Bovine Serum Albumin In Medium Research Articles

The Effect of Centrifugation Time on Sexing Spermatozoa with Bovine Serum Albumin Media on the Morphology of X-Y Spermatozoa in Simmental Cattle

Sexing and centrifugation of spermatozoa is one way to improve the quality of spermatozoa. This study aimed to obtain the right centrifugation time for sexing spermatozoa with Bovine Serum Albumin media on the morphology of X-Y spermatozoa in Simmental cattle. This study was carried out from October 2021 to November 2021 at the Laboratory of Animal Reproduction and Health at the Breeding Center for Superior Livestock and Forage, Sembawa. The design used in this study was a completely randomized design (CRD) with 3 treatments and 4 replications. Centrifugation treatments for sexing spermatozoa with BSA media P1: centrifugation time 4 minutes, P2: centrifugation time 8 minutes, and P3: centrifugation time 12 minutes. The parameters observed were the morphology of the X and Y spermatozoa. The results showed that the centrifugation time for sexing spermatozoa—which was different with Bovine Serum Albumin (BSA) media—indicates that the X spermatozoa abnormalities had no significant effect (P>0.05) but the Y spermatozoa abnormalities had a significant effect (P<0.05). The result of the right time use was in the P1 treatment with a centrifugation time of 4 minutes where the abnormalities value in the lower layer Y was 28.79%.

In vitro effects of aqueous extract of unfermented rooibos on human spermatozoa.

The inability to conceive is a baleful experience for thousands of couples worldwide. Among different well‐known reproductive techniques, medicinal plants have been utilized to treat male infertility. Medicinal plants, provide a therapeutic alternative, which is available and affordable for infertile couples. We investigated the direct effect of unfermented rooibos aqueous extract on human spermatozoa. Semen samples (n = 50) collected from donors and patients consulting for fertility were reassigned as normal (n = 22) and abnormal (n = 28) samples based on the outcome of the baseline semen analysis, using the World Health Organization (WHO) cut off value. Semen samples were allowed to liquefy and subsequently washed with human tubular fluid in bovine serum albumin medium. The samples were then treated with aqueous extracts of unfermented rooibos (0, 0.15, 1.5, 15, 150 μg/ml) at 37°C for 1 h and assessed thereafter. Sperm motility, vitality, DNA fragmentation, intracellular reactive oxygen species and mitochondrial membrane potential in both groups remained unchanged (p > 0.05). However, aqueous extract of unfermented rooibos (only at 1.5 μg/ml) significantly increased capacitation and acrosome reaction in the abnormal sample group (p > 0.05). Unfermented rooibos aqueous extract had no deleterious impact on human spermatozoa's function and might be attributed to its antioxidant properties.

P–208 Oleic acid rescues altered autophagy induced by palmitic acid during mouse preimplantation development

Abstract Study question The aim of the study is to identify the autophagic profile and the effects of fatty acid treatments on autophagic activity in preimplantation mouse embryos. Summary answer Autophagic activity varies significantly in early stages of mouse preimplantation development; exposure to fatty acids alters the embryonic autophagy profile. What is known already Obesity is one of the top comorbidities for infertility, and obese individuals have elevated fatty acid levels. In serum, palmitic acid (PA) and oleic acid (OA) are the most abundant saturated and unsaturated fatty acids, respectively. We recently reported that PA impairs blastocyst development, affects mitochondrial reactive oxygen species, triacylglycerol levels, and endoplasmic reticulum stress pathways during mouse preimplantation development. Interestingly, the addition of OA counteracts those effects. Autophagy plays an essential role in embryo development, as knock-out of a key autophagy protein is embryonic lethal. Little is known about the autophagic profile in fatty acid treated mouse preimplantation embryos. Study design, size, duration Pools of 20 – 25 mouse embryos were collected from gonadotrophin super-ovulated and mated CD1 female mice. Two-cell stage embryos were treated with 100 µM PA and 250 µM OA, alone and in combination, and 1.5% bovine serum albumin media (control) within KSOMaa media for 18, 24, and 48 hours in vitro. The detection of various autophagic markers were evaluated by immunofluorescence microscopy and RT-qPCR. Participants/materials, setting, methods mRNA levels of autophagic markers were measured using RT-qPCR with the Taqman primers and Universal PCR Mix. Immunofluorescence staining of LC3 puncta (marker for autophagosome formation) was performed using LC3A/B polyclonal antibody (Invitrogen PA1–16931) and DAPI (4′,6-Diamidino–2-phenylindole dihydrochloride) was used to stain for cell nuclei. Analysis of LC3 puncta was performed using ImageJ software. Images were acquired using an LSM 800 laser scanning confocal microscope. Data analysis was completed by GraphPad Prism software. Main results and the role of chance Mouse preimplantation embryos showed no change in mRNA levels of autophagic markers (Bcln1, ATG3, ATG5, and LC3) relative to the control group after 48-hours exposure of 100 µM PA and 250 µM OA treatments, alone and in combination. The number of LC3 puncta was measured and analyzed as a reflection of autophagic activity in mouse preimplantation embryos. Under the fatty acid-free condition, the average number of LC3 puncta per blastomere was significantly decreased after 18 hours of development (p < 0.005). However, the average number of LC3 puncta per blastomere at 18, 24, and 48 hours were not significantly different from each other (p = 0.2724). Following 100 µM PA and 250 µM OA treatments, alone and in combination, autophagic activity was impacted by the presence of fatty acids. Mouse preimplantation embryos exposed to control and fatty acid treatment groups demonstrated no significant differences in LC3 puncta per blastomere at 18- and 24-hours treatment time (p = 0.5381; p = 0.7829). However, embryos exposed to 48 hours of PA treatment had a significantly greater number of LC3 puncta per blastomere than embryos exposed to 48 hours of OA and PA and OA combination treatments (p < 0.05). Limitations, reasons for caution Although LC3 puncta count (autophagosome formation) is impacted by fatty acid treatment, autophagic flux must be measured to fully investigate autophagic activity during mouse preimplantation development. These processes need to be measured in human embryos cultured in vitro. Wider implications of the findings: Profiling autophagic activity in fatty acid treated mouse preimplantation embryos would guide future investigations on pharmacological modulation of autophagy as a therapeutic intervention for developmentally delayed embryos. With the information gained, we aim to develop strategies to assist overweight and obese patients with their fertility needs. Trial registration number Not applicable

High detection of virulence factors by Candida species isolated from bloodstream of patients with candidemia

ObjectiveCandida species are the normal inhabitants of the skin and mucosa that cause a wide range of debilitating diseases in immunocompromised patients and other susceptible individuals. The present study aimed to evaluate the production of exoenzymes and the biofilm formation capacity of Candida species isolated from candidemia. Materials and methodsIn this study, a total of 100 stock Candida species isolates consist of 50 Candida albicans and 50 non-Candida albicans Candida species (24 C. glabrata, 15 C. parapsilosis, 5 C. dubliniensis, 3 C. tropicalis, 2 C. krusei and 1 C. fabianii) which previously were recovered from patients with candidemia were used. The enzymatic activity tests for hemolysin, proteinase, and phospholipase were performed by using blood Sabouraud dextrose agar, bovine serum albumin medium and egg yolk agar, respectively. Biofilm formation was determined by microplate assay method. ResultAll of the Candida albicans species could produce hemolysin. The predominant enzyme activity of species included strong and very strong levels of phospholipase, proteinase and hemolysin activity were belonged to Candida albicans isolates. There were statistically significant differences in hemolysin (P < 0.001), proteinase (P = 0.003) and phospholipase (P < 0.001) activity between two groups of albicans and non-albicans species. The biofilm formation was seen in 30 (60%) of C. albicans and 49 (98%) of non-C. albicans species. There was significant statistical differences between the two groups of isolates in biofilm formation (P < 0.001). ConclusionIt is clear that Candida species have ability to produce several enzymes as virulence factors to contribute its pathogenicity. There were significant differences in virulence factors between the two C. albicans and non- C. albicans group. The ability for biofilm formation and producing exo-enzyme were an important virulence factors in Candida species isolates. This differences found in this report might have role in severity of disease caused by different species.

Changes in the Functional Activity of Horseradish Peroxidase and Bovine Serum Albumin in Media with Different Isotope 2H/1H Compositions

Abstract—It is established that a medium with a reduced content of deuterium has no effect on the secondary structure of horseradish peroxidase and bovine serum albumin and caused no conformational changes in the structures of these proteins. The placement of these proteins in a buffer solution prepared based on deuterium-depleted water led to a decrease in the intensity of intrinsic tryptophan fluorescence, while the circular dichroism spectra remained virtually unchanged. A decrease in the content of deuterium in the reaction medium led to a decrease in the activity of the peroxidase oxidation reaction of o-dianisidine and luminal with hydrogen peroxide.

Predicting tubular reabsorption with a human kidney proximal tubule tissue-on-a-chip and physiologically-based modeling

Kidney is a major route of xenobiotic excretion, but the accuracy of preclinical data for predicting in vivo clearance is limited by species differences and non-physiologic 2D culture conditions. Microphysiological systems can potentially increase predictive accuracy due to their more realistic 3D environment and incorporation of dynamic flow. We used a renal proximal tubule microphysiological device to predict renal reabsorption of five compounds: creatinine (negative control), perfluorooctanoic acid (positive control), cisplatin, gentamicin, and cadmium. We perfused compound-containing media to determine renal uptake/reabsorption, adjusted for non-specific binding. A physiologically-based parallel tube model was used to model reabsorption kinetics and make predictions of overall in vivo renal clearance. For all compounds tested, the kidney tubule chip combined with physiologically-based modeling reproduces qualitatively and quantitatively in vivo tubular reabsorption and clearance. However, because the in vitro device lacks filtration and tubular secretion components, additional information on protein binding and the importance of secretory transport is needed in order to make accurate predictions. These and other limitations, such as the presence of non-physiological compounds such as antibiotics and bovine serum albumin in media and the need to better characterize degree of expression of important transporters, highlight some of the challenges with using microphysiological devices to predict in vivo pharmacokinetics.

Anti-fungal susceptibility and virulence factors of Candida spp. isolated from blood cultures

Candidemia is one of the most common fungal nosocomial infections worldwide. It causes high mortality and morbidity rate with significant hospital costs due to increased length of hospital stay and costs for anti-fungal treatment. This study aims to investigate anti-fungal drug susceptibility, enzymatic activity and biofilm formation of the Candida spp. isolated from blood cultures. In 2016, a total of 84 clinical Candida isolates were analyzed for minimum inhibitory concentration (MIC) against fluconazole and amphotericin B by agar diffusion E-test (E-strips). Three enzymatic activity tests for phospholipase, proteinase and esterase were performed by using egg yolk agar, bovine serum albumin medium and Tween 80 opacity medium, respectively. Biofilm formation was determined by crystal violet staining. To describing the various Candida distributions cultured, C. albicans was the most frequent species (n=37, 44.1%), followed by C. tropicalis (n=30, 35.7%), C. parapsilosis (n=8, 9.5%), C. glabrata (n=6, 7.1%) and C. guilliermondii (n=3, 3.6%). Regarding anti-fungal drug susceptibility, C. albicans was susceptible to fluconazole (100%). In addition, all clinical Candida isolates were fully susceptible to amphotericin B (100%). The predominant enzyme activity of C. albicans included medium to high levels of phospholipase, proteinase and esterase activities. C. tropicalis displayed esterase activity, while C. glabrata and C. guilliermondii had no phospholipase and proteinase activity. Non-albicans Candida (NAC) i.e. C. tropicalis formed a biofilm at a higher rate than C. albicans. This study revealed the production of virulent factors in Candida strains from candidemia patients.

Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro.

Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

Exoenzyme activity and possibility identification of Candida dubliniensis among Candida albicans species isolated from vaginal candidiasis.

Vulvovaginal candidiasis (VVC) or vaginal candidiasis is a common fungal infection of the genitals causing inflammation, irritation, itching, and vaginal discharge. Common yeast infections are caused by the yeast species C.albicans. However, there are other species of Candida such as C.dubliniensis which are considered as the causative agents of this infection. Hydrolytic enzymes such as proteinase and coagulase are known as virulence factors. The aim of this study was the molecular confirmation and differentiation of C.dubliniensis among C.albicans strains isolated from women with vulvovaginal candidiasis by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and the evaluation of proteinase and coagulase activities. A total of 100 C.albicans strains isolated from women with vulvovaginal candidiasis referred to Shiraz medical clinics were enrolled in the study. All the isolates were primarily identified by conventional methods. PCR-RFLP method was used for the confirmation and identification of C.albicans and C.dubliniensis. Moreover, invitro proteinase and coagulase activities of these isolates were evaluated using bovine serum albumin media and classical rabbit plasma tube test. As a result, PCR-RFLP identified 100% of the isolates as C.albicans, and no C.dubliniensis could be identified in this study. 84% of the isolates showed proteinase activity, whereas coagulase activity was only detected in 5% of the isolates. This study reveals that C.dubliniensis plays no role in vaginal candidiasis in Iranian patients. Proteinase production could be an essential virulence factor in C. albicans pathogenicity, but coagulase activity has less potential in this matter.

Unbound monomers do diffuse through the dentin barrier

ObjectivesAssessing the role of dentinal fluid proteins in trans-dentinal diffusion of free monomers in vitro. MethodsAn artificial pulp chamber (APC) topped human dentin disks was used. A simplified two-step etch-and-rinse adhesive was formulated with 2-hydroethyl-methacrylate (HEMA), Bisphenol-A-diglycidyl-methacrylate (BisGMA), using Camphorquinone/tertiary amine as initiators. Two extraction media were used: buffered saline (Control), buffered saline with 1% bovine serum albumin (BSA). Samples were acid-etched, rinsed, air dried. Simplified primer was used, adhesive applied then light cured with a LED curing. Monomer diffusion was assessed by reverse phase HPLC. ResultsQuantifiable amounts of HEMA were detected in both extraction media while BisGMA was present in quantifiable amounts in BSA medium only. Diffused monomers concentrations were significantly higher for both monomers in BSA extraction medium. SignificanceAlbumin is sometimes referred to as taxi protein for its ability to bind and transport hydrophobic ligands. From our results, we hypothesized that albumin can also transport unbound monomers released from dental adhesive through the dentin barrier. However, dentinal fluid proteins like albumin could have significant effect on monomer diffusion through dentin to the dental pulp transporting highly hydrophobic molecules like BisGMA and enhancing diffusion of more hydrophilic ones like HEMA. These results demonstrate a new possible mechanism for cytotoxicity of resin monomers.

Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients.

In vitro generation of type-II pneumocytes can be initiated in human CD34(+) stem cells.

Human CD34(+) stem cells differentiated into type-II pneumocytes in Dulbecco's modified Eagle medium (DMEM) having hydrocortisone, insulin, fibroblast growth factor (FGF), epidermal growth factor (EGF) and bovine serum albumin (BSA), expressing surfactant proteins-B (SP-B) and C (SP-C), alkaline phosphatase (ALP) and lysozyme. FACS-enumerated pure CD34(+) cells, isolated from human peripheral blood, were cultured in DMEM and showed positive reaction with anti-human CD34 monoclonal antibodies in immunocytochemistry. These cells were cultured in DMEM having hydrocortisone, insulin, FGF, EGF and BSA (HIFEB-D) medium having an air-liquid interface. They differentiated into type-II pneumocytes with expression of SP-B and SP-C genes and disappearance of CD34 expression as assessed using real-time PCR. In reverse transcription-PCR amplicons showed 208 and 907 bp confirming SP-B and SP-C expressions. These cells expressed ALP with an activity of 1.05 ± 0.09 mM ml(-1) min(-1) and lysozyme that killed E. coli. The successful differentiation of human CD34(+) stem cells into type-II pneumocytes, and transplantation of such cells obtained from the patient's stem cell could be the futuristic approach to regenerate diseased lung alveoli.

A new fluorescent pyrene–pyridine dithiocarbamate probe: A chemodosimeter to detect Hg2+ in pure aqueous medium and in live cells

A new pyrene–pyridine dithiocarbamate based fluorescent chemodosimeter, potassium (pyren-1-ylmethyl)(pyridin-2-ylmethyl)dithiocarbamate (L1) has been designed and synthesized. The chemodosimeter shows high selectivity and sensitivity (5.2ppb) for Hg2+ in pure aqueous medium in which emission intensity was quenched by ≈80% due to the formation of new cyclized species, 1. The probe behaves as a chemodosimeter for Hg2+ ions and forms Hg2+ triggered cyclised imidazoline species with approximate detection time of 50s and exhibits both colorimetric and fluorometric changes on detection of Hg2+ ion. Color of the probe (L1) changed from green to colorless visible to the naked eye and from green to dark blue upon the addition of Hg2+ ions under UV light. The Hg2+ triggered cyclization reaction was confirmed by spectral data analysis and a single crystal structure determination of the cyclised entity 2 obtained from the model compound potassium benzyl(pyridin-2-ylmethyl) dithiocarbamate (L2). L1 finds its application for detection of Hg2+ ions on paper strips, and in BSA (bovine serum albumin) medium. L1 is also applicable for the monitoring of Hg2+ ion in NIH3T3 live cells.

Recognition Versus Adaptive Up-regulation and Degradation of CC Chemokines by the Chemokine Decoy Receptor D6 Are Determined by Their N-terminal Sequence

The chemokine decoy receptor D6 controls inflammatory responses by selective recognition and degradation of most CCR1 to CCR5 agonistic ligands. CCL14 is a homeostatic chemokine present at high concentrations in the serum with a weak agonist activity on CCR1. Under inflammatory conditions, plasmin and UPA-mediated truncation of 8 amino acids generates the potent CCR1/CCR3/CCR5 isoform CCL14(9-74), which is further processed and inactivated by dipeptidyl peptidase IV/CD26 that generates CCL14(11-74). Here we report that D6 efficiently binds both CCL14 and its truncated isoforms. Like other D6 ligands, the biologically active CCL14(9-74) induces adaptive up-regulation of D6 expression on the cell membrane and is rapidly and efficiently degraded. In contrast, the D6-mediated degradation of the biologically inactive isoforms CCL14(1-74) and CCL14(11-74) is very inefficient. Thus, D6 cooperates with CD26 in the negative regulation of CCL14 by the selective degradation of its biologically active isoform. Analysis of a panel of CC chemokines and their truncated isoforms revealed that D6-mediated chemokine degradation does not correlate with binding affinity. Conversely, degradation efficiency is positively correlated with D6 adaptive up-regulation. Sequence analysis indicated that a proline residue in position 2 of D6 ligands is dispensable for binding but crucial for D6 adaptive up-regulation and efficient degradation.

Two‐dimensional chromatography method applied to the enantiomeric determination of lansoprazole in human plasma by direct sample injection

A two-dimensional HPLC method based on the direct injection of biological samples has been developed and validated for the determination of lansoprazole enantiomers in human plasma. The lansoprazole enantiomers were extracted from the biological matrix using an octyl restricted access media bovine serum albumin column (C8 RAM BSA) and the enantioseparation was performed on an amylose tris(3,5-dimethoxyphenylcarbamate) chiral column using acetonitrile:water (35:65 v/v) and UV detection at 285 nm. Analysis time was 25 min with no time spent on sample preparation. The method was applied to the analysis of the plasma samples obtained from nine Brazilian volunteers who received a 30 mg oral dose of racemic lansoprazole and was able to quantify the enantiomers of lansoprazole in the clinical samples analyzed.

Functional Characterization of Three G Protein-coupled Receptors for Pigment Dispersing Factors in Caenorhabditis elegans

Here, we report the identification, cloning, and functional characterization of three Caenorhabditis elegans G protein-coupled pigment dispersing factor (PDF) receptors, which we designated as Ce_PDFR-1a, -b, and -c. They represent three splice isoforms of the same gene (C13B9.4), which share a high degree of similarity with the Drosophila PDF receptor and are distantly related to the mammalian vasoactive intestinal peptide receptors (VPAC2) and calcitonin receptors. In a reverse pharmacological screen, three bioactive C. elegans neuropeptides, which were recently identified as the Drosophila PDF orthologues, were able to activate these receptors in a dose-dependent manner with nanomolar potency (isoforms a and b). Integrated green fluorescent protein reporter constructs reveal the expression of these PDF receptors in all body wall muscle cells and many head and tail neurons involved in the integration of environmental stimuli and the control of locomotion. Using a custom data analysis system, we demonstrate the involvement of this newly discovered neuropeptide signaling system in the regulation of locomotor behavior. Overexpression of PDF-2 phenocopies the locomotor defects of a PDF-1 null mutant, suggesting that they elicit opposite effects on locomotion through the identified PDF receptors. Our findings strengthen the hypothesis that the PDF signaling system, which imposes the circadian clock rhythm on behavior in Drosophila, has been functionally conserved throughout the protostomian evolutionary lineage.

Triiodothyronine production by isolated rat hepatocytes: characterization and lack of glucoregulatory hormone effects.

Triiodothyronine (T3) production from thyroxine (T4) was studied in isolated rat hepatocytes. With an initial T4 concentration of 0.56 microM, hepatocyte T3 production was 0.029 +/- 0.003 (SEM) pmoles/min/mg protein. T3 production was greater in hepatocytes than in homogenates from the same liver prepared either before or after liver perfusion with collagenase. Most T3 produced remained within the cells under the conditions employed. Hepatocyte T3 production was dependent on cell number, medium bovine serum albumin concentration and temperature. It was stimulated by dithiothreitol, and inhibited by propylthiouracil, 3,3',5'-triiodothyronine and dinitrophenol; glutathione and ouabain had no effect. Alterations in medium glucose concentration and exposure to insulin or glucagon at several glucose concentrations in vitro did not alter T3 production. These results indicate that in hepatic tissue T3 production is enhanced when intact cellular organization is present and that insulin and glucagon do not acutely influence cell production of T3 in vitro.

Quantification of cephalexin as residue levels in bovine milk by high-performance liquid chromatography with on-line sample cleanup

A multidimensional, selective and precise high performance liquid chromatography (HPLC) method based on direct injection of biological samples has been developed for the determination of cephalexin in skimmed and whole bovine milk. The cephalosporin antibiotic was extracted from bovine milk using an octyl restricted access medium bovine serum albumin column (C 8-RAM-BSA) and analyzed on an octadecyl column using phosphate buffer (pH 7.5, 0.01 M): ACN (92:8, v/v) and ultraviolet detection at 260 nm. The calibration graph was linear in the concentration range of 25–1600 ng/mL and this is in accordance with the tolerance level of 100 ng/mL set by the FDA (Food and Drug Administration) and EU (European Union) for cephalexin as residue in bovine milk. The intra- and inter-day coefficients of variation for the replicate analysis at the quality control levels were less than 15% while the transfer efficiency was in the range of 90.2–92.3%. The limits of detection and quantification were 10 and 20 ng/mL, respectively.

Tropoelastin Interacts with Cell-surface Glycosaminoglycans via Its COOH-terminal Domain

Using a biochemical and cell biological approach, we have identified a cell interaction site at the carboxyl terminus of tropoelastin. Cell interactions with the COOH-terminal sequence are not through the elastin-binding protein (EBP67) because neither VGVAPG-like peptides nor galactoside sugars altered adhesion. Our results also show that cell adhesion to tropoelastin is not promoted by integrins. Through the use of mutant Chinese hamster ovary cell lines defective in glycosaminoglycan biosynthesis, as well as competition studies and enzymatic removal of specific cell-surface glycosaminoglycans, the tropoelastin-binding moieties on the cell surface were identified as heparan and chondroitin sulfate-containing glycosaminoglycans, with heparan sulfate being greatly preferred. Heparin affinity chromatography combined with cell adhesion assays identified the last 17 amino acids as the sequence element at the carboxyl terminus of tropoelastin responsible for the adhesive activity.

Perilipin Targets a Novel Pool of Lipid Droplets for Lipolytic Attack by Hormone-sensitive Lipase

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.