The muscle-bone unit of peripheral and central skeletal sites in children and young adults.

UNLABELLED: Changes and gender differences in the muscle bone unit at different skeletal sites were investigated during pubertal development. Females accrued greater BMC in relation to muscle compared to males; these gender differences were greater after adjustment for height and regional fat mass. PURPOSE: To describe changes and gender differences in the muscle-bone unit at different skeletal sites during pubertal development. METHODS: Four hundred forty-two children aged 5-18 years were studied. Measurements of bone mineral content (BMC), lean mass (LM) and fat mass of the whole body (WB), legs, arms and lumbar spine were obtained from dual-energy X-ray absorptiometry. Peripheral quantitative computed tomography was used to measure BMC of the radius diaphysis and cross-sectional muscle area (CSMA) of the mid-forearm. These measurements were used to describe differences between, and within, genders at each pubertal stage in BMC accrual relative to muscle, both before and after adjustment for height, regional fat and muscle at central and peripheral skeletal sites. RESULTS: In males, there were significant increases in adjusted WB and leg BMC at the end of pubertal development. Unadjusted and adjusted lumbar spine BMC increased at the onset of, and at the end, of puberty. Radius BMC increased at most pubertal stages. In females, there were increases in unadjusted and adjusted whole body BMC at late puberty, in leg BMC at the onset of puberty and at pubertal stage four. Unadjusted arm BMC increased at most pubertal stages; however, after adjustment, an increase occurred at pubertal stage four. Both adjusted and unadjusted lumbar spine BMC increased at pubertal stage four. Unadjusted radius BMC increased at most pubertal stages. Females had greater BMC at all skeletal sites, compared to males, except at the radius, where adjusted BMC was greater in males at pubertal stage four. CONCLUSIONS: Males and females accrue more BMC in relation to lean mass at multiple skeletal sites as puberty proceeds. Females accrue more BMC in relation to lean mass, in comparison to males, at most skeletal sites.

Body composition and bone status of children born to mothers with type 1 diabetes mellitus.

BACKGROUND: Altered growth, body composition and abnormalities of skeletal mineralisation have been reported in offspring of mothers with type 1 and type 2 diabetes mellitus. AIMS: The authors hypothesised that children born to mothers with type 1 diabetes mellitus (CDM) would be taller, have higher body mass index (BMI), greater fat mass, thicker diaphyseal bone cortices and reduced trabecular bone mineral density (BMD), as compared to those born to non-diabetic mothers. METHODS: Anthropometric, body composition and bone parameters were assessed using dual-energy x-ray absorptiometry (DXA) and peripheral quantitative CT in 67 white Caucasian CDM (35 boys; age 5-18 years) and in 246 (121 boys) age-matched controls. RESULTS: CDM were taller (p<0.0001), heavier (p<0.0001) and had higher BMI (p=0.02), and had 32% more total body fat mass and 7.5% more total body lean mass than controls. At the total body and lumbar spine (L1-L4) sites, CDM had significantly higher bone area and bone mineral content compared with controls. However, areal BMD at both these sites and lumbar spine bone mineral apparent density were not significantly different in the two groups, indicating that CDM have bigger bones compared with controls but their mineral content per unit area or volume is not substantially different. The distal radial trabecular and total volumetric BMD in CDM was not demonstrably different from controls. At the mid-radius, both periosteal (2.4%; p=0.03) and endosteal circumferences (5.7%; p=0.02) were bigger in CDM compared with controls. CONCLUSION: The authors speculate that the intrauterine diabetic environment is associated with an increase in linear growth, adiposity and larger bone dimensions during childhood and adolescence.

A reference database for the Stratec XCT-2000 peripheral quantitative computed tomography (pQCT) scanner in healthy children and young adults aged 6-19 years.

SUMMARY: We have produced paediatric reference data for forearm sites using the Stratec XCT-2000 peripheral quantitative computed tomography scanner. These data are intended for clinical and research use and will assist in the interpretation of bone mineral density and bone geometric parameters at the distal and mid-shaft radius in children and young adults aged between 6-19 years. INTRODUCTION: Peripheral quantitative computed tomography (pQCT) provides measurements of bone mineral content (BMC), density (BMD) and bone geometry. There is a lack of reference data available for the interpretation of pQCT measurements in children and young adults. The aim of this study was to provide reference data at the distal and mid-shaft radius. METHODS: pQCT was used to measure the 4% and 50% sites of the non-dominant radius in a cohort of healthy white Caucasian children and young adults aged between 5 and 25 years. The lambda, mu, sigma (LMS) technique was used to produce gender-specific reference centile curves and LMS tables for calculating individual standard deviations scores. RESULTS: The study population consisted of 629 participants (380 males). Reference centile curves were produced; total and trabecular BMD for age (distal radius) and for age and height, bone area (distal and mid-shaft radius), cortical area, cortical thickness, BMC, axial moment of inertia, stress-strain index and muscle area (mid-shaft radius). CONCLUSIONS: We present gender-specific databases for the assessment of the distal and mid-shaft radius by pQCT. These data can be used as control data for research studies and allow the clinical interpretation of pQCT measurements in children and young adults by age and height.

Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans.

The phaZ gene of Pseudomonas resinovorans codes for a poly(hydroxyalkanoates) (PHA) depolymerase. Two phaZ mutants of Pseudomonas resinovorans NRRL B-2649, FOAC001 and FOAC002, were constructed by an in vitro transposition procedure followed by chromosomal integration via homologous recombination. A detailed mapping of the transposon insertion sites and an analysis of the resultant sequences showed that putative fusion polypeptides PhaZ(FOAC001) (239 amino-acid residues) and PhaZ(FOAC002) (85 amino-acid residues) could result from the mutant phaZ genes of FOAC001 and FOAC002, respectively. In vivo PHA degradation data indicated that PhaZ(FOAC001) might still retain a partial PHA depolymerization activity, while PhaZ(FOAC002) is completely devoid of this function. The cell yields and PHA contents of B-2649, FOAC001, and FOAC002 were similar when the cells were grown either under a limiting nitrogen-source (low-N) condition for up to 5 days or in excess N-source (high-N) for 3 days. A dramatic decrease in PHA content was observed in the PhaZ-active B-2649 and FOAC001 cells during prolonged cell growth (5 days) in high-N medium or in response to a shift-up in nitrogen-source. The repeat-unit compositions of the PHAs from FOAC001 and FOAC002 contained slightly lower amounts of beta-hydroxyoctanoate and higher beta-hydroxytetradecenoate than that of the wild-type B-2649 when grown under a high-N condition. While the molecular masses of the PHAs from FOAC001 and FOAC002 did not vary under any conditions used in this study, those of the wild-type B-2649 were markedly increased in cells either grown for 5 days under a high-N condition or subjected to a nitrogen-source shift-up. These phaZ mutants thus provide a valuable system to study the influence of PHA depolymerase on the accumulation and properties of medium-chain-length PHA.

Poly(ethylene glycol)-mediated molar mass control of short-chain- and medium-chain-length poly(hydroxyalkanoates) from Pseudomonas oleovorans.

Three strains of Pseudomonas oleovorans, a well known poly(hydroxyalkanoate) (PHA) producer, were tested for the ability to control PHA molar mass and end group structure by addition of poly(ethylene glycol) (PEG) to the fermentation medium. Each strain of P. oleovorans - NRRL B-14682 (B-14682), NRRL B-14683 (B-14683), and NRRL B-778 (B-778) - synthesized a different type of PHA from oleic acid when cultured under identical growth conditions. Strain B-14682 produced poly(3-hydroxybutyrate) (PHB), while B-14683 synthesized a medium-chain-length PHA ( mcl-PHA) with a repeat unit composition ranging from C4 to C14 and some mono-unsaturation in the C14 alkyl side chains. Strain B-778 synthesized a mixture of PHB (95 mol%) and mcl-PHA (5 mol%). The addition of 0.5% (v/v) PEG (M(n) =200 g/mol, PEG-200) to the fermentation broth of strains B-14682 and B-778 resulted in chain termination through esterification at the carboxyl terminus of the PHB with PEG chain segments, thus reducing the molar mass by 54% and 23%, respectively. The molar mass of the mcl-PHA produced by strains B-14683 and B-778 also showed a 34% and 47% reduction in the presence of PEG-200, respectively, but no evidence of esterification was present. PEG-400 (M(n) =400 g/mol) had a reduced effect on PHA molar mass. In fact, the molar masses of the mcl-PHA derived from strain B-14683 and both the PHB and mcl-PHA from B-778 were unchanged by PEG-400. In contrast, the PHB produced by B-14682 showed a 35% reduction in molar mass in the presence of PEG-400.

The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown Pseudomonas oleovorans.

Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (Tm) between 177 and 179 degrees C, enthalpies of fusion (AHf) of 20 cal/g and glasstransition temperatures (Tg) of 3-4 degrees C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the Tm values were 161 degrees C, deltaHf values were 7 cal/g and Tg values were - 3 degrees C.

Glucose/lipid mixed substrates as a means of controlling the properties of medium chain length poly(hydroxyalkanoates).

Glucose-triacylglycerol (TAG) mixed substrates were used to modulate the physical and mechanical properties of medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs). Pseudomonas resinovorans NRRL B-2649 grew and produced mcl-PHAs on glucose and TAGs (coconut oil, C; soybean oil, S) after 24 h in a shake flask culture. However, with the exception of coconut oil, maximum cell productivity was not reached in any of the cultures until 72 h post-inoculation. Here, 50:50 mixtures of glucose and coconut oil (glc/C) or glucose and soybean oil (glc/S) resulted in intermediate cell productivities with a maximum of 57% and 48% of the CDW at 72 h, respectively. In addition, mixed substrates resulted in mcl-PHAs with compositions that varied slightly over time. PHA-glc/C and PHA-glc/S were composed of 7 mol % and 8 mol % 3-hydroxydodecenoic acid (C(12:1)), respectively at 72 h. These concentrations were intermediate to the C(12:1) concentration of PHA-glc and respective PHA-TAG. Also, significant amounts of 3-hydroxytetradecanoic acid (C(14:0)), 3-hydroxytetradecenoic acid (C(14:1)), and 3-hydroxytetradecadienoic acid (C(14:2)) were present in PHA-glc/C and PHA-glc/S, which were derived from the respective TAG, as glucose resulted in almost no C(14:)(X) monomers. The molar masses of each of the polymers remained relatively constant between 24 and 96 h. At 72 h, the number-average molar masses (M(n)) of PHA-glc/C and PHA-glc/S were 178,000 and 163,000 g/mol, respectively, which were also intermediate to the M(n) of PHA-glc (225,000 g/mol) and the respective PHA-TAG (PHA-C = 153,000 g/mol; PHA-S = 75,000 g/mol). These physical differences caused variations in the mechanical properties of mcl-PHA films, thus providing a new and effective method of modifying their properties.

Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas.

Pseudomonas putida and P oleovorans have been extensively studied for their production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). These bacteria are incapable of metabolizing triacylglycerols (TAGs). We have constructed recombinant P. putida and P. oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis. A recombinant plasmid, pCN51lip-1, carrying Pseudomonas lipase genes was used to electrotransform these organisms. The transformants expressed TAG-hydrolyzing activity as shown by a rhodamine B fluorescence plate assay. The genetically modified organisms grew in TAG-containing medium to a cell dry weight of 2-4 g/l. The recombinant P. putida produced mcl-PHA at a crude yield of 0.9-1.6 g/l with lard or coconut oil (Co) as substrate. While P. oleovorans transformant did not produce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield of 0.5 g/l. Compositional analysis by gas chromatography/mass spectrometry showed that beta-hydroxyoctanoate (31-45 mol %) and beta-hydroxydecanoate (28-35 mol %) were the dominant repeat units of the TAG-based PHA. The number-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82-170 x 10(3) g/mol and 464-693 x 10(3) g/mol, respectively. The recombinant approach can greatly increase the number of organisms that can be used to produce PHA from fat and oil substrates.

Viscoelastic properties of linseed oil-based medium chain length poly(hydroxyalkanoate) films: effects of epoxidation and curing.

Medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers derived from linseed oil (PHA-L) have a relatively small molar mass and contain a high concentration of unsaturated side-chains. As such, these polymers are amorphous and take on the consistency of a viscous liquid at room temperature. In order to increase the application potential of this material, the side-chain olefinic groups of PHA-L were converted to epoxy derivatives (PHA-LE) using m-chloroperoxybenzoic acid (m-CPBA). Epoxidation resulted in a 37% conversion of olefinic to epoxy groups. The epoxy groups enhanced the PHA-LE film susceptibility to crosslinking upon exposure to air. PHA-LE films began to crosslink and stiffen in less than 25 days, whereas PHA-L films began to crosslink between days 50 and 75. The PHA-LE films showed an increase in tensile strength (TS, from 4.8 to 20.7 MPa) and Young's modulus (YM, from 12.9 to 510.6 MPa) between 25 and 100 days. In contrast, PHA-L had a TS of 25.0 MPa and YM of 767.8 MPa after 100 days. Epoxidation helped induce crosslink formation; however, aging for 100 days ultimately resulted in crosslinked films from both PHA-L and PHA-LE with higher strength and durability than the original materials.

Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction.

A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding for type II polyhydroxyalkanoate (PHA) synthases. The primer-pair, I-179L and I-179R, was based on the highly conserved sequences found in the coding regions of Pseudomonas phaC1 and phaC2 genes. Purified genomic DNA or lysate of colony suspension can serve equally well as the target sample for the PCR, thus affording a simple and rapid screening of phaC1/C2-containing microorganisms. Positive samples yield a specific 540-bp PCR product representing partial coding sequences of the phaC1/C2 genes. Using the PCR method, P. corrugata 388 was identified for the first time as a medium-chain-length (mcl)-PHA producer. Electron microscopic study and PHA isolation confirmed the production of mcl-PHA in P. corrugata 388. The mcl-PHA of this organism has a higher molecular weight than that of similar polymers produced by other pseudomonads.

Urine is a saturated equilibrium and not a metastable supersaturated solution: evidence from crystalluria and the general composition of calcium salt and uric acid calculi.

A computer algorithm is described which allows urine to be modelled as a saturated equilibrium solution with respect to any combination of the solids calcium oxalate, calcium hydrogen phosphate (brushite), amorphous calcium phosphate, uric acid, sodium hydrogen urate and ammonium hydrogen urate. It is demonstrated that this model of urine, unlike the widely accepted metastable supersaturated solution model, explains the long-known calcium salt crystalluria versus pH curves of both non-stone-forming and stone-forming urine. Further, the saturation model accounts for why most "infection" stones do not contain calcium oxalate and why most "urate" stones are composed solely of uric acid and not admixed with alkali metal hydrogen urate salts. The supersaturation model of urine cannot explain satisfactorily these well-known phenomena. For example, the supersaturation model predicts that virtually all "infection" stones should contain calcium oxalate along with calcium phosphate and, perhaps, struvite.

A tunable switch to regulate the synthesis of low and high molecular weight microbial polyesters.

The addition of poly(ethylene glycol) (Mn = 200 g/mol) (PEG-200) to the fermentation media of Alcaligenes eutrophus and Alcaligenes latus at various stages of growth resulted in the synthesis of poly(3-hydroxybutyrate) (PHB) with bimodal molecular weight distributions. The presence of 2% w/v-PEG-200 did not have deleterious effects on PHB volumetric yields and cell productivity. In general, the Mn values of the high (H) and low (L) fractions showed little variability as a function of the time at which PEG-200 was added to the cultures. By this approach, the H:L ratios (w/w) of the PHB synthesized by A. eutrophus and A. latus were varied from 9:91 to 76:24 and from 16:84 to 88:12, respectively. It is believed that the H fractions were formed prior to the addition of PEG-200 to the cultures. Also, once PEG-200 was made available to the cells, PEG-200 acted as a switch so that the reduced molecular weight fraction was formed. In addition, a necessary requirement for the above is that the frequency of transesterification reactions during polymer synthesis was small. The efficiency that PEG-200 reduced the molecular weight of the PHBs formed by both bacteria appears similar. Indirect evidence suggests that the PHB L fractions formed by A. latus subsequent to PEG-200 addition consist primarily of chains that have PEG terminal groups. This terminal chain structure was not observed for PHB formed by A. eutrophus.

Medium-chain-length poly(beta-hydroxyalkanoate) synthesis from triacylglycerols by Pseudomonas saccharophila.

Pseudomonas saccharophila NRRL B-628 is capable of utilizing agricultural lipids for growth. The organism exhibited good growth with triacylglycerol substrates that contained saturated fatty acyl moieties such as coconut oil (CO; C10-12 fatty acids) and tallow (T; C16-18 fatty acids). Electron micrographs of the triacylglycerol-grown cells showed the presence of intracellular granules indicative of poly(beta-hydroxyalkanoate) (PHA) production. Cells grown in a 250-ml CO-containing medium produced ca. 0.2 g of medium-chain-length (mcl)-PHA. Gas chromatographic analysis showed that beta-hydroxyoctanoic acid (30%), beta-hydroxydecanoic acid (40%), and beta-hydroxydodecanoic acid (16%) were the major monomer repeat-units of the CO-derived polymer. The estimated mean molecular mass of the CO-derived mcl-PHA as determined by gel permeation chromatography was 13.1 x 10(4) g/mol with a polydispersity of 3.16.

Radiation crosslinking of a bacterial medium-chain-length poly(hydroxyalkanoate) elastomer from tallow.

Pseudomonas resinovorans produces a medium-chain-length poly(hydroxyalkanoate) (MCL-PHA) copolymer when grown on tallow (PHA-tal). This polymer had a repeat unit composition ranging from C4 to C14 with some mono-unsaturation in the C12 and C14 alkyl side chains. Thermal analysis indicated that the polymer was semi-crystalline with a melting temperature (T(m)) of 43.5 +/- 0.2 degrees C and a glass transition temperature (Tg) of -43.4 +/- 2.0 degrees C. The presence of unsaturated side chains allowed crosslinking by gamma-irradiation. Irradiated polymer films had decreased solubility in organic solvents that indicated an increase in the crosslinking density within the film matrix. The addition of linseed oil to the gamma-irradiated film matrix enhanced polymer recovery while minimizing chain scission. Linseed oil also caused a decrease in the enthalpy of fusion (delta Hm) of the films (by an average of 60%) as well as enhanced mineralization. The effects of crosslinking on the mechanical properties and biodegradability of the polymer were determined. Radiation had no effect on the storage modulus (E') of the polymer. However, radiation doses of 25 and 50 kGy did increase the Young modulus of the polymer by 129 and 114%, and the tensile strength of the polymer by 76 and 35%, respectively. Finally, the formation of a higher crosslink density within the polymer matrix decreased the biodegradability of the PHA films.

A thermodynamic equilibrium model for calcium salt urolithiasis: clinical application.

It is proposed that urine is better modelled as a true equilibrium rather than in a supersaturated/metastable state and that the free citrate3- ion plays a major role in maintaining dispersion of the solid particles (reduced agglomeration). Published urinary chemistries, in conjunction with the computer programme SEQUIL, have been used to formulate a novel risk index for calcium stone formation independent of the traditional clinical classification of the stone former. Applying the risk index to three consecutive 24-hour urine samples of 39 untreated Ca stone formers showed that 35 (90%) patients produced at least one abnormal urine. Traditional single urinary parameter assessment, Ca/Cr, oxalate/Cr or citrate/Cr ratios, showed that only 17, 14, and 18% of the patients, respectively, had an abnormality, while taking all three together 24 (70%) had some abnormality and thus 30% were 'idiopathic' stone formers. Treatment regimens have been devised using the computer programme to return an abnormal urine to the normal according to the proposed risk index. In most urines two or more factors had to be changed simultaneously. Clinically there has been only one recurrence in 36 months, whereas before they had 4.4/3 years.