A comparison of cardiovascular and autonomic adjustments to three types of cold stimulation tasks. Two types of 'cold pressor' tasks are used frequently in research settings: immersing the hand or foot in ice water, and applying an icebag to the forehead. Both tasks have commonly been selected as 'alpha-adrenergic' tasks due to expected increases in blood pressure and peripheral resistance. However, the forehead cooling task has been used by others to produce increased vagal tone due to the elicitation of the 'diving reflex' and subsequent bradycardia. This differs from the prototypical increase in heart rate during hand immersion. The present study directly compared the cardiovascular adjustments of hand immersion and forehead stimulation. As stimulation of the trigeminal nerve is thought to increase vagal activity during forehead cooling, a third condition in which most of the face was covered with an icebag was included to ostensibly stimulate more of the trigeminal. 18 males had counterbalanced exposures to hand immersion, forehead cooling, and facial cooling for 90 s each. Cardiovascular variables derived from impedance cardiography and the ECG were measured. Respiration was paced at 14 breaths/min to facilitate comparisons of respiratory sinus arrhythmia across conditions. No differences among baseline or tasks were found for respiratory rate or cardiac output. The pattern of results for hand immersion was that of increased heart rate with moderate blood pressure increases. In contrast, the forehead and facial cooling tasks elicited small heart rate decreases with little change in blood pressure. Facial cooling elicited significantly more vagal activation than hand cooling as indexed by respiratory sinus arrhythmia. The patterns of response for facial and forehead cooling were almost identical. Probable reasons for the lack of significant blood pressure responses during the facial and forehead cooling are discussed.


Temporal relationships between changes in phantom limb pain intensity and changes in surface electromyogram of the residual limb. Previous studies of relationships between surface EMG of the residual limb and phantom pain have not shown which changed first. Thus, predictive relationships could not be demonstrated. 24 male (20) and female (4) amputees between the ages of 33 and 71 who reported either burning (3), cramping (8), shocking-shooting-stabbing (6), or a combination of these descriptions of phantom pain (7) participated in one or two recording sessions. Raw surface EMG from the major muscles of the residual limb was recorded while subjects activated an event marker to indicate changes in pain. All eight subjects with cramping phantom pain reported changes in pain after the recording showed sharply demarcated increases in EMG. Subjects reporting either shocking-shooting or burning pain did not show any consistent relationships between EMG and pain. Three of the four subjects reporting experiencing both shocking-shooting and cramping phantom pain simultaneously during recordings showed changes in EMG preceding changes in pain. Sensations of cramping phantom pain were preceded by increases in muscle tension in the residual limb in almost every instance for each of our subjects showing changes in cramping phantom pain. Thus, changes in muscle tension in the residual limb are likely to either be causes or close intermediaries for the cause of cramping phantom pain but not necessarily of other common descriptors.


Paranoid-nonparanoid differences--psychophysiological parallels. It was predicted that the psychological differences between paranoids and nonparanoids would be parallelled by differences in psychophysiological responses to stimuli varying along both informational and motivational dimensions. Skin conductance and heart rate responses to stimuli in a guessing task were measured in a group of paranoid patients and compared with those from non-paranoid and non-patient controls. A group of nonschizophrenic patients on phenothiazine medication served as drug-matched controls. Skin conductance level, response frequency and amplitude data are reported; heart rate response changes were examined by means of times series analysis and reported as first and second deceleration and first and second acceleration components. It was predicted that paranoid patients would show a greater increase in skin conductance measures with cognitive demand, and an increase in acceleratory components of the heart rate to increases in motivational variables. The normal pattern of greater electrodermal responding to the negative stimuli than the positive, and greater heart rate acceleration to the positive stimuli but not the negative (Fowles, 1980) was predicted to be reversed in schizophrenic patients, and possibly more marked in paranoids. Cognitive manipulations showed the predicted increase in electrodermal and heart rate responding in normal controls. The paranoid subjects showed a similar increase as a function of task demands, but there was no significant difference between the other groups. Motivational manipulations produced increased skin conductance responding and increases in heart rate acceleration components in normal controls, this was not parallelled in the patient groups. All subject groups failed to show greater electrodermal activity to negative than to positive stimuli. Normal controls showed significant heart rate acceleration to positive as compared to negative stimuli, this pattern was reversed in paranoid patients.


Does the N100 evoked potential really habituate? Evidence from a paradigm appropriate to a clinical setting. This study examined the N100 component of the event related potential in a habituation paradigm with short interstimulus intervals. The paradigm was designed to be relatively brief in duration (approx. 4 min for each of two conditions), so that it could be used for clinical populations with cognitive dysfunction, in which compliance may be a problem with long paradigms. Two conditions - Ignore and Attend - were employed with normal subjects. In each condition, 15 stimulus trains, each consisting of 10 innocuous tones, were presented. The eighth tone was a change stimulus. There was a fixed interstimulus interval of 1.1 s and an inter-train interval of 5 s. From the perspective of traditional Orienting Response theory, evidence was sought for within-train habituation in terms of diminished N100 amplitude to repeated stimuli, response recovery to the change stimulus, and dishabituation of the response to the following standard stimuli. Habituation was suggested by significant decreases of approx. 50% with stimulus repetition, and response recovery to the change stimulus in both conditions. However, there was no evidence of dishabituation following the change stimulus. These results confirm that N100 fails to meet the formal requirements of response habituation, suggesting instead that it may index an earlier process than the Orienting Response.


Structural biology of epithelial tissue in histophysiologic gradient culture. Epithelial cells proliferate, forming organized tissues, when positioned in the lumen of a thin-walled, transparent, elongated cylindrical, cystlike culture chamber. The closed chamber, 2.5 mm in diameter and 25 mm long, bathed in medium, incubated with continuous gentle agitation, enables the inoculum to exchange metabolites including oxygen by diffusion across the thin, nylon filament-reinforced collagen membrane wall of the chamber. After periods of culture of a week or more, using inocula derived from urothelium, the inner surface of the cystic chamber is lined by a stratified epithelium. Proliferation of cells is seen in the basal zone, which is attached to the collagen substrate. The development of the model is briefly described. Some of the applications of the procedure are illustrated using cell lines, chick embryo tissues, and clinical tissues. Implications of the procedure are considered for studying categories of tissue biology, e.g., problems of aging, neoplasia, and toxicology.


Establishment of primary cultures of rat and human parotid epithelial cells for transfection experiments. The molecular mechanisms that regulate the synthesis of salivary proteins are unknown. The paucity of homogeneous cell populations of parotid acinar cells has become a limiting factor for such a study. Therefore, the establishment of immortalized clones of acinar cells is essential. This study has established primary cultures of rat and human parotid epithelial cells that are suitable for transfection with plasmid vectors, pSV2, pSV3, and pSV5 to generate immortalized cells in vitro. Among various techniques used, the rat and human parotid tissue or cellular clumps when restrained in chicken plasma clot allowed the outgrowth of epithelial cells that maintained epithelial cell morphology for over 4 wk. However, the initial growth requirements for rat and human parotid cells were different. The presence of 10% heat inactivated fetal bovine serum in supplemented MCDB-LB medium was essential for the outgrowth of rat parotid epithelial cells, but this was not needed for the outgrowth of human parotid epithelial cells. The growth of both human and rat parotid epithelial cells can be maintained in serum-free supplemented MCDB-LB. These primary cultures contained amylase-producing cells as demonstrated by immunofluorescent technique, and they were transfected with pSV2, pSV3, and pSV5 using primarily the calcium phosphate-DNA co-precipitation technique. After initial extensive cell death, many cells with epithelial cell morphology survived.


An in vitro model for cell-cell interactions. Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues. In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition. However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane, permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8 microns maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through pores of 2.0 microns. Vascular cell co-culture across membranes with 0.8-microns pores resulted in the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited endothelial cell growth The arrangement of the cells in this co-culture system mimics the in vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study of cell-cell interactions in a variety of systems.


Interaction of mouse ectoplacental cone trophoblast and uterine decidua in vitro. During the peri-implantation stages of mouse development, the secondary trophoblast invades into the uterine decidua. This uniquely controlled invasive process results in the formation of the placenta. We have analyzed this process in vitro using cultures of decidua and microdissected ectoplacental cones from Day 7 embryos. The results showed that the interaction between these two cell types is comparable to that seen in vivo. Morphologically, the decidua maintained close contact with the spreading trophoblast, limiting its invasion and producing a multilayered trophoblast outgrowth. Attachment to the decidua was not mediated through cell-matrix binding, but the subsequent invasion into the decidua was dependent on normal matrix interactions. Secretion of proteinases by the trophoblast also seemed to be a requirement for successful invasion, but not attachment.


Purine nucleosides and nucleotides stimulate proliferation of a wide range of cell types. Presumptive astrocytes isolated from 10-day white Leghorn chick embryos, Factor VIII-positive human brain capillary endothelial cells, meningeal fibroblasts from 10-day chick embryos, Swiss mouse 3T3 cells, and human astrocytoma cell lines, SKMG-1 and U373, were rendered quiescent when placed in culture medium that contained 0 or 0.2% serum for 48 h; their proliferation was markedly reduced and they incorporated [3H]thymidine at a low rate. [3H]Thymidine incorporation and cell proliferation were induced in all types of cells by addition of guanosine, GMP, GDP, GTP, and to a lesser extent, adenosine, AMP, ADP or ATP to the culture medium. The stimulation of proliferation by adenosine and guanosine was abolished by 1,3-dipropyl-7-methylxanthine (DPMX), an adenosine A2 receptor antagonist, but not by 1,3-dipropyl-8-(2-amino-4-chorophenyl)xanthine (PACPX), an A1 antagonist. Stimulation of proliferation by the nucleotides was not abolished by either DPMX or PACPX. The P2 receptor agonists, alpha, beta-methyleneATP and 2-methylthioATP, also stimulated [3H]thymidine incorporation into the cells with peak activity at approximately 3.5 and 0.03 nM, respectively. These data imply that adenosine and guanosine stimulate proliferation of these cell types through activation of an adenosine A2 receptor, and the stimulation of cell proliferation by the nucleotides may be due to the activation of purinergic P2y receptors. As the primary cultures grew older their growth rate slowed. The capacity of the purine nucleosides and nucleotides to stimulate their growth diminished concomitantly. The 3T3 cells showed neither decreased growth with increased passages nor reduced response to the purines. In contrast, although the doubling time of the immortalized human astrocytoma cell lines SKMG-1 and U373 remained constant, the responsiveness to purinergic stimulation of the U373 cells decreased but that of the SKMG-1 did not. These data are compatible with a decrease in the number, or the ligand-binding affinity of the purinergic receptors, or a decreased coupling of purinergic receptors to intracellular mediators in primary cells aged in tissue culture.


Growth, morphologic, and invasive characteristics of early and late passages of a human endometrial carcinoma cell line (RL95-2). Two in vitro passages of a human endometrial adenocarcinoma continuous cell line (RL95-2), an early (subcultured less than 30 times) and a late passage (subcultured greater than 200 times) have provided an interesting model to study the growth, morphologic, and invasive properties of endometrial tumors. The early passage, which has been shown to be estrogen-receptor positive, has characteristics closely resembling a primary tumor, whereas the estrogen receptor negative late passage exhibits several features of the metastatic phenotype. Compared to the early passage cells, the late passage cells were less serum dependent, formed foci, demonstrated a faster rate of growth (due to their shorter doubling times), and attained higher saturation densities. The late passage cells also displayed an altered morphology which was accompanied by alterations in the distribution of F-actin. Even though early and late passages showed similar invasive potential in an in vitro invasion assay, the late passage cells, by virtue of their several transformed characteristics, maintain distinctive properties compared with their early passage counterparts.


Bronchiolitis in the history of the asthmatic child. In a retrospective analysis of the history of 1,700 asthmatic children, 167 (9.8%) were found to have had bronchiolitis during the first 2 years of life. These 167 cases with asthma and bronchiolitis were matched against 215 selected cases of asthma without bronchiolitis. Both groups attended our Ambulatory Care Unit for Asthma. Data were collected from the clinical cards of the children. Asthmatics who had bronchiolitis were found to have an earlier onset of asthma (p less than 0.001), earlier resolution of symptoms (p less than 0.05 total; p less than 0.01 females), and less evidence of atopy (p less than 0.005). It is suggested therefore that the bronchial hyperreactivity in asthma following bronchiolitis may have different underlying mechanisms than in the typical atopic case, without preceding bronchiolitis. Long-term follow-up studies in infants with bronchiolitis are still necessary in order to clarify the process by which bronchiolitis predisposes to bronchial hyperreactivity and asthma. The consequences of the viral infection itself may lead to pathophysiological processes that promote bronchial hyperreactivity. On the contrary, an underlying condition of bronchial hyperreactivity could be the "primum movens" of the bronchiolitis itself.


Application of highly polymorphic DNA markers to the identification of HeLa cell sublines. Polymorphic DNA markers were used to identify eight sublines derived from HeLa. Using five highly polymorphic minisatellite DNA probes, these cell lines were distinguished and classified into six groups by Southern blot analysis. Polymorphic DNA markers, therefore, can provide a useful tool for monitoring genetic changes of a cell line during culture and for distinguishing sublines derived from the same origin.


Replication of Autographa californica baculovirus (AcMNPV) in a coleopteran cell line. A coleopteran cell line (AGE) derived from the cotton boll weevil Anthonomus grandis supported replication of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). The titer of extracellular virus (ECV) and the number of occlusion bodies (OB) produced in AGE cells were approximately equal to those produced by a Trichoplusia ni cell line (TN-CL1), and the OB produced by both cell lines were equally infectious for T. ni larvae. The identity of the AGE cell line was established by chromosome and isoenzyme analyses.


Perception of bronchoconstriction in chronic asthma. Twenty-nine asthmatic children were studied to assess their ability to detect the severity of their bronchoconstriction. First (STEP 0): each child was asked if he "felt asthma," inviting him to give a self-estimated obstruction score (SEOS) from 0 (no asthma) to 3 (severe asthma) and a baseline FEV1 was recorded. Then (STEP 1): methacholine was administered to all except 9 children with an FEV1 less than 80% of predicted who received saline nebulization and a second SEOS was recorded. Finally: salbutamol aerosol was administered and a third SEOS and FEV1 were obtained (STEP 2). Spearman's correlation coefficients r between SEOS and FEV1 were for the overall population -0.602, -0.517, and -0.104 at STEP 0, STEP 1, and STEP 2, respectively. The r values reduction during the trial can be due either to a decrease of children's concentration during the study or to a real difficulty in recognizing repeated changes in airway status. Some children tend to underestimate their bronchospasm. For these patients an accurate assessment of the severity of the bronchoconstriction requires an objective measurement during acute changes in asthmatic children.


Development of lung function in relation to increased degree of bronchial responsiveness. To study the relationship between development of lung function and bronchial responsiveness, we examined 106 subjects recruited from a random sample of 527 subjects, aged 8-18 years, from Copenhagen. Lung function and bronchial responsiveness to inhaled histamine were measured at two occasions, in 1986 and 1988. The participating subjects (n = 106) were divided into three groups: (a) 20 asthmatics with bronchial hyperresponsiveness (BHR), (b) 42 nonasthmatic subjects with BHR in 1986, and (c) 44 controls without BHR. In 1986, FEV1 expressed as percentage of predicted value, was found to be similar in the three groups (91%, 94%, and 99%, respectively). The increase in height during the observation period was found to be 5, 6, and 6 cm, respectively, in the three groups. However, at the second examination, in 1988, FEV1 was found to be significantly reduced in both asthmatics (87%) and nonasthmatic subjects (85%) compared with the controls (103%). In 1988, 16 asthmatics (80%) and 24 (57%) nonasthmatic subjects were found to have BHR, whereas none of the controls were found to have BHR. A multiple regression analysis was used to determine the correlation between change in FEV1 and potential factors of importance. The change in FEV1 was highly correlated with the presence of BHR in 1986, however, no correlation was found between change in FEV1 and change in bronchial responsiveness. In conclusion, nonasthmatic subjects with former BHR showed signs of airflow obstruction and less increase in lung function during growth irrespective of the change in level of bronchial responsiveness, which may suggest a risk for subsequent development of obstructive lung disease.


An increased level of specific IgG4 antibodies against Candida albicans in patients with bronchial asthma. Specific IgG4 antibodies against Candida albicans in sera were measured in 76 asthmatics. The increased level of specific IgG4 was found in cases 10-40 years old sensitive to house dust mite and/or Candida albicans, in cases with steroid-dependent intractable asthma (SDIA) and in elderly cases. The frequency of SDIA was the highest in cases 41 to 60 years old with higher frequency of increased IgG4 antibodies. The results show that specific IgG4 increases in relation to IgE-mediated immune response, long-term steroid therapy, and aging. All of these conditions may induce depressed cell-mediated immunity.


Pulsus paradoxus in acute asthma in children. One hundred children suffering from acute asthma were studied. Both pulsus paradoxus (PP) and CO2 pressure (PCO2) of the arterialized blood sample were measured in each child. The correlation between the PP and the PCO2 was not statistically significant when the latter was less than 35 mmHg (p less than 0.05). When the PCO2 was equal or higher than 35 mmHg, a directly proportional as well as statistically significant relationship with PP was found (p less than 0.00001). PP is proposed as an indirect indicator of the PCO2 on patients suffering from acute asthma, when the necessary equipment to measure the PCO2 is not available.


Cell surface modifications induced by calcium ion in the myxobacterium Stigmatella aurantiaca. Calcium ion induces in the myxobacterium Stigmatella aurantiaca the ability to glide on solid surfaces and to become cohesive (D. F. Gilmore and D. White, J. Bacteriol. 161:113-117, 1985; B. J. Womack, D. F. Gilmore, and D. White, J. Bacteriol. 171:6093-6096, 1989). The addition of calcium ion to the growth medium resulted in the formation of extracellular fibrils, the appearance in the membrane fractions of a 30-kDa protein, and the accumulation in a low-speed centrifugal pellet of 10 polypeptides that cross-reacted with affinity-purified antibody to one of the polypeptides. One of the polypeptides, a 55-kDa protein, was present in the membrane fraction of control cells not incubated with calcium ion and was apparently translocated to the extracellular matrix during incubation in medium containing calcium ion. The 55-kDa protein was immunologically related to a 65-kDa protein located on the fibrils of another myxobacterium, Myxococcus xanthus.


Anaerobic growth of Rhodopseudomonas palustris on 4-hydroxybenzoate is dependent on AadR, a member of the cyclic AMP receptor protein family of transcriptional regulators. The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris converts structurally diverse aromatic carboxylic acids, including lignin monomers, to benzoate and 4-hydroxybenzoate under anaerobic conditions. These compounds are then further degraded via aromatic ring-fission pathways. A gene termed aadR, for anaerobic aromatic degradation regulator, was identified by complementation of mutants unable to grow anaerobically on 4-hydroxybenzoate. The deduced amino acid sequence of the aadR product is similar to a family of transcriptional regulators which includes Escherichia coli Fnr and Crp, Pseudomonas aeruginosa Anr, and rhizobial FixK and FixK-like proteins. A mutant with a deletion in aadR failed to grow on 4-hydroxybenzoate under anaerobic conditions and grew very slowly on benzoate. It also did not express aromatic acid-coenzyme A ligase II, an enzyme that catalyzes the first step of 4-hydroxybenzoate degradation, and it was defective in 4-hydroxybenzoate-induced expression of benzoate-coenzyme A ligase. The aadR deletion mutant was unaffected in other aspects of anaerobic growth. It grew normally on nonaromatic carbon sources and also under nitrogen-fixing conditions. In addition, aerobic growth on 4-hydroxybenzoate was indistinguishable from that of the wild type. These results indicate that AadR functions as a transcriptional activator of anaerobic aromatic acid degradation.


Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci. Degenerate oligonucleotides based on the published Escherichia coli glutamate decarboxylase (GAD) protein sequence were used in a polymerase chain reaction to generate a DNA probe for the E. coli GAD structural gene. Southern blots showed that there were two cross-hybridizing GAD genes, and both of these were cloned and sequenced. The two GAD structural genes, designated gadA and gadB, were found to be 98% similar at the nucleotide level. Each gene encoded a 466-residue polypeptide, named, respectively, GAD alpha and GAD beta, and these differed by only five amino acids. Both GAD alpha and GAD beta contain amino acid residues which are highly conserved among pyridoxal-dependent decarboxylases, but otherwise the protein sequences were not homologous to any other known proteins. By restriction mapping and hybridization to the Kohara miniset library, the two GAD genes were located on the E. coli chromosome. gadA maps at 4046 kb and gadB at 1588 kb. Neither of these positions is in agreement with the current map position for gadS as determined by genetic means. Analysis of Southern blots indicated that two GAD genes were present in all E. coli strains examined, including representatives from the ECOR collection. However, no significant cross-hybridizing gene was found in Salmonella species. Information about the DNA sequences and map positions of gadA and gadB should facilitate a genetic approach to elucidate the role of GAD in E. coli metabolism.


Localization of a Porphyromonas gingivalis 26-kilodalton heat-modifiable, hemin-regulated surface protein which translocates across the outer membrane. We recently identified a 26-kDa hemin-repressible outer membrane protein (Omp26) expressed by the periodontal pathogen Porphyromonas gingivalis. We report the localization of Omp26, which may function as a component of a hemin transport system in P. gingivalis. Under hemin-deprived conditions, P. gingivalis expressed Omp26, which was then lost from the surface after a shift back into hemin-rich conditions. Experiments with 125I labeling of surface proteins to examine the kinetics of mobilization of Omp26 determined that it was rapidly (within less than 1 min) lost from the cell surface after transfer into a hemin-excess environment. When cells grown under conditions of hemin excess were treated with the iron chelator 2,2'-bipyridyl, Omp26 was detected on the cell surface after 60 min. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses using purified anti-Omp26 monospecific polyclonal immunoglobulin G antisera established that Omp26 was heat modifiable (39 kDa unheated) and consisted of a single protein species. Immunogold labeling of negatively stained and chemically fixed thin-section specimens indicated that Omp26 was associated with the cell surface and outer leaflet of the P. gingivalis outer membrane in hemin-deprived conditions but was buried in the deeper recesses of the outer membrane in hemin-excess conditions. Analysis of subcellular fractions of P. gingivalis grown either in hemin-excess or hemin-deprived conditions detected Omp26 only in the cell envelope fraction, not in the cytoplasmic fraction or culture supernatant. Limited proteolytic digestion of hemin-deprived P. gingivalis with trypsin and proteinase K verified the surface location of Omp26 as well as its susceptibility to proteolytic digestion. Heat shock treatment of hemin-excess-grown P. gingivalis also resulted in Omp26 translocation onto the outer membrane surface even in the presence of hemin. Furthermore, hemin repletion of heat-shocked, hemin-deprived P. gingivalis did not result in Omp26 translocation off the outer membrane surface, suggesting that thermal stress inactivates this transmembrane event. This newly described outer membrane protein appears to be associated primarily with the outer membrane, in which it is exported to the outer membrane surface for hemin binding and may be imported across the outer membrane for intracellular hemin transport.


Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus. A highly thermostable beta-xylosidase, exhibiting similarly high activities for arylxylose and arylarabinose, was purified (72-fold) to gel electrophoretic homogeneity from the ethanologenic thermophilic anaerobe Thermoanaerobacter ethanolicus. The isoelectric point is pH 4.6; the apparent molecular weight is around 165,000 for the native enzyme (gel filtration and gradient polyacrylamide gel electrophoresis) and 85,000 for the two subunits (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The enzyme exhibited the highest affinity towards p-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82 degrees C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside. T(opt), 5 min, the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93 degrees C; however at 65 and 82 degrees C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82 degrees C (pH 5.0 to 5.5). The pH curves and temperature curves for arylxylosides as substrates differed significantly from those for arylarabinosides as substrates. An incubation for 3 h at 82 degrees C in the absence of substrate reduced the activity to around 75%. At 86 degrees C the half-life was around 15 min. With pNPX as the substrate, an Arrhenius energy of 69 kJ/mol was determined. The N-terminal sequence did not reveal a high similarity to those from other published enzyme sequences.


Molecular characterization of the sor gene, which encodes the sulfur oxygenase/reductase of the thermoacidophilic Archaeum Desulfurolobus ambivalens. A 5.8-kbp HindIII fragment containing the sor gene which encodes the aerobically induced sulfur oxygenase/reductase of the thermoacidophilic, chemolithoautotrophic, and facultatively anaerobic archaeum Desulfurolobus ambivalens, was cloned in pUC18 by using an oligonucleotide derived from the N-terminal amino acid sequence for identification (pSOR-1/17). The native enzyme is a 550,000-molecular-weight oligomer composed of single 40,000-molecular-weight subunits; this oligomer is capable of the simultaneous oxidation and reduction of sulfur (A. Kletzin, J. Bacteriol. 171:1638-1643, 1989). From the fragment, 3,025 bp that contained the entire sor gene were sequenced. The sor gene encoded a protein with 309 amino acid residues (molecular weight, 35,317). The transcript length was determined by Northern RNA hybridization to be 960 to 1,020 nucleotides, and the transcriptional start site was mapped by primer extension analysis. The transcript of the sor gene in aerobically grown cells was amplified 38- to 42-fold relative to that in anaerobically grown cells. An initial transcriptional characterization of three neighboring genes of unknown function is also reported.


Regulation of the macrolide-lincosamide-streptogramin B resistance gene ermD. The erythromycin resistance gene ermD, which encodes an rRNA methylase protein, has an unusually long leader region (354 nucleotides). Previously, a single promoter-proximal leader peptide coding sequence was recognized from the nucleotide sequence, and erythromycin-induced ribosome stalling in this sequence was proposed to be required for the induction of methylase translation. We characterized spontaneously occurring and in vitro-constructed leader region mutations in an effort to understand the function of various segments of the long ermD leader region. A second leader peptide coding sequence was identified, and the location of insertion and point mutations that expressed ermD methylase constitutively suggested that translation of the second leader peptide is controlled by ribosome stalling in the first leader peptide. From Northern RNA blot analysis of ermD transcription, it appears that regulation of ermD expression is not by transcriptional attenuation.


Oligopeptidase A is required for normal phage P22 development. The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing.


Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco. Pseudomonas syringae pv. phaseolicola produces the tripeptide N delta(N'-sulfo-diaminophosphinyl)-ornithylalanyl-homoarginin e (phaseolotoxin), which functions as a chlorosis-inducing toxin in the bean halo blight disease by inhibiting ornithine carbamoyltransferase (OCT). The bacterium possesses duplicate OCT genes, one of which, argK, encodes a toxin-resistant enzyme (ROCT) and imparts resistance to phaseolotoxin. We sequenced the argK gene from strain NPS3121, defined its promoter region, analyzed its regulation, and characterized its transcripts. The gene probably originated from another organism, since it is very distantly related to the argF gene encoding the housekeeping toxin-sensitive OCT and has low G+C content compared with the bacterial genome as a whole and with other protein-coding genes from P. syringae pv. phaseolicola. Optimized alignments of 13 OCT sequences allowed us to define key residues that may be responsible for toxin resistance and to identify a distinct prokaryotic amino acid signature, in ROCT, which argues for a prokaryotic origin of argK. An in-frame fusion of the argK coding region with the chloroplast transit peptide segment of the pea rbcS gene was introduced in Nicotiana tabacum by Agrobacterium-mediated transformation. The presence of an ROCT activity in transgenic plants was demonstrated by in vitro and in vivo assays. Some plants were toxin resistant, suggesting that pathogen-derived resistance to the toxin should be feasible in the pathogen's host.


Expression of Vi antigen in Escherichia coli K-12: characterization of ViaB from Citrobacter freundii and identity of ViaA with RcsB. The Vi antigen in Salmonella typhi is stably expressed and may act to protect the strain against the defensive system of the host. Citrobacter freundii, not usually a common human pathogen, also expresses the Vi antigen but expresses it unstably, exhibiting a reversible transition between the Vi+ and Vi- states. Two widely separated chromosomal regions, ViaA and ViaB, are needed for Vi synthesis. Escherichia coli K-12 harboring a functional ViaB plasmid can also express Vi antigen, but the cloned ViaB sequence can only be stably maintained and expressed in recA hosts. Vi- derivatives arise either through IS1-like insertional events occurring in ViaB sequences or by chromosomal mutations at the ViaA region. P1vir mapping indicates that the ViaA mutations are located at min 47.75 on the E. coli chromosome. All the spontaneous viaA mutants isolated from E. coli and S. typhi were identified as rcsB mutants by complementation tests using plasmid pJB100. Introduction of rcsA::Tn10 into E. coli harboring functional ViaB sequences eliminates the expression of Vi antigen. These results indicate that Vi antigen synthesis is regulated by the same regulatory proteins involved in colanic acid synthesis in E. coli.


Characterization of the Campylobacter fetus sapA promoter: evidence that the sapA promoter is deleted in spontaneous mutant strains. Wild-type Campylobacter fetus cells possess S-layer proteins (S+ phenotype), whereas after laboratory passage, spontaneous stable mutants that do not express these proteins (S- phenotype) arise. To determine the molecular mechanisms by which C. fetus changes to the S- phenotype, we studied wild-type strain 23D, from which the sapA gene encoding the 97-kDa S-layer protein has been cloned, and strain 23B, a spontaneous S- mutant. We compared these strains with another pair of strains, LP (S+) and HP (S-). Southern analysis with the cloned sapA gene as a probe indicated that both pairs of strains have multiple sapA homologs. Using gene disruption and replacement techniques, we constructed an isogenic strain of 23D that differed only in sapA expression (strain 23D:401:1). A 6.0-kb HindIII fragment from 23D:401:1 containing 3.4 kb of sapA upstream region then was cloned into pBluescript to produce pBG101. Nucleotide sequence analysis of sapA upstream region revealed a consensus promoter at -121 bp from the translational start site. Primer extension analysis placed a single in vivo transcription initiation site at the -114-bp position of sapA. A DNA probe derived from the sapA promoter region hybridized to a 5.5-kb HindIII fragment of chromosomal DNA from strain 23D but not to DNA from strain 23B. Northern RNA blot analysis showed no sapA mRNA in strain 23B. These data indicate that the lack of S-layer protein expression in spontaneous mutant strains is caused by the deletion of promoter sequences.


Determination of the mechanism of retrotransfer by mechanistic mathematical modeling. Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that is, the ability of conjugative plasmids to mobilize genes into the cell containing the conjugative plasmid, were developed. This study deals with retromobilization of nonconjugative plasmids (Tra-Mob+). Plasmid transfer was modeled by two mass action models. The first is based on the hypothesis that retromobilization of the Tra-Mob+ vector occurs in one step, by means of the pilus formed by the Tra+ plasmid in the original host. In the second model, retromobilization is considered to be a two-step process involving two transfer events. The first step involves the transfer of the Tra+ plasmid from the recipient cell to the donor of the nonconjugative vector, and during the second encounter the nonconjugative vector is mobilized toward the recipient. Since the relationships between the number of transconjugants and the number of recipients for the two models are different, filter matings were performed for short time periods with different initial densities of the recipient population. Comparison of the numbers of transconjugants with the results of the mathematical equations confirmed the hypothesis that retromobilization is a one-step conjugation process.


Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100. The Escherichia coli gene coding for dihydropteroate synthase (DHPS) has been cloned and sequenced. The protein has 282 amino acids and a compositional molecular mass of 30,314 daltons. Increased expression of the enzyme was realized by using a T7 expression system. The enzyme was purified and crystallized. A temperature-sensitive mutant was isolated and found to express a DHPS with a lower specific activity and lower affinities for para-aminobenzoic acid and sulfathiazole. The allele had a point mutation that changed a phenylalanine codon to a leucine codon, and the mutation was in a codon that is conserved among published DHPS sequences.


Role of pilA, an essential regulatory gene of Neisseria gonorrhoeae, in the stress response. Sequence analysis has shown that PilA, a transcriptional regulator of pilin gene expression in Neisseria gonorrhoeae, has extensive homology with the 54-kDa protein of the signal recognition particle of eukaryotes and its receptor, as well as with two proteins of Escherichia coli, FtsY and Ffh, which have been proposed to be a part of a signal recognition particle-like apparatus. We tested the putative role of PilA in protein export in N. gonorrhoeae and did not find any effect. However, we did observe induction of a heat shock response and a previously described slow-growth phenotype when PilA function was impaired. We also examined the interference of pilA expression in E. coli with the function of the products of ftsY and ffh and observed an accumulation of pre-beta-lactamase. We argue against a direct role for PilA in protein export in gonococci and propose instead that PilA is involved in the modulation of cell growth rate in response to different environmental conditions.


The cytoplasmic peptidoglycan precursor of vancomycin-resistant Enterococcus faecalis terminates in lactate. Vancomycin resistance plasmids in enterococci carry the genes vanH and vanA, which encode enzymes catalyzing, respectively, the reduction of 2-keto acids to 2-D-hydroxy acids and the addition of D-hydroxy acids to D-alanine. It has therefore been postulated that resistant cells produce peptidoglycan precursors that terminate in the depsipeptide D-alanine-2-D-hydroxy acid rather than the dipeptide D-alanine-D-alanine, thus preventing vancomycin binding (M. Arthur, C. Molinas, T. D. H. Bugg, G. D. Wright, C. T. Walsh, and P. Courvalin, Antimicrob. Agents Chemother. 36:867-869, 1992). In the present work, a cytoplasmic peptidoglycan precursor was isolated from vancomycin-resistant Enterococcus faecalis and analyzed by mass spectrometry, which suggested the structure UDP-N-acetyl-muramyl-L-Ala-D-Glu-L-Lys-D-Ala-D-lactate.


An ordered clone bank for chromosome I of Saccharomyces cerevisiae. Chromosome I of Saccharomyces cerevisiae DC5 rho 0 was dissected into segments with an average size of 14.0 kb and cloned into lambda phage vectors. The physical maps of the resultant clones, totaling 205.9 kb, were used to construct an ordered clone bank of this chromosome.


Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains. A model system for one-step gene disruption for an asporogenous methylotrophic yeast, Candida boidinii, is described. In this system, the 3-isopropylmalate dehydrogenase gene (C. boidinii LEU2) was selected as the target gene for disruption to derive new host strains for transformation. First, the C. boidinii LEU2 gene was cloned, and its complete nucleotide sequence was determined. Next, the LEU2 disruption vectors, which had the C. boidinii URA3 gene as the selectable marker, were constructed. Of the Ura+ transformants obtained with these plasmids, more than half showed a Leu- phenotype. Finally, the double-marker strains of C. boidinii were derived. When vectors with repeated flanking sequences of the C. boidinii URA3 gene were used for gene disruption, Leu- Ura+ transformants changed spontaneously to a Leu- Ura- phenotype ca. 100 times more frequently than they did when plasmids without the repeated sequences were used. Southern analysis showed that these events included a one-step gene disruption and a subsequent popping out of the C. boidinii URA3 sequence from the transformant chromosome.


Characterization of a light-regulated gene encoding a new phycoerythrin-associated linker protein from the cyanobacterium Fremyella diplosiphon. Cyanobacteria utilize multimeric protein complexes, the phycobilisomes, as their major light-harvesting antennae. Associated with the chromophorylated phycobiliproteins in these complexes are nonpigmented proteins, designated linker proteins. These linker proteins are believed to mediate assembly of the phycobilisome and energy transfer to the photosynthetic reaction center. We cloned and sequenced a gene, cpeE, encoding a previously uncharacterized linker protein which is expressed in green light in Fremyella diplosiphon. This gene is part of an operon containing two other phycoerythrin-associated linker genes, cpeC and cpeD. Transcription of the cpeCDE operon in green light results in two predominant species of mRNA of approximately 2,100 and 3,200 nucleotides. The shorter transcript encodes only CpeC and CpeD, while the longer contains the coding regions for all three linker proteins. By altering the pH of the resolving gel and the running buffer during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this third linker protein CpeE can be resolved from the rod-core linker and the other rod linker proteins. The three proteins have an overall similarity of approximately 62%, and the genes encoding the three proteins are approximately 59% identical.


The medical consequences of the selection of an antidepressant. Patients who take antidepressants risk developing adverse drug reactions with medical sequelae. I will review noncardiac adverse drug reactions to antidepressant organized around (1) the effect of antidepressants on neurotransmitter systems and monoamine oxidase along with effects on concomitant physiologic systems, (2) side effects that are seen clinically but may or may not be related to antidepressant effects on neurotransmitter systems, (3) medical conditions that affect the choice of certain antidepressants, (4) antidepressant toxicity with overdose, and (5) recommendations for using antidepressants with the least probability of causing severe adverse drug reactions.


Treating the depressed patient with cardiovascular problems. A multidimensional relationship exists between cardiovascular disease and affective disorder that includes the observations that (1) there is a high rate of depression in the postmyocardial infarction period, (2) the presence of depressive illness adversely affects the prognosis of cardiac disease, and (3) depressed patients have a higher-than-expected rate of sudden cardiovascular death. The authors discuss these topics and the clinical management of depression in patients with significant preexisting heart disease. The cardiovascular effects of the tricyclic antidepressants and recently introduced nontricyclic antidepressants are reviewed with a focus on how the clinician can safely and effectively treat affective disorder in patients with severe cardiac disease.


Long-term treatments of recurrent depressive disorders. Many, if not most, depressive disorders become recurrent conditions. It is now clear that virtually all recently remitted patients should receive 4 to 6 months of continuation therapy. Moreover, this phase of treatment should continue until the patient has achieved a clear-cut, durable period of recovery. Further, an extended course of maintenance pharmacotherapy is recommended for those patients at risk for subsequent recurrent episodes. In this paper, the rationale for long-term treatment is presented and common strategies are reviewed. Depressive disorders appear to become more autonomous, severe, and potentially refractory with each new episode. Thus, prevention of recurrent depression remains the best available strategy to ensure an optimal long-term outcome for patients with recurrent affective disorders.


Treatment of depression in special populations. The purpose of this review is to set forth guidelines for the treatment of depression in several special populations: (1) the elderly (both ambulatory and institutionalized); (2) patients with concurrent neurologic disorders (Alzheimer's disease, Parkinson's disease, and stroke) and depression; and (3) patients with bereavement-related depression. This is a selective review of studies published in the past 10 years that have utilized structured psychiatric interviewing, randomized clinical trials, and/or monitoring of plasma antidepressant levels. Published data support specific efficacy and safety claims for both pharmacotherapeutic and psychotherapeutic approaches to the treatment of major depression in elderly ambulatory and institutionalized patients. In the case of depression associated with Alzheimer's, Parkinson's, and stroke, there is also evidence of efficacy for antidepressant medication. Finally, bereavement-related syndromal depression appears to respond to antidepressant medication, but further controlled evaluation is desirable. As emphasized by the 1991 National Institutes of Health Consensus Development Conference on the Diagnosis and Treatment of Depression in Late Life, depression in the elderly should be recognized as treatable and should be treated vigorously. Rather than being dismissed as a normal reaction to the multiple medical and psychosocial burdens of late life, it should be treated appropriately to reduce an important source of excess disability.


Somatic therapy for major depressive disorder: selection of an antidepressant. Major depressive disorder is a major health concern. It adversely affects the patient, the family, and society. While the consequences can be devastating and life-threatening, this condition has an excellent prognosis when properly identified and treated. Somatic therapy plays a pivotal role in inducing and maintaining a remission and preventing recurrent attacks. There are now five major classes of antidepressants which differ in terms of their clinical spectra of antidepressant activity, their safety and tolerability, their likelihood of pharmacodynamic and pharmacokinetic interactions with concomitantly prescribed drugs, ease of administration, and physician confidence, which is in large measure a reflection of the extent and quality of their human exposure database. The proper selection and management of antidepressant pharmacotherapy is based on an understanding of these differences.


Effect of non-surgical periodontal therapy in an undergraduate dental clinic. Results one year following treatment. The aim of the present study was to evaluate the effectiveness of non-surgical treatment of moderately to severely advanced periodontitis in patients at the Department of Periodontology of the Aristotle University of Thessaloniki. The patients were treated by senior students of the Dental School under the supervision of specialized personnel. The conservative treatment consisted of oral hygiene instructions and repeated instrumentation (scaling and root planing) for a period of about 3 months. Seventeen patients with advanced periodontitis participated in the study. The following data were recorded: 1. Gingival index (Silness and Löe, 1963), 2. Plaque index (Löe and Silness 1964), 3. Bleeding on probing and 4. Pocket depth and probing attachment level. The measurements were taken by the same examiner four times, that is at baseline, immediately after completion of the conservative therapy, 6 months and 12 months after treatment. Measurements from single and multi-rooted teeth were divided into three groups with respect to the initial pocket depth (less than or equal to 3 mm, 4-6 mm, greater than or equal to 7 mm). The findings of the present study demonstrated that non-surgical therapy can achieved a marked reduction in bleeding on probing scores and pocket depth 12 months after treatment. A gain in probing attachment levels was found for sites with initially deep (greater than or equal to 7 mm) and moderate (4-6 mm) pockets, while in sites with initially shallow pockets (less than or equal to 3.0 mm), a loss of attachment level was established.


Growth of the skeletal units of the rat mandible in acute renal failure experimental model. A study of the effect of acute renal failure (ARF) on the growth of the skeletal units of the mandible was performed. Male Wistar rats weighing 35-40 g were assigned to three groups. One of the groups received a basal choline-deficient diet ad libitum. A second group was pair-fed with the first one and was given a basal diet supplemented with 0.35 g choline chloride/100 g of diet. The third group was fed a basal diet plus 0.35 g choline chloride/100 g of diet ad libitum and used as the normal control. Body length, renal function parameters and measurements of the mandible were recorded at the end of a 12-day period on the different diets. Food intake and body weight were recorded every day. Plasma urea and creatinine concentrations markedly increased over the first 12 days of exposure to a choline-deficient diet. Significant increases in urine volume and significant depressions in urine osmolality were also observed. The average body weight and body length gains for rats given the choline-deficient diet were lower than those of control rats. There were no significant differences between the average body weight and body length of pair-fed and normal rats. All skeletal measurements of the mandible of the choline-deficient group differed significantly from those of the control group. These differences varied between 7% and 30%. The skeletal units of the mandible of the pair-fed group did not differ significantly from ad libitum controls. This study suggests that this rat model can be used for the investigation of the different alterations observed in uremia.(ABSTRACT TRUNCATED AT 250 WORDS)


[Gingival hyperplasia secondary to the use of calcium antagonists: analysis]. Calcium antagonists are drugs restricting transmembrane calcium delivery. They possess a wide range of action against vasoconstriction and spastic reactions and were therefore initially recommended for the treatment of angina pectoris. With the increasing number and classes of calcium antagonists new therapeutic indications have emerged. Cases of gingival hyperplasia associated with their use are repeatedly reported, therefore the question deserves to be restated. The aim of the present study was to discuss the clinical, pathologic and pathogenetic features bases on an investigation carried out in a Department of Cardiology, on a case observation and on review of published cases in the international literature.


[Antifungal varnish in the treatment of denture stomatitis]. The efficacy of a single-dose application of miconazole varnish in the treatment of denture stomatitis was compared with miconazole gel applied three times/day for 15 days. Among 288 wearers of complete dentures 50 patients severely affected by denture stomatitis and heavily colonized by Candida, were selected. The patients were assigned randomly to two groups: a miconazole varnish group and a gel group. All patients were examined 7 times: before starting the treatment (day 0); during treatment (day 3, 7 and 14); after treatment (day 21, 28 and 35). At each examination a photograph of the palatal mucosa was obtained and quantitative cultures of Candida from the lesions and the fitting denture surface were performed. At day 14 the inflammation was reduced to the same degree in the two groups of patients. A comparison of the antifungal effect, i.e. reduction of the yeast score, showed no significant difference between the two groups. Maximum reduction of the number of yeasts was obtained at day 14. From that moment, recolonization started; thus, about 60% of the treated patients whether it was with the gel or the varnish was positive of Candida by day 35. In conclusion, the present study showed no difference in the clinical and antifungal effect of a single-dose application of miconazole varnish compared with miconazole gel. The varnish seemed to be better with respect to the posology as the total dose of miconazole is minimal and only one application is necessary. Treatment with the miconazole varnish seems particularly indicated in debilitated and non-cooperative patients suffering from Candida-associated denture stomatitis.


Penetration of glutaric dialdehyde into human dentine as measured by changes in dentine microhardness and dentine dimensions. Microhardness indentation lengths were measured on human dentine sections unfixed and fixed with glutaric dialdehyde (GDA) at pH 7. Fixed dentine was found to be softer than unfixed dentine. Indentation length increased by approximately 10% after fixation in a buffered 5% GDA solution at pH 7. Using this effect the variation of indentation length in the direction of the penetrating GDA as a function of fixation time was measured. Describing the penetration in the mineralized matrix as diffusion in a plane sheet, a diffusion coefficient of 0.5.10(-12); m2/s was calculated. Dimensional changes of the dentine due to fixation were also measured. Dentine was found to expand in both the direction parallel (approximately 0.4%) and perpendicular (approximately 2%) to the tubules.


Differences between primary and permanent teeth in posteruptive age dependency of radiological changes in enamel during the development of approximal caries. A longitudinal radiological study of children (N = 549) who participated in a comprehensive preventive maintenance program showed that caries related events in the approximal surfaces of permanent teeth differed from those in deciduous teeth. Changes in the approximal surfaces of the younger permanent teeth were more pronounced than of the older primary teeth and differed significantly from 1 year to 2.5 years. These findings can be explained by posteruptive maturation of tooth enamel.


The effect of an experimental mouthrinse on enamel lesion remineralization in vitro. This paper describes the effect of a 2 ppm fluoride containing, slightly acidic mouthrinse (pH = 6) on the remineralization of enamel lesions in vitro. The rinse was applied twice daily for 2 min, on about 85 microns in depth artificial lesions. Artificial enamel lesions were formed at pH = 5 in a 6% CMC gel. The lesions were subsequently remineralized in vitro in a neutral remineralizing solution (1.5 mM Ca and 0.9 mM phosphate) with or without the application of the rinse. After 3 weeks the samples were evaluated by means of quantitative microradiography. The results showed that a statistically significant reduction in lesion depth occurred of 48 and 29% with or without rinse application, with respect to the original lesion depth. The effect of the rinse compared with no rinse decreased the lesion depth by 40%. The data indicate that application of a slightly acidic rinse with 2 ppm fluoride applied twice a day, stimulates the remineralization at the lesion front and does not lead to blocking of enamel pores observed after acidic, high level F applications (APF).


A quantitative evaluation of periprosthetic bone-remodeling after cementless total hip arthroplasty. Dual-energy x-ray absorptiometry analysis was used to determine the periprosthetic bone-mineral content of ten femora that had been obtained at the autopsies of five elderly patients who had had an AML (anatomic medullary locking) prosthesis in situ for seventeen to eighty-four months. Clinical radiographs showed resorptive remodeling changes characteristic of femora containing this implant. Before the absorptiometry was performed, an identical prosthesis had been inserted into the contralateral, normal femur of each cadaver. The mean difference in the periprosthetic bone-mineral content between the remodeled femora and the femora in which the prosthesis had been implanted post mortem ranged from 7 to 52 per cent, with the bone-mineral content always less in the remodeled femora. The greatest mean decrease in bone-mineral content (45 per cent) occurred adjacent to the proximal one-third of the remodeled femora. The percentage decrease in periprosthetic bone-mineral content in the remodeled femora had an inverse linear relationship with the corresponding bone-mineral content of the contralateral control femora. Preoperative analysis of bone density may therefore be useful for prediction of the extent of resorptive bone-remodeling after total hip replacement.


The use of computerized tomography in the measurement of glenoid version. Computerized tomography was done preoperatively on twenty shoulders (thirteen patients) in which there were severe arthritic changes, to measure glenoid version. Ten of the twenty shoulders had osteoarthrosis; eight, rheumatoid arthritis; and two, gouty arthritis. To help determine normal values, computerized tomographic scans of the chest of sixty-three patients who did not have roentgenographic evidence of disease of the shoulder were studied retrospectively for comparison as a control group. In the group of patients who had severe arthritis, the mean glenoid orientation was 11 degrees of retroversion (range, 2 degrees of anteversion to 32 degrees of retroversion). The computerized tomographic scans showed uneven wear of the glenoid surface, osteophytes, large cysts, and posterior displacement of the humeral head. In the control group, the mean orientation of the glenoid was 2 degrees of anteversion (range, 14 degrees of anteversion to 12 degrees of retroversion). The difference between the groups was significant (p less than 0.0001). Glenoid retroversion was increased in the patients who had severe arthritis, and the computerized tomographic scans accurately revealed the extent and pattern of erosion of the bone.


Transfer of the tibialis anterior for calcaneus deformity in myelodysplasia. We evaluated the results of transfer of the tibialis anterior in the management of calcaneus deformity in young patients who had myelodysplasia; fifteen patients (twenty-two feet) were operated on between 1978 and 1985. The neural deficit was at the fourth and fifth lumbar levels. The average age at the time of the operation was seven years and two months (range, two to nineteen years). The average age at the latest follow-up was thirteen years (range, five to twenty-four years). The average duration of follow-up was five years and ten months (range, two to eleven years). Seventeen feet (twelve patients) had a good result (no ulceration of the heel or osteomyelitis and correction of the calcaneus deformity), and five feet (three patients) had a poor result (persistent ulceration, signs of osteomyelitis, recurrent or persistent calcaneus deformity, or the need for additional operative intervention). Children who were less than five years old had a better outcome, as determined by the Fisher exact test (p less than 0.5).


Stability of an ankle arthrodesis fixed by cancellous-bone screws compared with that fixed by an external fixator. A biomechanical study. Twenty-three fresh-frozen human specimens were subjected to ankle arthrodesis and fixation with two cancellous-bone screws. The specimens were then subjected to four newton-meters of manually applied tibial torque, plantar flexion-dorsiflexion moment, and medial-lateral bending moment; relative rotation between the tibia and the talus was recorded for each mode of testing. A Calandruccio triangular compression device was then applied to threaded pins penetrating the tibia and talus, the screws were removed, and the test sequence was repeated. Tibiotalar motions recorded with both systems of fixation were markedly affected by the quality of the bone. When less than 2 degrees of total tibiotalar rotation was recorded in response to four newton-meters of manually applied internal-external tibial torque, all specimens demonstrated less torsional rotation with the screw fixation than with the external fixator. The mean rotations produced by medial-lateral bending moment were equivalent for both systems of fixation. When torsional rotations with the use of screw fixation were greater than 2 degrees, all specimens demonstrated more torsional rotation with the screws than with the external fixator, and all but one specimen had more medial-lateral rotation with the screws than with the fixator. For applied plantar flexion-dorsiflexion moment, twenty-two of the twenty-three specimens demonstrated more rotation with the Calandruccio fixator than with the screws; this was due in part to motion permitted at the hinge points of the frame itself.


Cotrel-Dubousset instrumentation for adolescent idiopathic scoliosis. We evaluated the results of segmental fixation of the spine with Cotrel-Dubousset instrumentation in ninety-five patients who had adolescent idiopathic scoliosis. The instrumentation was used in an attempt to achieve three-dimensional correction of the scoliosis, maintain lumbar lordosis, create thoracic kyphosis, and avoid the need for a postoperative cast or brace. The patients were followed for twenty-four to sixty-four months (average, thirty-five months). Cotrel-Dubousset instrumentation provided an average correction of the coronal curve of 48 per cent at the time of the most recent follow-up. The normal sagittal curves at the thoracolumbar junction and in the lumbar spine were maintained, and the thoracic kyphosis was increased slightly (average, +7 degrees). Apical translation improved an average of 60 per cent, and apical rotation improved an average of 11 per cent. Forced vital capacity improved an average of 21 per cent, and the one-second forced expiratory volume improved an average of 18 per cent. There were no major neurological deficits. A symptomatic pseudarthrosis developed in one patient. Postoperatively, decompensation of the spine developed in five of the first twenty-six patients who had a Type-II or Type-III curve. This complication was avoided in the last twenty-four patients who had a Type-II or Type-III curve by means of a stricter adherence to the definition of a Type-II curve, and reversal of the bend of the rod and the hooks between the caudal neutral and stable vertebrae. The major advantages of Cotrel-Dubousset instrumentation are the stable fixation that is achieved and the preservation of segmental lumbar lordosis.


The effect of malunion on functional outcome after plate fixation of fractures of both bones of the forearm in adults. Fifty-five adults who had a fracture of both bones of the forearm were managed with plating and were followed for a mean of six years (range, one year to sixteen years and two months) with functional and radiographic assessment. Malunion was quantified by measurement of the amount and location of the maximum radial bow in relation to the contralateral, normal forearm. Fifty-four of the radial and fifty-four of the ulnar fractures united. Eighty-four per cent of the patients had an excellent, good, or acceptable functional result, according to the criteria of Grace and Eversmann. Bone-grafting did not affect the rate of union. Restoration of the normal radial bow was related to the functional outcome. A good functional result (more than 80 per cent of normal rotation of the forearm) was associated with restoration of the normal amount and location of the radial bow (p less than 0.05 and p less than 0.005). Similarly, the recovery of grip strength was associated with restoration of the location of the radial bow toward normal (p less than 0.005).


Osteosarcoma as a second malignant neoplasm in children. Nine patients who had an osteosarcoma that had developed as a second malignant neoplasm in a previously irradiated site were managed at a major center for the treatment of tumors in children. The doses of radiation had averaged 4144 centigray (range, 2300 to 8000 centigray) and chemotherapy had been administered, when appropriate, for the primary malignant lesion (Ewing sarcoma, malignant fibrous histiocytoma, Hodgkin lymphoma, neuroblastoma, neurofibrosarcoma, rhabdomyosarcoma, and Wilms tumor). The interval between the initial treatment and the diagnosis of the secondary sarcoma averaged ten years and one month (range, five years and ten months to twenty-one years and nine months). Three patients were alive, two of them with active disease, at the time of writing. The other six had died within three years (average, fifteen months) after the second diagnosis. The prevalence of secondary osteosarcoma is increasing as the survival of children who have a malignant lesion increases. Plans for tumor therapy should take into account the risk of this complication, which is usually fatal.


The effect of a ligament-augmentation device on allograft reconstructions for chronic ruptures of the anterior cruciate ligament. A prospective study was performed to determine the effect of a combination of a ligament-augmentation device with a bone-patellar ligament-bone allograft for the treatment of chronic rupture of the anterior cruciate ligament. One hundred and fifteen knees in 110 patients were divided into two groups. Group BLB consisted of sixty-six knees in sixty-four patients who were managed with a bone-patellar ligament-bone allograft only, and Group BLB-LAD consisted of forty-nine knees in forty-six patients who were managed with both the allograft and a ligament-augmentation device. Preoperatively, there were no statistically significant differences between the two groups with regard to fifteen variables. All patients were managed with the same postoperative program of immediate motion and rehabilitation of the knee. All patients returned for evaluation at a mean of thirty-four months (range, twenty-three to fifty-three months) postoperatively. The results were evaluated with a comprehensive rating system that assessed twenty factors. Both of these procedures significantly decreased functional limitations and symptoms and improved the level of sports activity and the over-all score. However, the use of the ligament-augmentation device did not improve the efficacy of the reconstruction with regard to any of the individual variables that were assessed or in terms of the over-all score. All but one of the patients regained an arc of 0 to 135 degrees of motion. Although the augmentation device reduced anterior-posterior displacement effectively for the first twenty weeks postoperatively (p less than 0.05), there was no difference between the groups in terms of the percentage of knees that had abnormal displacement at the latest follow-up. A new classification system was developed to determine rates of failure. The over-all rate of failure was 28 per cent (thirty-two) of the 115 knees: 29 per cent (nineteen) of the sixty-six knees in Group BLB and 27 per cent (thirteen) of the forty-nine knees in Group BLB-LAD. The difference between the two groups was not statistically significant. The addition of the ligament-augmentation device did not improve the results of allograft reconstruction in the treatment of chronic rupture of the anterior cruciate ligament. The use of either an allograft alone or an allograft combined with a ligament-augmentation device did not reduce the amount of anterior-posterior displacement satisfactorily in all of the knees.


Posterior stabilized prosthesis. Results after follow-up of nine to twelve years. We reviewed, nine to twelve years postoperatively, the records on an original cohort of 289 arthroplasties (218 patients) in which a posterior stabilized knee prosthesis with an all-polyethylene tibial component had been inserted at The Hospital for Special Surgery. One hundred and eighty intact prostheses in 139 patients were available for this analysis. Fourteen knees in fourteen patients had had a revision procedure. Five of these fourteen patients had had a bilateral arthroplasty, but only one knee of each of the five patients had been revised. Forty-eight of the patients (sixty-six knees) had died less than nine years after the operation. Twenty-nine other knees (twenty-two patients) had been lost to follow-up before a nine-year evaluation could be performed. Considering all 194 knees (including the fourteen that had been revised), the result with the system of The Hospital for Special Surgery was excellent for 117 knees (61 per cent), good for fifty-one (26 per cent), fair for twelve (6 per cent), and poor for the fourteen knees (7 per cent) that had been revised. The 180 knees in which the prosthesis was intact were also rated with the new scoring system of The Knee Society: the average postoperative knee score was 92 points (range, 35 to 100 points), and the average score for function was 66 points (range, 0 to 100 points). Survivorship analysis showed that the average annual rate of failure was 0.4 per cent and that the over-all rate of success at thirteen years was 94 per cent.(ABSTRACT TRUNCATED AT 250 WORDS)


Revision of the acetabular component with an uncemented Harris-Galante porous-coated prosthesis. One hundred and forty revisions for loosening of the acetabular component were performed in 124 patients, with the use of an uncemented Harris-Galante acetabular prosthesis. A component was considered loose if there was a change in its position or vertical or horizontal migration, or both, of four millimeters or more, as demonstrated on serial radiographs. All patients were followed prospectively for a mean of forty-one months (range, twenty-four to seventy-six months). Bone-grafting was performed at the time of the revision in 127 of the hips. Identifiable failure of fixation of the acetabular component occurred in only two hips (1 per cent), which had both severe acetabular bone loss and pelvic discontinuity. In one of these hips, fixation of the component could not be achieved during the revision, and the component subsequently migrated. No other components migrated. Only one patient had a revision of the index acetabular operation. A continuous radiolucency developed at the bone-mesh interface of five acetabular components, and in one other hip a small portion of the mesh separated. This hip was reoperated on for a problem with the femur, and the socket was found to be rigidly fixed. No other evidence of loosening was identified. All of the bone grafts united, but partial resorption of the graft occurred in thirty-nine hips. Although there were substantial osseous defects that necessitated major bone-grafting, revision with the uncemented Harris-Galante porous-coated acetabular component provided superior fixation compared with that reported in other series in which cemented acetabular components were used for revision. Of the 140 hips, eighty-nine (64 per cent) had a postoperative score of good or excellent, according to the Harris hip-rating system. Twenty-eight (70 per cent) of the forty hips that had revision of the acetabular component alone were rated as good or excellent.


A cap binding protein that may mediate nuclear export of RNA polymerase II-transcribed RNAs. It has previously been shown that efficient export of U1 snRNA or of microinjected, in vitro synthesized, RNA transcripts from the nucleus of Xenopus oocytes is facilitated by their monomethyl guanosine cap structures. Nuclear exit of these transcripts could be competitively inhibited by microinjection of an excess of a cap analog, the dinucleotide m7GpppG (Hamm, J., and I. W. Mattaj. 1990. Cell. 63:109-118). We have now analyzed the ability of several other related cap analogs to inhibit the export of U1 snRNA from the nucleus. The results define the recognition specificity of a factor(s) involved in RNA transport, and indicate that the cap binding activity (CBA) involved in RNA export is different from cap binding proteins (CBPs) involved in the initiation of translation. A CBP, whose specificity for different analogs correlates with the ability of the analogs to inhibit U1 snRNA export, is identified in nuclear extracts prepared from HeLa cells. We propose that this protein may have a role in the export of capped RNAs from the nucleus.


In vitro assembly of prenucleolar bodies in Xenopus egg extract. Nuclei assembled in Xenopus egg extract from purified DNA or chromatin resemble their natural counterparts in a number of structural and functional features. However, the most obvious structural element of normal interphase nuclei, the nucleolus, is absent from the in vitro reconstituted nuclei. By EM, cytological silver staining, and immunofluorescence microscopy employing antibodies directed against various nucleolar components we show that nuclei assembled in vitro contain numerous distinct aggregates that resemble prenucleolar bodies (PNBs) by several criteria. Formation of these PNB-like structures requires pore complex-mediated nuclear transport of proteins but is independent of the genetic content of the in vitro nuclei as well as transcriptional and translational events. Our data indicate that nuclei assembled in vitro are capable of initiating early steps of nucleologenesis but that the resulting PNBs are unable to fuse with each other, probably due to the absence of a functional nucleolus organizer. With appropriate modifications, this experimental system should be useful to define and analyze conditions promoting the site-specific assembly of PNBs into a coherent nucleolar body.


Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment. Expression of hepatitis B surface antigen (HBsAg), the major envelope protein of the virus, in the absence of other viral proteins leads to its secretion as oligomers in the form of disk-like or tubular lipoprotein particles. The observation that these lipoprotein particles are heavily disulphide crosslinked is paradoxical since HBsAg assembly is classically believed to occur in the ER, and hence in the presence of high levels of protein disulphide isomerase (PDI) which should resolve these higher intermolecular crosslinks. Indeed, incubation of mature, highly disulphide crosslinked HBsAg with recombinant PDI causes the disassembly of HBsAg to dimers. We have used antibodies against resident ER proteins in double immunofluorescence studies to study the stages of the conversion of the HBsAg from individual protein subunits to the secreted, crosslinked, oligomer. We show that HBsAg is rapidly sorted to a post-ER, pre-Golgi compartment which excludes PDI and other major soluble resident ER proteins although it overlaps with the distribution of rab2, an established marker of an intermediate compartment. Kinetic studies showed that disulphide-linked HBsAg dimers began to form during a short (2 min) pulse, increased in concentration to peak at 60 min, and then decreased as the dimers were crosslinked to form higher oligomers. These higher oligomers are the latest identifiable intracellular form of HBsAg before its secretion (t 1/2 = 2 h). Brefeldin A treatment does not alter the localization of HBsAg in this PDI excluding compartment, however, it blocks the formation of new oligomers causing the accumulation of dimeric HBsAg. Hence this oligomerization must occur in a pre-Golgi compartment. These data support a model in which rapid dimer formation, catalyzed by PDI, occurs in the ER, and is followed by transport of dimers to a pre-Golgi compartment where the absence of PDI and a different lumenal environment allow the assembly process to be completed.


The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation. An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.


Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells. Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.


Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium. To investigate the intracellular role of the clathrin heavy chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in clathrin heavy chain expression. Immunoblots stained with an anti-clathrin heavy chain antiserum revealed that mutant cells contained undetectable amounts of clathrin heavy chain protein. Similarly, Northern blots showed an absence of clathrin heavy chain mRNA. Clathrin heavy chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of clathrin heavy chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.


The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin-sensitive cells. Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.


Localization of dystrophin COOH-terminal domain by the fracture-label technique. The precise localization of dystrophin in the skeletal muscle cell should contribute to a better understanding of the yet unclear functional role of this protein, both in normal and in Duchenne muscular dystrophy. Immunocytochemical studies did not give conclusive results on the localization of dystrophin with respect to the sarcolemma and to the cytoskeletal components. To improve the reliability of the electron microscopic immunocytochemical localization of dystrophin, a mAb against the COOH-terminus of the molecule has been used in association with the fracture-label technique, which, causing a partition of the membrane in protoplasmic and exoplasmic halves, allows a more precise dystrophin localization. The results obtained indicate that dystrophin is associated with the protoplasmic half of the plasmalemma, and the observation that it does not randomly follow the partition of the membrane is consistent with a stable association with the cytoskeleton.


Costameres are sites of force transmission to the substratum in adult rat cardiomyocytes. Costameres, the vinculin-rich, sub-membranous transverse ribs found in many skeletal and cardiac muscle cells (Pardo, J. V., J. D. Siciliano, and S. W. Craig. 1983. Proc. Natl. Acad. Sci. USA. 80:363-367.) are thought to anchor the Z-lines of the myofibrils to the sarcolemma. In addition, it has been postulated that costameres provide mechanical linkage between the cells' internal contractile machinery and the extracellular matrix, but direct evidence for this supposition has been lacking. By combining the flexible silicone rubber substratum technique (Harris, A. K., P. Wild, and D. Stopak. 1980. Science (Wash. DC). 208:177-179.) with the microinjection of fluorescently labeled vinculin and alpha-actinin, we have been able to correlate the distribution of costameres in adult rat cardiac myocytes with the pattern of forces these cells exert on the flexible substratum. In addition, we used interference reflection microscopy to identify areas of the cells which are in close contact to the underlying substratum. Our results indicate that, in older cell cultures, costameres can transmit forces to the extracellular environment. We base this conclusion on the following observations: (a) adult rat heart cells, cultured on the silicone rubber substratum for 8 or more days, produce pleat-like wrinkles during contraction, which diminish or disappear during relaxation; (b) the pleat-like wrinkles form between adjacent alpha-actinin-positive Z-lines; (c) the presence of pleat-like wrinkles is always associated with a periodic, "costameric" distribution of vinculin in the areas where the pleats form; and (d) a banded or periodic pattern of dark gray or close contacts (as determined by interference reflection microscopy) has been observed in many cells which have been in culture for eight or more days, and these close contacts contain vinculin. A surprising finding is that vinculin can be found in a costameric pattern in cells which are contracting, but not producing pleat-like wrinkles in the substratum. This suggests that additional proteins or posttranslational modifications of known costamere proteins are necessary to form a continuous linkage between the myofibrils and the extracellular matrix. These results confirm the hypothesis that costameres mechanically link the myofibrils to the extracellular matrix. We put forth the hypothesis that costameres are composite structures, made up of many protein components; some of these components function primarily to anchor myofibrils to the sarcolemma, while others form transmembrane linkages to the extracellular matrix.


The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain. The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions.


The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins. 60% of the peripheral membrane skeleton of Euglena gracilis consists of equimolar amounts of two proteins (articulins) with M(r)s in SDS gels of 80 and 86 kD. To understand eventually how these proteins assemble and function in maintaining cell form and membrane integrity we have undertaken a molecular characterization of articulins. A lambda gt11 expression library constructed from Euglena gracilis mRNAs was screened with antibodies against both articulins. Two sets of cDNAs were recovered, and evidence from three independent assays confirmed that both sets encoded articulins: (a) Anti-articulin antibodies recognized a high molecular weight beta-galactosidase (beta-gal) fusion protein expressed in bacteria infected with lambda gt11 cDNA clones. (b) Antibodies generated against the bacterially expressed beta-gal fusion protein identified one or the other articulin in Western blots of Euglena proteins. These antibodies also localized to the membrane skeletal region in thin sections of Euglena. (c) Peptide maps of the beta-gal fusion protein were similar to peptide maps of Euglena articulins. From the nucleotide sequence of the two sets of cDNAs an open reading frame for each articulin was deduced. In addition to 37% amino acid identity and overall structural similarity, both articulins exhibited a long core domain consisting of over 30 12-amino acid repeats with the consensus VPVPV--V--. Homology plots comparing the same or different articulins revealed larger, less regular repeats in the core domain that coincided with predicted turns in extended beta-sheets. Outside the core domain a short hydrophobic region containing four seven-amino acid repeats (consensus: APVTYGA) was identified near the carboxy terminus of the 80-kD articulin, but near the amino terminus of the 86-kD articulin. No extensive sequence similarities were found between articulins and other protein sequences in various databanks. We conclude that the two articulins are related members of a new class of membrane cytoskeletal proteins.


Identification of an epithelial protein related to the desmosome and intermediate filament network. Using a mAb, referred to as 08L, we have identified a protein, of M(r) approximately 140,000, associated with desmosomes of epithelial cells. The 08L antibody stained the intracellular side of lateral cell margins of monolayer epithelial cells but did not stain cell margins free of cell contact. Immunoelectron microscopy revealed that the 08L antigen was localized to the cytosolic surface of the desmosomal plaque near points of intermediate filament convergence with apparently little staining of the desmosomal plaque proper. Western blots revealed the 08L antigen to be a protein, of M(r) approximately 140,000, found in the Triton-X 100 insoluble pellet. High salt-containing buffers extracted the 08L antigen from the insoluble material. Examination of the assembly of 08L to the desmosome complex, in cells grown in low confluent culture or in calcium-switch assays, by double immunofluorescence with 08L and anti-desmoplakin antibody, revealed that 08L was recruited to morphologically identifiable desmosomes. 08L antigen may exist in a cytosolic pool prior to assembly to the cell surface. The solubility of 08L in low calcium and normal calcium conditions, however, was similar. 08L association to the desmosome was correlated with increased organization of the intermediate filament network. We suggest that the 08L antigen may be involved in the organization and stabilization of the desmosome-IF complexes of epithelia.


Serum response factor p67SRF is expressed and required during myogenic differentiation of both mouse C2 and rat L6 muscle cell lines. The 67-kD serum response factor (p67SRF) is a ubiquitous nuclear transcription factor that acts by direct binding to a consensus DNA sequence, the serum response element (SRE), present in the promoter region of numerous genes. Although p67SRF was initially implicated in the activation of mitogen-stimulated genes, the identification of a sequence similar to SRE, the CArG box motif, competent to interact with SRE binding factors in many muscle-specific genes, has led to speculation that, in addition to its function in cell proliferation, p67SRF may play a role in muscle differentiation. Indirect immunofluorescence using affinity-purified antibodies specifically directed against p67SRF reveals that this factor is constitutively expressed and localized in the nucleus of two skeletal muscle cell lines: rat L6 and mouse C2 myogenic cells during myogenic differentiation. This result was further confirmed through immunoblotting and Northern blot analysis. Furthermore, specific inhibition of p67SRF in vivo through microinjection of purified p67SRF antibodies prevented the myoblast-myotube transition and the expression of muscle-specific genes such as the protein troponin T. We further showed that anti-p67SRF injection also inhibited the expression of the myogenic factor myogenin, implying an early requirement for p67SRF in muscle differentiation. These results demonstrate that p67SRF is involved in the process of skeletal muscle differentiation. The potential action of p67SRF via CArG sequences is discussed.