Results: Commitment to publicize the work of the Task Force (Foundation Center) Effective methods... more Results: Commitment to publicize the work of the Task Force (Foundation Center) Effective methods to leverage change in eRA systems through foundations to their vendors “Buy in” on “Institutional Approval” – the need to require grantee institutional approval and signoff on all applications to a sponsor, was acknowledged for quality assurance of compliance issues and data provided. Major foundation staff persons agreed to serve as panelists for the NCURA Annual Meeting Task Force chairs invited to present to: Grants Managers Network – New York region in November 2003.
We have applied the Quartz Crystal Microbalance (QCM) technique to continuously record the proces... more We have applied the Quartz Crystal Microbalance (QCM) technique to continuously record the processes of endothelial cell (EC) adhesion, spreading and cellular mass distribution changes during initial cell to surface contact and homeostatic attachment. ECs (50,000) were layered onto a set volume of media in the QCM device and simultaneously in a mock cell used for photomicroscopy. As cells were observed in the mock cell device to contact and attach to the surface over 45-55 min, we measured in the QCM device a continuous decrease in frequency and continuous increase in resistance, achieving a maximum at about one hr (1400 Ω frequency change and 1400 Ω motional resistance change). These frequency and resistance values stabilized over the next 24 hrs and were unchanged out to 72 hr by QCM measurement (to ∼700 Hz, ∼700 Ω), as the cells were observed to spread in the mock device. Both bovine aortic (BAE) and bovine capillary (BCE) endothelial cells were studied and found to exhibit simil...
Different antisera raised against various regions of the human c-myc protein were used to identif... more Different antisera raised against various regions of the human c-myc protein were used to identify four human c-myc proteins with apparent molecular masses in sodium dodecyl sulfate-polyacrylamide gels ranging from 64 to 68 kilodaltons (phosphoproteins pp64 and pp67 and nonphosphorylated proteins p65 and p68). pp64 and p65 were the major detectable c-myc proteins, and pp67 and p68 were minor but specific components of the immunoprecipitates. The c-myc proteins were all localized in the cell nucleus. Accumulation of [35S]methionine-labeled p65 was observed after pulse-labeling and chase, suggesting that the stable p65 c-myc protein is generated posttranslationally from short-lived precursors. pp64, pp67, and p68 possessed short half-lives and may therefore be precursors of the stable p65. Confirmation of the nuclear localization of the human c-myc proteins was obtained by immunofluorescent staining. The human c-myc proteins were revealed as a pattern of punctate nuclear staining with...
The response of the microvasculature to ionizing radiation is thought to be an important factor i... more The response of the microvasculature to ionizing radiation is thought to be an important factor in the overall response of both normal tissues and tumours. It has recently been reported that basic fibroblast growth factor (bFGF), a potent mitogen for endothelial cells, protects large vessel endothelial cells from radiation-induced apoptosis in vitro. Microvessel cells are phenotypically distinct from large vessel cells. We studied the apoptotic response of confluent monolayers of capillary endothelial cells (ECs) to ionizing radiation and bFGF. Apoptosis was assessed by identifying changes in nuclear morphology, recording cell detachment rates and by detecting internucleosomal DNA fragmentation. Withdrawal of bFGF alone induces apoptosis in these monolayers. The magnitude of this apoptotic response depends upon the duration of bFGF withdrawal. Irradiation (2-10 Gy) induces apoptosis in a dose-dependent manner. Radiation-induced apoptosis occurs in a discrete wave 6-10 h after irradi...
It is known that radiation therapy results in some form of damage to the microcirculation. In sup... more It is known that radiation therapy results in some form of damage to the microcirculation. In support of this view, we found that capillary endothelial cells (EC) treated with X-rays (8 Gy) were defective in their ability to recover a denuded area. A scrape wound of 2 mm width was produced in monolayers 30 min after X-ray or sham treatment. After 48 h, the number of cells migrating into each of five successive 125 microns zones from both sides of the original wound were determined. Greater numbers of sham-treated EC entered zones 3 and 4, compared with irradiated cultures, and only sham-treated EC entered the most distant zone 5. We examined actin fibre orientation within migrating irradiated and sham-treated EC using 2-(D-2-aminobutanoic acid)-7-(N6-((((3,6-bis(dimethylamino)xanthylium-9-yl) carboxyphenyl) amino)thioxomethyl)-L-lysine), chloride (NBD)-phalloidin, immunofluorescent microscopy and computer image analysis. After 48 h, sham-treated, but not irradiated EC, contained act...
Normal endothelial cells (ECs), lining the blood vessels, are influenced by their interaction wit... more Normal endothelial cells (ECs), lining the blood vessels, are influenced by their interaction with the underlying potentially piezoelectric extracellular matrix (ECM). That this interaction may affect the EC metabolic state and functions in vivo prompted us to study the subsequent response of cultured ECs on indium-tin oxide (ITO) glass electrodes subjected to 1 hr of constant DC surface potential ranging from -0.3 to +0.6 V (vs. Ag/AgCl). We measured, relative to controls, cellular viability, growth rate and changes in actin microfilament organization in ECs over a subsequent 6 days in culture. The growth rate of ECs was stimulated by negative potential and inhibited by positive potential. Differences could be detected as early as three days post-potential. We also observed a potential dependent cellular shape change and actin microfilament rearrangement at positive potentials within four days of treatment. ECs changed in average cell surface area and assumed a polygonal cell shape...
Basic fibroblast growth factor (bFGF) and nitric oxide (NO) are expressed by endothelial cells (E... more Basic fibroblast growth factor (bFGF) and nitric oxide (NO) are expressed by endothelial cells (EC) and are involved in regulation of endothelial functions. In vivo, bFGF has a hypotensive effect which is mediated, in part, through activation of nitric oxide synthase (NOS) and the subsequent generation of NO. Thus we hypothesized that regulation of NOS in EC might be modulated by bFGF. bFGF treatment of EC in vitro resulted in increased NADPH diaphorase staining, a histochemical marker associated with the presence of NOS. Using cGMP generation in a reporter cell as a bioassay for NO release, we demonstrated that bFGF treatment of EC leads to increased production of biologically active NO. Furthermore, bFGF treatment of EC resulted in an increase in cellular content of the endothelial form of NOS as shown by Western blot analysis. Finally, Northern blot analysis was used to demonstrate that message levels of the constitutive, calcium-dependent, endothelial form of NOS is increased in...
We injected two drugs that modify the epigenome, the DNA methyltransferase inhibitor 5-aza-2'... more We injected two drugs that modify the epigenome, the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor trichostatin A (TSA), alone or in combination, into C57Bl/6 mice subjected to amputation through the mid-second phalanx of the third digit. Wound-site tissue was collected. We observed increased staining of the stem cell markers Rex1 (Zfp42) and stem cell antigen-1 at digit amputation sites from drug-treated mice. Samples from 5-aza-dC plus TSA and TSA treated mice also showed increased proliferating cell nuclear antigen staining, a measure of cell proliferation. Drug treatments increased Msx1, but not Cyp26a1 or ALDH1a2 (RALDH2) mRNA. 5-aza-dC and TSA treatments stimulated cell proliferation at the amputation site, possibly via increased expression of genes involved in digit development and regeneration.
Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to n... more Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to nanotoxicology and environmental health and safety is evaluating toxicity of ENMs before they become widely utilized. Cellular assays remain the predominant test platform yet these methods are limited by using discrete time endpoints and reliance on organic dyes, vulnerable to interference from ENMs. Label-free, continuous, rapid response systems with biologically meaningful endpoints are needed. We have developed a device to detect and monitor in real time responses of living cells to ENMs. The device, a living cell quartz crystal microbalance biosensor (QCMB), uses macrophages adherent to a quartz crystal. The communal response of macrophages to treatments is monitored continuously as changes in crystal oscillation frequency (Δf). We report the ability of this QCMB to distinguish benign from toxic exposures and reveal unique kinetic information about cellular responses to varying doses ...
We have previously reported that vitamin A inhibits the growth of capillary endothelial cells (EC... more We have previously reported that vitamin A inhibits the growth of capillary endothelial cells (EC) (Braunhut, S.J., and Palomares, M. (1991) Microvasc. Res. 41, 47-62). In this study, we have analyzed the effect of retinoids on the production and activity of enzymes involved in the proteolytic degradation of extracellular matrix (ECM) by EC. Substrate gel electrophoresis (zymography) revealed several major matrix metalloproteinases (MMP), of approximately 98-96, 72-68, and 46-45 kDa, whose activities were altered in their amounts in the conditioned media (CM) of EC following retinol or retinoic acid treatment when compared to amounts detected in CM of control cells. All of these gelatinases were inactivated by 1,10-phenanthroline, indicating that they were MMPs. MMP inhibitors (MMPI) were also present in these CM and were separated by gel filtration. Four distinct peaks of MMPIs were detected in the CM of EC. Chromatographic profiles indicated that an approximately 27-kDa MMPI was s...
Results: Commitment to publicize the work of the Task Force (Foundation Center) Effective methods... more Results: Commitment to publicize the work of the Task Force (Foundation Center) Effective methods to leverage change in eRA systems through foundations to their vendors “Buy in” on “Institutional Approval” – the need to require grantee institutional approval and signoff on all applications to a sponsor, was acknowledged for quality assurance of compliance issues and data provided. Major foundation staff persons agreed to serve as panelists for the NCURA Annual Meeting Task Force chairs invited to present to: Grants Managers Network – New York region in November 2003.
We have applied the Quartz Crystal Microbalance (QCM) technique to continuously record the proces... more We have applied the Quartz Crystal Microbalance (QCM) technique to continuously record the processes of endothelial cell (EC) adhesion, spreading and cellular mass distribution changes during initial cell to surface contact and homeostatic attachment. ECs (50,000) were layered onto a set volume of media in the QCM device and simultaneously in a mock cell used for photomicroscopy. As cells were observed in the mock cell device to contact and attach to the surface over 45-55 min, we measured in the QCM device a continuous decrease in frequency and continuous increase in resistance, achieving a maximum at about one hr (1400 Ω frequency change and 1400 Ω motional resistance change). These frequency and resistance values stabilized over the next 24 hrs and were unchanged out to 72 hr by QCM measurement (to ∼700 Hz, ∼700 Ω), as the cells were observed to spread in the mock device. Both bovine aortic (BAE) and bovine capillary (BCE) endothelial cells were studied and found to exhibit simil...
Different antisera raised against various regions of the human c-myc protein were used to identif... more Different antisera raised against various regions of the human c-myc protein were used to identify four human c-myc proteins with apparent molecular masses in sodium dodecyl sulfate-polyacrylamide gels ranging from 64 to 68 kilodaltons (phosphoproteins pp64 and pp67 and nonphosphorylated proteins p65 and p68). pp64 and p65 were the major detectable c-myc proteins, and pp67 and p68 were minor but specific components of the immunoprecipitates. The c-myc proteins were all localized in the cell nucleus. Accumulation of [35S]methionine-labeled p65 was observed after pulse-labeling and chase, suggesting that the stable p65 c-myc protein is generated posttranslationally from short-lived precursors. pp64, pp67, and p68 possessed short half-lives and may therefore be precursors of the stable p65. Confirmation of the nuclear localization of the human c-myc proteins was obtained by immunofluorescent staining. The human c-myc proteins were revealed as a pattern of punctate nuclear staining with...
The response of the microvasculature to ionizing radiation is thought to be an important factor i... more The response of the microvasculature to ionizing radiation is thought to be an important factor in the overall response of both normal tissues and tumours. It has recently been reported that basic fibroblast growth factor (bFGF), a potent mitogen for endothelial cells, protects large vessel endothelial cells from radiation-induced apoptosis in vitro. Microvessel cells are phenotypically distinct from large vessel cells. We studied the apoptotic response of confluent monolayers of capillary endothelial cells (ECs) to ionizing radiation and bFGF. Apoptosis was assessed by identifying changes in nuclear morphology, recording cell detachment rates and by detecting internucleosomal DNA fragmentation. Withdrawal of bFGF alone induces apoptosis in these monolayers. The magnitude of this apoptotic response depends upon the duration of bFGF withdrawal. Irradiation (2-10 Gy) induces apoptosis in a dose-dependent manner. Radiation-induced apoptosis occurs in a discrete wave 6-10 h after irradi...
It is known that radiation therapy results in some form of damage to the microcirculation. In sup... more It is known that radiation therapy results in some form of damage to the microcirculation. In support of this view, we found that capillary endothelial cells (EC) treated with X-rays (8 Gy) were defective in their ability to recover a denuded area. A scrape wound of 2 mm width was produced in monolayers 30 min after X-ray or sham treatment. After 48 h, the number of cells migrating into each of five successive 125 microns zones from both sides of the original wound were determined. Greater numbers of sham-treated EC entered zones 3 and 4, compared with irradiated cultures, and only sham-treated EC entered the most distant zone 5. We examined actin fibre orientation within migrating irradiated and sham-treated EC using 2-(D-2-aminobutanoic acid)-7-(N6-((((3,6-bis(dimethylamino)xanthylium-9-yl) carboxyphenyl) amino)thioxomethyl)-L-lysine), chloride (NBD)-phalloidin, immunofluorescent microscopy and computer image analysis. After 48 h, sham-treated, but not irradiated EC, contained act...
Normal endothelial cells (ECs), lining the blood vessels, are influenced by their interaction wit... more Normal endothelial cells (ECs), lining the blood vessels, are influenced by their interaction with the underlying potentially piezoelectric extracellular matrix (ECM). That this interaction may affect the EC metabolic state and functions in vivo prompted us to study the subsequent response of cultured ECs on indium-tin oxide (ITO) glass electrodes subjected to 1 hr of constant DC surface potential ranging from -0.3 to +0.6 V (vs. Ag/AgCl). We measured, relative to controls, cellular viability, growth rate and changes in actin microfilament organization in ECs over a subsequent 6 days in culture. The growth rate of ECs was stimulated by negative potential and inhibited by positive potential. Differences could be detected as early as three days post-potential. We also observed a potential dependent cellular shape change and actin microfilament rearrangement at positive potentials within four days of treatment. ECs changed in average cell surface area and assumed a polygonal cell shape...
Basic fibroblast growth factor (bFGF) and nitric oxide (NO) are expressed by endothelial cells (E... more Basic fibroblast growth factor (bFGF) and nitric oxide (NO) are expressed by endothelial cells (EC) and are involved in regulation of endothelial functions. In vivo, bFGF has a hypotensive effect which is mediated, in part, through activation of nitric oxide synthase (NOS) and the subsequent generation of NO. Thus we hypothesized that regulation of NOS in EC might be modulated by bFGF. bFGF treatment of EC in vitro resulted in increased NADPH diaphorase staining, a histochemical marker associated with the presence of NOS. Using cGMP generation in a reporter cell as a bioassay for NO release, we demonstrated that bFGF treatment of EC leads to increased production of biologically active NO. Furthermore, bFGF treatment of EC resulted in an increase in cellular content of the endothelial form of NOS as shown by Western blot analysis. Finally, Northern blot analysis was used to demonstrate that message levels of the constitutive, calcium-dependent, endothelial form of NOS is increased in...
We injected two drugs that modify the epigenome, the DNA methyltransferase inhibitor 5-aza-2'... more We injected two drugs that modify the epigenome, the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor trichostatin A (TSA), alone or in combination, into C57Bl/6 mice subjected to amputation through the mid-second phalanx of the third digit. Wound-site tissue was collected. We observed increased staining of the stem cell markers Rex1 (Zfp42) and stem cell antigen-1 at digit amputation sites from drug-treated mice. Samples from 5-aza-dC plus TSA and TSA treated mice also showed increased proliferating cell nuclear antigen staining, a measure of cell proliferation. Drug treatments increased Msx1, but not Cyp26a1 or ALDH1a2 (RALDH2) mRNA. 5-aza-dC and TSA treatments stimulated cell proliferation at the amputation site, possibly via increased expression of genes involved in digit development and regeneration.
Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to n... more Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to nanotoxicology and environmental health and safety is evaluating toxicity of ENMs before they become widely utilized. Cellular assays remain the predominant test platform yet these methods are limited by using discrete time endpoints and reliance on organic dyes, vulnerable to interference from ENMs. Label-free, continuous, rapid response systems with biologically meaningful endpoints are needed. We have developed a device to detect and monitor in real time responses of living cells to ENMs. The device, a living cell quartz crystal microbalance biosensor (QCMB), uses macrophages adherent to a quartz crystal. The communal response of macrophages to treatments is monitored continuously as changes in crystal oscillation frequency (Δf). We report the ability of this QCMB to distinguish benign from toxic exposures and reveal unique kinetic information about cellular responses to varying doses ...
We have previously reported that vitamin A inhibits the growth of capillary endothelial cells (EC... more We have previously reported that vitamin A inhibits the growth of capillary endothelial cells (EC) (Braunhut, S.J., and Palomares, M. (1991) Microvasc. Res. 41, 47-62). In this study, we have analyzed the effect of retinoids on the production and activity of enzymes involved in the proteolytic degradation of extracellular matrix (ECM) by EC. Substrate gel electrophoresis (zymography) revealed several major matrix metalloproteinases (MMP), of approximately 98-96, 72-68, and 46-45 kDa, whose activities were altered in their amounts in the conditioned media (CM) of EC following retinol or retinoic acid treatment when compared to amounts detected in CM of control cells. All of these gelatinases were inactivated by 1,10-phenanthroline, indicating that they were MMPs. MMP inhibitors (MMPI) were also present in these CM and were separated by gel filtration. Four distinct peaks of MMPIs were detected in the CM of EC. Chromatographic profiles indicated that an approximately 27-kDa MMPI was s...
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Papers by Susan Braunhut