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Biomedical subjects

A MacKenzie

Publications and source records attributed to A MacKenzie.

At least 19 recordsLinked to original sources
PubMed2002

Human vascular endothelial growth factor B: characterization of recombinant isoforms and generation of neutralizing monoclonal antibodies.

1. The vascular endothelial growth factor (VEGF) family is a focus of interest with respect to novel therapies for cardiovascular disease. Members of this family bind differentially to three receptor tyrosine kinases, namely VEGF-R1, -R2 and -R3, and to the semaphorin receptors neuropilin 1 and 2. The role of VEGF-R1 and the factors that interact exclusively with this receptor (VEGF-B and placenta growth factor) has remained controversial. 2. To further elucidate the role of VEGF-B in blood vessel formation and function, we have expressed, purified and refolded both naturally occurring VEGF-B isoforms and a truncated amino acid 10-108 form. All refolded proteins have been demonstrated to bind to VEGF-R1 with appropriate kinetics in biosensor-based analysis. 3. Robust cell assays for VEGF-R1 ligands, such as VEGF-B, have been problematic. We have developed an assay based on a chimeric receptor consisting of extracellular domains 1-4 of VEGF-R1 and the transmembrane and intracellular domains of gp130. The cell line expresses luciferase to high levels 24 h after exposure to VEGF-A and both refolded VEGF-B167 and the short 10-108 isoform have been demonstrated to be active in this assay. 4. The novel cell-based assay, in combination with a variety of immunochemical approaches, has been used to identify and characterize monoclonal antibodies that neutralize VEGF-B activity.

Angiogenesis Inducing Agents↗
PubMed2002

Glutathione peroxidase levels throughout normal pregnancy and in pre-eclampsia.

OBJECTIVE: Evidence suggests that hemoglobin, in addition to its function as a carrier of oxygen, also serves to transport nitric oxide, as S-nitroso cysteine, from the lungs to the peripheral circulation, where it can be released. Glutathione peroxidase, besides being an important antioxidant, is known to catalyze the release of nitric oxide from smaller carrier molecules, and may play a role in the distribution of nitric oxide throughout the body. In light of these findings, we sought to determine whether glutathione peroxidase levels differed throughout gestation, and specifically between pre-eclamptic and normal women. METHODS: A nested case-control study of women receiving routine prenatal care was conducted. Pre-eclampsia was defined by a blood pressure of at least 140 mmHg systolic and/or 90 mmHg diastolic as well as proteinuria > 300 mg/24 h or > 2+ by dipstick, both occurring on two occasions at least 6 h apart. Blood was collected in heparinized tubes and was then centrifuged in a clinical centrifuge for 10 min. Plasma was frozen promptly at -80 degrees C for later enzyme-linked immunosorbent assay (ELISA), with which plasma glutathione peroxidase was determined. RESULTS: The maternal demographics of the pre-eclamptic and non-pre-eclamptic study groups did not significantly vary with respect to mean maternal age, gravidity, parity and gestational age at the time of delivery. The median maternal ages were 33 and 34 years, and the median gestational ages at the time of birth were 37.5 and 38.1 weeks, respectively. In evaluating the glutathione peroxidase levels of all patients across the three trimesters, we found that there was essentially no difference in mean levels (83.7, 81.0 and 89.5 ng/ml, respectively). There was no difference between the pre-eclamptic and non-pre-eclamptic patients, again stratified by trimester. A linear regression analysis indicated that the plasma glutathione peroxidase concentration did not correlate with gestational age or the presence of pre-eclampsia. CONCLUSIONS: Plasma glutathione peroxidase expression is similar across all trimesters. There is no change in the glutathione peroxidase levels in pre-eclamptic patients.

Adult↗
PubMed2001

Rapid secretion of interleukin-1beta by microvesicle shedding.

The proinflammatory cytokine interleukin-1beta (IL-1beta) is a secreted protein that lacks a signal peptide and does not follow currently known pathways of secretion. Its efficient release from activated immune cells requires a secondary stimulus such as extracellular ATP acting on P2X(7) receptors. We show that human THP-1 monocytes shed microvesicles from their plasma membrane within 2-5 s of activation of P2X(7) receptors. Two minutes after such stimulation, the released microvesicles contained bioactive IL-1beta, which only later appeared in the vesicle-free supernatant. We conclude that microvesicle shedding is a major secretory pathway for rapid IL-1beta release from activated monocytes and may represent a more general mechanism for secretion of similar leaderless secretory proteins.

Cell Line↗
PubMed2001

Characterization of murine monoclonal antibody to tumor necrosis factor (TNF-MAb) formulation for freeze-drying cycle development.

PURPOSE: This study was designed to characterize the formulation of protein pharmaceuticals for freeze-drying cycle development. Thermal properties of a protein formulation in a freezing temperature range are important in the development of freezing and primary drying phases. Moisture sorption properties and the relationship between moisture and stability are the bases for the design of the secondary drying phase. METHODS: We have characterized the formulation of TNF-MAb for the purpose of freeze-drying cycle development. The methods include: DTA with ER probes, freeze-drying microscopy, isothermal water adsorption, and moisture optimization. RESULTS: The DTA/ER work demonstrated the tendency to "noneutectic" freezing for the TNF-MAb formulation at cooling rates of -1 to -3 degrees C/min. The probability of glycine crystallization during freezing was quite low. A special treatment, either a high subzero temperature holding or annealing could promote the maximum crystallization of glycine, which could dramatically increase the Tg' of the remaining solution. The freeze-drying microscopy further indicated that, after the product was annealed, the cake structure was fully maintained at a Tp below -25 degrees C during primary drying. The moisture optimization study demonstrated that a drier TNF-MAb product had better stability. CONCLUSIONS: An annealing treatment should be implemented in the freezing phase in order for TNF-MAb to be dried at a higher product temperature during primary drying. A secondary drying phase at an elevated temperature was necessary in order to achieve optimum moisture content in the final product.

Antibodies, Monoclonal↗
PubMed2001

NAIP protects the nigrostriatal dopamine pathway in an intrastriatal 6-OHDA rat model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder of the basal ganglia, associated with the inappropriate death of dopaminergic neurons of the substantia nigra pars compacta (SNc). Here, we show that adenovirally mediated expression of neuronal apoptosis inhibitor protein (NAIP) ameliorates the loss of nigrostriatal function following intrastriatal 6-OHDA administration by attenuating the death of dopamine neurons and dopaminergic fibres in the striatum. In addition, we also addressed the role of the cysteine protease caspase-3 activity in this adult 6-OHDA model, because a role for caspases has been implicated in the loss of dopamine neurons in PD, and because NAIP is also a reputed inhibitor of caspase-3. Although caspase-3-like proteolysis was induced in the SNc dopamine neurons of juvenile rats lesioned with 6-OHDA and in adult rats following axotomy of the medial forebrain bundle, caspase-3 is not induced in the dopamine neurons of adult 6-OHDA-lesioned animals. Taken together, these results suggest that therapeutic strategies based on NAIP may have potential value for the treatment of PD.

Amphetamine↗
PubMed2001

Cytologic and biochemical changes associated with inoculation of amniotic fluid and meconium into lungs of neonatal rats.

OBJECTIVE: To evaluate the effect of homologous amniotic fluid and meconium inoculated intratracheally into the lungs of neonatal rats. ANIMALS: 153 male 7-day-old Fischer-344 rats. PROCEDURE: Amniotic fluid was obtained by cesarean section from the uterus of pregnant rats and meconium was collected at the time of birth from the gastrointestinal tract of neonatal rats. Neonatal rats were randomly allocated into 5 treatment groups. Two groups received 0.05 ml of saline (0.9% NaCl) solution; the third and fourth groups received 0.05 ml of 50% or 100% amniotic fluid, respectively; the fifth group was inoculated with 0.05 ml of a 20% suspension of meconium. Six or 7 rat pups/group were euthanatized by exsanguination under halothane anesthesia at postinoculation days 1, 3, 7, and 14. The magnitude of injury and inflammatory response was determined by biochemical and cytologic analyses of bronchoalveolar lavage fluid. RESULTS: Inoculation with saline solution and amniotic fluid did not induce pulmonary injury or inflammatory response. Inoculation with meconium induced significant (P < 0.01) injury and inflammatory response, characterized by the release of cytosolic enzymes and recruitment of neutrophils in the lung. CONCLUSIONS: Saline solution is an innocuous vehicle that can be safely used in intratracheal inoculations in neonatal rats. Homologous amniotic fluid, despite containing keratin and epidermal cells, does not cause acute injury or inflammation in the lung. In contrast, meconium acts as a toxic substance injuring respiratory cells and causing a vigorous but transient leukocytic inflammatory reaction in the lungs.

Alkaline Phosphatase↗
PubMed2000

The neuronal apoptosis inhibitory protein (Naip) is expressed in macrophages and is modulated after phagocytosis and during intracellular infection with Legionella pneumophila.

Legionella pneumophila is an intracellular pathogen that causes Legionnaires' disease in humans. Inbred mouse strains are uniformly resistant to L. pneumophila infection with the notable exception of A/J, where the chromosome 13 locus Lgn1 renders A/J macrophages permissive to L. pneumophila replication. The mouse Lgn1 region is syntenic with the spinal muscular atrophy (SMA) locus on human chromosome 5 and includes several copies of the neuronal apoptosis inhibitory protein (Naip) gene. We have analyzed a possible link among Lgn1, Naip, and macrophage function. RNA expression studies show that Naip (mostly copy 2) mRNA transcripts are expressed in macrophage-rich tissues, such as spleen, lung, and liver and are abundant in primary macrophages. Immunoblotting and immunoprecipitation analyses identify Naip protein expression in mouse macrophages and in macrophage cell lines RAW 264.7 and J774A. Interestingly, macrophages from permissive A/J mice express significantly less Naip protein than their nonpermissive C57BL/6J counterpart. Naip protein expression is increased after phagocytic events. Naip protein levels during infection with either virulent or avirulent strains of L. pneumophila increase during the first 6 h postinfection and remain elevated during the 48-h observation period. This enhanced expression is also observed in macrophages infected with Salmonella typhimurium. Likewise, an increase in Naip protein levels in macrophages is observed 24 h after phagocytosis of Latex beads. The cosegregation of Lgn1 and Naip together with the detected Naip protein expression in host macrophages as well as its modulation after phagocytic events and during intracellular infection make it an attractive candidate for the Lgn1 locus.

Animals↗
PubMed2000

The human preprotachykinin-A gene promoter has been highly conserved and can drive human-like marker gene expression in the adult mouse CNS.

Toward an understanding of the mechanisms controlling Preprotachykinin-A (PPTA) transcription, we introduced a 380-kb human yeast artificial chromosome containing the PPTA gene tagged with the beta-galactosidase gene into transgenic mice. This resulted in a pattern of LacZ expression in the central nervous system (CNS) remarkably similar to that reported for PPTA mRNA in the rat. However, the human gene drove expression in areas of the mouse CNS not associated with strong PPTA expression in rodents but which have been shown to express PPTA in the human. This study clearly demonstrates the high degree of conservation of the mechanisms involved in PPTA transcription that has occurred throughout 100 million of divergent human and rodent evolution. This study also defines the maximum linear extent of the human PPT-A promoter. We believe these findings constitute the removal of a significant obstacle in studying the transcriptional regulation of the human PPTA gene in vivo.

Age Factors↗
PubMed2000

An evaluation of the implementation of case management in the community psychiatric nursing service.

This paper reports the preliminary results of a study on the implementation of case management in the community psychiatric nursing service (CPNS). The purpose of the study is to compare the outcome of case management service with the conventional practice of CPNS in the care of chronic schizophrenic clients. The impact of case management service on clients' clinical status, functional level and satisfaction was measured. A matched, pre-post, case-control group design was used to compare case management and conventional care groups. A specific case management model based on the practice at Carondelet Saint Mary's Hospital and Health Centre in Tucson has been developed to care for the schizophrenic clients in the community. Subjects in the experimental group were cared for by CPNs using this case management model. Subjects in the control group were cared for by CPNs based on current practices in the CPNS. Pre- and post-measures were taken upon recruitment and at 5 months later. Findings showed that the experimental group had better outcome in terms of their mental status and functional level when compared with the control group. They were generally more satisfied with the service. When comparing case managers' role with that of conventional CPNs, case managers performed more in areas such as assessment, liaison, case discussion and life skills training. This study demonstrates that this particular model of case management can be effective in caring for clients with long-term mental health problems in Hong Kong.

Adult↗
PubMed2000

One year's clinical experience with unenhanced spiral computed tomography for the assessment of acute loin pain suggestive of renal colic.

OBJECTIVE: To assess the use of unenhanced spiral computed tomography (CT) as the primary investigation of choice for suspected acute renal colic in clinical urological practice. Patients and Methods Between 1 August 1997 and 31 July 1998, all patients attending a hospital accident and emergency department with acute loin pain suggestive of renal colic underwent a physical examination, urine analysis, plain abdominal radiography (if clinically indicated) and unenhanced spiral CT. The effective radiation dose and financial cost of unenhanced spiral CT and standard three-film emergency intravenous urography (IVU) were calculated. RESULTS: In all, 116 patients were assessed, 63 of whom had calculi and related secondary phenomena of obstruction identified on unenhanced spiral CT. There were two false-positive and one false-negative result. An alternative urinary tract diagnosis was made in four patients, including two with renal cell carcinoma and one ureteric transitional cell carcinoma. Causes other than in the urinary tract were diagnosed in three patients, i.e. two with ovarian cyst and one with sigmoid diverticulitis. The effective radiation dose of unenhanced spiral CT was 4.7 mSv and that for three-film IVU was 1.5 mSv. The costs of both IVU and unenhanced spiral CT were identical. CONCLUSIONS: Unenhanced spiral CT allows a rapid, contrast-medium-free, anatomically accurate diagnosis of urinary tract calculi and in the present series had a sensitivity of 98% and a specificity of 97%. CT provided an alternative diagnosis in 6% of patients. These advantages must be weighed against the threefold greater radiation dose of unenhanced spiral CT than with three-film IVU, and in practice the requirement for a radiologist to interpret routine axial scans.

Adult↗
PubMed2000

Molecular models to analyse preprotachykinin-A expression and function.

Towards an understanding of the mechanisms controlling Preprotachykinin A (PPT) expression we have generated a variety of molecular models to determine the mechanisms regulating both the tissue-specific and stimulus-inducible expression of the PPT gene. The approaches used include transgenic and virus vector models complementing biochemical analysis of promoter interactions with transcription factors. We have identified and characterised a yeast artificial chromosome (YAC) containing the human PPT gene and generated transgenic mouse lines containing multiple copies of this chromosome on a normal mouse genetic background. This resulted in a pattern of expression in the nervous system remarkably similar to that reported for PPT mRNA in rodents. In addition, this transgenic model has been constructed in such a manner to allow for over expression of tachykinins based on the number of extra alleles in the transgenic mouse. These animals allow us to further examine the function of the tachykinins and acts as a useful complement to existing PPT ablated mice. In vitro we have introduced the proximal PPT promoter in reporter gene constructs into adult neurones in both DRG and the CNS by an adenoassociated virus (AAV) vector or by biolistic transfection respectively. Using the AAV vector we have demonstrated that the proximal promoter can mediate the effects of NGF in adult rat DRG. These models allow us to delineate transcriptional domains involved in the physiological and pathological expression of the PPT gene.

Animals↗
PubMed2000

Use of injectable potassium chloride for euthanasia of American lobsters (Homarus americanus).

Potassium chloride (KCl: 330 mg/ml) was assessed as an euthanasia agent in American lobsters (Homarus americanus). Two groups of 10 lobsters (408.2 to 849.9 g) were maintained at 11.9 to 12.1 degrees C ('warm') and 1.5 to 2.5 degrees C ('cold') to evaluate the possible effect of ambient temperature on response to KCl. Death was defined as time of cardiac arrest, as viewed and measured by use of ultrasound. The KCl solution was injected (100 mg of KCl/100 g of body weight) at the base of the second walking leg to flood the hemolymph sinus containing the ventral nerve cord with potassium. Disruption of this 'central nervous system' was immediate, followed by cardiac arrest within 60 to 90 seconds. Group median ( +/- SD) baseline heart rate was 42 +/- 14 'warm' and 36 +/- 5 'cold' beats per minute. Time until cardiac arrest ranged from 35 to 90 (57 +/- 18) seconds in the 'warm' group and from 40 to 132 (53 +/- 34) seconds in the 'cold' group. There was no significant difference between group medians for either parameter. Histologic lesions were limited to mild to moderate acute degeneration, characterized by cell swelling, loss of contraction bands, and occasional mild cytoplasmic vacuolation of skeletal muscle at the injection site. Injectable KCl solution was an effective, reliable method for euthanasia of H. americanus.

Abdominal Muscles↗
PubMed1999

A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo.

Polymorphic regions consisting of a variable number of tandem repeats within intron 2 of the gene coding for the serotonin transporter protein 5-HTT have been associated with susceptibility to affective disorders. We have cloned two of these intronic polymorphisms, Stin2.10 and Stin2.12, into an expression vector containing a heterologous minimal promoter and the bacterial LacZ reporter gene. These constructs were then used to produce transgenic mice. In embryonic day 10.5 embryos, both Stin2.10 and Stin2.12 produced consistent beta-galactosidase expression in the embryonic midbrain, hindbrain, and spinal cord floor plate. However, we observed that the levels of beta-galactosidase expression produced by both the Stin2.10 and Stin2.12 within the rostral hindbrain differed significantly at embryonic day 10.5. Our data suggest that these polymorphic variable number of tandem repeats regions act as transcriptional regulators and have allele-dependent differential enhancer-like properties within an area of the hindbrain where the 5-HTT gene is known to be transcribed at this stage of development.

Animals↗
PubMed1999

Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor.

1. Extracellular ATP acting on P2X7 receptors opens a channel permeable to small cations, creates an access pathway for the entry of larger molecular weight dyes, and causes cell death. We used whole-cell recording and fluorescence microscopy to measure the time courses of ionic currents, uptake of the propidium dye YO-PRO-1, and membrane disruption, in human embryonic kidney (HEK293) cells expressing the rat P2X7 receptor. 2. The ATP analogue 2', 3'-O-(benzoyl-4-benzoyl)-ATP (30 microM) induced membrane blebbing within 30-40 s of sustained application; this was 5-10 times slower when extracellular sodium was replaced by larger cations. 3. Fluorescence of YO-PRO-1 was detectable within 3 s, and the uptake reached a steady rate within 10-20 s; YO-PRO-1 uptake was greatly enhanced by removing extracellular sodium. 4. Electrophysiological measurements of current reversal potentials with intracellular sodium and extracellular cations of different sizes showed that the ionic channel progressively t'2+LE0i%-i"dilated during 10-20 s to a diameter greater than 1 nm (10 A). With short agonist applications (3-5 s) the pore dilatation and YO-PRO-1 uptake were reversible and repeatable. 5. Polyethylene glycols having molecular weights >= 5000 blocked the increase in cation permeability, YO-PRO-1 uptake and membrane blebbing. 6. We conclude that maximum P2X7 receptor activation causes an exponential dilatation of the ion channel with a time constant of 25 s to a final diameter of 3-5 nm from an initial minimum pore diameter of 0.8 nm.

Adenosine Triphosphate↗
PubMed1999

Avian species differences in susceptibility to noise exposure.

Previous studies of hair cell regeneration and hearing recovery in birds after acoustic overstimulation have involved relatively few species. Studies of the effects of acoustic overexposure typically report high variability. Though it is impossible to tell, the data so far also suggest there may be considerable species differences in the degree of damage and the time course and extent of recovery. To examine this issue, we exposed four species of birds (quail, budgerigars, canaries, and zebra finches) to identical conditions of acoustic overstimulation and systematically analyzed changes in hearing sensitivity, basilar papilla morphology, and hair cell number. Quail and budgerigars showed the greatest susceptibility to threshold shift and hair cell loss after overstimulation with either pure tone or bandpass noise, while identical types of overstimulation in canaries and zebra finches resulted in much less of a threshold shift and a smaller, more diffuse hair cell loss. All four species showed some recovery of threshold sensitivity and hair cell number over time. Canary and zebra finch hearing and hair cell number recovered to within normal limits while quail and budgerigars continued to have an approximately 20 dB threshold shift and incomplete recovery of hair cell number. In a final experiment, birds were exposed to identical wide-band noise overstimulation under conditions of artificial middle ear ventilation. Hair cell loss was substantially increased in both budgerigars and canaries suggesting that middle ear air pressure regulation and correlated changes in middle ear transfer function are one factor influencing susceptibility to acoustic overstimulation in small birds.

Animals↗