A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs.

Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as KD, kon, koff, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand-p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in Tm values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal.

Controlling and monitoring polymyositis under steroid therapy.

This paper illustrates how considerable information may be extracted from the easily obtained serum creatine kinase levels as a function of prednisolone intake in controlling and monitoring polymyositis effectively. A simple process is developed to enable a clinician to determine readily the optimum prednisolone intake to give the minimum average steroid levels within the patient with an effective reduction in the muscle damage arising from the polymyositis. As part of the process it is demonstrated how the effect of other drugs or natural products may be ascertained and monitored. The effects of specific Mannatech products are detailed as an example.

The effect of surgery and severe injury on blood vessels.

This paper evolves from our previous work that developed a blood vessel model based on the way in which the total tension within the blood vessel wall varies with pressure due to the interaction of specific ions. We use the previous outcomes to examine the diameter of rat middle cerebral arteries (MCAs) as a function of pressure. The MCAs were isolated at 2 h, 1 and 5 days following sham injury and severe traumatic brain injury (TBI). First, we are able to quantify the diameter versus pressure curve in a way that yields three parameters. One of these parameters is the diameter at zero pressure that incorporates properties of the blood vessel walls and the vascular smooth muscle cells. The other two parameters are associated with the myogenic response and the myogenic tone. These parameters enable us to characterize, from the MCAs as a function of pressure in a calcium buffer, each blood vessel into one of three distinct distributions. One of these distributions reflects the optimum conditions. From our data with our blood vessel model, we demonstrate the effect of sham injury on the way in which blood vessels respond to pressure change that appears to recover over time with a half-life of about 40 h. In contrast, severe TBI greatly affects the blood vessel with no indication of recovery over the five-day monitoring period.

Potentiated endothelium-derived hyperpolarizing factor-mediated dilations in cerebral arteries following mild head injury.

Evidence in the literature suggests that endothelium-derived hyperpolarizing factor (EDHF) may act in a compensatory manner such that during conditions of compromised nitric oxide (NO), EDHF serves as a back-up mechanism. Given that constitutive NO synthase is chronically downregulated after head trauma, we tested the hypothesis that EDHF is potentiated following injury. Male adult rats were subjected to either sham injury (n = 27) or mild controlled cortical impact (CCI) injury (n = 26). Branches of the middle cerebral artery (MCA) directly within the contusion site were harvested either 1 or 24 h later, pressurized to 60 mm Hg in a vessel chamber and allowed to develop spontaneous tone. Relaxation to luminal application of adenosine triphosphate (ATP) was similar in all groups. Relaxation to ATP in the presence of L-NAME (N(G)-nitro-L-arginine methyl ester) and indomethacin was similar in all groups except for vessels isolated at 24 h following mild CCI injury. In this case, L-NAME and indomethacin had no effect on the ATP-mediated dilation. The ATP-mediated dilation in L-NAME and indomethacin-treated MCA branches was inhibited by charybdotoxin, an inhibitor of large conductance Ca2+-sensitive K+ channels. These findings suggest that there is a significant potentiation of the EDHF-mediated dilation to ATP in cerebral arteries isolated at 24 h following mild CCI injury.

An insight into cortisol and polymyositis control with steroid therapy.

In the present report we have developed a mathematical model to describe the processes involved during high steroid therapy. We have shown that the model can predict clinical observations as well as determine the optimum steroid regimen without relying on trial and error methods. The model incorporates rate processes that simplify the physiological complexity as a few representative steps. In this way, it is possible to simulate clinical observations and then to predict clinical outcomes due to drug concentrations or additional reactions within the body. Specific examples are provided from patients with polymyositis and Becker's disease. For the first time, we provide an explanation into the control of cortisol production, and determine an optimum prednisolone concentration in order to minimize steroid side effects while maintaining adequate control of muscle degradation.

Role of estrogen in modulating EDHF-mediated dilations in the female rat middle cerebral artery.

We tested the hypothesis that endothelium-derived hyperpolarizing factor (EDHF) plays a less dominant role in the female cerebrovasculature. The contribution of EDHF to the ATP-mediated dilation was determined in middle cerebral arteries (MCAs) isolated from male and female rats. Four groups of rats were tested: intact male (n = 12), intact female (n = 13), estrogen-treated ovariectomized female (n = 13), and vehicle-treated ovariectomized female (n = 20) rats. Maximal dilation to ATP was similar in all groups. However, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-5) M) and indomethacin (10(-5) M), the maximal dilation to ATP was significantly reduced in intact female (24 +/- 9%) and estrogen-treated ovariectomized female (29 +/- 9%) rats compared with intact male (95 +/- 4%) and vehicle-treated ovariectomized female (96 +/- 2%) rats. The ATP-mediated dilation in L-NAME- and indomethacin-treated MCAs isolated from male and ovariectomized female rats was inhibited by charybdotoxin (10(-7) M), an inhibitor of large-conductance Ca2+-sensitive K+ channels. We have defined EDHF as the L-NAME- and indomethacin-insensitive component of the ATP-mediated dilation. Our findings indicate that EDHF-mediated dilations are negligible in the female rat MCA; these dilations can be significantly enhanced after ovariectomy, suggesting that this effect is mediated by estrogen.

A locus affecting obesity in human chromosome region 10p12.

AIMS/HYPOTHESIS: Obesity is a complex trait influenced by multiple genes. We evaluated linkage in three regions of human chromosome 10 previously linked to obesity-related phenotypes. METHODS: We conducted non-parametric linkage analysis of obesity-related phenotypes in cohorts of 170 European-American and 43 African-American families having extremely obese and normal weight subjects. RESULTS: We found support for linkage of an obesity phenotype (BMI > or = 27 kg/m2) in both cohorts, as well as in a combined analysis (European-American cohort, Z = 1.90, p = 0.03; African-American cohort, Z = 2.25, p = 0.014; combined cohort, Z = 2.55, p = 0.005). CONCLUSION/INTERPRETATION: These results confirm previous reports of linkage in French and German families. The consistency of results across these four cohorts supports the localization of a quantitative trait locus influencing obesity to human chromosome region 10p12.

Nitric oxide in the potassium-induced response of the rat middle cerebral artery: a possible permissive role.

In the middle cerebral artery (MCA), the presence of nitric oxide (NO) is responsible for maintaining a more dilated state than in its absence during increases in extracellular K(+) and osmolality. The purpose of the present study was to determine whether the involvement of NO was due to (a) a direct effect of the K(+)/osmolality (K(hyper)) on the endothelium or (b) a 'permissive' role of NO. MCAs (approximately 210 microm o.d.) were isolated, cannulated with glass micropipettes, and pressurized to 85 mmHg. When K(+) (KCl) in the extraluminal bath was increased to 21 mM, the diameter increased by 15-20% with the magnitude of dilation diminishing with further increases in K(hyper). The addition of N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-5) mM), an inhibitor of nitric oxide synthase, had no significant effect on dilations at lower K(hyper) concentrations but constricted the arteries relative to the control at 51, 66, and 81 mM K(hyper). In the presence of L-NAME, the addition of an exogenous NO donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-8) M) or an analog of cGMP, 8-bromo-cGMP (6x10(-5) M), tended to restore the response of K(hyper)to near the original response. We conclude that the basal release of NO from the endothelium plays a permissive role in the K(hyper)-induced response.

Mathematical modelling of responses of cerebral blood vessels to changing intraluminal pressure.

The authors have designed a mathematical model to investigate the influences of the physical and chemical properties of the cerebral blood vessel resistance on vessel diameter. The model is based on the way the total tension within the blood vessel walls varies due to specific ions interacting and affecting the vascular smooth muscle cells and the vascular walls. In particular, we shall model a series of calcium sites and derive a generalized equation of the diameter as a function of pressure. The model includes the action of the vascular smooth muscle cells and the elasticity of the vascular walls, the pressure exerted on the walls by the blood and the effect of alterations to their properties within the blood vessel. They are formulated in terms of three parameters: the diameter at zero pressure, the myogenic response as the pressure tends to zero and a term associated with the myogenic tone. All three parameters may be reliably extracted from diameter-pressure measurements. The model was successfully used in quantifying diameter oscillations and dynamic myogenic responses that are frequently observed both in vivo and in vitro. Finally, we tested the model on experimental data obtained from the resistance of cerebral vessels that have been isolated from rats. In particular, we have first shown that the blood vessel characteristics are such that the diameter change due to calcium ion variations is at a maximum value. Second, we have shown that blood flow affects the myogenic response and third, we can explain the affect of ATP on the vessel diameter.

The effects of potassium on the rat middle cerebral artery.

After traumatic brain injury, extracellular K(+) in brain can dramatically increase. We studied the effects of increased K(+) on the isolated pressurized rat middle cerebral artery (MCA). MCAs (200-250 microm OD) were isolated, cannulated with glass micropipettes, and pressurized. K(+) was increased in the extraluminal bath using three paradigms: (1) isotonic K(+) (K(iso)) where increases in K(+) were offset by decreases in Na(+), (2) hypertonic K(+) (K(hyper)) where K(+) was increased without a concomitant adjustment of Na(+), and (3) K(suc), a solution using K(iso) but with the addition of sucrose to obtain a hypertonic solution. Increases in K(+) in the extraluminal bath produced significant dilations (approximately 20%) at 21 mM K(+) in all three groups (K(iso), K(hyper), and K(suc)). With the K(hyper) and K(suc) groups, the magnitude of the dilation diminished with further increases in K(+). L-NAME (10(-5) M), an inhibitor of nitric oxide synthase, had no effect on the response of the K(hyper) and K(suc) groups at 21 mM but significantly enhanced constrictions of the MCAs above 40 mM K(+) compared to the control. The K(iso) group was not affected by L-NAME at any K(+) concentration and showed profound constrictions above 40 mM K(+). We conclude that changes in the K(+) concentration and osmolality of the extracellular fluid may have profound effects on the cerebral vasculature.

L-arginine partially restores the diminished CO2 reactivity after mild controlled cortical impact injury in the adult rat.

Using an open cranial window technique, the authors investigated the mechanisms associated with the suppressed CO2 reactivity after mild controlled cortical impact (CCI) injury in rats. The dilation of arterioles (n = 7) to hypercapnia before injury was 38 +/- 12%, which was significantly reduced both at 1 hour (23 +/- 15% dilation) and at 2 hours after injury (11 +/- 19% dilation). In the presence of L-arginine (10 mmol/L topical suffusion, 300 mg/kg intravenous infusion), the dilation of pial arterioles (n = 6) to hypercapnia was partially restored to 30 +/- 6% at 2 hours after injury. In the presence of the nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine (SNAP) (10(-8) mol/L topical suffusion), the dilation of pial arterioles (n = 5) to hypercapnia remained diminished (5 +/- 7%) at 2 hours after injury. The dilation of pial arterioles (n = 4) to hypercapnia also remained suppressed (5 +/- 6%) with topical suffusion of the free radical scavengers, polyethylene glycol-superoxide dismutase (60 units/mL) and polyethylene glycol-catalase (40 units/mL). The authors have shown that L-arginine at least partially restores the diminished response to hypercapnia after mild CCI injury. Furthermore, these data suggest that the beneficial effects of L-arginine are mediated by a combination of providing substrate for NO synthase and scavenging free radicals.

Cerebrovascular reactivity to CO(2) and hypotension after mild cortical impact injury.

Cerebrovascular reactivity to CO(2) or hypotension was studied in vivo and in vitro [pressurized arteries ( approximately 82 micrometer) and arterioles ( approximately 30 micrometer)] at 1 h after mild controlled cortical impact (CCI) injury in rats. The cortical perfusion response [assessed using laser-Doppler flowmetry (LDF)] to altered CO(2) was diminished (up to 81%) after mild CCI injury. The responses to CO(2) alterations in arteries and arterioles isolated from the injured cortex were similar to responses in vessels isolated from sham-injured animals. After mild CCI injury, the autoregulatory response to hypotension (measured using LDF) was maintained or even enhanced, depending on the method used to measure the response. Vessels isolated from the injury site showed a response to changes in pressure similar to that in vessels isolated from sham-injured rats. We conclude that mild CCI injury produces complicated alterations in cerebrovascular control. Whereas the autoregulatory response to hypotension was maintained or even enhanced, the in vivo vascular response to CO(2) was severely compromised. The altered response to CO(2) was not caused by an intrinsic vascular perturbation but rather an altered milieu after mild CCI injury.

The consequences of traumatic brain injury on cerebral blood flow and autoregulation: a review.

In this decade, the brain argueably stands as one of the most exciting and challenging organs to study. Exciting in as far as that it remains an area of research vastly unknown and challenging due to the very nature of its anatomical design: the skull provides a formidable barrier and direct observations of intraparenchymal function in vivo are impractical. Moreover, traumatic brain injury (TBI) brings with it added complexities and nuances. The development of irreversible damage following TBI involves a plethora of biochemical events, including impairment of the cerebral vasculature, which render the brain at risk to secondary insults such as ischemia and intracranial hypertension. The present review will focus on alterations in the cerebrovasculature following TBI, and more specifically on changes in cerebral blood flow (CBF), mediators of CBF including local chemical mediators such as K+, pH and adenosine, endothelial mediators such as nitric oxide and neurogenic mediators such as catecholamines, as well as pressure autoregulation. It is emphasized that further research into these mechanisms may help attenuate the prevalence of secondary insults and therefore improve outcome following TBI.

Temporal effect of severe controlled cortical impact injury in the rat on the myogenic response of the middle cerebral artery.

The present study examined the effect of severe traumatic brain injury (TBI) on the myogenic response in the rat middle cerebral artery (MCA). Rats were subjected to severe controlled cortical impact (CCI; 5 m/sec, 130-msec duration, 3-mm deformation) injury over the right parietal cortex. At 2, 24, and 120 h postinjury, ipsilateral and contralateral segments of MCAs were isolated, mounted in a vessel chamber, and pressurized. After equilibration, the myogenic tone, the difference in vessel diameter in the presence and absence of calcium for a given pressure, and the myogenic response (the active contractile response elicited by a vessel to increasing pressure), were measured. At 24 h postsurgery, there was a significant interaction between myogenic tone and pressure in the ipsilateral and contralateral MCAs when TBI was compared to shams. However, this was not apparent, at the 2- and 120-h time points. At 2- and 24-h postsurgery, there was a significant interaction between myogenic response and pressure in the ipsilateral MCAs when TBI was compared to shams. While the response of the vascular smooth muscle was altered following injury, it was still functioning, suggesting that these vessels compensate, perhaps through alternate mechanisms or by relying on those remaining intact mechanisms.

Endothelial-mediated dilations following severe controlled cortical impact injury in the rat middle cerebral artery.

The mechanisms associated with dysfunction of the cerebral vasculature following head trauma have not yet been fully elucidated. In an attempt to shed more light on the matter, we investigated the endothelial-mediated dilations in the rat middle cerebral artery (MCA) following severe traumatic brain injury (TBI). Rats were subjected to severe controlled cortical impact injury (CCI; 5 m/s, 130 ms duration, 3 mm deformation) over the right parietal cortex. At 24 h postinjury, ipsilateral segments of MCA and corresponding contralateral segments were isolated, mounted in a vessel chamber, and pressurized. The responses to 2 methylthio-ATP (2MeSATP), a selective agonist for the P2Y1 purinoceptors, N(omega)-nitro-L-arginine (L-NAME), an NO synthase inhibitor, and S-nitroso-N-acetylpenicillamine (SNAP), an exogenous NO donor, were determined. 2MeSATP elicited concentration dependent dilations in all MCAs studied. Ipsilateral MCAs harvested following TBI or sham-TBI, showed similar maximum dilations to 2MeSATP [70 +/- 4% (n = 17) and 72 +/- 6% (n = 13), respectively]. However, TBI reduced the concentration of 2MeSATP necessary to elicit one-half of the maximum dilation (EC50) from 15 to 9 nM (p < 0.05). Inhibition of NO synthase with 10(-5) M L-NAME abolished the dilation to 2MeSATP in both TBI and sham-TBI MCAs. The constriction to L-NAME was significantly reduced in TBI MCAs compared to sham vessels. Dilations to SNAP, an NO donor, were not altered by TBI indicating that the mechanisms of dilation involving NO in the vascular smooth muscle were not affected. Unlike other pathological conditions which often diminish endothelial-mediated responses, severe TBI enhanced the sensitivity to 2MeSATP without altering the maximum response.

Comparison of the myogenic response in rat cerebral arteries of different calibers.

The myogenic response, the characteristic of blood vessels to contract with increasing pressure, was studied at three different locations along the middle cerebral artery (MCA) vascular tree. We hypothesized that smaller caliber vessels would have a more pronounced myogenic response at lower pressures than larger diameter arteries, corresponding to pressures normally experienced in vivo. Cerebral vessels (MCAs, branches of the MCA, and penetrating arterioles) were isolated from male rats, cannulated with glass micropipettes, and pressurized. Changes in diameter were measured as the transmural pressure was increased from 20-100 mmHg. The MCAs, which had a resting diameter of 202 +/- 10 micron (n = 9) at 50 mmHg, showed its greatest myogenic response between 60-100 mmHg (8+/-2% constriction, n = 9, p < 0.001). The penetrating arterioles [58 +/- 4 micron (n = 8) at 50 mmHg], on the other hand, showed its greatest myogenic response between 20-60 mmHg (10 +/- 4% constriction, n = 8, p < 0.05). Branches of the MCA [118 +/- 14 micron (n = 8) at 50 mmHg] showed a slight constriction over the entire pressure range (5 +/- 9% constriction between 20-100 mmHg, p=ns). Our results suggest that the myogenic response appears to be best developed in the range of pressures found during physiological conditions for a given vessel in the MCA territory. This characteristic is fundamental in the overall control of cerebrovascular resistance.

A mathematical assessment of the precision of parameters in measuring resonance spectra.

The accurate interpretation of in vivo magnetic resonance spectroscopy (MRS) spectra requires a complete understanding of the associated noise-induced errors. In this paper, we address the effect of complex correlated noise patterns on the measurement of a set of peak parameters. This is examined initially at the level of a single spectral analysis followed by addressing the noise-induced errors associated with determining the signal parameters from the peak parameters. We describe a relatively simple method for calculating these errors for any correlated noise pattern in terms of the noise standard deviation and correlation length. The results are presented in such a way that an estimate of the errors may be made from a single MRS spectrum. We also explore how, under certain circumstances, the lineshape of the signal may be determined. We then apply these results to reexamine a set of in vivo 31P MRS spectra obtained from rat brain prior to and following moderate fluid percussion injury. The approach outlined in this paper will demonstrate how meaningful results may be obtained from spectra where the signal-to-noise ratio (SNR) is quite small and where knowledge of the precise shape of the signal and the detail of the noise pattern is unknown. In essence, we show how to determine the expected errors in the spectral parameters from an estimate of the SNR from a single spectrum, thereby allowing a more discriminative interpretation of the data.

Continuous venovenous rewarming: results from a juvenile animal model.

OBJECTIVE: To compare a standard and an experimental method of rewarming in 5-wk-old goats with induced moderate hypothermia. DESIGN: Hypothermia was induced in ten juvenile Nubian goats. Five goats were randomly assigned to be rewarmed using standard techniques, and five were assigned to the experimental rewarming technique of a modified continuous venovenous hemofiltration circuit. SETTING: Animal research facility, Greenville Hospital System/Clemson University Biomedical Cooperative, Clemson, S.C. SUBJECTS: Ten 5-wk-old goats. INTERVENTIONS: Hypothermia to a body temperature of 29.4 degrees C was induced in the goats. Each of the control group of five goats was rewarmed using standard methods. Each of the experimental group of five goats was rewarmed using a modified continuous venovenous hemofiltration circuit. MEASUREMENTS AND MAIN RESULTS: At 2 hrs, the median temperature increase in the experimental group was 6.5 degrees C, compared with an increase of only 1.5 degrees C in the control group (p=.02). The mean increase in core body temperature over time (from baseline to 150 mins) was also significantly greater in the experimental group (p=.006). CONCLUSIONS: The use of a modified continuous venovenous hemofiltration circuit for rewarming in a juvenile goat model after induction of moderate hypothermia is more effective than are standard methods.

Similar incidence of K-ras mutations in lung carcinomas of FVB/N mice and FVB/N mice carrying a mutant p53 transgene.

Mutated p53 genes are capable of complementing activated ras genes in the transformation of primary rat embryo fibroblasts in vitro. Mutations in both genes have also been found in several human cancers, including lung carcinomas. We generated transgenic mice containing a p53 construct with a missense mutation in exon 5 (ala135val) to study the role of p53 mutations in lung tumorigenesis, and to facilitate identification of other genetic events that might complement p53 mutations in mouse lung carcinogenesis. The p53 transgenic lines exhibited a higher frequency of lethal lung tumors than the parental FVB/N strain. We examined the spontaneously-arising lung carcinomas from mice expressing the mutated p53 transgene for K-ras mutations using single-stranded conformation polymorphism (SSCP) and/or direct sequencing approaches. Fifteen of 29 (52%) carcinomas contained mutations in the K-ras oncogene. Six of 15 of the K-ras mutations were in codon 61 and 9/15 were in codon 12. Subsequent analysis of spontaneous lung carcinomas from mice of the FVB/N parental strain showed that 9/12 (75%) carcinomas examined contained K-ras mutations. Two of these were in codon 12, one in codon 13, and 6 were in codon 61. These results demonstrate that the frequency of ras mutations does not differ between the p53 FVB/N transgenic mice and their parental FVB/N strain but suggest that a high frequency of mutations K-ras can be correlated with lung tumorigenesis in both groups of mice.