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Biomedical subjects

J M Richardson

Publications and source records attributed to J M Richardson.

At least 19 recordsLinked to original sources
PubMed2003

Synchrony of nutrient supply to the rumen and dietary energy source and their effects on the growth and metabolism of lambs.

The objective of the current series of experiments was to assess the effects of dietary synchrony of OM and N supply to the rumen, achieved by altering the sequence of feeding individual ingredients and in diets with different energy sources, on the metabolism and performance of growing lambs. In Exp. 1, the in situ degradability coefficients of OM and N were determined for five feed ingredients and subsequently was used to formulate two diets, based either on barley or sugar beet pulp, to have a similar predicted nutrient content. Within each diet, specific ingredients were shifted between the 0900 and 1600 feeding to provide either a synchronous, intermediate, or asynchronous supply of OM and N to the rumen. In Exp. 2, these diets were fed at a restricted level to 48 growing lambs with an initial live weight of 25.1 +/- 4.22 kg and a slaughter weight of 41.4 +/- 1.94 kg. There was no significant effect of dietary treatment on live weight gain or feed conversion efficiency. Lambs fed the synchronous diets deposited more kidney knob and channel fat than lambs on the asynchronous or intermediate diets (P < 0.05), whereas lambs fed the barley-based diets deposited more carcass (P < 0.05) and noncarcass (P < 0.001) fat than lambs on the sugar beet-based diets. Lambs fed the asynchronous diets retained less energy over the course of the experiment than lambs on the intermediate or synchronous diets (P < 0.05), and had a lower energy efficiency (0.079, 0.097, and 0.093 MJ retained/ MJ of intake, respectively, P < 0.05). Lambs fed the barley-based diets retained more energy than lambs on the sugar beet-based (P < 0.001) and had a higher energy balance (0.095 vs. 0.084 MJ retained/MJ intake, respectively; P < 0.01). Plasma ammonia concentrations mirrored ruminal ammonia concentrations on the barley-based diets, but not sugar beet-based diets. In Exp. 3, lambs fed the sugar beet-based diets had a higher digestibility of OM and NDF (P < 0.001). By contrast, lambs on the barley-based diets had a higher level of purine derivative excretion and microbial N production (P < 0.001). The results indicate that neither dietary synchrony nor energy source significantly influenced growth rate. However, both the asynchronous and sugar beet pulp-based diets resulted in a lower efficiency of dietary energy use, and the avoidance of asynchronous patterns of nutrient release within the rumen can improve energy efficiency in growing lambs.

Ammonia↗
PubMed2001

Expression, purification, and characterization of active recombinant prostate-specific antigen in Pichia pastoris (yeast).

BACKGROUND: Prostate-specific antigen (PSA), a member of the kallikrein family of serine proteases, is a chymotrypsin-like glycoprotein produced by the prostate epithelium. Elevated serum PSA (> 4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA over time is indicative of metastatic disease. It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular matrix glycoproteins, as well as cleavage of IGF binding protein-3, a modulator of IGF-1. To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective source of enzymatically active protein. Previous efforts to express recombinant PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide. We describe the expression of active recombinant mature PSA in yeast. METHODS: Stable chromosomal integration of a construct consisting of the yeast alpha-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA. The rPSA was purified from the yeast cell culture supernatant to homogeneity by strong cation-exchange chromatography, and characterized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-glycanase digestion, N-terminal amino acid sequencing, and inactivation by a PSA-specific inhibitor. RESULTS: We report the production of active, mature rPSA in Pichia pastoris. Two forms of rPSA varying slightly in glycosylation were identified. The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 micromol/min mg) as determined using a chromogenic substrate. CONCLUSIONS: Large-scale production of active rPSA will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis. In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing.

Amidohydrolases↗
PubMed2000

Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies.

The thermodynamic stability and temperature induced structural changes of oxidized thioredoxin h from Chlamydomonas reinhardtii have been studied using differential scanning calorimetry (DSC), near- and far-UV circular dichroism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded protein is unable to refold due to the formation of soluble high-order oligomers. In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protein to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced denaturation of thioredoxin h can be well approximated by a two-state transition. The unfolding of thioredoxin h is accompanied by a large heat capacity change [6.0 +/- 1.0 kJ/(mol.K)], suggesting that at low pH a cold denaturation should be observed at the above-freezing temperatures for this protein. All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold denaturation of thioredoxin h cannot, however, be fitted to a two-state model of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is fully unfolded at pH 2.0 and 0 degrees C, whereas the other three methods (near-UV CD, fluorescence, and DSC) indicate that under these conditions 20-30% of the protein molecules are still in the native state. Several alternative mechanisms explaining these results such as structural differences in the heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed.

Animals↗
PubMed2000

Primary structure determinants of the pH- and temperature-dependent aggregation of thioredoxin.

Thioredoxins are small proteins found in all living organisms. We have previously reported that Chlamydomonas reinhardtii thioredoxin h exhibited differences both in its absorption spectrum and its aggregation properties compared to thioredoxin m. In this paper, we demonstrate, by site-directed mutagenesis, that the particularity of the absorption spectrum is linked to the presence of an additional tryptophan residue in the h isoform. The pH and temperature dependence of the aggregation of both thioredoxins has been investigated. Our results indicate that the aggregation of TRX is highly dependent on pH and that the differences between the two TRX isoforms are linked to distinct pH dependencies. We have also analyzed the pH and temperature dependence of 12 distinct variants of TRX engineered by site-directed mutagenesis. The results obtained indicate that the differences in the hydrophobic core of the two TRX isoforms do not account for the differences of aggregation. On the other hand, we show the importance of His-109 as well as the second active site cysteine, Cys-39 in the aggregation mechanism.

Amino Acid Sequence↗
PubMed2000

A method for the dispersal and characterization of leukocytes from the human female reproductive tract.

PROBLEM: The isolation of human female reproductive tract (RT) cells that maintain viability and are representative of the entire population is essential for a thorough evaluation of mucosal immunity in the reproductive tract mucosa. Here, we describe the isolation of RT cells in high yields and with high viability from the Fallopian tube, uterine endometrium, endocervix, ectocervix and vagina. METHOD OF STUDY: This cell dispersion method uses an enzyme cocktail composed of pancreatin, hyaluronidase, and collagenase (PHC), and employs a 250-microm mesh screen to facilitate cell dispersion. RESULTS: The yields of cells isolated per gram of tissue in the presence of this PHC cocktail were compared and found to be strikingly higher relative to the yields obtained with other enzyme cocktails or in the absence of enzymes. Flow cytometry was used to characterize leukocyte subsets isolated from uterine endometrium in the presence of the various enzyme cocktails. The common leukocyte antigen marker CD45, pan T-cell marker CD3, monocyte/macrophage marker CD14 and B-cell marker CD19 were retained after exposure to the PHC cocktail of enzymes. The expression of CD8 and CD4 was lost after exposure to added enzymes but regained after culture overnight. CONCLUSION: These studies demonstrate the feasibility of using enzymatic digestion for the isolation of whole populations of Fallopian tube, endometrial, cervical and vaginal cells, including leukocyte subsets in high yields, and provide a foundation for investigating mucosal immune cell function in the human female RT.

CD4 Antigens↗
PubMed2000

Impact of insecticides and surfactant on lettuce physiology and yield.

Insecticides are used extensively on lettuce, Lactuca sativa L., grown in southwestern Arizona because of heavy insect pressure that can potentially reduce lettuce productivity. Multiple sprays are made per season to manage these insects in lettuce. One of the major concerns related to extensive insecticide applications in lettuce is the potential subtle impact of insecticides that may reduce lettuce photosynthesis and yield. We conducted field and greenhouse experiments to examine the impact of multiple insecticides and surfactant spray applications on lettuce photosynthesis and yield. Lettuce was planted in the field in 1998, insecticides and surfactant were applied, and lettuce gas-exchange and dry weights were determined. Treatments were arranged in a split-plot consisting of insecticides as main plot and surfactant as subplot treatments in a randomized complete block design with four replications. Photosynthetic rates of lettuce were significantly reduced by endosulfan, methomyl, acephate, and surfactant at seedling stage 4 h and 2 d after the spray application was made. However, the reduction in lettuce photosynthesis by these insecticides and surfactant was only transient, and lettuce photosynthesis recovered 5 d after the spray application was made. Photosynthetic rates were not altered by zeta-cypermethrin, emamectin benzoate, and spinosad at the seedling stage. Insecticides or surfactant (Kinetic, a nonionic surfactant) did not significantly affect lettuce photosynthesis after rosette formation. In addition, lettuce dry weight was not significantly altered. These studies suggest that lettuce photosynthesis may be susceptible to some insecticides at the seedling stage. Consequently, we found that biorational insecticides, introduced to manage insect pests in lettuce, have no influence on lettuce physiology at the seedling stage, unlike the chlorinated hydrocarbons, organophosphates, or carbamates tested in this study. In a greenhouse study, we found that lettuce photosynthesis and yield were not altered by Bacillus thuringiensis application. Our results indicate that B. thuringiensis and the newer insecticides, particularly biorationals, can be used to manage lettuce insect pests without significantly altering lettuce gas-exchange and yield.

Animals↗
PubMed1999

MEARA sequence repeat of human CstF-64 polyadenylation factor is helical in solution. A spectroscopic and calorimetric study.

The primary structure of the human CstF-64 polyadenylation factor contains 12 nearly identical repeats of a consensus motif of five amino acid residues with the sequence MEAR(A/G). No known function has yet been ascribed to this motif; however, according to secondary structure prediction algorithms, it should form a helical structure in solution. To validate this theoretical prediction, we synthesized a 31 amino acid residue peptide (MEARA(6)) containing six repeats of the MEARA sequence and characterized its structure and stability by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). No effects of concentration on the CD or DSC properties of MEARA(6) were observed, indicating that the peptide is monomeric in solution at concentrations up to 2 mM. The far UV-CD spectra of MEARA(6) indicates that at a low temperature (1 degrees C) the MEARA(6) peptide has a relatively high helical content (76% at pH 2.0 and 65% at pH 7.0). The effects of pH and ionic strength on the CD spectrum of MEARA(6) suggest that a number of electrostatic interactions (e.g., i, i + 3 Arg/Glu ion pair, charge-dipole interactions) contribute to the stability of the helical structure in this peptide. DSC profiles show that the melting of MEARA(6) helix is accompanied by positive change in the enthalpy. To determine thermodynamic parameters of helix-coil transition from DSC profiles for this peptide, we developed a new, semiempirical procedure based on the calculated function for the heat capacity of the coiled state for a broad temperature range. The application of this approach to the partial molar heat capacity function for MEARA(6) provides the enthalpy change for helix formation calculated per amino acid residue as 3.5 kJ/mol.

Amino Acid Sequence↗
PubMed1998

Conserved cysteines in the type 1 deiodinase selenoprotein are not essential for catalytic activity.

The iodothyronine deiodinases are a family of oxidoreductases that catalyze the removal of iodide from thyroid hormones. Each of the three isoforms contain selenocysteine at its active site and several cysteine residues that may be important for catalytic activity. Of particular interest in the type I deiodinase (D1) is Cys124, which is vicinal to the selenocysteine at position 126, and Cys194, which has been conserved in all deiodinases identified to date. In the present studies, we have characterized the functional properties of C124A, C194A, and C124A/C194A D1 mutants, which were prepared by site-directed mutagenesis and expressed in COS-7 cells. In broken cell preparations, the sensitivity of the mutants to the selective D1 inhibitors propylthiouracil and aurothioglucose were unaltered. Mutagenesis at the Cys124 position was associated with a 7-11-fold increase in the Km of dithiothreitol, whereas Vmax values remained largely unchanged. However, both mutations resulted in marked decreases in Vmax values when glutathione or a reconstituted thioredoxin cofactor system were used in the assay. In contrast to the results of these in vitro studies, no impairment in deiodinating capability was noted in intact cells expressing equivalent levels of the mutant constructs. These studies demonstrate that Cys124 and Cys194 influence the reactivity of the D1 with thiol cofactors in in vitro assay systems but are not determinants of the sensitivity of the enzyme to propylthiouracil and aurothioglucose. Furthermore, the observation that the cysteine mutants are fully active in intact cells demonstrates that the results of commonly used broken cell assays do not accurately predict the activity of the D1 in intact cells and suggests that glutathione and thioredoxin are not the major thiols utilized in vivo to support D1 activity.

Animals↗
PubMed1998

Structure of the glycosylphosphatidylinositol membrane anchor glycan of a class-2 variant surface glycoprotein from Trypanosoma brucei.

The neutral glycan fraction of the glycosylphosphatidylinositol (GPI) membrane anchor of a class-2 variant surface glycoprotein (VSG) from Trypanosoma brucei was isolated following aqueous hydrogen fluoride dephosphorylation and nitrous acid deamination of the purified glycoprotein. The neutral glycans were fractionated by high-pH anion exchange chromatography and gel-filtration and six major glycan structures were solved by a combination of one and two-dimensional NMR, composition analysis, methylation linkage analysis and electrospray-mass spectrometry. The glycans were similar to those previously described for class-1 VSGs, in that they contained the linear trimannosyl sequence Manalpha1-2Manalpha1-6Man and a complex alpha-galactose branch of up to Galalpha1-2Galalpha1-6(Galalpha1-2)Gal, but most also contained an additional galactose residue attached alpha1-2 to the non-reducing terminal mannose residue and about one-third contained an additional galactose residue attached beta1-3 to the middle mannose residue. The additional complexity of the class-2 VSG GPI glycans is discussed in terms of a biosynthetic model that explains the full range of mature GPI structures that can be expressed on different VSG classes by the same trypanosome clone.

Animals↗
PubMed1998

The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei.

Trypanosoma brucei, in common with the other African trypanosomes, exhibits unusual cell-surface molecular architecture. The bloodstream form of the parasite is coated with a continuous layer of approximately five million variant surface glycoprotein (VSG) dimers that provide the parasite with a macromolecular diffusion barrier to guard against lysis by the alternative complement pathway. The procyclic form of the parasite has a more diffuse cell-surface coat made up of approximately 2.5 million copies of procyclic acidic repetitive protein (PARP). Within the VSG and PARP coats exist lower-abundance surface glycoproteins such as receptors and nutrient transporters. Both the VSG molecules and the PARP molecules are attached to the membrane via glycosylphosphatidylinositol (GPI) membrane anchors and the VSGs and one form of PARP are N-glycosylated. In this article, the structures of the N-glycans and the GPI anchors of T. brucei VSGs and PARPs are reviewed and simple models of the surfaces of bloodstream and procyclic trypomastigotes are presented.

Animals↗
PubMed1998

Cloning, overexpression, purification, and spectroscopic characterization of human S100P.

The calcium-binding protein S100P has been found to be associated with human prostate cancer. We have overexpressed S100P in Escherichia coli using a T7 expression system. A rapid two-step procedure for the isolation of overexpressed S100P leads to a preparation of >95% pure protein with a yield of approximately 150 mg per liter of culture. The structural integrity of recombinant S100P was analyzed using CD and fluorescence spectroscopic techniques. The far-UV CD shows that secondary structure of recombinant S100P consists predominantly of a-helical structure. Both near-UV CD and tyrosine fluorescence spectra show that aromatic residues are involved in the formation of a specific, well packed structure, indicating that the recombinant S100P protein adopts a compact folded conformation. Ca2+ has a profound effect on S100P structure. Near-UV CD and fluorescence intensity of both internal (tyrosine) and external (ANS) probes suggest significant structural rearrangements in the tertiary structure of the molecule. The similarity of far-UV CD spectrum of S100P in the presence and in the absence of Ca2+ suggests that Ca2+ binding has only minor effects on secondary structure.

Amino Acid Sequence↗
PubMed1998

Anion binding to the ubiquitin molecule.

Effects of different salts (NaCl, MgCl2, CaCl2, GdmCl, NaBr, NaClO4, NaH2PO4, Na2SO4) on the stability of the ubiquitin molecule at pH 2.0 have been studied by differential scanning calorimetry, circular dichroism, and Tyr fluorescence spectroscopies. It is shown that all of the salts studied significantly increase the thermostability of the ubiquitin molecule, and that this stabilization can be interpreted in terms of anion binding. Estimated thermodynamic parameters of binding for Cl- show that this binding is relatively weak (Kd = 0.15 M) and is characterized by a negative enthalpy of -15 kJ/mol per site. Particularly surprising was the observed stabilizing effect of GdmCl through the entire concentration range studied (0.01-2 M), however, to a lesser extent than stabilization by NaCl. This stabilizing effect of GdmCl appears to arise from the binding of Cl- ions. Analysis of the observed changes in the stability of the ubiquitin molecule in the presence of GdmCl can be adequately described by combining the thermodynamic model of denaturant binding with Cl- binding effects.

Animals↗
PubMed1998

Influence of the estrous cycle on the presence and distribution of immune cells in the rat reproductive tract.

PROBLEM: Previous studies have shown that the uterus and vagina contain cells that can present antigen to ovalbumin-specific T-cells. The objective of the present study was to systematically characterize the immune cells [major histocompatibility complex (MHC) class-II+, macrophages, granulocytes, dendritic cells, and CD8+ cells] present in the uterus and vagina of the rat and to examine their distribution at various stages of the estrous cycle. METHOD OF STUDY: Uterine and vaginal tissues from female rats were selected at various stages of the estrous cycle and were examined by immunohistochemical analysis. MHC class-II (Ia)-positive cells were detected using the OX-6 monoclonal antibody; macrophages, granulocytes, and dendritic cells were detected by OX-41 monoclonal antibody and CD8-positive T-cells were identified by OX-8 monoclonal antibody. RESULTS: Immunohistochemical analysis showed cycle-dependent changes in the immune cell populations in the uterus and vagina. Ia+ cells, macrophages, granulocytes, and dendritic cells were present in large numbers in the stroma of the endometrium and around the glandular epithelium in the uterus at estrus, the stage of the reproductive cycle when estradiol levels are known to be high, relative to those seen at diestrus, when estrogen levels are low and progesterone is the predominant hormone. CD8+ cells were observed in the uterus interspersed between glandular epithelial cells at estrus. Immune cells were more numerous in the vagina, relative to the uterus. OX-6 and OX-41-positive cells were present in greater numbers in the subepithelial layers of the vagina at diestrus, in contrast to estrus. CONCLUSION: This study demonstrates that a variety of immune cells are present in the reproductive tract and that their number and distribution vary in a tissue-specific manner with the stage of the estrous cycle.

Animals↗
PubMed1997

Synthesis and characterization of insulin-fluorescein derivatives for bioanalytical applications.

Human insulin was labeled with fluorescein isothiocyanate (FITC) and fully characterized to yield four distinct insulin-FITC species. High-performance liquid chromatography and electrospray mass spectrometry were used to determine the extent and location of fluorescein conjugation. By changing the reaction conditions (i.e., pH, time, and FITC/insulin ratio) the selectivity of the fluorescein conjugation was altered, and all conjugates could be separated. The isolated species of insulin-FITC were labeled at the following residues: A1(Gly), B1(Phe), A1(Gly)B1(Phe), and A1(Gly)B1(Phe)B29(Lys). All four insulin-FITC conjugates were then used to develop fluorescence polarization binding assays with monoclonal and polyclonal anti-insulin antibodies. The assay sensitivity differed between the conjugates depending on the site of modification (B1 > A1 > A1B1 > A1B1B29). Also, the type of antibody used had an important role in the binding of insulin-FITC conjugates. Finally, for the first time the biological activity of the four conjugates was demonstrated by an autophosphorylation assay. The positional substitution dramatically affected the biological activity, confirming insights into the residues responsible for the insulin binding region. The B1 conjugate was found to retain almost all biological activity while the A1 and A1B1 conjugates had approximately 10 times lower activity. The trisubstituted species (labeled at A1, B1, and B29) was determined to be least active.

Amino Acid Sequence↗
PubMed1997

Leptin is a four-helix bundle: secondary structure by NMR.

Leptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin (13C/15N -labeled) have been assigned and its secondary structure reveals it to be a four-helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short-helix cytokine family. A three-dimensional model was built verifying the mechanical consistency of the identified elements with a short-helix cytokine core.

Amino Acid Sequence↗
PubMed1997

Uterine stromal cell suppression of pIgR production by uterine epithelial cells in vitro: a mechanism for regulation of pIgR production.

Previous studies have shown that the polymeric Ig receptor (pIgR) is produced by rat uterine epithelial cells both in vivo and vitro. The expression of the pIgR is regulated by sex hormones and/or cytokines at mucosal sites, however the mechanism of regulation in the uterus is not clear. In these studies, co-culture of stromal cells from mature rat uteri with uterine epithelial cells decreased epithelial cell pIgR production. Conditioned supernatants from stromal cells incubated with epithelial cells also decreased pIgR production. Immunohistochemical studies confirmed that expression of pIgR on uterine epithelial cells decreased in the presence of stromal cells. Viability of epithelial cells was sustained during these experiments, as evidenced by the maintenance of high transepithelial resistance. These studies are the first report of stromal cell regulation of pIgR production by epithelial cells at any site in the body and suggest that stromal cells can provide a signal that leads to the regulation of pIgR production.

Animals↗