Immunocytochemical localization of L-α-hydroxyacid oxidase in dense bar of dumb-bell-shaped peroxisomes of monkey kidney

Localization of the B of L-hydroxyacid oxidase (HOX-B) in monkey kidney peroxisomes was investigated by immunoelectron microscopic techniques. Kidneys of Japanese monkeys,Macaca fuscata, were fixed with 4% paraformaldehyde+0.25% glutaraldehyde and embedded in LR White resin. Thin sections were stained for HOX-B and catalase by the immunogold technique. HOX-B was localized in the marginal plates of normal peroxisomes and the dense bar of dumb-bellshaped peroxisomes. Catalase was detected in the matrix of normal peroxisomes and in the terminal dilatations of dumb-bell-shaped peroxisomes. There were no gold particles indicating presence of catalase associated with the marginal plates or with the dense bars. Immunoblot analysis of monkey kidney homogenate showed that HOX-B has a molecular mass of 42 kDa that was slightly larger than that of rat kidney HOX-B (39 kDa). The results show that the dense bar of dumb-bell-shaped peroxisomes in monkey kidney is composed of at least HOX-B and is a variation of the marginal plates.     

Different metastatic modes of malignant melanoma implanted in the ear of young and old mice

Summary An attempt has been made to determine (a) whether aging plays an important role in resistance against metastasis and (b) whether dithiothreitol, an effective in vitro mitogenic potentiator of splenic cells of young and old mice, can modulate the occurrence of pulmonary metastasis. B16-F10 melanoma cells were injected into the outer ear of young and old female C57BL/6 mice; and the growth of the primary tumor, the palpable size of the cervical lymph node, and the number of lung metastases were then determined at various intervals. The ear was amputated when the primary tumor reached 4 mm in mean diameter. The following results were obtained. (a) The growth rate of the primary tumor in young mice is comparable to that in old mice. (b) Enlargement of the cervical lymph node occurs earlier in old than in young mice. (c) Old mice are more vulnerable to pulmonary metastases, but small metastasized pulmonary colonies are more prominent in old than in young mice. (d) Dithiothreitol (100 g) injected every 2 days after the inoculation of tumor cells is effective in reducing the incidence of pulmonary metastases in old mice.     

Computer simulation and interpretation of45Ca efflux profile patterns

Summary Stimulations or inhibitions by various agents of⁴⁵Ca efflux from prelabeled cells or tissues display distinct and reproducible profile patterns when the results are plotted against time as fractional efflux ratios (FER). FER is the fractional efflux of⁴⁵Ca from stimulated cells divided by the fractional efflux from a control unstimulated group. These profile patterns fall into three categories: peak patterns, exponential patterns, and mixed patterns. Each category can be positive (stimulation) or negative (inhibition). The interpretation of these profiles is difficult because⁴⁵Ca efflux depends on three variables: the rate of calcium transport out of the cell, the specific activity of the cell compartment from which the calcium originates, and the concentration of free calcium in this compartment. A computer model based on data obtained by kinetic analyses of⁴⁵Ca desaturation curves and consisting of two distinct intracellular pools was designed to follow the concentration of the traced substance (⁴⁰Ca), the tracer (⁴⁵Ca), and the specific activity of each compartment before, during, and after the stimulation or the inhibition of calcium fluxes at various pool boundaries. The computer model can reproduce all the FER profiles obtained experimentally and bring information which may be helpful to the interpretation of this type of data. Some predictions of the model were tested experimentally, and the results support the views that a peak pattern may reflect a sustained change in calcium transport across the plasma membrane, that an exponential pattern arises from calcium mobilization from an internal subcellular pool, and that a mixed pattern may be caused by a simultaneous change in calcium fluxes at both compartment boundaries.     

On the antisymmetry of the amino acid code table

Antisymmetry of the amino acid code table in terms of codon degeneracy is pointed out, and it is related to a physico-chemical problem of codon-anticodon interaction energy. A strong negative correlation between molecular weight of an amino acid and its codon degeneracy is pointed out, and its implication to the origin of the amino acid code table is discussed. Finally, an earlier form of the amino acid code table is proposed.     

Block of acid secretion by amytal and its partial reversal by menadione with ascorbate in the gastric mucosa of the guinea-pig

The effect of amytal on energy metabolism and acid secretion in an isolated gastric mucosa of the guinea-pig were studied. Determination of adenine nucleotides, creatine phosphate, pyruvate and lactate in the gastric mucosa showed that amytal depressed the levels of ATP, creatine phosphate and energy charge with elevation of the AMP and pyruvate levels. This treatment inhibited concomitantly acid secretion and active chloride transport detected by short circuit current. The addition of menadione with ascorbate to the medium in the presence of amytal partially restored ATP and energy charge levels and also induced a partial recovery of acid secretion and active chloride transport. These results suggest that ATP is a direct energy donor for acid secretion in the gastric mucosa of the guinea-pig.     

Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver

There are two 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat liver, one in mitochondria (type I enzyme), and another in peroxisomes (type II enzyme). In a series of the studies on the properties and the physiological roles of fatty acid oxidation systems in both organelles, the two enzymes were purified and compared for their properties. The final preparations obtained were judged to be homogeneous based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Type I enzyme was composed of two identical subunits of molecular weight of 32,000, whereas type II enzyme was a monomeric enzyme having a molecular weight of 70,000–77,000. These subunit structures were confirmed by the results of fluorescence studies. Both enzymes were different in amino acid compositions, especially in the contents of tryptophan and half-cystine. Antibodies against them formed single precipitin lines for the corresponding enzymes, but not for the others when subjected to an Ouchterlony double-diffusion test. The K_m values of type II enzyme for various substrates were lower than those of type I enzyme except those for acetoacetyl-CoA. As for 3-hydroxyacyl-CoA substrates, both enzymes had lower K_m's for longer-chain substrates. The V for the substrates of C₄C₁₀ were similar for each enzyme, though the value of type II enzyme for C₁₀ substrate was rather lower. The results of fluorescence studies suggested that their dissociation constants for NADH were lower and those for NAD⁺ were higher at lower pH. Both enzymes were specific to l-form of 3-hydroxyacyl-CoA substrate. The optimal pH of the forward reaction of type I and type II enzymes was 9.6 and 9.8, and of the reverse reaction, 4.5 and 6.2, respectively. From these results they were concluded to be completely different enzymes.     

Achromobacter protease I-catalyzed conversion of porcine insulin into human insulin

We have established a procedure for converting porcine insulin into human insulin using a serine protease from $\text{Achromobacter}\text{lyticus}$ M497-1 which shows unique specificity against lysine residues on the carboxyl side of the splitting point. Desalanine-(B30)-insulin (DAI) was prepared by digestion of porcine insulin with $\text{Achromobacter}$ protease. The coupling between DAI and Thr-OBu^t was performed by the same enzyme at pH 6.5 with a large excess of the amine component (Thr-OBu^t) in the presence of high concentrations of organic co-solvents. The highest yield was 85% by 20 h reaction at 37°C. The synthesized [Thr-OBu^t-B30]-insulin was isolated, then deprotected with trifluoroacetic acid in the presence of anisole to obtain semisynthetic human insulin.     

Relationship between hormone content and autonomy in various autonomous tobacco cells cultured in suspension

Various autonomous cultured tobacco cells including crown gallwere examined for their contents of growth regulators by meansof Avena curvature test, cell-division induction test, and tobaccopith callus test. The crown gall cells derived from cv. Hicks produced auxin andcytokinin in the high levels of 300–500 µg IAA equivalentsand 40–80 µg kinetin equivalents per kg, respectively.The major auxin was identified as indole-3-acetic acid basedon mass spectrometry and gas chromatography. These cells alsoproduced methyl indole-3-acetate as a minor component. One ofthe cytokinins was identified as ribosyl-trans-zeatin by meansof both gas chromatography-mass spectrometry and high performanceliquid chromatography. Auxin and cytokinin activities were not detected in the followingthree suspension cultured tobacco cells: cells requiring neithercytokinin nor auxin derived from the callus of N. tabacum cv.Bright Yellow and cells requiring auxin but not cytokinin derivedfrom the calluses of cv. Bright Yellow and cv. Hicks. Theirauxin and cytokinin contents per kg were less than 1 µgIAA equivalent and less than 0.1 µg kinetin equivalent,respectively. The results obtained in this study indicate that enhanced hormonalcontent is not the only reason for autonomous growth. (Received August 16, 1979; )     

Distribution of growth regulators in relation to the light-induced geotropic responsiveness in Zea roots

Growth regulators were measured in extracts from the upper and lower halves of 7-mm apical segments of horizontally oriented, red-light-irradiated and non-irradiated roots of Zea mays L. cv. Golden Cross Bantam 70 which exhibit a georesponse only after an exposure to light. Abscisic acid (ABA) was measured by gas-liquid chromatography, auxin (indole-3-acetic acid, IAA) by the Avena straight-growth assay, and an unidentified growth inhibitor by a Zea root-growth assay. The ratio of ABA in the upper and lower halves was 1.6 in the irradiated roots and 1.0 in the non-irradiated ones. The total amount of ABA after irradiation was increased by a factor of ca. 1.8. The ratio of IAA in the upper and lower halves of irradiated and non-irradiated roots was 1:3.4 and 1:2.9, respectively. The content (or activity) of an unidentified growth inhibitor was highest in the lower halves of horizontally oriented roots which had been irradiated with red light. The unidentified growth inhibitor, rather than IAA or ABA, may be the major factor in the light-induced geotropic responsiveness in Zea roots.     

A self-organizing neural network model for the development of complex cells

On the basis of recent neurophysiological findings on the mammalian visual cortex, a selforganizing neural network model is proposed for the understanding of the development of complex cells. The model is composed of two kinds of connections from LGN cells to a complex cell. One is direct excitatory connections and the other is indirect inhibitory connections via simple cells. Inhibitory synapses between simple cells and complex cells are assumed to be modifiable. The model was simulated on a computer to confirm its behavior.