Biomedical subjects

David M Keller

Publications and source records attributed to David M Keller.

15 recordsLinked to original sources

PubMed2005

CK2 phosphorylates SSRP1 and inhibits its DNA-binding activity.

We have previously shown that CK2 associates with the human high-mobility group protein SSRP1 and that this association increases in response to UV irradiation. CK2 also phosphorylates SSRP1 in vitro. Here we extend this work by investigating CK2 regulation of SSRP1 function through phosphorylation. Phosphorylation of SSRP1 by CK2 inhibited the nonspecific DNA-binding activity of SSRP1 and FACT (facilitating chromatin-mediated transcription) complex in vitro. Using a serine/threonine-scanning Auto-spot peptide array coupled with a filter-based kinase assay with synthetic peptides as substrates, we identified serines 510, 657, and 688 as phosphorylation targets of CK2 in vitro. Mutagenesis of the three serines revealed that serine 510 was more important for the regulation of SSRP1 DNA-binding activity. Furthermore, we found that SSRP1 was phosphorylated in cells in response to UV (but not gamma) irradiation. These results suggest that CK2 regulates the DNA-binding ability of SSRP1 and that this regulation may be responsive to specific cell stresses.

Casein Kinase II↗

PubMed2004

Dynamic cerebral autoregulation during exhaustive exercise in humans.

We investigated whether dynamic cerebral autoregulation is affected by exhaustive exercise using transfer-function gain and phase shift between oscillations in mean arterial pressure (MAP) and middle cerebral artery (MCA) mean blood flow velocity (V(mean)). Seven subjects were instrumented with a brachial artery catheter for measurement of MAP and determination of arterial Pco(2) (Pa(CO(2))) while jugular venous oxygen saturation (Sv(O(2))) was determined to assess changes in whole brain blood flow. After a 10-min resting period, the subjects performed dynamic leg-cycle ergometry at 168 +/- 5 W (mean +/- SE) that was continued to exhaustion with a group average time of 26.8 +/- 5.8 min. Despite no significant change in MAP during exercise, MCA V(mean) decreased from 70.2 +/- 3.6 to 57.4 +/- 5.4 cm/s, Sv(O(2)) decreased from 68 +/- 1 to 58 +/- 2% at exhaustion, and both correlated to Pa(CO(2)) (5.5 +/- 0.2 to 3.9 +/- 0.2 kPa; r = 0.47; P = 0.04 and r = 0.74; P < 0.001, respectively). An effect on brain metabolism was indicated by a decrease in the cerebral metabolic ratio of O(2) to [glucose + one-half lactate] from 5.6 to 3.8 (P < 0.05). At the same time, the normalized low-frequency gain between MAP and MCA V(mean) was increased (P < 0.05), whereas the phase shift tended to decrease. These findings suggest that dynamic cerebral autoregulation was impaired by exhaustive exercise despite a hyperventilation-induced reduction in Pa(CO(2)).

Adult↗

PubMed2004

Cell-type-specific binding of the transcription factor CREB to the cAMP-response element.

The cAMP-response element-binding protein (CREB) transcription factor was initially identified as a mediator of cAMP-induced gene expression. CREB binds to a target sequence termed the cAMP-response element (CRE) found in many cellular and viral gene promoters. One of the best-characterized CREs resides in the promoter of the gene encoding the neuropeptide somatostatin, and this element has served as a model for studies of CREB function. Phosphorylation of CREB by protein kinase A allows recruitment of the coactivator CREB-binding protein (CBP). A central tenet of the CREB-CBP model is that CREB binds constitutively to the CRE and that regulation occurs through the phosphorylation-dependent recruitment of CBP. In this report, we use chromatin immunoprecipitation assays to show that CREB does not interact in vivo with the somatostatin CRE, or similar elements in several other genes, in PC12 cells, a standard model for studies of CREB function. Rather, CREB binding in vivo is regulated in a cell-specific manner, a finding that was confirmed by using in vivo genomic footprinting assays. The CREs in other genes were also found to interact differentially with CREB in PC12 cells, hepatoma cells, and cortical neurons. We conclude that the family of CREB target genes differs from one cell type to another and that the ability of CREB to bind to a particular CRE represents an important component of gene regulation.

Animals↗

PubMed2004

Periods of intermittent hypoxic apnea can alter chemoreflex control of sympathetic nerve activity in humans.

Obstructive sleep apnea is associated with sustained elevation of muscle sympathetic nerve activity (MSNA) and altered chemoreflex control of MSNA, both of which likely play an important role in the development of hypertension in these patients. Additionally, short-term exposure to intermittent hypoxic apneas can produce a sustained elevation of MSNA. Therefore, we tested the hypothesis that 20 min of intermittent hypoxic apneas can alter chemoreflex control of MSNA. Twenty-one subjects were randomly assigned to one of three groups (hypoxic apnea, hypercapnic hypoxia, and isocapnic hypoxia). Subjects were exposed to 30 s of the perturbation every minute for 20 min. Chemoreflex control of MSNA was assessed during baseline, 1 min posttreatment, and every 15 min throughout 180 min of recovery by the MSNA response to a single hypoxic apnea. Recovery hypoxic apneas were matched to a baseline hypoxic apnea with a similar nadir oxygen saturation. A significant main effect for chemoreflex control of MSNA was observed after 20 min of intermittent hypoxic apneas (P <0.001). The MSNA response to a single hypoxic apnea was attenuated 1 min postexposure compared with baseline (P <0.001), became augmented within 30 min of recovery, and remained augmented through 165 min of recovery (P <0.05). Comparison of treatment groups revealed no differences in the chemoreflex control of MSNA during recovery (P=0.69). These data support the hypothesis that 20 min of intermittent hypoxic apneas can alter chemoreflex control of MSNA. Furthermore, this response appears to be mediated by hypoxia.

Adult↗

PubMed2004

Dynamic carotid baroreflex control of the peripheral circulation during exercise in humans.

We sought to determine the dynamic relationship between carotid baroreflex (CBR)-mediated control and local control of the skeletal muscle vasculature during dynamic exercise. In 12 subjects (18-35 years old), oscillatory neck pressure (NP, +40 mmHg) was applied at 0.1 Hz (i.e. 5 s on, 5 s off) for 5 min to determine the degree of CBR control over heart rate (HR), arterial blood pressure (ABP), muscle sympathetic nerve activity (MSNA), femoral blood velocity and skeletal muscle tissue oxygenation at rest and during 7 W dynamic knee-extension exercise. Skeletal muscle tissue oxygenation measurements of both the exercising and nonexercising leg were evaluated. Fast Fourier transformation was performed on 5 min segments to calculate spectral power of the R-R interval (RRI), ABP, MSNA, femoral blood velocity and tissue oxygenation time series, and the low-frequency (LF, 0.085-0.115 Hz) power spectra were compared to evaluate the degree of CBR-mediated entrainment for each variable. At rest, sinusoidal NP significantly increased LF spectral power of RRI, ABP, MSNA and femoral blood velocity. During exercise, sinusoidal NP provoked a similar increase in spectral power for RRI and MSNA, while CBR-mediated changes in ABP and femoral blood velocity were attenuated compared to rest. Changes in spectral power of skeletal muscle tissue oxygenation during sinusoidal NP were similar between the exercising and nonexercising leg at rest. However, during exercise the changes in skeletal muscle tissue oxygenation power were significantly less in the exercising leg, while changes in the nonexercising leg were similar to rest. We have demonstrated simultaneous entrainment of all CBR end-organ measurements, ranging from cardiac chronotropic effects to alterations at the level of the skeletal muscle microcirculation. Moreover, we have identified a significant and specific attenuation of end-organ responsiveness to CBR-mediated sympathoexcitation in the vasculature of the exercising muscle. However, despite a shift towards more predominant local control over the exercising muscle vasculature, systemic arterial blood pressure was well preserved.

Adolescent↗

PubMed2003

Noninvasive assessment of sympathetic vasoconstriction in human and rodent skeletal muscle using near-infrared spectroscopy and Doppler ultrasound.

The precise role of the sympathetic nervous system in the regulation of skeletal muscle blood flow during exercise has been challenging to define in humans, partly because of the limited techniques available for measuring blood flow in active muscle. Recent studies using near-infrared (NIR) spectroscopy to measure changes in tissue oxygenation have provided an alternative method to evaluate vasomotor responses in exercising muscle, but this approach has not been fully validated. In this study, we tested the hypothesis that sympathetic activation would evoke parallel changes in tissue oxygenation and blood flow in resting and exercising muscle. We simultaneously measured tissue oxygenation with NIR spectroscopy and blood flow with Doppler ultrasound in skeletal muscle of conscious humans (n = 13) and anesthetized rats (n = 9). In resting forearm of humans, reflex activation of sympathetic nerves with the use of lower body negative pressure produced graded decreases in tissue oxygenation and blood flow that were highly correlated (r = 0.80, P < 0.0001). Similarly, in resting hindlimb of rats, electrical stimulation of sympathetic nerves produced graded decreases in tissue oxygenation and blood flow velocity that were highly correlated (r = 0.93, P < 0.0001). During rhythmic muscle contraction, the decreases in tissue oxygenation and blood flow evoked by sympathetic activation were significantly attenuated (P < 0.05 vs. rest) but remained highly correlated in both humans (r = 0.80, P < 0.006) and rats (r = 0.92, P < 0.0001). These data indicate that, during steady-state metabolic conditions, changes in tissue oxygenation can be used to reliably assess sympathetic vasoconstriction in both resting and exercising skeletal muscle.

Adult↗

PubMed2003

Hypoxia-mediated prolonged elevation of sympathetic nerve activity after periods of intermittent hypoxic apnea.

Obstructive sleep apnea (OSA) is associated with transient elevation of muscle sympathetic nerve activity (MSNA) during apneic events, which often produces elevated daytime MSNA in OSA patients. Hypoxia is postulated to be the primary stimulus for elevated daytime MSNA in OSA patients. Therefore, we studied the effects of 20 min of intermittent voluntary hypoxic apneas on MSNA during 180 min of recovery. Also, we compared MSNA during recovery after either 20 min of intermittent voluntary hypoxic apneas, hypercapnic hypoxia, or isocapnic hypoxia. Consistent with our hypothesis, both total MSNA and MSNA burst frequency were elevated after 20 min of intermittent hypoxic apnea compared with baseline (P < 0.05). Both total MSNA and MSNA burst frequency remained elevated throughout the 180-min recovery period and were statistically different from time control subjects throughout this period (P < 0.05). Finally, MSNA during recovery from intermittent hypoxic apnea, hypercapnic hypoxia, and isocapnic hypoxia were not different (P = 0.50). Therefore, these data support the hypothesis that short-term exposure to intermittent hypoxic apnea results in sustained elevation of MSNA and that hypoxia is the primary mediator of this response.

Adult↗

PubMed2003

Developing culturally competent community faculty: a model program.

The Liaison Committee on Medical Education recently set standards for cultural diversity training as part of the medical school curriculum. To the authors' knowledge, this is the first description of a faculty-development program designed to develop the capacity of the clinical faculty to integrate culture and advocacy education into clinical training. The paper describes the first two years of the development of an ongoing cultural competence curriculum that has been integrated into the training of community preceptors from 13 medical schools in New England and New York. The training, entitled"Teaching the Culture of the Community," consists of four 2.5-hour modules that include interactive lectures and small-group role-play exercises on cultural needs assessment, patient-centered interviewing, feedback on cultural issues and use of the community to enhance cultural understanding. The 137 participants in the first two years of the program (1999-00 and 2000-01) reported a high level of acceptance of the curriculum. In the second year, the program began to document participants' self-reported"intention to change" in relation to the cultural competence curriculum. Many participants reported plans to change aspects of their clinical care and their teaching practices. Intentions to change were most frequently expressed in the context of content on effective communication skills. In summary, cultural competency training has been successfully integrated into an existing faculty-development program for community-based preceptors.

Clinical Competence↗

PubMed2003

Does pulsatile and sustained neck pressure or neck suction produce differential cardiovascular and sympathetic responses in humans?

Although square-wave sustained and R wave-triggered pulsatile stimuli have been used to assess carotid baroreflex (CBR) function in humans, it remains unclear whether these different stimulus protocols elicit comparable responses and whether CBR responses adapt during prolonged stimulation. Thus, we measured muscle sympathetic nerve activity (MSNA), heart rate (HR) and mean arterial pressure (MAP) in response to +30 Torr neck pressure (NP) and -30 Torr neck suction (NS) delivered for 20 s either as a sustained or pulsatile stimulus. CBR-mediated changes in MSNA, HR and MAP were similar with sustained and pulsatile stimuli. The time course of MSNA and HR responses identified that significant changes occurred during the initial 5 s and were better maintained over 20 s with NP than with NS. Changes in MAP exhibited a slower onset with the peak increase during NP occurring at 10 s (sustained, 7 +/- 1 mmHg; pulsatile, 7 +/- 1 mmHg; P > 0.05) and the nadir during NS occurring at 20 s (sustained, -7 +/- 1 mmHg; pulsatile, -9 +/- 2 mmHg; P > 0.05). These data demonstrate that sustained and pulsatile NP and NS produce comparable CBR-mediated responses. Furthermore, despite MSNA and HR returning towards baseline during NS, CBR-mediated changes in MAP are well maintained over 20 s of NS and NP.

Adaptation, Physiological↗

PubMed2003

Recent insights into carotid baroreflex function in humans using the variable pressure neck chamber.

The variable pressure neck chamber has provided an invaluable research tool for the non-invasive assessment of carotid baroreflex (CBR) function in human investigations. The ability to construct complete stimulus-response curves and define specific parameters of the reflex function curve permits statistical comparisons of baroreflex function between different experimental conditions, such as rest and exercise. Results have convincingly indicated that the CBR stimulus-response curve is reset during exercise in an intensity-dependent manner to functionally operate around the prevailing pressure elicited by the exercise workload. Furthermore, both at rest and during exercise, alterations in stroke volume do not contribute importantly to the maintenance of arterial blood pressure by the carotid baroreceptors, and therefore, any reflex-induced changes in cardiac output (Q) are the result of CBR-mediated changes in heart rate. However, more importantly, the CBR-induced changes in mean arterial pressure (MAP) are primarily mediated by alterations in vascular conductance with only minimal contributions from Q to the initial reflex MAP response. Thus, the capacity of the CBR to regulate blood pressure depends critically on its ability to alter vascular tone both at rest and during exercise. This review will emphasize the utility of the variable pressure neck chamber to assess CBR function in human experimental investigations and the mechanisms by which the CBR responds to alterations in arterial blood pressure both at rest and during exercise.

Baroreflex↗

PubMed2003

Interaction of p53 with cellular proteins.

Cellular proteins that interact with p53 play a major role in both positive and negative regulation of this tumor suppressor and can fine-tune its response to specific cellular stresses. As a consequence, p53 biology will not be complete until these interacting proteins are fully characterized. This chapter outlines two methods for identifying and characterizing p53-binding proteins: (i) glutathione-S-transferase (GST) protein-protein interaction assay; and (ii) co-immunoprecipitation (co-IP) assay. These two methods are ideal for any laboratory to perform; the assays are short in duration, do not require specialized expertise to establish in the laboratory, give reliable and reproducible data, and are cost-efficient, because few reagents are needed. This chapter gives a basic description of these two techniques and provides tips that are not found in other protocol manuals on how to achieve the best results.

Animals↗

PubMed2002

p53 serine 392 phosphorylation increases after UV through induction of the assembly of the CK2.hSPT16.SSRP1 complex.

Previously, we purified a UV-responsive p53 serine 392 kinase from F9 and HeLa cells and found that its activity is attributed to a high molecular weight protein complex containing the protein kinase CK2, along with the chromatin-associated factors hSPT16 and SSRP1. Here we determine that these proteins interact in vitro and in cells via non-overlapping domains and provide evidence consistent with the idea that hSPT16 and SSRP1 change the conformation of CK2 upon binding such that it specifically targets p53 over other substrates. Also, UV irradiation apparently induces the association of the complex, thereby increasing the specificity of CK2 for p53 at the expense of other cellular CK2 substrates and leading to an overall increase in p53 serine 392 phosphorylation.

Animals↗

PubMed2002

Carotid baroreflex control of leg vascular conductance at rest and during exercise.

We sought to test the hypothesis that the carotid baroreflex (CBR) alters mean leg blood flow (LBF) and leg vascular conductance (LVC) at rest and during exercise. In seven men and one woman, 25 +/- 2 (SE) yr of age, CBR control of LBF and LVC was determined at rest and during steady-state one-legged knee extension exercise at approximately 65% peak O(2) uptake. The application of 5-s pulses of +40 Torr neck pressure and -60 Torr neck suction significantly altered mean arterial pressure (MAP) and LVC both at rest and during exercise. CBR-mediated changes in MAP were similar between rest and exercise (P > 0.05). However, CBR-mediated decreases in LVC (%change) to neck pressure were attenuated in the exercising leg (16.4 +/- 1.6%) compared with rest (33 +/- 2.1%) and the nonexercising leg (23.7 +/- 1.9%) (P < 0.01). These data suggest CBR control of blood pressure is partially mediated by changes in leg vascular tone both at rest and during exercise. Furthermore, despite alterations in CBR-induced changes in LVC during exercise, CBR control of blood pressure was well maintained.

Adult↗

PubMed2002

SSRP1 functions as a co-activator of the transcriptional activator p63.

The p53 homolog p63 is a transcriptional activator. Here, we describe the identification of an HMG1-like protein SSRP1 as a co-activator of p63. Over expression of wild-type, but not deletion mutant, SSRP1 remarkably enhanced p63gamma-dependent luciferase activity, G1 arrest, apoptosis and expression of endogenous PIG3, p21(Waf1/cip1) and MDM2 in human p53-deficient lung carcinoma H1299 cells and mouse embryonic fibroblasts. Also, SSRP1 interacted to p63gamma in vitro and in cells, and resided with p63gamma at the p53-responsive DNA element sites of the cellular endogenous MDM2 and p21(Waf1/cip1) promoters. Moreover, N-terminus-deleted p63 (DeltaN-p63) bound to neither SSRP1 nor its central domain in vitro. Accordingly, SSRP1 was unable to stimulate DeltaN-p63-mediated residual luciferase activity and apoptosis in cells. Finally, the ectopic expression of the central p63-binding domain of SSRP1 inhibited p63-dependent transcription in cells. Thus, these results suggest that SSRP1 stimulates p63 activity by associating with this activator at the promoter.

Animals↗

PubMedDate not supplied

Toward the development of advocacy training curricula for pediatric residents: a national delphi study.

BACKGROUND: Training in child advocacy is now required in pediatric residency program curricula. No national consensus exists regarding the content of such advocacy training. OBJECTIVE: To identify an operational definition of advocacy, as well as knowledge, skills, and attitude objectives for advocacy training in pediatric residency programs. METHODS: Professionals experienced in pediatric advocacy and training (n = 53) were invited to participate in a sequence of surveys to define the content of a pediatric residency advocacy curriculum that would result in acquisition of appropriate knowledge, skills, and attitudes related to advocacy for children. Three rounds of surveys were distributed, collected, and analyzed using a modified Delphi technique, in which the results from an antecedent survey were used to refine responses in a subsequent survey. RESULTS: Participants (n = 36), comprising a group of experienced leaders with diverse training and experience in child advocacy and resident education, created a consensus definition for advocacy. They initially identified 179 possible objectives for advocacy curricula. Through the iterative process of the Delphi technique, 32 of those objectives were identified as necessary for inclusion in a child advocacy curriculum for pediatric residents. CONCLUSIONS: Using a modified Delphi technique, a group of experienced leaders in pediatric advocacy were able to reach consensus on an operational definition of child advocacy and a set of objectives for a resident advocacy curriculum. Programs may use these findings to assist in developing an advocacy curriculum based on their own faculty assets and community resources.

Adult↗