Bovine Serum Albumin Group Research Articles

347 EFFECTS OF NEWBORN CANINE SERUM ON IN VITRO MATURATION OF CANINE OOCYTES

Research on in vitro maturation (IVM) of canine oocytes has been scant, due to the fact that canine oocytes are at an immature stage (geminal vesicle stage) during ovulation. The most commonly used protein source for IVM of canine oocytes is fetal bovine serum (FBS), estrus bitch serum (EBS), or bovine serum albumin (BSA). However, the IVM rate of canine oocytes is still low. One of the reasons may be that the culture medium is not appropriate enough for canine oocytes in IVM. Therefore, we evaluated the effects of four different protein sources, BSA, FBS, EBS, and newborn canine serum (NBCS), on IVM of canine oocytes. The NBCS was collected from 3 puppies that had induced euthanasia within 30 min after delivery. The entire volume of blood was collected by heart puncture, through which an 18 G needle was attached to a 10-mL sterile plastic syringe (without anticoagulant), and then transferred into a 10-mL plastic conical tube. The samples were stored overnight at 4�C to allow clotting to occur. To separate the serum, the sample was centrifuged for 10 min at 3000 rpm; the NBCS was heat inactivated at 56�C for 30 min and stored at -70�C for later use. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and the oocytes collected were cultured in NCSU37 supplemented with 1 mg mL-1 cysteine, 0.2 mM pyruvic acid, 20 �g mL-1 E2, and various protein sources (0.3% BSA, 10% FBS, 10% EBS, and 10% NBCS) at 38.5�C, 5% CO2 in air for 72 h. Cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were then fixed in 4% paraformaldehyde supplemented with 10 �g mL-1 Hoechst 33342 at room temperature for 40 min. Nuclear status was observed using fluorescence under a UV light. The proportion of oocytes at the MII stage was significantly higher in the NBCS group than in the EBS, FBS, and BSA groups (14.6 vs. 10.4, 8.8, and 3.3%; P < 0.05). These results indicate that the supplementation of NBCS in IVM medium can enhanced the development of canine oocytes to the MII stage. This work was supported by KOSEF (Grant M10525010003-05N2501-00310) and ARPC (Grant 203121-03-2-SB010).

Modulation of renal kallikrein by a high potassium diet in rats with intense proteinuria

Injury of the renal tubulointerstitial compartment is recognized to play an important role in hypertension. Its damage may in turn, impair the activity of vasodepressor systems, like the kallikrein-kinin, in blood pressure regulation. The overload proteinuria model induces tubulointerstitial injury with activation of the renin-angiotensin system, but renal kallikrein and the development of hypertension have not received special attention. Sprague-Dawley rats received seven intraperitoneal doses of bovine serum albumin (BSA) 2 g/day under normosodic diet and were hydrated ad libitum. A second group received a high potassium diet to stimulate kallikrein production during the previous four weeks and while under BSA administration. A third one received potassium and BSA in the same schedule, but with the kinin B2 receptor antagonist, HOE140, added during the protein load phase. A control group received seven saline injections. Kallikrein protein was detected by immune labeling on renal sections and enzymatic activity in the urine. The BSA group showed massive proteinuria followed by intense tubulointerstitial damage. Blood pressure increased after the third dose in BSA animals, remaining elevated throughout the experiment, associated with significant reductions in renal expression and urinary activity of kallikrein, compared with controls. An inverse correlation was found between blood pressure and immunohistochemistry and urinary activity of kallikrein. Potassium induced a significant increase in both urinary activity and renal kallikrein expression, associated with significant reduction in blood pressure. The HOE140 antagonist blunted the antihypertensive effect of kallikrein stimulation in proteinuric rats. Loss of renal kallikrein, produced by tubulointerstitial injury, may participate in the pathogenesis of the hypertension observed in this model.

Growth at high pH increases Enterococcus faecalis adhesion to collagen

To evaluate the effect of growth at pH levels from 7.1 to 9.5 on the adherence of Enterococcus faecalis to bovine serum albumin (BSA) and collagen type I. Enterococcus faecalis strain A197A was grown in broth of adjusted pHs varying between 7.1 and 9.5. Aliquots of bacterial suspensions were added to wells coated either with BSA or with collagen type I. Bacteria adhering to the surfaces were stained with crystal violet. Spectrophotometric measurements of the dissolved stain were used to assess the number of bacteria adhering to the surfaces. The data obtained were analysed using the Kolmogorov-Smirnov test, Levene's test and Student's t-test, with alpha = 0.05 as the level for statistical significance. The adhesion of E. faecalis to BSA-coated surfaces decreased inversely with alkalinity of the growth medium. The pH 7.1-grown bacteria bound to BSA significantly more than the other BSA groups. On the contrary, the adhesion to collagen type I-coated surfaces of bacteria grown at pH 8.0 and 8.5 was significantly greater than for those grown at pH 7.1. A minor increase in pH up to 8.5, which may be a consequence of insufficient treatment with alkaline medicaments such as calcium hydroxide, increases the collagen-binding ability of E. faecalis, in vitro. This can be a critical mechanism by which E. faecalis predominates in persistent endodontic infections.

Exogenous Bone Morphogenetic Protein-7 Fails to Attenuate Renal Fibrosis in Rats with Overload Proteinuria

Background: Bone morphogenetic protein-7 (BMP-7) plays a critical role in renal development, accelerates recovery from acute renal injury, and more recently it has been shown to delay progressive renal disease. The present study was designed to investigate the effect of BMP-7 on interstitial fibrosis in the rat protein-overloaded model. Methods: Renal disease was induced in 26 rats by daily intraperitoneal injections of bovine serum albumin (BSA); controls (n = 28) were injected with saline. Half of the rats in each group were treated with human recombinant BMP-7 (300 µg/kg i.p. 3 times weekly) and half with placebo. Animals were killed after 3 or 6 weeks. Results: Compared to the saline control groups, the BSA groups had evidence of chronic renal disease: significantly increased urinary protein excretion rates; total kidney collagen content, and increased fibronectin and collagen III interstitial areas. By 6 weeks the BSA + BMP-7 group compared to the BSA + placebo group had a nonsignificant decrease in blood urea nitrogen (40 ± 13 vs. 46 ± 11 mg/dl), total kidney collagen (10.8 ± 2.1 vs. 12.2 ± 3.5 µg/kidney), fibronectin interstitial area (23 ± 4 vs. 25 ± 8%) and collagen III interstitial area (22 ± 6 vs. 28 ± 7%). Despite these results, renal gene expression profiles actually predicted worse fibrosis in the BSA + BMP-7 group with significantly higher total kidney mRNA levels for α₁(III) procollagen (2.8 ± 0.5 vs. 1.6 ± 0.6, p < 0.05) and fibronectin at 6 weeks (1.9 ± 0.3 vs. 1.2 ± 0.5, p < 0.05). Renal BMP-7 mRNA levels at 6 weeks were significantly increased in the BSA + placebo group compared to the saline + placebo group with no difference between the BSA + BMP-7 and the BSA + placebo groups. Both cortical and medullary tubules expressed BMP-7 protein but BMP-7 was only detected in the tubular lumina and urine of proteinuric animals. Conclusions: In rats with protein-overload proteinuria, renal tubules continue to express BMP-7 but some of the endogenous protein is secreted into the urinary space. Administration of exogenous recombinant BMP-7 had no effect on proteinuria but was associated with a nonsignificant trend towards less interstitial fibrosis at 6 weeks despite significantly higher kidney extracellular matrix gene mRNA levels. These findings suggest that BMP-7 treatment may have anti-fibrotic effects through enhancement of matrix turnover, although overall these effects are modest in proteinuric states in the absence of significant tubular epithelial cell apoptosis and epithelial-mesenchymal transition.

Injectable Fibrin Scaffold Improves Cell Transplant Survival, Reduces Infarct Expansion, and Induces Neovasculature Formation in Ischemic Myocardium

In this study, we determined whether fibrin glue improves cell transplant retention and survival, reduces infarct expansion, and induces neovasculature formation. Current efforts in restoring the myocardium after myocardial infarction (MI) include the delivery of viable cells to replace necrotic cardiomyocytes. Cellular transplantation techniques are, however, limited by transplanted cell retention and survival within the ischemic tissue. The left coronary artery of rats was occluded for 17 min followed by reperfusion. One week later, bovine serum albumin (BSA), fibrin glue, skeletal myoblasts in BSA, or skeletal myoblasts in fibrin glue were injected into the infarcted area of the left ventricle. The animals were euthanized five weeks after injection, and their hearts were excised, fresh frozen, and sectioned for histology and immunohistochemistry. After five weeks, the mean area covered by skeletal myoblasts in fibrin glue was significantly greater than the area covered by myoblasts injected in BSA. Myoblasts within the infarct were often concentrated around arterioles. The infarct scar size and myoblasts in the fibrin group were significantly smaller than those in the control and BSA groups. Fibrin glue also significantly increased the arteriole density in the infarct scar as compared with the control group. This study indicates that fibrin glue increases cell transplant survival, decreases infarct size, and increases blood flow to ischemic myocardium. Therefore, fibrin glue may have potential as a biomaterial scaffold to improve cellular cardiomyoplasty treat and MIs.

BSA Immobilization on Amine-Functionalized Superparamagnetic Iron Oxide Nanoparticles

Immobilization of bovine serum albumin (BSA) on surface-modified superparamagnetic iron oxide nanoparticles (SPION) has been performed by two different double-step immobilization approaches. The first approach consists of preparation of SPION by controlled chemical coprecipitation in the presence of BSA solution, whereas the second approach includes preliminary surface modification of SPION with an amine group using a coupling agent of 3-aminepropyltrimethoxysilane (APTMS). Both procedures are followed by 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide hydrochloride (EDC) activation with sequential immobilization of the layer of BSA. Additionally, an attempt to modify the surface of SPION with amine and carboxylic groups is undertaken by using l-aspartic acid (LAA). TEM shows that the particle size varies in the range 10−15 nm and does not change significantly after the coating process. The presence of BSA and amine groups on the surface of SPION is confirmed by FT-IR. Magnetic properties are investigated by VSM and results indicate that the superparamagnetic properties are retained for BSA-coated SPION while reducing the value of saturation magnetization (Ms). The binding capacity is estimated from thermogravimetric and chemical analyses. APTMS-coated SPION show higher BSA binding capacity compared to that of coprecipitated SPION in the presence of BSA. In vitro tests have been performed after the functionalization of SPION with LAA and BSA. Human dermal fibroblasts are incubated with the surface-modified SPION for 6 and 24 h to observe cell behavior, morphology, cytoskeletal organization, and interactions between cell and SPION. BSA-coated SPION incubated with cells demonstrated a cell response similar to that of control cells, with no adverse cell damage and no endocytosis, whereas LAA-coated SPION show partial endocytosis without cytoskeletal disorganization.

Fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction.

Current efforts in cardiac tissue engineering center around the use of scaffolds that deliver cells to the epicardial surface. In this study, we examined the effects of fibrin glue as an injectable scaffold and wall support in ischemic myocardium. The left coronary artery of rats was occluded for 17 min, followed by reperfusion. Echocardiography was performed 8 days after infarction. One to 2 days later, either 0.5% bovine serum albumin (BSA) in phosphate-buffered saline, fibrin glue alone, skeletal myoblasts alone, or skeletal myoblasts in fibrin glue were injected into the ischemic left ventricle. Echocardiography was again performed 5 weeks after injection. The animals were then sacrificed and the hearts were fresh frozen and sectioned for histology and immunohistochemistry. Both the fractional shortening (FS) and infarct wall thickness of the BSA group decreased significantly after 5 weeks (p = 0.0005 and 0.02, respectively). In contrast, both measurements for the fibrin glue group, cells group, and cells in fibrin glue group did not change significantly (FS: p = 0.18, 0.89, and 0.19, respectively; wall thickness: p = 0.40, 0.44, 0.43, respectively). Fibrin glue is capable of preserving infarct wall thickness and cardiac function after a myocardial infarction in rats and may be useful as a biomaterial scaffold for myocardial cell transplantation.

Isolated rat kidney perfused with dextran and bovine serum albumin: A stable model for investigating renal drug handling

Introduction: The rat isolated perfused kidney (IPK) preparation is a very useful model for pharmacokinetic and pharmacologic studies. Bovine serum albumin (BSA) is the oncotic agent used most commonly in IPK models, but the protein is expensive and varies significantly in quality. The present study evaluated the use of dextran to replace a large proportion of BSA as the oncotic agent, to establish a more reliable and economic IPK model for pharmacokinetic studies. Methods: The right kidneys of male Sprague–Dawley rats were isolated and perfused in recirculating mode with Krebs–Henseleit pH 7.4 buffer containing amino acids, glucose and 65 g/l BSA (BSA group, n=11) or 6.5 g/l BSA plus 36 g/l dextran (dextran/BSA group, n=6). 14C-Inulin was added to the perfusate to permit estimation of glomerular filtration rate (GFR). An antiviral guanosine analogue, AM188, was administered to the IPK perfusate to investigate its renal disposition. During the 130-min experimental period, urine was collected in 10-min intervals and perfusate was collected at the midpoint of these intervals. Results: The kidney functional parameters were generally better and more stable in the dextran/BSA IPKs when compared to the BSA group. At a similar perfusate flow rate, the IPKs in the dextran/BSA group exhibited lower renal artery perfusion pressure, a higher GFR, and more extensive tubular reabsorption of water, glucose, and sodium. These functional parameters were acceptable and stable throughout the whole experimental period in the dextran/BSA group. The renal clearance of AM188 was higher in the dextran/BSA group compared with that in the BSA group. Discussion: Using a large proportion of dextran and a small proportion of BSA as oncotic agent in perfusate provides an improved IPK preparation. This offers a reliable and economic rat IPK model for pharmacokinetic studies.

Recombinant human interleukin-11 prevents mucosal atrophy and bowel shortening in the defunctionalized intestine

Background: Mucosal atrophy and bowel shortening are the hallmark of proximal intestinal diversion for extensive necrotizing enterocolitis (NEC) or Thiry-Vella fistulas (TVF), in which the ends of a defunctionalized loop of intestine are exteriorized as stomas. Recombinant human interleukin-11 (rhIL-11) is a pleiotropic cytokine that promotes epithelial regeneration and enhances adaptation after bowel resection. The authors hypothesized that rhIL-11 may prevent mucosal atrophy and bowel shortening in rats with TVF. Methods: After creation of ileal TVF, Sprague-Dawley rats were selected randomly to receive either rhIL-11 or equal volume of 0.1% bovine serum albumin (BSA) subcutaneously daily. On day 14, the TVF were excised and examined morphologically. Enterocyte apoptosis was measured using the TUNEL assay. Mucosal DNA and protein content were measured. Results: Administration of rhIL-11 resulted in a significantly greater weight gain and less shortening of TVF than BSA treatment. TVF from the rhIL-11-treated group showed evidence of hyperplasia and hypertrophy and increased crypt to villus ratio. The BSA group had substantial mucosal atrophy. There was a qualitative decrease in the incidence of apoptosis in the rhIL-11 group. Conclusions: Recombinant human IL-11 prevents mucosal atrophy and shortening of defunctionalized intestinal loops. It may help reduce the incidence of short gut syndrome in infants with extensive NEC. J Pediatr Surg 35:1079-1083. Copyright © 2000 by W.B. Saunders Company.

Effects of physiologic albumin and hespan levels on hepatocytes in vitro.

Although albumin and hydroxyethyl starch (HES) are routinely used in critically ill, hypoalbuminemic patients, no studies have tested the effect of supplemental albumin and HES on hepatocyte function. In this study, the effects of these agents were evaluated by using stable, rat hepatocyte cultures in a collagen sandwich configuration. Hepatocyte synthesis of albumin, urea, and intracellular triglycerides was monitored in Dulbecco's modified Eagle medium (supplemented with fetal bovine serum, hydrocortisone, L-proline, gentamycin, and insulin) without supplemental colloid (control cultures) and with supplemental 2% bovine serum albumin (BSA), 4% BSA, 2% HES, or 4% HES. The albumin secretion in control cultures rose from 31.03 microg/day per 10(6) cells on day 3 to 154.17 microg/day per 10(6) cells by day 12 and remained constant. In contrast, the level of albumin synthesis in the 2% and 4% BSA groups rose from significantly higher initial values (p < 0.05) of 71.25 microg/day per 10(6) cells and 73.27 microg/day per 10(6) cells, respectively, to 127.61 microg/day per 10(6) cells and 107.95 microg/day per 10(6) cells by day 7, then declined rapidly to 58.98 microg/day per 10(6) cells and 41.28 microg/day per 10(6) cells by day 12 when cell disruption was present. HES also reduced albumin synthesis. The urea genesis in the control groups and in the treatment groups was found to be comparable throughout the study. The BSA supplemented groups accumulated large amounts of intracellular lipid droplets during the experiment. The intracellular triglycerides analysis found the 4% BSA group to be significantly (p < 0.05) higher than the 4% HES. BSA, added to a collagen sandwich hepatocyte preparation, causes reduced hepatocyte synthesis by day 8, probably a result of intracellular triglyceride accumulation, whereas HES reduces synthesis through unidentified mechanisms.

Immunization of Low-density Lipoprotein Receptor Deficient (LDL-RD) Mice with Heat Shock Protein 65 (HSP-65) Promotes Early Atherosclerosis

Heat shock proteins are a family of approximately 25 highly conserved proteins upregulated in response to various forms of stress. They play an active role in the development autoimmune diseases in animals, and have been incriminated in human autoimmune diseases (i.e. rheumatoid arthritis, multiple sclerosis). It has been previously shown, that an induced immune response against Heat shock protein 65 (HSP-65) results in atherosclerotic lesions in normocholesterolemic rabbits. We have supported these findings showing that C57BL/6 mice immunized with HSP-65 and fed a high-fat diet develop enhanced fatty streaks. To create a model that will eliminate the need for exogenous supplementation of a high-fat diet, we have immunized LDL receptor deficient (LDL–RD) mice with HSP-65 or with heat-killed Mycobacterium tuberculosis (Mt). Seven groups of LDL-RD mice (n=10), were immunized subcutaneously with different concentrations of HSP-65, Mt or bovine serum albumin (BSA). All mice were fed a normal chow-diet for 3 months. The mice immunized with the higher doses of Mt developed significantly larger fatty streaks when compared with their BSA immunized littermates. The size of the lesions in the aortic sinus were: 31,562±5,994μm2in the 10μg Mt and 52,777±5,245μm2in the 100μg Mt vs. 11,500±3,750μm2in the BSA group (P<0.05). In the HSP-65-immunized mice, only the group injected with the highest dose (5μg, twice) developed significantly larger fatty streaks when compared with the BSA-immunized group (28,611±4,716μm2vs. 11,500±3,750μm2respectively, (P<0.05). The HSP-65-but not the Mt- or BSA-immunized mice developed high titers of anti HSP-65 antibodies, beginning 10 days after the immunization, which persisted until they were killed. Immunohistochemical staining showed CD3-positive lymphocytes in the aortic sinus of mice immunized with Mt or HSP-65, but not in the control group. Thus, we established a mouse model of HSP-65 immune mediated atherosclerosis devoid of high fat diet supplementation. This model will enable us to further study the role of the immune system in atherosclerosis, via HSP-65 and raise novel immunomodulatory therapeutic modalities.

Alpha-1-proteinase inhibitor prolongs small intestinal graft preservation and survival

Organ preservation solutions currently used for cold storage of human donor organs are less effective in preserving small intestinal grafts than other organ grafts. The maximal safe period of cold preservation for human small intestinal graft is only about 6 hours. The pathology of preserved and reperfused small intestinal grafts is characterized by mucosal autolysis and sloughing. The authors speculated that the preservation/reperfusion injury results from a proteinase/proteinase-inhibitor imbalance in the graft that favors tissue degradation. This study evaluates whether the addition of an alpha-1-proteinase inhibitor (alpha-1-PI) to the preservation solution can improve graft survival after small bowel transplantation. Forty small intestinal grafts (20 cm long) were harvested from Lewis rats. The grafts were divided randomly into three groups and were preserved in one of the following solutions: normal saline (NS) (n = 10), alpha-1-Pl (25 mg/mL; n = 20), or proteinase-free bovine serum albumin (BSA) (25 mg/mL; n = 10). After 12-hour cold storage in the respective solutions, the grafts were transplanted orthotopically into syngeneic recipients. Full-thickness graft biopsies were performed before and 1 hour after revascularization. The effectiveness of preservation was judged by graft histopathology and recipient survival. Histological studies showed that there was less muscosal autolysis and sloughing of the grafts in the alpha-1-Pl group than in the other two groups. All recipients in the NS and BSA groups died of graft failure within 7 days (NS: median, 4 days; range, 2 to 5 days; BSA: median, 6 days; range, 4 to 7). However, 60% (12 of 20) of the recipients in the alpha-1-Pl group survived more than 90 days (median, >90 days; range, 4 to >90 days; P < .005 v NS or BSA groups, log-rank method). These data suggest that the inclusion of alpha-1-Pl in the preservation solution may enhance graft integrity and improve the surgical outcome after small bowel transplantation.

Trophic effects of interleukin-11 in rats with experimental short bowel syndrome

Interleukin-11 (IL-11) is a multifunctional cytokine, derived from bone marrow stromal cells, that stimulates proliferation of stem/progenitor precursor cells in the small intestinal crypts and accelerates recovery of intestinal mucosa after cytoablative therapy. This study evaluates whether IL-11 can improve the function and structure of the small intestine and enhance adaptation in an experimental model of short bowel syndrome. After 90% small bowel resection, 32 Sprague-Dawley rats were divided randomly into eight experimental groups of four animals each. Four groups were treated with IL-11 (125 μg/kg twice daily, subcutaneously), and the four control groups were treated with a similar volume (0.1%) of bovine serum albumin (BSA). The animals were weighed daily and were killed on day 2, 4, 6, or 8; remnant small bowel was evaluated for villus height and crypt cell mitosis. The body weight of the animals that received IL-11 was significantly greater at the beginning of postoperative day 4 in comparison to that of the BSA groups (P < .01 during days 5 to 7). The rats that had IL-11 also had significantly greater villus height and crypt cell mitotic rates (P < .05). These observations suggest that IL-11 has a trophic effect on the small bowel during the adaptive phase that follows massive bowel resection and may be useful in the treatment of short bowel syndrome.

Scanning force microscopic studies of surface structure and protein adsorption behavior of organosilane monolayers

Alkyltrichlorosilanes, [2-(perfluorooctyl)ethyl]trichlorosilane (FOETS) and their mixed monolayers were polymerized on the water subphase and were subsequently immobilized onto the silicon wafer surface by covalent bonding. Atomic force microscopic (AFM) observation of the [n-octadecyltrichlorosilane(OTS)/FOETS] mixed monolayer revealed that the crystalline OTS formed circular domains of ∼1–2 μm in diameter that were surrounded by an amorphous FOETS matrix. Lateral force microscopic (LFM) and scanning viscoelasticity microscopic (SVM) observations of the (OTS/FOETS) mixed monolayer could also distinguish the difference in the physical and mechanical properties between domain and matrix. The (alkylsilane/FOETS) mixed monolayers with a shorter alkyl chain such as n-dodecyltrichlorosilane (DDTS) did not show the phase-separated structure maybe due to the lack in crystallinity of DDTS component. AFM observation of the [crystalline fatty acid (nonreactive)/FOETS (reactive)] mixed monolayer also revealed the phase-separated structure. The crystalline fatty acid domains in the mixed monolayer were extracted by solvent and then, the monolayer of FOETS with the holes of ∼1–2 μm in diameter, that is, the (Si/FOETS) monolayer was finally obtained. The Si phase of (Si/FOETS) was backfilled with the another organosilane by chemisorption. Then, the [(3-mercaptopropyl) trimethoxysilane (MTS)/FOETS] mixed monolayer was newly obtained by the chemisorption of MTS. The absorption of bovine serum albumin (BSA) onto mixed monolayers was studied by exposure of the mixed monolayers to a BSA solution. BSA was preferentially adsorbed onto the MTS part due to a specific reaction between disulfide bond (–S–S–) in BSA and SH groups in MTS. On the other hand, in the case of (OTS/FOETS) mixed monolayer, BSA adsorbed preferentially onto the FOETS matrix in order to minimize the interfacial free energy in the aqueous environment.

In vitro culture of preimplantation mouse embryos and day 12 limb-buds: Effects of serum and albumin

The effects of three different protein sources at different concentrations on the growth and development of preimplantation mouse embryos and day 12 mouse limb-buds in culture were studied. Mouse embryos and forelimb-buds were cultured with a range of concentrations (5.5 to 42%) of either donor bovine serum (DBS) or fetal bovine serum (FBS), or (0.2 to 0.8%) bovine serum albumin (BSA). After 48 h in culture, the rate of embryo development was significantly higher in 5.5% DBS than in all other groups ( P < 0.05). The embryo hatching rate was higher in 21% FBS, 42% FBS, and all DBS groups than in serum-free medium, and all BSA groups ( P < 0.05). Morphologic analysis of cultured limb-buds at 72 h revealed that total, paw, and cartilage area were greater ( P < 0.05) in the serum-free medium than in all other groups. Shape factor analysis suggested that 5.5% DBS was most beneficial to mouse limb-bud development. No differences were seen in DNA or protein content of limb-buds among groups. Results suggest that mouse forelimb-buds can be succesfully cultured in serum-free medium and that high concentrations of FBS and DBS may be detrimental for preimplantation embryo and/or limb-bud growth and development.

Chemistry of Succinimido Esters. III.

The chemical modification (N-acylation) of bovine serum albumin (BSA) by N-acyloxysuccinimides (1) (CN=28) was studied kinetically at 25°C (7.0≤pH ≤9.0). N-Acylation was competitive with the decomposition of (1) and the rate of which is expressed as V= (k1, d+k2 [BSA]) [(1), ] where K1, d is the first order rate constant for the decomposition of (1) and k2 is the second order rate constant for the N-acylation of BSA by (1).The reactivity of (1) with BSA was in the order of CN=2>CN=3>CN=4 C5H11COONa>C3H7COONa. This order corresponded tentatively to the fluorescence difference spectra of BSA by the addition of sodium carboxylate. These findings indicate hydrophobic interaction between BSA and alkyl group in (1) to possibly accelerate the N-acylation of BSA. This was supported by the chemical modification of BSA in a preparative scale. The rate ratio, k2/k1, d, maximized at about pH 8.0 and increased with increasing CN in (1) for CN≥4 and 10-3k2/k1, d larger than unity.From the above observations, (1) is concluded to be applicable as an N-acyl reagent, to the chemical modification of Lys residues in proteins.

701—The interaction between bovine serum albumin and molybdate ions

The interaction between molybdate ions and bovine serum albumin (BSA) has been studied employing transmittance measurements. In the acidic range, the stoichiometry of the combination between molybdate and protonated nitrogen groups of protein was found to be one-to-one. The pH-dependent binding result has been ascribed to the existence of anionic as well as cationic species of molybdenum(VI). The pH-metric titrations and ultraviolet absorption spectra of molybdenum(VI)-bovine serum albumin mixtures also supported the existence of polymerisation-depolymerisation of molybdenum as well as deprotonation and conformational changes in the protein molecule. The free energy, enthalpy and entropy of binding have been determined and the effect of molybdate ion on the heat of ionization and p K int. of BSA groups has' been discussed.

Lactate dehydrogenase isoenzymatic analysis of murine lymphocyte populations: A parameter of cellular differentiation

The lactate dehydrogenase isoenzyme pattern has been determined in different murine lymphocytic cell populations. In each cell population, the LDH activity was predominantly found in the LDH-4 and LDH-5 fractions. The percentage LDH-5 activity was significantly higher in B cells than in T cells. The same is true for lymphocytes from the spleen versus lymph node lymphocytes. The percentage LDH-5 activity is significantly higher in peripheral T lymphocytes than in thymocytes. Enrichment of the more mature thymocytes of the thymocyte cell pool by either cortisone treatment in vivo or gradient centrifugation on bovine serum albumin (BSA) results in a decrease of LDH-1 and LDH-2 fractions. In the cortisone-treated group, the shift in the LDH pattern is accompanied by a significant increase of LDH-5 and LDH-4 fractions, whereas in the BSA group only the LDH-4 fraction increases.