Fracture toughness and work of fracture of hydrated, dehydrated, and ashed bovine bone

Bone, a tri-phase composite, consists of nano-sized apatite minerals, an organic component, and water. Heat-treated bovine cortical bone has been proposed as a candidate for void-filling bone substitute. However, the toughness of heat-treated bone is not yet fully studied. Fracture toughness (K(c)) and work of fracture (W(f)) of hydrated, dehydrated, and ashed bovine bone were estimated using a single-edge V-notched beam method. Thermal gravimetric analysis and differential thermal analysis were used to determine the temperature at which the organics and water were removed. Dehydrated specimens were obtained by placing the samples in a 60 degrees C vacuum oven for 24h or a 110 degrees C furnace for 2h. Ashed specimens were obtained by heat-treating samples at 600 degrees C for 24h. K(c) of bovine specimens decreased from 5.5MPa.m(1/2) for hydrated bone, to 3.8MPa.m(1/2) for dehydrated specimens, and to 0.36MPa.m(1/2) for ashed specimens. W(f) decreased from 7.1 to 1.1kJ/m(2) for dehydrated specimens, and to 0.04kJ/m(2) for ashed specimens. The main reasons for the significant decreases in K(c) and W(f) may be attributed to water's ability in stabilizing collagen structure and to the organics' ability in making bone more ductile. Because of the large decrease in fracture toughness and work of fracture, we suggest that ashed bone is not appropriate for load-bearing bone substitute in areas where bone experiences loadings in flexure.     

Metabolic and thermal physiology of pigeons and doves

Pigeons and doves (Columbidae) are an interesting group to examine for physiological adaptations to climate and diet because this cosmopolitan family comprises more than 300 species that are mostly granivores, although some are specialized frugivores. We determined allometric and phylogenetic effects on body temperature (T(b)), basal metabolic rate (BMR; J h(-1)), and wet thermal conductance (C(wet); J h(-1) C(-1)), and we examined mass (M) and phylogenetically corrected residuals for further effects of climate, diet, and landmass size (mainland or island). Independent contrasts, correlograms, autoregression, and phylogenetic eigenvector regression (PVR) were used to examine phylogenetically related effects. We found a small but significant phylogenetic pattern for body mass of columbids. For T(b), there was no significant effect of mass or phylogeny. There was a significant effect of climate on T(b) and no significant effects of diet or landmass without mass or phylogenetic correction, but after mass and phylogenetic correction, there were no effects of climate, diet, or landmass. For BMR, there was a strong allometric effect, and residuals were significantly lower for arid and tropical species but not for temperate species, compared to predictions for nonpasserine birds. There was a nearly significant autoregressive phylogenetic relationship for BMR parl0;r=0.44), and the strong allometry of BMR remained for independent contrasts (slope=0.731), autoregressive residuals (0.698), and PVR (0.705). Residuals, from regression of autoregression and PVR residuals of M and BMR, were significantly associated with climate: arid pigeons had a lower BMR residual than tropical and temperate pigeons. PVR residuals were significantly affected by landmass (island columbids had a smaller residual than mainland columbids), but autoregression residuals were not. There was no association of autoregression or PVR residuals with diet. For C(wet), there was a strong allometric effect, and residuals for columbids were significantly higher compared to other birds. There was no significant relationship for C(wet) of columbids to climate, diet, or landmass. There was no significant autoregressive or PVR relationship for C(wet), and the strong allometry remained after phylogenetic analysis by independent contrasts (slope=0.501), autoregression (0.509), and PVR (0.514). Residuals from autoregression and PVR were not significantly correlated with climate, diet, or landmass (mainland/island).     

Interconversion and tissue distribution of pentoxifylline and lisofylline in mice

The aim of this study was to assess the interconversion pharmacokinetics and tissue distribution of pentoxifylline and the active (R)-enantiomer of its metabolite M1, lisofylline in male CD-1 mice. Both compounds were administered intravenously at a dose of 50 mg/kg on two separate occasions. Serum and tissues were collected at different time points following drug administration. In addition, the (S)-enantiomer of M1 was administered to a group of mice and serum samples were obtained. Analyte concentrations were measured by chiral HPLC. All serum concentration versus time data were fitted simultaneously to a pharmacokinetic model incorporating interconversion processes of parent drug and metabolites. The estimated conversion clearance of (-)-(R)-M1 to pentoxifylline (CL21) was six times greater than that for the reverse process (CL12). The interconversion of pentoxifylline and (+)-(S)-M1 was faster as reflected by the values of conversion clearances CL13 and CL31 which were approximately 16 and 7 times greater in comparison with the corresponding clearances for the interconversion of pentoxifylline and (-)-(R)-M1. When fitting pharmacokinetic data of both parent compounds to a one-compartment model, the values of elimination clearances assessed were close to those obtained on the basis of the interconversion model. After administration of pentoxifylline, tissue-to-serum AUC ratios ranged from 0.1 for liver and lungs to 0.32 for brain tissue. Serum levels of its metabolite, (-)-(R)-M1 were very low, whereas its tissue levels exceeded serum concentrations. The highest value of metabolite-to-parent AUC ratio (4.98) was observed in lungs. When (-)-(R)-M1 was given as a parent drug, tissue-to-serum AUC ratios in liver, kidney, and lungs were very close and ranged from 0.64 to 0.72. At the same time, levels of its metabolite, pentoxifylline were relatively low both in serum and all tissues studied. In consequence, metabolite-to-parent AUC ratios did not exceed the value of 0.27. In conclusion, reversible metabolism plays a modest role in the disposition of pentoxifylline and (-)-(R)-M1. It seems that pentoxifylline has less favourable pharmacokinetic properties than (-)-(R)-M1 due to lower concentrations attained in target organs. High levels of (-)-(R)-M1 observed after pentoxifylline administration in certain tissues such as liver or lungs suggest that pentoxifylline may constitute an effective prodrug for (-)-(R)-M1 in these organs.     

The influence of blinding, olfactory bulbectomy and pinealectomy on plasma and anterior pituitary prolactin levels and on uterine and anterior pituitary weights in normal and neonatally androgenized rats

Lactating Sprague-Dawley rats had their litters adjusted to 8-10 pups on day 3 of lactation favoring females and some litters were injected with 1.25 mg of testosterone propionate to neonatally androgenize (NA) them. At 22-25 days of age both normal and NA animals were ovariectomized (OVX) and subjected to either sham olfactory bulbectomy (ANOS) + sham pinealectomy (PX), blinding (BLD) + ANOS or BLD + ANOS + pinealectomy (PX). At 13 weeks of age all animals were injected with 0.5 mg of polyestradiol phosphate. At 15 weeks of age the animals were fitted with atrial catheters and at 16 weeks of age blood samples (0.3 ml) were obtained every 3 hours over a 24 hour period. Uterine and anterior pituitary (AP) weights were recorded at sacrifice. Plasma and AP were assayed for prolactin (PRL) by RIA and AP were also assayed for PRL using the Nb2 lymphoma cell PRL bioassay. In both normal and NA animals, BLD + ANOS suppressed plasma PRL levels and PX partially prevented this response. Uterine weights were similar among groups while AP weights were significantly lower for sensory deprived animals. Anterior pituitaries extracted at pH 7.6 had a PRL concentration that was higher for the BLD + ANOS groups when estimated by either RIA or BA, a result that was not observed when the AP were extracted at pH 10.6. The amount of PRL extracted at pH 10.6 was twice that obtained at pH 7.6. Sensory deprived animals that were OVX prepubertally and administered estrogen as adults had a small but significant increase in mean plasma prolactin at 1700 hr. Both normal and NA animals responded in a similar manner to experimental manipulation.     

Thermal responses during arm and leg and combined arm-leg exercise in water

Thermal and metabolic responses were examined during exposures in stirred water at approximately 20, 26, and 33 degrees C while subjects were performing 45 min of either arm (A), leg (L), or combined arm-leg (AL) exercise. Eight males immersed to the neck completed a low exercise intensity for A exercise and both a low and high exercise intensity for L and AL exercise. During low-intensity exercise, final metabolic rate (M) for A, L, and AL exercise was not different (P greater than 0.05) between exercise type for each water temperature (Tw). In contrast final rectal temperatures (Tre) for A and AL exercise were significantly lower than L values for each Tw during low-intensity exercise. These findings were supported by both mean weighted skin temperature (Tsk) and mean weighted heat flow (Hc) values, which were greater during A than L for each Tw. During high-intensity exercise, final Tre values were lower (P less than 0.05) during AL compared with L exercise across all Tw. Final Tsk and Hc values were not different between each type of exercise, although M was significantly lower during L exercise in 20 degrees C water. These data suggest a greater conductive and convective heat loss during exercise utilizing the arms when compared with leg-only exercise.     

Serum LDL- and HDL-cholesterol determined by ultracentrifugation and HPLC

Simple and precise methods for LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) measurements are essential for assessment of cardiovascular disease (CVD) risks and for lipid and lipoprotein studies. We report here an ultracentrifugation (UC) and HPLC method that requires substantially less specimen volume and provides the necessary reliability and throughput required by large-volume, high-quality research and clinical studies. 2-Mercaptoethanol (ME) was used to dissociate serum lipoprotein [a] (Lp[a]) into apolipoprotein [a] and Lp[a] remnant (Lp[a-]) and eliminated the contamination of Lp[a] in HDL separated by UC. Serum aliquots were centrifuged at a density of 1.006 kg/l for the separation of HDL plus LDL, and in the presence of ME at a density of 1.063 kg/l for the separation of HDL. Cholesterol concentrations of the bottom fractions were analyzed by HPLC. LDL-C and HDL-C determined using this method were equivalent to those with β-quantification and the designated comparison method of the Centers for Disease Control. The total coefficient of variations for LDL-C and HDL-C were 0.65-1.12% and 0.96-2.07%, respectively. This method requires a small amount of specimen and is easy to operate. This method may be used in research or in clinical laboratories where precise and specific lipoprotein cholesterol analysis is needed.     

Cloning and expression of cynomolgus monkey and baboon zona pellucida proteins

Partial clones for the three cynomolgus monkey (Macaca fasicularis) zona pellucida genes (cmZPA, cmZPB, and cmZPC) have previously been isolated. These partial clones contained the sequences for the C-terminal portion of each rcmZP protein. To obtain full-length clones for each cmZP, a fresh cynomolgus monkey ovarian cDNA library was constructed. PCR methodology was employed to speed the isolation of full-length clones for each cmZP cDNA. The 3' primers were designed based on sequence information from the previously identified clones; the 5' primers were designed using the human ZP sequences. The PCR technique yielded full-length clones of cmZPA and cmZPC, but not of cmZPB. Therefore, a genomic clone of cmZPB was isolated and the sequence determined. The exon/intron structure is nearly identical to the human ZPB exon/intron structure. New PCR primers were designed based on the cynomolgus monkey ZPB genomic sequence, and a full-length cmZPB cDNA was obtained. The same primers that were used to generate the cmZPB were also used to generate a baboon (Papio cynocephalus) ZPB (bZPB) cDNA. As was done previously for the human zona pellucida (hZP) cDNAs, the cmZP, and bZPB cDNAs were transferred to shuttle vectors for transfection into Chinese Hamster Ovary (CHO) cells. Stable cell lines for producing each ZP protein were isolated. Each cell line secreted the desired recombinant zona pellucida (rZP) protein into the culture medium, and each protein was purified using an established protocol. In terms of size and purity, the purified recombinant cmZP (rcmZP) and rbZPB proteins resemble the rhZP proteins.     

Quantitative light and electron microscopic correlations of nuclear features in papillary thyroid and ovarian carcinomas

The light microscopic (LM) and electron microscopic (EM) features of nuclear bodies and nucleoli were quantitatively compared in ten papillary carcinomas each of thyroid (PC-Thy) and ovarian (PC-Ovar) origins, along with eight nonthyroid, nonovarian papillary neoplasms from other organs (PN-Oth). In each neoplasm, 100 randomly selected nuclei were scored for the presence of characteristic nuclear bodies; these were defined at the LM level by the presence of a central density surrounded by a clear halo, which corresponded to four distinct quantifiable images at the EM level. Means (+/- standard deviations) of the nucleolar frequency factor, the nucleolar area and the computed total nucleolar area (the product of the nuclear frequency factor and the mean nucleolar area) were assessed for the EM images. The number of nuclear bodies was 11.9 +/- 10.7 for PC-Thy, 5.2 +/- 5.3 for PC-Ovar and 5.9 +/- 7.4 for PN-Oth; the means for PC-Thy and PC-Ovar were significantly different (P less than .03), as were the means for PC-Thy and PN-Oth (P less than .04). The nucleolar frequency factor was 0.60 +/- 0.19 for PC-Thy, 1.19 +/- 0.51 for PC-Ovar and 0.99 +/- 0.22 for PN-Oth; these means were significantly different for PC-Thy versus PC-Ovar (P less than .01) and for PC-Thy versus PN-Oth (P less than .001). The mean nucleolar area was 1.19 +/- 0.45 for PC-Thy, 1.91 +/- 0.92 for PC-Ovar and 1.94 +/- 0.76 for PN-Oth; the means were significantly different for PC-Thy and PC-Ovar (P less than .05) and for PC-Thy and PN-Oth (P less than .05). The computed total nucleolar area was 0.73 +/- 0.41 for PC-Thy, 2.17 +/- 1.09 for PC-Ovar and 1.94 +/- 1.00 for PN-Oth; these means were significantly different for PC-Thy versus PC-Ovar (P less than .001) and for PC-Thy versus PN-Oth (P less than .01). A comparison of the total number of nuclear bodies, as determined by both LM and EM, indicated a significant correlation for the PC-Ovar (P less than .01) and PN-Oth (P less than .001) groups using linear regression analysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relative biological effectiveness measurements using murine lethality and survival of intestinal and hematopoietic stem cells after fermilab neutrons compared to JANUS reactor neutrons and 60Co gamma rays

The relative biological effectiveness (RBE) of the 25-MeV (average energy) neutron beam at the Fermi National Accelerator Laboratory was measured using murine bone marrow (LD50/30) and gut (LD50/6) lethality and killing of hematopoietic colony forming units (CFU-S) or intestinal clonogenic cells (ICC). The reference radiation was 60Co gamma rays. The LD50/30 and LD50/6 for mice exposed to the Fermilab neutron beam were 6.6 and 8.7 Gy, respectively, intermediate between those of JANUS neutrons and 60Co gamma rays. The D0 values for CFU-S and ICC were 47 cGy and 1.05 Gy, respectively, also intermediate between the lowest values found for JANUS neutrons and the highest values found after 60Co gamma rays. The split-dose survival ratios for CFU-S at intervals of 1-6 hr between doses were essentially 1.0 for both neutron sources, while the corresponding split-dose survival ratio for 60Co gamma rays was consistantly above 1, reaching a maximum of 1.7 with a 1-hr interval between doses. The 3-hr split-dose survival ratios for ICC were 1.0 for JANUS neutrons, 1.85 for Fermilab neutrons, and 6.5 for 60Co gamma rays. The RBE estimates for LD50/30 were 1.5 and 2.3 for Fermilab and JANUS neutrons, respectively. Based on LD50/6, the RBEs were 1.9 (Fermilab) and 3.0 (JANUS). The RBEs for CFU-S D0 were 1.4 (Fermilab) and 1.9 (JANUS) and for jejunal microcolony D0 1.4 (Fermilab) and 2.8 (JANUS).     

Differential effects of haloperidol and olanzapine on the expression of erythropoietin and its receptor in rat hippocampus and striatum

Compared with first-generation antipsychotics (FGAs), second-generation antipsychotics (SGAs) seem to be neuroprotective and trigger neuroplasticity. Because neuroplasticity is regulated by a variety of neurotrophic factors we studied differential effects of haloperidol (HAL, a FGA) and olanzapine (OLZ, a SGA) on temporal expression of erythropoietin (EPO), a potent neuroprotective factor and its receptor (EPOr) in rat brain. Rats (8-10/group) were treated with HAL or OLZ for 14 days (HAL-14 or OLZ-14) or 45 days (HAL-45 or OLZ-45). Animals were killed by decapitation or by perfusion to collect brains for immunoblotting and immunohistochemical analysis respectively. In hippocampus, the levels of both EPO and EPOr were significantly increased in HAL-14 (p < 0.001) and OLZ-14 (p < 0.001) groups. Their levels decreased in HAL-45 compared with levels in HAL-14 (EPO, p < 0.001; EPOr, p < 0.05), whereas the levels were further increased (EPO, p < 0.05) in OLZ-45 compared with OLZ-14. In striatum, the levels of both EPO and EPOr were unchanged in HAL-14 and EPO levels significantly decreased in HAL-45 (p < 0.05), whereas their levels were significantly increased in OLZ-14 and OLZ-45 compared with the vehicle-treated control (p < 0.001). Both EPO and EPOr were primarily expressed by neurons and endothelial cells. These data suggest that SGAs such as OLZ may have neuroprotective effects through expression of EPO that may be clinically relevant for long-term safe and beneficial management of psychotic patients.     

In situ intestinal digestibility of dry matter and crude protein of cereal grains and rapeseed in sheep

The ruminal degradation and intestinal digestibility (ID) of dry matter (DM) and crude protein (CP) of different feed samples were measured in two trials by using nylon bag and rumen outflow rate techniques in three wethers cannulated in the rumen and in the duodenum. In trial 1, three samples of grains of wheat, barley, and corn treated by cooking (TW, TB, and TC, respectively) were studied together with a sample of untreated corn grains (CG) of different origin. In trial 2, these studies were carried out on a sample of rapeseed (RS) and on a mix of this same sample and rapeseed meal (in proportions 70:30) treated by cooking (TR). In both trials, the animals were fed at the same intake level (40 g DM x kg(-1) LW0.75) with 2:1 (DM basis) forage to concentrate diets. Rumen degradation rates of DM were high in the treated cereals (between 11.0 and 14.2% x h(-1)) and low in the CG (6.35% x h(-1)), whereas for CP these rates were low in all cereals. For DM, in all cereals, ID decreased linearly as the ruminal incubation time increased. The values of intestinal effective digestibility (IED), calculated from these functions and from the rumen outflow, were respectively: 86.4, 62.1, 51.5, and 67.9%. For CP, ID was unaffected by the ruminal incubation time in corn samples, whereas in TW and TB a reduction of these values was only observed for the time of 48 h. The values of IED of CP for CG, TW, TB and TC were: 82.6, 88.9,82.5, and 91.6%, respectively. Rumen degradation rates of the RS and TR samples were 8.35 and 8.23% x h(-1) for DM and 12.0 and 9.59% x h(-1) for CP. In RS, the ID of DM and CP showed a downward trend with an increase of the ruminal incubation time, as modelled according to an exponential function. This same trend was observed for TR after a lag period estimated at 7.53 and 6.51 h for DM and CP, respectively. The values of IED of RS and TR were respectively 56.5 and 50.8% for DM and 71.9 and 80.1% for CP. These same results were also determined by a simplified method using a sample pooled to be representative of the rumen outflow of undegraded feed. The respective values for RS and TR were 54.8 and 51.6 for DM and 65.8 and 78.9% for CP. This method seems to be a promising technique to estimate IED, although more studies are needed to improve its accuracy.     

淹水和干旱条件下水稻根系形成的QTLs及候选基因分析

为了研究不同水分条件下组成型根系性状和适应性根系性状的遗传机制,利用由IR64／Azucena发展的双单倍体（DH）群体分析了淹水和干旱条件下水稻幼苗种子根长（SRL）、不定根数（ARN）、总根干重（RW）及其对应的相对参数（干旱和淹水条件下根系性状的比值）的QTLs。淹水与干旱条件下检测到一个共同的种子根长QTL和一个共同的总根干重QTL。同时对前人发表的遗传群体定位的根系性状QTLs进行比较分析,检测到几个共同的根系性状QTLs。对与细胞伸长、分裂相关的候选基因进行了定位,其中4个细胞壁相关的ESTs（OsEXP2,OsEXP4,EXT和Xet）被定位在与不同水分条件下检测出的根系性状QTLs的相同区间。     

Intramolecularly quenched BODIPY-labeled phospholipid analogs in phospholipase A(2) and platelet-activating factor acetylhydrolase assays and in vivo fluorescence imaging

Phospholipase substrate analogs containing both a fluorescent BODIPY group and a quenching 2,4-dinitrophenyl (DNP) group were synthesized. They showed little fluorescence, but upon hydrolysis became fluorescent as the quenching group was removed. Two substrates were phosphatidylethanolamine analogs with a BODIPY-pentanoyl group at the sn-2 position and DNP linked to the amino head group. The third was a phosphatidylcholine analog with a BODIPY-labeled alkyl ether at the sn-1 position and a N-(DNP)-8-amino-octanoyl group at the sn-2 position. These compounds were evaluated as substrates for cytosolic (85 kDa) phospholipase A(2) (cPLA(2)) and plasma platelet-activating factor acetylhydrolase (rPAF-AH). Two were good substrates for cPLA(2) (specific activities: 18 and 5 nmol min(-1) mg(-1)) and all were good for rPAF-AH (specific activities: 17, 11, and 6 micro mol min(-1) mg(-1)). The minimal amount of enzyme detectable was 50 ng for cPLA(2) and 0.1 ng for rPAF-AH. These substrates were active in assays of PLA(2) in zebrafish embryo extracts and one was well suited for imaging of PLA(2) activity in living zebrafish embryos. Embryos were injected with substrate at the one- to four-cell stage and allowed to develop until early somitogenesis when endogenous PLA(2) activity increases dramatically; substrate persisted (12 h) and specifically labeled cells of the developing notochord.     

Sensor and biosensor based on Prussian Blue modified gold and platinum screen printed electrodes

Gold (Au) and platinum (Pt) screen-printed electrodes were modified with Prussian Blue (PB) for the development of amperometric sensors selective for hydrogen peroxide detection. The sensors exhibited sensitivities towards H(2)O(2) equal to 2 A M(-1) cm(-2) for Au and 1 A M(-1) cm(-2) for Pt electrodes. The sensors were also employed as the basis for construction of glucose biosensors through further modification with crystallised glucose oxidase immobilised in a Nafion membrane. In order to improve the operational stability of the modified electrodes a buffer solution containing tetrabutylammonium toluene-4-sulfonate was used. The long-term performance of the sensors and biosensors were evaluated by continuous monitoring of hydrogen peroxide and glucose solutions (50 microM and 1 mM, respectively) in the flow-injection mode for 10 h.     

Synthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin analogs

Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50 degrees C, l h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC(1)-AngII and TOAC(0)-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes.     

Form and function of fetal and neonatal pulmonary arterial bifurcations

Bifurcation is a basic form of vascular connection. It is composed of a parent vessel of diameter d(0), and two daughter vessels, d(1) and d(2), where d(0) > d(1) >/= d(2). Optimal values for the bifurcation area ratio, beta = (d(1)(2) + d(2)(2))/d(0)(2), and the junction exponent, x, in d(0)(x) = d(1)(x) + d(2)(x), are postulated to be universal in nature. However, we have hypothesized that the perinatal pulmonary arterial circulation is an exception. Arterial diameters were measured in pulmonary vascular casts of a fetal lamb (140 days gestation/145 days term) and a neonatal lamb (1 day old). The values for beta and x were evaluated in 10,970 fetal and 846 neonatal bifurcations sampled from the proximal and intermediate arterial regions. Mean values and confidence intervals (CI) for the fetus were beta = 0.890 (0.886-0.895 CI) and x = 1.75 (1.74-1.76 CI); and for the newborn were beta = 0.913 (0.90-0.93 CI) and x = 1. 79 (1.75-1.82 CI). These values are significantly different from Murray's law (beta > 1, x = 3) or the West-Brown-Enquist law (beta = 1, x = 2). Therefore, perinatal pulmonary bifurcation design appears to be distinctive and exceptional. The decreasing cross-sectional area with branching leads to the hemodynamic consequence of shear stress amplification. This structural organization may be important for facilitating vascular development at low flow rates; however, it may be the origin of unstable reactivity if elevated blood flow and pressure occurs.     

Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branches

Phylogenetic relationships in Cornales were assessed using sequences rbcL and matK. Various combinations of outgroups were assessed for their suitability and the effects of long branches and outgroups on tree topology were examined using RASA 2.4 prior to conducting phylogenetic analyses. RASA identified several potentially problematic taxa having long branches in individual data sets that may have obscured phylogenetic signal, but when data sets were combined RASA no longer detected long branch problems. t(RASA) provides a more conservative measurement for phylogenetic signal than the PTP and skewness tests. The separate matK and rbcL sequence data sets were measured as not containing phylogenetic signal by RASA, but PTP and skewness tests suggested the reverse [corrected]. Nonetheless, the matK and rbcL sequence data sets suggested relationships within Cornales largely congruent with those suggested by the combined matK-rbcL sequence data set that contains significant phylogenetic signal as measured by t(RASA), PTP, and skewness tests. Our analyses also showed that a taxon having a long branch on the tree may not be identified as a "long-branched" taxon by RASA. The long branches identified by RASA had little effect on the arrangement of other taxa in the tree, but the placements of the long-branched taxa themselves were often problematic. Removing the long-branched taxa from analyses generally increased bootstrap support, often substantially. Use of non-optimal outgroups (as identified by RASA) decreased phylogenetic resolution in parsimony analyses and suggested different relationships in maximum likelihood analyses, although usually weakly supported clades (less than 50% support) were impacted. Our results do not recommend using t(RASA) as a sole criterion to discard data or taxa in phylogenetic analyses, but t(RASA) and the taxon variance ratio obtained from RASA may be useful as a guide for improved phylogenetic analyses. Results of parsimony and ML analyses of the sequence data using optimal outgroups suggested by RASA revealed four major clades within Cornales: (1) Curtisia-Grubbia, (2) Cornus-Alangium, (3) Nyssa-Camptotheca-Davidia-Mastixia-Diplopanax, and (4) Hydrangeaceae-Loasaceae, with clades (2) and (3) forming a monophyletic group sister to clade (4) and clade (1) sister to the remainder of Cornales. However, there was not strong bootstrap support for relationships among the major clades. The placement of Hydrostachys could not be reliably determined, although most analyses place the genus within Hydrangeaceae; ML analyses, for example, placed the genus as the sister of Hydrangeeae. Our results supported a Cornales including the systematically problematic Hydrostachys, a Cornaceae consisting of Cornus and Alangium, a Nyssaceae consisting of Nyssa and Camptotheca, a monogeneric Davidiaceae, a Mastixiaceae consisting of Mastixia and Diplopanax, and an expanded Grubbiaceae consisting of Grubbia and Curtisia, and two larger families, Hydrangeaceae and Loasaceae.     

Melting behaviour of D-sucrose, D-glucose and D-fructose

The melting behaviour of d-sucrose, d-glucose and d-fructose was studied. The melting peaks were determined with DSC and the start of decomposition was studied with TG at different rates of heating. In addition, melting points were determined with a melting point apparatus. The samples were identified as d-sucrose, alpha-d-glucopyranose and beta-d-fructopyranose by powder diffraction measurements. There were differences in melting between the different samples of the same sugar and the rate of heating had a remarkable effect on the melting behaviour. For example, T(o), DeltaH(f) and T(i) (initial temperature of decomposition) at a 1 degrees Cmin(-1) rate of heating were 184.5 degrees C, 126.6Jg(-1) and 171.3 degrees C for d-sucrose, 146.5 degrees C, 185.4Jg(-1) and 152.0 degrees C for d-glucose and 112.7 degrees C, 154.1Jg(-1) and 113.9 degrees C for d-fructose. The same parameters at 10 degrees Cmin(-1) rate of heating were 188.9 degrees C, 134.4Jg(-1) and 189.2 degrees C for d-sucrose, 155.2 degrees C, 194.3Jg(-1) and 170.3 degrees C for d-glucose and 125.7 degrees C, 176.7Jg(-1) and 136.8 degrees C d-fructose. At slow rates of heating, there were substantial differences between the different samples of the same sugar. The melting point determination is a sensitive method for the characterization of crystal quality but it cannot be used alone for the identification of sugar samples in all cases. Therefore, the melting point method should be validated for different sugars.     

Revisited and large-scale synthesis and purification of the mutated and wild type neu/erbB-2 membrane-spanning segment.

Solid-phase syntheses of the hydrophobic peptides Neu(TM35) ((1)EQRASPVTFIIATVVGVLLFLILVVVVGILIKRRR(35)) and Neu*(TM35) ((1)EQRASPVTFIIATVEGVLLFLILVVVVGILIKRRR(35)), corresponding to the native and mutated (V15E) transmembrane domain of the neu/erbB-2 tyrosine kinase receptor, respectively, were accomplished using Fmoc chemistry. The use of a new resin and cleavage and purification conditions led to large increases in yields and peptide purity. Two (15)N-labelled versions of both wild type and mutated peptides were also synthesized. Approximately 20-40 mg of peptide was obtained using a small-scale synthesis, whereas ca 100 mg of pure peptide was collected on a medium scale. Peptide purity, as monitored by HPLC and mass spectrometry, ranged from 95 to 98% for the six peptides synthesized. Secondary structure as determined by UV circular dichroism (CD) in trifluoroethanol (TFE) showed ca 74% alpha-helical content for the native peptide and ca 63% for that bearing the mutation. Secondary structure of Neu(TM35) was retained in DMPC (dimyristoylphosphatidylcholine)/DCPC (dicaproylphosphatidylcholine) membrane bicelles, and evidences for dimers/oligomers in the lipid bilayer were found.