Fine mapping of the circling (cir) gene on the distal portion of mouse chromosome 9

Circling mice manifest profound deafness, head-tossing, and bi-directional circling behavior, which they inherit in autosomal recessive manner. Histologic examination of the inner ear reveals abnormalities of the region around the organ of Corti, spiral ganglion neurons, and outer hair cells. A genetic linkage map was constructed for an intraspecific backcross between cir and C57BL/6J mice. The cir gene was mapped to a region between D9Mit116/D9Mit15 and D9Mit38 on mouse chromosome (Chr) 9. Estimated distances between cir and D9Mit116, and between cir and D9Mit38 were 0.70 +/- 0.40 and 0.23 +/- 0.23 cM, respectively. Order of the markers was defined as follows: centromere - D9Mit182 - D9Mit51/D9Mit79/D9Mit310 - D9Mit212/D184 - D9Mit116/D9Mit15 - cir - D9Mit38 - D9Mit20 - D9Mit243 - D9Mit16 - D9Mit55/D9Mit125 - D9Mit281. On the basis of genetic mapping, we constructed a yeast artificial chromosome (YAC) contig across the cir region. The cir gene is located between the lactotransferrin (ltf) and microtubule-associated protein (map4) genes. The distal portion of mouse Chr 9 encompassing the cir region is homologous with human chromosome 3p21, which contains the Deafness, form B: Autosomal Recessive Deafness (DFNB6) locus. Therefore, the circling mouse is a potential animal model for DFNB6 deafness in humans.     

Hepatosplanchnic clearance of interleukin-6 in humans during exercise

The cytokine interleukin (IL)-6 can increase markedly in the circulation during exercise, but whether the liver is a source of this increase is unknown. The aim of this study was to measure IL-6 flux across the hepatosplanchnic tissues in humans. To elevate systemic concentrations of IL-6, six healthy male subjects performed 120 min of semirecumbent cycling, and blood samples were simultaneously obtained from a brachial artery and the hepatic vein before and during exercise for the analysis of IL-6. Hepatosplanchnic blood flow (HBF) was measured using the indocyanine green infusion technique. Net hepatosplanchnic IL-6 balance was calculated from these measures. HBF was 1.3 +/- 0.1 l/min at rest and was not reduced throughout exercise, averaging 1.1 +/- 0.2 l/min. Arterial plasma IL-6 markedly increased (P < 0.05) from 1.8 +/- 0.6 ng/l at rest to 14.3 +/- 3.2 ng/l after 120 min of exercise. The hepatosplanchnic viscera did not contribute to this increase, since there was a net hepatosplanchnic IL-6 uptake (0.8 +/- 0.3 vs. 5.5 +/- 1.9 ng/min, rest vs. 120 min; P < 0.05). These data demonstrate that the hepatosplanchnic viscera remove IL-6 from the circulation in humans. This removal may constitute a mechanism limiting the negative chronic metabolic action of chronically elevated circulating IL-6.     

Effects of GH on urea,glucose and lipid metabolism,and insulin sensitivity during fasting in GH-deficient patients

Fasting-related states of distress pose major health problems, and growth hormone (GH) plays a key role in this context. The present study was designed to assess the effects of GH on substrate metabolism and insulin sensitivity during short-term fasting. Six GH-deficient adults underwent 42.5 h of fasting on two occasions, with and without concomitant GH replacement. Palmitate and urea fluxes were measured with the steady-state isotope dilution technique after infusion of [9,10-3H]palmitate and [13C]urea. During fasting with GH replacement, palmitate concentrations and fluxes increased by 50% [palmitate: 378 +/- 42 (GH) vs. 244 +/- 12 micromol/l, P < 0.05; palmitate: 412 +/- 58 (GH) vs. 276 +/- 42 microM, P = 0.05], and urea turnover and excretion decreased by 30-35% [urea rate of appearance: 336 +/- 22 (GH) vs. 439 +/- 43 micromol. kg-1. h-1, P < 0.01; urea excretion: 445 +/- 43 (GH) vs. 602 +/- 74 mmol/24 h, P < 0.05]. Insulin sensitivity (determined by a euglycemic hyperinsulinemic clamp) was significantly decreased [M value: 1.26 +/- 0.06 (GH) vs. 2.07 +/- 0.22 mg. kg-1. min-1, P < 0.01] during fasting with GH replacement. In conclusion, continued GH replacement during fasting in GH-deficient adults decreases insulin sensitivity, increases lipid utilization, and conserves protein.     

Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport

Bicaudal D is an evolutionarily conserved protein, which is involved in dynein-mediated motility both in Drosophila and in mammals. Here we report that the N-terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. Here, we use the BICD2 N-terminal domain as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N-terminal domain is reduced within the full-length molecule, indicating that the C-terminal part of the protein might regulate the interaction between BICD2 and the motor complex. Our findings provide a novel model system for dissection of the molecular mechanism of dynein motility.     

Enzyme kinetic characterization of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta, PTPepsilon, CD45, LAR, PTP1B and SHP-1), using pNPP as substrate. Most noticeable is the increase in the turnover number for PTPbeta with increasing pH and the weak pH-dependence of the turnover number of CD45. The kinetic data for PTPalpha-D1 and PTPalpha-D1D2 suggest that D2 affects the catalysis of pNPP. PTPepsilon and the closely homologous PTPalpha behave differently. The K(m) data were lower for PTPepsilon than those for PTPalpha, while the inverse was observed for the catalytic efficiencies.     

Hox genes from the earthworm Perionyx excavatus

The Hox genes of the oligochaete, Perionyx excavatus, were surveyed using PCR and phylogenetic analysis. We were able to identify 11 different Hox gene fragments. Comparative and phylogenetic analyses revealed that this oligochaete would have at least five Hox genes of the anterior group, including three copies of labial-type, five of the central group and one of the posterior group. This is the first report regarding sequence information and phylogenetic analysis of Hox genes in the earthworm.     

EGF receptor residues leu(679), leu(680) mediate selective sorting of ligand-receptor complexes in early endosomal compartments

Dileucine-based motifs have been shown to regulate endosomal sorting of a number of membrane proteins. Previously, we have shown that the dileucine motif Leu(679), Leu(680) in the juxtamembrane domain of the human epidermal growth factor receptor is involved in the endosome-to-lysosome transport of ligand-receptor complexes. Substitution of alanine residues for Leu(679), Leu(680) led to a reduction in ligand-induced receptor degradation without affecting internalization. In the current study, we have further characterized ligand-dependent intracellular sorting of EGF receptors containing a L679A, L680A. Immunocytochemical studies reveal that although mutant receptors redistribute from the cell surface to transferrin receptor-positive endocytic vesicles similar to wild-type following ligand stimulation, their accumulation in Lamp-1-positive late endosomes/lysosomes is retarded compared to wild-type. Kinetic analysis of (125)I-EGF trafficking shows that reduced accumulation of internalized mutant receptors in Lamp-1-positive vesicles is due to rapid recycling of ligand-receptor complexes from early endocytic compartments. In addition, the fraction of intracellular (125)I-EGF that is transported to late endocytic compartments in cells with mutant receptors is not as efficiently degraded as it is in cells with wild-type receptors. Furthermore, wild-type receptors in endocytic vesicles isolated by Percoll gradient fractionation are more resistant to in vitro digestion with proteinase K than mutant receptors. We propose that mutant receptors interact inefficiently with lysosomal sorting machinery, leading to their increased recycling. Our results are consistent with a model in which the Leu(679), Leu(680) signal facilitates sequestration of ligand-receptor complexes into internal vesicles of multivesicular endosome-to-lysosome transport intermediates.     

Cloning and Bacterial Expression of Sesquiterpene Cyclase, a Key Branch Point Enzyme for the Synthesis of Sesquiterpenoid Phytoalexin Capsidiol in UV-Challenged Leaves of Capsicum annuum

Sesquiterpene cyclase, a branch point enzyme in the generalisoprenoid pathway for the synthesis of phytoalexin capsidiol,was induced in detached leaves of Capsicum annuum (pepper) byUV treatment. The inducibility of cyclase enzyme activitiesparalleled the absolute amount of cyclase protein(s) of pepperimmunodetected by monoclonal antibodies raised against tobaccosesquiterpene cyclase. A cDNA library was constructed with poly(A)⁺RNA isolated from 24 h UV-challenged leaves of pepper. A cDNAclone for sesquiterpene cyclase in pepper was isolated by usinga tobacco 5-epi aristolochene synthase gene as a hetero-logousprobe. The predicted protein encoded by this cDNA was comprisedof 559 amino acids and had a relative molecular mass of 65,095.The primary structural information from the cDNA clone revealedthat it shared 77%, 72% and 49% identity with 5-epi aristolochene,vetispiradiene, and cadinene synthase, respectively. The enzymaticproduct catalyzed by the cDNA clone in bacteria was identifiedas 5-epi aristolochene, as judged by argentation TLC. RNA blothybridization demonstrated the induction of an mRNA consistentwith the induction of cyclase enzyme activity in UV-treatedpepper. (Received March 2, 1998; Accepted June 15, 1998)     

Nitrogen distribution and leaf area indices in relation to photosynthetic nitrogen use efficiency in savanna grasses

Leaf photosynthetic characteristics, distribution patterns of nitrogen content per unit leaf area (n_L) and leaf area production per unit n_Lwere measured in natural stands of a C₄ grass (Hyparrhenia rufa) from the seasonal savannas and of a C₄grass (Paspalum fasciculatum) and two C₃grasses (Leersia hexandra and Hymenachne amplexicaulis) from the flooded savannas in central Venezuela. Daily rates of canopy photosynthesis (P_cD) as well as the optimal leaf area production per unit n_Lat which P_cDfor a given total amount of nitrogen in the canopy (i.e., canopy-PNUE) is maximized were also calculated. The C₃and C₄species from the flooded savannas had similar light saturated rates of photosynthesis per unit n_L(i.e. leaf-PNUE) and similar canopy-PNUEs which was in strong contrast with previous studies. Especially H. rufa but also L. hexandra and H. amplexicaulis had leaf- and canopy-PNUEs which were considerably higher than the values calculated for most other species with the same photosynthetic pathway (i.e., C₃or C₄). In contrast to previous studies, differences in the light gradient in the canopy between stands only partially explained differences in N distribution. Measured leaf area indices were greater and the average n_L values were consequently smaller than the calculated optima. There was, however, a very strong linear correlation between the optimal and actual average n_Lindicating that even though the model overestimated average n_L, it did predict the differences in leaf area production per unit nitrogen – the inverse average n_L– very well. This result strongly indicates that leaf area production per unit of leaf nitrogen increases with leaf-PNUE and decreases with the extinction coefficient for light. Grass species from seasonal savannas have extremely high leaf-PNUEs and thus optimally produce large amounts of leaf area per unit n_L. This helps explain how stands of these species may have high leaf area indices and achieve high photosynthetic productivity despite the very low nutrient availability at which they grow.