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Dissociated embryonic rat myocardial cells and chick myocardial cells labeled with radioactive isotope coaggregate and establish intercellular junctions. These bispecific cells reconstruct synchronously beating myocardial tissue within 24 hours of culture.
Neutron activation analysis was employed in a survey of weathering abilities of rock surface microorganisms. A yeast isolated from an amphibolite of a megalithic grave was found actively to concentrate, in media and in or on cells, iron and other elements when grown in the presence of ground rock. This was demonstrated by comparing a spectrum of neutron-activated amphibolite powder (particle size, 50 to 100 mum) with the spectra of neutron-activated, lyophilized yeast cells which had grown with or without amphibolite powder added to different media. The most active yeast (IFAM 1171) did not only solubilize Fe from the rock powder, but significant amounts of Co, Eu, Yb, Ca, Ba, Sc, Lu, Cr, Th, and U were also mobilized. The latter two elements occurred as natural radioactive isotopes in this amphibolite. When the yeast cells were grown with neutron-activated amphibolite, the cells contained the same elements. Furthermore, the growth medium contained Fe, Co, and Eu which had been solubilized from the amphibolite. This indicates the presence, in this yeast strain, of active rockweathering abilities as well as of uptake mechanisms for solubilized rock components.
Photosynthetic rates of phytoplankton were significantly lower after analysis by flow cytometry (FCM) than before. Exposure to the laser beam during the sorting process caused significant physiological damage. The cellular content of a radiolabel accumulated prior to FCM was not affected by FCM. Although it may not be possible to use FCM to preconcentrate cells for further physiological studies, samples may be incubated with stable or radioactive isotopes and then analyzed by FCM.
Information on the biosynthesis of phospholipids in bacteria has been derived principally from the study of Escherichia coli and other gram-negative organisms. We have now carried out a detailed study of the pathways of phospholipid biosynthesis in the gram-positive organism Bacillus megarterium KM in relation to investigations on the biogenesis of lipid asymmetry in membranes. Radioactive precursors such as 32Pi and [3H]palmitate initially label phosphatidylethanolamine much more than phosphatidylglycerol. This raised the possibility that phosphatidylglycerol may be the precursor of phosphatidylethanolamine in a pathway different from that in E. coli. Phosphatidylglycerol is known to be highly reactive metabolically, since it functions as a donor of phosphatidyl residues in the synthesis of cardiolipin and as a donor of glycerophosphate residues in the synthesis of teichoic acids and of membrane-derived oligosaccharides. The large pool of phosphatidylglycerol would dilute the radioactive isotope, slowing the initial rate of incorporation of label into phosphatidylethanolamine. However, assays of cell-free extracts revealed no evidence for such a novel pathway. Instead, phosphatidylserine synthase (cytidine 5'-diphosphate-diglyceride:L-serine phosphatidyl transferase) and phosphatidylserine decarboxylase were detected, although at low levels. These results suggest that the pathway in B. megaterium is the same as that in E. coli in which phosphatidylserine, derived from cytidine 5'-diphosphate-diglyceride, is the precursor of phosphatidylethanolamine. The lag in the appearance of label in phosphatidylethanolamine appears to be the effect of a considerable pool of phosphatidylserine (ca. 5 to 10% of the total phospholipid) in certain strains of B. megaterium. The lag in labeling can be correlated with the size of the pool of phosphatidylserine. Pulse-chase experiments in vivo support the conclusion that in B. megaterium phosphatidylserine is not derived from phosphatidylglycerol. Rates of turnover of the membrane phospholipids of B. megaterium have also been studied.
We have developed a simplified method for unambiguously typing herpes simplex virus. The method depends on the production of cell-associated virus at 34 degrees C and subsequently, on the separation of cellular DNA and viral DNA by Dounce homogenization and the removal of nuclei by centrifugation. Viral nucleic acid was prepared from the cytoplasmic fraction and analyzed after restriction endonuclease cleavage. The method obviates the use of radioactive isotopes, and the viral DNA is effectively free of interfering cellular DNA.
The present study describes a colony hybridization setup for identification of enterotoxigenic Escherichia coli obviating the need for advanced equipment and radioactive isotopes. With a modest laboratory arrangement, polynucleotide gene probes were produced in large quantities. The probes were labeled with digoxigenin and, after hybridization, detected with an antidigoxigenin alkaline phosphatase conjugate. With an established isotope-based oligonucleotide hybridization assay as reference, a blinded study on a large battery of enterotoxigenic and nonenterotoxigenic bacteria revealed a satisfactory sensitivity and specificity of the nonradioactive assay.
A simple and economical method was developed for using biotinylated DNA probes to hybridize with bacterial colonies belonging to the various categories of diarrhea-causing Escherichia coli. Simplification and cost containment were achieved by using Whatman no. 541 filter papers instead of nitrocellulose, by minimizing the concentration of proteinase K (an expensive but necessary reagent used to pretreat the colony blots prior to hybridization with biotin-labeled DNA probes) and by reusing hybridization solution containing labeled probe DNA. After exposing the colony blots to lysing solution and steam, followed by lysozyme (1.5 mg/ml), sucrose (25%), and proteinase K (10 micrograms/ml) treatments, biotinylated probes were used to detect enterotoxigenic, enteropathogenic, enterohemorrhagic, diffuse adherence, and enteroinvasive categories of diarrhea-causing E. coli with a high level of sensitivity and specificity. Three independent observers who were experienced in reading DNA blots recorded remarkably similar results, while less satisfactory results were obtained when the blots were read by an inexperienced observer. This technique will be useful in laboratories in which radioactive isotopes are unavailable or impractical and in which budgets are restricted.
We examined the synthesis and turnover of individual proteins in the Saccharomyces cerevisiae cell cycle. Proteins were pulse-labeled with radioactive isotope (35S or 14C) in cells at discrete cycle stages and then resolved on two-dimensional gels and analyzed by a semiautomatic procedure for quantitating gel electropherogram-autoradiographs. The cells were obtained by one of three methods: (i) isolation of synchronous subpopulations of growing cells by zonal centrifugation.; (ii) fractionation of pulse-labeled steady-state cultures according to cell age; and (iii) synchronization of cells with the mating pheromone, alpha-factor. In confirmation of previous studies, we found that the histones H4, H2A, and H2B were synthesized almost exclusively in the late G1 and early S phases. In addition, we identified eight proteins whose rates of synthesis were modulated in the cell cycle, and nine proteins (of which five, which may well be related, were unstable, with half-lives of 10 to 15 min) that might be regulated in the cell cycle by periodic synthesis, modification, or degradation. Based on the time of maximal labeling in the cell cycle and on experiments with alpha-factor and hydroxyurea, we assigned the cell cycle proteins to two classes: proteins in class I were labeled principally in early G1 phase and at a late stage of the cycle, whereas those in class II were primarily synthesized at times ranging from late G1 to mid S phase. At least one major control point for the cell cycle proteins occurred between "start" and early S phase. A set of stress-responsive proteins was also identified and analyzed. The rates of synthesis of these proteins were affected by certain perturbations that resulted during selection of synchronous cell populations and by heat shock.
(1) Subchondral avascular necrosis is an important cause of joint pain and disability and accounts for upwards of 20% of total hip replacements done in our hospital. (2) Early diagnosis may be made with the aid of magnetic resonance imaging and radioactive isotope studies. (3) Although the signs and symptoms are similar to those of osteoarthritis, there are significant differences--namely, (a) a history of sudden onset of pain, present in more than half the patients; (b) a younger age group; (c) a shorter duration of symptoms at time of surgery; (d) clinically the limiting factor is pain rather than actual joint deformity to account for restriction of movement; (e) a high incidence of multiple sites of involvement. (4) The disease is commonly associated with steroid treatment or alcohol abuse. Although many other causes are recognised, they are rare in Western urban practice. (5) Patients with stage I-II subchondral avascular necrosis, especially of the knee, are better treated conservatively. (6) Surgical treatment gives less satisfactory results than the treatment of osteoarthritis by similar modalities.
(99)Tc(m)-pertechnetate is concentrated in the thyroid in the same way as iodide but it does not become organically bound. The uptake of (99)Tc(m) is a measure of the thyroid trap, and this measurement is satisfactory as a routine test in the diagnosis of thyrotoxicosis. The reproducibility of the method is such that suppression tests can be carried out when necessary even when uptake is within the normal range of 0.4-3%. (99)Tc(m) gives a low radiation dose to the thyroid and has certain other advantages over radioactive isotopes of iodine for early thyroid uptake measurements. It is particularly suitable when serial tests of thyroid function are required during treatment with an antithyroid drug.
The sensitivity of digoxigenin and biotin labelled DNA probes for the detection of human papillomavirus (HPV) by dot blotting and in situ hybridisation was compared in tissues from cervical, laryngeal, and anogenital neoplasia. Probes were either labelled with digoxigenin by the random primer technique and detected with anti-digoxigenin antibody, or labelled with biotin by nick translation and detected with streptavidin, both methods having a common final visualisation procedure using alkaline phosphatase. Digoxigenin labelled probes proved two to 10-fold more sensitive by quantitative dot blotting and four-fold more sensitive in detecting HPV 16 DNA in a series of 31 anal carcinomas, compared with biotinylated probes. The digoxigenin method also produced less non-specific background staining of tissue sections than biotin labelled probes. It is concluded that digoxigenin DNA labelling and detection provides a simple, reliable, and efficient alternative to the use of biotin or radioactive isotopes for the detection of HPV DNA by in situ hybridisation. Digoxigenin labelled probes also offer the possibility of double labelling in situ hybridisation procedures when used with biotin labelled probes to provide simultaneous identification of different DNA sequences.
This paper reviews the current literature concerning the different investigative modalities available to assess the microcirculation in diabetic microangiopathy. The advantages and disadvantages of the different invasive and noninvasive methods available are presented objectively. We have concentrated on the tests that provide a quantitative assessment of the microcirculation, including laser Doppler fluxmetry, capillary microscopy, plethysmography, transcutaneous oximetry and radioactive isotope clearance. Some of the invasive methods described are now being replaced by noninvasive equivalents, providing similar information with less discomfort and risk to the patient.
A spontaneous mutant of Streptococcus salivarius ATCC 25975 was isolated by inoculating an agar medium containing 11 mM lactose and 0.5 mM 2-deoxyglucose. This mutant grew poorly on 5 mM glucose but almost as well as the parental strain on 110 mM glucose. Uptake of 2-deoxyglucose was abolished by the mutation, and phosphoenolpyruvate: glucose phosphotransferase activity could not be detected with toluenized cells under normal conditions when the glucose concentration was below 5 mM. Data from growth experiments, glycolysis, and uptake studies indicated the presence of a second phosphoenolpyruvate: glucose phosphotransferase system that could catalyze the phosphorylation of alpha-methyl glucoside. The activity of this system was detected by a spectrophotometric assay coupled with lactate dehydrogenase and by a radioactive isotope method using methyl alpha-D-[U-14C] glucoside. The phosphorylation was phosphoenolpyruvate dependent. The apparent Km of the system for glucose and alpha-methyl glucoside was approximately 20 mM. Studies with energy poisons ruled out the possibility of an active transport system, and accumulation of alpha-methyl glucoside argued against facilitated diffusion. It was concluded that the other glucose transport system which allowed growth of the mutant strain of S. salivarius was a second phosphoenolpyruvate: glucose phosphotransferase system.
A simple device constructed of common laboratory material served as a minifermenter for the growth of Clostridium perfringens. A constant flow of nitrogen gas into a culture tube containing C. perfringens assured agitation of the culture and a mechanism for dispensing small volumes of liquid from the culture without disturbing the growth environment. The method was applied to examining the growth-inhibiting effect of bacteriocins of C. perfringens where a very economical use of radioactive isotopes was possible. The activity of some bacteriocins differed when compared with previous data obtained with stationary cultures. Two major categories of bacteriocin appear to exist for this species: those bacteriocins which block the incorporation of DNA, RNA, and protein precursors and those which interfere with the organism's cell wall.
A new method is described for the measurement of cholesteryl sulfate (CS) in plasma. Taking advantage of the use of beta-sitosteryl sulfate (SS) as an internal standard it is simpler and more rapid than the methods currently in use. It does not require the use of radioactive isotopes. A potential contamination of the CS band by free cholesterol is excluded by developing the thin-layer chromatography plate on which the sterols are first separated with a second solvent system which pushes free cholesterol to the solvent front without appreciably moving the CS band. The method is sensitive and reproducible and may be advantageous for the screening of large populations. The values obtained for plasma CS concentrations in normal subjects compare well with those measured by more elaborate techniques.
A focus of current diabetes research is the development of insulinomimetic compounds for oral treatment of diabetes and its associated cardiac complications. Screening compounds for their potential insulinomimetic effects usually involves the use of radioactive isotopes. The focus of this study was to investigate a nonradioactive fluorescent compound for its use in screening insulinomimetic compounds. The indicator 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) has been used by some workers to measure glucose uptake in Escherichia coli and Candida albicans. We propose that 2-NBDG will also be a suitable indicator for mammalian cell lines, in particular rat cardiomyocytes. We found that the indicator could give a reliable reproducible standard curve following appropriate dilution and is taken up by isolated cardiomyocytes. The insulinomimetic compounds vanadyl sulfate and sodium molybdate showed rates of glucose uptake similar to that of insulin. Furthermore, the rate of uptake measured for insulin using this technique (0.04 +/- 0.003 nmol x min(-1) x 10(6) cells(-1) is comparable with previous literature using 2-deoxyglucose uptake measurements on isolated myocytes (0.040 nmol x min(-1) x 10(6) cells(-1), demonstrating the validity of this fluorescent compound for glucose uptake studies.
The intra- and extracellular chloride concentration dependencies of the rate of Cl-Cl exchange in human promyelocytic leukemic HL-60 cells were studied by means of radioactive isotope (36Cl) efflux measurements. Efflux of isotope from cells follows an exponential time course. The Cl-Cl exchange flux follows Michaelis-Menten kinetics as a function of both intra- and extracellular chloride concentrations. The ratio of the maximum exchange velocity to the apparent Michaelis constant for both extracellular and intracellular substrate increases as a function of trans Cl concentration, indicating that Cl-Cl exchange in the HL-60 cell does not follow ping-pong kinetics. A kinetic scheme in which extracellular and intracellular chloride ions bind in random order to the transporter and are then translocated simultaneously can adequately model the experimental data.
Effects of Na+ and K+ in plasma on the protein adsorption on red blood cells (RBCs) were studied using fresh human blood and human plasma albumin labeled with a radioactive isotope. Crystalline NaCl and/or KCl were added to blood samples containing labeled albumin so that the plasma osmolarity was increased to final values of 350, 400, and 450 mosmol. The number of albumin molecules adsorbed on the RBCs increased markedly with increases in Na+ but remained unaltered or increased only slightly with increases in K+. The distribution of Na+ and K+ around the RBC membrane was considered to contribute to increasing the adsorption of albumin on the outer surface and to restricting the deposition of hemoglobin on the inner surface with a consequent positive effect on maintaining the stiffness and flexibility of the membrane. In support of this hypothesis the ouabain treatment of RBCs resulted in a decrease in the cellular deformability.