Purine metabolism in germinating wheat embryos

1. Both the acid-soluble fraction and the nucleic acid fraction of wheat embryos were extensively labelled after incubation for 6hr. in the presence of [8-(14)C]adenine. Subsequent incubation in the absence of labelled adenine resulted in no loss of radioactivity to the medium during a 48hr. period. Radioautography indicated that during this period there was a continuous increase in the radioactivity present in the acid-insoluble fractions of the root and leaf tissues relative to that present in the coleorhiza and coleoptile. 2. During incubation at 25 degrees there was a 26-fold increase in the activity of 3'-nucleotidase between 4hr. and 24hr.; the activities of enzymes hydrolysing AMP and IMP increased to a smaller extent. The activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase increased three- to five-fold during incubation at 25 degrees for 24hr. 3. Adenosine kinase, inosine phosphorylase and 5-phosphoribosyl pyrophosphate synthetase activities were high in extracts from dry embryos and did not increase during 48hr. at 25 degrees . 4. The increase in 3'-nucleotidase activity was prevented by cycloheximide, cryptopleurine or incubation at 4 degrees , but not by actinomycin D; these treatments did not depress the activity of the other enzymes measured. 5. The results are discussed in relation to RNA translocation within the wheat embryo during germination.     

Acetylation of serotonin in vitro by a human N-acetyltransferase

1. There is a well-recognized genetic polymorphism for the N-acetylation of isoniazid and sulphamethazine by human livers. 2. Serotonin was found to be acetylated by human liver enzyme preparations and the N-acetylserotonin formed was identified and determined quantitatively. 3. In 13 livers examined there was a wide variability in the capacity to acetylate serotonin that did not correlate with the capacity of the same livers to acetylate isoniazid and sulphamethazine. The results suggest that serotonin is not a natural substrate for the polymorphic N-acetyltransferase and that it may be acetylated by a different enzyme.     

Effect of ethylenediaminetetraacetic acid-Tris(hydroxymethyl)aminomethane on release of the acid-soluble nucleotide pool and on breakdown of ribosomal ribonucleic acid in Escherichia coli

Brief treatment of Escherichia coli with 2 x 10(-4)m ethylenediaminetetraacetic acid (EDTA)-0.12 m tris(hydroxymethyl)aminomethane (Tris), pH 8.0, or 0.12 m Tris alone resulted in the release of the acid-soluble nucleotide pool at 3 or 23 C. Exposure to EDTA-Tris for up to 90 min at 3 C did not result in the release of increasing amounts of 260-mmu-absorbing material. At 23 and 37 C, EDTA-Tris resulted in a steady increase in acid-soluble 260-mmu-absorbing material. Previous growth environment did not alter the release. There appeared to be degradation of 23S ribonucleic acid (RNA) after 10 min of exposure at 23 C. In addition, there was degradation of nucleotides to nucleosides and bases. This occured either within the cells with altered permeability or in the periplasmic space. This occurred in the presence of EDTA and Tris but was not seen with EDTA-phosphate. The mechanism of this degradation is unclear, since it occurs in ribonuclease I-deficient strains. Exposure to Tris buffer for long periods of time at 23 C resulted in release of the nucleotide pool and in degradation of RNA and nucleotides. These studies point out that the EDTA-Tris effect on E. coli must be divided into two parts, an early (4 to 5 min) change in permeability and a later phase of actual RNA breakdown and nucleotide degradation. Studies utilizing EDTA and Tris as agents altering permeability must thus be viewed with caution. Although the cells are viable, they have lost their acid-soluble nucleotide pool and have undergone degradation of some ribosomal RNA.     

Vitamin K and oxidative phosphorylation

1. Oxidative phosphorylation was studied in a cell-free preparation of Mycobacterium phlei and in rat-liver mitochondria. Phosphorylation was destroyed in both systems by long-wave ultraviolet radiation and restored by the addition of small amounts of [2-Me-(14)C,(3)H]phylloquinone. When the radioactive quinones were recovered from the phosphorylating system and chromatographed with carrier phylloquinone and menaquinone-4 in adsorption and partition systems, only the phylloquinone band was labelled, and its isotopic ratio was identical with that of the original [2-Me-(14)C,(3)H]phylloquinone. This result does not support the contention that the role of vitamin K in oxidative phosphorylation involves a cyclic mechanism with intermediate formation of a quinone methide. 2. When the [2-Me-(14)C,(3)H]phylloquinone was given intravenously to rats and radioactive phylloquinone isolated from their liver mitochondria and microsomes 20hr. later, its isotopic ratio was unchanged. There was thus no evidence for quinone methide formation in vivo. No measurable conversion of phylloquinone into menaquinone-4 was observed. 3. When [(14)C]menadione was given intraperitoneally to rats whose alimentary tract had been treated with neomycin, conversion into menaquinone-4 was found in the liver mitochondria and microsomes, but there was also some indication that there had been synthesis of phylloquinone.     

Altered biodistribution of indium-111-labeled monoclonal antibody 96.5 to tumors and normal tissues of nude mice bearing human melanoma xenografts in visceral organs

Summary Human melanoma xenografts were produced in the subcutis, kidney, cecum and liver of different nude mice. An¹¹¹In-labeled anti-(human melanoma) monoclonal antibody (96.5) or an¹¹¹In-labeled nonspecific control monoclonal antibody (ZCE-025) was injected intravenously in separate groups of mice. Radioactive antibody accumulation was measured in tumor, blood, viscera, and carcasses. mAb 96.5 targeted specifically to tumor tissue regardless of site of growth. Tumors in the liver exhibited significantly (P <0.05) higher tumor-to-blood ratios (45±6, mean ±SEM) than xenografts at other visceral organs, the lowest value being found for subcutaneous melanoma (2.6±0.5). The differences in tumor-to-blood ratio were due to significant alterations of antibody biodistribution, since the actual antibody concentration in the different tumor sites was similar. The percentage of recovered anti-melanoma antibody per milliliter of blood in mice with visceral lesions (4.6±1.1%/ml) was significantly lower than that found in mice with subcutaneous tumors (9.5±1.4%/ml,P <0.05). Moreover, significantly higher levels (18.2±3.2%/g, 31.0±5.1%/g, respectively) of the melanoma mAb 96.5 were found in normal liver and spleen tissue recovered from mice with visceral tumors as compared to tissue from mice with subcutaneous tumors (9.2±0.9%/g, 13.5±1.9%/g, respectively;P <0.05). These results demonstrate that the presence of visceral tumor can significantly affect tumor-to-blood ratios, blood levels, and biodistribution of¹¹¹In-labeled mAb 96.5.This work was supported in part by funds from the National Institutes of Health, National Cancer Institute, Grant R35-CA42107 and Core Grant CA16672     

Purification of a hepatic S6 kinase from cycloheximide-treated Rats

Cycloheximide injection of rats results in the activation of a protein kinase that phosphorylates 40 S ribosomal protein S6. This Ca2+/cyclic nucleotide-independent kinase exhibits chromatographic properties that are indistinguishable from the S6 kinase in H4 hepatoma cells whose activity is stimulated by insulin and growth factors and the S6 kinase that is activated during liver regeneration. The enzyme has been purified 50,000-fold to near homogeneity: a critical step in purification employs a peptide affinity column using a synthetic peptide corresponding to the carboxyl-terminal 32-amino acid residues of mouse liver S6, which encompasses all S6 phosphorylation sites. The purified enzyme is a 70,000-dalton polypeptide that is reactive with azido-ATP. In addition to 40 S ribosomal S6 and the synthetic peptide, the S6 kinase catalyzes rapid phosphorylation of a number of other protein substrates including histone H2b, glycogen synthase, and ATP citrate lyase; this last protein is phosphorylated by S6 kinase in vitro on the same serine residue that is phosphorylated in response to insulin and epidermal growth factor in intact hepatocytes. Moreover, the S6 kinase catalyzes the phosphorylation of a number of hepatic nonhistone nuclear proteins. This S6 kinase probably underlies the increased hepatic S6 phosphorylation observed after cycloheximide treatment, which in turn corresponds to the mitogen-activated S6 kinase.     

Carbon Oxysulfide Is an Inhibitor of Both CO(2) and HCO(3) Uptake in the Cyanobacterium Synechococcus PCC7942

Carbon oxysulfide (COS) was reinvestigated as an inhibitor of active inorganic carbon transport in cells of Synechococcus PCC7942 adapted to growth at low inorganic carbon. COS inhibited both CO₂ and HCO₃⁻ transport processes in a reversible (in the short term) and mixed competitive manner. The inhibition of COS was established using both silicone oil centrifugation experiments and O₂-evolution studies. The K_i for COS inhibition was 29 micromolar for CO₂ transport and 110 micromolar for HCO₃⁻ transport. These results support a model of inorganic carbon transport with a central CO₂ pump and an inducible HCO₃⁻ utilizing accessory protein which supplies CO₂ to the primary pump.     

Ovarian response to hCG treatment during the oestrous cycle in heifers

The aims of this study were to investigate whether treatment with a single ovulatory dose of hCG, between the day of oestrus and the end of the luteal phase, could induce extra ovulations in heifers and whether the presence of an existing corpus luteum (CL) affected the response. Heifers (N = 32) were injected with 1500 i.u. hCG or saline on a given day of the oestrous cycle. Treatments were repeated during subsequent cycles to provide a total of 71 observations, 57 of which followed an injection of hCG, given between Day 0 (oestrus) and Day 16, and 14 of which followed saline injections as controls. Ovulatory responses were noted by laparoscopy 2 days after hCG treatment. No heifers injected with saline produced additional CL. Of the hCG-treated cycles, 23 resulted in the formation of an additional CL, and this was significantly affected by the stage of the oestrous cycle when hCG was given; a greater response was observed during the early (Days 4-7) and late (Days 14-16) stages of the luteal phase than at the mid-luteal phase of the oestrous cycle. Two heifers were also treated with hCG on Days 17 or 18 of the oestrous cycle, but before oestrus; both had induced CL. There were no significant differences between the left-right orientation of the existing CL or the hCG-induced CL. These results demonstrate that the large, luteal-phase follicle of the cow is capable of ovulating in response to hCG and that the induced CL is not affected by the presence of an existing CL.     

Neuroendocrine and behavioral effects of dietary tryptophan restriction in healthy subjects

The neuroendocrine and behavioral effects of gradual dietary tryptophan (TRP) depletion, utilizing two magnitudes of a 10-day TRP-restriction diet (700 mg/day and 200 mg/day), were studied in 22 healthy subjects. The prolactin response to a 7 gm L-TRP infusion was measured prior to and on day 10 of the diet. Both diets significantly reduced fasting total plasma TRP by 15 to 20%, but only the 200 mg/day TRP diet led to an enhancement of the prolactin response to intravenous L-TRP. Female subjects demonstrated a more robust increase in plasma prolactin following L-TRP infusion pre-diet and exhibited a larger decrease in plasma TRP following dietary TRP restriction compared to males. There were no significant behavioral effects of either diet. Gradual dietary TRP depletion leads to an enhancement of the prolactin response to L-TRP infusion, suggestive of postsynaptic serotonin receptor supersensitivity.