Evaluation of lignin contents in tropical forages using different analytical methods and their correlations with degradation of insoluble fiber

We compared the lignin contents of tropical forages by different analytical methods and evaluated their correlations with parameters related to the degradation of neutral detergent fiber (NDF). The lignin content was evaluated by five methods: cellulose solubilization in sulfuric acid [Lignin (sa)], oxidation with potassium permanganate [Lignin (pm)], the Klason lignin method (KL), solubilization in acetyl bromide from acid detergent fiber (ABLadf) and solubilization in acetyl bromide from the cell wall (ABLcw). Samples from ten grasses and ten legumes were used. The lignin content values obtained by gravimetric methods were also corrected for protein contamination, and the corrected values were referred to as Lignin (sa)p, Lignin (pm)p and KLp. The indigestible fraction of NDF (iNDF), the discrete lag (LAG) and the fractional rate of degradation (kd) of NDF were estimated using an in vitro assay. Correcting for protein resulted in reductions (P < 0.05) in the lignin contents as measured by the Lignin (sa), Lignin (pm) and, especially, the KL methods. There was an interaction (P < 0.05) of analytical method and forage group for lignin content. In general, LKp method provided the higher (P < 0.05) lignin contents. The estimates of lignin content obtained by the Lignin (sa)p, Lignin (pm)p and LKp methods were associated (P > 0.05) with all of the NDF degradation parameters. However, the strongest correlation coefficients for all methods evaluated were obtained with Lignin (pm)p and KLp. The lignin content estimated by the ABLcw method did not correlate (P > 0.05) with any parameters of NDF degradation. There was a correlation (P < 0.05) between the lignin content estimated by the ABLadf method and iNDF content. Nonetheless, this correlation was weaker than those found with gravimetric methods. From these results, we concluded that the gravimetric methods produce residues that are contaminated by nitrogenous compounds. Adjustment for these contaminants is suggested, particularly for the KL method, to express lignin content with greater accuracy. The relationships between lignin content measurements and NDF degradation parameters can be better determined using KLp and Lignin (pm)p methods. (C) 2011 Elsevier B.V. All rights reserved.

Long-Term Maternal Separation Differentially Alters Serum Corticosterone Levels and Blood Neutrophil Activity in A/J and C57BL/6 Mouse Offspring

Objectives: In this work, we searched for maternal separation effects on serum corticosterone levels and blood neutrophil activity in adult male A/J and C57BL/6 mouse offspring. Methods: 40 male A/J mice and 40 male C57BL/6 mice were divided within each strain into two groups. Mice in the maternal separation group were separated from their mothers (1 h/day) on postnatal days 0-13. Mice in the control group were left undisturbed. On postnatal day 45, blood was drawn from all mice and used to assess neutrophil activity by flow cytometry and serum corticosterone levels by radioimmunoassay. Results: The results showed that each mouse strain responded differently to maternal separation, but in both cases, serum corticosterone levels were affected. In both strains, adult mice that experienced maternal separation showed lower serum corticosterone levels than control mice. In relation to control mice kept together with their mothers, the levels of serum corticosterone were 72.7 and 36.36% lower in A/J and C57BL/6 mice submitted to maternal separation, respectively. The current findings showed that maternal separation increased neutrophil activity in mice after reaching adulthood. The observed effects, although in the same direction, differed between A/J and C57BL/6 mice. Maternal separation increased both the percentage and intensity of phagocytosis in C57BL/6 mice, but had no effects on A/J mice. Furthermore, maternal separation increased basal and propidium iodide-labeled Staphylococcus aureus-induced oxidative burst in A/J mice but did not affect oxidative burst in C57BL/6 mice. Finally, phorbol myristate acetate-induced oxidative burst increased in both strains. Conclusion: These results indicate that early maternal separation increases innate immunity, most likely by modifying hypothalamus-pituitary-adrenal axis activity. This suggests that maternal separation is a good model for stress which produces long-term neuroimmune changes whatever the animal species and strain used. Copyright (C) 2011 S. Karger AG, Basel

Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre-exposure of oocytes to Ricinus communis (RCA-1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm-egg binding and fertilization. In vitro-matured bovine oocytes were incubated (39 degrees C, 5% CO(2) in air) for 2 h in the following treatments: (i) 500 mu l of fertilization medium (FM); (ii) 250 mu l of FM with 0.25 ml of non-luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 mu l of FM with 250 mu l of NLAUTF and 4 mu l of RCA-1 lectin; (iv) 250 mu l of FM with 250 mu l of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA-1 lectin; (v) 500 mu l of FM and RCA-1 lectin. Following incubation, oocytes were washed, placed in FM with 2 mu g heparin, and incubated with 1 x 10(5) frozen-thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean +/- SEM) when oocytes were incubated in treatment 3 (59.0 +/- 5.5) than in treatments 2 (46.4 +/- 5.6), 4 (18.1 +/- 5.4), 5 (33.4 +/- 5.6) or 1 (32.5 +/- 5.6). More oocytes were fertilized when incubated in treatment 3 (91% +/- 3.0) than in 2 (84% +/- 3.0), 4 (40% +/- 3.0), 5 (77% +/- 3.0) or 1 (76% +/- 3.0). As in previous studies, this study suggests that RCA-1 lectin enhances binding of UTF-derived OPN to bovine oocytes, resulting in increased sperm-egg binding and fertilization in vitro and a possible role in fertilization.

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO2 in air) for I It in the following treatments either 500 mu L of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for I h either FM or FM with a L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) a L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) a L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 X 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha. L-PGDS: (1) 26.4 +/- 3.0; (2) 25.6 +/- 3.0; (3) 59.7 +/- 3.0; (4) 56.4 +/- 3.0; and (5) 57.1 +/- 3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P < 0.05) compared with the control: (1) 89.2 +/- 2.0%; (2) 87.5 +/- 2.0%; (3) 19.4 +/- 2.0%; (4) 27.2 +/- 3.1%; and (5) 14.1 +/- 3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization. (c) 2007 Elsevier B.V. All rights reserved.

Comparison of gonadorelin products in lactating dairy cows: Efficacy based on induction of ovulation of an accessory follicle and circulating luteinizing hormone profiles

This study evaluated whether the four gonadorelin products that are commercially available in the United States produce comparable ovulation responses in lactating cows. Dairy cows at 7 d after last gonadotropin-releasing hormone (GnRH) treatment of Ovsynch (Day 7), with a corpus luteum (CL) >= 15 mm and at least one follicle >= 10 mm, were evaluated for response to GnRH treatment. Selected cows were randomized to receive (100 mu g; im): (1) Cystorelin (n = 146): (2) Factrel (n = 132): (3) Fertagyl (n = 140); or (4) Ovacyst (n = 140). On Day 14, cows were examined for Ovulation by detection of an accessory CL. Circulating luteinizing hormone (LH) concentrations were also evaluated in some cows after treatment with 100 mu g (n = 10 per group) or 50 mu g (n = 5 per group) GnRH. Statistical analyses were performed with the procedures MIXED and GLIMMIX of the SAS program. Percentage of cows ovulating differed (P < 0.01) among groups, with that for Factrel being lower (55.3%) than that for Cystorelin (76.7%), Fertagyl (73.6%), or Ovacyst (85.0%), There was no effect of batch, parity, or follicle size on ovulation response. but increasing body condition score decreased Ovulation response. There was a much greater LH release in cows treated with 100 mu g than in those treated with 50 mu g, but there were no detectable differences among products in time to LH peak, peak LH concentration, or area under the LH curve and no treatment effects nor treatment by time interactions on circulating LH profile. Thus, ovulation response to Factrel on Day 7 of the cycle was lower than that for other commercial GnRH products, although a definitive mechanism for this difference between products was not demonstrated. (C) 2009 Elsevier Inc. All rights reserved.

Fluoride uptake and acid resistance of enamel irradiated with Er : YAG laser

This study evaluated the resistance to demineralization and fluoride incorporation of enamel irradiated with Er:YAG. A total of 110 bovine teeth were selected and divided into eight groups: unlased, 37% phosphoric acid, and samples irradiated with the Er:YAG laser at several fluences (31.84 J/cm(2), 25.47 J/cm(2), 19.10 J/cm(2), 2.08 J/cm(2), 1.8 J/cm(2), and 0.9 J/cm(2)). The application of acidulated phosphate fluoride was performed after treatments. All samples were immersed in 2 ml of 2.0 M acetic-acetate acid solution at pH 4.5 for 8 h, and fluoride, calcium, and phosphorus ions dissolved were analyzed by atomic absorption spectrometry and spectrophotometry. The phosphoric acid and 31.84 J/cm(2) groups presented the lowest dissolution of calcium and phosphorus ions. Higher fluoride incorporation was observed on 1.8 J/cm(2) and 0.9 J/cm(2) groups. Based on these results, Er:YAG laser was able to decrease acid dissolution and increase fluoride uptake and can be a promissory alternative for preventive dentistry.

Low-fluence CO(2) laser irradiation decreases enamel solubility

This study investigated whether subablative-pulsed CO(2) laser (10.6 mu m) irradiation, using fluences lower than 1 J/cm(2), was capable of reducing enamel acid solubility. Fifty-one samples of bovine dental enamel were divided into three groups: control group, which was not irradiated (CG); group laser A (LA) irradiated with 0.3 J/cm ; and group laser B (LB) irradiated with 0.7 J/cm(2). After irradiation, the samples were subjected to demineralization in an acetate buffer solution and were then analyzed by SEM. A finite-element model was used to calculate the temperature increase. The calcium and phosphorous content in the demineralization solution were measured with an ICP-OES. ANOVA and the t-test pairwise comparison (p < 0.016) revealed that LB showed significantly lower mean Ca and P content values in the demineralization solution than other groups. A reduction in the enamel solubility can be obtained with pulsed CO(2) laser irradiation (0.7 J/cm(2), 135 mJ/pulse, 74 Hz, 100 mu s) without any surface photomodification and a less than 2 degrees C temperature increase at a 3-mm depth from the surface.

Early dental plaque formation on toothbrushed titanium implant surfaces

Purpose: To evaluate the qualitative and quantitative differences on dental plaque formation on two different roughness titanium implant surfaces, i.e. machined and titanium plasma sprayed, as well as the amount of plaque removal by regular toothbrushing after 72-hour plaque accumulation. Methods: Eight systemically healthy subjects were recruited from the patient pool of a private dental practice. All patients underwent oral hygiene instruction and full mouth prophylaxis. Subsequently, maxillary casts from all patients were obtained and removable 0.7 mm-thick acetate stents without occlusal contact points were fabricated to support four titanium specimens of 4x2x2 mm divided into two groups (machined and plasma sprayed). Subjects were instructed to wear the stents for 72 hours, full time, removing them only during regular oral hygiene. Subsequently, the appliances were immediately repositioned and then the test side was brushed for 20 seconds. At the end of the 72-hour period, the stents were removed and prepared for microbiological analysis. Results: Both machined and plasma sprayed brushed surfaces presented statistically significant fewer bacteria than non-brushed surfaces. Similarly, regarding surface roughness, machined surfaces presented a total number of bacteria significantly smaller than those presented by plasma sprayed surfaces (P< 0.05). Statistically, the non-brushed machined turned surfaces presented a greater amount of Streptoccocus sp. when compared to the brushed machined surfaces. It was concluded that rough surfaces accumulated more dental plaque than polished surfaces. Both brushed surfaces presented less plaque accumulation, however, implant brushing was more effective on machined surfaces. (Am J Dent 2008;21:318-322).

Comparison of Cross-Sectional Hardness and Transverse Microradiography of Artificial Carious Enamel Lesions Induced by Different Demineralising Solutions and Gels

The aims of this study were: (1) to correlate surface (SH) and cross-sectional hardness (CSH) with microradiographic parameters of artificial enamel lesions; (2) to compare lesions prepared by different protocols. Fifty bovine enamel specimens were allocated by stratified randomisation according to their initial SH values to five groups and lesions produced by different methods: MC gel (methylcellulose gel/lactic acid, pH 4.6, 14 days); PA gel (polyacrylic acid/lactic acid/hydroxyapatite, pH 4.8, 16 h); MHDP (undersaturated lactate buffer/methyl diphosphonate, pH 5.0, 6 days); buffer (undersaturated acetate buffer/fluoride, pH 5.0, 16 h), and pH cycling (7 days). SH of the lesions (SH(1)) was measured. The specimens were longitudinally sectioned and transverse microradiography (TMR) and CSH measured at 10- to 220-mu m depth from the surface. Overall, there was a medium correlation but non-linear and variable relationship between mineral content and root CSH. root SH(1) was weakly to moderately correlated with surface layer properties, weakly correlated with lesion depth but uncorrelated with integrated mineral loss. MHDP lesions showed the highest subsurface mineral loss, followed by pH cycling, buffer, PA gel and MC gel lesions. The conclusions were: (1) CSH, as an alternative to TMR, does not estimate mineral content very accurately, but gives information about mechanical properties of lesions; (2) SH should not be used to analyse lesions; (3) artificial caries lesions produced by the protocols differ, especially considering the method of analysis. Copyright (C) 2009 S. Karger AG, Basel

Effect of a 4% titanium tetrafluoride (TiF4) varnish on demineralisation and remineralisation of bovine enamel in vitro

Objectives: This in vitro study assessed the effect of a 4% TiF4 varnish on demineralisation and remineralisation of sound enamel and artificial carious enamel lesions, respectively. Methods: Bovine sound and carious enamel (n = 110) were randomly allocated to each type of varnish: Duraphat (R))-D (NaF, 2.26%F, pH 4.5, Colgate-Brazil, n = 30), Duofluorid (R)-F (NaF, 2.71%F, pH 8.0, FGM-Brazil, n = 30), TiF4-T (2.45%F, pH 1.0, FGM-Brazil, n = 30) and no-fluoride-P (FGM-Brazil, pH 5.0, n = 20). For the formation of artificial enamel caries, half of the blocks were immersed in 32 mL buffer acetate solution (16 h), whereas the other half was maintained sound. The varnishes were applied onto the enamel surfaces. Thus, the samples were subjected to pH cycles (37 degrees C) for 7 days. The response variables tested were surface and cross-sectional hardness. Data were tested using Kruskal-Wallis test (p < 0.05). Results: All F varnishes significantly reduced demineralisation and increased remineralisation in comparison to placebo. The TiF4 did not significantly reduce the surface enamel softening when compared with the other F varnishes, but it decreased the loss of subsurface hardness to the same extent. In enamel blocks with previous artificial carious lesions, the TiF4 significantly improved the rehardening compared to the other varnishes up to 30 mu m depth. Conclusions: The TiF4 varnish was able to decrease the demineralisation and increase the remineralisation of previously sound and carious enamel, respectively. It was equally effective compared to NaF varnishes on reducing the demineralisation at subsurface, but it was more effective on improving the remineralisation at surface and subsurface. (c) 2007 Elsevier Ltd. All rights reserved.

Prevalence of Upper Cervical Vertebrae Anomalies in Patients With Cleft Lip and/or Palate and Noncleft Patients

Objectives: To investigate the prevalence of upper cervical vertebrae anomalies in patients with isolated cleft lip, isolated cleft palate, and complete cleft lip and palate, as well as to compare the prevalence of these anomalies between groups, between genders, and with noncleft patients. Design: Retrospective cross-sectional study of randomly selected patients. Setting: Radiology Section, Hospital for Rehabilitation of Craniofacial Anomalies and Department of Orthodontics, Bauru School of Dentistry, University of Sao Paulo, Sao Paulo, Brazil. Participants: The sample comprised 300 lateral cephalograms of cleft patients, aged 12 to 13 years, of both genders, from the files of the Hospital for Rehabilitation of Craniofacial Anomalies-University of Sao Paulo and 300 lateral cephalograms from noncleft patients of the Department of Orthodontics, Bauru School of Dentistry. Cephalograms of patients with syndromes were not included. Method: Radiographs were interpreted on a film viewer by a single examiner; the profiles of vertebrae were traced on acetate paper, and cervical vertebrae anomalies were registered and categorized into posterior arch deficiencies, fusion, and association of both. Main Outcome Measures: Statistical comparison of groups using the chi-square test. Results: In the cleft group, 38.67% of the patients had cervical vertebrae anomalies. Of those in the noncleft sample, 31% showed anomalies of the cervical spine. This difference was statistically significant. There was no statistically significant difference when the types of clefts were compared with each other or when both genders were compared in both samples. Conclusions: This study confirms the association between clefts and cervical anomalies. Additional research on this topic is necessary.

Lead Deposition in Bovine Enamel during a pH-Cycling Regimen Simulating the Caries Process

Like fluoride, lead (Pb) accumulates on the enamel surface pre-eruptively, but it is not yet known whether it also deposits on enamel while dental caries is developing. This study evaluates Pb distribution in bovine enamel slabs submitted to a pH-cycling regimen simulating the caries process. The slabs were subjected to 8 cycles of de- and remineralizing conditions, and Pb (as acetate salt) was added to the de- and remineralized solutions at concentrations of 30 mu g/l (experimental group, E1) and 300 mu g/l (experimental group, E2). The control group (C) consisted of solutions to which Pb was not added. After the pH cycling, 100-mu m sections of the slabs were analyzed by polarizing microscopy, to observe the extent of caries-like lesions, and these sections were used for Pb estimation by Synchrotron radiation X-ray microfluorescence. Caries lesions were observed along all superficial enamel surfaces to an extent of 120 mu m. A Pb concentration gradient was observed in enamel, which decreased toward dentine. The highest Pb signals were observed for group E2, and the differences were statistically significant at enamel depths of 0 (C vs. E2; p = 0.029) and 50 mu m (C vs. E2 and E1 vs. E2; p = 0.029). In conclusion, this study suggests that if Pb is present in the oral environment, it may deposit in enamel during the caries process. Copyright (C) 2011 S. Karger AG, Basel

Propyretic role of the locus coeruleus nitric oxide pathway

Nitric oxide has been reported to modulate fever in the brain. However, the sites where NO exerts this modulation remain somewhat unclear. Locus coeruleus (LC) neurons express not only nitric oxide synthase (NOS) but also soluble guanylyl cyclase (sGC). In the present study, we evaluated in vivo and ex vivo the putative role of the LC NO-cGMP pathway in fever. To this end, deep body temperature was measured before and after pharmacological modulations of the pathway. Moreover, nitrite/nitrate (NOx) and cGMP levels in the LC were assessed. Conscious rats were microinjected within the LC with a non-selective NOS inhibitor (NG-monomethyl-l-arginine acetate), a NO donor (NOC12), a sGC inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) or a cGMP analogue (8-bromo-cGMP) and injected intraperitoneally with endotoxin. Inhibition of NOS or sGC before endotoxin injection significantly increased the latency to the onset of fever. During the course of fever, inhibition of NOS or sGC attenuated the febrile response, whereas microinjection of NOC12 or 8-bromo-cGMP increased the response. These findings indicate that the LC NO-cGMP pathway plays a propyretic role. Furthermore, we observed a significant increase in NOx and cGMP levels, indicating that the febrile response to endotoxin is accompanied by stimulation of the NO-cGMP pathway in the LC.

Effects of statins on matrix metalloproteinases and their endogenous inhibitors in human endothelial cells

Statins exert anti-inflammatory effects and downregulate matrix metalloproteinases (MMPs) expression, thus contributing to restore cardiovascular homeostasis in cardiovascular diseases. We aimed at comparing the effects of different statins (simvastatin, atorvastatin, and pravastatin) on MMP-2, MMP-9, tissue inhibitors of metalloproteinases (TIMP)-1, TIMP-2, and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios released by human umbilical vein endothelial cells (HUVEC) stimulated by phorbol myristate acetate (PMA). HUVECs were incubated with statins (0.1-10 mu M) for 12 h before stimulation with PMA 100 nM. Monolayers were used to perform cell viability assays and the supernatants were collected to determine MMPs and TIMPs levels by gelatin zymography and/or enzyme immunoassay. While treatment with PMA increased MMP-9 and TIMP-1 levels (by 556% and 159%, respectively; both P < 0.05), it exerted no effects on MMP-2 and TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, attenuated PMA-induced increases in MMP-9 levels (P < 0.05). Only atorvastatin decreased baseline MMP-2 levels significantly (P < 0.05). We found no effects on TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, decreased MMP-9/TIMP-1 ratio significantly (both P < 0.05), whereas atorvastatin and pravastatin, but not simvastatin, decreased MMP-2/TIMP-2 ratio significantly (both P < 0.05). Our data support the notion that statins with different physicochemical features exert variable effects on MMP/TIMP ratios (which reflect net MMP activity). Our results suggest that more lipophilic statins (simvastatin and atorvastatin), but not the hydrophilic statin pravastatin, downregulate net MMP-9 activity. However, atorvastatin and pravastatin may downregulate net MMP-2 activity. The clinical implications of the present findings deserve further investigation.

Studies of solute self-association by sedimentation equilibrium: allowance for effects of thermodynamic non-ideality beyond the consequences of nearest-neighbor interactions

A sedimentation equilibrium study of a-chymotrypsin self-association in acetate-chloride buffer, pH 4.1 I 0.05, has been used to illustrate determination of a dimerization constant under conditions where thermodynamic non-ideality is manifested beyond the consequences of nearest-neighbor interactions. Because the expressions for the experimentally determinable interaction parameters comprise a mixture of equilibrium constant and excluded volume terms, the assignment of reasonable magnitudes to the relevant virial coefficients describing non-associative cluster formation is essential for the evaluation of a reliable estimate of the dimerization constant. Determination of these excluded volume parameters by numerical integration over the potential-of-mean-force is shown to be preferable to their calculation by approximate analytical solutions of the integral for this relatively small enzyme monomer with high net charge (+ 10) under conditions of low ionic strength (0.05 M). (C) 2001 Elsevier Science B.V. All rights reserved.