The predicted role of steric specificity in crowding-mediated effects on reversible biomolecular association.

A fundamental question in biology is whether the presence of non-reacting macromolecules in the cytoplasm affects the rates and extents of reversible association reactions, a phenomenon often referred to as 'macromolecular crowding.' Under certain conditions, crowding has been proposed to dramatically alter the kinetics and thermodynamics of chemical reactions, making it difficult to quantitatively relate rates and extents of reactions measured in vitro to those occurring in vivo. In this work, we use Brownian dynamics simulation and Monte Carlo methods to (1) quantify the overall thermodynamic and kinetic effects of crowding by independently investigating each step of reversible bimolecular association (i.e. translational diffusion, steric specific binding, and dissociation), and (2) provide an explicit, quantitative investigation of how the degree of steric specificity of protein dimerization influences crowding-mediated effects on association and dissociation. We find that k on decreases by ∼2-fold for non-steric specific reactions, and increases by ∼3-fold for highly steric specific reactions. In addition, k off decreases by only ∼30%-60% in the presence of crowders, depending on the strength of the bond between the reactant pair, so that the equilibrium constant is increased by ∼4-fold, at most. These results suggest that crowding-mediated effects on globular protein dimerization reactions in the cytoplasm are modulated by the steric specificity of the reactants, and that reversible protein-protein association is relatively insensitive to the physical presence of crowders (i.e. steric repulsion effects in the cytoplasm) for crowders of similar size and shape to reactants over a range of volume fractions (0-0.3).

Phenotype and genotype in 17 patients with Goltz-Gorlin syndrome.

BACKGROUND: Goltz-Gorlin syndrome or focal dermal hypoplasia is a highly variable, X-linked dominant syndrome with abnormalities of ectodermal and mesodermal origin. In 2007, mutations in the PORCN gene were found to be causative in Goltz-Gorlin syndrome. METHOD: A series of 17 patients with Goltz-Gorlin syndrome is reported on, and their phenotype and genotype are described. RESULTS: In 14 patients (13 females and one male), a PORCN mutation was found. Mutations included nonsense (n = 5), frameshift (n = 2), aberrant splicing (n = 2) and missense (n = 5) mutations. No genotype-phenotype correlation was found. All patients with the classical features of the syndrome had a detectable mutation. In three females with atypical signs, no mutation was found. The male patient had classical features and showed mosaicism for a PORCN nonsense mutation in fibroblasts. Two affected sisters had a mutation not detectable in their parents, supporting germline mosaicism. Their father had undergone radiation for testicular cancer in the past. Two classically affected females had three severely affected female fetuses which all had midline thoracic and abdominal wall defects, resembling the pentalogy of Cantrell and the limb-body wall complex. Thoracic and abdominal wall defects were also present in two surviving patients. PORCN mutations can possibly cause pentalogy of Cantrell and limb-body wall complexes as well. Therefore, particularly in cases with limb defects, it seems useful to search for these. CONCLUSIONS: PORCN mutations can be found in all classically affected cases of Goltz-Gorlin syndrome, including males. Somatic and germline mosaicism occur. There is no evident genotype-phenotype correlation.

Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome.

BACKGROUND: Recurrent 15q13.3 microdeletions were recently identified with identical proximal (BP4) and distal (BP5) breakpoints and associated with mild to moderate mental retardation and epilepsy. METHODS: To assess further the clinical implications of this novel 15q13.3 microdeletion syndrome, 18 new probands with a deletion were molecularly and clinically characterised. In addition, we evaluated the characteristics of a family with a more proximal deletion between BP3 and BP4. Finally, four patients with a duplication in the BP3-BP4-BP5 region were included in this study to ascertain the clinical significance of duplications in this region. RESULTS: The 15q13.3 microdeletion in our series was associated with a highly variable intra- and inter-familial phenotype. At least 11 of the 18 deletions identified were inherited. Moreover, 7 of 10 siblings from four different families also had this deletion: one had a mild developmental delay, four had only learning problems during childhood, but functioned well in daily life as adults, whereas the other two had no learning problems at all. In contrast to previous findings, seizures were not a common feature in our series (only 2 of 17 living probands). Three patients with deletions had cardiac defects and deletion of the KLF13 gene, located in the critical region, may contribute to these abnormalities. The limited data from the single family with the more proximal BP3-BP4 deletion suggest this deletion may have little clinical significance. Patients with duplications of the BP3-BP4-BP5 region did not share a recognisable phenotype, but psychiatric disease was noted in 2 of 4 patients. CONCLUSIONS: Overall, our findings broaden the phenotypic spectrum associated with 15q13.3 deletions and suggest that, in some individuals, deletion of 15q13.3 is not sufficient to cause disease. The existence of microdeletion syndromes, associated with an unpredictable and variable phenotypic outcome, will pose the clinician with diagnostic difficulties and challenge the commonly used paradigm in the diagnostic setting that aberrations inherited from a phenotypically normal parent are usually without clinical consequences.

Clinical features in a family with an R460H mutation in transforming growth factor beta receptor 2 gene.

OBJECTIVES: To describe the clinical findings and natural history in 22 carriers of an R460H mutation in the transforming growth factor beta receptor 2 gene (TGFbetaR2) from a five-generation kindred ascertained by familial aortic dissection. METHODS: 13 of the confirmed carriers were interviewed and examined, and information about the remaining carrier was obtained from medical records. Clinical information about deceased individuals was obtained, when possible, from postmortem reports, death certificates and medical records. RESULTS: There have been eight sudden deaths; the cause of death was aortic dissection in all six cases in which a postmortem examination was performed. Three individuals had undergone aortic replacement surgery. Dissection had occurred throughout the aorta, and in one case in the absence of aortic root dilatation. Subarachnoid haemorrhage, due to a ruptured berry aneurysm, had occurred in two individuals. Four gene carriers and one deceased family member who were investigated had tortuous cerebral blood vessels. One had tortuous vertebral arteries, two had tortuous carotid arteries and one a tortuous abdominal aorta. Two individuals were found to have a brachiocephalic artery aneurysm and a subclavian artery aneurysm, respectively. CONCLUSIONS: Despite the predisposition to aortic dilatation and dissection, individuals did not frequently manifest the skeletal features of Marfan syndrome, with the exception of joint hypermobility. No one individual had ocular lens dislocation. Striae and herniae were common. There was some overlap with Ehlers-Danlos syndrome type 4, OMIM 130050, with soft translucent skin, which is easily bruised. Other features were arthralgia, migraine and a tendency to fatigue easily, varicose veins and prominent skin striae. This family provides further evidence that mutations in TGFbetaR2 cause a distinct syndrome that needs to be distinguished from Marfan syndrome to direct investigation and management of patients and shows the natural history, spectrum of clinical features and variable penetrance of this newly recognised condition.

CD40-mediated signaling in monocytic cells: up-regulation of tumor necrosis factor receptor-associated factor mRNAs and activation of mitogen-activated protein kinase signaling pathways.

The biochemical pathways involved in CD40 signaling have been extensively studied in B cells and B cell lines, and appear to be primarily initiated by recruitment of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) signaling proteins to the CD40 cytoplasmic domain. Signaling pathways activated through CD40 in monocytes/macrophages have not been characterized as well as in B cells. Using human monocytes and the human monocytic cell line THP1, we examined signal transduction events induced by CD40 engagement with its ligand, CD154. In human monocytes, all TRAF mRNAs were expressed constitutively and CD40 ligation resulted in a strong up-regulation of TRAF1 mRNA. In THP1 cells, CD40 ligation induced expression of TRAF1 and TRAF5 mRNAs. Engagement of CD40 in both monocytes and THP1 cells led to the rapid and transient activation of the extracellular signal-regulated kinases (ERK) 1 and 2, and to low levels of JNK activation. No CD40-dependent activation of p38 mitogen-activated protein kinase (MAPK) was found. In CD154-stimulated monocytes and THP1 cells the upstream ERK1/2 activator, MAPK kinase (MEK) 1/2, and downstream substrate, c-Myc, were activated. By blocking activation of ERK1/2 with a MEK-specific inhibitor, PD98059, CD40-dependent secretion of the pro-inflammatory cytokines, TNF-alpha, IL-6 and IL-8, was demonstrated to be linked to the ERK1/2 pathway. The ERK1/2 pathway did not appear to be involved in up-regulating TRAF1 and TRAF5 mRNAs in THP1 cells. Collectively, these results suggest distinct differences between B cells and monocytic cells in CD40-dependent activation of MAPK pathways.

Gout is on the increase in New Zealand.

OBJECTIVE: To determine the current prevalence of hyperuricaemia and gout in New Zealand Maori and Europeans for comparison with previous studies. METHODS: 342 Maori and 315 European men and women aged 15 years and older were studied by personal interview and a musculoskeletal system examination. The 1977 ARA criteria for gout in a survey setting were used and serum uric acid was determined by a uricase method. The data were compared with those of previous New Zealand studies. RESULTS: Gout was significantly more common in Maori (6.4%) than Europeans (2.9%) (delta = 3.6%, 95% confidence interval 0.4 to 6.8) and in Maori men (13.9%) than in European men (5.8%) (delta = 8.1%, 95% CI 1.0 to 15.2). Hyperuricaemia was significantly more common in Maori men (27.1%) than in European men (9.4%) (delta = 17.7%, 95% CI 8.3 to 27.1) and in Maori women (26.6%) than in European women (10.5%) (delta = 16.1%, 95% CI 8.5 to 23.7). At least 14% of hyperuricaemic individuals were receiving diuretics, of whom 78% were women. Comparison with previous studies shows that the prevalence of gout has increased in both Maori and Europeans, particularly in men. In Maori men the prevalence of gout has risen from 4.5-10.4% previously to 13.9%, and in European men from 0.7%-2.0% previously to 5.8%. Clinical differences included a stronger family history, earlier age at onset, and a higher frequency of tophi and polyarticular gout in Maori than Europeans. Of those with gout, 62% of Maori and 63% of Europeans were hyperuricaemic on the day surveyed and six (19.4%) were on diuretics. Treatment of gout was inadequate in most cases. CONCLUSIONS: Hyperuricaemia and gout remain common among Maori. Of concern is that the prevalence of gout appears to be on the increase, not only in Maori but also in Europeans in New Zealand.

Outcomes management: an interdisciplinary approach to improving patient outcomes.

Outcomes management provides a means for interdisciplinary collaboration in improving patient outcomes. The benefits of an outcomes management program are numerous, including decreasing healthcare costs, decreasing the length of stay, improving clinical outcomes, improving system processes, and fostering outcomes research. The outcomes manager is responsible for the development, implementation, and evaluation of an outcomes management program. One of the functions of an outcomes manager is developing collaborative practice teams to serve as vehicles of change through their analysis of outcomes data and the identification of best practice patterns. Interdisciplinary collaboration is a key component of an outcomes management program. Without an atmosphere os shared responsibility among disciplines for outcomes noted in a patient population, and recognition of individual expertise regarding care, efforts to produce change will move very slowly, if at all, and optimal improvements in care will be forfeited.

Multivalent, but not divalent, antigen receptor cross-linkers synergize with CD40 ligand for induction of Ig synthesis and class switching in normal murine B cells. A redefinition of the TI-2 vs T cell-dependent antigen dichotomy.

A number of previous studies have suggested that cross-linkage of the B cell Ag receptor may be critical for induction of humoral immune responses to T cell-dependent (TD) Ags in vivo. Previous work also indicated a critical role, in these responses, for CD40-mediated signaling mediated by binding of the inducible T cell membrane protein, CD40 ligand (CD40L). Data in this manuscript demonstrate that concentrations of bivalent anti-IgD or anti-IgM Ab as high as 30 micrograms/ml induced little if any enhancement of CD40-dependent Ig secretion by resting murine B cells. In contrast, concentrations as low as 3 pg/ml of multivalent, dextran-conjugated, anti-IgD (alpha delta-dex) or anti-IgM (alpha mu-dex) were strongly synergistic with CD40L for induction of B cell proliferation, viable cell outgrowth, Ig isotype switching, and maturation to Ig secretion. As many as 30% of the B cells became membrane IgG1+ after stimulation with CD40L, anti-Ig-dextran, and IL-4 + IL-5, with a concomitant three- to fivefold increase in numbers of viable cells as compared with control cultures. High Ig secretory responses were obtained in response to the combined actions of CD40L and alpha delta-dex or alpha mu-dex, utilizing concentrations of B cell activator that when acting alone induced only modest Ig secretion. Surprisingly, although we previously demonstrated that alpha delta-dex selectively and strongly suppressed IgE production by T cell-activated B cells, it strikingly augmented IgE expression by CD40L-activated B cells. These data suggest 1) a key role for Ag receptor cross-linkage in CD40-dependent induction of humoral immune responses, 2) that to achieve a membrane Ig-dependent enhancing effect in the presence of activated T cells, TD Ags must be displayed to the B cell as a multivalent array of epitopes, 3) that picomolar concentrations of Ag can mediate this effect, and 4) that at least for induction of IgE responses, B cell stimulation via CD40L or via activated T cells may lead to a qualitatively different pathway of activation.

Regulation of CD40 ligand expression and use of recombinant CD40 ligand for studying B cell growth and differentiation.

Helper T cell activation leads to transient expression of a ligand for the B cell surface protein, CD40. CD40 ligand can deliver helper T cell-derived contact signals to B lymphocytes that drive B cell activation and proliferation. Regulation of expression of CD40 ligand is analogous to that of other helper T cell-derived lymphokines. A soluble form of CD40 ligand is capable of delivering proliferative signals to B cells only when cross-linked or when IL-4 is added. Recombinant CD40 ligand in membrane vesicles can be used to develop in vitro systems for studying class switching, clonal selection, and affinity maturation in normal B lymphocytes.