Galactomannan antigenemia in invasive aspergillosis. Galactomannan (GM) extracted from mycelia of Aspergillus fumigatus with cold dilute alkali reacted with antiserum specific for an antigen that circulated in invasive aspergillosis in rabbits and humans. The GM was purified by its affinity for concanavalin A and was separated from a nonantigenic glucan by gel permeation on Sephacryl S-200. The GM molecular weight of between 25,000 to 75,000 was smaller than the antigen present in infected rabbit serum which was retained by an ultrafiltration membrane that had a nominal molecular weight limit of 125,000. The ratio of galactose to mannose present in GM was 1:1.17. The serological activity of GM was stable to boiling but labile to 0.01 N HCl, implicating galactofuranose as an antigenic determinant. Analysis of purified GM by methylation-gas chromatography suggested a structure consisting of a 1 leads to 6-linked mannan backbone with oligogalactoside side chains 3 units long, terminating in galactofuranose. The presence of mannose as a side chain component was also inferred. Another antigen of A. fumigatus, which did not bind to concanavalin A, was isolated after tandem chromatography on diethylaminoethyl- and carboxymethyl-Sephadex and was identified as a galactan. The galactan inhibited the immune precipitation of GM was specific antiserum.


Immunological behavior after mycobacterial infection in selected lines of mice with high or low antibody responses. Resistance and susceptibility to mycobacterial infection in the Biozzi high and low lines of mice which were genetically selected for their responses to heterologous erythrocytes have been found to be related to the innate ability of nonimmune macrophages to kill or inhibit the growth of the organisms during the first two weeks after infection and to their ability to mount specific and nonspecific immune responses. High antibody-producer mice were more capable of expressing cell-mediated immune parameters than low antibody-producer mice. A direct relationship was observed between the ability of bacteria (BCG vaccine) to multiply inside the reticuloendothelial system and the development of cell-mediated immunity, as measured by the delayed local reaction at the injection site, the lymphoproliferative response in the draining nodes, the tuberculin delayed-type hypersensitivity, the acquired resistance, and the adjuvant effect after BCG inoculation. In high line mice, apart from the inability of their macrophages to inhibit the early growth of bacteria, their lymphocytes in spleen and thymus were more capable of being stimulated in vitro by varying concentrations of living BCG. The data presented in this report are compatible with the hypothesis that a group of genes segregated in each line during the selective breeding controls the innate microbicidal activity.


Virulence of Escherichia coli in experimental hematogenous pyelonephritis in mice. Differences in nephropathogenicity between Escherichia coli strains were studied by following the kinetics of the viable count in the mouse kidney during 8 h after intravenous injection. Assuming as a reference point that at zero time 0.1% of the inoculum was lodged in the kidney, we found that strains fell into three main groups with different behavior patterns: in group I, the viable count fell and remained low; in group II, the viable count first fell and then rose after 4 h, reaching the level of the reference point within 8 h; in group III, the viable count rose rapidly and remained high. The kinetics of the viable counts were also studied in blood, spleen, and liver: group I and II strains behaved similarly; only in the kidney did group II strains show higher counts than group I strains. These data suggest that group II strains are specifically virulent for the mouse kidney. Group III strains also gave higher viable counts in blood and spleen but comparable counts in the liver, suggesting that group III strains are more generally virulent. Fifty percent lethal dose measurements confirmed the conclusion that group I strains are avirulent and group III strains are the most virulent. Possible relationships between behavior pattern and serotype are discussed.


[Experience with sisomicin in pediatrics (author's transl)]. Sisomicin, an aminoglycoside antibiotic, is especially effective against Escherichia coli, Klebsiella, Enterobacter, Citrobacter, Serratia, indole-positive and indole-negative Proteus species, Pseudomonas aeruginosa, Salmonella and Staphylococcus aureus. It has a bactericidal action. Although sisomicin is similar to the other aminoglycoside antibiotics, there is not complete cross-resistance to them. Our own pharmacokinetic investigations showed that a dose of 2--3 mg/kg body weight of sisomicin twice daily is necessary in the neonatal period. Infants should be given 2.5 mg/kg body weight three times daily, and school children 1.5--20 mg/kg body weight, likewise three times daily. Excretion of sisomicin in the urine is lower in children than in adults, amounting within 24 hours to only 10--20% in newborns, and 30--40% in school-children. Sisomicin induces excretion of some enzymes in higher quantities from the tubular part of the kidneys, especially alaninaminopeptidase. A report is given on 58 patients, especially newborns and prematures, who were treated for about seven days with sisomicin. The results obtained with a wide variety of infections (such as omphalitis, aspiration of amniotic fluid with broncho-pneumonia, phlegmons of the galea, and also pyelonephritis and mucoviscidosis with pulmonary complications) can be described as good, with a success rate of 85%. On only seven occasions were insignificant transitory side-effects, such as slight increase in transaminases, toxic-allergic exanthema and pain in the region in injection, observed.


Rheumatoid arthritis: review of searches for an infectious cause. Part I. No distinctive pattern has yet emerged from the accumulated mass of results that would provide a generally acceptable hypothesis of the etiology of rheumatoid arthritis. A number of immunologic aberrations have been described, but there has been no identification of a key immunologic defect that might link together the various components of the immune response into an agreed pattern. The possibility of a persistent antigenic stimulus arising from an infection cannot be confirmed or refuted. If a virus is involved, it would seem more likely to be a "slow" virus rather than a commonly recognized form, but there is no strong candidate of this type in view. Despite the fact that mycoplasmas are undoubtedly arthritogenic in other species, their role as an etiologic agent in rheumatoid arthritis has not been proven. The idea that bacterial cell wall peptidoglycan may provide a persistent stimulus has much to offer, but it is not possible at this stage to accept peptidoglycan as a recognized etiologic factor. This suggestion will, however, indoubtedly stimulate much further investigation.


Hemodynamic, mechanical and renal effects during "apneic oxygenation" with extracorporeal carbon dioxide removal, at different levels of intrapulmonary pressure in lambs. It has been shown that apneic oxygenation can be safely performed for many days when metabolic carbon dioxide is removed by an extracorporeal membrane lung, and 100% oxygen is supplied directly into the trachea to keep the intrapulmonary pressure at 5 cm. H2O. The study was designed to find "best intrapulmonary pressure (IP)", analogous to "best PEEP" during continuous positive pressure ventilation. In the present study we have shown that when IP was progressively raised from 5cm H2O to 20 cm H2O the PaO2 rose significantly due to progressive decrease in QVA/Q. A linear positive correlation was found between CO and QVA/Q. There was a negative correlation between QVA/Q and FRC. Total static lung compliance and FRC increased significantly at 15--20 cm H2O IP. There was no significant change in urinary flow, urea and creatinine clearances. We conclude that in apneic oxygenation a more "optimal IP" is attained at an IP pressure of 20 cm H2O than at 5 cm H2O.


Remote densitometric analysis of stenotic lesions. In conventional densitometric evaluation of stenoses the digitized angiogram is displayed on a multi-grey-level scope to facilitate operator interaction. With a remote terminal, linked to the computer with regular telephone lines, such display is unacceptably slow. To circumvent this difficulty, the projection of the original image is used for interactions through a sonic pen digitizer interfaced to the computer. A coordinate system can be defined on the projected image, in addition to the blood vessel segment to be analyzed by the operator. The computer can retrieve these features on the stored-pre-digitized image to carry out the quantitative evaluation of the stenosis. This method reduces the time of analysis and allows several institutions to share the same computing facility.


Computer image analysis of kidney histopathological sections. Human renal histological sections have been photographed on a black and white transparency and digitized. The digitizing camera, an image dissector, converts a 2 cm2 picture into 400 x 400 numbers each representing the grey level value of sampled point. The grey levels are represented in the computer by number ranging between 0-255. In this scale 'O' stands for black and 255, for white. The program provides 2 grey level thresholds, which outline a window through which the picture is scanned. Only structures, whose grey level value lies in the window range, are taken into consideration. The thresholds are set during digitization from the console switches. Image analysis is performed on-line in real time.


Indirect leukocyte migration assay in patients with malignant melanoma. Leukocytes from 72 melanoma patients and 75 control donors were examined in a two-stage (indirect) leukocyte migration assay, using formalin-fixed melanoma cells (FMC) and control, normal or non-melanoma tumour cells (FCC) as the source of antigen. Inhibitory supernatants were produced significantly more often by cultures of melanoma leukocytes and FMC than by melanoma leukocytes and FCC or control leukocytes and FMC or FCC. Reactive leukocytes were most frequently derived from stage II patients (65%), followed by stage I patients with tumour present (58%), stage III patients (29%) and stage I patients without detectable tumour (20%). The likelihood that a melanoma patient's leukocytes would react increased progressively with the number of different FMC preparations tested. A similar, though less steep increase in reactivity was seen with control donor leukocytes and different FMC preparations. No significant increase in reaction frequency was seen with melanoma or control leukocytes exposed to increasing numbers of different FCC preparations. Significant discrimination between melanoma patients' and control donors' leukocytes was achieved with FMC from both primary and metastatic tumours. Concordance of positivity or negativity was seen in 60% of concurrent one-stage and two-stage leukocyte migration assays.


Personality characteristics of heroin addicts: a review of the empirical literature with critique--part II. This paper is the second part of a two-part series reviewing the empirical literature on personality characteristics of heroin addicts. It focuses on MMPI-based studies, personality differences between addict subgroups, and changes during the addiction cycle and following treatment. Several personality characteristics of heroin addicts have been consistently validated experimentally, but it is impossible to determine whether these traits comprise the "addictive personality" and predated drug use, or whether they are the result of drug addiction. Most research in this area has proceeded without the benefit of a theoretical premise on which to generate hypotheses and build models of heroin addiction. It is recommended that more study-based research occur that tests postulates.


Free radical reactions with proteins and enzymes: the inactivation of pepsin. The reactions of free radicals produced by ionizing radiation with pepsin have been studied by steady-state inactivation measurements and by pulse radiolysis. In de-aerated solutions thehydroxyl radical has been found to be the most efficient of the primary free radicals generated from water in causing inactivation. The reactions of the more selective oxidizing inorganic radical anions Br2-. and (SCN)2-., with pepsin have also beenexamined. In the case of the thiocyanate radical anion (SCN)2-., the inactivation efficiency is found to depend on SCN- concentration, an effect shown to arise from a reversible redox reaction involving the tryptophan and (SCN)2-. radicals. The results demonstrate that tryptophan residue plays an essential role in the enzyme activity of pepsin.


Influence of unsaturated fatty acids, membrane fluidity and oxygenation on the survival of an E. coli fatty acid auxotroph following gamma-irradiation. Escherichia coli K1060, a fatty acid auxotroph unable to either synthesize or degrade unsaturated fatty acids (uFAs), was used to study the effect of membrane fluidity on survival after exposure to ionizing radiation. Using this strain of E. coli, significant alterations in the fatty acid composition of the membrane have been produced and verified by gas chromatography. Linolenic, oleic, elaidic and palmitelaidic acids were the uFAs used. Survival above the transition temperature (Tt) (liquid crystal in equilibrium gel) was comparable for these fatty-acid-supplemented membranes after exposure to gamma-irradiation, whereas gamma-irradiation below Tt resulted ina significant decrease in survival. An oxygen enhancement effect was observed for each experimental condition employed.


Human trabecular cells. I. Establishment in tissue culture and growth characteristics. After a careful dissection of trabecular tissue from recent postmortem specimens, human trabecular cells were established in tissue culture with 250 ng/ml fibroblast growth factor (FGF), Dulbecco's modified Eagle's medium (DME), and 10% human serum. These conditions have allowed propagation of human trabecular cells for a number of passages at high density without apparent cellular degeneration. FGF increased the rate of cell division and the plating efficiency for trabecular cells but was not needed after cells had achieved confluency. Human trabecular cells had a pattern of growth which differed from human corneal keratocytes and human corneal endothelial cells compared at a similar passage. Propagation of human trabecular cells in vitro may provide a valuable source of experimental material to study the functional aspects of these cells which line the trabecular meshwork.


Scanning electron microscopy and the surface morphology of human leukocytes: current status. This illustrated review summarizes the current status of knowledge relating to the surface morphology of leukocytes as seen by scanning electron microscopy (SEM). Problems of SEM interpretation and controversial issues are discussed. In addition, this report briefly reviews the factors that are known to influence the surface architecture of these cells and summarizes SEM findings in the leukemias.


Immunocytochemical studies of human myeloma cells by light and electron microscopy. Demonstration of atypical plasma cells in bone marrow and extramedullary sites is the conventional cytological criterion for the diagnosis of human myeloma and assessment of its development. By immunocytochemical methods, it is possible to demonstrate in the blood of myeloma patients the occurrence of mononuclear cells, which manufacture and express idiotypic determinants (Id+) of the patient's own myeloma protein. These circulating Id+ cells have a polymorphic ultrastructure ranging from that of atypical lymphocytes to lymphoblastic-lymphoplasmacytoid and plasmacytoid cells. Our observations in several patients indicate that monitoring the blood levels of Id+ and Ig+ cells is a sensitive probe for following the development of the disease.


Immunoelectron microscopy and immunocytochemistry in pathology, with special reference to immunoglobulin-producing cells. The advantage of immunoelectron microscopy (immuno-EM) is that it allows the simultaneous detection of surface and internal cell components. The immunoperoxidase method is often more suitable than immunoferritin. There are no major difficulties in staining surface antigens by immuno-EM, provided sufficient amounts of pure antibodies are available for coupling to peroxidase. Prior fixation of cells, with faxatives that preserve the antigenicity of surface components, avoids ligand-induced alterations of the surface components. It is believed that, unlike the surface, intracellular antigens are difficult to stain by immuno-EM because of the poor penetration of conjugates into fixed cells; thus, various technical approaches have been proposed by workers involved in tissue immuno-EM. In fact, the method that we initially devised for the surface staining of fixed cell suspensions has proved to detect specifically intracellular immunoglobulins in B cells obtained from patients with proliferative diseases. Thus, conjugates do penetrate into fixed cells, although by an unknown mechanism. On this basis, it is possible to study both surface and intracellular immunoglobulins at the EM level and to determine the precise localization synthesis.


The present status of imaging of the gallbladder. Oral cholecystography is still the basic modality for the diagnosis of gallbladder disease. Now, for the first time since the introduction of this procedure in 1924, this status is being challenged by another diagnostic technique, ultrasonography of the gall bladder. Ultrasound studies have major advantages over conventional cholecystography. These are sufficiently important that it can be safely predicted that ultrasonography will eventually become the primary diagnostic tool for evaluating the gallbladder. Just as the use of colonoscopy has provoked a new analysis of the barium enema examination in the evaluation of the colon, the introduction of ultrasound examination of the gallbladder has revealed the fact that false-negative oral cholecystograms are not rare. Before ultrasonography this information was unobtainable, because of the surgeon's proper reluctance to perform a cholecystectomy in patients with normal oral cholecystograms. Indeed, the availability of an accurate alternate diagnostic procedure has stimulated a reevaluation of other difficulties associated with oral cholecystography. These include problems involved with non-visualization and impaired visualization of the gallbladder after a single dose of contrast material, misunderstandings concerning the uses of oral cholecystography compared to those for intravenous cholangiography and uncertainty regarding the value of cholecystokinin cholecystography in the diagnosis of acalculous cholecystitis. Knowledge of the pharmacokinetics of the oral cholecystographic contrast agents has been long overdue, considering the extensive use of these compounds in clinical practice for more than 50 years. However, information on this subject has finally reached a stage where the data can be applied to ensure that cholecystography is done with maximun efficiency and safety and with the least radiation exposure. The purpose of this report is to review the problems involved with imaging of the gallbladder by oral cholecystography and to discuss the indications, interpretation and accuracy of gallbladder imaging with ultrasonography. Data concerning the use of computerized tomography for evaluation of the gallbladder are still preliminary but are discussed.


The utilization of xeroradiography for radiography of cross-section of thin cadaveric slices. Although xeroradiography has had many medical applications, its utilization and the advantages it offers in the study of cadaveric sections has not previously been fully explored. It was our purpose to determine possible advantages or disadvantages this modality might have for this purpose. The edge enhancement which xeroradiography allows is a distinct advantage since the various anatomic structures become more clearly defined. On the other hand, the hollow viscera and the lumina of blood vessels are not enhanced, but rather blend with the walls of these "hollow" structures. Also, there is some loss of detail in the depiction of some of the glandular structures, such as the seminal vesicles. An artist's assistance to define these by comparison with the gross specimen gives us an optimum result. An x-ray enhanced image is thereby attained which exceeds the usual line drawing or artist's depiction of the cross-sectional specimen, and both can be similarly labeled for study.


[Cosmus Conrad Cuno (1652-1745) on a human ectoparasite: the head louse]. Cosmus Conrad Cuno, a less well known optician and inventor of microscopes from the second half of the 17th century, published in 1734 at Augsburg his Observationes durch dessen verfertigte Microscopia where along with various observations he communicated salient details pertaining to the biology of the head louse.


Consecutive demonstration of catecholamines and dopamine-beta-hydroxylase within the same specimen. A combined method was developed for characterization and differentiation of catecholamines in neuron populations containing more than one catecholamine, e.g. dopamine and norepinephrine. Its application to small intensely fluorescent (SIF-) cell clusters in sympathetic ganglia allows the successive demonstration of glyoxylic acid-induced catecholamine fluorescence and dopamine-beta-hydroxylase by indirect immunofluorescence within the same tissue section. Applying this technique to an example, two types of SIF-cells were demonstrated in the guinea pig superior cervical ganglion.


Gut-type glucagon immunoreactivity in nerves of the rat brain. Nerve fibers reacting with antisera demonstrating gut-type glucagon were numerous in certain areas of hypothalamus and thalamus but absent from neocortex and hippocampus. They did not react with glucagon antisera specific for pancreatic type glucagon. Immunoreactive cell bodies were not observed.


Calcium, zinc and other elements in islet and exocrine tissue of the rat pancreas as measured by histochemical methods and electron-probe micro-analysis, Effects of fasting and tolbutamide. Fasting for 24 or 72 h causes a strong decrease of pancreatic islet calcium content as detected by glyoxal-bis-(2-hydroxyanil), (GBHA). There is strong evidence that GBHA only detects ionized calcium and not total calcium (Wolters et al., 1979). Fasting does not influence the zinc content as detected by dithizone (DZN), and aldehyde-fuchsin (AF) staining intensity is only slightly decreased. After degranulation of islets by tolbutamide (which reduced the insulin content of the pancreas to 10% of the control value) the staining intensities of GBHA, DZN and AF were strongly depressed. Calcium (as well as other elements) were also measured by electron-probe micro-analysis (EPMA). It appeared that 24 or 72 h of fasting did neither affect the total content of Ca nor of Na, P, S, and K of the islets significantly. In exocrine tissue the Ca content increased gradually as a result of fasting. Thus, after 72 h of fasting the Ca content was 25% higher than in fed controls. On the other hand after 72 h of fasting the K content appeared to be decreased. EPMA revealed that after degranulation of islets the Ca content decreased markedly (35%). S appeared to be decreased by only 14%, whereas the content of the other elements was not changed. The results show that GBHA-detectable Ca is only a part of EPMA-detectable Ca. The GBHA-Ca "pool" which contains ionized Ca, is subjected to changes when the animals are fasted, the total Ca content as measured by EPMA does not change. Thus, at least two distinguishable pools of Ca exist within the islets (GBHA-detectable and not-GBHA-detectable). It is suggested that as a result of fasting Ca passes from one pool to another.


Immunocytochemical localization of renin in mouse kidney. The distribution of renin in mouse kidney was examined in immunohistochemical studies by using an antiserum against pure mouse submaxillary renin and the peroxidase-antiperoxidase (PAP) technique. At antibody dilutions from 1:10(4) to 1:10(6), renin was found in high concentrations in the epitheloid cells of the vasa afferentia and, in lower concentrations, in the wall of some of the vasa efferentia. Renin was also detected in most of the interlobular arteries. Mesangial cells and Goormaghtigh cells were always free of specific staining. At high antiserum concentrations (i.e., dilutions from 1:10(2) to 1:10(4)) specific reaction product was also observed in the apical part of proximal tubule cells. This staining may represent filtered and pinocytozed renin.


Comparative investigation of the mixed aggregation immunocytochemical technique and the indirect peroxidase technique for the detection of prostate specific acid phosphatase in paraffin or paraplast sections. The results obtained with the indirect peroxidase technique for the identification of prostate specific acid phosphatase in formalin fixed, paraffin or paraplast embedded autopsy material are compared with the results obtained with the mixed aggregation immuno-cytochemical technique. When using a monospecific antiserum the former technique is prefered. However, when a monospecific antiserum is not available, one has to balance the advantages of the mixed aggregation immuno-cytochemical technique against the disadvantages of having to prepare a monospecific antiserum, necessary for the indirect peroxidase technique. Both methods appeared positive in 20 prostatic carcinomas and in 36 metastases of prostatic carcinomas. In the epithelium of the seminal vesicles and in osteoclasts no acid phosphatase could be detected with the antiserum. A comparison of both techniques, as well as different types of preincubation to diminish nonspecific background staining are discussed.


Effect of fixation on the antigenicity of human lactoferrin in paraffin-embedded tissues and cytocentrifuged cell smears. Immunoperoxidase techniques were used to study the preservation of the antigenicity of human lactoferrin (LF) of polymorphonuclear neutrophils (PMN) and various exocrine glandular cells in paraffin-embedded tissue blocks and cytocentrifuged cell smears. Tissues fixed in Carnoy's fluid in contrast to other fixatives used, showed good preservation of LF antigenicity irrespective of the fixation time. Cell smears fixed in Carnoy's fluid showed diffuse nuclear and cytoplasmic staining for LF, although morphologic integrity was poorly preserved. Granular cytoplasmic staining for LF with no staining of nuclei was seen in cell smears fixed in buffered formol acetone for 2--10 min. The nature of nuclear LF staining and future applications of the present methods are discussed.


[Hearing results following tympanoplasty (author's transl)]. Tympanoplasty can produce a stable hearing improvement in many patients with chronically inflamed ears. In our series of patients, severe damage to the inner ear following surgery occurred in 3% patients and was associated with a hearing loss of greater than 15 dB. Complete hearing loss occurred in 0.2% of the patients reviewed. In patients with an intact ossicular chain, simple myringoplasty is associated with the best results. Loss of the stapedial arches affects the postoperative air-bone gap most significantly, while loss of the manubrium mallei or the creation of a radical mastoid cavity are additional negative factors. Extent or localization of ossicular fixation has no direct influence on hearing results when appropriate ossicular reconstruction is performed.


[Echinococcus cysticus infections in the head and neck (author's transl)]. An unusual case of Echinococcus cysticus infection of the neck, face and skull is reported. The disease was misdiagnosed for several years as tuberculosis of the lymph nodes and consequently incorrectly treated. Definitive therapy required radical surgery.


The McCannel suture: a bimanual technique. The bimanual technique utilizing the iris manipulator and a full air bubble allows excellent control of anterior chamber depth and IOL position. It also minimizes the risk of trauma to the underlying lens, posterior capsule or vitreous during placement of iris sutures; and its use may prevent the tragedy of severe endothelial damage during insertion of the McCannel suture.


Development of culture vessel for sterility testing of bandages and other medical devices. A cylindrical glass culture vessel equipped with a vented nylon closure was developed and evaluated for sterility testing. The sterility testing cylinder (STC), made in 2 sizes, accommodates 250 or 400 mL culture broth. A tight-fitting, skirted nylon cap protects the upper part of the cylinder, and a membrane filter in a recessed opening allows venting during autoclaving and ensures sterility during removal and replacement of the cap. The configuration of the vessel in terms of the opening size and ratio of the horizontal to vertical cross section provides depths desirable for testing many shapes of medical devices, including elongated and narrow ones, without excessive waste of medium. They are easy to charge with medium, and to autoclave, store, inoculate, and observe for growth.


Small arrays of electron-dense cylinders in Escherichia coli cells. Electron microscopy of unstained Escherichia coli cells from cultures kept near 0 degrees C after incubation at 37 degrees C revealed small areas of geometrically arranged electron-dense cylinders. Their morphology, organization, and occurrence are described.


New tool for studying interactions of components of ribonucleic acid polymerase: rifampin-dependent mutants. Mutants of Escherichia coli showing a rifampin-dependent phenotype were isolated from cells of strain CP78 mutagenized with ethyl methane sulfonate or nitrosoguanidine when an antibiotic underlay technique was used. The mutants varied greatly in their rifampin requirement. The minimum necessary concentration ranged from 1 to 50 micrograms/ml. The mutants could be divided into four phenotypic classes. These dependent mutants and their revertants should be a useful tool for probing interactions between the component polypeptides of ribonucleic acid polymerase and for studying the linkage of transcription with other cellular processes.


Effect of transcription on RecBC- and RecF-mediated recombination within the tryptophan operon of Escherichia coli K-12. Recombination between tryptophan gene mutations within the trp operon was determined among transductants for an outside linked cysB marker under conditions of repression and derepression. These studies, carried out with recipient strains utilizing the RecBC or RecF pathway, or a combination of these pathways of recombination, demonstrate that transcription of trp genes as regulated by the trp repressor has no significant effect on RecBC- or RecF-mediated recombination within the trp operon.


his-Linked hydrogen sulfide locus of Salmonella typhimurium and its expression in Escherichia coli. A his-linked H2S locus of Salmonella typhimurium has been further defined by direct isolation of H2S mutants. Expression of this locus in Escherichia coli has been demonstrated.


Interaction between mutant alleles of araC of the Escherichia coli B/r L-arabinose operon. Strains were constructed that contain mutational alterations affecting two distinct functional domains within the araC gene protein. The araCi (catabolite repression insensitivity) and araCh (catabolite repression hypersensitivity) mutations were used to alter the catabolite repression sensitivity domain, and mutation to D-fucose resistance was used to alter the inducer binding domain. araCh, D-fucose-resistant double mutants never exhibited constitutive ara operon expression, whereas all of the araCi, D-fucose-resistant double mutants did exhibit constitutivity. When L-arabinose was used as an inducer, most of the double mutants exhibited the sensitivity to catabolite repression associated with the araCi or araCh mutation. However, when D-fucose was used as an inducer, changes in sensitivity to catabolite repression were observed that were attributed to interactions between the two protein domains. The roles of catabolite activator protein and araC gene protein in the induction of the araBAD operon were discussed.


Growth-rate-dependent alteration of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase levels in Escherichia coli K-12. The levels of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase are subject to metabolic regulation; they increased three- to fivefold with increasing growth rate.


The ends of Tn10 are not IS3.

The ends of Tn10 are not IS3. By heteroduplex and hybridization analysis we showed that the inverted repetition (here called IS10) at the ends of the translocatable tetracycline resistance element Tn10 is not IS3, as had previously been reported by Ptashne and Cohen (J. Bacteriol. 122:776--781, 1975). Further analysis confirmed the homology between IS3 and the alpha beta sequence of F and demonstrated that IS10 was not present in the genomes of Salmonella typhimurium LT2 or Escherichia coli K-12.


Two Escherichia coli chromosomal cistrons, sfrA and sfrB, which are needed for expression of F factor tra functions. Twelve mutants of Escherichia coli K-12 have been isolated which carry chromosomal mutations that exhibit pleiotropic effects on the expression of F factor tra cistrons. F pilus synthesis, deoxyribonucleic acid transfer, and surface exclusion are all inhibited. Six of the mutants carry sfrA mutations, and six carry sfrB mutations. sfrA and sfrB are cistrons mapping near thr and metE, respectively. Several F-like plasmids are dependent on sfrA and on sfrB for expression of tra cistrons. Plasmids of incompatibility groups C and S are only dependent on sfrB,and other conjugative plasmids are dependent on neither. sfrB mutations also result in changes in certain cell envelope properties, including change sensitivity to certain bacteriophages which use lipopolysaccharide as a receptor, synthesis of nonfunctional flagella, and altered sensitivity to antibiotics.


Escherichia coli murein-DD-endopeptidase insensitive to beta-lactam antibiotics. A novel endopeptidase degrading the peptide cross-links in sacculi has been isolated from Escherichia coli and purified to homogeneity. The enzyme has a molecular weight of 30,000 and, in contrast to already known enzymes of similar specificity, remains fully active in the presence of beta-lactam antibiotics. In addition, it is exceptional in being inhibited by single-stranded deoxyribonucleic acid and by some polynucleotides. The possible role of the enzyme in cell division is discussed.


Isolation and characterization of Escherichia coli K-12 mutants unable to induce the adaptive response to simple alkylating agents. When Esherichia coli cells are exposed to a low level of simple alkylating agents, they induce the adaptive response which renders them more resistant to the killing and the mutagenic effects of the same or other alkylating agents. This paper describes the isolation of one strain that was deficient in mutagenic adaptation and five that were deficient in both mutagenic and killing adaptation, confirming previous suggestions that killing and mutagenic adaptation are, at least to some extent, separable. These six strains have been called Ada mutants. They were more sensitive to the killing and mutagenic effects of N-methy-N'-nitro-N-nitrosoguanidine (MNNG) than the unadapted Ada+ parent. Thus, the adaptation pathway is responsible for circumventing some alkylation-induced damage even in cells that are preinduced. The increase in mutation frequency seen in Ada cells treated with MNNG was the same whether the cells were lexA+ or lexA, showing that the extra mutations found in Ada- strains do not depend upon the SOS pathway. Ada strains accumulated more O6-methyl guanine lesions than the Ada+ parent on prolonged exposure to MNNG, and this supports the idea that O6-methyl guanine is the most important lesion for MNNG-induced mutagenesis. The ada mutations have been shown to map in the 47 to 53-min region of the E. coli chromosome.


Incompatibility group P-1 bla+ plasmids do not increase penicillin resistance of Pseudomonas acidovorans. Incompatibility group P-1 plasmids with the bla+ genotype were transferred from various Escherichia coli strains to Pseudomonas acidovorans strain 29. When resistance to ampicillin was used as the criterion, none of these plasmids appeared able to express their Bla+ phenotype in this host. When the plasmids were subsequently transferred back from these ampicillin-sensitive P. acdiovorans transcipients to E. coli strains, it was found that the Bla+ phenotype was again expressed. Although beta-lactamase was not detected in cultures of P. acidovorans transcipients, macroiodometric determinations of beta-lactamase activity made on broken cell suspensions revealed that beta-lactamase was indeed synthesized. It was concluded that P. acidovorans strain 29 allows expression of the bla gene within the cell but that this organism is unable to excrete the enzyme.


Properties of Escherichia coli mutants altered in calcium/proton antiport activity. Mutants sensitive to growth inhibition by CaCl2 were found to have alterations in calcium uptake in everted membrane vesicles. These mutations map at different loci on the Escherichia coli chromosomes. A mutation at the calA locus results in vesicles which have two- to threefold higher levels of uptake activity than vesicles from wild-type cells. The calA mutation is phenotypically expressed as increased sensitivity to CaCl2 in a strain also harboring a mutation in the corA locus, which is involved in Mg2+ transport. The calA locus maps very close to purA and cycA at about min 97. The calB mutation results both in sensitivity to CaCl2 at pH 5.6 and in vesicles with diminished calcium transport capability. The CalB phenotype is also expressed only in a corA genetic background; the calB locus appears to map very near, yet separately from, the calA locus. When the cor+ allele is present, calA and calB mutations still result in a defect in calcium transport in vesicles. In addition, both calC and calD mutations result in vesicles with impaired calcium transport activity. calC is cotransducible with kdp and nagA, whereas calD is cotransducible with proC.


Protein K: a new major outer membrane protein found in encapsulated Escherichia coli. The protein composition of purified outer membranes of 47 Escherichia coli strains was examined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. Of 33 encapsulated strains, all contained an outer membrane protein distinguishable from previously reported proteins. The 14 non-encapsulated strains with one exception lacked this protein. Because of its apparent association with encapsulation (K antigen) we have named it K protein. The protein was purified nearly to homogeneity by chromatography in the presence of detergents, and its composition was determined. Its amino acid composition does not differ significantly from that reported for protein I, another E. coli major outer membrane protein. Furthermore, the N-terminal amino acid sequence of protein K indicates that it is related to protein I.


Ribosomal ribonucleic acid isolated from Salmonella typhimurium: absence of the intact 23S species. Ribonucleic acid (RNA) isolated by four distinct methods and from a variety of Salmonella typhimurium strains lacked intact 23S ribosomal RNA (rRNA). On sucrose gradients which minimize aggregation, the vast majority of S. typhimurium rRNA sedimented as a 16S peak with a 14S shoulder. RNA from this region of the gradient was resolved into three discrete bands by electrophoresis in formamide. Two very minor S. typhimurium RNA peaks were resolved at 21S and 10S on sucrose gradients, and each peak formed discrete bands in electrophoresis. It is concluded that if S. typhimurium does possess an intact 23S rRNA species, this species is extremely "labile." The absence of isolatable S. typhimurium 23S rRNA possibly reflected in vivo processing of the rRNA before isolation. Under certain conditions, S. typhimurium rRNA formed discrete aggregates which sedimented similarly to intact Escherichia coli 23S rRNA.


Cistrons encoding Escherichia coli heat-labile toxin. The structure and products of the two cistrons encoding the Escherichia coli heat-labile toxin (LT) were studied. The LT deoxyribonucleic acid (DNA) region had been isolated as part of a DNA fragment from the plasmid P307, and this fragment was joined to the cloning vector pBR313. Deletion mutations of various lengths were introduced into the LT DNA region and into the adjacent DNA sequences. Analysis of the deletions indicated that the maximum size of the LT DNA region was 1.2 x 10(6) daltons. Two proteins of 11,500 daltons and 25,500 daltons had been shown to be encoded by the LT DNA region. The functions of these LT gene products were investigated. The 11,500-dalton protein had an adsorption activity for Y-1 adrenal cells, and this protein was shown to form aggregates of four or five monomers. The 25,500-dalton protein was shown to have an adenylate cyclase-activating activity. The two cistrons encoding for each of the LT proteins have been located on a genetic map of the LT DNA region. Both cistrons are probably transcribed from the same promoter.


Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants. Dominant mutations at two newly identified loci, designated SRS1 and SRS2, that metabolically suppress the trimethoprim sensitivity of rad6 and rad18 strains, have been isolated from trimethoprim-resistant mutants arising spontaneously in rad6-1 rad18-2 strains of the yeast Saccharomyces cerevisiae. The SRS2 mutations also efficiently suppress the ultraviolet light sensitivity of the parent strains. They do not, however, suppress their sensitivity to ionizing radiation or their deficiency with respect to induced mutagenesis and sporulation. Such observations support the hypothesis that RAD6-dependent activities can be separated into two functionally distinct groups: a group of error-free repair activities that are responsible for a large amount of the radiation resistance of wild-type strains and also for their resistance to trimethoprim, and a group of error-prone activities that are responsible for induced mutagenesis and are also important in sporulation, but which account at best for only a very small amount of wild-type recovery.


Two mutations which affect the barrier function of the Escherichia coli K-12 outer membrane. Two genetically distinct classes of novobiocin-supersensitive mutants were isolated from Escherichia coli K-12. One class, given the phenotypic name NbsA, lies at 10 min on the E. coli chromosome. The order of the genes in this region, based on transductional analyses, is proC NbsA plsA purE. The second, NbsB, lies at 80 min. The order of the genes in this region, based on transduction analyses, is xyl cysE NbsB pyrE. Both classes of mutants show increased sensitivity to hydrophobic drugs but are different: NbsA cells tend to be more sensitive to cationic agents, whereas NbsB cells show the opposite tendency. The sole detectable biochemical alteration in NbsA strain is greater than 90% reduction in the phosphate content of the lipid A region of the lipopolysaccharide. The NbsB mutation results in lipopolysaccharide that contains primarily the stereoisomer D-glycero-D-mannoheptose, rather than L-glycero-D-mannoheptose, and which contains very little of the distal sugars. Since NbsA strains have apparently normal outer membrane proteins and total cellular phospholipids, changes solely in lipopolysaccharide can increase permeability to certain hydrophobic antibiotics. Complementation studies indicate that the NbsA marker is probably allelic with acrA. In addition, the NbsB marker is genetically and phenotypically similar to the rfaD locus of Salmonella typhimurium. For this reason, the phenotypic designations NbsA and NbsB have been changed to the genotypic designations acrA and rfaD, respectively.


Oxalurate transport in Saccharomyces cerevisiae. Oxalurate, the gratuitous inducer of the allantoin degradative enzymes, was taken into the cell by an energy-dependent active transport system with an apparent Km of 1.2 mM. Efflux of previously accumulated oxalurate was rapid, with a half-life of about 2 min. The oxalurate uptake system appears to be both constitutively produced and insensitive to nitrogen catabolite repression. The latter observations suggest that failure of oxalurate to bring about induction of allophanate hydrolase in cultures growing under repressive conditions does not result from inducer exclusion, but rather from repression of dur1,2 gene expression.


Specificity in formation of type II F' plasmids. Eight new F' plasmids derived from Hfr strains in which F is integrated at the chromosomal element alpha 3 beta 3 have been isolated and subjected to restriction enzyme, hybridization, and electron microscope heteroduplex analysis. Plasmids carrying extensive amounts of bacterial deoxyribonucleic acid were produced even though they were obtained by selection for transfer of lac, which is closely linked to F in the parental Hfr strains. Seven plasmids were type II Flac+ proC+ purE+ plasmids, and one was a type I Flac+ proC+ plasmid. Five of the Flac+ proC+ purE+ plasmids contain approximately 284 kilobases of bacterial deoxyribonucleic acid, which is identical for all five within the resolution of the restriction enzyme analysis. Theses results indicate that type II F' plasmids are the predominant tra+ F' type from this region of the Escherichia coli K-12 chromosome and that the recombination events leading to formation of these plasmids exhibit site specificity.


Mutation spoT of Escherichia coli increases expression of the histidine operon deleted for the attenuator. F'-episomes carrying the Salmonella typhimurium wild-type or attenuator-deleted histidine (his) operons were introduced into Escherichia coli strains containing relA or spoT single and double mutations known to affect guanosine 3'-diphosphate 5'-diphosphate (ppGpp) and guanosine 3'-triphosphate 5'-diphosphate (pppGpp) levels. Expression of the his operon and expression of the gene for 6-phosphogluconate dehydrogenase (gnd) were measured during balanced growth in amino acid-rich and minimal media. The data were consistent with the interpretation that ppGpp is a positive effector of his operon expression, whereas pppGpp is not an essential effector. The conclusion that his operon expression is maximally stimulated at a lower than maximum intracellular ppGpp concentration was further confirmed. Neither ppGpp nor pppGpp appeared to influence gnd gene expression. The metabolic regulation of the E. coli his operon was found to be similar to the ppGpp-meidated metabolic regulation of the S. typhimurium his operon.


Trazodone, a new antidepressant: efficacy and safety in endogenous depression. The effectiveness of trazodone was assessed over a 4-week period under double-blind conditions. Twenty-eight inpatients with a diagnosis of endogenous depression received either trazodone, imipramine, or placebo. Trazodone was significantly better than placebo and frequently better than imipramine according to analyses of the results of the Hamilton Psychiatric Scale for Depression, severity of illness and clinical global improvement ratings, and the Global Ward Behavior Scale. Significant improvement was evident in the trazodone group by the end of the first week of therapy, particularly in those symptoms associated with depression and accompanying anxiety. There were fewer side effects with trazodone than with imipramine.


Phosphoenolpyruvate carboxylase of Escherichia coli. Effect of proteolytic modification on the catalytic and regulatory propties. Phosphoenolpyruvate carboxylase from Escherichia coli W was treated with ten proteases, and the effects of the treatments on the enzyme activity and sensitivity to effectors were investigated. Proteases such as trypsin, alpha-chymotrypsin, papain, and subtilisin inactivated the enzyme, whereas elastase, carboxypeptidase Y and leucine aminopeptidase had no effect on the enzyme activity. Elastase and carboxypeptidase Y, however, inactivated the enzyme in the presence of 1 m urea. Subtilisin and alpha-chymotrypsin caused not only inactivation of the enzyme but also a significant desensitization to the effectors. DL-Phospholactate, a potent competitive inhibitor, markedly protected the enzyme from inactivation by subtilisin but did not protect it from desensitization to the effectors. Acetyl-CoA, fructose 1, 6-bisphosphate, and GTP-the allosteric activators--protected the enzyme from subtilisin inactivation, while laurate, the other allosteric activator, accelerated the inactivation. These activators did not protect the enzyme from desensitization to themselves. In contrast, modification with subtilisin in the present of l-aspartate, the allosteric inhibitor, caused an apparent transient activation of the enzyme. The enzyme modified in the presence of L-aspartate retained its sensitivity to L-aspartate, but the sensitivities to the other effectors were reduced to about one-half their initial values. Based on these results, a possible mode of desensitization of the enzyme by subtilisin modification and the possible existence of a multiplicity of conformational states of the enzyme, induced upon binding with the various effectors, are discussed.


Purification and properties of glyoxylate reductase I from baker's yeast. The purification and properties of NADPH-linked glyoxylate reductase [EC 1. 1. 1. 79] from baker's yeast were studied. Two active fractions (peak I and peak II) were isolated by DEAE-cellulose column chromatography. The peak I fraction was purified to homogeneity by the criteria of disc gel electrophoresis and tentatively designated glyoxylate reductase I. Its molecular weight was calculated to be 31,000 from gel filtration measurements. The enzyme reduced glyoxylate 7 times faster than hydroxypyruvate and was specific for NADPH. The enzyme showed optimum activity between pH 5.5 and 7.2. The Michaelis constants for glyoxylate and NADPH were found to be 13 mM and 4 microM, respectively. The enzymic activity was not significantly affected by anions, except for nitrate and iodide, which were inhibitory.


Distribution of phospholipid molecular species in outer and cytoplasmic membrane of Escherichia coli. The outer membrane of Escherichia coli K-12 contained a smaller proportion of phospholipid molecular species with two unsaturated fatty acyl chains than did the cytoplasmic membrane. Proportions of phospholipid molecular species in the outer and cytoplasmic membranes changed in response to temperature changes. As the temperature increased, the content of 1-palmitoyl-2-cis-9,10-methylenehexadecanoyl species increased. Translocation of phospholipids from the cytoplasmic membrane to the outer membrane and synthesis of various molecular species were observed.


Studies on bacterial chemotaxis. IV. Interaction of maltose receptor with a membrane-bound chemosensing component. Highly purified maltose receptor of Escherichia coli was bound to Sepharose 4B via a long spacer and affinity chromatography was performed to isolate the membrane-bound proteins having affinity for the maltose receptor. The experiments were carried out either in the presence of maltose or in the absence of maltose and the proteins absorbed on the mattix were identified by two-dimensional gel electrophoresis. The results showed that the maltose receptor interacted with the product of tar gene, one of the methyl-accepting chemotaxis proteins, only in the presence of maltose.


Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16 S ribosomal RNA of Escherichia coli. II. The effect of the absence of the methyl groups on initiation of protein biosynthesis. The effect of the presence or absence of methyl groups on the N6 atoms of two adjacent adenosines near the 3' end of 16 S rTNA of Escherichia coli on initiation of protein biosynthesis has been studied using wild type (methylated) and kasugamycin-resistant (unmethylated) E. coli ribosomes (see preceding paper (Poldermans, B., Goosen, N., and Van Knippenberg, P. H. (1979) J. Biol. Chem. 254, 9085--9089)). Conditions of pH, temperature, and ionic strength at which binding of fMet-tRNA to ribosomes proceeds maximally are the same for wild type and mutant ribosomes. Mg2+- and factor-dependent dissociation of ribosomes as well as the association of the subunits is also the same for methylated and unmethylated ribosomes. Binding of fMet-tRNA to wild type and to mutant 70 S ribosomes requires the same amount of the three initiation factors. However, optimal fMet-tRNA binding to unmethylated 30 S ribosomes needs more of initiation factor 3 than does binding to methylated 30 S ribosomes, provided that initiation factor 1 is absent. This difference is completely abolished when mutant 30 S ribosomes are methylated using purified methylase from the wild type strain and the methyl donor S-adenosylmethionine.


Kinetics of the utilization of medium and long chain fatty acids by mutant of Escherichia coli defective in the fadL gene. Experiments were performed to assess the role of the fadL gene in Escherichia coli. These studies have revealed that this organism requires a functional fadL gene in order to (i) transport optimally the fatty acids C10 to C18:1 into the cell, (ii) optimally grow on and oxidize C10 to C18:1 fatty acids, and (iii) incorporate efficiently C12 to C18:1 fatty acids into its membrane phospholipids. A defect in the fadL gene does not prevent E. coli from optimally utilizing fatty acids with chain lengths less than 10 carbon atoms. These results suggest that the fadL gene governs a transport component(s) which is required for the optimal transport of fatty acids with chain lengths greater than 9 carbon atoms.


Isolation of recombinant plasmids bearing cDNA to hen ovomucoid and lysozyme mRNAs. A large library of hen oviduct cDNA-pCR1 recombinant plasmids has been established in Escherichia coli X1776. From this library, ovomucoid cDNA and lysozyme cDNA-bearing plasmids have been identified. One of these plasmids, pMu7, yielded the sequence of the 3'-untranslated region of ovomucoid mRNA.


Bone ingrowth and stress shielding with a porous surface coated fracture fixation plate. The extent of bone remodelling under metal fixation plates attached to bone by (i) normal screw fixation and (ii) bone ingrowth into a porous metallic surface underlayer sintered to the metal plate was investigated. Extensive bone remodelling was observed under the integrally bonded porous surface layered plate. This remodelling could be attributed to the more extensive stress transfer from bone to metal plate because of the good bone-to-implant bonding. The importance of stress shielding with high stiffness implants is demonstrated.


Osteonecrosis after renal transplantation in children. Osteonecrosis occurred in nine of thirty-six children following renal transplantation. The distal femoral condyle was the most common location but the femoral head was the most symptomatic and required total hip replacement in three of five patients. Total steroid dosage did not correlate with the development of osteonecrosis. There were no cases of osteonecrosis in patients under the skeletal age of ten years or in patients who did not have rejection reactions. In three children non-progressive focal lesions developed, similar to those of osteochondritis dissecans.


Plate fixation versus conservative treatment of tibial shaft fractures. A randomized trial. One hundred consecutive patients with displaced fractures of the tibial shaft were randomly assigned to two groups, one treated with AO-plate fixation and the other with conservative methods. Strictly conservative treatment failed in two patients because of the interposition of muscles. Comparison of the groups showed that a longer duration of hospital stay was needed in the AO-plate group, and there were also more complications in that group. However, the median healing time was shorter and the anatomical end results were better than in the conservatively treated patients. Closed longitudinal fractures were found to be suitable and open fractures, unsuitable for AO-plate fixation. In the group that was treated conservatively most fractures that healed in malalignment were located in the distal third of the tibial shaft.


Inheritance and spondylolisthesis: a radiographic family survey. A radiographic suvey has been carried out of 147 first-degree relatives of forty-seven patients treated in Edinburgh for spondylolisthesis of the fifth lumbar vertebra; twelve patients had the dysplastic (congenital) type and thirty-five an isthmic defect. The survey identified 19 per cent of relatives with spondylolysis, and index patients with each type of spondylolisthesis had relatives with the opposite type. Index patients with the dysplastic form had a higher proportion of affected relatives (33 per cent) than had those with the isthmic type (15 per cent), but both figures were significantly in excess of the estimated frequency for the general population of under 1 per cent and 5 per cent respectively. Spina bifida occulta at the fifth lumbar or first sacral level or both, and lumbosacral segmental defects were commoner amongst all individuals with spondylolysis than amongst unaffected relatives (dysplastic form 94 per cent, isthmic type 32 per cent, unaffected relatives 7 per cent). However, there was no single instance of a neural tube defect (anencephaly, spina bifida with or without meningocele, other generalised vertebral anomalies or spinal dysraphism) amongst 826 first-, second- or third-degree relatives. It is concluded that the developmental defects of the vertebrae associated with spondylolysis are not aetiologically related to the neural tube defects. The one in three risk of spondylolysis to near relatives of patients with the dysplastic form of spondylolisthesis is emphasised in order that the deformity in their sibs and children can be recognised at any early age.


Binding of Clostridium botulinum neurotoxin to the presynaptic membrane in the central nervous system. Large synaptosome fractions were isolated from the cerebellar and cerebral cortices of rats and were incubated with Clostridium botulinum type A neurotoxin in vitro. The binding of the neurotoxin to the synapses was observed by electron microscopy, using the double-sandwich immunocytochemical method. Botulinum neurotoxin was preferentially bound to the presynaptic membrane in the large synaptosome fraction. The binding regions for the neurotoxin were localized on both the extrajunctional and junctional areas of the presynaptic membranes and appeared as patches of various sizes. However, they did not exist on the postsynaptic membranes. Botulinum neurotoxin is proposed to be a useful analytical tool for understanding the characteristics of the presynaptic membranes in the central nervous system.


Isolation of the pericellular matrix of human fibroblast cultures. The pericellular matrix of human fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. The matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the major glycosaminoglycan components and fibronectin and procollagen as major polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix.


Cellular tumorigenicity in nude mice. Test of associations among loss of cell-surface fibronectin, anchorage independence, and tumor-forming ability. Fibronectin (FN; also called large external transformation-sensitive [LETS] protein or cell-surface protein [CSP]) is a large cell-surface glycoprotein that is frequently observed to be either absent or greatly reduced on the surfaces of malignant cells grown in vitro. Because FN may be a useful molecular marker of cellular malignancy, we have carried out an extensive screening to test the specific association among the degree of expression of FN, anchorage-independent growth, and tumorigenicity in the athymic nude mouse. A variety of diploid cell strains and established cell lines were tested for the expression of surface FN by indirect immunofluorescence using rabbit antisera against human cold insoluble globulin, rodent plasma FN, or chicken cell-surface FN. Concomitantly, the cells were assayed for tumor formation in nude mice and for the ability to form colonies in methylcellulose. Tumorigenic cells often showed very low surface fluorescence, confirming earlier reports. However, many highly tumorigenic fibroblast lines from several species stained strongly with all three antisera. In contrast, the anchorage-independent phenotype was nearly always associated with tumorigenicity in approximately 35 cell lines examined in this study. In another series of experiments, FN-positive but anchorage-independent cells were grown as tumors in nude mice and then reintroduced into culture. In five of the six tumor-derived cell lines, cell-surface FN was not significantly reduced; one such cell line showed very little surface FN. Our data thus indicate that the loss of cell-surface FN is not a necessary step in the process of malignant transformation and that the growth of FN-positive cells as tumors does not require a prior selection in vivo for FN-negative subpopulations.


Role of tropomyosin in actin filament formation in embryonic salamander heart cells. Recessive mutant gene c in Ambystoma mexicanum embryos causes a failure of the heart to function even though initial heart development appears normal. An analysis of the constituent proteins of normal and mutant hearts by SDS-poly-acrylamide gel electrophoresis shows that actin (43,000 daltons) is present in almost normal amounts, while myosin heavy chain (200,000 daltons) is somewhat reduced in mutants. Both SDS-polyacrylamide gel electrophoresis and immunofluorescence studies reveal that tropomyosin is abundant in normal hearts, but very much reduced in mutants. Electron microscope studies of normal hearts show numerous well-organized myofibrils. Although mutant cardiomyocytes contain a few 60- and 150-A filaments, organized sacromeres are absent. Instead, amorphous proteinaceous collections are prominent. Previously reported heavy meromyosin (HMM)-binding experiments on glycerinated hearts demonstrate that most of the actin is contained within the amorphous collections in a nonfilamentous state, and the addition of HMM causes polymerization into F actin (Lemanski et al., 1976, J. Cell. Biol. 68:375-388). In the present study, glycerol-extracted hearts are incubated with tropomyosin, purified from rabbit or chicken skeletal muscle. This treatment causes the amorphous collections to disappear, and large numbers of distinct thin actin (60- to 80-A) filaments are seen in their place. Negative staining experiments corroborate this observation. These results suggest that the nonfilamentous actin located in the amorphous collections of mutant heart cells is induced to form into filaments with the addition of tropomyosin.


Expression of microtubule networks in normal cells, transformed cells, and their hybrids. Microtubules play an important role in several cellular functions including cellular architecture and chromosome movement in cell division. Tubulin which polymerizes to form mictobules can be purified to homogeneity and used to raised antisera. Antisera prepared against porcine or chicken tubulin reacts well with mammalian tubulin. We have examined normal and transformed cells of mouse and human origin for microtubules by indirect immunofluorescence methods. Extensive networks of microtubules (MN) are easily detectable in normal and some transformed cells. The fixation procedure employed and the morphology and the cellular attachment properties seem to determine the ease of detection of MN in these cells. Cells derived from tumors and exhibiting several transformed phenotypes contained MN comparable to those of normal cells. Hybrids between transformed mouse cells and normal human cells were examined. They showed a variability in morphology, but all contained MN. These hybrids exhibited several transformed phenotypes. We conclude that in the cell lines we have examined there is no correlation between the transformed phenotypes and the organization of tubulin.


Changing patterns of plasma membrane-associated filaments during the initial phases of polymorphonuclear leukocyte adherence. By utilizing a combination of several ultrastructural techniques, we have been able to demonstrate differences in filament organization on the adherent plasma membranes of spreading and mobile PMN as well as within the extending lamellipodia. To follow the subplasmalemmal filaments of this small amoeboid cell during these kinetic events, we sheared off the upper portions of cells onto glass and carbon surfaces for 30 s--5 min. The exposed adherent membranes were immediately fixed and processed for high-resolution SEM or TEM. Whole cells were also examined by phase contrast microscopy, SEM, and oriented thin sections. Observed by SEM, the inner surface of nonadherent PMN membranes is free of filaments, but within 30 s of attachment to the substrate a three-dimensional, interlocking network of globular projections and radiating microfilaments--i.e., a subplasmalemmal filament complex--is consistently demonstrable (with or without postfixation in OsO4). Seen by TEM, extending lamellipodia contain a felt of filamentous and finely granular material, distinct from the golbule/filament complex of the adjacent adherent membrane. In the spread cell, this golbule-filament complex covers the entire lower membrane and increases in filament-density over the next 2--3 min. By 3--5 min after plating, as the PMN rounds up before the initiation of amoeboid movements, another pattern emerges--circumferential bands of anastomosing filament bundles in which thick, short filaments resembling myosin are found. This work provides structural evidence on the organization of polymerized contractile elements associated with the plasma membrane during cellular adherence.


Ultrastructural identification of muscle fiber types by immunocytochemistry. In a fast-twitch muscle, three types of fibers (red, intermediate, and white) can be distinguished on the basis of mitochondrial content. Red fibers, identified by abundant mitochondria, can be further differentiated on the basis of a positive or negative response to antibodies specific for white ("fast") myosin. Because there is also a difference in Z-line width among fibers of the same muscle, the possibility existed that the two red fibers, which differ in type of myosin, might also differ in dimensions of the Z line. We therefore examined, with the electron microscope, fibers which had been exposed to antibody against white myosin. In those fibers which react with the antibody, an electron-opaque band is evident in the H-band region, thereby distinguishing reactive from unreactive fibers. The red fiber can now be subdivided on the basis of a positive or negative response to anti-white myosin visualized directly with the electron microscope. Both categories of red fibers ("fast" and "slow") have wide Z lines, and thus are distinguished from white and intermediate fibers, which react with the antibody but which have narrow Z lines. On the basis of combined immunocytochemical and ultrastructural characteristics, four types of fibers can be recognized in a single muscle. Moreover, it is demonstrated here that a wide Z line does not necessarily imply a slow speed of contraction.


Localization of an endogenous lectin in chicken liver, intestine, and pancreas. Extracts of adult chicken liver, pancreas, and intestine contain high levels of a lectin which appears to be identical to one previously purified from embryonic chick muscle. This lectin is virtually absent from adult muscle, but is highly concentrated in cells lining liver sinusoids, intestinal goblet cells, and the extracellular spaces surrounding pancreatic acini. These findings suggest that the lectin may play different roles in different tissues and at different times in the life of a chicken.


Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro. The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K). Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm. With maturation, staining of myotubes by anti-58K diminished and eventually disappeared. Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation--from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I--Z region of myofibrils. However, the emergence of a cross-striated anti-55K pattern did not coincide temperally with the emergence of striated myofibrils, but occurred over a period of days thereafter.


Arrest of cell membrane movements by in vitro incubation with polycation reversed by polyanion. A total arrest of cell membrane movement occurred after incubation for 2 h of cultured fibroblasts with the polycation DEAE-dextran. The immobilized cells resumed normal membrane undulations after exposure for 5 min to the polyanion dextran-sulphate. The influence of DEAE-dextran on the electrophoretic mobility, which was seen after 10 min of incubation was also erased by a subsequent incubation with dextran-sulphate. No influence on growth of the culture was seen.


The effect of local anaesthetics and barbiturates on myogenesis and myotube integrity in rat skeletal muscle cultures. Several cationic anaesthetics and barbiturates including bupivacaine, lignocaine and pentobarbital reversibly inhibit myoblast fusion in rat primary muscle cultures. The same drugs also cause disruption of myotube structure, which is normally complete within 2 h; fusion of myoblasts and reformation of myotubes takes place on removal of the drugs. The disruption of myotubes caused by tertiary amine anaesthetics observed in vitro appears to mimic muscle fibre disruption caused by the same drugs in vivo. The effects of temperature, culture age, medium constitution and calcium flux on anaesthetic-induced myotube breakdown have been examined.


Cell-to-substratum contacts in living cells: a direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and alpha-actinin. Rat mammary cells growing on glass coverslips were photographed first using interference-reflexion microscopy and then after processing for indirect-immunofluorescence microscopy with antibodies to actin or to alpha-actinin. A comparison of the images of the same cell given by the 2 microscopical procedures indicates that the focal contacts between the cell and the substratum correspond to distal ends of microfilament bundles, and the these bundles are only in limited areas close to the substratum. The focal contracts are rich in alpha-actinin which has been proposed as a membrane-anchorage protein for microfilament bundles. Use of stereo immunofluorescence microscopy allows a direct comparison between the interference-reflexion image, and the underside of the cell after staining with antibodies to actin or alpha-actinin.


A morphological study of the interaction between Trypanosoma cruzi and rat eosinophils, neutrophils and macrophages in vitro. The interaction between trypomastigotes of T. cruzi and purified rat eosinophils, neutrophils and macrophages has been studied. Macrophages induced by dextran, became infected and the parasites multiplied as amastigotes, eventually being released as trypomastigotes into the medium. Eosinophils show an antibody-dependent phagocytosis, while neutrophil phagocytosis is enhanced by antibody. Electron microscopy showed that once inside the granulocyte phagosome, the trypomastigotes transform to amastigotes as a prelude to degeneration. This occurs as material apparently of granule origin accumulates in the phagosome. Eosinophils have the capacity to eject incompletely degenerated amastigotes into the medium. This phenomenon is discussed in relation to previous time-lapse cinematography studies of the lysis of chick erythrocytes by eosinophils, and reports that eosinophils release granule contents by means of a secretory vesicle.


Capping of variable antigen on Trypanosoma brucei, and its immunological and biological significance. Pathogenic trypanosomes undergo antigenic variation, whereby the glycoprotein molecules constituting the cell coat are changed, the parasite thus evading the host's immune response. On application of homologous antiserum in indirect immunofluorescence to a given variable antigen type of Trypanosoma brucei, the surface variable antigen moves to the flagellar pocket region, which overlies the Golgi apparatus. This redistribution, or capping, is temperature-dependent, occurring at 37 degrees C but not at 0-4 degree C. Patching does not occur at either temperature. Immediately after capping no homologous or heterologous variable antigen, or host plasma or blood cell antigens, can be detected by immunofluorescence on the cell surface outside the cap; only trypanosome membrane common antigens can be found. It seems unlikely for two reasons that this antibody-induced redistribution is relevant to antigenic variation. Capping of the coat requires the indirect, rather than the direct, immunofluorescent method; a single layer of antibody, in nature, would appear to be ineffective. Also, capping of variable antigen of one type is followed within 3 h by appearance of antigen of the same, and not another, type. The necessity for 2 antibody layers is usually thought of as meaning that the individual molecules of the cell surface antigen are spaced further apart than the binding sites of an individual antibody molecule, so that the necessary cross-linked lattice cannot be formed, but on T. brucei the surface variable antigen molecules are very closely packed. It is proposed that one layer of antibody is ineffective for steric reasons; the dimensions of the exposed face of each variable antigen molecule may not permit the binding of more than one molecule of immunoglobulin, or perhaps the antigen molecules are so closely packed that most of the antigenic determinants are hidden from antibodies. To test this hypothesis, an attempt was made to cap variable antigen on trypanosomes transforming in vitro from the bloodstream to the procyclic (insect midgut) stage; such forms have a much less densely packed surface coat. Patching was observed, indicative of lattice formation, but these trypanosomes did not survive the in vitro manipulation long enough to permit any possible capping. T. brucei differs structurally from most other eukaryotic cells. It has no detectable microfilaments under the plasma membrane, except at the desmosomes in the region of flagellar binding, and it also has a pellicular cortex of microtubules. Capping of its surface antigen would appear then to differ from that on mammalian cells, either in the cellular components involved or in that specialized areas of the plasma membrane are involved.


Transfer of mouse nuclear envelope specific proteins to nuclei of chick erythrocytes during reactivation in heterokaryons with mouse A9 cells. When chick erythrocyte nuclei are introduced into the cytoplasm of mouse A9 cells by cell fusion, proteins present in a fraction of the mouse nuclear envelope begin to appear in the envelope of the chick erythrocyte. The protein uptake was examined using antisera raised in chickens against the 3 major polypeptides of the nuclear pore complex-fibrous lamina fraction from rat liver nuclei. In indirect immunofluorescence studies these antisera give a strong envelope-specific staining with various mammalian but not chicken cells. Eighteen hours after cell fusion the first murine antigens can be observed in the erythrocyte nucleus. Two days after cell fusion the vast majority of the erythrocyte nuclei in cell hybrids contain some antigen and by 3 days the fluorescence of the reactivated erythrocyte nuclei reaches a level comparable to that of the mouse A9 nuclei. The rate of appearance of fluorescence in the chick nuclei depends upon the ratio of A9 cytoplasm to chick nuclei. Antigen uptake by the erythrocyte envelope is inhibited when protein synthesis is blocked suggesting that synthesis of mouse antigen, rather than a redistribution, determines the velocity or erythrocyte envelope reactivation. The early uptake of nucleospecific protein into the reactivating chick erythrocyte may not require any alteration in the nuclear envelope.


[Effect of commonly practiced manoeuvres in vascular microsurgery. Study under the scanning electron microscope (author's transl)]. The authors studied in 100 Wistar rats the manoeuvres that may commonly be carried out during vascular microanastomosis of diameter less than 3 mm (aorta and posterior vena cava): clamping for 30 to 45 minutes, bipolar electrocoagulation of the collateral arteries, more or less marked pinching of the vascular wall. The risk of thrombosis seems all the greater when: --the surgeon is inexperienced: viz. trauma with the forceps, parietal tears at the level of the stitches, two many threads passed for hemostasis (all these factors accentuate the fibrinoid and platelet deposits); --bipolar electrocoagulation of a collateral artery applied too close to the vessel to be anastomosed (less than 1 mm.)The formation of a smooth and regular fibrinous pseudo-intima appears within 48 to 72 hours which may explain the elective onset of thromboses before this delay.


Effect of LRH on gonadotropin secretion in newborn male infants. The concentrations of LH and FSH were measured in eight male newborn babies, aged 4-6 h, before and after the administration of 25 micrograms synthetic LHR. A comparison was performed with six control newborns receiving normal saline. Both LH and FSH rose significantly in all subjects after LHR administration. Their values were significantly higher than those observed during the control study. These data demonstrate sensitivity of the pituitary gonadotropes to synthetic LHR and a pubertal-type response to LH in the early hours of human life.


beta-Endorphin in the human pancreas.

beta-Endorphin in the human pancreas. Using a specific antiserum, beta-endorphin was quantitated in 8 human pancreas obtained at autopsy by radioimmunoassay and localized by immunocytochemistry. The mean (+/- SE) concentration of beta-endorphin in pancreatic extracts of 5 non-diabetic adults was 13.5 +/- 9.8 ng/gm with a range of 2.1 to 52.8 ng/gm of tissue. Pancreatic beta-endorphin concentration in two premature infants were within the range found in adults. In one diabetic pancreas, there was no measurable beta-endorphin. Specific beta-endorphin immunofluorescence is localized within the pancreatic islets. This finding suggests that beta-endorphin may participate in intraislet regulation of pancreatic hormone secretion.


Pulsatile subcutaneous nocturnal administration of GnRH by portable infusion pump in hypogonadotropic hypogonadism: initiation of gonadotropin responsiveness. GnRH was administered subcutaneously in hourly pulses for 10 consecutive nights to two immature males with Kallmann's Syndrome using a portable, battery-operated infusion pump adapted for home use. Pulsatile GnRH produced a progressive increase in urinary gonadotropin excretion, a significant increase in mean basal plasma FSH, pulsatile LH release, and an increased LH response to a standard 3 hour GnRH infusion test. One subject developed a striking increment in plasma testosterone in response to GnRH pulses, as well as a biphasic LH response to the 3 hour infusion.


Evaluation of the modified API 20C system for identification of clinically important yeasts. The modified API 20C system (Analytab Products, Inc.) containing 19 carbohydrate assimilation tests was used to identify stock cultures of clinical isolates and routine clinical isolates from the Mayo Clinic mycology laboratory. The system provided correct identifications for 96% of the 505 organisms tested. The API 20C represents a commercial system useful for the identification of yeasts from clinical specimens. Although reliable, it is not a complete system and must be used in conjunction with microscopic morphological features for definitive identification. Since the system requires 72 h for identification, it is not designed for the rapid presumptive identification of such organisms as Cryptococcus neoformans; other biochemical tests must be used for this purpose.


Methods for serotyping nasopharyngeal isolates of Haemophilus influenzae: slide agglutination, Quellung reaction, countercurrent immunoelectrophoresis, latex agglutination, and antiserum agar. Nasopharyngeal isolates of H. influenzae were typed by the slide agglutination test, the Quelling reaction, the latex agglutination test, countercurrent immunoelectrophoresis, and the antiserum agar test. These tests gave essentially comparable results, with countercurrent immunoelectrophoresis and latex agglutination being slightly more sensitive. Cross-reactive problems encountered with latex agglutination and the expense of performing countercurrent immunoelectrophoresis or the antiserum agar test made these tests less practical than the slide agglutination test to identify single strains that were already isolated. The Quellung reaction and slide agglutination were the most rapid tests used to type an organism. For mass screening of multiple samples, countercurrent immunoelectrophoresis was the simplest technique. The antiserum agar test was slow but was the best technique to screen nasopharyngeal swab cultures to identify the presence of any encapsulated strains in the mixed flora. Whether any of the above techniques were as sensitive as the immunofluorescence test was not evaluated in this study.


Detection of Legionella pneumonophila antigen in urine by enzyme-linked immunospecific assay. An enzyme-linked immunospecific assay "sandwich" technique was developed for detecting soluble antigen from the Legionnaires disease bacterium (Legionella pneumophila). With this technique, antigen was detected in urine specimens from guinea pigs inoculated intraperitoneally with heat-killed Legionnaires disease bacteria and in urine specimens from three of four patients who attended the American Legion Convention in Philadelphia in 1976. Urine from a fifth pneumonia patient who attended the Eucharistic Congress (but who was a dubious seroconverter) was negative. Presumably, the test could also be used for detecting antigen in sputum or respiratory aspirates, but this has not been tried to date.


Practical method to facilitate estimation of Streptococcus mutans levels in saliva. A method was developed to facilitate the estimation of Streptococcus mutans levels in saliva. Saliva-contaminated wodden spatulas were pressed directly against an elevated agar plate containing a selective medium. The results were compared with the number of S. mutans per 1 ml of paraffin-stimulated saliva. It was shown that the spatula method gave a good estimation of the level of S. mutans infection. The incubation was also made in expired air instead of 95% N2-5% CO2. The outgrowth was in good agreement with that after conventional incubation. The method is useful in epidemiological studies or in selecting persons at a high caries risk, and when ordinary saliva sampling cannot be done, for example in small children. Compared with conventional saliva sampling, this method requires less time and material at sampling as well as at the laboratory.


Reliability of a single urine culture in establishing diagnosis of asymptomatic bacteriuria in adult males. Fifty-nine asymptomatic men without catheters of ileal-loop bladders, who were attending a urology clinic and were incidentally discovered to have 100,000 or more Enterobacteriaceae per ml ("significant bacteriuria") in a clean voided urine sample, were prospectively evaluated. To identify these 59 patients, 5,876 urine samples, collected exclusively from men, had been subjected to quantitation and identification. A repeat urine culture performed on these patients invariably confirmed the results of the initial culture. The reproducibility of a single urine culture containing significant bacteriuria occurred independently of the tissue source of infection, as determined by the antibody-coated-bacteria immunofluorescence test. We conclude that a single urine culture obtained from a cooperative man can establish the diagnosis of asymptomatic bacteriuria.


Comparison of three methods for identification of pathogenic Neisseria species. A radiometric procedure was compared with the Minitek and Cystine Trypticase Agar sugar degradation methods for identification of 113 Neisseria species (58 Neisseria meningitidis, 51 Neisseria gonorrhoeae, 2 Neisseria lactamica, 2 Neisseria sicca). Identification of meningococci and gonococci was confirmed by agglutination and fluorescent antibody techniques, respectively. The Minitek method identified 97% of meningococci, 92% of gonococci, and 100% of other Neisseria after 4 h of incubation. The radiometric (Bactec) procedure identified 100% of gonococci and 100% of miscellaneous Neisseria after 3 h, but problems were encountered with meningococci: 45% of the later strains yielded index values for fructose between 20 and 28 (recommended negative cut-off point, less than 20), with strongly positive (greater than 100) glucose and maltose and negative o-nitrophenyl-beta-D-galactopyranoside reactions in all 58 strains. The Cystine Trypticase Agar method identified 91% of meningococci, 90% of gonococci, and 100% of other Neisseria after 24 to 48 h. Prolongation of the Cystine Trypticase Agar incubation period led to abnormal lactose/sucrose reactions in some meningococci and gonococci. Radiometric and Minitek systems are more accurate and convenient than Cystine Trypticase Agar techniques, but, on the basis of these results, radiometric fructose sensitivity levels for meningococci need reevaluation.


Comparison of Micro-ID and API 20E systems for identification of Enterobacteriaceae. The Micro-ID 4-h identification system for Enterobacteriaceae was compared to the API 20E overnight method, using 230 fresh clinical isolates and 74 stock cultures. Agreement was 97.8% for the clinical isolates and 93.2% for the stock cultures. Eighty-seven percent of primary culture plates containing gram-negative rods yielded sufficient growth to perform the 4-h Micro-ID identification on the same day the organisms were isolated.


Evidence for the synthesis and release of strongly immunosuppressive, noncytotoxic substances by Streptococcus intermedius. Products secreted by Streptococcus intermedius were studied for their effects on the immune response. Three different preparations of crude extracellular products from S. intermedius (CEP-Si) were found to have powerful suppressor activity in vitro as shown by inhibition of human lymphocyte proliferation (uptake of [3H]thymidine) and protein synthesis in response to a wide variety of stimulants, including mitogens and antigens, and suppression of plaque formation by human cells in response to sheep erythrocytes. CEP-Si was noncytotoxic, because cells incubated with high concentrations of CEP-Si and subsequently washed were viable and recovered their ability to respond to mitogens, and because leukocyte migration was not inhibited by CEP-Si, nor was the release of leukocyte migration inhibitory factor from sensitized lymphocytes. The possibility of antigen or mitogen competition was excluded. The effects of CEP-Si in vitro were time dependent and did not require the presence of monocytes. Cells pretreated with CEP-Si and then washed suppressed plaque formation by fresh autologous cells in highly stimulated cultures. CEP-Si injected into C57BL/6 mice also strongly suppressed their immune response to sheep erythrocytes, and the in vivo suppression was correlated with the effects of CEP-Si in vitro.


Clinical evaluation of hypnotic drugs: contributions from sleep laboratory studies. The most thorough and clinically relevant approach to hypnotic drug evaluation is one that balances the strengths and weaknesses of clinical trials and sleep laboratory evaluations. Advantages of clinical trials include the ability to evaluate large numbers of subjects and specific target groups and to thoroughly assess and quantify a drug's side effects, whereas sleep laboratory studies are very limited in all of these areas. Sleep laboratory studies however provide a rigorous, precise, and comprehensive profile of a drug's activity since there is more control over experimental variables and measurements are objective as well as continuous throughout the night. These benefits offset the shortcomings of clinical trials, which include a lack of objective measurements, less control over experimental variables, failure to evaluate a drug's effectiveness with continued use, and inattention to drug interaction and withdrawal effect. Several basic principles derived from sleep laboratory findings have been incorporated into both the clinical trials and sleep laboratory evaluations recommended in the new FDA Guidelines for the Clinical Evaluation of Hypnotic Drugs. These principles include provision for adequate baseline and withdrawal periods, use of multiple consecutive drug nights to assess a drug's effectiveness with continued use, and inclusion of an adequate washout period when a cross-over design is used. The guidelines do not emphasize either clinical trials or sleep laboratory studies at the expense of each other, but rather stress their complementary utilization.


Immunopathologic and histologic studies on skin from pemphigus patients in tissue culture. Explants of skin from patients with pemphigus vulgaris and pemphigus foliaceus taken in the active stages of the disease had in vivo bound IgG in the intercellular area. After 24-48 h incubation of these explants in normal sera acantholytic bullae developed above the stratum basale, thus indicating that the bound IgG is probably in vivo bound pemphigus antibody. In both cases, skin from the inactive stage of the disease contained no in vivo bound pemphigus antibodies. Explants of these skin specimens failed to develop acantholysis in culture.


Immunohistochemical demonstration of basement membrane collagen in normal human skin and in psoriasis. Antibodies prepared against the collagen C-chain derived from human placenta specifically strain the epidermal basement membrane of normal human skin as well as the basement membrane surrounding the skin appendages and capillaries. Using the antibodies, it was observed that all basement membranes in untreated psoriatic plaques appear fragmented and separted into several layers. The results suggest that psoriatic lesions are associated with a general loss of basement membrane integrity which may play a crucial role in the pathogenesis of the disease.


Immunotherapy of melanoma.

Immunotherapy of melanoma. Malignant melanoma is a disease characterized by clinical evidence of host defense, possibly immunologically mediated. It is a disease which tends to be refractory to both radiotherapy and chemotherapy. Immunotherapy has been used in three phases of the disease. 1. Intralesional immunotherapy with a nonspecific immune adjuvant in patients with local intradermal or soft tissue recurrence. This treatment produces approximately 15% regression of both injected and uninjected lesions, and about 60% regression of injected lesions only. Both clinical and laboratory evidence suggests that this regression is immunologically mediated. 2. Patients with surgical removal of all clinically demonstrable tumor, either primary disease alone or regional node recurrence, active nonspecific, and specific, immunotherapy has been used in an adjuvant setting. There is considerable controversy about the benefits accruing to such immunotherapy, but most large scale prospective and randomized studies have suggested that if benefit does result it is modest in degree and probably cannot be measured in terms of increase in cure rate. 3. Immunotherapy has also been used as a nonspecific active adjuvant to single drug or polychemotherapy in patients with disseminated melanoma. Whereas complete response rate may be slightly increased by this maneuver there is no convincing evidence that immunotherapy markedly increases the total objective response rate to polychemotherapy, and survival is only marginally superior when immunotherapy is added to chemotherapy in this setting. Further studies need to be done with active specific immunotherapy with tumor cell membrane extracts; as an adjuvant in patients with minimal body burden of tumor cells; and to study the inaction between chemotherapy and immunotherapy in this disease. Furthermore, studies of chemically defined fractions of either bacterial cell wall or tumor cell extracts must be evaluated both in terms of their ability to augment cell mediated immune responses in the melanoma patient, and also in terms of their ability to induce objective benefit for the patient. The possible use of immunotherapy in patients with primary melanoma has been briefly explored but needs further study. Possible additive effects with radiotherapy and immunotherapy should also be looked at in this disease utilizing high dose fractions and other new forms of radiotherapeutic technique.


The future of dental specialization: policy issues. There has been a shift in the fundamental values placed by society on its health care system, as reflected by public insistence on greater accountability, a more influential role in policy decisions, and a more effective use of system resources. This change in values will profoundly influence the future of dental specialization. The role of the generalist, expanding as a result of various public and internal pressure, will shape the future role of the specialist more than anything else. No new dental specialties are likely to emerge, but there will be changes in emphasis and consolidation among the specialties. Graduate dental education will require a reorganization and a variety of sharing arrangements for its survival. Some form of demonstrating continuing competence will be required for maintenance of specialty status.


Correction of a defect on the dorsum of the nose. A particular defect on the dorsum of the nose, which was traumatically incurred, was successfully corrected after two previous failures.


Quality and standards--the dietitian's heritage. 16th Lenna Frances Cooper Memorial Lecture. Since 1917, when The American Dietetic Association was founded, dietitians have been charged with accountability for defining and maintaining quality and standards related to food. This heritage continues to be a challenge to the profession. With current methods of food production, processing, preparation, and distribution, and varied and changing consumer preferences and demands, the need for monitoring the effect of these changes on quality as it relates to improving the nutrition of people is critical. Quality must be defined with regard for values and goals for individual situations and under varying circumstances. Standards by which that specific quality can be measured need to be described. Tian's heritage" their "hallmark" of quality.


Fissure sealants in primary molars: retention of colored sealants with variable etch times, at twelve months. There is no significant difference in sealant retention on primary molars etched for sixty seconds or for 120 seconds, twelve months after application. No caries was found on any sealed surface. Decreasing the etch-time for primary molars has been found to decrease the chance of contamination, during etching. Additionally, the shorter etch-time was far more acceptable to three and four-year-old children. This study is part of a continuing investigation of the retention, caries-reduction and overall cost-effectiveness of sealing permanent and primary teeth in children.


Overview of extended wear contact lenses. Extended wear of contact lenses is an attractive alternative to daily wear regimens. Immediate application has been for therapeutic purposes. Recently, cosmetic investigations have occurred, primarily outside of the United States. Regardless of the application, extended wear requires careful patient selection, fitting and post-fitting care to insure that ocular integrity has not been disturbed.


The effect of tooth preparation on the retention of stainless steel crowns. Preparations maintaining the greatest amount of buccal and lingual tooth structure were the most retentive. Cement significantly increased the retention capacity of all preparations. Mechanical retention did not contribute significantly to the separation-resistance of the stainless steel crown. Factors other than retention may be of greater importance to the clinical success of stainless steel crowns.


Metatarsal lengthening by use of autogenous bone graft and internal wire compression fixation: a preliminary report. As a preliminary report, our paper is designed to show another procedure to be used by podiatrists in lengthening metatarsals by the use of an autogenous bone graft. The procedure described should be considered when metatarsal length problems arise. Discussed were the basic physiologic properties of grafting, and the actual procedure of doing these grafts.