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Biomedical subjects

N Hawkins

Publications and source records attributed to N Hawkins.

At least 37 records · Page 2Linked to original sources
PubMed2000

Hormonal induced lactation in transgenic goats.

The aim of this study was to hormonally induce lactation in prepubertal, nulliparous, and male goats both transgenic and non-transgenic. Analysis of milk quality, recombinant protein expression levels, total amount of recombinant protein produced, and the affect on long-term reproductive capability was assessed. Fifty-one goats (Saanen, Alpine, and Toggenburg), male and non-pregnant females, 2-31 months of age, either non-transgenic or transgenic were evaluated with a total of 10 transgenes (constructs) represented. Animals were given estradiol (0.25 mg/kg, i.m.) and progesterone (0.75 mg/kg, i.m.) on days 1, 3, 5, 7, 9, 11 and 13, while prednisilone (0.4 mg/kg, i.m.) was administered on days 14-16 with mammary massage occurring daily from day 5 onward. Forty of 51 animals, (36 of 38 females and 4 of 13 males) produced milk with total volumes in the 30-day experiment, ranging from 20 microl to 530 mls per day, or approximately 500 microl to 6.8 liters total. Milk composition was analyzed for various parameters (total protein, fat content, total solids and somatic cell count) with no significant differences found between induced and natural milk. Expression levels of recombinant proteins from transgenic animals that were analyzed during the induced lactation, and subsequently during normal lactations, were found to have no significant differences. Total amount of recombinant protein produced was evaluated at different expression levels with no statistical significance seen. While over 90% of the females placed in the regimen became pregnant, there was a correlation between increased age at time of induction and an increase in number of breedings, or reproductive cycles needed to establish a pregnancy after induction. For males, 100% placed in the regimen settled females after hormonal induction of lactation. Semen quality was evaluated prior to, during, and after hormonal treatments. Semen volume and sperm number did not differ; however, for a small percentage of males, there was a decrease in sperm and post thaw motility after hormonal treatments. These levels returned to normal within 4-5 weeks. Subsequent natural lactations showed total milk volumes within breed standards. These findings indicate that hormonal induction of lactation in the caprine species is a viable alternative to pregnancy for initiating lactation and milk production, does not adversely impact reproductive performance long-term, and can benefit the early assessment of recombinant proteins produced in a transgenic founder program.

Animals↗
PubMed1999

Haemoglobin and ferritin concentrations in children aged 12 and 18 months. ALSPAC Children in Focus Study Team.

AIMS: To define the normal ranges and investigate associated factors for haemoglobin and ferritin in British children at 12 and 18 months of age, and to estimate correlations between both haemoglobin and ferritin concentrations at 8, 12, and 18 months of age. SUBJECTS AND METHODS: Subjects were part of the "children in focus" sample, randomly selected from the Avon longitudinal study of pregnancy and childhood. Capillary blood samples were taken from 940 children at 12 months and 827 children at 18 months of age. RESULTS: Haemoglobin was distributed normally and ferritin was distributed log normally at 12 and 18 months of age. Ninety five per cent reference ranges were established from empirical centiles of haemoglobin and ferritin. Haemoglobin concentrations at 18 months were associated with sex and maternal education. Concentrations of ferritin at 12 and 18 months of age were associated with birth weight and current weight. Girls at 12 months, but not at 18 months, had 8% higher ferritin concentrations than boys. Haemoglobin and ferritin concentrations were significantly correlated over time (8-12 months: rHb = 0.26, rFer = 0.46; 12-18 months: rHb = 0.37, rFer = 0.34; 8-18 months: rHb = 0.22, rFer = 0.24). CONCLUSION: Iron stores are depleted by rapid growth in infancy. A definition of anaemia based on the fifth centile gives cut off points at 12 and 18 months of age of haemoglobin < 100 g/l, and for iron deficiency of ferritin < 16 micrograms/l and < 12 micrograms/l, respectively. Because children below the fifth centile at one time point differ from those six months later, it is unclear whether screening would be effective.

Age Factors↗
PubMed1998

Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.

The ligand for osteoprotegerin has been identified, and it is a TNF-related cytokine that replaces the requirement for stromal cells, vitamin D3, and glucocorticoids in the coculture model of in vitro osteoclastogenesis. OPG ligand (OPGL) binds to a unique hematopoeitic progenitor cell that is committed to the osteoclast lineage and stimulates the rapid induction of genes that typify osteoclast development. OPGL directly activates isolated mature osteoclasts in vitro, and short-term administration into normal adult mice results in osteoclast activation associated with systemic hypercalcemia. These data suggest that OPGL is an osteoclast differentiation and activation factor. The effects of OPGL are blocked in vitro and in vivo by OPG, suggesting that OPGL and OPG are key extracellular regulators of osteoclast development.

Amino Acid Sequence↗
PubMed1998

Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.

The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.

Adenocarcinoma↗
PubMed1997

Putative folding pathway of insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) has three disulfide bonds and refolding of the fully reduced molecule generates varying ratios of correctly (PII) and incorrectly (PI) folded forms via several intermediates. All of the intermediates have the disulfide bond between Cys18 and 61 formed, indicating that formation of this disulfide is the first step in refolding. In order to further understand the refolding pathway, two intermediate froms, PIII with the additional disulfide Cys(6/47) formed and PIIIa with Cys(6/48) formed, were isolated. The oxidation of the remaining Cys48 and 52 in PIII and Cys47 and 52 in PIIIa would lead to PI and PII, respectively; however, air oxidation of these resulted in a rapid reshuffling into other intermediates as well as folding into the fully oxidized forms, and this occurred whether refolding was started with PIII or PIIIa. When oxidation occurred in the presence of an excess of oxidized glutathione, the predominant species generated were various glutathione adducts regardless of the initial intermediate form, indicating that formation of the last disulfide bond is not a favorable process relative to disulfide exchange when excess disulfides from oxidized glutathione are present. Interestingly, if 80 microM copper sulfate, an oxidant, is added to the refolding buffer, PIII resulted in formation of the PI form alone, whereas PIIIa resulted in the PII form alone. It was concluded from these results that the intermediate forms of IGF-1 can rapidly reshuffle between different disulfide structures, and that formation of the last disulfide bond is not as favorable a process as the conversion to other intermediates. The oxidation to form the last disulfide bond in PIII or PIIIa is accelerated and hence the interconversion to other intermediates is kinetically minimized only in the presence of copper sulfate. It appears, therefore, that the two intermediate forms, PIII and PIIIa, are the precursors of the corresponding fully oxidized forms, but their conversions are not energetically a favorable process.

Chromatography, High Pressure Liquid↗
PubMed1997

Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system.

We have generated and characterized a multi-functional polyclonal anti-brain-derived neurotrophic factor antibody. Western blot analysis, dorsal root ganglion neurite outgrowth and dorsal root ganglion neuron survival assays showed that this antibody specifically recognized brain-derived neurotrophic factor and not the other neurotrophins. Furthermore, it was capable of blocking the functional effects of brain-derived neurotrophic factor. Using this antibody, we examined the expression of brain-derived neurotrophic factor in adult rat brains by immunohistochemistry. We found distinct brain-derived neurotrophic factor immunoreactivity in several structures of the brain. These included the neocortex, piriform cortex, amygdaloid complex, hippocampal formation, claustrum, some thalamic and hypothalamic nuclei, the substantia nigra and some brainstem structures. In contrast to brain-derived neurotrophic factor messenger RNA expression, brain-derived neurotrophic factor immunoreactivity was also found in the lateral septum, bed nucleus of the stria teminalis, medial preoptic nucleus, olivery pretectal nucleus, lateral paragigantocellular nucleus and the dorsal horn of the spinal cord. In normal adult rat brains, there was little or no staining in the CA1 region or the granule cell layer of the dentate gyrus of the hippocampus. However, kainate treatments greatly increased brain-derived neurotrophic factor immunoreactivity in the pyramidal cells of the CA1 region, as well as in the dentate gyrus, CA2 and CA3 hippocampal regions. We present evidence for both the subcellular localization and anterograde transport of endogenous brain-derived neurotrophic factor in the central nervous system. The detection of brain-derived neurotrophic factor protein in several discrete regions of the adult brain, and brain-derived neurotrophic factor's dramatic up-regulation following kainate treatment, strongly supports a role of brain-derived neurotrophic factor in the maintenance of adult neurons and synapses. Since several populations of neurons lost during neurodegenerative diseases synthesize brain-derived neurotrophic factor protein, modulation of brain-derived neurotrophic factor levels may be clinically beneficial. The antibody described in this paper will be helpful in determining more precisely the functional activities of brain-derived neurotrophic factor in the adult.

Animals↗
PubMed1997

Comparison of solution properties of human and rat ciliary neurotrophic factor.

The solution structure and stability of rat and human ciliary neurotrophic factor (CNTF) were examined by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopy and sedimentation equilibrium analyses. The secondary structure of both proteins, as assessed by CD and FTIR, consists primarily of alpha-helix, consistent with CNTF being a member of the four-helical bundle family of cytokines and neurokines, with rat CNTF containing slightly less helix (about 10% less) and slightly more disordered structure. The environment of the tyrosine and tryptophan residues, assessed by intrinsic fluorescence emission spectroscopy, appears to be the same in both proteins. Binding of anilinonaphthalene sulfonate is identical for both proteins, indicating that these two proteins have similar surface hydrophobicities in the native state. The thermal stability of the human CNTF is significantly less than that of the rat CNTF, yet their stabilities to guanidine HCl-induced denaturation are equivalent. This apparent discrepancy in stability between the two proteins may be explained by solubility differences upon thermal unfolding. Although the human protein precipitates as it is denatured by heat, the rat protein does not. It thus appears that the unfolded state of human CNTF is less soluble and more prone to aggregation than that of the rat protein upon heating, although their conformational stability is similar. Both proteins remain largely folded at pH 3.0. Sedimentation equilibrium analysis demonstrates that both rat and human CNTF exist primarily as monomers; however, significant dimer formation occurs as the protein concentrations are increased to greater than 3 mg/mL, particularly in the presence of ammonium sulfate.

Anilino Naphthalenesulfonates↗
PubMed1997

Pathological and genetic correlates of apoptosis in the progression of colorectal neoplasia.

PURPOSE: Apoptosis is commonly observed in a variety of human tumors, and some of the genetic events which control this process have been identified in vitro. The aim of this study was to determine the frequency of apoptosis in colorectal neoplasms and to examine its relationship to a number of pathological parameters, to the presence of mutations in the p53 tumor suppressor gene, and to overexpression of the bcl-2 oncoprotein. METHODS: A total of 109 colorectal neoplasms (26 adenomas 83 carcinomas) were examined. An in situ end-labelling assay was used to detect apoptosis in paraffin-embedded tumor sections, and scores were determined by light microscopy. The p53 and bcl-2 status were determined by immunohistochemistry. RESULTS: Apoptotic frequency increased with tumor progression. Normal mucosa contained significantly fewer apoptotic cells than adenomas or carcinomas. Similarly adenomas showed less apoptosis than carcinomas, and the frequency of apoptosis increased with Dukes' stage. Overall, changes in apoptotic frequency were inversely related to the level of bcl-2 expression, but were not related to the p53 status of the tumors. CONCLUSIONS: The frequency of apoptosis in colorectal neoplasia appears to increase in the course of tumor progression in association with a decline in bcl-2 expression, but is not influenced by p53 gene mutations.

Adenocarcinoma↗
PubMed1996

Native and non-native structure in a protein-folding intermediate: spectroscopic studies of partially reduced IGF-I and an engineered alanine model.

The structure of a metastable folding intermediate of human insulin-like growth factor I (IGF-I) and an engineered model are investigated by circular dichroism and two-dimensional 1H NMR spectroscopy. The intermediate, which contains two of three native disulfide bonds, was trapped by acid quenching and isolated by reverse-phase HPLC. The reduced cysteine residues were mapped to residues 47 and 52 (corresponding to A6-A11 in insulin). In the native state this disulfide bridge anchors an adjoining amphipathic alpha-helix (helix 2; residues 42 to 49) against the hydrophobic core. Comparison of CD and 1H-NMR spectra demonstrates that the acid-quenched intermediate is partially folded and contains elements of native secondary and tertiary structure. Spectra are similar to those of an equilibrium model in which the reduced cysteine residues are replaced by alanine. Complete 1H-NMR sequential assignment of the alanine model has been obtained and demonstrates that removal of the disulfide bond is associated with local unfolding of the adjoining alpha-helix. Native secondary structure (including helices 1 and 3) is otherwise retained and defines a folded subdomain. Long-range nuclear Overhauser effects (NOE) within this subdomain are similar to those of native IGF-I; no non-native NOE is observed. Our results support the hypothesis that folding of the insulin motif is directed by a subset of native structural elements and that these elements form at an early step in the pathway. Formation of helix 2, despite its prominence in the native state, is likely to represent a late step. Hydrophobic collapse of this segment appears to precede helix formation.

Alanine↗
PubMed1996

Haemoglobin and ferritin concentrations in infants at 8 months of age.

AIM: To identify the optimum age to screen for iron deficiency, the normal distribution of haemoglobin and ferritin in a representative population sample was investigated. METHODS: Normal values for haemoglobin and ferritin were measured from heel prick capillary samples obtained from a representative cohort of 1175 infants at 8 months old who were randomly selected from children taking part in the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC). RESULTS: Haemoglobin was normally distributed: mean (SD) 117 (11) milligrams, 95% confidence interval (CI) 116 to 118, and range 72-153 milligrams. Ferritin was log normally distributed: geometric mean 38.5 micrograms/l, 95% CI 37.0 to 39.9, range 7.1-224 micrograms/l. The 5th centile for haemoglobin was 97 milligrams and for ferritin 16.9 micrograms/l. No correlation was found between haemoglobin and ferritin. Multiple regression analysis showed ferritin concentrations to be positively related to birth weight (p < 0.0001) and the sex of the child (girls with higher concentrations) (p < 0.0001) but negatively with the child's weight at 8 months (p < 0.0001). Haemoglobin concentrations were positively related to the child's weight at 8 months (p = 0.04). Neither haemoglobin nor ferritin concentrations were related to social class as measured by maternal education level. CONCLUSION: These data define the normal range for haemoglobin and ferritin in capillary samples in the UK population, and suggest that anaemia is common in infancy. Using current recommendations, 23% of infants would be identified as anaemic. For British infants at 8 months of age, a more representative 'cut off' for anaemia would be haemoglobin concentration < 97 milligrams and for iron deficiency ferritin < 16 micrograms/l.

Age Factors↗
PubMed1995

A survey of the administration of drugs to young infants. The Alspac Survey Team. Avon Longitudinal Study of Pregnancy and Childhood.

Medication which is given to young infants during the first months of life, an important period of development, may have effects on development which would not be observed in adults receiving the same drugs. The aim of this study was to estimate the numbers of children receiving various types of medication, including both prescription and non-prescription drugs, during the first 6 months of life. Self-completion questionnaires were posted to mothers participating in the Avon Longitudinal Study of Pregnancy & Childhood (ALSPAC) when their children were 6 months of age. These questionnaires included enquiries about the administration of drugs to the study children. The study was based in the three Bristol-based health districts of Avon in the United Kingdom. The study population comprised of 6973 children born in the 12 month period between the 1st July 1991 and the 30th June 1992. The majority of mothers, 96%, reported that their children had received medication, excluding vaccines, during the first 6 months of life. 35% had been given drugs from four or more different classes. Paracetamol had been given to 84% of the children, antibiotics to 30%. In view of potential effects of drug exposure on long term development, it is important that drugs which are administered to children are carefully assessed to ensure that they are not harmful.

Acetaminophen↗
PubMed1993

Crystallization and preliminary X-ray analysis of leukemia inhibitory factor.

Leukemia inhibitory factor (LIF) is a polyfunctional molecule with significant and diverse biological activities. LIF is a glycoprotein secreted by a number of different cell types in vitro. It is induced in fibroblasts, lymphocytes, monocytes and astrocytes by various inducers such as serum, TNF, interleukin-IP and EGF. Due to extensive and variable glycosylation the molecular weight can range from 38 to 67 kDA. The biological functions of LIF are mediated through a receptor and a signal transducer, gp130, which is also used by factors like interleukin-6 (IL-6), cilliary neurotropic factor (CNTF), and oncostatin M (OSM). Here, we report the crystallization of the non-glycosylated human-like LIF expressed in E. coli. The present crystals diffract to 2.0 A using synchrotron radiation. They belong to the monoclinic space group C2, and the cell dimensions are a = 61.5 A, b = 45.3 A, c = 77.7 A and beta = 112.3 degrees.

Cloning, Molecular↗
PubMed1993

Dose dependency and wound healing aspects of the use of tissue plasminogen activator in the prevention of intra-abdominal adhesions.

Intra-abdominal adhesions have been shown to result from the impairment of peritoneal fibrinolysis by inhibitors present in ischemic tissue. A reproducible model for the formation of intra-abdominal adhesions was utilized for the evaluation of the effectiveness of intraperitoneal applications of recombinant tissue plasminogen activator (rtPA) in adhesion prevention. Concentrations of rtPA required to overcome the inhibition of fibrinolysis in this model were estimated by titration of that amount of rtPA required to lyse blood clot in the presence of a measured amount of ischemic tissue. Adhesions were graded, and the hydroxyproline content of the abdominal wounds was analyzed. The effect of intraperitoneal administration of rtPA on adhesion formation was strongly dose related. Levels of rtPA of 0.01 mg/mL showed no effect (p < 0.75) on adhesion formation, whereas levels of 0.1 mg/mL either prevented or significantly modified the formation of intra-abdominal adhesions (p < 0.05). Concomitantly, wound hydroxyproline content was significantly reduced (p = 0.004). Prior investigations have shown a strong correlation between wound bursting strength and hydroxyproline content. The results of this study indicated that the levels of rtPA required to alter or prevent intra-abdominal adhesion formation also produce a significant impairment of the early phase of wound healing as measured by the wound content of hydroxyproline.

Animals↗
PubMed1993

A new outlook for rehabilitation: creative art therapy.

Each year illness, accidents, and age leave thousands of people homebound and feeling as though their lives are over. Within each person, however, is the seed of creativity that, when nurtured through creative art therapy, can bloom to vastly improve the quality of health and life, regardless of the individual's physical limitations.

Art Therapy↗
PubMed1991

The predicted protein sequence of a fish testis-specific alpha-tubulin cDNA shows conservation of isotype-specific amino acid substitutions.

The alpha-tubulin cDNA clone, pTUB5 (1536 bp), contains the entire coding sequence for an alpha-tubulin of 450 amino acids (mol. wt. 50,000) and is the first tubulin sequence to be reported for a fish. The transcript encoded by pTUB5 showed testis-specific expression. Its gene appears to be present at a single copy number in the trout tubulin multigene family. Comparison of the TUB5 amino acid sequence to the murine testis-specific alpha-tubulin (M alpha 3/7) revealed that eight of ten murine isotype-specific amino acid substitutions were in common. Certain of these substitutions are also conserved in other testis-specific alpha-tubulins and in alpha-tubulins of certain protozoans with flagella. The conservation of specific amino acids at these positions suggests that they may have important structural and or functional roles in the microtubules of axonemes.

Amino Acid Sequence↗