Postpartum aHUS secondary to a genetic abnormality in factor H acquired through liver transplantation.

We report here a young female who underwent a successful deceased donor liver transplant for hepatic vein thrombosis. Five years after transplantation she developed postpartum atypical hemolytic uremic syndrome (aHUS). She did not recover renal function. Mutation screening of complement genes in her DNA did not show any abnormality. Mutation screening of DNA available from the donor showed a nonsense CFH mutation leading to factor H deficiency. Genotyping of the patient showed that she was homozygous for an aHUS CD46 at-risk haplotype. In this individual, the development of aHUS has been facilitated by the combination of a trigger (pregnancy), an acquired rare genetic variant (CFH mutation) and a common susceptibility factor (CD46 haplotype).

Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II.

Akt activation supports survival of cardiomyocytes against ischemia/reperfusion, which induces cell death through opening of the mitochondrial permeability transition pore (PT-pore). Mitochondrial depolarization induced by treatment of cardiomyocytes with H(2)O(2) is prevented by activation of Akt with leukemia inhibitory factor (LIF). This protective effect is observed even when cardiomyocytes treated with LIF are permeabilized and mitochondrial depolarization is elicited by elevating Ca(2+). Cell fractionation studies demonstrate that LIF treatment increases both total and phosphorylated Akt in the mitochondrial fraction. Furthermore, the association of Akt with HK-II is increased by LIF. HK-II contains consensus sequences for phosphorylation by Akt and LIF treatment induces PI3K- and Akt-dependent HK-II phosphorylation. Addition of recombinant kinase-active Akt to isolated adult mouse heart mitochondria stimulates phosphorylation of HK-II and concomitantly inhibits the ability of Ca(2+) to induce cytochrome c release. This protection is prevented when HK-II is dissociated from mitochondria by incubation with glucose 6-phosphate or HK-II-dissociating peptide. Finally LIF increases HK-II association with mitochondria and dissociation of HK-II from mitochondria attenuates the protective effect of LIF on H(2)O(2)-induced mitochondrial depolarization in cardiomyocytes. We conclude that Akt has a direct effect at the level of the mitochondrion, which is mediated via phosphorylation of HK-II and results in protection of mitochondria against oxidant or Ca(2+)-stimulated PT-pore opening.

Problems in the coupling of eye and hand in the sequential movements of children with Developmental Coordination Disorder.

BACKGROUND: Shifting gaze and attention ahead of the hand is a natural component in the performance of skilled manual actions. Very few studies have examined the precise co-ordination between the eye and hand in children with Developmental Coordination Disorder (DCD). METHODS: This study directly assessed the maturity of eye-hand co-ordination in children with DCD. A double-step pointing task was used to investigate the coupling of the eye and hand in 7-year-old children with and without DCD. Sequential targets were presented on a computer screen, and eye and hand movements were recorded simultaneously. RESULTS: There were no differences between typically developing (TD) and DCD groups when completing fast single-target tasks. There were very few differences in the completion of the first movement in the double-step tasks, but differences did occur during the second sequential movement. One factor appeared to be the propensity for the DCD children to delay their hand movement until some period after the eye had landed on the target. This resulted in a marked increase in eye-hand lead during the second movement, disrupting the close coupling and leading to a slower and less accurate hand movement among children with DCD. CONCLUSIONS: In contrast to skilled adults, both groups of children preferred to foveate the target prior to initiating a hand movement if time allowed. The TD children, however, were more able to reduce this foveation period and shift towards a feedforward mode of control for hand movements. The children with DCD persevered with a look-then-move strategy, which led to an increase in error. For the group of DCD children in this study, there was no evidence of a problem in speed or accuracy of simple movements, but there was a difficulty in concatenating the sequential shifts of gaze and hand required for the completion of everyday tasks or typical assessment items.

Early dislocation after total hip arthroplasty: are postoperative restrictions necessary?

We studied prospectively 499 cases of primary total hip arthroplasty done through an anterolateral approach to establish the early dislocation rate when restrictions on postoperative mobilization were not imposed. There were 3 early dislocations (within 6 weeks of surgery). All were reduced closed, and every patient subsequently achieved a stable hip without further intervention. Our results suggest that a low early dislocation rate can be achieved using an anterolateral approach without the need to restrict patients' postoperative mobilization. It may not be appropriate, however, to remove these restrictions when using other surgical approaches to the hip.

Penetration of linezolid into bone, fat, muscle and haematoma of patients undergoing routine hip replacement.

Twelve patients undergoing total hip replacement were given 600 mg of linezolid as a 20 min iv infusion along with conventional prophylaxis of 1 g of cefamandole immediately before surgery. Routine total hip arthroplasty was carried out, and at timed intervals during surgery samples of bone, fat, muscle and blood were collected for assay by high-performance liquid chromatography analysis. Samples of the haematoma fluid that formed around the operation site and further blood samples for assay were also collected at timed intervals following the operation. The penetration of linezolid into bone was rapid, with mean concentrations of 9.1 mg/L (95% CI 7.7-10.6 mg/L) achieved at 10 min after the infusion, decreasing to 6.3 mg/L (95% CI 3.9-8.6 mg/L) at 30 min. Correction for the simultaneous blood concentrations gave mean values for bone penetration of 51% at 10 min, 60% at 20 min and 47% at 30 min. Although the penetration of linezolid into fat was also rapid, mean concentrations and degree of penetration were c. 60% of those in bone; at 10 min they were 4.5 mg/L (95% CI 3.0-6.1 mg/L; penetration 27%); at 20 min they were 5.2 mg/L (95% CI 4.0-6.4 mg/L; penetration 37%); and at 30 min, 4.1 mg/L (95% CI 3.3-4.8 mg/L; penetration 31%). For muscle the corresponding values were 10.4 mg/L (95% CI 8.1-12.7 mg/L; penetration 58%) at 10 min, 13.4 mg/L (95% CI 10.2-16.5 mg/L; penetration 94%) at 20 min and 12.0 mg/L (95% CI 9.2-14.8 mg/L; penetration 93%) at 30 min. Mean concentrations of linezolid in the haematoma fluid drained from around the operation site were 8.2 mg/L at 6-8 h and 5.6 mg/L at 10-12 h after the infusion, and 7.0 mg/L at 2-4 h following a second 600 mg infusion given 12 h post-operatively. We conclude that linezolid exhibits rapid penetration into bone, fat and muscle of patients undergoing hip arthroplasty, to achieve levels in excess of its MIC for susceptible organisms (< or=4 mg/L); therapeutic concentrations were maintained in the haematoma fluid that surrounds the operation site for >16 h.

Localization, cDNA sequence and genomic organization of the rat seipin gene (Bscl2) and sequence analysis in inbred rat models of Type 2 diabetes mellitus.

Mutations in the gene encoding seipin cause Berardinelli-Seip congenital lipodystrophy 2, with symptoms including near-absence of adipose tissue and altered glucose tolerance. Radiation hybrid analysis localized the seipin gene (Bscl2) in rat to a major quantitative trait locus in rat chromosome 1 linked to glucose intolerance in the Goto-Kakizaki (GK) rat model of Type 2 diabetes. We determined the genomic organization of Bscl2 and screened coding exons and flanking intron sequences for mutations in GK, Wistar and Brown Norway rats, as well as in the Otsuka Long-Evans Tokushima Fatty (OLETF) diabetic rat. Two silent single nucleotide polymorphisms that were identified also were found in non-diabetic rat strains. We conclude that mutations in the gene for seipin are unlikely to contribute to diabetes in GK and OLETF rats.

A general model for ontogenetic growth.

Several equations have been proposed to describe ontogenetic growth trajectories for organisms justified primarily on the goodness of fit rather than on any biological mechanism. Here, we derive a general quantitative model based on fundamental principles for the allocation of metabolic energy between maintenance of existing tissue and the production of new biomass. We thus predict the parameters governing growth curves from basic cellular properties and derive a single parameterless universal curve that describes the growth of many diverse species. The model provides the basis for deriving allometric relationships for growth rates and the timing of life history events.

Crystal structure of the central region of bovine fibrinogen (E5 fragment) at 1.4-A resolution.

The high-resolution crystal structure of the N-terminal central region of bovine fibrinogen (a 35-kDa E(5) fragment) reveals a remarkable dimeric design. The two halves of the molecule bond together at the center in an extensive molecular "handshake" by using both disulfide linkages and noncovalent contacts. On one face of the fragment, the Aalpha and Bbeta chains from the two monomers form a funnel-shaped domain with an unusual hydrophobic cavity; here, on each of the two outer sides there appears to be a binding site for thrombin. On the opposite face, the N-terminal gamma chains fold into a separate domain. Despite the chemical identity of the two halves of fibrinogen, an unusual pair of adjacent disulfide bonds locally constrain the two gamma chains to adopt different conformations. The striking asymmetry of this domain may promote the known supercoiling of the protofibrils in fibrin. This information on the detailed topology of the E(5) fragment permits the construction of a more detailed model than previously possible for the critical trimolecular junction of the protofibril in fibrin.

Effects of size and temperature on metabolic rate.

We derive a general model, based on principles of biochemical kinetics and allometry, that characterizes the effects of temperature and body mass on metabolic rate. The model fits metabolic rates of microbes, ectotherms, endotherms (including those in hibernation), and plants in temperatures ranging from 0 degrees to 40 degrees C. Mass- and temperature-compensated resting metabolic rates of all organisms are similar: The lowest (for unicellular organisms and plants) is separated from the highest (for endothermic vertebrates) by a factor of about 20. Temperature and body size are primary determinants of biological time and ecological roles.

Increased expression and activity of RhoA are associated with increased DNA synthesis and reduced p27(Kip1) expression in the vasculature of hypertensive rats.

We have previously shown that the function of the small G protein Rho is required for vascular smooth muscle cell proliferation and migration. We hypothesized that changes in Rho or Rho signaling might contribute to enhanced vascular proliferative responses associated with hypertension. Western blot analysis revealed that total RhoA expression was approximately 2-fold higher in aortas, tail arteries, and aortic smooth muscle cells (ASMCs) obtained from adult male spontaneously hypertensive rats (SHR) compared with those from Wistar Kyoto rats (WKY). An increase in active GTP-bound RhoA was detected in aortic homogenates by affinity precipitation with the RhoA effector rhotekin and by examining RhoA-[(35)S]GTPgammaS binding. RhoA protein and activity were also increased in vessels from rats treated with N-nitro-L-arginine methyl ester to increase blood pressure. Thrombin-stimulated RhoA activation was also significantly greater in ASMCs from SHR. As a functional correlate of these changes in Rho signaling, thrombin-stimulated DNA synthesis was enhanced in tail arteries and ASMCs from SHR. Expression of the cyclin-dependent kinase inhibitor p27(Kip1) was decreased by two thirds in SHR, and this decrease was mimicked in ASMCs by expression of a constitutively active (GTPase-deficient) mutant of RhoA. Wortmannin (10 nmol/L) fully inhibited the decrease in p27(Kip1) induced by RhoA, and a membrane-targeted catalytic subunit of phosphatidylinositol-3 kinase (PI3K [p110(CAAX)]) decreased p27(Kip1) expression, suggesting that RhoA signals through PI3K. These data provide evidence that RhoA brings about changes in DNA synthesis through reduced expression of p27(Kip1), mediated in part via PI3K, and suggest that increases in RhoA expression and activity contribute to the enhanced vascular responsiveness observed in hypertension.

Complex species interactions and the dynamics of ecological systems: long-term experiments.

Studies that combine experimental manipulations with long-term data collection reveal elaborate interactions among species that affect the structure and dynamics of ecosystems. Research programs in U.S. desert shrubland and pinyon-juniper woodland have shown that (i) complex dynamics of species populations reflect interactions with other organisms and fluctuating climate; (ii) genotype x environment interactions affect responses of species to environmental change; (iii) herbivore-resistance traits of dominant plant species and impacts of "keystone" animal species cascade through the system to affect many organisms and ecosystem processes; and (iv) some environmental perturbations can cause wholesale reorganization of ecosystems because they exceed the ecological tolerances of dominant or keystone species, whereas other changes may be buffered because of the compensatory dynamics of complementary species.

Selective loss of sphingosine 1-phosphate signaling with no obvious phenotypic abnormality in mice lacking its G protein-coupled receptor, LP(B3)/EDG-3.

Sphingosine 1-phosphate (S1P) exerts diverse physiological actions by activating its cognate G protein-coupled receptors. Five S1P receptors have been identified in mammals: LP(B1)/EDG-1, LP(B2)/H218/AGR16/EDG-5, LP(B3)/EDG-3, LP(B4)/NRG-1/EDG-8, and LP(C1)/EDG-6. One of these receptors, LP(B1), has recently been shown to be essential for mouse embryonic development. Here we disrupted the lp(B3) gene in mice, resulting in the complete absence of lp(B3) gene, transcript, and LP(B3) protein. LP(B3)-null mice were viable and fertile and developed normally with no obvious phenotypic abnormality. We prepared mouse embryonic fibroblast (MEF) cells to examine effects of LP(B3) deletion on S1P-induced signal transduction pathways. Wild-type MEF cells expressed lp(B1), lp(B2), and lp(B3) but neither lp(B4) nor lp(C1), and they were highly responsive to S1P in phospholipase C (PLC) activation, adenylyl cyclase inhibition, and Rho activation. Identically prepared LP(B3)-null MEF cells showed significant decreases in PLC activation, slight decreases in adenylyl cyclase inhibition, and no change in Rho activation. Retrovirus-mediated rescue of the LP(B3) receptor in LP(B3)-null MEF cells restored S1P-dependent PLC activation and adenylyl cyclase inhibition. These results indicate a nonessential role for LP(B3) in normal development of mouse but show nonredundant cellular signaling mediated by a single type of S1P receptor.

Deciphering the design of the tropomyosin molecule.

The crystal structure at 2.0-A resolution of an 81-residue N-terminal fragment of muscle alpha-tropomyosin reveals a parallel two-stranded alpha-helical coiled-coil structure with a remarkable core. The high alanine content of the molecule is clustered into short regions where the local 2-fold symmetry is broken by a small (approximately 1.2-A) axial staggering of the helices. The joining of these regions with neighboring segments, where the helices are in axial register, gives rise to specific bends in the molecular axis. We observe such bends to be widely distributed in two-stranded alpha-helical coiled-coil proteins. This asymmetric design in a dimer of identical (or highly similar) sequences allows the tropomyosin molecule to adopt multiple bent conformations. The seven alanine clusters in the core of the complete molecule (which spans seven monomers of the actin helix) promote the semiflexible winding of the tropomyosin filament necessary for its regulatory role in muscle contraction.

A high-resolution consensus linkage map of the rat, integrating radiation hybrid and genetic maps.

We have constructed a high-resolution consensus genetic map of the rat in a single large intercross, which integrates 747 framework markers and 687 positions of our whole-genome radiation hybrid (RH) map of the rat. We selected 136 new gene markers from the GenBank database and assigned them either genetically or physically to rat chromosomes to evaluate the accuracy of the integrated linkage-RH maps in the localization of new markers and to enrich existing comparative mapping data. These markers and 631 D-Got- markers, which are physically mapped but still uncharacterized for evidence of polymorphism, were tested for allele variations in a panel of 16 rat strains commonly used in genetic studies. The consensus linkage map constructed in the GK x BN cross now comprises 1620 markers of various origins, defining 840 resolved genetic positions with an average spacing of 2.2 cM between adjacent loci, and includes 407 gene markers. This whole-genome genetic map will contribute to the advancement of genetic studies in the rat by incorporating gene/EST maps, physical mapping information, and sequence data generated in rat and other mammalian species into genetic intervals harboring disease susceptibility loci identified in rat models of human genetic disorders.

Stereodynamics of N-isopropyl-N-methylpropargylamine. Dynamic NMR studies. Molecular mechanics calculations.

N-Isopropyl-N-methylpropargylamine (N-isopropyl-N-methyl-2-propyn-1-amine; IMPA) is chiral at the pyramidal nitrogen. Racemization occurs via an inversion-rotation process. Both 13C(1H) and 1H dynamic NMR (DNMR) spectra decoalesce in response to slowing inversion-rotation (delta G++ = 7.7 +/- 0.1 kcal/mol). While aspects of the DNMR spectra suggest the presence of minor conformations, the spectrum at 100 K shows a strong preference for one conformation. The NMR data suggest that the preferred conformation has both the isopropyl methine proton and the ethynyl group anti to the lone pair. Both isopropyl methyl groups are gauche to the lone pair. This conformational preference is in significant contrast to N-ethyl-N-methyl-2-aminopropane in which the population of that conformation having the ethyl methyl group and the isopropyl methine proton both anti to the lone pair is only 5% at 95 K. The NMR data, supported by molecular mechanics (MMX) calculations, suggest a special stabilization for the ethynyl group being oriented anti to the lone pair.

Youth, drugs and resilience education.

Billions of dollars are spent annually on school-based drug education programs, with youthful drug use remaining near peak levels since the 1980s. Institutional, policy, and program evidence presented here suggests that although primarily delivered in schools, the educational community rarely participates in the development of drug education; and despite the finding that "no-use" programs such as Drug Abuse Resistance Education (D.A.R.E.) or Life-Skills Training (LST) are likely ineffective, they continue to thrive in schools. This may be explained by insufficient educational participation and scientific discourse considering these issues; and the role of interest group politics, such as the symbiotic relationship between government, researchers, and the tobacco industry, in drug education. In addition to this evidence, resilience based program alternatives, evaluation, and implications are discussed.

G-proteins in growth and apoptosis: lessons from the heart.

The acute contractile function of the heart is controlled by the effects of released nonepinephrine (NE) on cardiac adrenergic receptors. NE can also act in a more chronic fashion to induce cardiomyocyte growth, characterized by cell enlargement (hypertrophy), increased protein synthesis, alterations in gene expression and addition of sarcomeres. These responses enhance cardiomyocyte contractile function and thus allow the heart to compensate for increased stress. The hypertrophic effects of NE are mediated through Gq-coupled alpha(1)-adrenergic receptors and are mimicked by the actions of other neurohormones (endothelin, prostaglandin F(2alpha) angiotensin II) that also act on Gq-coupled receptors. Activation of phospholipase C by Gq is necessary for these responses, and protein kinase C and MAP kinases have also been implicated. Gq stimulated cardiac hypertrophy is also evident in transgenic mouse models. In contrast, stimulation of G(s)-coupled beta-adrenergic receptors or G(i)-coupled receptors do not directly effect cardiomyocyte hypertrophy. Apoptosis is also induced by G-protein-coupled receptor stimulation in cardiomyocytes. Sustained or excessive activation of either Gq- or Gs-signaling pathways results in apoptotic loss of cardiomyocytes both in vitro and in vivo. Apoptosis is associated with decreased ventricular function in the failing heart. Cardiomyocytes provide an ideal model system for understanding the basis for G-protein mediated hypertrophy and apoptosis, and the mechanisms responsible for the transition from compensatory to deleterious levels of signaling. This information may prove critical for designing interventions that prevent the pathophysiological consequences of heart failure.

Delayed compensation for missing keystone species by colonization.

Because individual species can play key roles, the loss of species through extinction or their gain through colonization can cause major changes in ecosystems. For almost 20 years after kangaroo rats were experimentally removed from a Chihuahuan desert ecosystem in the United States, other rodent species were unable to compensate and use the available resources. This changed abruptly in 1995, when an alien species of pocket mouse colonized the ecosystem, used most of the available resources, and compensated almost completely for the missing kangaroo rats. These results demonstrate the importance of individual species and of colonization and extinction events in the structure and dynamics of ecosystems.

Physical and functional interactions of Galphaq with Rho and its exchange factors.

Recent reports have shown that several heterotrimeric protein-coupled receptors that signal through Galpha(q) can induce Rho-dependent responses, but the pathways that mediate the interaction between Galpha(q) and Rho have not yet been identified. In this report we present evidence that Galpha(q) expressed in COS-7 cells coprecipitates with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, Galpha(q) expression enhances Rho-dependent responses. Coexpressed Galpha(q) and Lbc have a synergistic effect on the Rho-dependent rounding of 1321N1 astrocytoma cells. In addition, serum response factor-dependent gene expression, as assessed by the SRE.L reporter gene, is synergistically activated by Galpha(q) and Rho GEFs. The synergistic effect of Galpha(q) on this response is inhibited by C3 exoenzyme and requires phospholipase C activation. Surprisingly, expression of Galpha(q), in contrast to that of Galpha(12) and Galpha(13), does not increase the amount of activated Rho. We also observe that Galpha(q) enhances SRE.L stimulation by activated Rho, indicating that the effect of Galpha(q) occurs downstream of Rho activation. Thus, Galpha(q) interacts physically and/or functionally with Rho GEFs; however this does not appear to lead to or result from increased activation of Rho. We suggest that Galpha(q)-generated signals enhance responses downstream of Rho activation.