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Biomedical subjects

T J Gillespie

Publications and source records attributed to T J Gillespie.

At least 19 recordsLinked to original sources
PubMed2004

Electronic leaf wetness duration sensor: why it should be painted.

The purpose of this study was to compare and evaluate the performance of electronic leaf wetness duration (LWD) sensors in measuring LWD in a cotton crop canopy when unpainted and painted sensors were used. LWD was measured with flat, printed-circuit wetness sensors, and the data were divided into two periods of 24 days: from 18 December 2001 to 10 January 2002, when the sensors were unpainted, and from 20 January to 13 February 2002, when the sensors were painted with white latex paint (two coats of paint). The data analysis included evaluating the coefficient of variation (CV%) among the six sensors for each day, and the relationship between the measured LWD (mean for the six sensors) and the number of hours with dew point depression under 2 degrees C, used as an indicator of dew presence. The results showed that the painting markedly reduced the CV% values. For the unpainted sensors the CV% was on average 67% against 9% for painted sensors. For the days without rainfall this reduction was greater. Comparing the sensor measurements to another estimator of LWD, in this case the number of hours with dew point depression under 2 degrees C, it was also observed that painting improved not only the precision of the sensors but also their sensitivity, because it increases the ability of the sensor to detect and measure the wetness promoted by small water droplets.

Environmental Monitoring↗
PubMed2001

Degradation of insulin-like growth factors in small intestine of suckling rats.

Insulin-like growth factors (IFGs), IGF-I and IGF-II, present in mammalian milk, play an important role during gastrointestinal tract development. In this study we identified and localized the activities of the common intestinal proteolytic enzymes and investigated their degradation effect on IGFs. Results indicated that the enzymatic activities of chymotrypsin, trypsin, and elastase progressed from the lowest in the duodenum, to the highest in the midjejunum, and declined in the ileum. Chymotrypsin exhibited the greatest IGFs degradation activities in neonatal intestinal lumen followed by elastase. These data furnish a potential strategic design to supplement IGFs into milk formulas.

Animals↗
PubMed2001

Peptide drug modifications to enhance bioavailability and blood-brain barrier permeability.

Peptides have the potential to be potent pharmaceutical agents for the treatment of many central nervous system derived maladies. Unfortunately peptides are generally water-soluble compounds that will not enter the central nervous system, via passive diffusion, due to the existence of the blood-brain barrier. Peptides can also undergo metabolic deactivation by peptidases, thus further reducing their therapeutic benefits. In targeting peptides to the central nervous system consideration must be focused both on increasing bioavailability and enhancing brain uptake. To date multiple strategies have been examined with this focus. However, each strategy comes with its own complications and considerations. In this review we assess the strengths and weaknesses of many of the methods currently being examined to enhance peptide entry into the central nervous system.

Biological Availability↗
PubMed1999

Microclimate and habitat in relation to Ixodes scapularis (Acari: Ixodidae) populations on Long Point, Ontario, Canada.

The impact of microclimate and density of hosts for adult ticks on the density of Ixodes scapularis Say was evaluated within 4 habitats on Long Point, Ontario, from 1989-1992. During the period from May to September, mean weekly vapor pressure deficits were greater within the oak savannah and cottonwood dune habitats than at the maple forest and white pine habitats, which were similar. Vapor pressure deficit was likely the major factor affecting the survivorship of eggs and immature tricks in these habitats. Based on drag sampling, I. scapularis adults demonstrated peak activity in April and October of each year. The mean number of I. scapularis adults collected by dragging during the fall or in the spring did not differ significantly within each habitat. The mean number of adults collected also did not differ among tick cohorts within each habitat; however, significantly more adults were collected within the maple forest than in the white pine habitat. The mean number of I. scapularis adults per white-tailed deer, Odocoileus virginianus (Zimmerman), increased from 1989 to 1991 and then decreased in 1992. Significantly more adult I. scapularis infested deer were observed in 1990 than in 1989. Removal of deer in 1989 and 1990 resulted in a calculated decrease of > 100,000 fed female ticks. Although seasonal variation in microclimate within habitats was closely linked with tick survival and partly explains the differences in abundance of I. scapularis among habitats on Long Point, habitat utilization by deer was also a primary factor governing the local abundance of I. scapularis populations.

Animals↗
PubMed1998

Neurotensin is metabolized by endogenous proteases in prostate cancer cell lines.

The formation and processing of neurotensin (NT) by three prostate cancer cell lines was investigated. Neurotensin (NT) immunoreactivity was detected in conditioned media and extracts of LNCaP cells. Using HPLC techniques, the immunoreactivity extracted from LNCaP cells coeluted with synthetic NT standard. Metalloendopeptidase 3.4.24.15 activity was detected in PC-3, DU-145 and LNCaP cells, whereas high levels of neutral endopeptidase 3.4.24.1 1 activity was detected only in LNCaP cells. NT was relatively stable when incubated with PC-3 or D-145 cells but was rapidly degraded by LNCaP cells to NT1-11 and NT1-10. Phosphoramidon inhibited the metabolism of NT by LNCaP cells. These data suggest that NT is present in and metabolized by LNCaP cellular enzymes.

Chromatography, High Pressure Liquid↗
PubMed1998

Survival and development of the different life stages of Ixodes scapularis (Acari: Ixodidae) held within four habitats on Long Point, Ontario, Canada.

From November 1989 to April 1993, blood-fed females and unfed adults and nymphs of lxodes scapularis Say were maintained in housings within 4 different habitats on Long Point, Ontario, Canada, to evaluate the effects of habitat on tick development. More fed females survived the winter within the maple forest (75.6%) than the cottonwood dune (36.1%), whereas 52.8-62.0% survived the winter within the 2 remaining habitats. The proportions of females that laid eggs within the maple forest (90.3%), oak savannah (83.9%), and white pine habitats (78.4%) were similar and greater than in the cottonwood dune (53.8%). In each habitat and all years, females began laying eggs during late April or early May. The time of egg deposition was consistent whether females fed in November and overwintered, or fed during April of the subsequent year. Significantly more eggs hatched within the maple forest (96.4%) and white pine (79.3%) than in the oak savannah (3.8%) or cottonwood dune habitats (0.0%). Hatch occurred in mid- to late July each year. The proportion of unfed I. scapularis adults that survived the winter was not significantly different among the 4 habitats. Unfed adults held in the oak savannah and cottonwood dune habitats died by early June, whereas ticks survived until late June or early July within the maple forest and white pine habitats. Unfed nymphs survived an average of 3.4 mo (range, 0.5-5.5) longer than unfed adults. Fed larvae placed in the field from 22 April to 3 July 1992 molted or died that year. In contrast, 66.7 and 100% of fed larvae placed in the field between 15 and 28 July, and after 28 July, respectively, overwintered before molting. More larvae successfully molted before overwintering (46.9%) than did those that overwintered (17.9%). The proportion of larvae that successfully molted was greatest within the maple forest and least within the cottonwood dune. Fed nymphs placed in the field from 22 April to 4 June molted or died in 1992, whereas 53.6 and 99.2% of fed nymphs placed in the field between 17 June and 28 July, and later than 28 July, respectively, overwintered before molting. Over all habitats, the proportion of nymphs that molted successfully was similar for those that overwintered (43.5%) and those that did not (36.0%). The proportion of nymphs that molted successfully was greatest in the maple forest (60.6%) and least within the cottonwood dune (13.3%). Differences in seasonal extremes of vapor pressure deficits among habitat types were likely responsible for habitat-specific differences in survival of I. scapularis. Based on observations on captive I. scapularis, the life cycle of this tick on Long Point is completed in 3 or 4 yr.

Animals↗
PubMed1998

Novel somatostatin analogs for the treatment of acromegaly and cancer exhibit improved in vivo stability and distribution.

The biodistribution of several radiolabeled somatostatin (SRIF) analogs was determined in the rat. Newly developed analogs BIM-23190 and BIM-23197 attained higher plasma levels and much greater target tissue concentrations than the clinically used BIM-23014 analog. Highest tissue concentrations of BIM-23190 and BIM-23197 were found in adrenal, kidney, pituitary and pancreas, tissues that are known to be abundant in mRNA for the somatostatin subtype 2 receptor. BIM-23190 and BIM-23197 associated radioactivity in these tissues was prolonged compared with that of BIM-23014, especially in the SRIF-receptor-rich pituitary. BIM-23190 and BIM-23197 were more stable in vivo and much less subject to biliary excretion than BIM-23014. These properties account for the elevated plasma and target tissue concentrations of these new SRIF analogs. Based on higher plasma levels, greater distribution to target tissues and longer in vivo stability, BIM-23190 and BIM-23197 may prove to be superior to BIM-23014 for the treatment of acromegaly and some types of cancer.

Acromegaly↗
PubMed1997

Structure-activity relationships of a series of [D-Ala2]deltorphin I and II analogues; in vitro blood-brain barrier permeability and stability.

[D-Ala2]deltorphins are enzymatically stable, amphibian heptapeptides that have a higher affinity and selectivity for delta-opioid receptors than any endogenous mammalian compound known. This study investigated the in vitro blood-brain barrier permeability, using primary bovine brain microvessel endothelium culture, and the resistance to enzymatic degradation, in mouse 15% brain membrane homogenates and 100% plasma, of [D-Ala2]deltorphin I, [D-Ala2]deltorphin II and several analogues. Derivatives were designed with the addition of N-terminal neutral and basic amino acids or with alterations of the amino acids present within the deltorphin sequences. The results indicated that the N-terminal sequence and the amino acids in position 4 and 5 are critical to deltorphin analogue BBB permeability and biological stability, i.e., t 1/2 brain; 4.8 hr- [D-Ala2]deltorphin I; > 15 hr- [D-Ala2, Ser4, D-Ala5]deltorphin. Although, no analogue was found to increase the BBB permeability coefficient (PC; x10(-4) cm/min) of the parent compounds ([D-Ala2]deltorphin II, PC = 23.49 +/- 2.42) analogues were identified: [Arg0, D-Ala2]deltorphin II, PC = 19.06 +/- 3.73 and [Pro-1, Pro0, D-Ala2]deltorphin II, PC = 22.22 +/- 5.93; which had similar permeability coefficients, even though they had larger molecular weights and, in the case of the cationic prodrug, a significantly lower lipophilicity. These analogues provide directions in the development of future pro-drugs for the treatment of pain and this study further clarifies the structure-activity relationship of the deltorphins.

Analgesics, Opioid↗
PubMed1996

Enkephalin analog prodrugs: assessment of in vitro conversion, enzyme cleavage characterization and blood-brain barrier permeability.

To improve the blood-brain barrier penetration of the delta-opioid receptor peptides [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Pen2, L-Cys5]enkephalin (DPLCE), various prodrug forms were synthesized to increase lipophilicity and drug delivery to the brain. The aims of this study were 3-fold, 1) to assess the metabolic conversion of various DPDPE and DPLCE prodrugs in vitro using mouse brain homogenate and mouse serum, 2)to characterize the proteolytic enzymes responsible for cleaving prodrugs to the parent compounds using select peptidase inhibitors and 3)to assess the blood-brain barrier permeability of prodrugs, compared with their parent compounds, using the in vitro bovine brain microvessel endothelial cell culture model. The prodrugs with carboxyl-terminal phenylalanine residues (DPDPE-Phe and DPLCE-Phe) had significantly longer metabolic conversion times in both mouse serum and brain homogenates than did the prodrugs with amino-terminal phenylalanine residues. Inhibition of leucine aminopeptidase with bestatin in the serum increased the conversion time of Phe0-DPDPE from 6.8 min to 92.2 min. Inhibition of aminopeptidase M with amastatin in the brain homogenate increased the conversion time of Phe0-DPDPE from 3.9 min to > 450 min. The long half-life of DPLCE-Arg-Pro-Ala in serum (317 min) vs. brain (9.2 min) can be explained by the high levels of the degradative endopeptidase 24.15 (EC 3.4.24.15) in the central nervous system but not in plasma. The data also showed that, for specific prodrugs of DPDPE such as Phe0-DPDPE and DPDPE-Arg-Gly, the prodrug shows a significant improvement in permeability, compared with the parent compound. Therefore, these data provide evidence that prodrugs or prodrug-enzyme inhibitor combinations may optimize the delivery of peptide and/or protein drugs to the central nervous system.

Analgesics↗
PubMed1995

In vitro stability of some reduced peptide bond pseudopeptide analogues of dynorphin A.

Eight analogues of DYN A(1-11)-NH2 incorporating the nonhydrolyzable psi [CH2-NH] peptide bond surrogate were tested for their in vitro enzymatic stability in mouse brain homogenates. Results show that the Leu(5)-Arg6 and to a lesser extent the Arg(7)-Ile8 and Ile(8)-Arg9 peptide bonds are the more susceptible to enzymatic cleavage in the native peptide. (Leu5 psi[CH(2)-NH]Arg6)DYN A(1-11)-NH2 exhibits an almost complete resistance to enzymatic cleavage with a half-life greater than 500 min in brain, compared to 42 min for the standard peptide, DYN A(1-11)-NH2.

Animals↗
PubMed1995

Survival and development of Ixodes scapularis (Acari: Ixodidae) under various climatic conditions in Ontario, Canada.

Distribution of the blacklegged tick, Ixodes scapularis Say, is poorly defined in Ontario. An endemic population is known on Long Point peninsula, Lake Erie, Ontario, but I. scapularis adults have also been collected from other localities within the province. To test the hypothesis that distribution of the blacklegged tick is limited by cold climatic extremes, 35 fed female, 70 unfed adult, and 70 unfed nymphal I. scapularis were held in containers within four natural habitats on Long Point (42 degrees 36' N; 80 degrees 5' W) and at northern localities near Ottawa (45 degrees 27' N; 75 degrees 42' W), Hearst (49 degrees 40' N; 83 degrees 41' W), and Kenora (49 degrees 47' N; 94 degrees 29' W), Ontario, from early December 1991 until May 1993. At the northern localities, 84.8 and 30.5% of fed females and unfed adults survived overwinter, respectively. On Long Point, 56.4% of fed females and 23.6% of unfed adults successfully overwintered. Longevity of fed females and unfed adults was increased by > 2 mo at the northern localities compared with Long Point, although survival rates for unfed nymphs at the northern sites and on Long Point were similar. Females within the four habitats on Long Point, and at Kenora and Ottawa, laid eggs from late April to mid-May, whereas eggs were deposited in late June at Hearst. Emergence of larvae from eggs began in late July or early August on Long Point and at Ottawa. Larvae were first observed in early October at Kenora, and no larvae emerged during 1992 at Hearst. Some eggs that overwintered during 1992-1993 at the northern sites were viable; however, hatching rate was < 10%. The minimum duration of the life cycle of I. scapularis is extended when ticks are introduced into regions of the province with seasonal degree-day accumulations lower than those observed on Long Point. Delays in deposition of eggs and emergence of larvae at Hearst and Kenora were likely a result of insufficient accumulation of degree-days above threshold temperatures for development in 1992. Though some eggs can overwinter successfully, suggesting that latitude-related reduction in seasonal temperature may not limit distribution of this tick in Ontario, hatchability was low. This factor, combined with innate incremental mortality at each instar, difficulty in finding a mate, and low density of medium to large mammal hosts for adults, may mitigate against establishment of I. scapularis by introduction of individual ticks into certain northern regions.

Animals↗
PubMed1994

Effect of peptidases at the blood brain barrier on the permeability of enkephalin.

The blood brain barrier (BBB) presents an enzymatic barrier to the passage of peptides, from blood to brain. The studies presented here used a well established in vitro model of the BBB to measure the presence of peptidases and the permeability of two opioid peptides. The in vitro BBB model consisted of confluent monolayers of bovine brain microvessel endothelial cells (BMECs). Enkephalin metabolizing enzymes, total aminopeptidase, aminopeptidase M (APM), angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) activities were measured in BMEC monolayers. The effect of specific inhibitors of APM, ACE and NEP on the permeability of [Met5]enkephalin (Met-Enk) and a conformationally constrained and enzymatically stable analog, DPDPE, also was determined. High levels of membrane-associated enzyme activity were measured for total aminopeptidase, APM and ACE. Interestingly, the permeability coefficient of Met-Enk was increased 4-fold in the presence of specific inhibitors of APM and ACE. Low levels of NEP activity were measured in BMEC monolayers and inhibition of NEP had no effect on Met-Enk permeability. The permeability coefficient for DPDPE was not increased with enzyme inhibitors but was 4-fold greater than Met-Enk alone. In the presence of APM or ACE inhibitors, there was no difference in the permeability of DPDPE and Met-Enk. These experiments demonstrate the presence of specific peptidases in BMECs and that the presence of inhibitors to Met-Enk inactivating peptidases significantly increased permeability of this biologically active peptide.

Amino Acid Sequence↗
PubMed1992

A specific enzyme assay for aminopeptidase M in rat brain.

A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin > MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6 microM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 microM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 microM) had only a slight effect on the Km of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2 mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The Km of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the Km of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.

Aminopeptidases↗
PubMed1992

Specificity of neurotensin metabolism by regional rat brain slices.

Regional differences in neurotensin metabolism and the peptidases involved were studied using intact, viable rat brain microslices and specific peptidase inhibitors. Regional brain slices (2 mm x 230 microns) prepared from nucleus accumbens, caudate-putamen, and hippocampus were incubated for 2 h in the absence and presence of phosphoramidon, captopril, N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, and o-Phenanthroline, which are inhibitors of neutral endopeptidase 24.11, angiotensin-converting enzyme, metalloendopeptidase 24.15, and nonspecific metallopeptidases, respectively. Neurotensin-degrading proteolytic activity varied by brain region. Significantly less (35.0 +/- 1.6%) neurotensin was lost from hippocampus than from caudate-putamen (45.4 +/- 1.0%) or nucleus accumbens (47.8 +/- 1.1%) in the absence of inhibitors. Peptidases responsible for neurotensin metabolism on brain slices were found to be predominantly metallopeptidases. Metalloendopeptidase 24.15 is of major importance in neurotensin metabolism in each brain region studied. The relative contribution of specific peptidases to neurotensin metabolism also varied by brain region; angiotensin-converting enzyme and neutral endopeptidase 24.11 activities were markedly elevated in the caudate-putamen as compared with the nucleus accumbens or hippocampus. Interregional variation in the activity of specific peptidases leads to altered neurotensin fragment formation. The brain microslice technique makes feasible regional peptide metabolism studies in the CNS, which are impractical with synaptosomes, and provides evidence for regional specificity of neurotensin degradation.

Animals↗
PubMed1991

Changes in opioid receptor selectivity following processing of peptide E: effect on gut motility.

Peptide E is a mu-selective opioid peptide derived from proenkephalin A which contains [Met5]-enkephalin at the amino end and [Leu5]-enkephalin at the carboxyl end. Peptide E is further processed both centrally and peripherally to a [Leu5]-enkephalin-containing fragment which was investigated to determine if processing leads to alterations in receptor selectivity. Peptide E-(15-25) inhibited electrically stimulated contractions in both the mouse vas deferens, longitudinal muscle, myenteric (IC50 = 459 nmol/L), and guinea pig ileum (IC50 = 2630 nmol/L), indicating a sixfold delta-receptor selectivity. When administered intracerebroventricularly to mice, peptide E-(15-25) also produced potent analgesia which was completely antagonized by naloxone pretreatment, but the peptide had no effect on intestinal transit as measured by the radiochromium geometric center method. This is consistent with earlier findings that intracerebroventricular delta-opioid-selective agents are analgesic but do not inhibit intestinal transit. In vitro radioligand binding assays were performed using male Sprague-Dawley rat whole brain homogenates. The IC50 for peptide E against [3H]naloxone was 1.8 nmol/L compared with the delta-opioid ligand, [3H] [D-Pen2, D-Pen5]-enkephalin of 38.8 nmol/L. The IC50 for peptide E-(15-25) against [3H]naloxone was 497 nmol/L, but for [3H] [D-Pen2, D-Pen5]-enkephalin it was 50.6 nmol/L. Therefore, peptide E loses mu-opioid receptor affinity (1.8-497 nmol/L) after proteolytic processing and the loss of the amino terminal tyrosine but maintains a high delta-opioid affinity (38.8-50.6 nmol/L). These studies demonstrate that enzymatic peptide processing of peptide E to peptide E-(15-25) leads to a shift from mu- to delta-receptor selectivity and a different spectrum of biological effects on gut motility.

Animals↗