Cloning and characterization of murine neuromedin U receptors

Neuromedin U (NmU) is a neuropeptide involved in various physiological functions such as feeding behavior, muscle contractile activity, and regulation of intestinal ion transport. Recently, two human G protein-coupled receptors have been identified as NmU-specific receptors, NmU-R1 and NmU-R2, which share 55% amino acid identity. It is unclear however, which of the two receptors mediates responses to NmU observed in rodent models. Attempts to define the pharmacological profile of the two receptors are confounded by overlapping expression of the two receptors and a lack of subtype-selective compounds. In order to establish a basis to further our understanding of the function of these receptors, we cloned and characterized the mouse homologues of the two human NmU receptors. Mouse NmU-R1 and mouse NmU-R2 are 79 and 81% identical to their respective human homologues. Expression of NmU-R1 was mainly observed in testis, gastrointestinal (GI) tract, and immune system, while NmU-R2 was primarily expressed in brain tissues. Each mouse receptor was independently expressed in HEK293 cells and demonstrated a dose-dependent calcium flux in response to NmU-8, NmU-23 and NmU-25. In an attempt to identify a synthetic NmU peptide that would exhibit selectivity at one of the two receptors, we examined the functional activity of eight alanine-substituted NmU-8 peptides. These experiments demonstrated that alanine substitution at positions 5 and 7 affects the functional activity of the peptide at both receptors. The arginine residue at position 7 is required for NmU-8 activity at either receptor while alanine substitution at position 5 selectively affects the potency and the efficacy at mNmU-R1. These experiments validate the use of rodent models to characterize NmU function relative to humans and suggest that substitution at Arginine-5 of NmU-8 may provide a receptor selective peptide.     

The physical location of fourteen RFLP markers in rice (Oryza sativa L.)

A biotin-labeled in situ hybridization technique was used in order to physically map RFLP markers to the chromosomes of rice (Oryza sativa L.). Fourteen RFLP markers, associated with the ends of the linkage groups on rice chromosomes 7, 8, 11, 12, were physically mapped onto specific regions of the chromosomes. The average detection rate of in situ hybridization was 5.91%. The markers were located on seven different chromosome arms. Ten of the fourteen markers were distributed near the chromosome ends. This demonstrated that the RFLP linkage groups involved covered a wide physical distance and that the centromeric region was bisected by all but one linkage group. Two markers covered a short genetic distance but were physically distant, while two covering a longer genetic distance were physically closer together. This indicates that considerable variation can, and does, exist between genetic and physical maps.This paper is a contribution of the U.S. Department of Agriculture, Agricultural Research Service, and Missouri Agricultural Experiment Station, Journal Series No. 11 882All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicapThis paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the U.S. Department of Agriculture or the University of Missouri     

Characterization of a new degradable polymer scaffold for regeneration of the dermis: In vitro and in vivo human studies

Full thickness skin wounds in humans heal with scars, but without regeneration of the dermis. A degradable poly(urethane urea) scaffold (PUUR), Artelon® is already used to reinforce soft tissues in orthopaedics, and for treatment of osteoarthritis of the hand, wrist and foot. In this paper we have done in vitro experiments followed by in vivo studies to find out whether the PUUR is biocompatible and usable as a template for dermal regeneration. Human dermal fibroblasts were cultured on discs of PUUR, with different macrostructures (fibrous and porous). They adhered to and migrated into the scaffolds, and produced collagen. The porous scaffold was judged more suitable for clinical applications and 4 mm Ø, 2 mm-thick discs of porous scaffold (12% w/w or 9% w/w polymer solution) were inserted intradermally in four healthy human volunteers. The implants were well tolerated and increasing ingrowth of fibroblasts was seen over time in all subjects. The fibroblasts stained immunohistochemically for procollagen and von Willebrand factor, indicating neocollagenesis and angiogenesis within the scaffolds. The PUUR scaffold may be a suitable material to use as a template for dermal regeneration.Key words: dermal regeneration, tissue engineering, polymer scaffold, wound healing, in vitro, in vivo, guided tissue regeneration, human, burns     

Bradykinin stimulates bone resorption and lysosomal-enzyme release in cultured mouse calvaria.

The effect of bradykinin on bone resorption was studied in cultures of newborn-mouse calvaria. Bradykinin (0.03 microM, 1 microM) stimulated the release of 45Ca2+ from bones dissected out from mice prelabelled in vivo with 45Ca. Bradykinin (1 microM) also augmented the release of stable calcium ( 40Ca ), Pi and the lysosomal enzyme beta-glucuronidase. The stimulatory effect of bradykinin on mineral mobilization and lysosmal -enzyme release could be blocked by indomethacin. It is speculated that concomitant generation of thrombin and bradykinin in areas of trauma and inflammation may induce resorption of nearby bone tissue.     

Annual variations in plasma sex steroid-binding protein and testosterone concentrations in the adult male little brown bat: relation to the asynchronous recrudescence of the testis and accessory reproductive organs

The annual reproductive cycle of the male little brown bat, in contrast to seasonal reproductive patterns of other mammals, is differentiated by an asynchronous recrudescence of the testis and the accessory reproductive glands. Spermatogenesis occurs during the summer, whereas fully stimulated accessory organs, stored epididymal spermatozoa, and sexual behavior are expressed later during a mating period that extends, albeit interrupted by hibernation, from late summer until early spring. To investigate whether changes in high affinity androgen-binding activity in the circulation are related to the delayed renewal of the accessory organs, plasma sex steroid-binding protein (SBP) and total testosterone (T) levels were measured throughout the year. From these data and determinations of association constants for T binding to SBP and albumin at both hibernating (4 degrees C) and active (40 degrees C) temperatures, estimates of the unbound ("free") and albumin-bound T fractions were made and correlated with changes in the accessory reproductive organs. Plasma SBP concentrations (mean +/- SEM) exhibited wide seasonal fluctuations: they were baseline in May (10 +/- 2 nM) following spring arousal, increased dramatically in June (184 +/- 24 nM), and reached peak levels in early July (262 +/- 29 nM), where they remained until August. In late August they began to fall (104 +/- 23 nM) and then returned to baseline during the hibernation period (October-April). Although total T levels were also elevated in June, it appeared that the unbound ("free") and the unbound plus albumin-bound T fractions did not increase until late July. Since the accessory gland weights did not begin to increase until late July as well, it was concluded that increases in the unbound and albumin-bound T fractions may be an important factor in the recrudescence of the accessories and that increased SBP activity in early summer may play a role in the regression and delayed renewal of these organs. However, what factor(s) maintain the accessory glands, epididymal spermatozoa, and sexual behavior during the breeding and hibernation periods when all T fractions were low are, as yet, undetermined.     

Photosynthesis and Activity of Ribulose Bisphosphate Carboxylase of Wheat and Maize Seedlings during and following Exposure to O(2)-Low, CO(2)-Free N(2)

Seven day old wheat and maize seedlings were exposed to 1300 or 2000 microeinsteins per square meter per second photosynthetically active radiation in CO₂-free air for 3 hours with either 1% O₂ in N₂ or N₂-only and then returned to normal air of 340 microliters per liter CO₂, 21% O₂ in N₂. Activity of the ribulose bisphosphate carboxylase and amount of the substrate, ribulose 1,5-bisphosphate, were measured during and following the CO₂-free treatments as was photosynthetic CO₂ fixation. Photoinhibition of photosynthesis was observed only with wheat seedlings following the N₂ only treatment. During the CO₂-free treatments, the levels of RuBP rose during all experiments except when wheat was photoinhibited. The activity of the ribulose bisphophate carboxylase, measured directly upon grinding the leaves, declined during the CO₂-free conditions. The carboxylase total activity increased in minutes in the leaf during and following the CO₂-free treatments. The specific activities of the wheat carboxylase went from 0.16 to 1.06 micromoles CO₂ fixed per milligram protein per minute while the maize carboxylase varied from 0.05 to 0.36 micromole CO₂ fixed per millogram protein per minute. This suggests that in these seedlings considerable inactive carboxylase must be stored in a form not activatable in extracts by CO₂ and Mg²⁺. Possible mechanisms of regulation of photosynthesis by the ribulose bisphosphate carboxylase must consider not only the amount of active enzyme, but the amount of enzyme which the plant can make activatable upon demand.     

Phosphopeptide binding by Sld3 links Dbf4‐dependent kinase to MCM replicative helicase activation

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45‐MCM‐GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK‐dependent manner. Sld3 binds specifically to DDK‐phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho‐MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK‐independent replication. Thus, Sld3 is an essential “reader” of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.