Effect of disulfiram on turnover of 5-hydroxytryptamine in rat brain.

Effect of disulfiram on 5-hydroxytryptamine (5-HT) turnover was studied. Treatment with disulfiram caused increases in 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in rat brain. Under the same condition, activity of brain mitochondrial aldehyde dehydrogenase was reduced, however, supernatant aldehyde dehydrogenase and monoamine oxidase activities remained unchanged. Disulfiram had no effect on synthesis rate of 5-HT, but decreased metabolism of 5-HT. Moreover, disulfiram impaired transport of 5-HIAA from brain tissue.     

Effect of tryptophol on pentylenetetrazol and picrotoxin induced convulsion in mice.

The effect of treatment of mice with tryptophol (TOL), a neutral metabolite of tryptophan, on drug-induced convulsion was studied. TOL effectively suppressed both pentylenetetrazol and picrotoxin induced convulsion. Diphenylhydantoin (DPH), a potent inhibitor of brain aldehyde reductase, significantly reduced the anticonvulsant effect of TOL, however, TOL level in brain of DPH-treated mice was rather higher than that of control one. These results strongly suggest that the manifestation of the anticonvulsant effect of TOL requires the conversion of TOL to its active metabolite, indoleacetaldehyde.     

Epidermal protein metabolism directed toward keratinization by hydrocortisone in the chick embryonic skin growing in a chemically defined medium.

The metabolism of the epidermal structural and nonstructural proteins was studied in hydrocortisone-induced in vitro keratinization of 13-day chick embryonic skin growing in a chemically defined medium. The protein metabolism of the epidermis was examined by determining the amounts of radioactivity incorporated into the fractions of reduced, S-carboxymethylated epidermal proteins (SCMEp) which were separated by polyacrylamide gel electrophoresis. A group of high molecular weight, glycine-rich derivatives of the epidermal fibrous protein called SCMEpA were found to be actively synthesized in the hydrocortisone-treated epidermis alone, while a group of undefined protein derivatives called SCMEpX was shown to be synthesized exclusively in the nontreated epidermis. Chase-culture of the prelabeled explants revealed that hydrocortisone accelerated the degradation of general proteins including SCMEpX while SCMEpA remained metabolically stable throughout the culture. Actinomycin D did not significantly affect the hydrocortisone-induced synthesis of SCMEpA but greatly inhibited that of SCMEpX of the nontreated epidermis, suggesting the induction by the steroid of relatively stable mRNA for SCMEpA. From these findings, it is concluded that hydrocortisone directed the cultured epidermis toward keratinization through acceleration of the synthesis of epidermal structural proteins and degradation of other proteins.     

Phycobilisome composition and possible relationship to reaction centers

The photosynthetic apparatus was studied in Anacystis nidulans wild type and in a spontaneous pigment mutant 85Y which had improved growth in far-red light (greater than 650 nm). Two phycobiliproteins, C-phycocyanin (lambda max 625) and allophycocyanin (lambda max 650), were present in a molar ratio of approximately 3:1 in the wild type and approximately 0.4:1 in the mutant. Phycobilisomes of wild type cells were larger (57 X 30 nm) than those of the mutant 85Y (28 X 15 nm). In the mutant they seemed to consist primarily of the allophycocyanin core. Fluorescence emission maxima of wild type and mutant 85Y phycobilisomes were at 680 nm (23 degrees C) and 685 nm (-196 degrees C). Excitation maxima of phycobilisomes were at 630 and 650 nm for the wild type and the mutant 85Y, respectively. The phycobilisomes of wild type cells whether grown in white or far-red light had the same size and pigment composition. A typical wild type cell in white light had a thylakoid area of 22.8 microns 2, but in far-red light the area was reduced to 13.5 microns 2, which was close to that of 85Y at 13.6 microns 2. Chlorophyll molecules per cell decreased in far-red light from 1.1 X 10(7) in wild type (white light) to 4.5 X 10(6) in mutant 85Y (far-red). The number of phycobilisomes per cell (approx 2 X 10(4)), calculated from the phycobiliprotein content and phycobilisome size, was about the same in wild type (white light) and mutant 85Y (far-red light), but the number of phycobilisomes per unit area of thylakoid was significantly greater in mutant 85Y than in wild type. The present results suggest that the phycobilisomes are linked with reaction centers and that the PSII complement (photo-system II and phycobilisome) was fully maintained in far-red light.     

Differential effect of UV irradiation on induction of intragenic and intergenic recombination during commitment to meiosis in Saccharomyces cerevisiae

A comparison was made between the induction of intragenic and intergenic recombinations during meiosis in a wild-type diploid of Saccharomyces cerevisiae. Under non-irradiated normal conditions, production of both intragenic and intergenic recombinants greatly increased in the cells with commitment to meiosis. The susceptibility of cells to the induction of both the spontaneous intra- and intergenic recombinations in meiotic cells was similar. However, under condition of UV irradiation, there were striking differences between intra- and intergenic recombinations. Susceptibility to induction of intragenic recombination by UV irradiation was not enhanced at meiosis compared with mitosis, and was not altered through commitment to meiotic processes. In contrast, however, susceptibility to the induction of intergenic recombination by UV irradiation was enhanced markedly during commitment to meiosis compared with mitosis. Genetic analysis suggested that the enhanced susceptibility to recombination during meiosis is specifically concerned with reciprocal-type recombination (crossing-over) but not non-reciprocal-type recombination (gene conversion). Hence it is concluded that the meiotic process appears to be intimately concerned with the mechanism(s) of induction of recombination, especially reciprocal-type recombination.     

Coordination chemical studies on metalloenzymes. IX. Properties of the ternary complex between cobalt(II)-bovine carbonic anhydrase and bidentate ligands

The spectrum, thermodynamic parameters, and proton longitudinal relaxation time of the ternary complex between various bidentate ligands (2-pyridinecarboxylate, 2-quinolincarboxylate, 8-quinolinecarboxylate, and 2-pyridylacetate) and cobalt(II)-bovine carbonic anhydrase were measured to clarify the nature of the ternary complex. The formation constants of the ternary complexes of bidentate ligands are in the order of (2-pyridinecarboxylate ? 8-quinolinecarboxylate ? 2-quinolinecarboxylate ≈2-pyridylacetate). The degree of the shift of the band characteristic of five-coordinate species at 13-15 kcm^-1 (cm^-1 × 10^-3) and that of the higher energy band at 21–22 kcm^-1 decrease almost in the same order. These results are explained on the basis of the contribution of the bond formation between the nitrogen atom of the heterocyclic ring of ligands and cobalt ion. The formation constants of the ternary complex of bidentate ligands were compared to the stability constants of various ligands with a cobalt ion but there is no correlation in these values. The rate constant of break-up of the ternary complex was discussed on the coordination geometry of the ternary complex on the basis of the degree of the distortion.     

Isolation and characterization of PZ-peptidase in developing rat brain

Changes in the activity of a collagen peptidase, PZ-peptidase, acting on a synthetic substrate [4-Phenylazobenzyloxycarbonyl(PZ)-l-Pro-l-Leu-Gly-l-Pro-l-Arg] for bacterial collagenase were examined in developing rat brain regions. The hypothalamus, pons-medulla, colliculi, cerebellum, ceerbrum, midbrain and pituitary gland were studied in rats ranging in age from 1 week to adult; PZ-peptidase activity continuously decreased with maturation in all of the brain regions examined except the hypothalamus. The pituitary gland showed the highest activity in all of the brain regions. PZ-peptidase activity in crude mitochondrial and supernatant fractions from rat whole brain had an optimum pH between 7.5–8.0. It was strongly inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide or EDTA. whereas iodoacetic acid did not affect the enzyme activity. Among various metal ions, the enzyme activity was inhibited by Zn⁺² or Cu⁺² but not by Mn⁺², Ca⁺², Mg⁺² or Na⁺. There is no inhibition of the activity by serine protease inhibitors, including diisopropylfluorophosphate and phenylmethylsulphonyl fluoride. An approximate molecular weight of this enzyme was estimated to be 68,000 by gel filtration. Since these properties of rat brain PZ-peptidase were similar to those of other peripheral PZ-peptidases, we suppose that PZ-peptidase in the brain may be the same molecule as the enzyme which hydrolyses collagen peptides in peripheral tissues, but it may have some different physiological roles.     

Hog thyroid peroxidase: physical, chemical, and catalytic properties of the highly purified enzyme.

Studies are reported on the purity and on the physical, chemical, and catalytic properties of a highly purified, stable, thyroid peroxidase (TPO). The enzyme was solubilized by treatment with deoxycholate and trypsin, and it was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. The final product, designated TPO VII, had a value for A₄₁₀/A₂₈₀ of 0.54, and its specific activity based on the guaiacol assay (794 μmol of guaiacol oxidized/min/mg) was considerably greater than that of any previously described TPO. Specific activity values based on other peroxidase-catalyzed reactions were also higher for TPO VII than for previous TPO preparations. Purity estimates for TPO VII, based on polyacrylamide disc gel electrophoresis and on isoelectric focusing in polyacrylamide gels, ranged from 80 to 95%. The molecular weight, determined by sedimentation equilibrium, was 93,000. Results of sodium dodecyl sulfate-gel electrophoresis also indicated a molecular weight of approximately 90,000. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions indicated that TPO VII is composed of two peptide chains of unequal size, with the larger about 2.5-fold the size of the smaller. Carbohydrate analysis revealed that TPO is a glycoprotein containing about 10% by weight of carbohydrate. The predominant sugars were mannose and N-acetyl glucosamine. A significant amount of glucose was also found, along with small amounts of galactose, fucose, and xylose. The amino acid composition of TPO VII showed a high proline content, a predominance of arginine over lysine, and a ratio of [Asp] plus [Glu] to [Lys] plus [Arg] of over 2. Isoelectric focusing in polyacrylamide gels indicated an isoelectric pH of 5.75. In agreement with observations made on earlier preparations of TPO, heme spectral data showed significant differences between the pyridine hemochromogens of TPO VII and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. Circular dichroism measurements indicated that approximately 40% of TPO VII involves α helix or β structure.     

Studies of the reaction products of adenosine with [Pt(NH3)3Cl]Cl and cis-Pt(NH3)2Cl2 by high performance liquid chromatography

The reaction products of adenosine with [Pt(NH₃)₃Cl]Cl or cis-Pt(NH₃)₂Cl₂ have been studied using high performance liquid chromatography and uv spectroscopy. The reaction of [Pt(NH₃)₃Cl]Cl with adenosine (pH = 7.0, Pt/base = 0.5) gives four products. Two of them, mononuclear complexes in which platinum is bound to adenosine through N(7) or N(1), comprise more than 90% of all the products. The N(1) and N(7) sites on adenosine indicate almost equal binding affinity for [Pt(NH₃)₃Cl]Cl. The reaction of cis-Pt(NH₃)₂Cl₂ with adenosine has been studied in the presence of a large excess of adenosine (Pt/base ? 0.05). The reaction gives four products. One is the monomeric 2:1 complex with cis-Pt(NH₃)₂²⁺ bound to two adenosine molecules through the N(7) site and the N(1) site, and another is the monomeric 2:1 complex with cis-Pt(NH₃)₂²⁺ bound to two adenosine molecules through the N(7) sites. cis-Pt(NH₃)₂Cl₂ is stronger affinity to the N(7) site than of adenosine to the N(1) site.     

Plasma 13, 14-dihydro-15-keto-PGF2 (PGFM) in pregnant and nonpregnant heifers prior to and during surgery and following intrauterine injection of PGF2

On day 17 postestrus or postmating, heifers were given intrauterine injections of saline (2 pregnant, 2 non-pregnant) or 200 μg PGF₂α (7 pregnant, 6 nonpregnant) through cannulae installed surgically into the uterine horn ipsilateral to the corpus luteum bearing ovary. Jugular blood samples were collected prior to the laparotomy at which the cannulae were installed during surgery, and for 90 min following the intrauterine injection. Plasma was assayed for progesterone and 13,14-dihyro-15-keto-PGF₂α )PGFM). Laparotomies were reopened to confirm proper cannula placement and to determine if blastocysts were present in mated heifers. Concentrations of PGFM were higher in pregnant compared to nonpregnant heifers during the presurgery (68 26 24 26 pg/ml; P < 0.25) and surgery (186 47 65 17 pg/ml; P < .05) periods. Pregnancy status did not alter the mean concentrations of PGFM (pregnant, 554 70 pg/ml; nonpregnant, 422 81 pg/ml) or the half-life of its decline in concentration (18 min) following intrauterine injection of PGF₂α. Pregnancy at 17 days in cattle does not appear to influence PGF₂α transport from the uterine lumen or its metabolism in the uterus or elsewhere in response to an acute intrauterine injection.     

Chloroplast and cytoplasmic enzymes 1, 2, 3. Subunit structure of pea leaf aldolases.

The pea leaf chloroplastic and cytoplasmic forms of aldolase are very similar in structure. The subunit molecular weights determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate are approximately 37.000. Both aldolases appear to terminate in the same sequence, SerAlaTyrCOOH, and the amino-terminal sequence H₂NGlySerTyrAla was obtained for each. The previously reported differences in kinetic properties and in isoelectric points of the two pea leaf enzymes probably are the consequence of minor differences in amino acid composition or conformation.     

Potential tumor or organ-imaging agents, 24. Radioiodinated pregnenolone esters

A series of radioiodinated pregnenolone esters was prepared in an effort to find an agent that would be rapidly and selectively taken up by adrenal cortical tissue. Achievement of such a goal would provide the basis for the development of an adrenal imaging agent having several advantages over those agents currently available for clinical use. The radioiodinated esters for this study were readily prepared by treating pregnenolone with the appropriate iodobenzoic acid in the presence of dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-pyridine (DMAP). The resulting esters were readily labeled with radioiodine by isotope exchange with sodium iodide-125 in pivalic acid. Subsequent tissue distribution studies in rats revealed that those esters displaying the most stability towards hydrolysis achieved the highest concentration in adrenal cortical tissue. For example, the 2,3,5-triiodobenzoate (6) showed an adrenal uptake of 23% of administered dose per gram of tissue at 0.5 hours. The achievement of high levels of radioactivity in the adrenal with this agent at early time periods warrants further evaluation of this agent in other animals.     

Positive correlation between the levels of natural killer cells and the in vivo resistance to syngeneic tumor transplants as influenced by various routes of administration of Corynebacterium parvum bacteria.

Depending on the route of administration, heat-killed Corynebacterium parvum (C. parvum) bacteria caused an increase or decrease of natural killer (NK)-cell activity in mice. We used a syngeneic tumor with known susceptibility to NK lysis in vitro. The tumor was administered to mice whose NK levels had been increased or decreased by previous inoculations of C. parvum bacteria. A positive correlation between changes in NK-cell activity as measured in vitro and changes in tumor resistance as measured in vivo was observed. Additional evidence was provided in support of the view that NK cells may play an important role in resistance to tumor growth. The route of administration of C. parvum was considered important for protection against tumor growth.     

Effect of taurine on ATP-dependent calcium transport in guinea-pig cardiac muscle

The effect of taurine on ATP-dependent calcium transport was examined in guinea-pig cardiac ventricle homogenates and in microsomal preparations enriched in sarcoplasmic reticulum. Taurine (5?50 mM) did not affect ATP-dependent calcium binding or uptake in either of these preparations or alter the rate of decay of calcium uptake activity. Taurine (20 mM) also did not affect the oxalate-dependent calcium uptake stimulation noted in the presence of cyclic AMP-dependent protein kinase and cyclic AMP. The mechanism by which taurine alters cardiac function remains to be elucidated.     

Rat brain aspartate β-decarboxylase. A comparative study with the liver enzyme

Aspartate β-decarboxylase (AspD), which catalyses the β-decarboxylation of aspartate (Asp) to alanine (Ala), was found in significant quantities only in the brain, kidney and liver. This enzyme has an optimum pH at 7.4. Addition of exogenous pyridoxal 5′-phosphate did not increase enzyme activity presumably because of firmly bound cofactor. However, aminooxyacetic acid is a potent inhibitor.There is an apparent 8-fold variation in AspD in the seven brain regions studied, with the highest activities in the cortex and the lowest in the striatum and hippocampus. In the presence of α-ketoglutarate, the production of ¹⁴CO₂ from [¹⁴C]Asp may no longer represent AspD activity due to active transamination of Asp, presumably by aspartate aminotransferase, to oxaloacetate. Under such conditions, comparable AspD activities were observed in all seven brain regions.Kinetic analysis showed that the liver and kidney enzymes have identical affinity for Asp (K_m = 3.5 mM) while the brain enzyme has a higher affinit (K_m = 1.3 mM). The V_max values obtained indicated that the enzyme populations in liver, kidney and brain are in the ratio 18:4:1. Various amino acids were found to inhibit both brain and liver AspD. Serine, however, activated the liver enzyme but inhibited competitively the kidney and brain enzymes. These results indicate that AspD may exist as two or more isozymes.     

Changes in adenosine 3',5'-monophosphate induced by parathyroid hormone and theophylline in the developing rat renal cortex

To test the biochemical responsiveness of developing rat renal cortex to parathyroid hormone (PTH), intracellular concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were measured. Renal cortical slices from 10-day-, 20-day-, and 12-week-old animals contained higher concentrations of cyclic AMP when incubated in the presence of theophylline than in its absence. In the absence of theophylline, tissue from all three age groups responded to PTH with dose-dependent increases in cyclic AMP. In the presence of theophylline the response of tissue from 10-day-old animals was greater than that of 12-week-old animals.It is suggested that the differential effect of theophylline with respect to age may be the result of higher turnover rates of cyclic AMP in the young animals.     

Changes in the binding of copper in the plasma of molybdenum supplemented rats

After incubating plasma of Mo-supplemented rats (Mo-plasma) with ⁶⁴Cu only part of it could be removed by dialysis against EDTA or histidine or by treatment with dithiocarbamate; this nondialyzable Cu was shown to be bound to albumin. The maximal amount of ⁶⁴Cu bound this way equaled the Mo-induced increase in total plasma Cu. After addition of stable Cu dialysis of Mo-plasma against a histidine solution showed that no extra Cu became tightly bound, suggesting that the ⁶⁴Cu binding was due to an exchange between added ⁶⁴Cu and stable Cu already present. Incubating Mo-plasma with Hg compounds prevented ⁶⁴Cu binding and released stable Cu, indicating that Cu in Mo-plasma was sulfhydryl bound. Part of the Mo in Mo-plasma was freely dialyzable. The remaining part was shown to be SH bound as well. The estimated atomic ratio of SH-bound Cu and Mo was unity. Molybdenum increased the number of SH groups in plasma, and for each Cu atom at least one SH group was calculated to be present.     

Sound localization by the Hawaiian squirrelfishes, Myripristis berndti and M. argyromus

Sound localization was investigated in a large pond open to a bay and similar to the normal environment of the animals. Observations were made of fish movements towards one of two underwater loud-speakers emitting squirrelfish alarm calls normally produced in response to predators. When the sound source was within 2·0 m of the test cage housing the fish, the subjects faced and moved toward the speaker. The animals responded some of the time when the source was within 3·0 m but generally did not orient to the sound source when the speaker was beyond 3·0 m. Response loss was correlated with the fish being in the acoustic far-field. Possible cues which release and direct localization remain unknown, but include particle velocity information alone, or some change in particle velocity: pressure relationships.