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Biomedical subjects

D R Brown

Publications and source records attributed to D R Brown.

At least 145 records · Page 8Linked to original sources
PubMed1998

The human oncoprotein MDM2 arrests the cell cycle: elimination of its cell-cycle-inhibitory function induces tumorigenesis.

The human oncoprotein MDM2 (hMDM2) overexpresses in various human tumors. If amplified, the mdm2 gene can enhance the tumorigenic potential of murine cells. Here, we present evidence to show that the full-length human or mouse MDM2 expressed from their respective cDNA can inhibit the G0/G1-S phase transition of NIH 3T3 and normal human diploid cells. The protein harbors more than one cell-cycle-inhibitory domain that does not overlap with the p53-interaction domain. Deletion mutants of hMDM2 that lack the cell-cycle-inhibitory domains can be stably expressed in NIH 3T3 cells, enhancing their tumorigenic potential. The tumorigenic domain of hMDM2 overlaps with the p53-interaction domain. Some tumor-derived cells, such as Saos-2, H1299 or U-2OS, are relatively insensitive to the growth-inhibitory effects of hMDM2. These observations suggest that hMDM2 overexpression in response to oncogenic stimuli would induce growth arrest in normal cells. Elimination or inactivation of the hMDM2-induced G0/G1 arrest may contribute to one of the steps of tumorigenesis.

3T3 Cells↗
PubMed1998

Cardiovascular responses to feeding in the neonate during the first four days of life.

Prior work has demonstrated acute cardiovascular responses associated with breast and bottle feeding of newborn infants that consist of increases in both blood pressure and heart rate. This current study sought to determine if the amplitude of these responses is related to the age of the infant and/or the amount of nutrient ingested during the study period. Results show that the magnitude of the feeding responses does not change over the first four days of life. In a second study it was found that changes in systolic blood pressure during feeding are positively correlated with volume of nutrient intake during the initial phase of feeding (r = +0.75, p < 0.01). These results suggest that some of the individual variability in the increase in blood pressure during feeding is related to the amount of nutrient ingested. It is not known whether these differences are due to differences in sucking effort or other aspects of feeding efficiency.

Blood Pressure↗
PubMed1998

A method for long term culture of murine type 2 astrocytes.

Producing cultures of mouse type 2 astrocytes is difficult as these cells have low proliferative ability when isolated as pure cultures. Often standard glial culture techniques yield mixed glial cultures from mouse which contain little or no type 2 glia. This has made studies of the nature and activity of type 2 astrocytes difficult. A co-culture technique has been established to allow long term culturing of type 2 astrocytes which can be grown in large number and isolated at very high purity. This technique uses co-culture of isolated type 2 astrocytes and their progenitor cells with microglia. Using this method, type 2 astrocytes can be grown to high density cultures and maintained in culture for over a year without noticeable change in basic phenotypic characteristics. Aged type 2 astrocytes show increased activity of anti-oxidant enzymes suggesting that these cells have increased resistance to oxidative stress. This method may allow analysis of the development of type 2 astrocytes from progenitor cells and may help to identify the in vivo equivalent of these cells.

Animals↗
PubMed1998

The intracellular expression pattern of the human papillomavirus type 11 E1--E4 protein correlates with its ability to self associate.

The function of the human papillomavirus type 11 (HPV 11) E1--E4 spliced protein is not known. E1--E4 protein in HPV-infected tissue is detected in the cytoplasm of differentiated epithelial cells and as immunoreactive bands corresponding to potential monomers, dimers and trimers in immunoblots. The yeast two-hybrid system was employed to test for self association of the HPV 11 E1--E4 protein. To confirm the results of the yeast two-hybrid experiments, coimmunofluorescence studies of a green fluorescent fusion protein (GFP-E1--E4) and a T7 epitope-tagged E1--E4 protein were performed in C33a keratinocytes. E1--E4 protein was shown to self associate in the yeast two-hybrid system, and this result was confirmed by colocalization of GFP-E1--E4 and T7-E1(wedge)E4 proteins in keratinocytes. Analysis of E1--E4 mutants established that the C-terminus was required for self association and that sequences in the N-terminus influenced the intracellular localization of E1--E4 protein. The intracellular expression patterns of GFP-E1--E4 and GFP-E1--E4 mutants were correlated with E1--E4 binding in the yeast two-hybrid system. Those E1--E4 mutants that did not self associate in the yeast two-hybrid system were detected as diffuse cellular fluorescence when expressed as GFP fusions. In contrast, GFP-E1--E4 was detected as a perinuclear aggregate. All E1--E4 mutants capable of associating with E1--E4 in the yeast two-hybrid system were detected as aggregates when expressed as GFP fusion proteins in keratinocytes.

Animals↗
PubMed1998

A critical, invariant chain-independent role for H2-M in antigen presentation.

Antigen presentation by MHC class II (class II) is facilitated by the accessory molecules, invariant chain (Ii) and H2-M. Ii associates with class II during biosynthesis and promotes transport of class II to Ag-loading compartments. One function of H2-M is the removal of Ii fragments from MHC class II. We have previously demonstrated that Ii-deficient mice, unlike class II-deficient mice, are resistant to L. major infection. In the present study, we found that H2-M-deficient (H2-M0) mice were susceptible to progressive infection with L. major. The dispensability of Ii for control of L. major allowed genetic analysis of whether H2-M functions by association with or independently of Ii. In contrast to Ii-deficient (Ii0) mice, Ii0H2-M0 mice were as susceptible to L. major as H2-M0 mice. Thus, H2-M has an essential, Ii-independent function during presentation of microbial pathogens.

Animals↗
PubMed1998

Control of Leishmania major by a monoclonal alpha beta T cell repertoire.

Little is known regarding the diversity of the host T cell response that is required to maintain immunologic control of microbial pathogens. Leishmania major persist as obligate intracellular parasites within macrophages of the mammalian host. Immunity is dependent upon activation of MHC class II-restricted T cells to an effector state capable of restricting growth and dissemination of the organisms. We generated alpha-beta Leishmania-specific (ABLE) TCR transgenic mice with MHC class II-restricted T cells that recognized an immunodominant Leishmania Ag designated LACK. Naive T cells from ABLE mice proliferated in vitro after incubation with recombinant LACK or with Leishmania-parasitized macrophages and in vivo after injection into infected mice. Infected ABLE mice controlled Leishmania infection almost as well as wild-type mice despite a drastic reduction in the T cell repertoire. ABLE mice were crossed to mice with disruption of the TCR constant region alpha gene to create animals with a single alpha beta T cell repertoire. Although mice deficient in all alpha beta T cells (TCR-C alpha 0 mice) failed to control L. major, mice with a monoclonal alpha beta T cell repertoire (ABLE TCR-C alpha 0 mice) displayed substantial control. The immune system is capable of remarkable efficiency even when constrained to recognition of a single epitope from a complex organism.

Amino Acid Sequence↗
PubMed1998

Medical costs and lost productivity from health conditions at volatile organic compound-contaminated superfund sites.

This paper estimates the health costs at Superfund sites for conditions associated with volatile organic compounds (VOCs) in drinking water. Health conditions were identified from published literature and registry information as occurring at excess rates in VOC-exposed populations. These health conditions were: (1) some categories of birth defects, (2) urinary tract disorders, (3) diabetes, (4) eczema and skin conditions, (5) anemia, (6) speech and hearing impairments in children under 10 years of age, and (7) stroke. Excess rates were used to estimate the excess number of cases occurring among the total population living within one-half mile of 258 Superfund sites. These sites had evidence of completed human exposure pathways for VOCs in drinking water. For each type of medical condition, an individual's expected medical costs, long-term care costs, and lost work time due to illness or premature mortality were estimated. Costs were calculated to be approximately $330 million per year, in the absence of any remediation or public health intervention programs. The results indicate the general magnitude of the economic burden associated with a limited number of contaminants at a portion of all Superfund sites, thus suggesting that the burden would be greater than that estimated in this study if all contaminants at all Superfund sites could be taken into account.

Anemia↗
PubMed1998

A prion protein fragment primes type 1 astrocytes to proliferation signals from microglia.

Giliosis is a hallmark of prion disease. A neurotoxic prion peptide (PrP106-126) induces astrocyte proliferation in the presence of microglia. This peptide also directly enhances microglial proliferation in culture. We have investigated this further to understand the method by which factors released by microglia and PrP106-126 work together to enhance astrocyte proliferation. PrP106-126 in the presence of microglia specifically enhanced type 1 astrocyte proliferation but not Type 2. Astrocytes that do not express the prion protein were more sensitive to oxidative stress and the toxicity of cytosine arabinoside. In the presence of cytosine arabinoside, PrP106-126 was toxic to pure astrocyte cultures. Using conditioned medium from microglia we have shown that PrPc-expressing astrocytes proliferate in response to factors released by microglia stimulated by granulocyte/macrophage colony-stimulating factor. This response is enhanced in the presence of PrP106-126. PrPc-deficient astrocytes do not show this response. These results suggest that astrocytes are primed by PrP106-126 to respond more to factors released by proliferating microglia. Astrocytes may proliferate in this system to escape entering the cell suicide pathway.

Animals↗
PubMed1998

Seasonal dynamics and variation among sheep in densities of the sheep biting louse, Bovicola ovis.

Cyclic patterns and variations among sheep in numbers of Bovicola ovis are described in Polypay and Columbia ewes that were initially infested with equal numbers of lice and penned indoors continuously for 2 years. Bovicola ovis populations were censused at 3-4-week intervals at 69 body sites on each animal. In the second year of the study, the ewes were reinfested and half were mated. Louse populations were monitored on the resulting lambs from birth until 25 weeks of age. Strong seasonal cycles in louse numbers were observed on the ewes, with peaks in spring and troughs in summer. These cycles occurred in the absence of shearing, direct solar radiation or rainfall. Populations began to decline when daily mean and maximum temperatures were 11.5 degrees C and 15 degrees C, respectively, well below temperatures thought to cause warm season decline. Louse densities on Polypay ewes were approximately 10 times higher than on Columbias at most inspections. There were also large differences among sheep within breeds and sheep counts were highly correlated among dates, both within and between years. One third of the ewes failed to become infested despite having lice applied on five separate occasions and being penned together with other infested sheep. Pregnancy and lactation did not significantly affect louse numbers on the ewes. There was a significant negative correlation between louse counts and weight gains in the lambs, and lamb counts were significantly correlated with those of their dams up until, but not after, weaning. It is suggested that sheep may exert regulatory influences on lice which contribute to cycles in B. ovis populations.

Animals↗
PubMed1998

Prion protein expression in muscle cells and toxicity of a prion protein fragment.

The prion protein (PrP) is a cell surface glycoprotein normally associated with neurones. Expression of the prion protein in cultured mouse myoblasts and myotubes suggests that the prion protein may play a physiological role in skeletal muscle. When myotubes differentiate from myoblasts prion protein expression is upregulated. Accompanying this increase is an upregulation of Cu/Zn superoxide dismutase (SOD-1) in myotubes. Muscle cells derived from mice deficient in cellular PrP (PrPc) show little increase in SOD-1 after differentiation from myoblasts to myotubes. Myoblasts and myotubes are resistant to the toxicity of a neurotoxic prion protein peptide (PrP106-126). However, in the presence of murine microglia, PrP106-126 causes a reduction in cell number. This effect is greater on myotubes than myoblasts. Even in the presence of microglia PrP106-126 is not toxic to muscle cells derived from PrP-deficient mice. Our results suggest that PrPc expression is associated with regulation of cellular resistance to oxidative stress in skeletal muscle.

Animals↗
PubMed1998

Helper T cell differentiation is controlled by the cell cycle.

Helper T (Th) cell differentiation is highly regulated by cytokines but initiated by mitogens. By examining gene expression in discrete generations of dividing cells, we have delineated the relationship between proliferation and differentiation. Initial expression of IL-2 is cell cycle-independent, whereas effector cytokine expression is cell cycle-dependent. IFNgamma expression increases in frequency with successive cell cycles, while IL-4 expression requires three cell divisions. Cell cycle progression and cytokine signaling act in concert to relieve epigenetic repression and can be supplanted by agents that hyperacetylate histones and demethylate DNA. Terminally differentiated cells exhibit stable epigenetic modification and cell cycle-independent gene expression. These data reveal a novel mechanism governing Th cell fate that initially integrates proliferative and differentiative signals and subsequently maintains stability of the differentiated state.

Animals↗
PubMed1998

The glio-toxic mechanism of alpha-aminoadipic acid on cultured astrocytes.

The mechanism of action of the glutamate analogue alpha-aminoadipic (AAA) acid was investigated in terms of its toxicity to cultured astrocytes. AAA was more toxic to type 1 astrocytes than type 2 astrocytes. Also the higher toxicity of the L-isomer as compared to the D-isomer was seen on type 1 astrocytes but not type 2. The toxicity of AAA can be reduced by co-culture of type 1 astrocytes with microglia. This inhibition may be due to glutamate release by microglia. No such effect is seen for type 2 astrocytes. The major uptake route for AAA by type 1 astrocytes is through the sodium dependent glutamate port. Both isomers of AAA are toxic to dividing astrocytes. The D-isomer appears to be toxic only for mitotic cells. The mechanism of this toxicity is protein synthesis dependent. It is suggested that AAA is toxic to mitotic astrocytes by interference with protein synthesis needed for cell division. D-AAA as opposed to L-AAA may prove a valuable tool for investigation of astrocyte proliferation in development and disease.

2-Aminoadipic Acid↗
PubMed1998

Delta-opioid receptor mRNA expression and immunohistochemical localization in porcine ileum.

Opiates have potent antidiarrheal actions that are mediated in part by delta-opioid receptors (DOR). We examined DOR localization within subregions of porcine ileum, a tissue analogous to human small bowel. A partial cDNA sequence for porcine DOR was obtained after reverse transcription-polymerase chain reaction cloning of forebrain RNA; it encoded the end of transmembrane domain 1 through the beginning of transmembrane domain 7 and exhibited 93% nucleotide identity with human DOR. Positive signals for DOR mRNA were found in all subregions of the porcine ileal wall. With an antiserum recognizing an N-terminal epitope in murine DOR, DOR-like immunoreactivity was localized in neurons within myenteric and submucous ganglia, longitudinal and circular smooth muscle, and villous lamina propria. The DOR agonist [D-Ser2, Leu5, Thr6]enkephalin (DSLET) attenuated circular smooth muscle contractions in porcine ileum that were evoked by electrical stimulation of myenteric cholinergic neurons. These results are consistent with previous reports of the DOR-mediated neuromodulation that underlies the antipropulsive and antisecretory effects of opioids in the intestinal tract.

Animals↗
PubMed1998

Effects of copper on survival of prion protein knockout neurons and glia.

The N-terminal region of the prion protein (PrP) contains an octameric repeat region suggested to bind copper. A 32-amino acid peptide (PrPOcta) based on this region in the protein was tested for its effects on cultured cerebellar cells. Cerebellar cells from mice deficient in cellular PrP (Prnp0/0 mice) are more sensitive to copper toxicity and oxidative stress. PrPOcta selectively promotes the survival of Prnp0/0 cerebellar cells. However, PrPOcta also reduces the toxicity of CuSO4 on cerebellar cells and abolishes the difference in increased sensitivity of Prnp0/0 cells to both copper toxicity and also oxidative stress from xanthine oxidase. PrPOcta does not promote the survival or proliferation of astrocytes or microglia. The survival-promoting effects of PrPOcta on neurons may be due to its ability to effectively chelate copper. The octameric repeat region of PrP may represent a functional domain of the native protein.

Animals↗
PubMed1998

Role of microglia in neuronal cell death in prion disease.

To elucidate the role played by the prion protein in scrapie pathogenesis, we performed experiments with PrP27-30 isolated from scrapie-infected hamster brains in cell culture and studied in vivo the temporal and spatial correlation between deposition of the disease-associated isoform of the prion protein (PrPSc), microglial activation and neuronal cell death in mice infected with scrapie strains 79A, ME7 and RML. The results presented here show that cellular expression of PrPc and the presence of microglia are necessary for the neurotoxicity of PrPSc in vitro. In vivo, accumulation of protease-resistant prion protein was detected early in the incubation period using the histoblot technique. Microglial activation was also detected early in the incubation period of all models studied. Both the time course and the spatial distribution of microglial activation closely resembled the pattern of PrPSc deposition. Microglial activation clearly preceded the detection of apoptotic neuronal cell death which was assessed using the in situ end-labeling technique (ISEL). Taken together, our results indicate that microglial activation is involved in the neurotoxicity of PrPSc both in vitro and in vivo.

Animals↗
PubMed1998

Roles of cardiac output and peripheral resistance in mediating blood pressure response to stress in rats.

The change in arterial blood pressure (BP) in response to presentation of an acute behavioral stress (i.e., classical conditioning) in rat includes an initial rapid rise (C1) followed by a delayed, but more sustained, pressor response (C2). The purpose of this experiment is to determine the patterns of change in cardiac output (CO) and total peripheral vascular resistance (TPR) that are associated with the behaviorally induced pressor response. A blood flow probe was implanted around the ascending aorta, and a catheter was implanted in a femoral artery in 10 male Sprague-Dawley rats. The rats were trained by a 15-s tone (CS+) followed by a 0.5-s tail shock; another tone (CS-), never followed by shock, served as a behavioral control. BP responded to the stressful stimulus (CS+) by a rapid C1 increase (8 +/- 1 mmHg; mean +/- SE) followed by the delayed C2 response (2 +/- 0.3 mmHg); the unconditioned response to shock was a 9 +/- 2 mmHg increase in BP. The C1 BP increase produced a significant increase in TPR (10 +/- 1 dyn.s/cm5); CO was not significantly changed. TPR decreased during C2 (-4 +/- 2 dyn.s/cm5), whereas CO was significantly increased (2 +/- 1 ml/min). These data contribute to our understanding of how the autonomic nervous system organizes the cardiovascular response to a suddenly perceived behavioral stress.

Animals↗
PubMed1998

Ablation of posterior atrial ganglionated plexus potentiates sympathetic tachycardia to behavioral stress.

The role of the posterior atrial ganglionated plexus (PAGP) in heart rate (HR) control was tested in unanesthetized dogs (n = 8). Resting HR was unchanged before (85 +/- 20 beats/min, mean +/- SD) versus after (87 +/- 18 beats/min) surgical ablation of these intrinsic cardiac ganglia (PAGPX). However, the peak tachycardia to a 30-s stressful stimulus was significantly increased (P < 0.05) from +53 +/- 22 beats/min before the denervation to +77 +/- 13 beats/min after PAGPX. Conversely, the peak HR increase during the stress after beta-adrenergic blockade was the same before (36 +/- 24 beats/min) versus after (38 +/- 14 beats/min) PAGPX. Moreover, the HR response to a neutral behavioral stimulus, which is mediated primarily by withdrawal of parasympathetic inhibition of the sinoatrial (SA) node, was unaltered by PAGPX. Thus the augmented tachycardia subsequent to PAGPX was attributable primarily to increased sympathetic action at the SA node. These findings indicate that a major role of PAGP parasympathetic neurons is to inhibit sympathoexcitatory effects on HR, probably either via interactions between neurons comprising the intrinsic plexus(es) or perhaps via presynaptic inhibition of sympathetic neurotransmitter release. This organization would allow parasympathetic ganglia within the PAGP to selectively modify sympathetic input to the SA node independent of direct vagal inhibition of pacemaker activity.

Adrenergic beta-Antagonists↗