Dinucleotide repeat polymorphism in the human alpha-cardiac actin gene, intron IV (ACTC), detected using the polymerase chain reaction.
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Actins↗
Biomedical subjects
K Nyberg
Publications and source records attributed to K Nyberg.
Phagolysosomal pH and location of particles in alveolar macrophages.
Fluorescein-labeled silica particles (FSP) were instilled into the tracheae of rabbits. Groups of four rabbits were killed after 24 hr, 1 week, 1 month, or 3 months and their lungs were lavaged. Phagolysosomal pH in the alveolar macrophages (AM) was measured using microscope fluorometry with FSP as a probe. Due to the marked decline of the fluorescence intensities from the FSP between 1 and 3 months after instillation, it was not possible to measure pH at 3 months, but the values from 24 hr, 1 week, and 1 month were quite similar, with group means of 4.8 and 4.9, respectively. Phagolysosomal pH in AM which phagocytized the FSP in vitro showed values about half a pH unit higher. AM from rabbits lavaged at 1 week had more lysosomes in contact with the FSP-containing phagolysosomes and a higher degree of vacuolization between the FSP and the phagolysosomal membrane than AM lavaged at 1 day. The location of the FSP in the AM appeared to be similar in rabbits lavaged after 1 week and 3 months. In histologic sections from the lungs the large majority of the FSP were within cells at all time points.
Animals↗
Opiate addiction in adult offspring through possible imprinting after obstetric treatment.
OBJECTIVE: To test the hypothesis that opiate addiction in adults might stem partly from an imprinting process during birth when certain drugs are given to the mother. DESIGN: Retrospective study by logistic regression of opiate addicts with siblings as controls. SETTING: Stockholm, Sweden. SUBJECTS: 200 Opiate addicts born in Stockholm during 1945-66, comprising 41 identified during interviews of probands for an earlier study; 75 patients whose death from opiate addiction had been confirmed during 1978-88; and 84 accepted for the methadone programme. 262 Siblings (controls) born in Stockholm during the same period, 24 of whom were excluded for drug addiction or being brought up outside the family. Birth records were unavailable for eight, leaving 230 siblings and 139 corresponding probands. MAIN OUTCOME MEASURES: Administration of opiates, barbiturates, and nitrous oxide (for greater than 1 h) to mothers of all subjects during labour within 10 hours before birth as a risk factor for adult opiate addiction. RESULTS: In subjects who had subsequently become addicts a significant proportion of mothers had received opiates or barbiturates, or both, compared with unmatched siblings (25% v 16%, chi 2 = 5.83, df = 1, p = 0.02), and these mothers had received nitrous oxide for longer and more often. After controlling for hospital of birth, order of birth, duration of labour, presentation other than vertex, surgical intervention, asphyxia, meconium stained amniotic fluid, and birth weight the relative risk for offspring subsequently becoming an adult opiate addict increased with the number of administrations of any of the three drugs. When the addicts were matched with their own siblings the estimated relative risk was 4.7 (95% confidence interval 1.8 to 12.4, p for trend = 0.002) for three administrations compared with when no drug was given. CONCLUSIONS: The results are compatible with the imprinting hypothesis. Therefore, for obstetric pain relief methods are preferable that do not permit substantial passage of drugs through the placenta.
Analgesia, Obstetrical↗
[Laughing gas and amphetamine abuse].
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Amphetamine↗
Energetics of complementary side-chain packing in a protein hydrophobic core.
The energetics of complementary packing of nonpolar side chains in the hydrophobic core of a protein were analyzed by protein engineering experiments. We have made the mutations Ile----Val, Ile----Ala, and Leu----Ala in a region of the small bacterial ribonuclease barnase where the major alpha-helix packs onto the central beta-sheet. The destabilization resulting from the creation of cavities was determined by measuring the decrease in free energy of folding from reversible denaturation induced by urea, guanidinium chloride, or heat. The different methods give consistent and reproducible results. The loss in free energy of folding for the mutant proteins is 1.0-1.6 kcal/mol per methylene group removed. This exceeds by severalfold the values obtained from model experiments of the partitioning of relevant side chains between aqueous and nonpolar solvents. Much of this discrepancy arises because two surfaces are buried when a protein folds--both the amino acid side chain in question and the portions of the protein into which it packs. These experiments directly demonstrate that the interior packing of a protein is crucial in stabilizing its three-dimensional structure: the conversion of leucine or isoleucine to alanine in the hydrophobic core loses half the net free energy of folding of barnase with a concomitant decrease in yield of the expressed recombinant protein.
Amino Acids↗
Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis.
Barnase, the ribonuclease from Bacillus amyloliquefaciens, has been cloned and expressed in Escherichia coli [Hartley, R. W. (1988) J. Mol. Biol. 202, 913-915], thus enabling the overproduction and site-directed mutagenesis of one of the smallest enzymes (Mr equals 12,382). As barnase is also composed of just a single polypeptide chain with no disulfide bridges and has a reversible folding transition, it affords a fine system for studying protein folding and design. We show here that the recombinant enzyme has properties identical with those of the authentic enzyme, characterize the basic kinetics and specificity of the enzyme, and, using site-directed mutagenesis, identify key residues involved in catalysis to provide evidence that supports the classic ribonuclease mechanism. The wild-type enzyme catalyzes the hydrolysis of dinucleotides of structure GpN. There is a prime requirement for G and a preference for A greater than G greater than C greater than U for N. The pH-activity curve for the transesterification step of dinucleotides is bell shaped with an optimum for kcat/KM and kcat at about pH 5. The enzyme is far more active toward long RNA molecules, and the pH optimum for kcat is at 8.5. The activity of barnase toward dinucleotide substrates is about 0.5% of that of the highly homologous T1 nuclease at pH 5.9, but barnase is twice as active as T1 toward RNA at pH 8.5. There must be important subsite interactions that contribute to catalysis in barnase in addition to those immediately on either side of the scissile bond.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacillus↗
Intraphagosomal pH in alveolar macrophages studied with fluorescein-labeled amorphous silica particles.
Intraphagosomal pH in rabbit alveolar macrophages was studied using amorphous silica particles (FSP) and yeast particles (FYP) labeled with fluorescein. The pH was estimated from the quotient between the fluorescence intensity at wavelength 519 nm with excitation at wavelengths 495 and 450 nm. Within a factor of 10, pH was independent of the number of FSP added to macrophages in vitro. In macrophages cultured for 24 h, the pH obtained with FSP and FYP was about 5. Three hours after lavage, pH was the same as after 24 h for the FSP but significantly higher for the FYP, 5.8. Both 3 and 24 h after lavage, more lysosomes were in contact with the FYP-containing phagosomes than with the FSP-containing ones. Most FSP were in tight contact with the phagosomal membrane, while there was a clear zone between most FYP and the phagosomal membrane. The differences in pH and morphology between cells containing FSP and FYP might be explained by the assumptions that the macrophages disintegrate the FYP, which results in higher pH, and that the disintegration of FYP is more efficient at 3 than at 24 h after the lavage. The intraphagosomal pH was lower when the macrophages were allowed to phagocytize the FSP in vivo. The pH values were 5.1 in vitro, 4.9 at 24 h, and 4.5 at 1 week after the FSP had been instilled via trachea. The FSP should be a useful tool for estimation of intraphagosomal pH at basic conditions, i.e., the milieu to which many inhaled inorganic particles will be exposed.
Animals↗
Estimation of pH in individual alveolar macrophage phagolysosomes.
A method for measurement of phagolysosomal pH in individual alveolar macrophages based on a cytofluorometric technique with fluorescein-labeled silica particles (FSP) as a probe was developed. The size of the FSP, 3.0 or 5.0 microns, did not affect the result of the pH measurements. The average pH values, range 5.1-5.5, of individual particles in macrophages from three rabbits agreed well with the pH values obtained in a macrophage population using a fluorescence spectrometer and the FSP. The variation of pH in phagolysosomes in alveolar macrophages from five rabbits was investigated. Measurements were performed 3, 6, and 24 h after addition of the FSP in cells containing one particle as well as in cells containing two particles. The variation in pH was small, with a coefficient of variation less than 10% in all rabbits at all times. There was a significant correlation between values obtained from two phagolysosomes in the same cell, indicating a cell factor responsible for 10-30% of the total variance. The fact that the variation of pH is small in normal, untreated rabbit alveolar macrophages should be of importance when estimating alveolar clearance of inorganic particles due to dissolution in the acid phagolysosomal milieu.
Animals↗
Contribution of hydrophobic interactions to protein stability.
A major factor in the folding of proteins is the burying of hydrophobic side chains. A specific example is the packing of alpha-helices on beta-sheets by interdigitation of nonpolar side chains. The contributions of these interactions to the energetics of protein stability may be measured by simple protein engineering experiments. We have used site-directed mutagenesis to truncate hydrophobic side chains at an alpha-helix/beta-sheet interface in the small ribonuclease from Bacillus amyloliquefaciens (barnase). The decreases in stability of the mutant proteins were measured by their susceptibility to urea denaturation. Creation of a cavity the size of a -CH2-group destabilizes the enzyme by 1.1 kcal mol-1, and a cavity the size of three such groups by 4.0 kcal mol-1.
Bacillus↗
Quantification of alpha-amylase mRNA in Bacillus subtilis by nucleic acid sandwich hybridization.
A novel hybridization test for quantification of mRNA from bacterial cells lysed directly in the culture medium was developed and optimized. The method uses two adjacent probes from a DNA fragment of interest in a sandwich hybridization. An unlabeled probe is immobilized on a solid support to capture homologous nucleic acids in the test solution. Hybrid detection is performed with the other, labeled, probe, which can bind to the filter only as a sample-mediated hybrid. We used the Bacillus amyloliquefaciens alpha-amylase gene and its mRNA as models for characterizing gene expression in Bacillus subtilis. To confirm the specificity of mRNA hybridization, nondenatured nucleic acid samples were allowed to react simultaneously with two filters: the mRNA-specific filter containing anti-sense single-stranded DNA and the background filter containing single-stranded DNA of the mRNA sense, respectively. We also developed a rapid enzymatic lysis procedure for B. subtilis, allowing complete degradation of late stationary phase cells grown in rich culture medium, while retaining mRNA molecules of high integrity. The applicability of this rapid lysis procedure and the hybridization method for mRNA analyses in long-term fermentations was demonstrated.
Bacillus subtilis↗
Obstetric pain medication and eventual adult amphetamine addiction in offspring.
Our purpose was to investigate whether obstetric analgesia, particularly by nitrous oxide, constitutes a risk that the infant might develop amphetamine addiction in later life. Of 200 current amphetamine addicts born between 1945 and 1966 in Stockholm, proportionately more were born at hospitals where pain medication had been administered in large doses (p less than 0.05). A blind matched comparison was made between 73 addicts and 109 non-addicted siblings by logistic regression, in which nitrous oxide administration was tested in competition with 12 other natal variables as possible confounders. The risk for amphetamine drug addiction in offspring was found to increase with duration of intermittent administration of pure nitrous oxide, i.e. it was estimated to be 5.6 times greater (95% confidence intervals 1.6-16.9, p = 0.005) when nitrous oxide had been given for greater than or equal to 4.5 h vis-à-vis less than or equal to 0.25 h. Calculated risks are probably underestimates. Results can be explained as an effect of imprinting. It is concluded that local or regional anesthesia are preferable to general anesthesia which allows substantial amounts of drugs to cross the placenta.
Adult↗
Intraphagosomal pH in alveolar macrophages after phagocytosis in vivo and in vitro of fluorescein-labeled yeast particles.
The pH in phagolysosomes of rabbit alveolar macrophages was studied using yeast particles labeled with fluorescein isothiocyanate (FITC). The yeast particles were added to the macrophages in vitro a few hours or 1 day after they had been lavaged from the lung and in vivo 3 h or 1 day before the lungs were lavaged. Intracellular pH was estimated from the ratio between the fluorescence intensity at wavelength 519 nm with excitation at wavelengths of 495 and 450 nm. In both the in vitro and in vivo experiments pH decreased significantly during the first hours after lavage, but after a few hours reached almost the day-2 levels, i.e., 4.9-5.4. The decrease in pH was related to time after lavage and not to time after phagocytosis of the particles. It is suggested that intracellular measurements of pH in alveolar macrophages should be combined with determinations of lung clearance of metal particles.
Animals↗
Uptake and metabolism of androgens by bovine spermatozoa.
Spermatozoa from bovine ejaculates and cauda epiditymidis were incubated with either tritiated 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol). Examination of the medium incubations demonstrated metabolic conversion of both DHT and 3 alpha-diol when these steriods were incubated with ejaculated sperm. In addition to this interconversion, the following metabolities were identified: 5 alpha-androstane-3 beta, 17 beta-diol, (3 beta-diol), androsterone and 5 alpha-androstane-3, 17-dione (5 alpha-A-dione). Incubations with cauda spermatozoa showed similar metabolic patterns. Androgen binding was exhibited by both sperm types. Examination of the washed cauda sperm pellet, following incubations with 3 alpha-diol showed that the incubated steroid was the most abundantly bound. DHT and 5 alpha-androst-16-en-3 alpha-ol (delta 16-3 alpha-ol1 were also detected. The major part of the radioactivity bound in the sperm pellet was identified as DHT when this steroid was used as the substrate; the remaining radioactivity consisted of 3 alpha-diol and delta 16-3 alpha-ol. Investigations of ejaculated sperm pellets gave similar results apart from the additional identification of 5 alpha-androst-16-en-3 one (delta 16-3-one) and 5 alpha-androst-16-en-3 beta-ol (delta 16-3 beta-ol (delta 16-3 beta-ol).
Androstane-3,17-diol↗
Studies of an acrosomal defect in spermatozoa from a platinum fox.
An acrosomal defect in the spermatozoa from a platinum fox was studied under light- and electron microscopy. The defect, occurring in about 40 % of the sperm cells appeared under phase-contrast and bright-field microscopy as a knob-like irregularity, mostly rounded or slightly triangular in outline and situated in the apical part of the sperm head. Electron microscopy of the sperm head revealed a marked deviation from the normal development of the acrosome. In most of the cases this structure contained a large membrane bounded vacuole filled with numerous small vesicles and some membranous material. In spermatids studied in the cap-phase, the developing acrosome exhibited a high degree of abnormal morphology, indicating that the development of the defect must have started rather early during the formation of the acrosome.
Acrosome↗
Alveolar macrophage response to yeasts and inert particles.
Interactions between alveolar macrophages (AM) from rats and a yeast with relatively high pathogenicity (Candida albicans), a yeast with low pathogenicity (Saccharomyces cerevisiae) and an inert control particle (amorphous silica) of similar diameters, 3-4 microns, were studied. Both yeasts were phagocytized significantly faster by AM than were the control particles and C. albicans significantly faster than S. cerevisiae. Quantitative nitroblue tetrazolium reduction by AM reflecting their oxidative metabolism was markedly increased in response to both fungi during the period of phagocytosis as well as 24 h after the phagocytosis. Macrophages with silica particles also showed a moderate but significant increase in oxidative metabolism 24 h after phagocytosis. Phagolysosomal pH was significantly higher for S. cerevisiae than the control particles after 3 and 24 h. pH in phagolysosomes with C. albicans tended to be higher after 3 h but was significantly lower after 24 h than in the phagolysosomes with silica particles. Both yeasts showed a considerable number (around 10%) of phagolysosomes with high pH > or = 6.5 after 3 h and a smaller percentage after 24 h. No such fraction could be seen for the control particles. Electron microscopy showed narrow passages from AM cell surface to phagolysosomes with particles. These passages might be more frequent in AM containing the yeasts and could explain the phagolysosomes with high pH.
Animals↗
Long-term effects of labor analgesia.
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Analgesia, Obstetrical↗
[Hormonal heat induction in anoestral blue fox vixens after recording ovarial and hormonal conditions].
An investigation on the ovarial status of blue fox vixens which had not come into oestrus until the 15 of April is described. The ovarial status was determined both by progesterone-analysis and by laparotomy and direct inspection of the ovaries. Afterwards, the vixens were treated with 100 I.U. PMSG (Antex, Leo) every second day for five treatments. One day after the last PMSG treatment, 200 I.U. HCG (Physex, Leo) was administered. Two days after the last treatment, the vixens were killed and two ovaries were reexamined. The investigation showed that 44% of the vixens had functioning corpora lutea in the ovaries by the time of the first inspection. One vixen had cystic degeneration of the ovaries, two had follicles, while the rest had inactive ovaries. The progesteron levels were in accordance with the findings by laparotomy. Thirty-one percent of the vixens with inactive ovaries responded to treatment with ovulation, and all of those with follicles ovulated, while no response of the vixens with corpora lutea could be registered.
Anestrus↗
[Crossing blue fox (Alopex lagopus) to red fox (Vulpes vulpes) using artificial insemination].
An experiment with breeding red fox to blue fox by artificial insemination is described. The conception rate obtained was low. Of five blue fox vixens three whelped, and average litter size was 6.5. None of the six red fox vixens whelped. Examination of semen from the hybrid males showed aspermia. The testosterone levels in the male hybrids were significantly lower than those in male blue fox, the difference was less outstanding between the hybrids and the red fox males. The histological investigation of testes of the hybrids indicated that the spermatogenesis was arrested in the later prophase of the first meiotic division.
Animals↗