UDP-glucuronate decarboxylase, a key enzyme in proteoglycan synthesis: cloning, characterization, and localization

UDP-glucuronate decarboxylase (UGD) catalyzes the formation of UDP-xylose from UDP-glucuronate. UDP-xylose is then used to initiate glycosaminoglycan biosynthesis on the core protein of proteoglycans. In a yeast two-hybrid screen with the protein kinase Akt (protein kinase B), we detected interactions with a novel sequence, which we cloned and expressed. The expressed protein displayed UGD activity but did not display the activities of homologous nucleotide sugar epimerases or dehydratases. We did not detect phosphorylation of UGD by Akt nor did we detect any influence of Akt on UGD activity. Effects of UGD on Akt kinase activity were also absent. Northern blot and Western blot analyses revealed the presence of UGD in multiple tissues and brain regions. Subcellular studies and histochemistry localized UGD protein to the perinuclear Golgi where xylosylation of proteoglycan core proteins is known to occur.     

Segment and joint angles of hind limb during bipedal and quadrupedal walking of the bonobo (Pan paniscus)

We describe segment angles (trunk, thigh, shank, and foot) and joint angles (hip, knee, and ankle) for the hind limbs of bonobos walking bipedally ("bent-hip bent-knee walking," 17 sequences) and quadrupedally (33 sequences). Data were based on video recordings (50 Hz) of nine subjects in a lateral view, walking at voluntary speed. The major differences between bipedal and quadrupedal walking are found in the trunk, thigh, and hip angles. During bipedal walking, the trunk is approximately 33-41 degrees more erect than during quadrupedal locomotion, although it is considerably more bent forward than in normal human locomotion. Moreover, during bipedal walking, the hip has a smaller range of motion (by 12 degrees ) and is more extended (by 20-35 degrees ) than during quadrupedal walking. In general, angle profiles in bonobos are much more variable than in humans. Intralimb phase relationships of subsequent joint angles show that hip-knee coordination is similar for bipedal and quadrupedal walking, and resembles the human pattern. The coordination between knee and ankle differs much more from the human pattern. Based on joint angles observed throughout stance phase and on the estimation of functional leg length, an efficient inverted pendulum mechanism is not expected in bonobos.     

Evaluation of progesterone receptor expression in eosinophils using real-time quantitative PCR

Progesterone has been shown in many instances to have immune-suppressant activities. Most of these activities have been investigated in the light of general immune suppression or with a focus on lymphocytes. However, many clinical and in vitro studies have shown that progesterone also has a suppressive effect on eosinophilia. This effect so far has not been thoroughly investigated. The purpose of this study was to evaluate whether the effect is mediated via the classical progesterone receptor (PR). We developed a new real-time quantitative PCR (RQ-PCR) for the analysis and quantification of expression of the classical PR. The test was first validated both on breast cancer cell lines and on breast cancer biopsies. Subsequently, when using eosinophils isolated from peripheral blood of healthy volunteers, we could not find evidence for the expression of PR. These data suggest that the effects of progesterone on eosinophils are not mediated by the classical PR.     

Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.     

Ontogenetic shift in mouth opening mechanisms in a catfish (Clariidae, Siluriformes): a response to increasing functional demands

During ontogeny, larval fish have to deal with increasing nutritional and respiratory demands as they grow. As early ontogeny is characterized by an increasing complexity of moving structural elements composing a fish skull, some constraints will have to be met when developing mechanisms, which enable feeding and respiration, arise at a certain developmental stage. This article focuses on the presence/absence of a possible functional response in mouth opening during ontogeny in Clarias gariepinus. Some reflections are given, based on morphological data, as well as related function-analysis data from the literature. Starting shortly after hatching, a total of up to five different mouth opening mechanisms may become functional. Of these, three may remain functional in the adult. As could be expected, the apparatuses that enable these mechanisms show an increase in complexity, as well as a putative improvement in mouth opening capacity. Initially, two consecutive mechanisms may allow a restricted depression of the lower jaw (both passively and actively). Synchronously, two more mechanisms may arise, which involve the coupling of the hyoid depression to the mouth opening. At about 11 mm SL a fifth mechanism becomes established, better known as the opercular mouth opening mechanism. An overlapping chronology of functionality of the different mechanisms, as well as differences in efficiencies, could be an indication of the absence of a true critical period in C. gariepinus (at least in relation to mouth opening), as well as the possible presence of a shift in feeding type. Finally, the coupling of the chronology of the shift in mouth opening mechanisms and several morphological, behavioral, and physiological changes during ontogeny, related to feeding and respiration, make it possible to distinguish five important phases in the early life history of C. gariepinus.     

19F Nuclear Magnetic Resonance as a Tool To Investigate Microbial Degradation of Fluorophenols to Fluorocatechols and Fluoromuconates

A method was developed to study the biodegradation and oxidative biodehalogenation of fluorinated phenols by ¹⁹F nuclear magnetic resonance (NMR). Characterization of the ¹⁹F NMR spectra of metabolite profiles of a series of fluorophenols, converted by purified phenol hydroxylase, catechol 1,2-dioxygenase, and/or by the yeast-like fungus Exophiala jeanselmei, provided possibilities for identification of the ¹⁹F NMR chemical shift values of fluorinated catechol and muconate metabolites. As an example, the ¹⁹F NMR method thus defined was used to characterize the time-dependent metabolite profiles of various halophenols in either cell extracts or in incubations with whole cells of E. jeanselmei. The results obtained for these two systems are similar, except for the level of muconates observed. Altogether, the results of the present study describe a ¹⁹F NMR method which provides an efficient tool for elucidating the metabolic pathways for conversion of fluorine-containing phenols by microorganisms, with special emphasis on possibilities for biodehalogenation and detection of the type of fluorocatechols and fluoromuconates involved. In addition, the method provides possibilities for studying metabolic pathways in vivo in whole cells.     

Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.     

Abnormal offspring following in vitro production of bovine preimplantation embryos: a field study

Data on 944 calves from 2228 in vitro-produced (IVP) bovine preimplantation embryos were compared with data on 2787 AI calves born in the same herds in 1995. Bovine preimplantation embryos were produced in vitro following ovum pick up (OPU) from donor cows and pregnant heifers in an open nucleus breeding program. After 7 d of in vitro culture on a BRL cell monolayer in the presence of 10% FCS, frozen-thawed expanded blastocysts and fresh morulae to expanded blastocysts were transferred into recipient heifers and cows at 119 contracted farms throughout the Netherlands. The pregnancy rate, as confirmed by palpation per rectum between 90 and 150 d after transfer was 43.5% for both fresh and frozen embryos. Data on IVP and AI calves were registered by the farmers. The percentage of calves with a congenital malformation and the percentage of male calves were related to the total number of calves born. Gestation length, birth weight (measured by a balance), perinatal mortality and ease of calving were analyzed in a subdataset (699 IVP and 2543 AI calves, respectively) by a comparative analysis of variance (ANOVA). The ANOVA model included herd, month of calving, sire nested within AI or IVP, parity and breed of the inseminated cow/embryo recipient, sex of calf, type of calf (AI or IVP) and two-way interactions between type of calf and sex, parity and breed. The percentage of calves with congenital malformations was 3.2% and 0.7% for IVP and AI calves, respectively. An increased incidence of hydro-allantois and abnormal spinal cords and limbs was observed in IVP calves. The percentage of male calves was significantly different between IVP and AI, 55.5% and 48.9%, respectively (Chi-square, 1 degree of freedom, P < 0.05). On the average, IVP calves showed a significant increase of birth weight by 10% (4-5 kg), a 3-d longer gestation period, 2.4% more perinatal mortality and a more difficult calving process compared to AI calves (P < 0.05). From these results it is concluded that calves produced by IVP deviate significantly from calves produced by AI.     

A QTL with major effect on milk yield and composition maps to bovine Chromosome 14

A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand²-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect. Received: 30 December 1997 / Accepted: 21 February 1998