression and protein synthesis during murine erythropoiesis in vivo. The major cytoskeletal proteins alpha-spectrin, beta-spectrin, and ankyrin are synthesized and assembled into a supportive membrane skeleton during erythroid differentiation. Information on the temporal appearance of mRNA and protein species is essential for understanding both the cytoskeletal assembly process and the function of various isoforms. We have isolated highly enriched populations of fetal erythroid cells at various stages of maturation. mRNAs for erythroid ankyrin, alpha-spectrin, and beta-spectrin were expressed at all stages but there were differences in transcript types and levels. The ratio of 9-kilobase (kb) to 7.5-kb erythroid ankyrin transcripts decreased markedly during differentiation, but there was no change in the ratio of the 10.1-kb and 9.3-kb erythroid beta-spectrin transcripts. The relative amounts of ankyrin, alpha-spectrin, and beta-spectrin mRNA increased during yolk sac cell differentiation, whereas only alpha-spectrin mRNA increased during differentiation of the fetal liver cells. The amounts of beta-spectrin mRNA exceeded the amounts of alpha-spectrin mRNA in the early precursors from both yolk sac and fetal liver; protein synthetic levels showed the same pattern. The 16-day fetal peripheral reticulocytes, on the other hand, had the adult mRNA and protein synthetic ratios with alpha/beta greater than 1. The data indicate that at least two mechanisms exist to meet changing erythroid membrane cytoskeletal requirements during development in utero: (i) stage-specific processing of the mRNA for the major cytoskeletal linker protein ankyrin and (ii) developmentally regulated alpha/beta-spectrin protein synthetic rates.


Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments. The chicken retina contains rhodopsin (a rod visual pigment) and four kinds of cone visual pigments. The primary structures of chicken red (iodopsin) and rhodopsin have been determined previously. Here we report isolation of three cDNA clones encoding additional pigments from a chicken retinal cDNA library. Based on the partial amino acid sequences of the purified chicken visual pigments together with their biochemical and spectral properties, we have identified these clones as encoding the chicken green, blue, and violet visual pigments. Chicken violet was very similar to human blue not only in absorption maximum (chicken violet, 415 nm; human blue, 419 nm) but also in amino acid sequence (80.6% identical). Interestingly, chicken green was more similar (71-75.1%) than any other known cone pigment (42.0-53.7%) to vertebrate rhodopsins. The fourth additional cone pigment, chicken blue, had relatively low similarity (39.3-54.6%) in amino acid sequence to those of the other vertebrate visual pigments. A phylogenetic tree of vertebrate visual pigments constructed on the basis of amino acid identity indicated that an ancestral visual pigment evolved first into four groups (groups L, S, M1, and M2), each of which includes one of the chicken cone pigments, and that group Rh including vertebrate rhodopsins diverged from group M2 later. Thus, it is suggested that the gene for scotopic vision (rhodopsin) has evolved out of that for photopic vision (cone pigments). The divergence of rhodopsin from cone pigments was accompanied by an increase in negative net charge of the pigment.


Antithrombotic effects of synthetic peptides targeting various functional domains of thrombin. To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol/ml, a systemic dose that failed to affect hemostasis. Thus, inhibiting both thrombin's catalytic and exosite domains increases antithrombotic potency by several orders of magnitude over the inhibition of either domain alone, particularly at sites of deep arterial injury.


Differential regulation of T helper phenotype development by interleukins 4 and 10 in an alpha beta T-cell-receptor transgenic system. To address the mechanisms controlling T helper (Th) phenotype development, we used DO10, a transgenic mouse line that expresses the alpha beta T-cell receptor from an ovalbumin-reactive T hybridoma, as a source of naive T cells that can be stimulated in vitro with ovalbumin peptide presented by defined antigen-presenting cells (APCs). We have examined the role of cytokines and APCs in the regulation of Th phenotype development. Interleukin 4 (IL-4) directs development toward the Th2 phenotype, stimulating IL-4 and silencing IL-2 and interferon gamma production in developing T cells. Splenic APCs direct development toward the Th1 phenotype when endogenous IL-10 is neutralized with anti-IL-10 antibody. The splenic APCs mediating these effects are probably macrophages or dendritic cells and not B cells, since IL-10 is incapable of affecting Th phenotype development when the B-cell hybridoma TA3 is used as the APC. These results suggest that early regulation of IL-4 and IL-10 in a developing immune response and the identity of the initiating APCs are critical in determining the Th phenotype of the developing T cells.


Phosphatidylinositol-glycan (PI-G)-anchored membrane proteins: requirement of ATP and GTP for translation-independent COOH-terminal processing. Placental alkaline phosphatase (PLAP) belongs to a class of proteins that are anchored to the plasma membrane by a COOH-terminal phosphatidylinositol-glycan (PI-G) moiety. Nascent forms of such proteins undergo NH2- and COOH-terminal processing to yield the mature PI-G-tailed proteins. We previously introduced a shortened engineered form of preproPLAP (preprominiPLAP) that permits monitoring in cell-free preparations its sequential processing to the pro form and then to the mature PI-G-tailed form. Previous studies were carried out by synthesizing the preproprotein cotranslationally in the presence of rough microsomal membranes (RM). Because of the complexity of the cotranslational system it was not possible to determine whether cofactors were required for processing. We have now prepared RM that are preloaded with prominiPLAP but contain little mature PI-G-tailed miniPLAP. Maximal processing requires supplementation with both ATP and GTP. Inhibitors of PI-G biosynthesis do not affect processing. Since cleavage and PI-G addition are presumably catalyzed by a transamidase, the nucleoside triphosphate requirements suggest that there are additional steps in prominiPLAP processing prior to transamidation with PI-G. These may involve translocation of the pro protein in a proper conformational state to the transamidase site.


Contraction characteristics and ATPase activity of skeletal muscle fibers in the presence of antibody to myosin subfragment 2. To investigate the role of the myosin hinge region in muscle contraction, we examined the contraction characteristics and Mg-ATPase activity of glycerinated muscle fibers prepared from rabbit psoas in the presence and absence of polyclonal antibody directed against the subfragment 2 (S-2) region of myosin. The antibody-induced reduction of Ca(2+)-activated isometric force was always accompanied by a parallel decrease of muscle fiber stiffness, so that the stiffness versus force relation remained unchanged by the antibody treatment. Force-velocity relations of the fibers, obtained by applying ramp decreases in force at steady isometric forces, indicated that the antibody had no effect on maximum shortening velocity or on the shape of force-velocity curves. Simultaneous measurements of Mg-ATPase activity and Ca(2+)-activated force showed that Mg-ATPase activity of the fibers remained unchanged despite the antibody-induced reduction of isometric force even to zero. These results indicate that when anti-S-2 antibody attaches to the S-2 region of myosin molecules, their heads still hydrolyze ATP but no longer contribute to both force generation and muscle fiber stiffness.


Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.


Sensitivity to vanadate and isoforms of subunits A and B distinguish the osteoclast proton pump from other vacuolar H+ ATPases. Analysis of proton (H+) transport by inside-out vesicles derived from highly purified chicken osteoclast (OC) membranes has revealed the presence of a newly discovered type of vacuolar H+ ATPase (V-ATPase). Unlike vesicles derived from any other cell type or organelle, H+ transport in OC-derived vesicles is sensitive to V-ATPase inhibitors (N-ethylmaleimide and Bafilomycin A1) and vanadate (IC50, 100 microM), an inhibitor previously found to affect only P-type ATPases. The OC H+ ATPase contains several V-like subunits (115, 39, and 16 kDa) but subunits A and B of the catalytic domain of the enzyme differ from that of other V-ATPases. In OCs, subunit A has a mass of 63 kDa instead of the 67-70 kDa expressed in monocytes, macrophages, and kidney microsomes, which contain a vanadate-insensitive H+ ATPase. Moreover, two types of 57- to 60-kDa B subunits are also found: one is expressed predominantly in OCs and the other is expressed in kidney microsomes. The OC H+ pump may therefore constitute a class of H+ ATPase with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme. This H+ ATPase is involved in resorption of bone and may be expressed in a cell-specific manner, thereby opening possibilities for therapeutic intervention.


Development of [125I]RB104, a potent inhibitor of neutral endopeptidase 24.11, and its use in detecting nanogram quantities of the enzyme by "inhibitor gel electrophoresis". Neutral endopeptidase 24.11, also known as the common acute lymphoblastic leukemia antigen, is a zinc metallopeptidase involved in the inactivation of biologically active peptides, such as the enkephalins and atrial natriuretic peptide. The highly potent radiolabeled inhibitor 2-((3-[125I]iodo-4-hydroxy)phenylmethyl)-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)propyl]amino-4-oxobutanoic acid ([125I]RB104; Ki = 30 pM) has been developed for the enzyme. [125I]RB104 is highly specific, its Ki for another widely distributed zinc peptidase, angiotensin-converting enzyme, being 15 microM. In binding studies using rat brain slices, [125I]RB104 was shown to have a high affinity (Kd = 300 +/- 20 pM) and high specific binding at the Kd concentration (90%). With rat brain homogenates the Kd of [125I]RB104 was 26.8 +/- 0.9 pM, close to the kinetically derived Kd, 7.0 +/- 0.8 pM. Using the inhibitor, we have developed a simple, rapid, and quantitative technique to detect low nanogram quantities of the endopeptidase directly from tissue extracts after SDS/PAGE. The method has been used to show the presence of low quantities of the enzyme in rabbit bone marrow. Apart from its sensitivity, "inhibitor gel electrophoresis" using [125I]RB104 has the advantage over immunohistochemical methods of being able to label the enzyme in all tissues and species. It will therefore be of great value in determining the exact role of this important regulatory peptidase in a number of biological systems. Moreover, this one-step characterization of neutral endopeptidase 24.11 could be extended to other zinc metallopeptidases such as angiotensin-converting enzyme or collagenases, and inhibitors with affinities as high as RB104 could open the way to visualization of zinc metallopeptidases in different tissues by electron microscopy.


T cells expressing variable elements of T-cell receptor beta 8 and beta 2 chain regulate murine IgE production. After sensitization to ovalbumin (Ova) by inhalation of nebulized antigen, BALB/c mice respond with an early rise in IgE but not in IgG anti-Ova antibody production. Our purpose here was to analyze the repertoire of T cells that may contribute to regulating this IgE response. Initial study of Ova-reactive T-cell hybridomas showed that they selectively express the T-cell receptor variable beta-chain (V beta) elements 2, 8.1/8.2, and 14. The frequency of T cells bearing these V beta elements in local draining lymph nodes of the airways and lungs (peribronchial-draining lymph nodes) after Ova inhalation was examined. Local sensitization increased the proportion of V beta 8.1/8.2 T cells in the peribronchial-draining lymph nodes, whereas expression of V beta 2 or V beta 14 was similar in sensitized and nonsensitized animals. In the presence of increased antigen concentrations, V beta 8 and V beta 2 T cells were equally reactive to Ova when cell proliferation was assayed. Coculture of Ova-selected V beta 8 T cells from peribronchial-draining lymph nodes and spleens of sensitized animals with primed splenic B cells increased IgE but not IgG production. The V beta 8 increase in IgE production was related to an increase in numbers of IgE-secreting B cells. In contrast, coculture of Ova-selected V beta 2 T cells with sensitized B cells had no stimulatory effect on either IgE or IgG production. Further, addition of V beta 2 cells to V beta 8 cells inhibited the V beta 8-induced augmentation of IgE production. These data indicate that T cells expressing different T cell receptors or, perhaps, different V beta elements may play different roles in IgE production in sensitized mice.


N-methyl-D-aspartate receptor-mediated neuroprotection in cerebellar granule cells requires new RNA and protein synthesis. Cerebellar granule cells are susceptible to the excitotoxin glutamate, which acts at N-methyl-D-aspartate (NMDA) receptors, as well as the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+), the active cytotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Paradoxically, preincubation of cultured cerebellar granule cells with low concentrations of NMDA or glutamate markedly antagonizes the neurotoxicity resulting from subsequent exposure to toxic concentrations of either MPP+ or glutamate. The neuroprotective effects of NMDA and glutamate against MPP+ toxicity are observed at agonist concentrations as low as 1 microM, are blocked by specific NMDA receptor antagonists, and require at least 30 min to develop fully. Moreover, NMDA receptor-mediated neuroprotection is prevented by the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide. Thus, in cerebellar granule cells activation of NMDA receptors by glutamate can result in either neurotoxicity or neuroprotection, depending on the apparent degree of receptor stimulation. NMDA receptor-mediated neuroprotection requires new RNA and protein synthesis and therefore appears to be mediated by the expression of a neuroprotective protein(s). These data demonstrate the presence of an active NMDA receptor-mediated and transcriptionally directed neuroprotective mechanism in cerebellar granule cells.


Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes. The ADP-ribosylation factor (ARF), a highly conserved low molecular weight GTP-binding protein, has been implicated to function in intracellular protein transport to and within the Golgi complex. In pancreatic acinar cells the ARF is confined to the cytoplasmic faces of trans-Golgi stack membranes, a compartment known to maintain a low intravesicular pH, which is established by a chloride-dependent MgATP-driven proton pump. The present study shows that MgATP (2mM), but neither adenosine 5'-[gamma-thio]triphosphate in the presence of Mg2+ nor ATP in the absence of Mg2+, increases transfer of ARF from the surrounding medium into the vesicle membranes. The specific vacuolar-type proton pump inhibitor bafilomycin B1 (10 nM), the protonophore carbonylcyanide m-chlorophenylhydrazone (10 microM), and replacement of chloride in the incubation buffer by acetate or nitrate resulted in an almost complete inhibition of the MgATP-dependent association of ARF to the vesicle membranes. The results demonstrate that redistribution of ARF to the vesicle membrane correlates with the intravesicular pH established by a vacuolar-type H(+)-ATPase. The intravesicular pH appears to be one mechanism by which certain low molecular weight GTP-binding proteins become relocated from the cytosol to their specific membrane vesicles.


Enhancement of rotational behavior induced by repeated administration of SKF38393 in rats with unilateral nigrostriatal 6-OHDA lesions. To clarify if the enhancement of rotational behavior induced by repeated administration of SKF38393 is mediated by upregulation of D1 and/or D2 receptors in the striatum, we investigated effects of SCH23390 and sulpiride on SKF38393-induced rotational behavior and the changes in striatal dopamine receptors in rats with unilateral nigrostriatal 6-hydroxydopamine lesions (1). Repeated weekly administration of SKF38393 markedly enhanced the number of rotations and shortened the latency of rotational behavior depending on the number of SKF38393 administrations 1 or 6 weeks after the treatment with 6-OHDA (2). A selective D1 antagonist, SCH23390, but not a selective D2 antagonist, sulpiride, suppressed SKF38393-induced rotation and inhibited the enhancement by the repeated administration (3). Repeated administration of SKF38393 did not modify the density and the affinity of either the striatal D1 or D2 receptors in the striatum. These results suggest that the enhancement of SKF38393-induced rotational behavior by the repeated administration is not associated with the upregulation of striatal D1 and D2 receptors.


Role of genotype in brain dopamine metabolism and dopamine-dependent behavior of mice. In mice of eight inbred strains--BALB/c, AKR/J, DBA/2, CBA, C57B1/6, DD, CC57Br, and C3H/He--brain dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in striatum and nucleus accumbens with tuberculum olfactorium, the structures of two main dopaminergic systems--nigrostriatal and mesolimbic--were determined. In both dopaminergic regions, no strain effect on either dopamine or DOPAC levels was found, while for HVA content a highly significant hereditary determination was shown. Influences of selective D1 and D2 dopamine receptor agonists--SK&F 38393 and quinpirole, respectively--as well as that of a mixed D1/D2 agonist, apomorphine, on general locomotor activity and stereotypic climbing were studied. By that, marked genotypic differences in dopamine-dependent behavior and dopamine receptor sensitivity were observed. Although both SK&F 38393 (5 mg/kg) and apomorphine (0.25 mg/kg) decreased locomotion, the effect being genotype dependent, in all strains of mice quinpirole (2.5 mg/kg) proved more potent in locomotor inhibition. SK&F 38393 (10 mg/kg) induced climbing, but 2.5 mg/kg apomorphine in most strains was much more effective. At the same time, quinpirole (up to 8 mg/kg) failed to induce this behavior. This suggests the crucial role of D1 receptors in the generation of climbing, attracting, at the same time, attention to the importance of D1/D2 interaction. The observed drastic interstrain differences in dopamine receptor sensitivity demonstrate the essential role of genotype in the effects of dopaminergic drugs.


Alterations of skin-associated lymphoid tissue in the carcinogenesis of arsenical skin cancer. We investigated the skin-associated lymphoid tissue in arsenical skin cancers, including 14 Bowen's disease, 6 basal cell carcinoma and 6 squamous cell carcinoma patients from an endemic area by immunohistochemical and morphometric methods. There was a progressive decrease of Langerhans cells in the order of normal skin, normal appearing edge and arsenical cancers. A disruption of the uniform Langerhans cell dendrites was also noticed. The Langerhans cell density in arsenical tumors did not correlate with the peritumoral infiltrates. The prominent infiltrated cells in the peritumoral area had T cell markers. The number of peritumoral T lymphocytes in squamous cell carcinoma was significantly less than that of Bowen's disease and basal cell carcinoma. Peritumoral mononuclear infiltrates in Bowen's disease and squamous cell carcinoma showed a higher helper/suppressor T cell ratio than that in basal cell carcinoma. This may be accounted for by a selective increased recruitment of helper T cells to the tumor infiltrates in Bowen's disease and squamous cell carcinoma.


[Laparoscopic cholecystectomy].

[Laparoscopic cholecystectomy]. Laparoscopic cholecystectomy has been accepted clinically in a very short time. Following an intensive training-course for manual dexterity clinical experience should be achieved in carefully selected cases. After a certain number of cholecystectomies the majority of cholecystectomies can be performed laparoscopically. The complication rate of laparoscopic cholecystectomy in the hand of a well trained surgeon seams to be comparable or even smaller than in conventional procedure. The patients have significantly less pain and bodily activity starts early. The postoperative time in hospital in our clinic is two to three days. The rehabilitation-time could be shortened.


[Hormonal assessment in a woman with acne and alopecia]. Acne, androgenogenetic alopecia, hyperseborrhea and hirsutism may result from hyperandrogenism in women. This may be peripheral "idiopathic" hyperandrogenism due to cutaneous metabolism of steroids, but in some cases hyperandrogenism is due to abnormal production or input of steroids with androgenic activity (hyperplasia, endocrine tumors, cysts, consumption of progestogens or other hormones with androgenic activity, menopause...). An assessment is useful only in cases of acne or alopecia if they are accompanied by other signs of peripheral hyperandrogenism and/or disturbed menstruation. The treatment is based on the administration of an anti-androgen (in France, usually cyproterone acetate), combined with other local or systemic treatments for the problem, depending on the age, dermatological signs and context.


[Lipomatous hypertrophy of the interauricular septum, fibrosis of the bundle of His and calcification of the mitral annulus associated with multiple rhythm and conduction disorders. The anatomicoclinical correlations]. We present the case of a 72 years old female with multiple episodes of atrial fibrillation, interatrial block and different degrees of atrioventricular block, that died because of stroke. Postmortem pathologic examination showed a lipomatous hypertrophy of the interatrial septum (previously suspected on echocardiogram), his bundle and right branch degeneration as well as mitral annular calcification extended to conduction system. According to these pathologic findings, rhythm and conduction disturbances are correlated with the two levels (atrial and atrioventricular) where anatomic abnormalities were found.


Permeability of protective gloves to (di)methacrylates in resinous dental materials. Dentists may develop contact allergy induced by handling resinous dental materials, and vinyl and latex gloves may provide protection against the monomers of these materials only during the time it takes for a monomer to permeate the glove. The passage times and rates of penetration of four commonly used (di)methacrylates--HEMA, TEGDMA, BISGMA, and UEDMA--for 11 protective gloves were measured. The passage time for HEMA and TEGDMA through vinyl gloves was 1-3 min, and around 20 min for BISGMA and UEDMA. The rate of penetration of HEMA and TEGDMA in vinyl gloves was 0.5-4.5 mumol.min-1.cm-2. The passage time of HEMA and TEGDMA through most of the latex or the modified latex gloves was 5-8 min and the rate of penetration 0.5-3.1 mumol.min-1.cm-2. Latex or modified latex gloves provide a protection against BISGMA and UEDMA for 80 min or more with one exception (Elastyrene), which burst after about 50 min in contact with the resins. Of the tested gloves Ansell, Neutralon, Mediglove, and Biogel D provide protection against HEMA and TEGDMA for at least 5 min.


Aspects on diagnosis and treatment of the foregut carcinoid syndrome. Eight patients with the foregut carcinoid syndrome (two gastric and six bronchial primary tumors) are reported. The patients presented with complex clinical symptoms including ectopic production of adrenocorticotrophic hormone and growth hormone-releasing factors. The most alarming symptoms were facial flush and edema, accompanied by severe bronchoconstriction, which easily was misinterpreted as asthmatic attacks. Conventional bronchodilatory drugs may be potentially dangerous in these patients, in whom combined blockade of histamine receptors and treatment with cortisone and octreotide are recommended. Owing to the patients' age and general condition individualized long-term therapy was instituted. Surgical therapy under optimal protection by drugs can be of substantial value also in patients with advanced disease. One patient with life-threatening hormonal symptoms underwent hyperthermic perfusion of the liver with cytotoxic drugs, resulting in good palliation.


Mutagenicity from ozonation of humic substances. Eight structural components of humic substances were ozonated. Mutagenic activity was found using TA100 with and without S9 mix for all ozonated components. p-Hydroxybenzaldehyde was chosen as an important component and ozonation products were determined by gas chromatography-mass spectrometry (GC-MS). Aldehydes, ketones and carboxylic acids were identified as the ozonation products. Among these products, acetaldehyde, formaldehyde, glyoxal, methylglyoxal and glyoxylic acid were recognized to be mutagenic. Furthermore, p-hydroxybenzaldehyde was first ozonated and then chlorinated. A great variety of chlorinated organic compounds, many of which are known mutagens, have been identified by GC-MS in the ether extract. The same compounds have previously been reported as chlorination products of humic substances. Aldehydic products by ozonation were identified from ozonation followed by chlorination of humic substances and p-hydroxybenzaldehyde.


Laparoscopic posterior truncal vagotomy and anterior highly selective vagotomy--a case report. Laparoscopic vagotomy provides a viable alternative to expensive long-term treatment with H2 antagonists in patients with intractable peptic ulcer disease. The minimally invasive procedure offers reduced postoperative discomfort and improved cosmesis. Here, we report our first case of a posterior truncal vagotomy and anterior highly selective vagotomy performed laparoscopically for the first time in Asia. The surgery was uneventful. Diet was resumed on day 3 and the patient was discharged on day 4. Post-vagotomy acid secretion tests on the third week revealed a dramatic decrease in acid production. With further experience, laparoscopic vagotomy can be an attractive alternative to long term medication in peptic ulcer disease.


Childhood psychological trauma correlates with unsuccessful lumbar spine surgery. In a retrospective study of 86 patients who underwent lumbar spine surgery, patients who had three or more of a possible five serious childhood psychological traumas (risk factors) had an 85% likelihood of an unsuccessful surgical outcome. Conversely, in patients with a poor surgical outcome, the incidence of these traumas was 75%. In the group of 19 patients with no risk factors, there was only a 5% incidence of failure. This study shows that a highly significant correlation exists between unsuccessful lumbar spine surgery and a history of childhood traumas. Although recognition of predictors for unsuccessful outcome can be useful in avoiding surgery in patients whose indications for surgery are borderline, the greater challenge is to help the patient who, despite being at high psychological risk for negative outcome, has severe spinal pathology that will likely require surgery. In such cases, psychiatric treatment is critical. In the group of 19 patients with no risk factors, single-level laminectomies and discectomies were performed on 6 patients. The other 13 cases were complex, involving a combination of repeat surgeries (n = 4) fusions (n = 3), and/or multilevel laminectomies and discectomies (n = 11).


Intraosseous vertebral body pressures.

Intraosseous vertebral body pressures. In an effort to determine the relationship between low-back pain and intraosseous hypertension, in vivo vertebral pressure measurements were performed on 19 patients. A cannulated screw was placed percutaneously into the middle of the vertebral body by a transpedicular route. Pressure measurements were recorded with the patient in various positions. Pressures were greatest in the sitting position, lowest in the prone position, and intermediate in the standing position. A correlation was found between intravertebral body pressure and patient position. Pressures were highest in the positions most commonly associated with low-back pain.


Pedicle diameter determined by computed tomography. Its relevance to pedicle screw fixation in the lumbar spine. Intraoperative technical complications of pedicle screw fixation include screw cutout or maldirection and pedicle fracture. The aims of this study were 1) to use computed tomography to determine the average pedicle diameter; and 2) to compare these measurements with the outer diameter measurements of commonly used pedicle screws. The pedicle diameters of L2, L3, L4, L5, and S1 were measured in 154 adult patients (81 men, 73 women) who had low-back pain. The distance across the isthmus of the pedicle was measured with the distance mode on the axial computed tomographic image in the bone window setting. The lateral computed tomographic scout view was used to select the axial section through the midportion of the pedicle. To standardize data retrieval, the lower four motion segments were denoted as L2-S1, even when there were more or fewer than five lumbar vertebrae. The pedicle diameter at each level was measured and averaged for all patients and for men and women separately. The percentage of pedicles that measured less than 7 mm was determined at each level. The average pedicle diameters were 8.13 mm for L2, 8.7 mm for L3, 10.88 mm for L4, 14.54 mm for L5, and 18.37 mm for S1. Twenty percent of the L2 pedicle diameters, 15.6% of L3, and 1.9% of L4 were less than 7 mm; none of the L5 or S1 pedicles measured less than 7 mm. The outer diameters of the most commonly used pedicle screws range from 5 mm to 7 mm. Screw pitch, tooth profile, outer diameter, and depth of penetration affect implant strength.(ABSTRACT TRUNCATED AT 250 WORDS)


Which disc as visualized by magnetic resonance imaging is actually a source of pain? A correlation between magnetic resonance imaging and discography. Sixty-three discs in 25 consecutive patients with nonradicular discogenic pain were studied with magnetic resonance imaging and lumbar discography. Sagittal T2 magnetic resonance imaging sequences were blindly classified according to the nuclear signal intensity and to the status of the posterior anulus. A positive discogram had to include abnormal morphology and a provocative pain response of significant intensity and familiar quality. Specific magnetic resonance imaging patterns with a high probability for positive and negative discograms are identified. Also identified are magnetic resonance imaging patterns with intermediate probability, ie, ones that should not be presumed either asymptomatic or symptomatic. In many cases, magnetic resonance imaging could not reliably predict or replace discography.


Intradiscal steroids. A prospective double-blind clinical trial. A prospective, randomized, double-blind study was performed to evaluate the clinical efficacy of intradiscal steroid injections. Criteria for entrance were one-level internal disc disruption or nonsequestered nuclear prolapse with or without sciatica and a positive pain response on awake discography. Exclusion criteria were multilevel disease, central or lateral stenosis, prior lumbar surgery, or medical disease requiring systemic steroids. A total of 25 patients were randomly assigned to Treatment Group A (methylprednisolone, Depo-Medrol 80 mg/ml, The Upjohn Co., Kalamazoo, Michigan) or Treatment Group B (bupivacaine, Marcaine .5% 1.5 ml, Sanofi Winthrop Pharmaceuticals, New York, New York). Fourteen patients received Depo-Medrol, with 21% showing subjective improvement and 79% no improvement; 0% were clinically worse. Eleven patients received intradiscal Marcaine, with 9% showing clinical improvement and 91% no improvement; 0% were clinically worse. To quantify clinical response, a pain diagram grid score, a visual analog scale, and the Oswestry Pain Questionnaire were used before injection and 10-14 days after injection. No statistically significant benefit was identified in the use of intradiscal steroids.


Effect of smoking and pulsed electromagnetic fields on intradiscal pH in rabbits. The adverse effect of cigarette smoking on human spines has been noted indirectly. There is correlation of increased back pain among individuals who smoke heavily. The hypothesis of this study was that an environment of cigarette smoking is an adverse event and will create a reduced pH in the rabbit intervertebral disc. Electromagnetic fields, however, can defend against this adverse event and reduce the tendency toward acidic pH. Rabbits were exposed to cigarette smoke for 2, 4, or 6 weeks and their intradiscal pH measured. Cigarette-smoke-exposed discs demonstrated a consistently lower pH than did the discs of the machine control rabbits. The second group of rabbits were exposed to cigarette smoke and pulsed electromagnetic fields. The cigarette-smoke-exposed rabbits that were exposed to the pulsed electromagnetic fields for 4 hr/day demonstrated no change in their intradiscal pH, in contrast to those who were exposed to smoke alone. In conclusion, cigarette smoke exposure in rabbits consistently produces a lower intradiscal pH and pulsed electromagnetic fields can defend against this adverse effect.


[Surgical treatment of atypical location of a clavus on the lower extremity]. Clavus is a very frequent condition of the toes which, despite the fact that it is usually underrated, can complicate the life of its carriers. The authors describe a surgical technique tested in a group of 12 female patients in the course of six years, which became a simple method of choice for the elimination of these complaints.