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Biomedical subjects

W L Kelly

Publications and source records attributed to W L Kelly.

7 recordsLinked to original sources
PubMed2001

Spectroscopic studies of substrate interactions with clavaminate synthase 2, a multifunctional alpha-KG-dependent non-heme iron enzyme: correlation with mechanisms and reactivities.

Using a single ferrous active site, clavaminate synthase 2 (CS2) activates O(2) and catalyzes the hydroxylation of deoxyguanidinoproclavaminic acid (DGPC), the oxidative ring closure of proclavaminic acid (PC), and the desaturation of dihydroclavaminic acid (and a substrate analogue, deoxyproclavaminic acid (DPC)), each coupled to the oxidative decarboxylation of cosubstrate, alpha-ketoglutarate (alpha-KG). CS2 can also catalyze an uncoupled decarboxylation of alpha-KG both in the absence and in the presence of substrate, which results in enzyme deactivation. Resting CS2/Fe(II) has a six-coordinate Fe(II) site, and alpha-KG binds to the iron in a bidentate mode. The active site becomes five-coordinate only when both substrate and alpha-KG are bound, the latter still in a bidentate mode. Absorption, CD, MCD, and VTVH MCD studies of the interaction of CS2 with DGPC, PC, and DPC provide significant molecular level insight into the structure/function correlations of this multifunctional enzyme. There are varying amounts of six-coordinate ferrous species in the substrate complexes, which correlate to the uncoupled reaction. Five-coordinate ferrous species with similar geometric and electronic structures are present for all three substrate/alpha-KG complexes. Coordinative unsaturation of the Fe(II) in the presence of both cosubstrate and substrate appears to be critical for the coupling of the oxidative decarboxylation of alpha-KG to the different substrate oxidation reactions. In addition to the substrate orientation relative to the open coordination position on the iron site, it is hypothesized that the enzyme can affect the nature of the reactivity by further regulating the binding energy of the water to the ferrous species in the enzyme/succinate/product complex.

Circular Dichroism↗
PubMed2001

Growth hormone is permissive for skeletal adaptation to mechanical loading.

The Lewis dwarf (DW) rat was used as a model to test the hypothesis that growth hormone (GH) is permissive for new bone formation induced by mechanical loading in vivo. Adult female Lewis DW rats aged 6.2 +/- 0.1 months (187 +/- 18 g) were allocated to four vehicle groups (DW), four GH treatment groups at 32.5 microg/100 g body mass (DWGH1), and four GH treatment groups at 65 microg/100 g (DWGH2). Saline vehicle or GH was injected intraperitoneally (ip) at 6:30 p.m. and 6:30 a.m. before mechanical loading of tibias at 7:30 a.m. A single period of 300 cycles of four-point bending was applied to right tibias at 2.0 Hz, and magnitudes of 24, 29, 38, or 48N were applied. Separate strain gauge analyses in 5 DW rats validated the selection of loading magnitudes. After loading, double-label histomorphometry was used to assess bone formation at the periosteal surface (Ps.S) and endocortical surface (Ec.S) of tibias. Comparing left (unloaded) tibias among groups, GH treatment had no effect on bone formation. Bone formation in tibias in DW rats was insensitive to mechanical loading. At the Ec.S, mechanically induced lamellar bone formation increased in the DWGH2 group loaded at 48N (p < 0.05), and no significant increases in bone formation were observed among other groups. The percentage of tibias expressing woven bone formation (Wo.B) at the Ps.S was significantly greater in the DWGH groups compared with controls (p < 0.05). We concluded that GH influences loading-related bone formation in a permissive manner and modulates the responsiveness of bone tissue to mechanical stimuli by changing thresholds for bone formation.

Adaptation, Physiological↗
PubMed1998

Localisation of prostaglandin endoperoxide H synthase (PGHS)-1 and PGHS-2 in bone following mechanical loading in vivo.

Recent data suggests that induction of prostaglandin endoperoxide H synthase-2 (PGHS-2) is critical for the anabolic response of lamellar bone elicited by mechanical strain in vivo. The aim of the present study was to localise PGHS-1 and PGHS-2 in rat tibiae following four-point bending in vivo. Right tibiae of 19 adult female rats were subjected to 300 cycles of bending or sham loading at 2.0 Hz with an applied load of 65 N. At 0, 6, and 24 hr postloading, rats were anaesthetised and perfused with Bouin's fixative. Left and right tibiae were dissected, postfixed for 4 hr at 4 degrees C, decalcified in EDTA, and embedded in paraffin. Serial 5 pM sections were stained for PGHS-1 and PGHS-2 using standard immunoperoxidase procedures. For the first time, immunoreactivity for both PGHS-1 and PGHS-2 was localised in bone cells in situ, in the rat tibia. PGHS-1 was distributed widely in all tibiae, while PGHS-2 showed sparse localisation. At the endocortical surfaces (EcS), osteoblasts, lining cells, and osteocytes close to the surface reacted strongly for PGHS-1, as did intracortical osteocytes. At the periosteal surface (PsS), osteoblasts and cells of the osteogenic region were immunopositive. Immediately after loading, the numerical density (n.mm(-2)) of osteocytes labeled with PGHS-1 was significantly greater in loaded tibiae compared to controls. This increase was not seen after sham loading. At 6 and 24 hr postloading, this difference was no longer evident. Staining for PGHS-2 was sparse compared to PGHS-1. Light to moderate reactivity was observed in osteocytes and canaliculae, but the numerical density of labeled cells was significantly less than that for PGHS-1. Moderate staining was seen in lining cells and osteoblasts at the EcS and PsS of some tibiae. Osteoclasts at the PsS reacted strongly for both PGHS-1 and PGHS-2. There was a similar load-related increase in the density of PGHS-2-labeled osteocytes 0 hr postloading. The labeled osteocyte density had decreased at 6 hr, but remained significantly greater in loaded bones. These results show that both forms of PGHS can be localised in bone cells, with PGHS-1 expressed to a greater extent than PGHS-2. The data also suggest that both PGHS-1 and PGHS-2 may play important roles in the early response of bone to mechanical loading in vivo.

Adaptation, Physiological↗
PubMed1985

Coexistence of peptide immunoreactivity in sensory neurons of the cat.

The coexistence of the neuropeptides substance P, cholecystokinin, somatostatin and vasoactive intestinal polypeptide in cat sensory neurons has been examined using peroxidase-anti-peroxidase immunocytochemistry. Attempts were also made to locate cells containing bombesin, neurotensin, [Met]enkephalin and [Leu]enkephalin but no immunoreactivity was found when antisera to these peptides was used. Cells in the dorsal root ganglia were studied by cutting 5 microns serial wax sections or 15 microns cryostat sections. Coexistence was established by applying the antiserum to each peptide to serially adjacent 5 microns sections and establishing the presence of peptide-like immunoreactivity in each of 4 different sections through a single cell. Results showed that the distribution and combinations of coexistence of these neuropeptides in the cat is extremely complex; three and sometimes all four antisera showing immunoreactivity with a single cell. About 21% of all ganglion cells contained some immunoreactivity but there were certainly some small cells which did not contain any immunoreactivity. The coexistence of these peptides differed markedly from that previously reported in the rat suggesting that interspecific differences in the neuropeptide content of cells might be much greater than they are for classical neurotransmitters. The results are discussed in relation to the possible role of neuropeptides and the regulation of their production by sensory neurons.

Animals↗
PubMed1983

A modified differential stain for cartilage and bone in whole mount preparations of mammalian fetuses and small vertebrates.

Fixation in formol-acetic-alcohol as a prelude to the staining of whole mount vertebrate skeletons with alcian blue and alizarin red S has greatly facilitated the enzyme clearing step of the method outlined by Dingerkus and Uhler. The modified method has been tested on fetal and neonatal mice, and on a variety of vertebrates including bony fish, reptiles, amphibia and birds, and shown to be rapid, reproducible and permanent. The method is not so rapid as that reported by Kimmel and Trammell but is superior at least in certain circumstances. In the present study, optimal results were obtained by fixing in formol-acetic-alcohol for 40 minutes, staining cartilage with alcian blue 8GX, then clearing with trypsin. The time taken to complete the latter step was reduced significantly by incubation at 37 C. The next step was to stain bone using alizarin red S in a weak solution of potassium hydroxide, followed by clearing in a potassium hydroxide-glycerol series.

Amphibians↗