Inhibition of kynureninase (L-kynurenine hydrolase, EC 3 . 7. 1 . 3) by oestrone sulphate: an alternative explanation for abnormal results of tryptophan load tests in women receiving oestrogenic steroids

1. A partial purification of kynureninase (L-kynurenine hydrolase, EC 3 . 7. 1 . 3) from rat liver and a total resolution of the apoenzyme have been achieved. The hypothesis that conjugates of oestrogenic steroids compete with pyridoxal phosphate for the cofactor binding site of the enzyme, and so disturb tryptophan metabolism, leading to apparent vitamin B6 deficiency, has been tested. 2. Kynureninase from rat liver was partially purified, and the cofactor-free apoenzyme was prepared. Oestrone sulphate inhibited the enzyme uncompetitively with respect to pyridoxal phosphate, and competitively with respect to kynurenine, with a mean (+/- SE) inhibitor constant (Ki) of 82 +/- 6 microM. 3. The addition of a saturating concentration of pyridoxal phosphate to unfractionated liver homogenates led to an approximately fivefold increase in kynureninase activity, indicating the presence of a relatively large amount of apo-kynureninase in the tissue. 4. It is suggested that the abnormal results of tryptophan load tests in women receiving oestrogens are the result of inhibition of kynureninase by oestrogen conjugates, and that there is no evidence for oestrogen-induced vitamin B deficiency in such cases

Probable mechanisms of regulation of the utilization of dietary tryptophan, nicotinamide and nicotinic acid as precursors of nicotinamide nucleotides in the rat

1. The regulation of the utilization of dietary tryptophan, nicotinamide and nicotinic acid as precursors of the nicotinamide nucleotides has been studied in groups of rats fed on diets providing only one of these precursors at a time, in amounts adequate to meet their requirements for nucleotide synthesis. 2. The concentration of nicotinamide nucleotides in the liver of rats receiving a high-tryptophan diet was 56% higher than in animals fed on a diet providing a minimum amount of tryptophan, together with nicotinic acid or nicotinamide. The excretion of N1-methyl nicotinamidc was three times higher in the tryptophan-fed animals than in the other two groups. 3. The concentration of quinolinic acid in the liver was significantly higher in animals receiving the high-tryptophan diet than in the other two groups; that of nicotinic acid was highest in those animals receiving the nicotinic-acid-containing diet. The concentration of nicotinamide was highest in the livers of those animals receiving the high-tryptophan diet, and lowest in those receiving the nicotinic-acid-contaning diet. 4. The values of the Michaelis constant Km of nicotinamide deamidase (nicotinamide amidohydrolase. EC 3.5,1,19) and nicotinamide phosphoribosylltransferase (nicotinamide nucleotide: pyrophosphate phosphoribosyltransferase, EC 2.4.2.12) were approximately equal, and approximately one-tenth of the concentration of nicotinamide was in the liver. This suggests that both these enzymes normally function at their maximum rate, and a change in the availability of nicotinamide would not affect the rate of its incorporation into nucleotides. 5. The maximum rate of reaction (Vmax) of nicotinamide deamidase was twice that of nicotinamide phosphoribosyltransferase; this suggests that unless compartmental or other factors are involved, the major route of nicotinamide utilization will be by way of deamidation. 6. The Km of nicotinate phosphoribosyltransferase (nicotinate nucleotide: pyrophosphate phosphoribosyltransferase, EC 2.4.2.11) was less than twice the concentration of nicotinic acid in the liver, so that a change in the availability of nicotinic acid might be expected to lead to a small change in the rate of its utilization. 7. The Km of quinolinate phosphoribosyltransferase (nicotinate nucleotide: pyrophosphate phosphoribosyltransferase (carboxylating) EC 2.4.2.19) was approximately 100 times greater than the concentration of quinolinic acid in the liver, so that any change in the availability of quinolinic acid would be expected to lead to a considerable change in the rate of its utilization. The Vmax of quinolinate phosphoribosyltransferase was relatively low, so that under conditions of high tryptophan flux, some accumulation of quinolinic acid might be expected. This was observed in animals receiving the high-tryptophan diet. 8. It is concluded that it is unlikely that the utilization of quinolinic acid, arising from tryptophan. for the synthesis of nicotinamide nucleotides is regulated, but that control over tissue concentrations of nucleotides is achieved by hydrolysis of NAD to nicotinamide. Incorporation of nicotinamide into nucleotides seems to be strictly limited by the activity of the enzymes involved

Measuring vascular reactivity with breath-holds after stroke: a method to aid interpretation of group-level BOLD signal changes in longitudinal fMRI studies

Blood oxygenation level dependent (BOLD) contrast fMRI is a widely used technique to map brain function, and to monitor its recovery after stroke. Since stroke has a vascular etiology, the neurovascular coupling between cerebral blood flow and neural activity may be altered, resulting in uncertainties when interpreting longitudinal BOLD signal changes. The purpose of this study was to demonstrate the feasibility of using a recently validated breath-hold task in patients with stroke, both to assess group level changes in cerebrovascular reactivity (CVR) and to determine if alterations in regional CVR over time will adversely affect interpretation of task-related BOLD signal changes. Three methods of analyzing the breathhold data were evaluated. The CVR measures were compared over healthy tissue, infarcted tissue, and the peri-infarct tissue, both sub-acutely (~two weeks) and chronically (~four months). In this cohort, a lack of CVR differences in healthy tissue between the patients and controls indicates that any group level BOLD signal change observed in these regions over time is unlikely to be related to vascular alterations. CVR was reduced in the peri-infarct tissue but remained unchanged over time. Therefore, although a lack of activation in this region compared to the controls may be confounded by a reduced CVR, longitudinal grouplevel BOLD changes may be more confidently attributed to neural activity changes in this cohort. By including this breath-hold based CVR assessment protocol in future studies of stroke recovery, researchers can be more assured that longitudinal changes in BOLD signal reflect true alterations in neural activity

The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal(1), Gal(2) and the less studied Gal(3) (GalR1–3 gene products). There is a wealth of data on expression of Gal(1–3) at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal(1) or Gal(2) receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal(1)-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal(1)-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal(1)-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal(2)-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal(1) in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs)

Altered mRNA expression of genes related to nerve cell activity in the fracture callus of older rats: A randomized, controlled, microarray study

BACKGROUND: The time required for radiographic union following femoral fracture increases with age in both humans and rats for unknown reasons. Since abnormalities in fracture innervation will slow skeletal healing, we explored whether abnormal mRNA expression of genes related to nerve cell activity in the older rats was associated with the slowing of skeletal repair. METHODS: Simple, transverse, mid-shaft, femoral fractures with intramedullary rod fixation were induced in anaesthetized female Sprague-Dawley rats at 6, 26, and 52 weeks of age. At 0, 0.4, 1, 2, 4, and 6 weeks after fracture, a bony segment, one-third the length of the femur, centered on the fracture site, including the external callus, cortical bone, and marrow elements, was harvested. cRNA was prepared and hybridized to 54 Affymetrix U34A microarrays (3/age/time point). RESULTS: The mRNA levels of 62 genes related to neural function were affected by fracture. Of the total, 38 genes were altered by fracture to a similar extent at the three ages. In contrast, eight neural genes showed prolonged down-regulation in the older rats compared to the more rapid return to pre-fracture levels in younger rats. Seven genes were up-regulated by fracture more in the younger rats than in the older rats, while nine genes were up-regulated more in the older rats than in the younger. CONCLUSIONS: mRNA of 24 nerve-related genes responded differently to fracture in older rats compared to young rats. This differential expression may reflect altered cell function at the fracture site that may be causally related to the slowing of fracture healing with age or may be an effect of the delayed healing