No description available.

No description available.

Factor H (FH) is a 155-kDa plasma protein that regulates the alternative pathway of the complement system. Its 20 CCP modules, of 51-62 amino acid residues each, are linked by short stretches (“linkers’) of three to eight residues. We set out to test the hypothesis that long linkers towards the middle of FH play a role in ensuring that its architecture allows binding sites near its N- and C-termini to engage cooperatively with the main target, C3b, which is the key complement pathway-triggering product of C3 cleavage. In initial work, site-directed mutagenesis was used to test whether two mutations, R53H and R78G, located within CCP 1 and linked to the kidney disease atypical hemolytic uremic syndrome, are functionally deficient. Mutant versions and a native-sequence version of CCPs 1-4 of FH (i.e. FH 1-4) were tested for their ability to act as a cofactor for the FI-mediated cleavage of C3b, and accelerate the decay of the C3 convertase. It was shown that FH 1-4 R53H binds normally to C3b but has no regulatory activity while FH 1-4 R78G binds very poorly and is also deficient in cofactor and decay-accelerating activities. In subsequent work, mutagenesis was used to make the eight-residue CCPs 12-13 linker shorter (SL), or more flexible through introduction of glycine residues (3xGLY), within recombinant (r) module pair FH 12-13, and in rFH 10-15 and rFH 8-15 as well as full-length rFH. NMR showed CCPs 12 and 13 remain intact following mutation of the linker but (in FH 12-13) are more flexibly mutually disposed, as expected. SAXS indicated that both FH 10-15 SL and FH 10-15 3xGLY nonetheless have similar compact structures to native sequence (WT) FH 10-15. On the other hand, FH linker mutants interact with C3b (according to surface plasmon resonance) somewhat less well than WT FH and in the case of FH SL, affinity is similar to that of FH 19-20, i.e. there is no evidence that both C3b-binding sites in this mutant bind to the target simultaneously. Nonetheless, the bacterial protein PspCN boosts binding of linker mutants to C3b by a similar factor (three-to-fivefold) to that observed for FH WT. Thus, while interactions between non-sequential CCPs are important for FH architecture, a bend at the 12-13 linker is needed for full-length FH to adopt a fully biological activity confirmation. The use of EPR for structural studies of rFH and its mutants was explored. Free cysteines were engineered in so they could have spin labels site-specifically attached. Alternatively, a recognition site for transglutaminase was introduced so a spin label could be incorporated. These strategies were applied to rFH 12-13 and rFH 10-15 as a prelude to studies of full-length FH. Several suitably engineered proteins were prepared but only one paramagnetically labeled sample (of FH 12-13) made it for EPR; this yielded results commensurate with the NMR-derived structure. Taken together, these promising data lay the groundwork for a future, potentially very insightful, combined mutagenesis and EPR study of FH architecture and its role in complement activation.

Equine spermadhesin HSP-7 is a 14 kDa protein isolated from stallion seminal plasma and present on the surface of spermatozoa. HSP-7 displays carbohydrate and zona-pellucida binding properties, but the physiological role in equine fertilization is not well defined. HSP-7 has 98% amino acid sequence homology with the well-studied boar spermadhesin, AWN. Currently, these two proteins are considered to have the same reproductive function. Immunofluorescence studies presented here show that the stallion and boar spermadhesins are localized to different segments on spermatozoa. The variation in molecular compartmentalization of spermadhesin molecules in different species suggests that these structurally related proteins could be involved in independent events of fertilization. While the variation in HSP-7 abundance was not statistically significant between fertile and subfertile stallions, capacitated spermatozoa displayed a marked increase in HSP-7 when compared to neat sperm (P < 0.05). These results indicate that rather than aiding in capacitation, HSP-7 is exposed with capacitation and may have a more significant role in the acrosome reaction and sperm-oocyte recognition than previously documented.

The objectives of these studies were to investigate the effects of two special proteins, spray-dried plasma protein (SDPP), a high quality protein source, and Peptiva®, a mixture of peptides manufactured from marine products, on growth performance, nitrogen balance and enzyme and nutrient transporter mRNA expression in the brushborder membrane in weanling pigs. The results indicated that 6 % SDPP increased ADG and ADFI in the first 10 d after weaning (P < 0.05) without carry-over benefits in subsequent phases. There were potential additive effects of SDPP and Cu on growth promotion. Trends for interaction of diet and pen sanitation were observed for G:F with more pronounced response to SDPP (P = 0.07) and Cu (P = 0.11) supplementation in the sub-sanitary pens. In the duodenum, reduced crypt depth with Cu supplementation (P < 0.01) and a trend for greater villous length with SDPP supplementation (P = 0.09) were observed. Pigs reared in the sub-sanitary pens had lower ADG (P < 0.05) as well as shorter villous length and less crypt depth (P < 0.05) than those from sanitary pens. To investigate the potential impact of dietary proteins on gene expression in the intestine, 54 weanling pigs were fed either 6 % SDPP, 0.5 % Peptiva®, or soy control diets, and were killed 3 or 10 d after weaning. Northern blot results revealed significant diet by intestinal segment interactions (P < 0.05) for aminopeptidase A and aminopeptidase N. Aminopeptidase A was evenly distributed along the small intestine in the Peptiva® group, but decreased dramatically in the ileum in other groups. Aminopeptidase N increased from the proximal to the distal intestine in the soy protein and SDPP groups, whereas in the Peptiva® group, relative abundance was highest in the jejunum and lowest in the duodenum. Most of the enzyme and nutrient transporter mRNA abundance was observed in the distal segements of the small intestine and changed as the animals matured. Due to the low abundance of cytokine mRNA expression in the intestine, mRNA levels of cytokine were quantified by Real-Time PCR. The results indicated that the pigs fed the SDPP diet tended to have lower pro-inflammatory cytokine IL-1-β and TNF-α compared to other treatments. Tumor necrosis factor--α and IL-10 mRNA abundance increased from the proximal to the distal intestine, and was higher (P < 0.05) in the ileum than in the duodenum and jejunum. The mRNA abundance of IL-1-β, IL-10, and TNF-α also increased as the animals matured (P < 0.01). In summary, SDPP increased growth performance of weanling pigs, which were associated with changes in intestinal morphology and function. Peptiva® influenced aminopeptidases distribution along the small intestine. The mRNA abundance for digestive enzymes, nutrient transporters, and cytokines were differentially regulated along the small intestine as pigs matured. / Ph. D.

Majority of medical decisions are based on results of diagnostic tests that help to differentiate normal from abnormal. The choice of appropriate test and its intepretation are neccessary steps for correct diagnosis and treatment strategy determination. Rapid prove of acute coronary syndrome (ACS) plays a key role in choice of optimal treatment strategy, because timing of intervention directly influences prognosis of the patient. Pregnancy-associated plasma protein-A (PAPP- A) has been studied as a promising marker of ACS. For PAPP-A evaluation in patients with coronary atherosclerosis, we have chosen commercially available system Kryptor that had been verified in prenatal screening of pregnancies in risk. PAPP-A belongs among metalloproteinases. It is important marker of physiological development of placenta and fetus. The only proven physiological role of PAPP- A is the enabling of bioavailability of insulin-like growth factor (IGF). IGF as a growth factor, plays significant role in atherosclerosis development, but also it might contribute to healing processes connected with tissue injury. Nevertheless, PAPP-A role in plaque destabilization has not been proven yet, although it was found in other metaloproteinases. In our pilot study, we confirmed the use of Kryptor system also for patients with coronary...

The purpose of this research was to evaluate whether serum prealbumin levels would serve as a predictor of hospital length of stay for elderly orthopedic patients who underwent hip replacement surgery. The study consisted of a set of 54 patients admitted to a hospital in Bristol, Tennessee. Patients with depleted prealbumin levels, low to low/normal prealbumin levels, or normal prealbumin levels were analyzed. Data collected from a retrospective chart review included: age, length of stay, serum glucose, sodium, potassium, hematocrit, hemoglobin, BUN, creatinine, WBC, prealbumin, and post operative diet consumption. Data were analyzed using analysis of variance for treatment effects. Because of the limited size of the data set, probabilities approaching p<0.10 were considered and levels of p<0.05 were considered significant. The research failed to show a significant relationship between prealbumin levels at admission and length of patient stay during post-operative recovery.

Atualmente a cucurbitacina E é considerada uma candidata a fármaco em razão de sua atividade anticâncer, reconhecimento de seus alvos moleculares e sinergismo com outros fármacos utilizados no tratamento do câncer. Porém, ainda não é possível seu uso na clínica devido a importantes lacunas na literatura relativas a ensaios de farmacocinética pré-clínicos e clínicos. A cucurbitacina E é hidrolisada a cucurbitacina I em plasma e em microssomas de fígado humano. O presente estudo visa avaliar a farmacocinética populacional e ligação as proteínas plasmáticas da cucurbitacina E e seu metabólito cucurbitacina I em plasma de ratos. O método de análise sequencial da cucurbitacina E e cucurbitacina I em plasma de ratos foi desenvolvido utilizando LCMS/ MS. Alíquotas de 50 ?L de plasma foram desproteinizadas com acetonitrila e os resíduos reconstituídos com acetonitrila:água (1:1, v/v) e adicionados do padrão interno clobazam. Os extratos foram injetados na coluna RP-18 com fase móvel constituída por mistura de acetonitrila:água:metanol (32:35:33, v/v/v). O método é preciso e exato com linearidade no intervalo de 1-100 ng de cucurbitacina E/mL de plasma e de 0,4-200 ng de cucurbitacina I/mL de plasma. O método foi aplicado na avaliação da farmacocinética da cucurbitacina E administrada a ratos machos Wistar em dose única oral (gavagem) e intravenosa de 1mg/kg dissolvida em DMSO e tampão fosfato salino pH 7,4 (5:95, v/v). As amostras seriadas de sangue foram coletadas até 24 h após a administração oral ou intravenosa. As concentrações plasmáticas de cucurbitacina E foram quantificadas até 16 h somente após a administração intravenosa, enquanto as concentrações plasmáticas de cucurbitacina I permaneceram abaixo dos valores de LIQ seguindo a administração oral ou intravenosa. O modelo de farmacocinética populacional foi desenvolvido para a cucurbitacina E administrada por via intravenosa com o auxílio do programa NONMEM com adequada qualidade de ajuste e desempenho preditivo. O perfil farmacocinético da cucurbitacina E administrada por via intravenosa foi descrito por modelo bicompartimental com distribuição e eliminação de primeira ordem com os seguintes parâmetros farmacocinéticos: tempo de liberação (D) de 0,45 h, volume de distribuição (Vd) de 27,22 L, clearance (Cl) de 4,13 L/h e meiavida de eliminação (t1/2) de 4,57 h. As interações da cucurbitacina E e cucurbitacina I com as albuminas séricas humana (HSA) e de rato (RSA) foram investigadas utilizando biossensor óptico baseado em ressonância de plasma de superfície (SPR) e espectroscopia de dicroísmo circular (CD). Os dados de ligação das cucurbitacinas a albuminas foram obtidos por experimentos de competição de CD com biliverdina. Os dados de SPR revelaram um evento de ligação inédito entre a cucurbitacina I e a HSA e as afinidades de ligação da cucurbitacina E e cucurbitacina I são maiores para a RSA do que para a HSA. A cucurbitacina E e a cucurbitacina I podem ser classificadas como substâncias de alta afinidade de ligação com a HSA e com a RSA. A análise de CD mostrou que a cucurbitacina E e a cucurbitacina I modificam a ligação da biliverdina as albuminas através de modulação alostérica oposta (positiva para HSA, negativa para RSA), confirmando a necessidade de cuidados na extrapolação de dados farmacocinéticos pré-clínicos para clínicos. / Cucurbitacin E is currently considered a drug candidate due to its anticancer activity, recognition of its molecular targets, and synergism with other drugs used for cancer treatment. However, the use of cucurbitacin E in clinical practice is not possible because of important research gaps regarding its preclinical and clinical pharmacokinetic characteristics. Cucurbitacin E is hydrolyzed to cucurbitacin I in plasma and in human liver microsomes. The aim of this study was to evaluate the population pharmacokinetics and plasma protein binding of cucurbitacin E and of its metabolite cucurbitacin I in rats. The method for the sequential analysis of cucurbitacins E and I in rat plasma was developed using LC-MS/MS. Plasma aliquots of 50 ?L were deproteinized with acetonitrile, the residues were reconstituted in acetonitrile:water (1:1, v/v), and clobazam was added as internal standard. The extracts were injected into an RP-18 column using a mobile phase consisting of a mixture of acetonitrile:water:methanol (32:35:33, v/v/v). The method was precise and accurate, showing linearities at a range of 1-100 ng cucurbitacin E/mL plasma and of 0.4-200 ng cucurbitacin I/mL plasma. The method was applied to the pharmacokinetic evaluation of cucurbitacin E administered to male Wistar rats at a single oral (gavage) or intravenous dose of 1 mg/kg dissolved in DMSO and phosphate-buffered saline, pH 7.4 (5:95, v/v). Serial blood samples were collected up to 24 h after oral or intravenous administration. The plasma concentrations of cucurbitacin E were quantified up to 16 h only after intravenous administration, while the plasma concentrations of cucurbitacin I remained below the limit of quantification after oral or intravenous administration. The population pharmacokinetic model was developed for administered intravenously cucurbitacin E using the NONMEM program, with adequate goodness of fit and predictive performance. The pharmacokinetic profile of intravenously administered cucurbitacin E was described by a two-compartment model with first-order distribution and elimination. The following pharmacokinetic parameters were obtained: release time (D) of 0.45 h, volume of distribution (Vd) of 27.22 L, clearance (Cl) of 4.13 L/h, and elimination half-life (t1/2) of 4.57 h. The interactions of cucurbitacin E and I with human (HSA) and rat (RSA) serum albumins were investigated using an optical biosensor by surface plasmon resonance (SPR) and circular dichroism (CD) spectroscopy. The binding data of the cucurbitacins to albumins were obtained by CD competition experiments with biliverdin. The SPR data revealed an undescribed binding event between cucurbitacin I and HSA and the binding affinities of cucurbitacin E and cucurbitacin I were higher for RSA than for HSA. Cucurbitacin E and cucurbitacin I can be classified as substances with high binding affinity for HSA and RSA. CD analysis showed that cucurbitacin E and cucurbitacin I modify the binding of biliverdin to albumins through opposite allosteric modulation (positive for HSA, negative for RSA), confirming that care should be taken when extrapolating the pharmacokinetic data between species.

Haemostasis is a complex physiological process involving cellular and plasma protein components that interact to keep the blood fluid under normal conditions and prevent blood loss after vessel injury by promoting clot formation. Primary haemostasis encompasses the activation and aggregation of platelets and is supported by secondary haemostasis, in which the coagulation factors of the plasma interact in a complex series of reactions. Secondary haemostasis is initiated by the exposure of tissue factor (TF) to the blood after vessel injury. TF forms a complex with activated factor VII (FVIIa), which in turn activates factor X (FXa) and ultimately results in fibrin formation. The TF-FVIIa complex and FXa are tightly regulated by tissue factor pathway inhibitor (TFPI), a trivalent Kunitz-type protease inhibitor. The endothelium, consisting of endothelial cells (ECs), constitutes the inner lining of all blood vessels. As such, it is in constant contact with the blood and plays a major role in haemostasis by synthesising and storing both pro- and anti- coagulant substances, including TF and TFPI. Release of TFPI from ECs is increased after exposure to both unfractionated and low molecular weight heparins, though the mechanisms are not clearly defined. TFPI circulates in plasma, predominantly bound to lipoproteins, though the effect of the three major lipoproteins [low density (LDL), very low density (VLDL) and high density (HDL)] on the release of TFPI from ECs is not well established. Furthermore, previous studies have not systematically investigated the effect of these lipoproteins on both TF and TFPI. The initial aim of this project was to establish assays for the measurement of TF activity and TFPI antigen to supplement the TFPI activity assay that is well established in our laboratory. / These assays were then used to determine the effects of heparin and the major lipoproteins on the expression of TF and the release of TFPI on/from ECs. Human umbilical vein endothelial cells (HUVECs) were used as the EC model because their collection and isolation is well established and they have biochemical and physiological properties representative of in vivo conditions. A TF activity assay, based on a previously published method, was successfully modified and validated for the measurement of cell surface TF (standard curve R2 = 0.997). Despite exhaustive attempts, adaptation of this assay for plasma TF was unsuccessful, raising doubts regarding the plasma fractionation procedure of the originally published assay [Fukuda, C., Iijima, K. and Nakamura, K. (1989). "Measuring tissue factor (factor III) activity in plasma." Clinical Chemistry 35(9): 1897‐1900]. A novel insect cell expression system was used to produce well defined recombinant TFPI standards for use in TFPI activity and antigen assays. For the first time, truncated TFPI variants, containing the first Kunitz domain only, the first and second Kunitz domains only, and the first through third Kunitz domains minus the carboxyl terminus, were successfully produced in insect cells, though the full length molecule was not. Possible reasons for this include codon bias, protein instability and/or the signal peptide used. An ELISA to measure TFPI antigen was designed using a monoclonal anti‐TFPI antibody directed against the N‐terminus for protein capture and a polyclonal anti‐ TFPI antibody for detection. The assay was successfully optimised (standard curve R2 = 0.978, intra‐assay CV = 4.8%), however it produced inaccurate results (normal range = 498.7 ± 156.3 ng/mL), probably due to the antibody combination used. / TF and TFPI activity assays were used to determine the effect of both unfractionated and low molecular weight heparins (UFH and LMWH, respectively) on the release of TFPI and the expression of TF from/on ECs. A significant increase in the secretion of functional TFPI from ECs due to heparin (0 U/ml vs 1 and 10 U/mL) was demonstrated only in the presence of serum (UFH: 9.0 mU/mL vs 18.3 and 18.4 mU/mL, p < 0.0001; LMWH: 8.8 mU/mL vs 13.3 and 21.4 mU/mL, p < 0.05), suggesting, for the first time, that a component of serum is required for the heparin‐dependent release of TFPI. The effect of LDL, VLDL and HDL on the release of TFPI and the expression of TF from/on ECs was also investigated. All three lipoprotein fractions increased the secretion of functional TFPI after one hour incubation (LDL: 12.5 μg/mL, p < 0.01; 25 μg/mL, p < 0.05; VLDL: 50 μg/mL, p < 0.01; HDL: 50 μg/mL, p < 0.05). This is the first data to demonstrate a HDL‐dependent increase in released TFPI. After 24 hours, both LDL and VLDL decreased levels of secreted functional TFPI (LDL: 25 μg/mL, p < 0.01; 50 μg/mL, p < 0.01; VLDL: 12.5 μg/mL, p < 0.01), probably due to the oxidation and subsequent association of both lipoprotein species with TFPI. Surprisingly, both LDL and VLDL decreased cell surface TF, though this effect was not dose dependent. These results suggest that the major lipoproteins have a short term anticoagulant effect which is reversed in the longer term due to lipid oxidation. In summary, this thesis describes the successful adaptation of a chromogenic assay for the measurement of cell surface TF activity and the production of truncated TFPI variants. / Both will be used for the measurement of TF and TFPI, their association with thrombus formation and propagation, and investigations into potential therapeutic applications of TFPI. The results presented in this thesis extend the current knowledge on the expression and release of TF and TFPI on/from ECs by heparin, highlighting the importance of serum in the heparin dependent release of TFPI in vitro. Furthermore, it describes for the first time the effects of the major lipoprotein fractions on TFPI release and TF expression. The data support novel mechanisms by which LDL and VLDL are procoagulant, and HDL anticoagulant. This study provides a foundation for future research of the TF pathway in cellular models, which is critical in increasing the understanding of the pathogenesis and treatment of thrombotic disease. vitro. Furthermore, it describes for the first time the effects of the major lipoprotein fractions on TFPI release and TF expression. The data support novel mechanisms by which LDL and VLDL are procoagulant, and HDL anticoagulant. This study provides a foundation for future research of the TF pathway in cellular models, which is critical in increasing the understanding of the pathogenesis and treatment of thrombotic disease.

The studies presented in this thesis were designed to elucidate whether the abundance of ovulation-inducing factor/nerve growth factor (OIF/NGF) in alpaca semen can be used as a biomarker to predict male fertility. The neurotrophin, OIF/NGF has been identified in camelid, cattle and human semen. It is only in camelids, however, that an ovulation-inducing role for OIF/NGF has been described. The information gathered from several studies clearly demonstrate that this protein is the stimulus responsible for initiating the ovulatory cascade in camelids. In addition, intramuscular administration of OIF/NGF resulted in a dose-dependent response in terms of ovulation rate, corpus luteum (CL) lifespan, luteinizing hormone (LH) and progesterone secretion. I hypothesized that the quantity of OIF/NGF differs among male alpacas and this abundance arbitrates ovulation and pregnancy rates as well as CL formation and function. To substantiate this hypothesis, the following questions were answered: 1) can OIF/NGF in alpaca semen be quantified using a radioimmunoassay; 2) does the concentration and total abundance of OIF/NGF in alpaca semen vary within and among male ejaculates; 3) what is the glandular source of OIF/NGF that contributes to the male ejaculate; 4) is OIF/NGF concentration or abundance related to parameters associated with male fertility; 5) can OIF/NGF concentration or total abundance in the ejaculate discriminate fertile and subfertile males using both retrospective and prospective approaches; and 6) can power Doppler ultrasonography be used to assess the luteotrophic effect of OIF/NGF in tissue vasculature of the developing CL? I discovered that the source and the amount of OIF/NGF varies among species. In llamas, OIF/NGF is produced by both the corpus and disseminate portions of the prostate gland. In rats, OIF/NGF was detected in testis interstitial cells and in the lumen of the coagulating gland (anterior prostate). Ovulation-inducing factor/NGF secretion by the ampullae and vesicular glands contributed to its presence in bull (cattle and bison) ejaculates. In elk and white tail deer, OIF/NGF was detected in the ampullae and prostate glands, respectively. To gain an understanding of the abundance of OIF/NGF in ejaculates and changes in its concentration within and among males, OIF/NGF levels in semen were quantified using the radioimmunoassay. The assay developed exhibited parallel displacement curves among recombinant NGF, OIF/NGF purified from llama seminal plasma, llama and bull (cattle) seminal plasma. Ovulation-inducing factor/NGF comprised a greater percentage of the total protein found in camelid ejaculates than in cattle. Ovulation-inducing factor/NGF concentration correlated positively with sperm concentration and negatively with pH and semen volume, while total abundance of OIF/NGF was related to total prostate area and OIF/NGF concentration. Although a correlation was found between sperm concentration, neither OIF/NGF concentration nor total abundance was associated with higher ovulation, pregnancy or live birth rates. A clear association of the quantity of OIF/NGF in the male ejaculate at breeding and CL form and function was not evident. The measurement of CL vasculature by power Doppler ultrasonography, however, was able to determine nonpregnancy in alpacas earlier than the assessment of changes in CL diameter. In summary, my results did not support the hypothesis that the measurement of OIF/NGF concentration or total abundance in alpaca semen can be used to predict fertility in male alpacas.