DNA and protein content of mouse sperm: Implications regarding sperm chromatin structure

A variety of biochemical and histochemical techniques have been used to compare the composition of chromatin in sperm nuclei isolated from the epididymides of five mouse strains. The DNA content was determined by phosphorus analysis, deoxyribose analysis, absorption spectroscopy at 260 nm, and cytomorphometry following gallocyanine chrome alum staining. All four methods indicate that the mouse sperm nucleus contains approx. 3.3 pg DNA and that the DNA content does not vary significantly among the strains tested. Three different techniques, quantitative amino acid analysis, absorption spectroscopy at 230 nm, and sperm head density analysis in cesium chloride, were used to determine the protein content. Sperm nuclei from each strain of mouse were found to have a protein to DNA ratio of 0.9 and a chromatin protein content of 3 pg/nucleus. Comparisons of the basic proteins by disc gel electrophoresis demonstrate that the sperm nuclei contain only protamine and lack significant levels of somatic histones or transition proteins. The sperm from each strain contained both mouse protamine variants and the relative distribution of the two proteins did not appear to differ among strains. Using this information, we have been able to draw certain conclusions regarding DNA-protamine interactions and the mode of DNA packaging in the sperm nucleus. The most important of these is that the DNA in the mouse sperm nucleus cannot be packaged in nucleosomes. The protamines in sperm chromatin do not function as structural proteins, providing a subunit core around which the DNA is wrapped, but appear to completely neutralize the phosphodiester backbone of the DNA molecule, thereby minimizing the repulsion between neighboring segments of DNA and allowing it to be condensed into a biochemically inactive particle of genetic information.     

Correlations between epidermal cell structure and endogenous hormone titers during the fifth larval instar of the tobacco hornworm, Manduca sexta

Epidermal cell morphology and cuticle production in Manduca sexta are directly influenced by both ecdysterone and juvenile hormone. Up to day 6 of the last larval instar, post-molt endocuticle is continuously deposited even though cells undergo a partial and temporary separation from the overlying cuticle at the time when a small ecdysteroid peak is detected (approximately day 3.5). At about days 6--7 when another, larger ecdysteroid peak is present, apolysis occurs accompanied by the appearance of edcysial droplets. Following apolysis, layers of pupal cuticle are deposited. Increased quantities of rough endoplasmic reticulum characterize the epidermis at times of peak endocuticle deposition (day 3, larval cuticle; day 9, pupal cuticle). Dense pigment inclusions are found in epidermis from the day of ecdysis to the last larval instar until they are eliminated 5 days later. These dense bodies migrate from cell apex to base in the absence of juvenile hormone (or in the presence of a negligible amount of juvenile hormone) and probably contain insecticyanin.     

In vitro metabolism of possible ecdysone precursors by the prothoracic glands of the tobacco hornworm, Manduca sexta

3 beta, 14 alpha-Dihydroxy-5 alpha-7-en-6-one (5 alpha-ketodiol) (1) is metabolized by the prothoracic glands to 2,22-dideoxy-5 alpha-ecdysone (4) and 2-deoxy-5 alpha-ecdysone (3) but not to ecdysone (5) or any other 5 beta-metabolites. Similarly, 3 beta,5 alpha,14 alpha-trihydroxy-cholest-7-en-6-one (5 alpha-ketotriol) (8) is hydroxylated at C-22 and C-25 (9,10) of the side chain. However, 3 beta,14 alhpa-dihydroxy-cholesta-4,7-diene-6-one (ketodienediol) (11) is not metabolized. The absence of 2 beta-hydroxymetabolites for substrates (1) and (8) implies that hydroxylation at C-2 can occur only when the A-B rings are cis fused (5 beta-configuration). By contrast, the enzyme complexes that introduce hydroxyls at C-22 and C-25 do not exhibit a preference for cis over trans fusion and appraently cannot recognize the planar A-B ring configuration.     

Effects of yohimbine on squid axons.

Yohimbine, an indolealkylamine alkaloid, reduces the amplitude of the sodium current in the squid giant axon. For doses that reduce sodium current amplitude by up to 50%, there is no significant change in the kinetics or in any of the voltage-dependent parameters associated with sodium channels. The effective equilibrium constant for yohimbine binding to the sodium channel is 3 x 10(-4) M. Repetitive depolarizing pulses increase the inhibition of squid axon sodium current by yohimbine. This use-dependent inhibition is enhanced by increasing the concentration of yohimbine, by increasing the frequency of pulsing, and by increasing the magnitude or the duration of depolarization. It is reduced by hyperpolarizing prepulses. This behavior can be explained by a model wherein yohimbine binds more readily to open sodium channels than to closed sodium channels and wherein the Hodgkin-Huxley kinetic parameters are modified by the binding of the drug. This type of model may also explain the tonic and use-dependent inhibition previously described by others for local anesthetics.     

Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta

The titer of ecdysone in whole animal extracts of Manduca sexta was determined by radioimmunoassay during the fifth (last) larval instar, pharate pupal development and pupation. A subtle peak in ecdysone concentration was noted at day 4 (just prior to the onset of the wandering stage) and a second and greater peak at day 8.5 (coincident with pharate pupal development). The titer fluctuations during development were a result of changes in tissue ecdysone and not of alterations in the ecdysone content of the gut. When prothoracic gland secretory activity was analyzed in vitro at the same stages, the most rapid rate of α-ecdysone secretion was shown to occur on day 7 (one day prior to the peak in whole-animal ecdysone concentration). An earlier peak in prothoracic gland activity may occur at day 4–5. Thin layer and gas-liquid chromatographic analyses revealed developmental changes in the ratio of β:α-ecdysone in hemolymph and whole-animal extracts. It is suggested that the steroid-hydroxylating capacity of the insect increases during the instar.     

Effect of concanavalin A on membrane-bound enzymes from mouse lymphocytes

The ionic influence and ouabain sensitivity of lymphocyte Mg²⁺-ATPase and Mg²⁺-(Na⁺ + K⁺)-activated ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5′-nucleotidase and Mg²⁺-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of (Na⁺ + K⁺)-ATPase was located inside the membrane.Concanavalin A induced an early stimulation of Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase both on intact cells and purified plasma membranes. In contrast, 5′-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg²⁺-ATPase activity was 3–5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20 %). (Na⁺ + K⁺)-ATPase activity was undetectable in thymocytes. However, in spleen lymphocytes (Na⁺ + K⁺)-ATPase activity can be detected and was 30 % increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg²⁺-ATPase, in lymphocyte stimulation.     

Modification of arginine and lysine in proteins with 2,4-pentanedione.

Primary amines react with 2,4-pentanedione at pH 6-9 to form enamines, N-alkyl-4-amino-3-penten-2-ones. The latter compounds readily regenerate the primary amine at low pH or on treatment with hydroxylamine. Guanidine and substituted guanidines react with 2,4-pentanedione to form N-substituted 2-amino-4,6-dimethylpyrimidines at a rate which is lower by at least a factor of 20 than the rate of reaction of 2,4-pentanedione with primary amines. Selective modification of lysine and arginine side chains in proteins can readily be achieved with 2,4-pentanedione. Modification of lysine is favored by reaction at pH 7 or for short reaction times at pH 9. Selective modification of arginine is achieved by reaction with 2,4-pentanedione for long times at pH 9, followed by treatment of the protein with hydroxylamine. The extent of modification of lysine and arginine side chains can readily be measured spectrophotometrically. Modification of lysozyme with 2,4-pentanedione at pH 7 results in modification of 3.8 lysine residues and less than 0.4 arginine residue in 24 hr. Modification of lysozyme with 2,4-pentanedione at pH 9 results in modification of 4 lysine residues and 4.5 arginine residues in 100 hr. Treatment of this modified protein with hydroxylamine regenerated the modified lysine residues but caused no change in the modified arginine residues. One arginine residue seems to be essential for the catalytic activity of the enzyme.     

Mitral stenosis with posterior diastolic movement of posterior leaflet.

The echocardiographic diagnosis of mitral stenosis depends in part on the demonstration of abnormal posterior leaflet movement to distinguish it from other conditions that similarly affect anterior leaflet motion. In mitral stenosis the posterior leaflet has been shown to move anteriorly in diastole rather than in the normal posterior direction. A patient presented with clinical evidence of moderate mitral stenosis. The anterior leaflet echo was typical but the posterior leaflet showed posterior diastolic movement. At catheterization moderate mitral stenosis was confirmed. To our knowledge this is the first report of the echocardiographic demonstration of posterior diastolic movement of the posterior mitral leaflet in documented mitral stenosis.