A method of DNA histogram analysis by computer and its evaluation by simultaneous combination with 3H-thymidine autoradiography

A method for determining the fractions of cells in the G1, S, and G2 + M phases of the cell life cycle, by quantifying DNA histograms derived from static fluorescence cytophotometry, was evaluated by simultaneous combination with 3H-thymidine autoradiography. DNA histograms were obtained by cytofluorometry on the Feulgen-stained autoradiographs of HeLa cells, and mouse and rat hepatocytes, after DNA labelling with 3H-thymidine. The synthetic histogram determined by "sum of discrete normal curves" technique was fitted to the experimental data according to a weighted least-squares method by a desk-top computer (HP 85F). The mean relative percent deviations of estimated cell cycle phase fractions from the actual phase fractions determined directly on an autoradiograph was 6.6 +/- 3.3%.     

The Ionic Permeability Changes during Acetylcholine-Induced Responses of Aplysia Ganglion Cells

ACh-induced depolarization (D response) in D cells markedly decreases as the external Na⁺ is reduced. However, when Na⁺ is completely replaced with Mg⁺⁺, the D response remains unchanged. When Na⁺ is replaced with Tris(hydroxymethyl)aminomethane, the D response completely disappears, except for a slight decrease in membrane resistance. ACh-induced hyperpolarization (H response) in H cells is markedly depressed as the external Cl^- is reduced. Frequently, the reversal of the H response; i.e., depolarization, is observed during perfusion with Cl^--free media. In cells which show both D and H responses superimposed, it was possible to separate these responses from each other by perfusing the cells with either Na⁺-free or Cl^--free Ringer's solution. High [K⁺]₀ often caused a marked hyperpolarization in either D or H cells. This is due to the primary effect of high [K⁺]₀ on the presynaptic inhibitory fibers. The removal of this inhibitory afferent interference by applying Nembutal readily disclosed the predicted K⁺ depolarization. In perfusates containing normal [Na⁺]₀, the effects of Ca⁺⁺ and Mg⁺⁺ on the activities of postsynaptic membrane were minimal, supporting the current theory that the effects of these ions on the synaptic transmission are mainly presynaptic. The possible mechanism of the hyperpolarization produced by simultaneous perfusion with both high [K⁺]₀ and ACh in certain H cells is explained quantitatively under the assumption that ACh induces exclusively an increase in Cl^- permeability of the H membrane.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of the pond snail, Cipangopaludina malleata Reeve, were fixed in 1% osmium tetroxide, 3% permanganate, or 4% formaldehyde followed by 1% osmium tetroxide, each being buffered to pH 7.2 with Veronal-acetate or Sörensen's phosphate buffer. On the other hand, testes fixed with 4% formaldehyde adjusted to pH 7.2 with 0.075 M Na-cacodylate were incubated in Novikoff-Goldfischer medium for demonstrating thiamine pyrophosphatase, uridine or inosine diphosphatase, uridine monophosphatase or adenosine triphosphatase. The specimens incubated were postfixed in 1% osmium tetroxide buffered to pH 7.2 with Veronal-acetate buffer. Thin sections of the epoxy Epon resin-embedded tissue were stained either singly with saturated aqueous uranyl acetate or doubly with saturated aqueous uranyl acetate followed by lead citrate.In a concentric lamellar structure consisting of the granular endoplasmic reticulum in the cytoplasm of early atypical spermatids, disappearance of ribosomes attached to the outer surface of cisternae seems to have initiated at the central part of the structure, and the cisterna-attached ribosomes seem to participate in the formation of dense granules appearing in the vesicles representing the endoplasmic reticulum of atypical spermatids.The Golgi apparatus of the atypical spermatids in the advanced stages of development is composed of at least three different layers, the central part consisting of an amorphous material, the following lamellar and vesicular elements, and the peripheral fine vesicles.It has been assumed that the mechanism by which the nucleic acid, especially DNA is converted into the polysaccharide might be attributed to the function of the Golgi apparatus, because the transformation of dense granules into less dense granules as well as diphosphatase activities have been detected within the Golgi apparatus.This study was supported by Grant GM-8327-06 from the United States Public Health Service.     

Multiple determinants for the high substrate specificity of an angiotensin II-forming chymase from the human heart

Human heart chymase, a chymotrypsin-like serine proteinase that hydrolyzes the Phe8-His9 bond in angiotensin I (Ang I) to yield the octapeptide hormone angiotensin II (Ang II) and His-Leu, is the most specific, efficient Ang II-forming enzyme described. Other mammalian chymases display a much broader substrate specificity. To better define its substrate specificity, we have mapped the extended substrate-binding site of human heart chymase using Ang I analogs. The enzyme has a preference for aromatic amino acids phenylalanine, tyrosine, and tryptophan at the P1 site. At the S2 subsite there is a significant preference for proline over hydrophobic or hydrophilic amino acids. There is no clear preference for hydrophobic or hydrophilic amino acids at the S'1 and S'2 subsites, but an Ang I analog containing a P'1 proline is not hydrolyzed and one with a P'2 proline is hydrolyzed poorly. An increasing reduction in reactivity occurs when the P position amino acids in Ang I are deleted sequentially from the N terminus. An increase or decrease in the length of the His-Leu leaving group also produces a marked decrease in reactivity. No single determinant in Ang I is preeminently required for efficient catalysis, but several factors acting synergistically appear to be important. Thus, we propose that ideal substrates for human heart chymase should contain the structure nXaa-Pro-[Phe, Tyr, or Trp]-Yaa-Yaa, where n greater than or equal to 6; Xaa = any amino acid; Yaa = any amino acid except proline. This structure exists in Ang I and neurotensin, both of which are good substrates for human heart chymase. These findings indicate that the selection of the scissile bond by the extended substrate-binding site of human heart chymase is more restricted than that in other chymases.     

Redox properties of membrane-bound b-type cytochromes and a soluble c-type cytochrome of nitrate reductase in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

In order to identify the b-type cytochrome involved in the nitrate reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, the b-type cytochromes in the spheroplast membranes were characterized. Difference spectra at 77K of spheroplast membranes indicated the presence of two b-type cytochromes with a bands at 556.5 and 562 nm. Three components considered to be of the b-type cytochrome were resolved by anaerobic potentiometric titration at 560-572 nm. Their midpoint potentials at pH 7, Em,7, were - 135 mV, +40 mV and +175 nm and their approximate reduced minus oxidized maxima were determined to be at 565 nm (562 nm at 77K), 560 nm (556.5 nm) and 560 nm (556.5 nm), respectively. These values are almost the same as those reported for R. sphaeroides. The Em,7 value of the cytochrome c involved in the nitrate reductase of this denitrifier was determined to be 250 mV. A b-type cytochrome reduced with NADH and FMN was oxidized by nitrate in chromatophore membranes. The possibility that cytochrome b (Em,7 = 175 mV) is involved in the nitrate reduction is discussed.     

Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons

During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (β-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.     

Design, synthesis, and biological evaluation of novel (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids as selective inhibitors for AKR1B1

New substituted (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids were designed as the inhibitor of AKR1B1 based upon the structure of rhetsinine, a minor alkaloidal component of Evodia rutaecarpa, and twenty derivatives were synthesized and evaluated. The most active compound of the series was (2-benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7m), which showed comparable inhibitory activity for AKR1B1 (IC(50)=0.15μM) with clinically used epalrestat (IC(50)=0.1μM). In the view of activity and selectivity, the most potent compound was (2-benzyl-6-carboxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7t), which showed strong inhibitory effect (IC(50)=0.17μM) and very high selectivity for AKR1B1 against AKR1A1 (311:1) and AKR1B10 (253:1) compared with epalrestat.