Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.

NMR investigations of the structural properties of the nodulation protein, NodF, from Rhizobium leguminosarum and its homology with Escherichia coli acyl carrier protein.

Heteronuclear NMR methods have been used to elucidate the secondary structure and the general tertiary fold of the protein NodF from Rhizobium leguminosarum. A similarity to acyl carrier proteins of the fatty acid synthase system had been suggested by the presence of a phosphopantetheine prosthetic group and a short stretch of sequence homology near the prosthetic group attachment site. NMR results suggest that the structural homology extends well beyond this region. Both proteins have three well-formed helices which fold in a parallel-antiparallel fashion and a prosthetic group attachment site near the beginning of the second helix.

Electron spin-nuclear spin cross-correlation effects on multiplet splittings in paramagnetic proteins.

The effects of cross-correlation between Curie spin-nuclear dipole and nuclear dipole-nuclear dipole interactions on the linewidths and resonance frequencies of the individual lines of an AX multiplet in paramagnetic systems have been calculated. The implication of the relaxation-induced frequency shift of the lines (dynamic frequency shift) for the accurate measurement of residual dipolar couplings in field-oriented systems has been discussed. Our simulations indicate that these effects may play a role in the precise measurement of residual dipolar couplings in systems which belong to the small and intermediate tumbling regime, i.e., correlation times less than 5 ns.

Tilt angle dependence of cross-relaxation in off-resonance ROESY.

We present an efficient experimental method to evaluate whether the effective cross-relaxation rate between a pair of spins vanishes when applying an off-resonance spin-lock field. It is shown that the cross-relaxation rate can be made to vanish even when the two spins concerned resonate at different offsets and experience significantly different tilt angles of their respective spin-lock fields. This is verified experimentally using a sample of 15N-labeled human ubiquitin, through selective excitation of chosen amide protons. The results are relevant for the quantitative interpretation of off-resonance ROESY experiments.

Improved estimation of CSA-dipolar coupling cross-correlation rates from laboratory-frame relaxation experiments.

We have investigated the underlying assumptions in estimating cross-correlation rates between chemical shift anisotropy (CSA) and dipolar coupling mechanisms in a scalar-coupled two-spin IS system, from laboratory frame relaxation experiments. It has ben shown that for an arbitrary relaxation delay, the difference in relaxation rates of the individual components of an in-phase (or antiphase) doublet is not related to the CSA-dipolar coupling cross-correlation rate in a simple way. This is especially true in the case where the difference in the decay rates of the in-phase and antiphase terms of the density matrix becomes comparable to the magnitude of the scalar coupling between the two spins. Improved means of extracting cross-correlation rates in these cases are presented.

Determination of the rotational diffusion tensor of macromolecules in solution from nmr relaxation data with a combination of exact and approximate methods--application to the determination of interdomain orientation in multidomain proteins.

In this paper we present a method for determining the rotational diffusion tensor from NMR relaxation data using a combination of approximate and exact methods. The approximate method, which is computationally less intensive, computes values of the principal components of the diffusion tensor and estimates the Euler angles, which relate the principal axis frame of the diffusion tensor to the molecular frame. The approximate values of the principal components are then used as starting points for an exact calculation by a downhill simplex search for the principal components of the tensor over a grid of the space of Euler angles relating the diffusion tensor frame to the molecular frame. The search space of Euler angles is restricted using the tensor orientations calculated using the approximate method. The utility of this approach is demonstrated using both simulated and experimental relaxation data. A quality factor that determines the extent of the agreement between the measured and predicted relaxation data is provided. This approach is then used to estimate the relative orientation of SH3 and SH2 domains in the SH(32) dual-domain construct of Abelson kinase complexed with a consolidated ligand.

Origin of the correlation time dependence of coherence transfer distortions in rotating frame cross-relaxation spectra.

HOHAHA distortions in conventional (CW) ROESY experiments are known to be prominent when the frequency of the spin-lock field is near the midpoint between the resonance frequencies of a pair of coupled spins. That the disappearance of these distortions with offset from the midpoint is more rapid for large molecules than smaller ones is less widely known, and less well understood. We provide a quantitative explanation of the latter phenomenon using a combination of theory and numerical simulations. The cause of this effect can be found in the differential relaxation of the various magnetization modes which are involved in HOHAHA transfer. These modes experience enhanced relaxation far from a Hartmann-Hahn match. This enhancement is larger for molecules which have long correlation times.

Measurement of cross correlation between dipolar coupling and chemical shift anisotropy in the spin relaxation of 13C, 15N-labeled proteins.

We present a simple method for extracting interference effects between chemical shift anisotropy (CSA) and dipolar coupling from spin relaxation measurements in macromolecules, and we apply this method to extracting cross-correlation rates involving interference of amide 15N CSA and 15N-1H dipolar coupling and interference of carbonyl 13C' CSA and 15N-13C' dipolar coupling, in a small protein. A theoretical basis for the interpretation of these rates is presented. While it proves difficult to quantitatively separate the structural and dynamic contributions to these cross-correlation rates in the presence of anisotropic overall tumbling and a nonaxially symmetric chemical shift tensor, some useful qualitative correlations of data with protein structure can be seen when simplifying assumptions are made.

Offset profiles of selective pulses in isotopically labeled macromolecules.

The experimental verification of offset profiles and calibration of selective pulses in NMR is usually carried out with doped water samples but not under conditions typical of macromolecules with short T2, long T1, and possibly homo- and heteronuclear couplings. A new method for selective excitation in isotopically labeled macromolecules is shown to be particularly suited to this purpose. This is illustrated for a backbone amide resonance in a sample of 15N-labeled human ubiquitin.

Rapid onset of action of oral labetalol in severe hypertension.

Nine patients with persistently elevated diastolic blood pressure, exceeding 130 mmHg, received an oral dose of labetalol ranging from 200 mg to 400 mg. In all patients, this produced a significant and gradual decline in recumbent systolic and diastolic blood pressure, unaccompanied by symptoms or complications, with only minor changes in heart rate. Individual variation in the rate of blood pressure fall suggested differences in absorption and first-pass metabolism. It is suggested that the efficacy of oral labetalol merits further investigation in the rapid control of severe hypertension.

The effect of histidine on the structure and antitumor activity of metal-5-halouracil complexes.

The ternary complexes of Mn(II), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) ions with 5-halouracils, viz., 5-fluorouracil (5FU), 5-chlorouracil (5ClU), and 5-bromouracil (5BrU), and the biologically important ligand L-histidine (HISD) have been synthesized and characterized by elemental analysis, conductance measurements, infrared spectra, electronic spectra, and magnetic moment (room temperature) measurements. On the basis of these studies, the structures of the complexes have been proposed. All these ternary complexes were screened for their antitumor activity against Dalton's lymphoma in C3H/He mice. It was found that only Mn(II)-5BrU-HISD, Co(II)-5BrU-HISD, Cu(II)-5ClU-HISD, Cu(II)-5BrU-HISD, Zn(II)-5FU-HISD, and Zn(II)-5BrU-HISD complexes have significant antitumor activity with T/C greater than 125% (where T and C represent mean lifespan of treated mice and control mice respectively). The Mn(II)-5FU-HISD, Co(II)-5FU-HISD, Co(II)-5ClU-HISD, Ni(II)-5ClU-HISD, Ni(II)-5BrU-HISD, and Zn(II)-5ClU-HISD complexes are also effective antitumor agents, with T/C greater than 115%. The complexes that showed effective antitumor action in vivo were also found to inhibit 3H-thymidine incorporation (DNA replication) in Dalton's lymphoma cells in vitro.

Post-gastrectomy bone disease undiagnosed for forty years.

Polya partial gastrectomy was performed for peptic ulcer in a previously healthy woman aged 28 years. She complained afterwards of a variety of non-specific symptoms including weakness, tiredness, debility, slowness of walking, poor appetite and constipation. Within ten years her back became bent. She was treated for intercurrent hypertension and epilepsy. Bone fractures on low-impact trauma occurred in her fifties. At 57 years, she was unable to care for herself and had to be admitted to a nursing home. She could still walk slowly with the aid of a stick. For three months at the age of 65 years, she was unable to rise from her chair. Investigations disclosed severe post-gastrectomy bone disease. At no time had she complained of bone pains.

Pharmacokinetics of oxytetracycline in presence of calcium gluconate in goats.

The concentration of oxytetracycline (OTC) in plasma, after single dose i.v. administration at 10 mg kg-1, was determined during pre and post induced-hypercalcemia in goats. The pharmacokinetic variables were then calculated. Hypercalcemia caused several changes in the determined variables. The CPmax and CPmin of OTC observed at 0.08 and 4 hr in normal goats were respectively 34.50 +/- 1.65 and 1.19 +/- 0.14 micrograms ml-1, while the CPmax and CPmin of OTC in presence of calcium at 0.08 and 8 hr were 20.81 +/- 2.18 and 1.04 +/- 0.05 micrograms ml-1 respectively. Hypercalcemic state in goats increased t1/2 (alpha) (0.19 +/- 0.02 hr), t1/2 (beta) (2.77 +/- 0.03 hr), AUC (37.67 +/- 0.83 micrograms x hr x ml vd (area) (1.07 +/- 0.03 L kg-1) and vd (ss) (0.95 +/- 0.04 L kg-1) values of OTC compared to normal goats. The semilogarithmic plot of plasma level-time profile of OTC administered i.v. showed biphasic decline suggestive of two compartment open model 'kinetics' in both normal and hypercalcemic animals.

Hypokalaemic paralysis in remote aboriginal communities.

Increasing weakness occurring over several days, involving the upper and lower limbs, and progressing to symmetrical paresis is a clinical presentation that raises many diagnostic issues. When faced with these circumstances most clinicians opt for a range of neurological possibilities. However, metabolic possibilities are equally important and are eminently treatable. There are a variety of reasons why potassium depletion presents as paralysis in Aboriginal Australians. The cause may not be apparent, although careful questioning may unearth clues that can lead to a diagnosis. Once the potassium level has been measured in the blood the diagnosis becomes obvious, but until that time the presentation can be confusing. Early serum potassium measurement is recommended with this clinical picture.

A blueprint for reducing turnover among nursing assistants: a Louisiana study.

For decades, various exposes and reports have painted an unflattering portrait of the nursing home industry across the nation. Nursing homes in Louisiana have endured their fair share of publicity and criticism. The industry in this state has been accused of being preoccupied with profits rather than quality resident care. And, while there is much debate as to the validity of this complaint, there is solid agreement that competent and stable nursing assistants are the key to quality resident care. Unfortunately, the annual turnover rate of these essential employees ranges from 50% to 400%, nationally. This research identified the factors most responsible for the rate of turnover of nursing assistants employed in Louisiana nursing homes. Based upon the results of this study, pay, benefits, workload, and employee-employer relations, are not related to turnover. The analysis revealed that only three issues are associated with turnover--the number of beds, the number of beds per registered nurse, and the number of beds per social service worker. The message is clear: nursing home administrators must be very careful in stretching such resources. The number of beds assigned to an RN, and, in particular, the number of beds per social service worker are management issues that, if overextended, risk the turnover of nursing assistants.

CYP3A-dependent drug metabolism is reduced in bacterial inflammation in mice.

BACKGROUND AND PURPOSE: Gene expression of Cyp3a11 is reduced by activation of Toll-like receptors (TLRs) by Gram-negative or Gram-positive bacterial components, LPS or lipoteichoic acid (LTA) respectively. The primary adaptor protein in the TLR signalling pathway, TIRAP, plays differential roles in LPS- and LTA-mediated down-regulations of Cyp3a11 mRNA. Here, we have determined the functional relevance of these findings by pharmacokinetic/pharmacodynamic (PK/PD) analysis of the Cyp3a substrate midazolam in mice. Midazolam is also metabolized by Cyp2c in mice. EXPERIMENTAL APPROACH: Adult male C57BL/6, TIRAP+/+ and TIRAP-/- mice were pretreated with saline, LPS (2 mg·kg⁻¹) or LTA (6 mg·kg⁻¹). Cyp3a11 protein expression, activity and PK/PD studies using midazolam were performed. KEY RESULTS: Cyp3a11 protein expression in LPS- or LTA-treated mice was reduced by 95% and 60% compared with saline-treated mice. Cyp3a11 activity was reduced by 70% in LPS- or LTA-treated mice. Plasma AUC of midazolam was increased two- to threefold in LPS- and LTA-treated mice. Plasma levels of 1'-OHMDZ decreased significantly only in LTA-treated mice. Both LPS and LTA decreased AUC of 1'-OHMDZ-glucuronide. In the PD study, sleep time was increased by ∼2-fold in LPS- and LTA-treated mice. LTA-mediated decrease in Cyp3a11 protein expression and activity was dependent on TIRAP. In PK/PD correlation, AUC of midazolam was increased only in LPS-treated mice compared with saline-treated mice. CONCLUSIONS AND IMPLICATIONS: LPS or LTA altered PK/PD of midazolam. This is the first study to demonstrate mechanistic differences in regulation of metabolite formation of a clinically relevant drug by Gram-negative or Gram-positive bacterial endotoxins.

Bystander CPR in south east Scotland increases over 16 years.

BACKGROUND: Out-of-hospital cardiac arrest (OHCA) remains a leading cause of mortality and serious neurological disability across Europe. Without immediate bystander cardiopulmonary resuscitation (CPR), chances of survival are minimal. Despite community initiatives to increase the number of trained CPR providers, the effectiveness of these measures remains unknown and the proportion of OHCA patients receiving bystander CPR in the United Kingdom yet to be established. We sought to identify the change in the rate of bystander CPR in south east Scotland over a 16-year period. METHODS: Retrospective cohort study of all adult non-traumatic OHCA in south east Scotland from 1 January 1992 to 31 December 2007 using the Heartstart Scotland database. RESULTS: 7928 OHCA were included. The proportion of patients receiving bystander CPR increased from 34% in 1992 to 52% in 2007 (p for trend <0.0001). The rate of CPR from bystanders, spouses and from relatives increased significantly over the study period. Patients arresting at home received significantly less bystander CPR than those arresting away from home (39% vs 52%, p<0.0001) regardless of age or sex. CONCLUSION: There has been a significant increase in bystander CPR in south east Scotland during the 16-year period. Bystander CPR is associated with an increased rate of survival and targeted CPR training for relatives of patients at risk of sudden cardiac death may be beneficial.