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Biomedical subjects

P J McMillan

Publications and source records attributed to P J McMillan.

At least 19 recordsLinked to original sources
PubMed2005

Plasmodium falciparum possesses organelle-specific alpha-keto acid dehydrogenase complexes and lipoylation pathways.

The human malaria parasite Plasmodium falciparum possesses a single mitochondrion and a plastid-like organelle called the apicoplast. Both organelles contain members of the KADH (alpha-keto acid dehydrogenase) complexes--multienzyme complexes that are involved in intermediate metabolism. In the asexual blood stage forms of the parasites, the alpha-ketoglutarate dehydrogenase and branched chain KADH complexes are both located in the mitochondrion, whereas the pyruvate dehydrogenase is exclusively found in the apicoplast. In agreement with this distribution, Plasmodium parasites have two separate and organelle-specific pathways that guarantee lipoylation of the KADH complexes in both organelles. A biosynthetic pathway comprised of lipoic acid synthase and lipoyl (octanoyl)-ACP:protein Nepsilon-lipoyltransferase B is present in the apicoplast, whereas the mitochondrion is supplied with exogenous lipoic acid, and ligation of the metabolite to the KADH complexes is accomplished by a lipoate protein ligase A similar to that of bacteria and plants. Both pathways are excellent potential targets for the design of new antimalarial drugs.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2001

Exhibits facilitate histology laboratory instruction: student evaluation of learning resources.

Some professional schools have replaced microscopes for histology laboratory instruction with printed and electronic media. It is recognized that these media cannot replace experience with the microscope and that there is a cognitive dissonance of completely replacing microscope study. In addition, students believe that their time is not optimally used in the traditional histology laboratory. Therefore, at Loma Linda University, nine weekly microscope exhibits consisting of 10-15 slides each were prepared. For each exhibited slide, a one page "atlas" is provided, consisting of labeled low- and high-power color micrographs taken from that slide and an informative legend. By referring to the atlas, the student can easily identify the exact field and the labeled features with little help from an instructor. A live or taped video demonstration of the microscope exhibit is available on the first day of the exhibit. During the eighth week of the quarter, students were asked to evaluate the various learning resources available to them. No resource was valued significantly more than the microscope exhibits, but the video demonstrations were valued significantly more than the printed black and white atlas or the color atlas on CD. These exhibits have been used for 2 years to instruct a class of 90 dental students. Advantages are (1) students' time is used efficiently, (2) only one slide set and a fourth as many microscopes need be maintained compared with a traditional laboratory, and (3) one-of-a-kind slides derived from research activities provide for high impact learning.

Computer-Assisted Instruction↗
PubMed2001

A simple stereologic method for analysis of cerebral cortical microvessels using image analysis.

Previous methods for determining morphological features of vascular networks in cerebral cortex were subject to arbitrary variation and bias. Unbiased estimates of vessel number, volume, surface area and length can be obtained using stereology but these techniques tend to be tedious and time-consuming. Stereologic protocols generally require micrographs that have to be analyzed manually for intersections of vessels on grid points or lines. In this report, we provide a simpler and more precise method for measuring morphological features of cerebral cortical microvessels. Images of microvessels in 1 microm toluidine blue stained sections were captured using a popular image analysis software package. Luminal surfaces of endothelial cells were automatically traced using commonly available features; the two-dimensional data of vessels (diameter, area, perimeter and number of vessels) were automatically computed and transferred to a spreadsheet. Three-dimensional features were then determined using basic stereologic equations. The method eliminates the need for manual measurements and is particularly time- and cost-effective for quantitative studies where numerous images have to be evaluated.

Animals↗
PubMed2001

Angiogenesis after stroke is correlated with increased numbers of macrophages: the clean-up hypothesis.

Brain cells manufacture and secrete angiogenic peptides after focal cerebral ischemia, but the purpose of this angiogenic response is unknown. Because the maximum possible regional cerebral blood flow is determined by the quantity of microvessels in each unit volume, it is possible that angiogenic peptides are secreted to generate new collateral channels; other possibilities include neuroprotection, recovery/regeneration, and removal of necrotic debris. If the brain attempts to create new collaterals, microvessel density should increase significantly after ischemia. Conversely, if angiogenic-signaling molecules serve some other purpose, microvessel densities may increase slightly or not at all. To clarify, the authors measured microvessel densities with quantitative morphometry. Left middle cerebral arteries of adult male Sprague-Dawley rats were occluded with intraluminal nylon suture for 4 hours followed by 7, 14, 19, or 30 days of reperfusion. Controls received no surgery or suture occlusion. Changes in microvessel density and macrophage numbers were measured by light microscopic morphometry using semiautomated stereologic methods. Microvessel density increased only in the ischemic margin adjacent to areas of pannecrosis and was always associated with increased numbers of macrophages. Ischemic brain areas without macrophages displayed no vascularity changes compared with normal animals. These data suggest that ischemia-induced microvessels are formed to facilitate macrophage infiltration and removal of necrotic brain.

Animals↗
PubMed2001

From first base: the sequence of the tip of the X chromosome of Drosophila melanogaster, a comparison of two sequencing strategies.

We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome of Drosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been sequenced already in the course of studying the biology of their gene products, and examples of 12 different transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a correlation between the number of bands on the polytene chromosomes and the 20 genes identified by conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the insertion sites of P-elements from 111 mutant lines, about half of which are in a position likely to affect the expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis. We compare the European Drosophila Genome Project sequence with the corresponding part of the independently assembled and annotated Joint Sequence determined through "shotgun" sequencing. Discounting differences in the distribution of known transposable elements between the strains sequenced in the two projects, we detected three major sequence differences, two of which are probably explained by errors in assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination codons; the remaining 9% show major differences in interpretation.

Animals↗
PubMed2000

Dose response of rat retinal microvessels to proton dose schedules used clinically: a pilot study.

PURPOSE: This preclinical rat pilot study quantifies retinal microvessel, endothelial, and pericyte population changes produced by proton irradiation METHODS AND MATERIALS: The left eyes of rats were irradiated with single doses of 8, 14, 20, and 28 Gy protons; right eyes, with two fractions. Animals were euthanized, and eyes were removed; elastase digests were prepared, and cell populations were counted in sample fields. Results were compared with unirradiated controls. RESULTS: Progressive time- and dose-dependent endothelial cell loss occurred following all schedules. Cell loss was significantly different from control values (p < 0.001) following 28 Gy and following 20 Gy (p < 0.05) in a single dose. Endothelial cell loss was the same for single- and split-dose schedules. Progressive endothelial cell loss produced vessel collapse and acellular vessel strands. Endothelial cells were in the G(0) phase of the mitotic cycle. 28 Gy produced photoreceptor cell loss. CONCLUSION: The retinal digest is an elegant bioassay to quantify the microvessel population response. Single- and split-dose schedules appear to yield similar outcomes, in terms of endothelial cell density.

Animals↗
PubMed2000

Specific downregulation of presenilin 2 gene expression is prominent during early stages of sporadic late-onset Alzheimer's disease.

Mutations in the presenilin genes PS1 and PS2 cause familial Alzheimer's disease (AD). In a previous study, we reported that PS2 mRNA levels are decreased in the hippocampus, frontal cortex and basal forebrain of subjects with late-onset sporadic AD. In this study, we examined whether this downregulation occurs as the disease progresses from mild to severe stages or whether downregulation of PS2 expression is an early event in AD. We used in situ hybridization histochemistry to quantify the level of expression of PS2 message in the hippocampus of normal subjects and subjects with mild, moderate or severe AD. Several regions of the hippocampus which are sequentially susceptible to AD neuropathology as the disease progresses in severity were analyzed. We demonstrate that specific downregulation of PS2 expression is as severe in subjects with mild AD as it is in subjects in late stages of the disease. In addition, we show that hippocampal regions that are relatively free of AD neuropathology during early stages of the disease exhibit severely compromised PS2 mRNA levels even in mild AD cases. In contrast, PS2 is expressed at normal levels in the cerebellum, a region which succumbs to significantly fewer AD-related insults even at very advanced stages of the disease. These results suggest that the specific downregulation of PS2 gene expression is an early event in sporadic late-onset AD.

Age of Onset↗
PubMed2000

From sequence to chromosome: the tip of the X chromosome of D. melanogaster.

One of the rewards of having a Drosophila melanogaster whole-genome sequence will be the potential to understand the molecular bases for structural features of chromosomes that have been a long-standing puzzle. Analysis of 2.6 megabases of sequence from the tip of the X chromosome of Drosophila identifies 273 genes. Cloned DNAs from the characteristic bulbous structure at the tip of the X chromosome in the region of the broad complex display an unusual pattern of in situ hybridization. Sequence analysis revealed that this region comprises 154 kilobases of DNA flanked by 1.2-kilobases of inverted repeats, each composed of a 350-base pair satellite related element. Thus, some aspects of chromosome structure appear to be revealed directly within the DNA sequence itself.

Animals↗
PubMed2000

Calcium-dependent ATPase unlike ecto-ATPase is located primarily on the luminal surface of brain endothelial cells.

Numerous cytochemical studies have reported that calcium-activated adenosine triphosphatase (Ca2+-ATPase) is localized on the abluminal plasma membrane of mature brain endothelial cells. Since the effects of fixation and co-localization of ecto-ATPase have never been properly addressed, we investigated the influence of these parameters on Ca2+-ATPase localization in rat cerebral microvessel endothelium. Formaldehyde at 2% resulted in only abluminal staining while both luminal and abluminal surfaces were equally stained following 4% formaldehyde. Fixation with 2% formaldehyde plus 0.25% glutaraldehyde revealed more abluminal staining than luminal while 2% formaldehyde plus 0.5% glutaraldehyde produced vessels with staining similar to 4% and 2% formaldehyde plus 0.25% glutaraldehyde. The abluminal reaction appeared unaltered when ATP was replaced by GTP, CTP, UTP, ADP or when Ca2+ was replaced by Mg2+ or Mn2+ or p-chloromercuribenzoate included as inhibitor. But the luminal reaction was diminished. Contrary to previous reports, our results showed that Ca2+-specific ATPase is located more on the luminal surface while the abluminal reaction is primarily due to ecto-ATPase. The strong Ca2+-specific-ATPase luminal localization explains the stable Ca2+ gradient between blood and brain, and is not necessarily indicative of immature or pathological vessels as interpreted in the past.

Adenosine Diphosphate↗
PubMed2000

Distribution and activation of uterine mononuclear phagocytes in peripartum endometrium and myometrium of the mouse.

The present study tested the hypothesis that macrophage distribution and activation are enhanced in the uterus before term. Mid-uterine horn tissue strips from mice on Days 15 and 18 of pregnancy, the day of birth (= Day 19), and one day postpartum were paraffin-embedded and then sectioned, stained with a monoclonal pan-macrophage marker (BM8), and processed for visualization and quantification of resident macrophages per nuclear area. Macrophages were dispersed throughout the endometrium and subluminal epithelium; cell numbers declined on the day before term, then increased postpartum. Within myometrium, macrophages congregated in stroma surrounding muscle bundles, and staining was enhanced near term. Macrophage numbers were similar in pregnant and postpartum uteri, enhanced more than 2-fold over those in nonpregnant controls. Uterine sections were also analyzed by laser-scanning cytometry to enumerate activated macrophages (i.e., those that express the intercellular adhesion molecule marker CD54+) and to determine cell cycle (propidium iodide fluorescence). Activated macrophages were directly proportional to cell numbers and, by cell cycle analysis, were not terminally differentiated. Highest cell numbers occurred on Day 15: 4-fold greater than those in nonpregnant controls and 2-fold higher than those at Day 18 or in postpartum groups. These findings indicate a decline in endometrial macrophage numbers at least one day before the onset of parturition and raise the possibility that trafficking of this immune cell may contribute to onset of labor.

Animals↗
PubMed2000

Luminal localization of blood-brain barrier sodium, potassium adenosine triphosphatase is dependent on fixation.

Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na(+), K(+)-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na(+), K(+)-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.

Animals↗
PubMed1999

Quantitative electron microscopic study of the hypoxic fetal sheep heart.

In order to determine the effects of chronic, high-altitude hypoxia on the ovine fetal heart, we exposed pregnant ewes to 3,820 m beginning at 30 days gestation. We previously showed that following approximately 110 days of hypoxia the fetal heart showed significant reduction in cardiac output (76% of control) and contractility, and elevated levels of citrate synthase and lactate dehydrogenase. To investigate ultrastructural influences on these observed physiologic changes at altitude, we hypothesized that the volume densities of myofibrils and mitochondria, and glycogen content would be reduced in the ovine fetal heart and that this may contribute to contraction and cardiac output deficits in hypoxia. Mitochondria and myofibril volume density were determined by standard point-counting techniques and glycogen content was determined by biochemical analysis. The glycogen content from the hypoxic right ventricle (4.8 +/- 0.3%) was significantly lower than in control right ventricle (6.8 +/- 0.5%) and both left ventricles (hypoxia, 7.2 +/- 0.5; control, 7.8 +/- 0. 4%). Total mitochondrial volume density was also significantly reduced following hypoxia (15.5 +/- 0.7%) compared to controls (16.9 +/- 0.4%). As is common in the ovine fetal heart, the myofibril volume density of the right ventricle from both groups was significantly higher than the left ventricle (RV, 58.6 +/- 1.6; LV 54.3 +/- 0.9%). However, it was not different between control and high altitude. In support of our hypothesis, we may speculate that deficits in the quantity of myocyte glycogen and mitochondria contribute to the observed reduction in cardiac output and contractility, despite the upregulation of citrate synthase and lactate dehydrogenase. In contrast, myofibril volume density was unchanged.

Altitude↗
PubMed1999

Effects of long-term, high-altitude hypoxia on the capillarity of the ovine fetal heart.

To determine the effect of chronic hypoxia on myocardial capillarity, we exposed pregnant ewes to an altitude of 3,820 m from day 30 to day 139 of gestation and compared the fetus to low-altitude (approximately 300 m) controls. We hypothesized that capillarity would increase in the hypoxic myocardium to optimize oxygen and metabolite flux to hypoxic tissues. Fetal hearts were fixed by retrograde aortic perfusion and processed for microscopy and stereological evaluation. Fiber cross-sectional area and capillary density were measured and standardized to sarcomere length. Capillary volume density and capillary diameter were measured, capillary-to-fiber ratio and capillary length density were calculated, and the capillary anisotropy coefficient was obtained from a table of known values. Capillary-to-fiber ratio, capillary volume density, and the capillary anisotropy coefficient were not different between hypoxia and control groups. Capillary diameter was significantly larger in the right compared with the left ventricle of hypoxic but not control hearts; fiber cross-sectional area tended to be larger in the right ventricle of both groups, but this was not significant. As a result of larger fiber size, capillary density and capillary length density were significantly smaller in the right ventricle of hypoxic but not control fetal hearts. Contrary to our hypothesis, the ovine fetus does not show morphological adaptation in the myocardium after approximately 109 days of high-altitude hypoxic stress.

Altitude↗
PubMed1998

Effects of estrogen replacement on choline acetyltransferase and trkA mRNA expression in the basal forebrain of aged rats.

The effects of one week of estrogen replacement on choline acetyltransferase (ChAT) and trkA mRNA expression are examined in young and aged rodents to determine whether estrogen continues to affect cholinergic neurons in aging brain. Significant increases in ChAT and trkA are observed in the nucleus basalis of Meynert (nBM) of both age groups. ChAT expression is also increased in the HDB without changes in trkA expression. Results indicate modulation of ChAT expression by estrogen is retained in the aged rodent brain and suggests the possibility that changes in ChAT expression may be dissociated from concurrent alterations in trkA.

Aging↗
PubMed1998

Development of periradicular lesions in immunosuppressed rats.

PROBLEM: The role of bacteria has been well established in pulpal and periapical diseases, but the contribution of the host defenses is less clear. OBJECTIVES: The purpose of this study was to compare periradicular lesion development in immunosuppressed rats with that in normal rats. STUDY DESIGN: Fifteen rats were given weekly injections of Cytoxan (Bristol Laboratories) to suppress their immune systems. The pulps of mandibular first molars of these animals and another 15 rats that had received no medications were exposed and left open to their oral flora. The rats were killed at 2, 4, and 6 weeks. Radiographic analysis was performed by means of a computer linked to a digitizing board and stylus. In addition, specimens were decalcified, sectioned, stained, and examined under a microscope with a grid to quantify relative percentages of surface areas of bone, root, periodontal ligament, marrow spaces, soft tissue, and inflammatory infiltrate. RESULTS: Statistical analysis showed a significantly greater radiographic bone loss in the immunosuppressed group only at 4 weeks. No significant histologic differences were found between the two groups. CONCLUSION: Our results suggest that reduction of circulating leukocytes may not significantly affect the development of periradicular pathosis in rats.

Alveolar Bone Loss↗
PubMed1996

The effects of ovariectomy and estrogen replacement on trkA and choline acetyltransferase mRNA expression in the basal forebrain of the adult female Sprague-Dawley rat.

Cognitive deficits associated with aging and with neurodegenerative diseases such as Alzheimer's disease have been attributed to degeneration of cholinergic neurons in the basal forebrain. Estrogen is known to provide trophic support to cholinergic neurons, although the mechanisms underlying the actions of estrogen have yet to be determined. Because cholinergic neurons require neurotrophic growth factors for their survival, it is possible that the trophic effects of estrogen on basal forebrain systems are caused by enhanced expression of neurotrophins or their receptors. To begin to examine this hypothesis, we used in situ hybridization analysis to determine the effects of ovariectomy (ovx) and estrogen replacement on trkA mRNA levels in the rat basal forebrain. Ten days of estrogen deprivation after ovx resulted in significant decreases in trkA mRNA levels in the horizontal limb of the diagonal band of Broca and the nucleus basalis of Meynert. Short-term estrogen replacement therapy restored trkA mRNA expression to a level comparable with ovary-intact animals. No changes in trkA mRNA levels were observed in the vertical limb of the diagonal band of Broca after ovx or estrogen replacement. To assess the functional status of cholinergic neurons in the absence and presence of estrogen, the effects of ovx and estrogen replacement on ChAT mRNA levels were also examined and found to reflect the changes observed in trkA mRNA expression. These studies suggest that the trophic effects of estrogen on basal forebrain cholinergic systems may be mediated, in part, through the signaling of neurotrophic growth factors through their receptors.

Animals↗
PubMed1996

Neuronal expression of STM2 mRNA in human brain is reduced in Alzheimer's disease.

Mutations in the STM2 gene cause familial Alzheimer's disease (AD) in Volga Germans. To understand the function of this protein and how mutations lead to AD, it is important to determine which cell types in the brain express this gene. In situ hybridization histochemistry indicates that STM2 expression in the human brain is widespread and is primarily neuronal. In addition, STM2 mRNA is expressed in a cell line with neuronal origins. Quantification of the level of expression of the STM2 message in the basal forebrain, frontal cortex, and hippocampus reveals a significant decrease in AD-affected subjects compared to normal age-matched controls. These data suggest that downregulation of neuronal STM2 gene expression may be involved in the progression of AD.

Aged↗