Argyrophilic nucleolar organizer regions

Silver staining techniques developed to demonstrate argyrophilic nucleolar organizer regions (Ag-NORs) have been widely applied in a variety of cell kinetic studies, using the mean number of AgNORs in tumour cells as a marker for malignancy of certain types of neoplasms. However, the AgNOR techniques currently available are not entirely satisfactory, as unspecific silver precipitates readily form in the sections. On the other hand, the contrast staining, may be so weak as to render identification of the AgNORs difficult. In the present study, some of the key factors influencing the outcome of AgNOR staining were evaluated in a more systematic way. A modified AgNOR staining procedure is now proposed, giving highly contrasting AgNORs with minimal unspecific silver precipitation, thus facilitating both manual and computerized counting. The new technique involves the use of microwave irradiation in order to shorten the processing time, the use of gelatin as a protective colloid, and a Farmer's solution to optimize the specificity of the technique.     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.     

Serum relaxin levels in prostaglandin E2 induced abortions

Relaxin, a peptide hormone produced only by the corpus luteum of pregnancy, can be used as a marker of luteal function in human pregnancy. Serum immunoreactive relaxin levels were measured serially in six women having second trimester abortions induced with intravaginal prostaglandin E2 (PGE₂) suppositories. All patients aborted within 17 hours of the first suppository. No significant changes were detectable in serum relaxin levels in any of the patients. It is concluded that PGE₂ does not interfere with the corpus luteum's ability to secrete relaxin in the second trimester of human pregnancy.     

Morphological and histochemical organization of the flexor carpi radialis muscle in the cat.

Most studies concerning the structure and function of skeletal muscle have utilized the hind limb of the experimental animal. However, it has been shown that the number of behavioral tasks performed by the cat's forelimb is greater than that of the hind limb. In addition, the forelimb muscles exhibit a functional complexity not observed in hind-limb musculature. The purpose of this study was to investigate the distribution of fast-twitch and slow-twitch muscle fibers and muscle spindles in the flexor carpi radialis muscle (FCR) and to correlate the distributional patterns in these structures with muscle tendon architecture and muscle function. It was found that the FCR, a wrist flexor, contains 37% slow-twitch fibers and 63% fast-twitch fibers. However, the slow-twitch fibers were concentrated in the deep region located between the tendons of origin and insertion, while the fast-twitch-glycolytic fibers were concentrated more peripherally. Muscle spindles were associated with the slow-twitch region and were never found in the region containing high concentrations of fast-twitch-glycolytic fibers. Fast-twitch-oxidative-glycolytic fibers were uniformly distributed throughout the muscle. It is proposed that the association of muscle spindles with slow-twitch fibers and the differential distribution of muscle fibers into slow-twitch and fast-twitch regions might allow these regions to function independently of one another when called upon to perform complex behavioral tasks.     

The isolation and characterization of TabR bacteria: Hosts that restrict bacteriophage T4 rII mutants

Summary We have isolated mutants of Escherichia coli B (called TabR) that restrict the growth of bacteriophage T4 rII mutants at high temperature. TabR strains lysed very rapidly after infection with rII mutants, and no progeny phage were produced. T4⁺-infected TabR cells also lysed quickly, but the cells remained intact long enough to give a small burst. We have selected pseudorevertants of rII deletion mutants that grow on TabR at high temperature; tk (thymidine kinase) is a component of one class of these pseudorevertants.T4 strains harboring mutations in genes 12, 16, 25, 34, 36, 45 and 63 were also specifically restricted on TabR strains at high temperature. Bacteriophages T2, T4, T5, T6, and T7 grew normally on TabR, while , 80, and P1 failed to grow at any temperature. The most restrictive TabR strains were auxotrophic for methionine at high temperature, and most spontaneous Met⁺ revertants had also lost the ability to restrict rII mutants, suggesting that the TabR phenotype and methionine auxotrophy result from the same mutation.Although the mechanism by which TabR strains exert their restriction has not been determined, one model is described. The potential uses of these and similar strains is discussed.     

Epithelial cell volume modulation and regulation

Summary Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cell swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cell shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described, and a number of experiments are discussed in which cell volume determination provided significant new information about the dynamic behavior of epithelia. The mechanism of volume regulation of epithelial cells exposed to anisotonic bathing solution is discussed and shown to involve the transient stimulation of normally dormant ion exchangers in the cell membrane.     

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium . This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO₂ concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum . Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO_2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10⁻⁶ g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10⁻⁶ g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.     

The effect of pH on the oxygen kinetics of cytochrome c oxidase proteoliposomes

The effect of pH on the oxygen kinetics of cytochrome c oxidase incorporated into phospholipid vesicles is studied. The pH profiles of the oxygen kinetics of energized and deenergized oxidase vesicles are similar. An effect of pH on the slope of the reciprocal plot of rate against oxygen concentration is observed, and this may indicate that protons are involved in the rate limiting step of the reaction between oxygen and reduced oxidase. In contrast to the pH dependence of the oxygen kinetics, the binding of CO to the oxidase is not pH dependent.