Oral spray wintertime vitamin D3 supplementation has no impact on inflammation in Gaelic footballers.

Vitamin D inadequacy [total 25(OH)D <50 nmol/L] is widespread in athletes. The biologically active metabolite, 1,25-dihydroxyvitamin D, may be involved in regulating inflammation although in vitro findings have not been consistently replicated in human intervention trials. This study, conducted at a latitude of 55°N, aimed to assess inflammatory biomarkers in Gaelic footballers before and after a wintertime vitamin D3 intervention. Samples from a 12-week double-blind, randomized, placebo-controlled trial, in which 42 Gaelic footballers received 3000 IU (75 µg) vitamin D3 daily or placebo via oral spray solutions, were analysed for a range of inflammatory biomarkers. Cytokines (interleukin-8 and tumor necrosis factor-α), cathelicidin and high sensitivity C-reactive protein were quantified by multiplex assay, enzyme-linked immunosorbent assay and clinical biochemistry, respectively. White blood cell, lymphocyte, and neutrophil concentrations were determined by full blood profile. Data on total 25-hydroxyvitamin D, measured by LC-MS/MS, were available from the previous study. Vitamin D3 supplementation significantly increased mean total 25-hydroxyvitamin D concentrations from 47 to 84 nmol/L (P = 0.006); yet this had no effect on white blood cell count (P = 0.699), lymphocyte (P = 0.694), neutrophil (P = 0.594), interleukin-8 (P = 0.334), tumor necrosis factor-α (P = 0.587), cathelicidin (P = 0.745) or high sensitivity C-reactive protein concentration (P = 0.621) compared to placebo. 12-weeks vitamin D3 supplementation did not impact the immune profile of Gaelic footballers. This is likely because biomarkers were within their respective normal range or at a concentration similar to that of the general population at baseline. Future studies are encouraged to use inflammation as their primary outcome measure and recruit athletes at risk of compromised immunity.

Lactide polymerization catalyzed by Mg and Zn diketiminate complexes with flexible ligand frameworks.

Diketimine ligands bearing N-benzyl, N-9-anthrylmethyl and N-mesitylmethyl substituents (nacnac(Bn)H, nacnac(An)H, and nacnac(Mes)H) were prepared from condensation of the amine with either acetyl acetone or its ethylene glycol monoketal. Chlorination with N-chlorosuccinimide in the 3-position yielded Clnacnac(Bn)H and Clnacnac(An)H. The ligands were reacted with Zn(TMSA)2 (TMSA = N(SiMe3)2) to yield nacnac(An)Zn(TMSA) and Clnacnac(Bn)Zn(TMSA). Protonation with isopropanol afforded nacnac(An)ZnOiPr and Clnacnac(Bn)ZnOiPr. Reaction of the diketimines with Mg(TMSA)2 afforded nacnac(An)Mg(TMSA), nacnac(Mes)Mg(TMSA), Clnacnac(Bn)Mg(TMSA) and Clnacnac(An)Mg(TMSA). Subsequent protonation with tert-butanol produced nacnac(Mes)MgOtBu and Clnacnac(Bn)MgOtBu, but only decomposition was observed with N-anthrylmethyl substituents. Most complexes were characterized by X-ray diffraction studies. TMSA complexes were monomeric and alkoxide complexes dimeric in the solid state. All alkoxide complexes, as well as nacnac(An)Mg(TMSA)/BnOH and Clnacnac(An)Mg(TMSA)/BnOH were moderately to highly active in rac-lactide polymerization (90% conversion in 30 s to 3 h). nacnac(An)ZnOiPr produced highly heterotactic polymers (P(r) = 0.90), Clnacnac(Bn)MgOtBu/BnOH produced slightly isotactic polymers at -30 °C (P(r) = 0.43), and all other catalysts produced atactic polymers with a slight heterotactic bias.

Square-planar Cu(II) diketiminate complexes in lactide polymerization.

Cu(OiPr)2 was reacted with several ß-diketimine ligands, nacnac(R)H. Sterically undemanding ligands with N-benzyl substituents afforded the dimeric heteroleptic complexes [nacnac(Bn)Cu(µ-OiPr)]2 and [3-Cl-nacnac(Bn)Cu(µ-OiPr)]2 (Bn = benzyl). With sterically more demanding amines, dimerization was not possible, and the putative nacnacCuOiPr intermediate underwent ligand exchange to the homoleptic bisdiketiminate complexes Cu(nacnac(ipp))2 and Cu(nacnac(Naph))2 (ipp = 2-isopropylphenyl, Naph = 1-napthyl). Homoleptic complexes were also prepared with N-benzyl ligands to yield Cu(nacnac(Bn))2 and Cu(3-succinimido-nacnac(Bn))2. All complexes were characterized by single-crystal X-ray diffraction. Even bulkier ligands with N-anthrylmethyl, N-mesitylmethyl, or N-methylbenzyl substituents failed to react with Cu(OiPr)2. In the case of nacnac(dipp)CuOiPr, putative nacnac(dipp)CuOiPr decomposed by ß-hydride elimination. Heteroleptic complexes [nacnac(Bn)Cu(µ-OiPr)]2 and [3-Cl-nacnac(Bn)Cu(µ-OiPr)]2 are very highly active rac-lactide polymerization catalysts, with complete monomer conversion at ambient temperature in solution in 0.5-5 min. In the presence of free alcohol, the homoleptic complexes seem to be in equilibrium with small amounts of the respective heteroleptic complex, which are sufficient to complete polymerization in less than 60 min at room temperature. All catalysts show high control of the polymerization with polydispersities of 1.1 and below. The obtained polymers were essentially atactic, with a slight heterotactic bias at ambient temperature and at -17 °C.

Exceptionally high lactide polymerization activity of zirconium complexes with bridged diketiminate ligands.

A cyclohexanediyl-bridged, bis(N-xylyl) diketiminate ligand, (±)-C6H10(nacnac(Xyl)H)2, LH2 (Xyl = 2,6-dimethylphenyl), was obtained from the reaction of [(2,6-dimethylphenyl)amino]-pent-3-en-2-one first with Meerwein's salt, then with (±)-cyclohexanediamine. The reaction of the ligand with Zr(NMe2)4 yielded LZr(NMe2)2. Protonation of the remaining diamide ligands with EtOH or [H2NMe2]Cl yielded LZr(OEt)2 and LZrCl2, respectively. The latter complex was also obtained by the reaction of LH2 first with nBuLi and then with ZrCl4(THF)2. The dichloride complex yielded LZr(OEt)2 and LZrMe2 upon reaction with NaOEt or MeLi/AlMe3, respectively. X-ray diffraction studies showed a trans-configuration of the ancillary ligands in LZrCl2 and LZrMe2, and a cis-configuration in LZr(NMe2)2 and LZr(OEt)2. LZr(OEt)2 was tested as a catalyst for the polymerization of rac-lactide. Kinetic investigations yielded a rate law first order in catalyst and monomer and a rate constant k = 14(1) L mol(-1) s(-1), the latter being orders of magnitude higher than typical activities for group 4 complexes in lactide polymerization. Analyses of the obtained polymer revealed an atactic polymer and broad polymer molecular weight distributions with sizeable fractions of cyclic oligomers. The influence of contaminants on the polymerization activity was examined: while lactic acid deactivates the catalyst, addition of up to 1 equiv. of water or para-toluenesulfonic acid revitalized catalysts not showing maximum activity.

Nacnac(Bn)CuOiPr: a strained geometry resulting in very high lactide polymerization activity.

N,N'-Dibenzyl diketiminate copper isopropanolate, (nacnac(Bn)CuOiPr)(2), polymerizes rac- and S,S-lactide in the presence or absence of isopropanol as a chain-transfer reagent with very high activity (k(2) = 32 M(-1) s(-1)), narrow polydispersities and without evidence of side reactions such as transesterification, epimerization or catalyst decomposition.

Nacnac(Bn)MgOtBu: a diketiminate-based catalyst for the polymerisation of rac-lactide with slight isotactic preference.

The reaction of N,N'-dibenzyl-2-amino-4-imino-pent-2-ene, nacnac(Bn)H, with 1 or 2 equiv. of MgnBu2 afforded the homoleptic complex nacnac(Bn)2Mg. The reaction of nacnac(Bn)H with Mg(N(SiMe3)2)2 yielded nacnac(Bn)MgN(SiMe3)2, which reacted with tert-butanol to form nacnac(Bn)MgOtBu. The latter complex crystallizes as an alkoxide bridge dimer and is active in the ring-opening polymerisation of rac-lactide. Polymerisations at room temperature afforded atactic polylactide, while polymerisations at -17 and -26 °C afforded polylactide with a small isotactic bias (P(m) = 0.52, and 0.55, respectively).

Observer accuracy in the detection of pulmonary nodules on CT: effect of cine frame rate.

AIM: To assess the effect of cine frame rate on the accuracy of the detection of pulmonary nodules at computed tomography (CT). MATERIALS AND METHODS: CT images of 15 consecutive patients with (n = 13) or without (n = 2) pulmonary metastases were identified. Initial assessment by two thoracic radiologists provided the "actual" or reference reading. Subsequently, 10 radiologists [board certified radiologists (n = 4) or radiology residents (n = 6)] used different fixed cine frame rates for nodule detection. Within-subjects analysis of variance (ANOVA) was used to evaluate the data. RESULTS: Eighty-nine nodules were identified by the thoracic radiologists (median 8, range 0-29 per patient; median diameter 9 mm, range 4-40 mm). There was a non-statistically significant trend to reduced accuracy at higher frame rates (p=0.113) with no statistically significant difference between experienced observers and residents (p = 0.79). CONCLUSION: The accuracy of pulmonary nodule detection at higher cine frame rates is reduced, unrelated to observer experience.

Was there sufficient justification for the 10-fold increase in the TLV for silica fume? A critical review.

In 1992, the Threshold Limit Value (TLV) for amorphous silica fume produced as a by-product of metallurgical processes was revised upwards from 0.2 mg/m3 (respirable dust) to 2.0 mg/m3. Comparison of the documentation justifying the lower TLV published by the ACGIH in 1989, with the subsequent documentation justifying the higher value published in 1992, does not support this increase. Following an outline of the problem areas existing in interpretational difficulties of experimental and review material in the silica fume bibliography, this paper provides a detailed examination of the six additional references cited in the 1992 documentation. All additional material suggests a need for extra caution, particularly with respect to recent experimental work in Australia on the sizing of silica fume. This paper concludes that the health evidence supports a TLV for silica fume closer to 0.3 mg/m3 rather than the current 2.0 mg/m3 now adopted in the U.S. and Australia.

Duplications That Suppress and Deletions That Restore Expression from a Chalcone Synthase Multigene Family.

Seed coat color in soybean is determined by four alleles of the classically defined / (inhibitor) locus that controls the presence or absence as well as the spatial distribution of anthocyanin pigments in the seed coat. By analyzing spontaneous mutations of the / locus, we demonstrated that the / locus is a region of chalcone synthase (CHS) gene duplications. Paradoxically, deletions of CHS gene sequences allow higher levels of CHS mRNAs and restore pigmentation to the seed coat. The unusual nature of the / locus suggests that its dominant alleles may represent naturally occurring examples of homology-dependent gene silencing and that the spontaneous deletions erase the gene-silencing phenomena. Specifically, mutations from the dominant ii allele (yellow seed coats with pigmented hila) to the recessive i allele (fully pigmented) can be associated with the absence of a 2.3-kb Hindlll fragment that carries CHS4, a member of the multigene CHS family. Seven independent mutations exhibit deletions in the CHS4 promoter region. The dominant / allele (yellow seed coats) exhibits an extra 12.1-kb Hindlll fragment that hybridizes with both the CHS coding region and CHS1 promoter-specific probes. Mutations of the dominant / allele to the recessive i allele (pigmented seed coats) give rise to 10.4- or 9.6-kb Hindlll CHS fragments that have lost the duplicated CHS1 promoter. Finally, gene expression analysis demonstrated that heterozygous plants (I/i) with yellow seed coats have reduced mRNA levels, indicating that the 12.1-kb Hindlll CHS fragment associated with the dominant / allele inhibits pigmentation in a trans-dominant manner. Moreover, CHS gene-specific expression in seed coats shows that multiple CHS genes are expressed in seed coats.

Prevention of pink-pigmented methylotrophic bacteria (Methylohacterium mesophilicum) contamination of plant tissue cultures.

Pink-pigmented facultative methylotrophic bacteria (PPFMs) have been found on the surfaces of leaves of most plants tested. We found PPFMs on the leaf surfaces of all 40 plants (38 species) tested and on soybean pods by pressing onto AMS medium with methanol as the sole carbon source. The abundance ranged from 0.5 colony forming unit (cfu) /cm(2) to 69.4 cfu/cm(2) on the leaf surfaces. PPFMs were found in homogenized leaf tissues of only 4 of the species after surface disinfestation with 1.05% sodium hypochlorite and were rarely found in cultures initiated from surface disinfested Datura innoxia leaves or inside surface disinfested soybean pods. Of 20 antibiotics tested for PPFM growth inhibition, rifampicin was the most effective and of seven others which also inhibited PPFM growth, cefotaxime should be the most useful due to the expected low plant cell toxicity. These antibiotics could be used in concert with common surface sterilization procedures to prevent the introduction or to eliminate PPFM bacteria in tissue cultures. Thus, while PPFMs are present on the surfaces of most plant tissues, surface disinfestation alone can effectively remove them so that uncontaminated tissue cultures can be initiated in most cases.

Chalcone synthase mRNA and activity are reduced in yellow soybean seed coats with dominant I alleles.

The seed of all wild Glycine accessions have black or brown pigments because of the homozygous recessive i allele in combination with alleles at the R and T loci. In contrast, nearly all commercial soybean (Glycine max) varieties are yellow due to the presence of a dominant allele of the I locus (either I or i) that inhibits pigmentation in the seed coats. Spontaneous mutations to the recessive i allele occur in these varieties and result in pigmented seed coats. We have isolated a clone for a soybean dihydroflavonol reductase (DFR) gene using polymerase chain reaction. We examined expression of DFR and two other genes of the flavonoid pathway during soybean seed coat development in a series of near-isogenic isolines that vary in pigmentation as specified by combinations of alleles of the I, R, and T loci. The expression of phenylalanine ammonia-lyase and DFR mRNAs was similar in all of the gene combinations at each stage of seed coat development. In contrast, chalcone synthase (CHS) mRNA was barely detectable at all stages of development in seed coats that carry the dominant I allele that results in yellow seed coats. CHS activity in yellow seed coats (I) was also 7- to 10-fold less than in the pigmented seed coats that have the homozygous recessive i allele. It appears that the dominant I allele results in reduction of CHS mRNA, leading to reduction of CHS activity as the basis for inhibition of anthocyanin and proanthocyanin synthesis in soybean seed coats. A further connection between CHS and the I locus is indicated by the occurrence of multiple restriction site polymorphisms in genomic DNA blots of the CHS gene family in near-isogenic lines containing alleles of the I locus.

Pigmented Soybean (Glycine max) Seed Coats Accumulate Proanthocyanidins during Development.

The dominant I gene inhibits accumulation of anthocyanin pigments in the epidermal layer of soybean (Glycine max) seed coats. Seed-coat color is also influenced by the R locus and by the pubescence color alleles (T, tawny; t, gray). Protein and RNA from cultivars with black (i,R,T) and brown (i,r,T) seed coats are difficult to extract. To determine the nature of the interfering plant products, we examined seed-coat extracts from Clark isogenic lines for flavonoids, anthocyanins, and possible proanthocyanidins by thin-layer chromatography. We show that yellow seed-coat varieties (I) do not accumulate anthocyanins (anthocyanidin glycosides) or proanthocyanidins (polymeric anthocyanidins). Mature, black (i,R,T) and imperfect-black (i,R,t) seed coats contained anthocyanins, whereas mature, brown (i,r,T) and buff (i,r,t) seed coats did not contain anthocyanins. In contrast, all colored (i) genotypes tested positive for the presence of proanthocyanidins by butanol/ HCl and 0.5% vanillin assays. Immature, black (i,R,T) and brown (i,r,T) seed coats contained significant amounts of procyanidin, a 3[prime],4[prime]-hydroxylated proanthocyanidin. Immature, black (i,R,T) or brown (i,r,T) seed-coat extracts also tested positive for the ability to precipitate proteins in a radial diffusion assay and to bind RNA in vitro. Imperfect-black (i,R,t) or buff (i,r,t) seed coats contained lesser amounts of propelargonidin, a 4[prime]-hydroxylated proanthocyanidin. Seed-coat extracts from these genotypes did not have the ability to precipitate protein or bind to RNA. In summary, the dominant I gene controls inhibition of not only anthocyanins but also proanthocyanidins in soybean seed coats. In homozygous recessive i genotypes, the T-t gene pair determines the types of proanthocyanidins present, which is consistent with the hypothesis that the T locus encodes a microsomal 3[prime]-flavonoid hydroxylase.