Protein Concentration In Supernatant Research Articles

PSII-3 Sheep Neutrophil Activation in Response to Garlic in Vitro

Abstract St. Croix hair sheep are known to resist parasitic infection. Parasitic resistance is in part due to the increased number and ability of neutrophil leukocytes to fight pathogens. Dietary supplements such as garlic (Allium sativum L.) are antimicrobial immune modulators that can enhance production. This study sought to evaluate the impact of fresh filtered garlic on sheep neutrophils through In vitro treatment. The study collected blood from the jugular vein of five clinically healthy female St. Croix sheep at NC A&T State University Farm and isolated neutrophils from 10 mL whole blood samples from each animal. The neutrophils were treated with fresh filtered garlic after the total cell count was measured, and 1 million cells were taken for treatment. The total protein concentration in the neutrophil supernatant was measured before and after treatment. Garlic (10 ug/mL PBS) was added to neutrophils, incubated (30 minutes, 37 °C) and centrifuged. Total RNA was extracted from the pellet using the TRIZOL RNA Isolation Protocol (Sigma-Aldrich). The RNA concentration and purity were assessed using a Nanodrop Spectrophotometer (Thermo Scientific Inc.). Pooled RNA from each group was used for cDNA synthesis, followed by qPCR analysis of the expression of 84 test genes related to the innate and adaptive immune response (RT2 profiler array) of the host. Total protein concentration in the supernatant was determined using the BCA Assay (Pierce BCA Protein Assay Kit). In vitro treatment with garlic extract resulted in increased total secreted protein concentrations from treated neutrophils, as well as upregulation of 10 genes involved in immune response and downregulation of 4 genes associated with inflammation and immune regulation. The study findings indicate that garlic stimulates gene transcription and protein secretion in neutrophils from St. Croix sheep in vitro. Studies are needed on the possible impacts on neutrophil function and animal response to infection for sustainable use of garlic in animal production.

Physicochemical and functional properties of buckwheat (Fagpopyrum esculentum Moench) albumin

Buckwheat albumin is a promising functional food material that can suppress postprandial blood glucose elevation. Here, we studied the physicochemical and functional properties of buckwheat albumin such as thermal resistance, solubility, and emulsifying and foaming properties to evaluate its potential for application in various foods such as mayonnaise, ice cream, meringues and sponge cake. The denaturation temperature of buckwheat albumin was measured using Differential Scanning Calorimetry (DSC) with and without the addition of dithiothreitol (DTT), and solubility in the pH range of 3.0–6.0 was measured as protein concentration in supernatant before and after heating. Emulsifying and foaming properties of buckwheat albumin were also evaluated before and after hydrolysis by protease. Buckwheat albumin exhibited high thermal resistance with a denaturation temperature of 148.4 °C. However, the peak temperature of DSC thermogram decreased and denaturation enthalpy increased with DTT addition. High solubility was observed at pH 3.0–6.0, which was retained even after heating at 80 °C for 20 min. Furthermore, buckwheat albumin showed high emulsifying and foaming properties in the pH range 3.0–6.0. Although its emulsifying property was decreased on protease treatment, high foaming capacity was maintained even after hydrolysis. These findings suggested that buckwheat albumin can be widely used in various functional foods.

Direct comparison of the tofu-like precipitate formation by adding different coagulants: magnesium chloride and glucono-δ-lactone

Tofu is produced by adding a coagulant such as MgCl2 and glucono-δ-lactone (GDL) in soymilk. However, the molecular mechanism of tofu formation by adding these coagulants has been compared between the results obtained under different conditions. In this study, the formation of a tofu-like precipitate (TLP) by adding GDL was directly compared with that formed by adding MgCl2 under the same conditions except for the coagulants. The effects of both the coagulants were almost the same on the changes in the precipitate weight, supernatant protein concentration, and urea-soluble protein concentration, indicating that a common contributing factor induces TLP formation. However, the effects of the coagulants on pH were largely different, suggesting that pH reduction is not an absolute requirement in TLP formation induced by adding MgCl2. Moreover, the findings of this study revealed that the decrease in the surface charge of soymilk proteins is a common initiation factor for TLP formation, whereas the intermolecular hydrophobic interaction is an important factor for the formation of urea-insoluble precipitates. Overall, these findings will be useful in discovering new coagulants to enhance the quality characteristics of tofu.

Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag.

In this study, we assessed the potential of arginine and lysine solubility-enhancing peptide (SEP) tags to control the solubility of a model protein, anti-EGFR VHH-7D12, in a thermally denatured state at a high temperature. We produced VHH-7D12 antibodies attached with a C-terminal SEP tag made of either five or nine arginines or lysines (7D12-C5R, 7D12-C9R, 7D12-C5K and 7D12-C9K, respectively). The 5-arginine and 5-lysine SEP tags increased the E. coli expression of VHH-7D12 by over 80%. Biophysical and biochemical analysis confirmed the native-like secondary and tertiary structural properties and the monomeric nature of all VHH-7D12 variants. Moreover, all VHH-7D12 variants retained a full binding activity to the EGFR extracellular domain. Finally, thermal stress with 45-minute incubation at 60 and 75 °C, where VHH-7D12 variants are unfolded, showed that the untagged VHH-7D12 formed aggregates in all of the four buffers, and the supernatant protein concentration was reduced by up to 35%. 7D12-C5R and 7D12-C9R did not aggregate in Na-acetate (pH 4.7) and Tris-HCl (pH 8.5) but formed aggregates in phosphate buffer (PB, pH 7.4) and phosphate buffer saline (PBS, pH 7.4). The lysine tags (either C5K or C9K) had the strongest solubilization effect, and both 7D12-C5K and 7D12-C9K remained in the supernatant. Altogether, our results indicate that, under a thermal stress condition, the lysine SEP tags solubilization effect is more potent than that of an arginine SEP tags, and the SEP tags did not affect the structural and functional properties of the protein.

High-fat meal increases peripheral blood mononuclear cell pro-inflammatory cytokine expression in African-American women.

African-American (AA) women have elevated predominance of inflammatory diseases concurrent with local inflammation resulting in compromised metabolic function. The purpose of the study was 2-fold: 1) to examine the gene and protein expression of pro- and anti-inflammatory cytokine secretion by peripheral blood mononuclear cells (PBMC) obtained from AA and Caucasian-American (CA) women in response to an acute high-fat meal; and 2) to explore the influence of race (AA vs. CA) on PBMC reactivity. Ten AA and 11 CA women consumed a high-fat meal with baseline and 4h postprandial venous blood draws. PBMCs were incubated for 3 h then messenger RNA expression and supernatant protein concentration was used to examine inflammatory profiles. All women had a postprandial increase in interleukin (IL)-8 gene expression, IL-8 protein concentration, and tumor necrosis factor alpha (TNF-α) protein concentration (P< 0.05). AA women had a postprandial increase in IL-6, IL-8, and TNF-α protein concentration (P< 0.05). AA women had higher postprandial IL-1β protein concentration and IL-8 gene expression compared with CA women (P< 0.05). Our data uncovers the specific impact of race and time on pro-inflammatory PBMC (IL-1β, IL-6, IL-8, and TNF-α) expression profiles in response to an acute high-fat meal challenge. Novelty: African Americans have higher predominance of inflammatory disease. We explored the potential race impact on peripheral blood mononuclear cell reactivity in response to a meal. A pro-inflammatory response to an acute high-fat meal with race impact was observed possibly contributing to health disparities impacting African-American women.

Protective effects of rolipram on endotoxic cardiac dysfunction via inhibition of the inflammatory response in cardiac fibroblasts

BackgroundCardiac fibroblasts, regarded as the immunomodulatory hub of the heart, have been thought to play an important role during sepsis-induced cardiomyopathy (SIC). However, the detailed molecular mechanism and targeted therapies for SIC are still lacking. Therefore, we sought to investigate the likely protective effects of rolipram, an anti-inflammatory drug, on lipopolysaccharide (LPS)-stimulated inflammatory responses in cardiac fibroblasts and on cardiac dysfunction in endotoxic mice.MethodCardiac fibroblasts were isolated and stimulated with 1 μg/ml LPS for 6 h, and 10 μmol/l rolipram was administered for 1 h before LPS stimulation. mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in fibroblasts and their protein concentrations in supernatant were measured with real-time PCR (rt-PCR) and enzyme-linked immunosorbent assay, respectively. The expression of dual specificity phosphatase 1 (DUSP1), an endogenous negative regulator that inactivates MAPK-mediated inflammatory pathways, was also measured by rt-PCR and western blotting. DUSP1-targeted small interfering RNA (siRNA) was used to examine the specific role of DUSP1. To evaluate the role of rolipram in vivo, an endotoxic mouse model was established by intraperitoneal injection of 15 mg/kg LPS, and 10 mg/kg rolipram was intraperitoneally injected 1 h before LPS injection. mRNA and protein levels of inflammatory cytokines and DUSP1 in heart, inflammatory cell infiltration and cardiac function were all examined at 6 h after LPS injection.ResultsThe results showed that LPS could increase the expression and secretion of inflammatory cytokines and decrease the transcription and expression of DUSP1 in cardiac fibroblasts. However, rolipram pretreatment significantly reversed the LPS-induced downregulation of DUSP1 and inhibited LPS-induced upregulation and secretion of TNF-α and IL-6 but not IL-1β. Moreover, DUSP1-targeted siRNA experiments indicated that the protective effect of rolipram on inflammatory response was specific dependent on DUSP1 expression. Moreover, rolipram could further reduce inflammatory cell infiltration scores as shown by pathological analysis and increase the ejection fraction (EF) detected with echocardiography in the hearts of endotoxic mice.ConclusionsRolipram could improve endotoxin-induced cardiac dysfunction by upregulating DUSP1 expression to inhibit the inflammatory response in cardiac fibroblasts, which may be a potential treatment for SIC.

Emergence of dominant toxigenic M1T1 Streptococcus pyogenes clone during increased scarlet fever activity in England: a population-based molecular epidemiological study

SummaryBackgroundSince 2014, England has seen increased scarlet fever activity unprecedented in modern times. In 2016, England's scarlet fever seasonal rise coincided with an unexpected elevation in invasive Streptococcus pyogenes infections. We describe the molecular epidemiological investigation of these events.MethodsWe analysed changes in S pyogenes emm genotypes, and notifications of scarlet fever and invasive disease in 2014–16 using regional (northwest London) and national (England and Wales) data. Genomes of 135 non-invasive and 552 invasive emm1 isolates from 2009–16 were analysed and compared with 2800 global emm1 sequences. Transcript and protein expression of streptococcal pyrogenic exotoxin A (SpeA; also known as scarlet fever or erythrogenic toxin A) in sequenced, non-invasive emm1 isolates was quantified by real-time PCR and western blot analyses.FindingsCoincident with national increases in scarlet fever and invasive disease notifications, emm1 S pyogenes upper respiratory tract isolates increased significantly in northwest London in the March to May period, from five (5%) of 96 isolates in 2014, to 28 (19%) of 147 isolates in 2015 (p=0·0021 vs 2014 values), to 47 (33%) of 144 in 2016 (p=0·0080 vs 2015 values). Similarly, invasive emm1 isolates collected nationally in the same period increased from 183 (31%) of 587 in 2015 to 267 (42%) of 637 in 2016 (p<0·0001). Sequences of emm1 isolates from 2009–16 showed emergence of a new emm1 lineage (designated M1UK)—with overlap of pharyngitis, scarlet fever, and invasive M1UK strains—which could be genotypically distinguished from pandemic emm1 isolates (M1global) by 27 single-nucleotide polymorphisms. Median SpeA protein concentration in supernatant was nine-times higher among M1UK isolates (190·2 ng/mL [IQR 168·9–200·4]; n=10) than M1global isolates (20·9 ng/mL [0·0–27·3]; n=10; p<0·0001). M1UK expanded nationally to represent 252 (84%) of all 299 emm1 genomes in 2016. Phylogenetic analysis of published datasets identified single M1UK isolates in Denmark and the USA.InterpretationA dominant new emm1 S pyogenes lineage characterised by increased SpeA production has emerged during increased S pyogenes activity in England. The expanded reservoir of M1UK and recognised invasive potential of emm1 S pyogenes provide plausible explanation for the increased incidence of invasive disease, and rationale for global surveillance.FundingUK Medical Research Council, UK National Institute for Health Research, Wellcome Trust, Rosetrees Trust, Stoneygate Trust.

Exploring in vitro gastric digestion of whey protein by time-domain nuclear magnetic resonance and magnetic resonance imaging

Gastric digestion is crucial for protein breakdown. Although it has been widely studied with in vitro models, verification in vivo remains a big challenge. Magnetic resonance imaging (MRI) has the potential to bridge this gap. Our objective was to use the transverse relaxation time (T2) and rate (R2 = T2−1) to monitor hydrolysis of protein-rich food during in vitro gastric digestion. Whey protein solution and heat-induced hydrogels were digested by means of simulated gastric fluid (SGF). Free amino groups (–NH2 groups) and protein concentration in the supernatant were measured. T2 and R2 of the digestion mixture were determined by time-domain nuclear magnetic resonance (TD-NMR) and MRI. Subsequently, relative amplitudes (TD-NMR) for different T2 values and T2 distribution (MRI) were determined. For the solution, protein concentration and T2 did not change during digestion. For the gels, water in supernatant and gel phase could be discriminated on the basis of their T2 values. During digestion, R2 of supernatant correlated positively with protein (–NH2 groups) concentration in SGF. Also, the decrease in relative amplitude of gel fraction correlated linearly with the increase of supernatant protein concentration. MRI T2-mapping showed similar associations between R2 of supernatant and protein (–NH2 groups) concentration. In conclusion, T2-measurements by TD-NMR and MRI can be used to monitor in vitro gastric digestion of whey protein gels; TD-NMR measurements contributed to interpreting the MRI data. Thus, MRI has high potential for monitoring in vivo gastric digestion and this should be further pursued.

Effect of polysaccharide concentration and charge density on acid-induced soy protein isolate-polysaccharide gels using HCl

Mixed biopolymer gels of soy protein isolate with κ-carrageenan, λ-carrageenan and alginate were produced via acidification with simulated gastric fluid (HCl) at pH 1.2. The gels could be formed instantaneously via dripping into the acid, or over 24 h via a dialysis membrane. The main factors studied were charge density of the polysaccharide and polysaccharide concentration (0.25 − 1.0% w/w). Their effects on the physical properties of the gel i.e. gel mass formed, protein concentration in supernatant, hardness, storage modulus (G′), water holding capacity (WHC) and microstructure were evaluated. Overall, with higher charge density of the polysaccharide (at pH 1.2) and/or increasing polysaccharide concentration, the gel formed more rapidly and had greater mechanical strength due to a denser network between the biopolymers. As a result, WHC was also increased with improved freeze-thaw stability as observed under confocal microscopy.

Transplantation of vascular endothelial growth factor 165‑transfected endothelial progenitor cells for the treatment of limb ischemia.

The present study aimed to investigate the effects of neovascularization in rabbits with limb ischemia transplanted with vascular endothelial growth factor (VEGF)165-transfected endothelial progenitor cells (EPC). Bone marrow mononuclear cells were isolated by gradient centrifugation, cultured in M199 culture medium and induced into EPCs using VEGF, basic fibroblast growth factor, and insulin-like growth factor-1, and subsequently identified. The EPCs were transfected with Adv-green fluorescent protein-VEGF165 and the proliferation potential of the cells was determined using an MTT assay. The protein expression levels of VEGF were measured by detecting its concentration levels in the supernatant using an ABC-ELISA assay. A rabbit hind limb ischemic model was established and randomly divided into three groups: (A) Control group, (B) EPC-transplanted group, and (C) Ad-VEGF165/EPCs-transplanted group. The effects of transplantation and the levels of recanalization were detected. Incorporation of the transplanted cells into the ischemic region was confirmed by 5-bromodeoxyuridine staining, and the levels of recanalization were measured by computer tomography ateriography and immunohistochemical staining. Bone marrow-derived EPCs were induced, cultivated, and successfully identified. The results of the present study determined the optimum transfection ratio that promoted the growth of EPCs. The EPCs were successfully transfected with VEGF165, and EPC proliferation was not affected by the transfection. The supernatant protein concentration levels of VEGF were markedly higher in the VEGF165-transfected group, as compared with those of the control group. Introduction of the transplanted cells into the ischemic region of group C occurred more efficiently, as compared with groups A and B. The recanalization capillary density in group C was significantly higher, as compared with groups A and B. VEGF gene transfection was able to improve the quality of EPCs, and the response of rabbits with limb ischemia to transplantation with VEGF-transfected EPCs was significantly better, as compared with transplantation with EPCs alone.

Production of Fungal Amylases Using Cheap, Readily Available Agriresidues, for Potential Application in Textile Industry

The study aimed at isolation and screening of fungal amylase producer, optimization of solid state fermentation conditions for maximum amylase production by the best amylase producer, and characterization of the crude amylases, so produced. Aspergillus fumigatus NTCC1222 showed the highest amylase activity (164.1 U/mL) in secondary screening under SSF conditions and was selected for further studies. The test strain showed maximum amylase production (341.7 U/mL) and supernatant protein concentration (9.7 mg/mL) for incubation period (6 days), temperature (35°C), initial pH (6.0), nutrient salt solution as moistening agent, and beef extract as nitrogen source. Pomegranate peel produced maximum amylase activity, but wheat bran (only slightly lesser amylase activity as compared to that of pomegranate peel) was chosen for further studies, keeping in mind the seasonal availability of pomegranate peel. TLC confirmed the amylase produced to be α-type and 60 kDa was the molecular weight of the partially purified amylase. The enzyme showed maximum enzyme activity at pH 6.0, temperature of 55°C, and incubation time of 60 minutes. UV (616.0 U/mL) and chemical (814.2 U/mL) mutation enhanced amylase activity as compared to wild test strain. The study indicates that Aspergillus fumigatus NTCC1222 can be an important source of amylase and the crude enzyme, hence obtained, can be cost effectively applied in multiple sections of textile wet processing.

Influence of Incubation Conditions on Hydrolysis Efficiency and Iodine Enrichment in Baker’s Yeast

The influence of incubation conditions, enzyme type, hydrolysis time, and potassium iodide concentration on hydrolysis and iodine enrichment were studied in supernatant and pellets of Saccharomyces cervisiae hydrolysates. The type of enzyme used and incubation time significantly influence hydrolysis efficiency and protein concentration in supernatant and pellet. The highest protein hydrolysis efficiency was obtained by 24-h incubation with papain. Significantly lower values were observed for pepsin and autolysis. The potassium iodide concentration influences the iodine content of supernatant and pellet, but not hydrolysis. Iodide enrichment of supernatant and pellet depends on the concentration of iodide using during incubation. High concentration of iodide and long incubation times were the conditions for optimal iodide enrichment and high-protein hydrolysates. The optimal hydrolysis efficiency and iodine enrichment were obtained during 24-h incubation with papain in a 4.5-mM potassium iodide medium. The efficiency reached 98.22% with iodine concentrations of 2,664.91 and 9,200.67 μg/g iodine in pellet and supernatant, respectively.

E93R Substitution of Escherichia coli FtsZ Induces Bundling of Protofilaments, Reduces GTPase Activity, and Impairs Bacterial Cytokinesis

Recently, we found that divalent calcium has no detectable effect on the assembly of Mycobacterium tuberculosis FtsZ (MtbFtsZ), whereas it strongly promoted the assembly of Escherichia coli FtsZ (EcFtsZ). While looking for potential calcium binding residues in EcFtsZ, we found a mutation (E93R) that strongly promoted the assembly of EcFtsZ. The mutation increased the stability and bundling of the FtsZ protofilaments and produced a dominating effect on the assembly of the wild type FtsZ (WT-FtsZ). Although E93R-FtsZ was found to bind to GTP similarly to the WT-FtsZ, it displayed lower GTPase activity than the WT-FtsZ. E93R-FtsZ complemented for its wild type counterpart as observed by a complementation test using JKD7-1/pKD3 cells. However, the bacterial cells became elongated upon overexpression of the mutant allele. We modeled the structure of E93R-FtsZ using the structures of MtbFtsZ/Methanococcus jannaschi FtsZ (MjFtsZ) dimers as templates. The MtbFtsZ-based structure suggests that the Arg(93)-Glu(138) salt bridge provides the additional stability, whereas the effect of mutation appears to be indirect (allosteric) if the EcFtsZ dimer is similar to that of MjFtsZ. The data presented in this study suggest that an increase in the stability of the FtsZ protofilaments is detrimental for the bacterial cytokinesis.

GEMCITABINE ENHANCES THE EFFECT OF ADENOVIRUS-MEDIATED GENE THERAPY

Backgrounds and Aims: Adenovirus-mediated gene therapy is a promising treatment for cancers, but its clinical efficacy is still low. So, more improved gene expression strategy is needed for gene therapy. Meanwhile, genotoxic stresses, such as those induced by chemotherapy, are known to enhance transgene expressions. Therefore, we investigated the effects of gemcitabine(GEM), now the first-line agent for treatment of pancreatic cancer, on the transgene expression of adenovirus vector. Methods: NK4 is an HGF-antagonist and inhibits invasion of cancer cells. In this study, we used Ad-NK4, which expresses NK4 under the control of CMV promoter. SUIT-2 cells were infected with Ad-NK4 and then treated with or without GEM for 24 hrs, and investigated the effects of GEM on Ad-NK4 by invasion assay. Results: The invasion of cancer cells was inhibited in a GEM-dose dependent manner (GEM 0, 1, 10, 100nM: 23, 27, 55, 79% inhibition on day3). Both of NK4 protein concentrations in supernatant of Ad-NK4 infected cells and of pcDNA transfected cells were increased in GEM-treated groups, and in vivo, NK4 expressions within subcutaneously implanted tumors in mice were increased in GEM-treated groups. Conclusion: These results indicate that GEM enhances the effects Ad-NK4 through GEM-enhanced CMV promoter. Combination of Ad-NK4 with GEM might be a new strategy for pancreatic cancer treatment.

The effect of Sargassum fusiforme polysaccharide extracts on vibriosis resistance and immune activity of the shrimp, Fenneropenaeus chinensis

Immunostimulants are valuable for control of shrimp diseases and the immunostimulatory effects of some polysaccharide additives for shrimp have been reported. In this study, the Sargassum fusiforme polysaccharide extract (SFPSE) was assessed as a feed additive when supplemented in the diet (0%, 0.5%, 1.0%, and 2.0%) for juvenile shrimp, Fenneropenaeus chinensis, in order to study the effects of SFPSE on vibriosis resistance and immune activity. Shrimp were cultured in the same pond with cages. The body weight, survival, the cumulative mortality after injection with Vibrio harveyi (30 μl V. harveyi suspension at 9.3 × 10 7 CFU ml −1 per shrimp), the total haemocyte counts (THCs), the protein concentration and the phenoloxidase (PO) activity in supernatant of haemolymph, the lysozyme (LSZ) and superoxide dismutase (SOD) activity in muscle of the shrimp were assayed after 14 days feeding period. The results indicated that shrimp survival under the stress of V. harveyi was affected by the dietary SFPSE. The shrimp treated with 1.0% and 0.5% SFPSE displayed significantly lower cumulative mortalities after being injected with V. harveyi suspension 24 and 30 h later, respectively, compared with that of the control. However, cumulative mortality of 2.0% SFPSE treatment was not significantly different from that of the control. There was no significant difference of cumulative mortality between 0.5% and 1.0% SFPSE treatment groups. The immune activities of the shrimp also were affected by dosage of dietary SFPSE. The THCs of the shrimp rose with increasing SFPSE dosage. The protein concentration and PO activity in supernatant of haemolymph as well as muscular LSZ activity first rose then dropped with increasing SFPSE dosage. The protein concentration in supernatant of haemolymph appeared a maximum of 167.46 mg ml −1 in 1.0% SFPSE treatment. The PO activity and LSZ activity reached the peaks as 13.20 U and 3.21 U mgprot −1 in 0.5% SFPSE treatment, respectively. SOD activity of the shrimp was not significantly affected by dietary SFPSE. It is therefore suggested that oral administration of SFPSE at an optimal level of 0.5% and 1.0% for 14 days effectively improved vibriosis resistance and enhanced immune activity of the shrimp in general.

Immediate and delayed effects of lead on AChE, GSH‐T and thiols in the substantia nigra, neostriatum and cortex of the rat brain

We studied the effects, at 10 and 30 min, of a single dose (10 mg kg(-1)) of lead chloride, administered by the intraperitoneal route, on the activities of acetylcholinesterase (AChE) and glutathione transferase (GSH-T) and on the concentrations of total and non-protein thiols in substantia nigra compacta (SNCO) and substantia nigra reticulata (SNRE), caudate putamen (CAU) and cerebral cortex (CC) from adult male rats in comparison with the effects of this metal at 24 and 72 h. The main immediate effects of lead consisted of decreased GSH-T activity and total and non-protein thiol concentrations in CAU and CC 10 min after administration. These effects were reversed after 30 min but with increased GSH-T activity in SNCO and AChE activity in SNRE along with diminished concentration of homogenate proteins in SNRE, CAU and CC. The GSH-T activity again was increased in SNCO but the AChE activity was decreased in CC 24 h after Pb administration; total and non-protein thiol concentrations were diminished but homogenate protein concentration was augmented in all areas. Finally, 72 h after Pb administration, AChE and GSH-T activities were decreased in CAU and CC, accompanied by an increased concentration of precipitate and supernatant proteins; supernatant protein concentration also was augmented in SNCO and SNRE; here, again, the concentrations of total and non-protein thiols were diminished and the homogenate protein concentration was augmented in all areas.

Some characteristics of protein precipitation by salts

The solubilities of lysozyme, alpha-chymotrypsin and bovine serum albumin (BSA) were studied in aqueous electrolyte solution as a function of ionic strength, pH, the chemical nature of salt, and initial protein concentration. Compositions were measured for both the supernatant phase and the precipitate phase at 25 degrees C. Salts studied were sodium chloride, sodium sulfate, and sodium phosphate. For lysozyme, protein concentrations in supernatant and precipitate phases are independent of the initial protein concentration; solubility can be represented by the Cohn salting-out equation. Lysozyme has a minimum solubility around pH 10, close to its isoelectric point (pH 10.5). The effectiveness of the three salts studied for precipitation were in the sequence sulfate > phosphate > chloride, consistent with the Hofmeister series. However, for alpha-chymotrypsin and BSA, initial protein concentration affects the apparent equillibrium solubility. For these proteins, experimental results show that the compositions of the precipitate phase are also affected by the initial protein concentration. We define a distribution coefficient kappa(e) to represent the equilibrium ratio of the protein concentration in the supernatant phase to that in the precipitate phase. When the salt concentration is constant, the results show that, for lysozyme, the protein concentrations in both phases are independent of the initial protein concentrations, and thus kappa(e) is a constant. For alpha-chymotrypsin and BSA, their concentrations in both phases are nearly proportional to the initial protein concentrations, and therefore, for each protein, at constant salt concentration, the distribution coefficient kappa(e) is independent of the initial protein concentration. However, for both lysozyme and alpha-chymotrypsin, the distribution coefficient falls with increasing salt concentration. These results indicate that care must be used in the definition of solubility. Solubility is appropriate when the precipitate phase is pure, but when it is not, the distribution coefficient better describes the phase behavior.

Dihydropyrimidine Dehydrogenase Activity in Human Blood Mononuclear Cells

Dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) catalyzes the rate-limiting reaction in the catabolism of endogenous uracil and thymine and exogenous fluoropyrimidines. DPD activity was studied in human blood mononuclear cell supernatants utilizing a new and sensitive radiochromatographic assay. Total DPD activity showed a linear correlation with supernatant protein concentration. The affinity constants (Km) for NADPH and thymine were approximately 10 and 1 mumol/l, respectively. Maximal activity (Vmax) was observed at 0.25 mmol/l NADPH and 10 mumol/l thymine, respectively. DPD activity in normal individuals was 8.0 +/- (SD) 2.2 nmol/mg protein/h, and ranged from 4.4 to 12.3 nmol/mg/h (n = 25). This activity range was quite similar to values obtained in patients with metastatic solid tumors treated with fluorodeoxyuridine (FUdR; n = 33, p = 0.57). No correlation was found to exist between mononuclear leucocyte DPD activity and the observed toxicity of FUdR in the tested patients. A bimodal distribution of DPD activity was observed in the patients and in normal individuals. The entire study population tested could be divided into two groups with respect to DPD activity; one group with high (greater than 8 nmol/mg/h) activity and another with low (less than 8 nmol/mg/h) activity. The possibility that sex differences may have been responsible for this distribution of DPD activity could not be excluded. The findings of this study are relevant to the pharmacogenetics of fluoropyrimidines in humans.

Elaboration of Bacillus anthracis antigens in a new, defined culture medium.

Improved culture conditions and a new, completely synthetic medium (R medium) were developed to facilitate the production of Bacillus anthracis holotoxin antigens. Levels of these antigens up to fivefold greater than the highest previously reported values were recovered with the described system. Cultures of Sterne, V770-NP1-R, and Vollum 1B strains of B. anthracis were monitored for growth, pH change, glucose utilization, supernatant protein concentration, lethal toxin activity, and protease activity.

Intestinal calcium-binding protein in the developing rat duodenum.

Summary: An immunoassay for rat intestinal calcium-binding protein (CaBP) employing antibody raised in chickens using purified rat intestinal CaBP has been used to measure immunoreactive CaBP in the duodenum of suckling and weanling rats. A 5-fold increase in immunoreactivity, associated with a 2 ½-fold increase in calcium-binding activity occurred at weaning, a period during which duodenal mucosal mass increased 2-fold and supernatant protein concentration increased 1 ½-fold. Speculation: The ontogeny of calcium-binding protein in the duodenum of young rats parallels the maturation of cholecalciferol hydroxylases and represents maturation of the intestine as an end organ responsive to the vitamin D endocrine system.