Biomedical subjects

J Engelke

Publications and source records attributed to J Engelke.

7 recordsLinked to original sources

PubMed2003

Bone loss detection in rats using a mouse densitometer.

Estrogen-depletion bone-loss studies often use ovariectomized (ovx) rats and measure bone mineral density in vivo or ex vivo using DXA. Recently, a portable densitometer (PIXImus) was developed for mouse research; however, its use in rats is unclear. This study compared the ability of PIXImus and a standard densitometer (DPXL) to detect ovx-induced bone loss in rats both in vivo and ex vivo. Additionally, instrument accuracy was assessed by comparing measured bone mass with ash weight. Finally, the use of two distal femur regions of interest (ROI) to detect ovx-induced bone loss was evaluated. Twenty-three 6-month-old nulliparous female Sprague-Dawley rats were randomly assigned to sham or ovx groups. Distal femur bone mineral density was assessed at baseline and at 1 and 2 months postoperatively, using a PIXImus and DPXL densitometer. At 3 months postoperatively, all animals were killed, and ex vivo femur scans obtained. Distal femur bone loss was demonstrable by 1 month post-ovx using either densitometer. With the PIXImus, a 4-mm ROI demonstrated greater bone loss (p < 0.05) than an 8-mm ROI. Using the 4-mm ROI, similar amounts of bone loss were detected by the PIXImus and DPXL: 22.2% and 22.4%, respectively, at 2 months post-ovx. Total femur bone mineral content was overestimated by the PIXImus but highly correlated with the DPXL measurement (r = 0.988) and ash weight (r = 0.998). Given its comparability to standard DXA plus its rapid scan speed and portability, the PIXImus is useful in evaluating ovx-induced osteopenia in rats.

Animals↗

PubMed2002

Vitamin K supplementation does not affect ovariectomy-induced bone loss in rats.

Vitamin K may be important in bone metabolism. Notably, high-dose menaquinone-4 (menatetrenone, MK4) has been reported to reduce ovariectomy (ovx)-induced bone loss in rats and to decrease osteoporotic fracture in postmenopausal women. However, it is unclear whether these beneficial effects reflect a physiologic effect of vitamin K, or indicate direct pharmacologic activity of MK4. To further evaluate this, 60 6-month-old nulliparous Sprague-Dawley rats were randomized by distal femur bone mineral density (BMD) in a 3:1 ratio to ovx or sham groups. The sham and one ovx group's diet contained 1% calcium and 1300 microg/kg of vitamin K1, phylloquinone. Diets of the other two ovx groups were supplemented with 882 mg phylloquinone or MK4 per kilogram chow. Distal femur bone mineral density (DFBMD) in an 8 mm region of interest was measured at baseline, 1 and 3 months postoperatively, utilizing dual-energy X-ray absorptiometry (DXA). All animals were killed at 3 months, their right femurs excised, ex vivo BMD measured by DXA, and biomechanical testing performed. No effect of phylloquinone or MK4 supplementation on ovx-induced bone loss was observed. Specifically, DFBMD declined 10.5%, 9.2%, and 11.2% at 1 month and 14.4%, 10.6%, and 13.9% at 3 months in the ovx control, high phylloquinone, and high MK4 groups, respectively. In addition, serum osteocalcin was elevated by ovx; this was not altered by phylloquinone or MK4. Finally, femoral biomechanical properties were not affected by phylloquinone or MK4. To conclude, in this study, neither high-dose phylloquinone nor MK4 reduced the ovx-associated increase in bone turnover or decline in DFBMD.

Animals↗

PubMed2000

Diet- or warfarin-induced vitamin K insufficiency elevates circulating undercarboxylated osteocalcin without altering skeletal status in growing female rats.

To further characterize the skeletal role of vitamin K (K), markers of bone turnover, density, and strength were evaluated in rats with diet- or warfarin (W)-induced K insufficiency. One hundred two, 7-week-old, female rats were randomly assigned to low K (phylloquinone [K1], 20 microg/kg diet), control K (K1, 1300 microg/kg diet), low-dose W (W, 1.5 mg/kg control diet), or high-dose W plus K (W/K1, 10/100 mg/kg diet). Femur bone mineral content (BMC) and bone mineral density (BMD), plasma prothrombin time (PT) and prothrombin concentration (PC), and serum total alkaline phosphatase (ALP) and skeletal alkaline phosphatase (sALP) were measured at baseline and days 20, 40, 60, and 80. Serum total osteocalcin (OC) and undercarboxylated osteocalcin (ucOC) and femur length (FL) were measured at baseline and day 80. Left femur OC was measured and biomechanical testing of the right femur and third lumbar vertebral body was performed at day 80. Low dietary K elevated circulating ucOC (17% higher than control; p < 0.0001) at day 80. Furthermore, in both W groups, essentially all circulating OC was undercarboxylated and femur OC was lower than control (p < 0.0001). However, there was no change in femur percent ucOC, suggesting deposition of less newly synthesized OC. No between group differences were observed in PT, ALP, sALP, FL, BMC, BMD, or bone strength. In conclusion, skeletal K insufficiency can be induced by W or diet manipulation. This does not hinder peak bone mass attainment in female rats; however, W causes less newly synthesized OC to be deposited in bone.

Absorptiometry, Photon↗

PubMed1998

Bound water in apo and holo bovine heart fatty-acid-binding protein determined by heteronuclear NMR spectroscopy.

Two- and three-dimensional heteronuclear NMR experiments have been performed to identify internally bound water molecules in the solution structure of bovine heart fatty-acid-binding protein (heart FABP). NOE and rotating-frame NOE (ROE) cross peaks between protein protons and protons of bound water molecules were observed in two-dimensional H2O-ROE/NOE-1H,15N-heteronuclear single quantum coherence spectra recorded from a uniformly 13C/15N-enriched sample of bovine heart FABP. Contacts between water protons and 23 NH protons of the protein backbone were identified. The protein structure consists of 10 antiparallel beta-strands (betaA-betaJ), forming two nearly orthogonal beta-sheets, and a short helix-turn-helix motif connecting beta-strands A and B. The spatial folding resembles a beta-barrel. Most of the water molecules are localized in the gap between beta-strands D and E, and near the two alpha-helices. In the delipidated heart FABP additional contacts between water molecules and NH protons could be observed using a three-dimensional rotating frame Overhauser 1H,15N heteronuclear single quantum coherence experiment obtained with a 15N-labeled sample of apo-heart FABP.

Animals↗

PubMed1987

Synthetic bovine prothrombin precursor 13-29 for studies of vitamin K dependent carboxylase.

The synthesis of the amino acid sequence found in bovine prothrombin precursor 13-29 (PTP 13-29) has been achieved by solid-phase synthesis of the bis(acetamidomethyl)-protected linear peptide followed by cyclization to the monomeric disulfide. Synthesis of the disulfide bond was achieved by deprotection with mercuric acetate in acetic acid followed by oxidation with potassium ferricyanide. Experimental conditions for closure of the disulfide bond were identified by obtaining the circular dichroism spectra of the linear precursor in a variety of solvent systems. Cyclization in organic solvent systems was not successful but led to the formation of insoluble polymers. Synthetic PTP 13-29 was tested as a substrate for the vitamin K dependent carboxylase. Neither the linear nor cyclic synthetic 17 amino acid peptides were carboxylated as well as the standard, Boc-Glu-Glu-Leu-OMe, at mM concentrations. The estimated Km of synthetic PTP 13-29 is greater than 1 mM. Thus, bovine prothrombin precursor 13-29 is not an unusually effective substrate for the carboxylase as reported by Soute et al.

Animals↗

PubMed1987

Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site.

The liver microsomal vitamin K-dependent carboxylase catalyzes the posttranslational conversion of specific glutamate residues to gamma-carboxyglutamate residues in a limited number of proteins. A number of these proteins have been shown to contain a homologous basic amino acid-rich "propeptide" between the leader sequence and the amino terminus of the mature protein. Plasmids encoding protein C, a vitamin K-dependent protein, containing or lacking a propeptide region were constructed and the protein was expressed in Escherichia coli. The protein products were assayed as substrates in an in vitro vitamin K-dependent carboxylase system. Only proteins containing a propeptide region were substrates for the enzyme. These data support the hypothesis that this sequence of the primary gene product is an important recognition site for this processing enzyme.

Animals↗

PubMed1983

Vitamin K-dependent carboxylase: synthesis of an inhibitor of the glutamyl binding site.

Liver microsomes contain a vitamin K and O2-dependent carboxylase that converts peptide-bound glutamyl residues to gamma-carboxyglutamate residues. The peptide Boc-O-phospho-Ser-O-phospho-Ser-Leu-OMe has now been synthesized. This peptide inhibits the carboxylation of endogenous protein precursors by a detergent-solubilized preparation of the carboxylase and is an apparent competitive inhibitor of the carboxylation of Phe-Leu-Glu-Glu-Leu.

1-Carboxyglutamic Acid↗