Instability of plasmid DNA sequences: Macro and micro evolution of the antibiotic resistance plasmid R6-5

Summary Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possibly other replicons occur more frequently than has hitherto been appreciated. The sequence changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existed in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous,and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.     

Class II tubulin, the major brain beta tubulin isotype is polyglutamylated on glutamic acid residue 435.

Protein sequencing shows that porcine brain tubulin retains the N-terminal sequences of alpha and beta tubulin after a mild treatment with subtilisin. C-terminal peptides released by subtilisin were purified and characterized by automated Edman degradation and mass spectrometry. We confirm the polyglutamylation of alpha tubulin on glutamic acid residue 445 reported by others and show in addition that class II beta tubulin, the major beta tubulin isotype of adult brain, is also polyglutamylated. The substitution is restricted to glutamic acid residue 435. Thus all major tubulin isotypes of adult brain are subjected to polyglutamylation.     

Radial Turgor and Osmotic Pressure Profiles in Intact and Excised Roots of Aster tripolium: Pressure Probe Measurements and Nuclear Magnetic Resonance-Imaging Analysis

High-resolution nuclear magnetic resonance images (using very short spin-echo times of 3.8 milliseconds) of cross-sections of excised roots of the halophyte Aster tripolium showed radial cell strands separated by air-filled spaces. Radial insertion of the pressure probe (along the cell strands) into roots of intact plants revealed a marked increase of the turgor pressure from the outermost to the sixth cortical layer (from about 0.1-0.6 megapascals). Corresponding measurements of intracellular osmotic pressure in individual cortical cells (by means of a nanoliter osmometer) showed an osmotic pressure gradient of equal magnitude to the turgor pressure. Neither gradient changed significantly when the plants were grown in, or exposed for 1 hour to, media of high salinity. Differences were recorded in the ability of salts and nonelectrolytes to penetrate the apoplast in the root. The reflection coefficients of the cortical cells were approximately 1 for all the solutes tested. Excision of the root from the stem resulted in a collapse of the turgor and osmotic pressure gradients. After about 15 to 30 minutes, the turgor pressure throughout the cortex attained an intermediate (quasistationary) level of about 0.3 megapascals. This value agreed well with the osmotic value deduced from plasmolysis experiments on excised root segments. These and other data provided conclusions about the driving forces for water and solute transport in the roots and about the function of the air-filled radial spaces in water transport. They also showed that excised roots may be artifactual systems.     

Survival and replication of male-specific bacteriophages in molluscan shellfish.

The survival and replication of male-specific bacteriophages in hard-shelled clams (Mercenaria mercenaria) and their homogenates were examined to further assess their potential utility as indicator organisms. Trials were conducted in the presence and absence of a suitable bacterial host, Escherichia coli HS[pFamp]R. Results of this study demonstrated that male-specific bacteriophages were unable to replicate in hard-shelled clams, with or without added host cells. In addition, the densities of these bacteriophages were stable for up to 7 days in shellfish held at ambient seawater temperatures (less than 25 degrees C). Evidence of replication, although not observed in live shellfish, was found to occur in temperature-abused shellfish homogenates and supernatants, but only when a suitable bacterial host was present.     

Circadian blood pressure rhythm in primary and secondary hypertension.

Circadian blood pressure variability was recorded in patients with primary hypertension and with different forms of secondary hypertension using ambulatory 24-h blood pressure measurement. A group of 20 patients with different forms of secondary hypertension was compared with a matched group of patients with primary hypertension. Although the mean 24-h blood pressure was not different between the two groups, the patients with secondary hypertension had significantly higher systolic blood pressure during sleep and higher systolic and diastolic blood pressure in the early morning, compared with the primary hypertension group. This nocturnal blood pressure fall was then investigated in various groups of patients with different forms of secondary hypertension and compared with normotensives and patients with primary hypertension. Patients with mild primary hypertension (n = 152) and with severe primary hypertension (n = 30) had the same blood pressure fall (14-16 mm Hg systolic and diastolic) during the night (23:00-05:00 h) as normotensives (n = 20). However, in patients with renoparenchymal hypertension (n = 29), renovascular hypertensions (n = 20), hyperaldosteronism (n = 6), and hyperthyroidism (n = 14), the nocturnal blood pressure fall was significantly (p less than 0.01) reduced. One patient with coarctation of the aorta and nine patients with primary hyperparathyroidism and elevated blood pressure had a normal circadian blood pressure profile with a normal nocturnal blood pressure fall. The heart rate decrease during the night was equal in all patient groups. Ambulatory blood pressure measurement allows blood pressure recording under everyday conditions, including nighttime. In primary hypertension the blood pressure variability exhibits the same circadian variation as in normotension, showing a marked nocturnal fall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical demonstration of villin in the normal human pancreas and in chronic pancreatitis

Summary Human pancreatic tissue was investigated by immunohistochemistry using a polyclonal antibody against the actin binding protein villin, which participates in the formation of actin filament bundles in the microvilli. In cells of the different parts of the pancreatic duct system as well as in the acinar cells villin immunoreactivity was located mainly at the apical cell surface. This was confirmed by the ultrastructural demonstration of microvilli on the surface of duct and acinar cells, which exhibited the typical actin bundles. In chronic pancreatitis the staining for villin in duct-like structures of degenerative pancreatic tissue was irregular or even absent. This correlated with the electron microscopic observation of duct-like structures known as tubular complexes composed of cells devoid of microvilli at the apical cell surface. At the light microscopical level degenerative structures without lumen and of unknown origin showed a strong staining for villin at their basal cell surface.     

ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane. Role of the ATP-dependent transport system for glutathione S-conjugates

The liver is the major organ which eliminates leukotriene C4 (LTC4) and other cysteinyl leukotrienes from the blood circulation into bile. Transport of LTC4 was studied using inside-out vesicles enriched in canalicular and sinusoidal membranes from rat liver. The incubation of canalicular membrane vesicles with [3H]LTC4 in the presence of ATP resulted in an uptake of LTC4 into vesicles. The initial rate of ATP-stimulated LTC4 uptake was about 40-fold higher in canalicular than in sinusoidal membrane vesicles. When liver plasma membrane vesicles were incubated in the absence of ATP, an apparent transient uptake of LTC4 was observed which was temperature-dependent and not affected by the osmolarity. This indicates that LTC4 was bound to proteins on the surface of plasma membrane vesicles. Two proteins with relative molecular weights of 17,000 and 25,000 were detected by direct photoaffinity labeling as major LTC4-binding proteins. One protein (Mr 25,000) was ascribed to subunit 1 (Ya) of glutathione S-transferase which was associated with the membrane. LTD4, LTE4, N-acetyl-LTE4, and omega-carboxy-N-acetyl-LTE4 were also transported into liver plasma membrane vesicles in an ATP-dependent manner with initial rates relative to LTC4 (1.0) of 0.46, 0.11, 0.35, and 0.22, respectively. Mutual competition between the cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione for uptake indicated that they are transported by a common carrier. Apparent Km values of the transport system for LTC4, LTD4, and N-acetyl-LTE4 were 0.25, 1.5, and 5.2 microM, respectively. The ATP-dependent transport of LTC4 into vesicles was not inhibited by doxorubicin, daunorubicin, or verapamil, or by the monoclonal antibody C219, suggesting that the transport system differs from P-glycoprotein. Liver plasma membrane vesicles prepared from mutant rats deficient in the hepatobiliary excretion of cysteinyl leukotrienes lacked the ATP-dependent transport of cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione. These results demonstrate that the ATP-dependent carrier system is responsible for the transport of cysteinyl leukotrienes and glutathione S-conjugates from the hepatocytes into bile.     

Inhibitory effect of irradiation products of cyclic adenosine-monophosphate on the relaxing ability of smooth muscle preparations

Summary In a previous publication it has been shown that the radiation induced physiological inactivation of dibutyryl-cAMP was far more pronounced than the chemical modification (Schachinger et al. 1981). In this paper it will be shown, that irradiation of dib-cAMP in solution resulted in the formation of monobutyryl cAMP and other not yet completely identified hydroxylated derivatives as well as a decomposition of the purine structure. Moreover, irradiated dib-cAMP inhibited the physiological activity of not irradiated dib-cAMP on the smooth muscle. From the data an effectiveK _m -value for dib-cAMP of 2.4 × 10^–5 M was determined and an effectiveK _I -value of 1.3 × 10^–6 M was found for the irradiation products, i.e., a tenfold affinity of the latter compared to unirradiated dib-cAMP. The results are discussed with respect to a better understanding of dose-response curves for chemical and physiological inactivation.