Elevated Wall Shear Stress in Aortic Type B Dissection May Relate to Retrograde Aortic Type A Dissection: A Computational Fluid Dynamics Pilot Study.

OBJECTIVE: Retrograde aortic type A dissection (RTAD) is a known complication in patients with aortic type B dissection. The purpose of this computational fluid dynamics (CFD) study was to identify haemodynamic risk factors for the occurrence of RTAD. METHODS: Computed tomographic angiography (CTA) images of 10 patients with type B dissections, who subsequently developed a RTAD, were retrospectively analysed together with patients constituting a control group (n = 10) where no further vascular events after the initial type B dissection occurred. CFD simulations were conducted based on 3D surface models of the aortic lumen derived from CTA datasets. For both groups, pressures, velocity magnitudes and wall shear stress (WSS) were compared at the site of the future RTAD entry tear and the surrounding aortic wall. RESULTS: WSS at the site of the future entry tear was significantly elevated compared with the surrounding wall (15.10 Pa vs. 5.15 Pa, p < .001) and was significantly higher in the RTAD group than in the control group (6.05 Pa, p < .002). Pressures and velocity magnitudes were not significantly elevated at the entry tear (3825.8 Pa, 0.63 m/s) compared with the aortic arch (3549.8 Pa, 0.50 m/s) or control group (3501.7 Pa, 0.62 m/s). CONCLUSIONS: Increased WSS accompanies the occurrence of RTAD. The results merit the design for a prospective study to confirm whether WSS is a risk factor for the occurrence of RTAD.

Rapid correction of low vitamin D status in nursing home residents.

UNLABELLED: This prospective study finds that ergocalciferol 50,000 IU three times weekly for four weeks effectively and safely corrects vitamin D inadequacy in nursing home residents. INTRODUCTION: Low vitamin D status is common among nursing home residents and contributes to bone loss, falls and fractures. The objective of this study was to evaluate the efficacy and safety of short course, high dose, oral vitamin D(2) (ergocalciferol) treatment. METHODS: This prospective study included 63 nursing home residents. The 25 with low vitamin D status (serum 25(OH)D < or = 25 ng/ml) received oral ergocalciferol 50,000 IU three times weekly for four weeks; the others received no change to their routine care. Serum total 25(OH)D, 25(OH)D(2), 25(OH)D(3), calcium, parathyroid hormone (PTH), bone turnover markers and neuro-cognitive assessments were obtained at baseline and four weeks. RESULTS: Mean total 25(OH)D concentration increased (p < 0.0001) from 17.3 to 63.8 ng/ml in the treated group and remained unchanged in the comparison group. Serum 25(OH)D(3) remained stable in the comparison group, but declined (p < 0.0001) with D(2) treatment from 15.4 to 9.1 ng/ml. Serum PTH trended down in the treatment group (p = 0.06). No treatment-induced improvement in ambulation, cognition or behavior was observed. No hypercalcemia or other adverse effects were observed with ergocalciferol treatment. CONCLUSION: Four weeks of oral vitamin D(2) supplementation effectively and safely normalizes serum 25(OH)D in nursing home residents.

Vitamin K-dependent carboxylase: utilization of decarboxylated bone Gla protein and matrix Gla protein as substrates.

The ability of des-gamma-carboxy bone Gla protein (dBGP) and des-gamma-carboxy matrix Gla protein (dMGP) to act as substrates for the rat liver vitamin K-dependent carboxylase has been investigated. An amino-terminal 'propeptide' is present on the intracellular form of BGP and is thought to interact with a recognition site on the enzyme. dBGP, lacking this extension, is a poor, high apparent Km, carboxylase substrate, but is a much better substrate when free propeptide is added. MGP lacks an amino-terminal propeptide, but contains a a homologous region in the mature protein. dMGP is an excellent substrate for the carboxylase with a low apparent Km and its carboxylation is inhibited by free propeptide.

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.

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.

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.

Vitamin K supplementation reduces serum concentrations of under-gamma-carboxylated osteocalcin in healthy young and elderly adults.

BACKGROUND: Subclinical vitamin K insufficiency, manifested by under-gamma-carboxylation of the bone matrix protein osteocalcin, may be common. OBJECTIVE: Our objective was to delineate the prevalence of submaximal gamma-carboxylation as assessed by response to phylloquinone supplementation and to evaluate the effect of this intervention on skeletal turnover in healthy North American adults. DESIGN: Healthy subjects (n = 219), approximately equally distributed by sex and age (18-30 y and >/=65 y), received daily phylloquinone (1000 microg) or placebo for 2 wk. Serum undercarboxylated osteocalcin (ucOC) and total osteocalcin, N:-telopeptides of type I collagen (NTx), bone-specific alkaline phosphatase (BSAP), and phylloquinone concentrations were measured at baseline and after weeks 1 and 2. RESULTS: At baseline, the mean serum phylloquinone concentration was lower in the young than in the old group; there was no effect of sex. Concomitantly, baseline %ucOC was highest in the young and lowest in the old men (P: < 0.0001) but did not differ significantly by age in women. After supplementation, serum phylloquinone concentration increased approximately 10-fold (P: < 0.0001) at week 1 (from 0.93 +/- 0.08 to 8.86 +/- 0.70 nmol/L, x+/- SEM); this was sustained through week 2. Among all supplemented groups, mean %ucOC decreased from 7.6% to 3. 4% without significant differences by age or sex; 102 of 112 subjects had a >1% decrease. Phylloquinone supplementation reduced serum osteocalcin but did not alter NTx or BSAP concentration. CONCLUSIONS: Usual dietary practices in this population did not provide adequate vitamin K for maximal osteocalcin carboxylation. Phylloquinone supplementation reduced serum osteocalcin concentration but did not alter other markers of serum bone turnover.

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.

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.

Derivatives of 2-methyl-1,4-naphthoquinone as substrates and inhibitors of the vitamin K dependent carboxylase.

A series of peptides that contain an N-terminal 2-methyl-1,4-naphthoquinone group or analogues of this structure have been prepared as potential substrates or inhibitors of the rat liver microsomal vitamin K dependent carboxylase. The parent compound, gamma-2-(methyl-1,4-naphthoquinonyl-3)butyryl-Glu-Glu-Leu-OMe, is a good substrate for the carboxylase at low concentrations and has a Km of about 50 microM. This is roughly 2 orders of magnitude lower than the Km of most simple peptide substrates that have been synthesized. Replacement of the 2-methyl-1,4-naphthoquinone group with its desmethyl analogue, a naphthyl, or a stearyl group decreased substrate effectiveness. At higher concentrations, the parent compound and its desmethyl analogue were potent inhibitors of the vitamin K dependent carboxylation reaction. The degree of inhibition exhibited by these peptides was dependent on the vitamin KH2 concentration of the incubation.

Insulin concentrations in aqueous humor after paracentesis and feeding of rabbits.

Although the lens has been shown to have the capacity to respond to insulin in vitro, little is known concerning the biochemical relationships of insulin to the lens in vivo. Therefore we have measured insulin in the aqueous humor of rabbits by a sensitive radioimmunoassay after paracentesis and feeding. The insulin concentration in aqueous humor was 3% of that in plasma. One hour after paracentesis the aqueous humor insulin concentration was increased sixfold, apparently due to breakdown of the blood-aqueous barrier, but 1 week after paracentesis it had returned to its original level, apparently because of restoration of the blood-aqueous barrier within that time. After feeding, the aqueous humor insulin concentration was increased by 30% compared to a 175% increase in plasma. Factors influencing the aqueous humor insulin concentration and the possibility of insulin influence on lens metabolism are discussed.

Prothrombin synthesis in rat hepatoma H4IIEC3 cells: response to the proposed "coagulopoietin factor".

Based on studies in intact animals, the presence of humoral factors, "coagulopoietins", which regulate the synthesis of vitamin K-dependent plasma proteins has been proposed. These proposed factors are produced in response to vitamin K deficiency, coumarin treatment, or specific antibody depletion of vitamin K-dependent clotting factors. The production of prothrombin by rat hepatoma H4IIEC3 cells has now been shown to be dependent on the source of bovine serum in the media. Cells grown in serum from cows treated with dicoumarol produce about 20% more prothrombin in 24 h than those cells grown in control serum. The humoral factor causing this response is present early in the course of dicoumarol treatment, and the increase in prothrombin production is dependent on the amount of serum from a dicoumarol-treated cow in the media. Based on membrane filtration studies, the factor appears to be associated with the protein fraction of serum.

Effect of N-methyl-thiotetrazole on rat liver microsomal vitamin K-dependent carboxylation.

The use of a number of antibiotics which contain an N-methyl-thiotetrazole (NMTT) side chain has been reported to be associated with an increased incidence of hypoprothrombinemia. The suggested role of NMTT as an inhibitor of the liver microsomal vitamin K-dependent carboxylase has been investigated. In standard incubations, NMTT had no effect on carboxylation when vitamin KH2 was a substrate but was a weak inhibitor when [vitamin K + NADH] was a substrate. Microsomal vitamin K reductases, however, were not inhibited by NMTT. Preincubation of the incubation mixture with NADH and NMTT resulted in inhibition of carboxylase activity when either vitamin KH2 or [vitamin K + NADH] was the substrate. A fraction of the microsomal membrane which was not readily solubilized by dilute detergent protected the enzyme from this inhibition. The data suggest that NMTT is metabolized to an active inhibitor or is able to covalently inactivate the enzyme in the presence of NMTT. The vitamin K responsiveness of the clinically observed hypoprothrombinemia suggests that it is not related to this in vitro inhibition of the vitamin K-dependent carboxylase.

Vitamin K-dependent carboxylase activity, prothrombin mRNA, and prothrombin production in two cultured rat hepatoma cell lines.

The presence of under-gamma-carboxylated forms of plasma prothrombin is a marker for human primary hepatocellular carcinoma. A rat hepatoma cell line (7777) which was previously shown to secrete undercarboxylated prothrombin when grown as a solid tumor has now been grown in monolayer culture. This cell line has a decreased activity of the microsomal vitamin K-dependent carboxylase when compared to a control (H4IIEC3) hepatoma line, does not increase intracellular prothrombin concentrations in response to vitamin K depletion, and secretes undercarboxylated prothrombin even when grown in vitamin K supplemented media. Prothrombin gene expression in the 7777 cell line, as measured by prothrombin mRNA levels, was not altered in the 7777 cell line. This cell line appears to be a model for assessing the cellular alterations responsible for undercarboxylated prothrombin excretion by human hepatocellular tumors.

Thin film MnGe grown on Si(111).

MnGe has been grown as a thin film on Si(111) substrates by molecular beam epitaxy. A 10 Å layer of MnSi was used as the seed layer in order to establish the B20 crystal structure. Films of a thickness between 45 and 135 Å have been prepared and structurally characterized using reflection high-energy electron diffraction, atomic force microscopy and x-ray diffraction. These studies provided evidence that MnGe forms in the cubic B20 crystal structure as islands exhibit a very smooth surface. The islands become larger with increasing film thickness. A magnetic characterization reveals that the ordering temperature of MnGe thin films is enhanced compared to that for bulk material. The properties of the helical magnetic structure obtained from magnetization and magnetoresistivity measurements are compared with those of films of the related compound MnSi. The much stronger Dzyaloshinskii-Moriya interaction in MnGe results in a higher rigidity of the spin helix.

Evaluation of ergocalciferol or cholecalciferol dosing, 1,600 IU daily or 50,000 IU monthly in older adults.

CONTEXT: Whether ergocalciferol (D(2)) and cholecalciferol (D(3)) are equally effective to increase and maintain serum 25-hydroxyvitamin D [25(OH)D] concentration is controversial. OBJECTIVE: The aim of the study was to evaluate the effect of daily and once monthly dosing of D(2) or D(3) on circulating 25(OH)D and serum and urinary calcium. DESIGN, SETTING AND PARTICIPANTS: In a university clinical research setting, 64 community dwelling adults age 65+ were randomly assigned to receive daily (1,600 IU) or once-monthly (50,000 IU) D(2) or D(3) for 1 yr. MAIN OUTCOME MEASURES: Serum 25(OH)D, serum calcium, and 24-h urinary calcium were measured at months 0, 1, 2, 3, 6, 9, and 12. Serum PTH, bone-specific alkaline phosphatase, and N-telopeptide were measured at months 0, 3, 6, and 12. RESULTS: Serum 25(OH)D was less than 30 ng/ml in 40% of subjects at baseline; after 12 months of vitamin D dosing, levels in 19% of subjects (n = 12, seven receiving daily doses and five monthly doses) remained low, despite compliance of more than 91%. D(2) dosing increased 25(OH)D(2) but produced a decline (P < 0.0001) in 25(OH)D(3). Substantial between-individual variation in 25(OH)D response was observed for both D(2) and D(3). The highest 25(OH)D observed was 72.5 ng/ml. Vitamin D administration did not alter serum calcium, PTH, bone-specific alkaline phosphatase, N-telopeptide, or 24-h urine calcium. CONCLUSIONS: Overall, D(3) is slightly, but significantly, more effective than D(2) to increase serum 25(OH)D. One year of D(2) or D(3) dosing (1,600 IU daily or 50,000 IU monthly) does not produce toxicity, and 25(OH)D levels of less than 30 ng/ml persist in approximately 20% of individuals. Substantial between-individual response to administered vitamin D(2) or D(3) is observed.

Dynamics of beta-CH and beta-CH2 Groups of Amino Acid Side Chains in Proteins.

The dynamics of amino acid side chains of uniformly 13C/15N-enriched ribonuclease T1 (RNase T1) have been investigated. Heteronuclear longitudinal relaxation rates, 1H/13C NOEs, and transverse cross-correlated cross-relaxation rates between the Sx and the SxIz1Iz2 operators (SIIS cross relaxation) [Ernst and Ernst (1994) J. Magn. Reson., A110, 202-213] have been determined in this study. New pulse sequences for measuring the longitudinal relaxation time and the heteronuclear NOE of aliphatic side chain carbon nuclei were developed using the CCONH type of magnetization transfer and 1HN detection. In addition, an improved pulse sequence for the determination of the SIIS cross relaxation is presented. For the analysis of the relaxation rates, the model of restricted rotational diffusion around the chi1 dihedral angle has been applied [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. These techniques were used in order to describe the side chain dynamics of the small globular protein RNase T1 (104 amino acids, MW about 11 kDa). Qualitative values of microdynamical parameters were obtained for 73 out of 85 amino acid side chains (glycine and alanine residues excepted) whereas more quantitative values were derived for 67 beta-CH and beta-CH2 groups.

Determination of (13)C (α) relaxation times in uniformly (13)C/ (15)N-enriched proteins.

Relaxation times of (13)C(α) carbons of uniformly (13)C/(15)N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T(1), T(2) and the heteronuclear NOE of (13)C(α) in uniformly (13)C/(15)N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T(1) and the heteronuclear NOE were similar to those of the corresponding (15)N experiments published previously. The determination of T(2) for the C(α)-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T(2) evolution period owing to the scalar coupling of C(α)-C(ß) must be suppressed. Various different pulse sequences for the T(2) evolution period were simulated in order to optimize the bandwidth for which reliable T(2) relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual (1)H-(13)C(α) cross peaks, in a series of ((1)H, (13)C)-ct-HSQC spectra with a modified CPMG sequence as well as a T(1p) sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.