Temporally resolved, independent stages of individual exocytotic secretion events.

The stages of the complex events involved in exocytotic secretion after vesicle-cell membrane fusion have been examined at the level of individual vesicles. Catecholamine flux from single bovine adrenal medullary cells was measured with carbon-fiber microelectrodes firmly touching the cell surface. The data reveal that secretion during exocytotic events has three distinct stages: a small increase in catecholamine flux, a rapid, but not instantaneous, rise to a maximum, followed by an exponential decrease in the flux. These stages are interpreted in the following ways. The initial stage corresponds to catecholamine secretion through a fusion pore. The rate of pore expansion appears to control the rise time of the flux to its maximum value. The final exponential stage is consistent with chemical dissociation of the intravesicular matrix or gel.     

Long form of latent TGF-beta binding protein 1 (Ltbp1L) is essential for cardiac outflow tract septation and remodeling

Latent TGF-beta binding protein 1 (LTBP1) is a member of the LTBP/fibrillin family of extracellular proteins. Due to the usage of different promoters, LTBP1 exists in two major forms, long (L) and short (S), each expressed in a temporally and spatially unique fashion. Both LTBP1 molecules covalently interact with latent TGF-beta and regulate its function, presumably via interaction with the extracellular matrix (ECM). To explore the in vivo role of Ltbp1 in mouse development, at the time when only the L isoform is expressed, we mutated the Ltbp1L locus by gene targeting. Ltbp1L-null animals die shortly after birth from defects in heart development, consisting of the improper septation of the cardiac outflow tract (OFT) and remodeling of the associated vessels. These cardiac anomalies present as persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA), which are associated with the faulty function of cardiac neural crest cells (CNCCs). The lack of Ltbp1L in the ECM of the septating OFT and associated vessels results in altered gene expression and function of CNCCs and decreased Tgf-beta activity in the OFT. This phenotype reveals a crucial role for Ltbp1L and matrix as extracellular regulators of Tgf-beta activity in heart organogenesis.     

Structural Basis for the Inverted Repeat Preferences of mariner Transposases

The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase. We have compared the preferences of two active mariner transposases, Mos1 and Mboumar-9, for their imperfect transposon IRs in each step of transposition: DNA binding, DNA cleavage, and DNA strand transfer. A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR. For both Mos1 and Mboumar-9, in vitro DNA transposition is most efficient when the preferred IR sequence is present at both transposon ends. We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end. We show that the efficiency of Mboumar-9 transposition is improved almost 4-fold by changing the 3′ base of the preferred Mboumar-9 IR from guanine to adenine. This preference for adenine at the reactive 3′ end for both Mos1 and Mboumar-9 may be a general feature of mariner transposition.     

The control of flowering by vernalization

The process by which vernalization, the exposure of a germinating seed or a juvenile plant to a prolonged period of low temperature, promotes flowering in the adult plant has remained a mystery for many years. The recent isolation of one of the key genes involved in vernalization, FLOWERING LOCUS C, has now provided an insight into the molecular mechanism involved, including the role of DNA methylation.     

Synovial membrane protein expression differs between juvenile idiopathic arthritis subtypes in early disease

Introduction

Juvenile idiopathic arthritis (JIA) is the most common rheumatological disease of childhood with a prevalence of around 1 in 1,000. Without appropriate treatment it can have devastating consequences including permanent disability from joint destruction and growth deformities. Disease aetiology remains unknown. Investigation of disease pathology at the level of the synovial membrane is required if we want to begin to understand the disease at the molecular and biochemical level. The synovial membrane proteome from early disease-stage, treatment naive JIA patients was compared between polyarticular and oligoarticular subgroups.

Methods

Protein was extracted from 15 newly diagnosed, treatment naive JIA synovial membrane biopsies and separated by two dimensional fluorescent difference in-gel electrophoresis. Proteins displaying a two-fold or greater change in expression levels between the two subgroups were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry with expression further verified by Western blotting and immunohistochemistry.

Results

Analysis of variance analysis (P ≤ 0.05) revealed 25 protein spots with a two-fold or greater difference in expression levels between polyarticular and oligoarticular patients. Hierarchical cluster analysis with Pearson ranked correlation revealed two distinctive clusters of proteins. Some of the proteins that were differentially expressed included: integrin alpha 2b (P = 0.04); fibrinogen D fragment (P = 0.005); collagen type VI (P = 0.03); fibrinogen gamma chain (P = 0.05) and peroxiredoxin 2 (P = 0.02). The identified proteins are involved in a number of different processes including platelet activation and the coagulation system.

Conclusions

The data indicate distinct synovial membrane proteome profiles between JIA subgroups at an early stage in the disease process. The identified proteins also provide insight into differentially perturbed pathways which could influence pathological events at the joint level.