Response of urinary purine derivatives excretion to different levels of ruminal glucose infusion in heifers

This study investigated the response of urinary purine derivatives (PD) excretion to increasing levels of intraruminal glucose infusion to evaluate how well this indicator reflects induced changes in microbial crude protein flow. Four rumen-cannulated heifers (482 ± 25 kg body weight) were fed at maintenance energy level with a basal diet (on fresh matter basis) of 4 kg/d hay, 1.5 kg/d concentrate and 60 g/d minerals in two equal meals. The trial comprised a control period (Control I) without glucose infusion followed by four consecutive periods in which all animals received 125 g, 250 g, 500 g or 1000 g/d of glucose, respectively. For this, daily dosages of glucose and urea (90 g/d during all periods) were divided into three portions that were dissolved in water and directly administered into the rumen during morning and afternoon feedings and once during noon. After the highest glucose dosage, a second control period was carried out (Control II). Urinary PD excretion increased with glucose infusion of 125 g/d (71.4 mmol/d) and 1000 g/d (74.2 mmol/d) over the level at Control I (53.9 mmol/d (standard error of the mean (SEM) 3.4; p = 0.012). After withdrawing glucose infusion, PD excretion (79.0 mmol/d) did not return to Control I level (p = 0.001). In contrast, faecal nitrogen (N) excretions linearly increased with incremental glucose infusion (p < 0.001) from 33.9 g/d at Control I to 39.7 g/d (SEM 0.5) at 1000 g/d of glucose and were similar in Control I and II (p = 0.086). The contradicting responses in the excretions of faecal N and urinary PD to increasing glucose infusions highlight the limited accuracy of the PD excretion as a non-invasive indicator when incremental dosages of rapidly fermentable carbohydrates are supplied.     

Physiological and biochemical responses of Theobroma cacao L. genotypes to flooding

Flooding is common in lowlands and areas with high rainfall or excessive irrigation. A major effect of flooding is the deprivation of O₂ in the root zone, which affects several biochemical and morphophysiological plant processes. The objective of this study was to elucidate biochemical and physiological characteristics associated with tolerance to O₂ deficiency in two clonal cacao genotypes. The experiment was conducted in a greenhouse with two contrasting clones differing in flood tolerance: TSA-792 (tolerant) and TSH-774 (susceptible). Leaf gas exchange, chlorophyll (Chl) fluorescence, chemical composition and oxidative stress were assessed during 40 d for control and flooded plants. Flooding induced a decrease in net photosynthesis, stomatal conductance and transpiration of both genotypes. In flood conditions, the flood-susceptible clone showed changes in chlorophyll fluorescence, reductions in chlorophyll content and increased activity of peroxidase and polyphenol oxidase. Flooding also caused changes in macro- and micronutrients, total soluble sugars and starch concentrations in different plant organs of both genotypes. Response curves for the relationship between photosynthetically active radiation (PAR) and net photosynthetic rate (P _N) for flooded plants were similar for both genotypes. In flood conditions, the flood-susceptible clone exhibited (1) nonstomatal limitations to photosynthesis since decreased in maximum potential quantum yield of PSII (F_v/F_m) values indicated possible damage to the PSII light-harvesting complex; (2) oxidative stress; (3) increased leaf chlorosis; and (4) a reduction in root carbohydrate levels. These stresses resulted in death of several plants after 30 d of flooding.     

ATP binding properties of the soluble part of the KdpC subunit from the Escherichia coli K(+)-transporting KdpFABC P-type ATPase

P-Type ATPases catalyze the transport of cations across the cell envelope via site-specific hydrolysis of ATP. Modulation of enzyme activity by additional small subunits and/or a second regulatory nucleotide binding site is still a subject of discussion. In the K(+)-transporting KdpFABC complex of Escherichia coli, KdpB resembles the catalytic P-type ATPase subunit, but ATP binding also occurs in the essential but noncatalytic subunit, KdpC. For further characterization, the soluble portion of KdpC (KdpC(sol), residues Asn39-Glu190) was synthesized separately and purified to homogeneity via affinity and size exclusion chromatography. Protein integrity was confirmed by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy, which revealed an alpha-helical content of 44% together with an 8% beta-sheet conformation consistent with the values deduced from the primary sequence. The overall protein structure was not affected by the addition of ATP to a concentration of up to 2 mM. In contrast, labeling of KdpC(sol) with the photoreactive ATP analogue 8-azido-ATP resulted in the specific incorporation of one molecule of 8-azido-ATP per peptide. No labeling could be observed upon denaturation of the protein with 0.2% sodium dodecyl sulfate, which suggests the presence of a structured nucleotide binding site. Labeling could be inhibited by preincubation with either ATP, ADP, AMP, GTP, or CTP, thus demonstrating a low specificity for nucleotides. Following 8-azido-ATP labeling and tryptic digestion of KdpC(sol), mass spectrometry showed that ATP binding occurred within the Val144-Lys161 peptide located within the C-terminal part of KdpC, thereby further demonstrating a defined nucleotide binding site. On the basis of these findings, a cooperative model in which the soluble part of KdpC activates catalysis of KdpB is suggested.     

FAS inhibitor cerulenin reduces food intake and melanocortin receptor gene expression without modulating the other (an)orexigenic neuropeptides in chickens

Cerulenin, a natural fatty acid synthase (FAS) inhibitor, and its synthetic analog C75 are hypothesized to alter the metabolism of neurons in the hypothalamus that regulate ingestive behavior to cause a profound decrease of food intake and an increase in metabolic rate, leading to body weight loss. The bulk of data exclusively originates from mammals (rodents); however, such effects are currently lacking in nonmammalian species. We have, therefore, addressed this issue in broiler chickens because this species is selected for high growth rate and high food intake and is prone to obesity. First, we demonstrate that FAS messenger and protein are expressed in the hypothalamus of chickens. FAS immunoreactivity was detected in a number of brain regions, including the nucleus paraventricularis magnocellularis and the nucleus infundibuli hypothalami, the avian equivalent of the mammalian arcuate nucleus, suggesting that FAS may be involved in the regulation of food intake. Second, we show that hypothalamic FAS gene expression was significantly (P < 0.05) decreased by overnight fasting similar to that in liver, indicating that hypothalamic FAS gene is regulated by energy status in chickens. Finally, to investigate the physiological consequences of in vivo inhibition of fatty acid synthesis on food intake, we administered cerulenin by intravenous injections (15 mg/kg) to 2-wk-old broiler chickens. Cerulenin administration significantly reduced food intake by 23 to 34% (P < 0.05 to P < 0.0001) and downregulated FAS and melanocortin receptors 1, 4, and 5 gene expression (P < 0.05). However, the known orexigenic (neuropeptide Y, agouti gene-related peptide, orexin, and orexin receptor) and anorexigenic (pro-opiomelanocortin and corticotropin-releasing hormone) neuropeptide mRNA levels remained unchanged after cerulenin treatment. These results suggest that the catabolic effect of cerulenin in chickens may be mediated through the melanocortin system rather than the other neuropeptides known to be involved in food intake regulation.     

The CORVET Subunit Vps8 Cooperates with the Rab5 Homolog Vps21 to Induce Clustering of Late Endosomal Compartments

Membrane tethering, the process of mediating the first contact between membranes destined for fusion, requires specialized multisubunit protein complexes and Rab GTPases. In the yeast endolysosomal system, the hexameric HOPS tethering complex cooperates with the Rab7 homolog Ypt7 to promote homotypic fusion at the vacuole, whereas the recently identified homologous CORVET complex acts at the level of late endosomes. Here, we have further functionally characterized the CORVET-specific subunit Vps8 and its relationship to the remaining subunits using an in vivo approach that allows the monitoring of late endosome biogenesis. In particular, our results indicate that Vps8 interacts and cooperates with the activated Rab5 homolog Vps21 to induce the clustering of late endosomal membranes, indicating that Vps8 is the effector subunit of the CORVET complex. This clustering, however, requires Vps3, Vps16, and Vps33 but not the remaining CORVET subunits. These data thus suggest that the CORVET complex is built of subunits with distinct activities and potentially, their sequential assembly could regulate tethering and successive fusion at the late endosomes.     

Glutamate levels and transport in cat (Felis catus) area 17 during cortical reorganization following binocular retinal lesions

Glutamate is known to play a crucial role in the topographic reorganization of visual cortex after the induction of binocular central retinal lesions. In this study we investigated the possible involvement of the glial high-affinity Na+/K+-dependent glutamate transporters in cortical plasticity using western blotting and intracortical microdialysis. Basal extracellular glutamate levels and the re-uptake activity for glutamate have been determined by comparing the extracellular glutamate concentration before and during the blockage of glutamate removal from the synaptic cleft with the potent transporter inhibitor l-trans-pyrrolidine-3,4-dicarboxylic acid. In cats with central retinal lesions we observed increased basal extracellular glutamate concentrations together with a decreased re-uptake activity in non-deprived, peripheral area 17, compared with the sensory-deprived, central cortex of the same animal as well as the topographically matching regions of area 17 in normal subjects. Western blotting experiments revealed a parallel decrease in the expression level of the glial glutamate transporter proteins GLT-1 and GLAST in non-deprived cortex compared with sensory-deprived cortex of lesion cats and the corresponding regions of area 17 of normal subjects. This study shows that partial sensory deprivation of the visual cortex affects the removal of glutamate from the synaptic cleft and implicates a role for glial-neuronal interactions in adult brain plasticity.     

The DNA tumor virus SV 40 induces gene mutations in human cells. Reversion of HPRT deficiency

Summary The mutagenic effect of papovavirus SV 40 on human cells could be demonstrated by reversion of HPRT deficiency in Lesch-Nyhan fibroblasts transformed by the virus. SV 40 seems to induce different gene mutations in individually selected cell clones, as was clearly shown by the respective HPRT enzyme properties. The consequences of our results for use of SV 40-derived vectors in gene substitution experiments are discussed.     

Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.     

How much non-coding DNA do eukaryotes require?

Despite tremendous advances in the field of genomics, the amount and function of the large non-coding part of the genome in higher organisms remains poorly understood. Here we report an observation, made for 37 fully sequenced eukaryotic genomes, which indicates that eukaryotes require a certain minimum amount of non-coding DNA (ncDNA). This minimum increases quadratically with the amount of DNA located in exons. Based on a simple model of the growth of regulatory networks, we derive a theoretical prediction of the required quantity of ncDNA and find it to be in excellent agreement with the data. The amount of additional ncDNA (in basepairs) which eukaryotes require obeys N_DEF=1/2 (N_C/N_P) (N_C−N_P), where N_C is the amount of exonic DNA, and N_P is a constant of about 10 Mb. This value N_DEF corresponds to a few percent of the genome in Homo sapiens and other mammals, and up to half the genome in simpler eukaryotes. Thus, our findings confirm that eukaryotic life depends on a substantial fraction of ncDNA and also make a prediction of the size of this fraction, which matches the data closely.