Fatty Acid-free Bovine Albumin Research Articles

Inhibition of Protein Glycation by Extracts of Culinary Herbs and Spices

We tested whether polyphenolic substances in extracts of commercial culinary herbs and spices would inhibit fructose-mediated protein glycation. Extracts of 24 herbs and spices from a local supermarket were tested for the ability to inhibit glycation of albumin. Dry samples were ground and extracted with 10 volumes of 50% ethanol, and total phenolic content and ferric reducing antioxidant potential (FRAP) were measured. Aliquots were incubated in triplicate at pH 7.4 with 0.25 M fructose and 10 mg/mL fatty acid-free bovine albumin. Fluorescence at 370 nm/440 nm was used as an index of albumin glycation. In general, spice extracts inhibited glycation more than herb extracts, but inhibition was correlated with total phenolic content (R(2) = 0.89). The most potent inhibitors included extracts of cloves, ground Jamaican allspice, and cinnamon. Potent herbs tested included sage, marjoram, tarragon, and rosemary. Total phenolics were highly correlated with FRAP values (R(2) = 0.93). The concentration of phenolics that inhibited glycation by 50% was typically 4-12 microg/mL. Relative to total phenolic concentration, extracts of powdered ginger and bay leaf were less effective than expected, and black pepper was more effective. Prevention of protein glycation is an example of the antidiabetic potential for bioactive compounds in culinary herbs and spices.

Herb and spice extracts inhibit protein glycation

We hypothesized that substances in herbs and spices may inhibit protein glycation. Extracts of 20 herbs and spices were tested for ability to inhibit glycation of albumin. Dry samples were ground and extracted with 50% ethanol (10% w/v), and total phenolic content and ferric reducing antioxidant potential (FRAP) were measured. Aliquots were incubated in triplicate at 37°C in 0.2 M potassium phosphate buffer, pH.7.4, containing 0.25 M fructose and 10 mg/ml fatty acid-free bovine albumin with final concentrations of total phenolics from 33 to 267 μg/mL. After 4 days, fluorescence was measured at 440 nm as an index of the formation of glycated albumin. In general, the spice extracts inhibited glycation more than the herb extracts. When tested at 267μg/mL total phenolics, the most potent inhibitors included clove (83.5%), ground Jamaican allspice (67.8%), and cinnamon (65.0%). Potent herbs tested included sage (49.1%), tarragon (48.1% ), and rosemary (47.7%). Inhibition of glycation by spice extracts was concentration dependent and correlated with total phenolic and FRAP values (R²=0.66 and R²=0.51, respectively) whereas the inhibition by herb extracts was not highly correlated (R²=0.14 and R²=0.004, respectively). Total phenolics were highly correlated with FRAP (R²=0.93). The evidence shows that compounds found in herb and spice extracts inhibit albumin glycation in vitro. Not all active substances appear to be polyphenolics. (Supported by AES Hatch project GE09622)

Optimized conditions for measuring lipolysis in murine primary adipocytes

The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells.

Docosahexaenoic Acid Supports Cell Growth and Expression of Choline Acetyltransferase and Muscarinic Receptors in NG108-15 Cell Line

A large body of evidence indicates that adequate intake of polyunsaturated fatty acids is essential for brain development in early ontogenesis and positively impacts various pathological states connected with aging, as well as other neurodegenerative diseases (Jump, 2002; Bazan, 2003; Ruxton et al., 2004). In the present experiments, we investigated the possible effects of polyunsaturated docosahexanoic acid (DHA [22:6, n = 3]) on the expression of cholinergic phenotype-represented by choline acetyltransferase (ChAT) activity and a number of surface muscarinic receptors-as well as on cell growth in the cholinergic cell line NG108-15(Hamprecht, 1977; Hamprecht et al., 1985). However, chemical composition of different batches of sera is neither stable nor defined, and this fact complicates investigations on in vitro effects of substances that are natural constituents of serum. To avoid this restraint we employed defined medium in which fatty acid-free bovine albumin as a carrier of DHA replaced serum. Growth of most cell lines, as well as cells in primary cultures, depends strictly on the presence of serum in growth medium. As expected, withdrawal of serum resulted in growth arrest exemplified by a decrease in protein content compared with control cells grown in the presence of serum and also caused a decrease in ChAT activity (Fig. 1, lower left). DHA, at a concentration of 10 mumol/L, largely prevented both growth arrest in defined medium with fatty acid-free bovine albumin as a carrier of DHA and the attenuation of ChAT activity. DHA at concentrations 10 times higher had no further effect. At a concentration of 100 mumol/L, DHA also significantly increased the number of surface muscarinic receptors compared with cells grown in serum-containing as well as serum-free medium (Fig. 1, upper right). These data demonstrate the ability of DHA at low micromolar concentrations to support cell growth and expression of ChAT activity. Although it is not possible to stipulate a mechanism of action on the expression of ChAT and muscarinic receptors, a plausible explanation could be prevention of apoptosis, evidenced by a sharp decrease in executive caspase-3 activity (Fig. 1, lower right). Apoptosis is a process with a high requirement for energy. An improved metabolic state of cells consequent to suppression of apoptosis might thus better fulfill requirements for protein synthesis and targeting.

240 GESTATION LENGTH AND BIRTH WEIGHT OF IN VITRO PRODUCED EMBRYOS FROM ZEBU DAIRY CATTLE

Gir cattle are a popular zebu dairy breed in tropical and subtropical regions because of their tolerance of heat stress and resistance to tick-borne disease. The use of in vitro embryo production (IVP) technology may help accelerate genetic improvement of this breed. However, in general, IVP systems have been implicated in the production of large offspring and a greater proportion of male calves. Natural breeding results in newborn Gir calves weighing around 25 kg despite the fact that dams may weigh over 500 kg. It is unknown whether in vitro-produced Gir embryos also result in large offspring. The aim of this work was to evaluate the effect of in vitro embryo production on gestation length, birth weight, and sex ratio in Gir cattle. COCs were harvested by oocyte pickup from mature non-lactating Gir cows and in vitro-matured in TCM 199 medium (Gibco, Sao Paulo, Brazil) with 10% inactivated estrous cow serum for 24 h under 5% CO2 at 38.5°C in air. Gir spermatozoa were obtained through the swim-up method and co-incubated with oocytes in Fert-TALP media (Parrish JJ et al. 1988 Biol. Reprod. 38, 1171–1180) with 10 μg/mL heparin (Sigma, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson RF et al. 1996 Theriogenology 45, 41–49) with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. Fresh Day 7 blastocysts were transferred to synchronized B. indicus × B. taurus crossbred recipients. Data of gestation length, birth weight, and gender ratio from 26 IVP calves (IVP group) were recorded and compared to data obtained from Gir calves produced by artificial insemination or natural mating (n = 24; control group) using ANOVA or chi-square analysis. There was no difference (P > 0.05) in gestation length between pregnancies of the IVP and control groups (mean ± SEM, 285.4 ± 1.5 vs. 284.4 ± 1.1 days, respectively). IVP and control calves showed similar (P > 0.05) weight at calving (29.6 ± 0.9 vs. 26.9 ± 1.2 kg for IVP and control male calves, and 27.0 ± 2.5 vs. 25.2 ± 0.5 kg for IVP and control female calves, respectively). The percentage of male calves was greater (P < 0.05) in the IVP group than in the control group (76.9% vs. 43.4%, respectively). IVP calves did not show abnormalities associated with Large Offspring Syndrome, such as breathing difficulty and perinatal death. These data suggest that in vitro production may affect the development of Gir embryos, biasing the sex ratio in a manner similar to previously reported for in vitro-produced embryos from Bos taurus breeds. This work was supported by FAPEMIG and CNPq.

272 DEVELOPMENTAL COMPETENCE OF OOCYTES OBTAINED FROM BOS TAURUS AND BOS INDICUS DAIRY COWS RAISED IN A TROPICAL CLIMATE

The effects of heat stress on Bos taurus reproductive performance in tropical and subtropical regions are well known, and have been associated with lower oocyte developmental capacity. The aim of this study was to evaluate the developmental competence of oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) dairy cows raised in a Brazilian tropical region, located at 21°35′S latitude, 43°51′W longitude, and 435 meters altitude. Cumulus–oocyte complexes (COCs) were recovered by oocyte pickup (OPU) from mature non-lactating Holstein (n = 9) and Gyr (n = 13) donor cows between the end of spring and the beginning of autumn, with at least two OPU sessions/cow. COCs were in vitro-maturated in TCM-199 (GIBCO, Grand Island, NY, USA) with 10% inactivated estrus cow serum for 24 h under 5% CO2 at 38.5°C in air. Bos taurus and Bos indicus semen with similar cleavage rates, previously evaluated by in vitro fertilization with oocytes obtained from slaughterhouse ovaries, were used to reduce bull effect. Holstein and Gyr spermatozoa were obtained through swim-up method and co-incubated with Holstein (n = 390) and Gyr (n = 505) oocytes, respectively, in Fert-TALP medium (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180) supplemented with 10 μg/mL heparin (Sigma-Aldrich, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson et al. 1996 Theriogenology 45, 41–49) supplemented with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. On Day 7 to 8 of co-culture, Gyr and Holstein blastocysts were assessed and those classified as grade 1 (IETS Manual) were transferred to synchronized Bos indicus × Bos taurus crossbred recipients managed under the same nutritional and environmental conditions. Pregnancy diagnosis was performed between 35 and 50 days after estrus. Cleavage, blastocyst, and pregnancy rates were analyzed by chi-square test. Cleavage and blastocyst rates were greater (P < 0.05) in Gyr than in Holstein (66.7% vs. 53.1% for cleavage and 19.6% vs. 10.8% for blastocyst, respectively), but the pregnancy rate was similar (P > 0.05; 44.5% vs. 60% for Gyr and Holstein, respectively). These results show that Gyr oocytes obtained in a tropical region have greater developmental capacity than Holstein oocytes, suggesting an interaction between genotype and environment that influences the success of an in vitro embryo production program; nevertheless, the blastocyst viability after transferring to recipients is similar for both breeds.

Albumin up-regulates the type II transforming growth factor-beta receptor in cultured proximal tubular cells

Clinical and experimental observations suggest that proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. Filtered proteins are taken up by tubular cells, and overwhelming this system may lead to tubular synthesis of various proinflammatory and profibrogenic cytokines, including transforming growth factor-beta (TGF-beta). TGF-beta acts by first binding to specific receptors. We studied in an in vitro system using a well-defined mouse proximal tubular cell line (MCT cells) whether fatty acid-free bovine albumin modulates expression of specific receptors for TGF-beta. MCT (and LLC-PK1) cells were challenged in serum-free medium with different concentrations of albumin. Activation of a local renin-angiotensin system was tested by real-time polymerase chain reaction (PCR) for renin and angiotensinogen transcripts and determination of secreted angiotensin II (Ang II) by enzyme-linked immunosorbent assay (ELISA). Some cells were also treated with the AT1 receptor antagonist losartan. TGF-beta receptor types I and II mRNA levels were determined by Northern analysis whereas protein abundance was measured by Western blots. To test for a functional consequence of up-regulated TGF-beta receptors, MCT cells were preincubated with albumin and subsequently treated with low-dose TGF-beta that normally does not induce collagen type IV expression by itself. Downstream signaling events were detected by Western blots for phosphorylated Smad2. Scatchard assays with [125I]TGF-beta1 were performed to estimate affinity and number of specific binding sites. Different length TGF-beta type II promoter constructs linked to CAT reporter were transiently transected into MCT cells to determine transcriptional activity. Incubation of MCT cells with 0.5 to 10 mg/mL albumin leads to an increase in type II TGF-beta receptor mRNA and protein expression without influencing type I receptors. An increase in type II TGF-beta receptor protein expression was detected after 12 hours of albumin incubation and was still detectable after 48 hours. The albumin-mediated increase in type II TGF-beta receptor mRNA was attenuated in the presence of 1 micromol/L losartan, suggesting involvement of a local renin-angiotensin system. MCT cells treated with albumin significantly increased expression of angiotensinogen and renin transcripts and also secreted more Ang II into the culture supernatant. Analysis of transcriptional activity showed that promoter segments containing activating protein (AP-1)-binding sites are necessary for albumin-induced transcription of the TGF-beta type II receptor. Binding assays revealed that albumin treatment significantly increased the overall binding sites as well as the affinity for TGF-beta. This effect had functional consequences because MCT cells pretreated with albumin reacted with a stronger TGF-beta-mediated phosphorylation of down-stream Smad2 and also increased collagen IV expression compared with control cells. Our findings indicate that albumin up-regulates ligand-binding TGF-beta receptors on cultured proximal tubular cells. Albumin-induced activation of local Ang II production appears to be responsible for this effect. This may amplify the matrix-stimulatory actions of TGF-beta on tubular cells and could be a novel mechanism for how proteinuria exhibits pathophysiologic effects on tubular cells ultimately leading to tubulointerstitial fibrosis.

Protection by albumin against ischaemia- and hypoxia-induced hepatic injury.

In previous studies using isolated perfused rat livers, we have shown that reactive oxygen species are involved in hypoxic and ischaemic liver damage. Since albumin was shown to possess strong antioxidant properties we now investigated the capacity of albumin to prevent ischaemic and hypoxic damage in isolated perfused rat livers. Both, partial ischaemia and hypoxia/reoxygenation, resulted in marked hepatic injury as evidenced by an increased release of hepatic enzymes (GPT, LDH), by a strong decline of bile flow and by a decrease in hepatic GSH levels. With partial ischaemia, hepatic ATP depletion and calcium accumulation were also observed. Bovine serum albumin, added to the perfusate at concentrations of 0.1 or 1%, provided nearly complete protection against both types of liver injury. The same level of protection was also afforded by sulfhydryl-blocked and fatty acid-free bovine albumin preparations and by human albumin. In conclusion, the protective effect of albumin in our models of oxidative liver injury is neither due to the thiol moiety nor to the presence of oxidizable fatty acids in the albumin fraction. More likely, albumin provides protection by an unspecific binding of redox-active transition metal ions capable of catalyzing reactions which yield hydroxyl or hydroxyl-like radicals. Besides, unspecific sacrifice reactions of albumin with highly reactive oxygen species or other endogenous compounds may also be implicated.

Renal mouse proximal tubular cells are more susceptible than MDCK cells to chemical anoxia.

To elucidate the mechanisms responsible for the resistance of continuous cell lines to anoxic injury, we have compared the effects of ATP depletion induced by chemical anoxia on primary cultures of mouse proximal tubular (MPT) cells and on Madin-Darby canine kidney (MDCK) cells. Inhibition of ATP production by cyanide and 2-deoxyglucose (CN+DOG) in the absence of dextrose reduced cell ATP content to < 5% of control values in MPT cells and caused progressive deterioration in mitochondrial function as well as loss of cell viability in these cells. Cell free fatty acid (FFA) content rose from 4.3 +/- 0.9 to 23.7 +/- 2.0 micrograms/mg of total lipid weight after 4 h of CN + DOG (P < 0.05). The mitochondrial injury and cell death induced by CN + DOG in MPT cells was ameliorated by the addition of fatty acid-free bovine albumin to the cell medium, which reduced cell FFA content during chemical anoxia from 25.0 +/- 3.0 to 10.4 +/- 2.0 micrograms/mg (P < 0.05). The phospholipase A2 (PLA2) inhibitor, mepacrine, also resulted in functional protection and reduction of cell FFA content from 20.2 +/- 2.3 to 15.9 +/- 1.7 micrograms/mg (P < 0.05). These data suggest a role for phospholipase activation and accumulation of toxic lipid metabolites in the pathophysiology of MPT cell injury. We then compared cell injury induced by CN + DOG in MPT and MDCK cells. Despite comparable reduction in cell ATP content in the two cell types, injury was far more severe in MPT than MDCK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoteichoic acid-binding and biological properties of T protein of group A streptococcus.

T protein was extracted with trypsin from an avirulent, M protein-deficient, type 1 group A Streptococcus and purified by ammonium sulfate precipitation and anion-exchange chromatography. The latter procedure removed contaminating lipoteichoic acid (LTA) from the T protein, which consisted of a heterogeneous mixture of polypeptides resistant to digestion by trypsin and ranged in molecular size from 160,000 to 200,000 daltons. Threonine, aspartic acid, glutamic acid, lysine, and valine were the most predominant amino acids. The binding of LTA to an affinity column of T protein was reversible with increasing concentrations of ethanol but not with increasing ionic strength. T protein bound less palmitic acid and LTA than did fatty acid-free bovine albumin and did not stimulate human peripheral lymphocytes. Because the surface and cell wall distribution of the T proteins and LTA appear similar, the possibility exists that T proteins and LTA may interact in situ by weakly hydrophobic bonds. Such ligand-ligand interaction may be indirectly involved in the adherence of group A streptococci to host cell membranes that is known to be mediated by LTA.

Subcellular Distribution of Steryl Ester Biosynthesis in Spinach Leaves

Higher steryl ester biosynthetic activities were obtained with Triton X-100-phosphatidylcholine-cholesterol mixed micelles than with Tween 80-phosphatidylcholine-cholesterol mixed micelles when incubated with spinach leaf (Spinacia oleracea L.) acetone powder preparations. The best incorporation of [4-(14)C]cholesterol into [4-(14)C]cholesteryl ester was obtained with a Triton X-100-phosphatidylcholine-cholesterol (10:1:1, w/w) mixed micelle system. This mixed micelle system, however, required 1,2-dipalmitin and fatty acid-free bovine albumin for optimal activity. The reaction exhibited a diglyceride specificity since the dipalmitin requirement could be replaced with neither 1-monopalmitin nor tripalmitin. Significant amounts of steryl ester biosynthetic activity were detected in the chloroplast (1,000g pellet), mitochondrial (3,000g pellet), and microsomal (20,000g and 88,000g pellet) fractions. Little activity was detected in the water-soluble (88,000g supernatant) fraction. The highest specific activity occurred in the 88,000g pellet. The 88,000g supernatant contained a heatstable, water-soluble substance that was required for optimal steryl ester biosynthesis in all of the pellet fractions. This factor was not lost during extensive dialysis but was destroyed by ashing, indicating that it was large and organic. Silver nitrate thin layer chromatography indicated that 60% of the biosynthesized steryl esters contained saturated fatty acids in the absence of 1,2-dipalmitin and that 83% contained saturated fatty acids in the presence of 1,2-dipalmitin.

Detection of Escherichia coli Antigens by a Latex Agglutination Test

A latex particle agglutination technique to detect ethylenediaminetetraacetate-solubilized extracts from Escherichia coli and whole E. coli cells is described. The sensitivity of the serological test was found to be 0.5 to 2.5 ng for the solubilized antigens and 1.5 x 10(6) to 5.7 x 10(6) cells per ml for the particulate antigens. The test was 100 to 1,000 times more sensitive than the standard bacterial agglutination test. Furthermore, it detected E. coli antigens during all phases of bacterial growth, whereas the bacterial test detected the antigens only after the mid-log phase. No significant cross-reactivity was observed between latex-anti-E. coli preparations and heterologous bacterial strains used in the experimental procedure. A buffer formula containing fatty acid-free bovine albumin prevented nonspecific aggregation of the latex particles.