Bajzer, Željko

To assess the reliability of electron beam computed tomography (EBCT), measurements of single-kidney renal blood flow (RBF), glomerular filtration rate (GFR), and intratubular contrast medium concentration (ITC) of radiographic contrast media were quantified in anesthetized pigs before and after acetylcholine-induced vasodilation and diuresis. EBCT measurements were compared with those obtained with intravascular Doppler and inulin clearance. The capability of EBCT to detect chronic changes in single-kidney function was evaluated in pigs with unilateral renal artery stenosis, and their long-term reproducibility in normal pigs was studied repeatedly at 1-mo intervals. EBCT-RBF (ml/min) correlated with Doppler-RBF as RBFEBCT= 45 + 1.07 ∗ RBFDoppler, r = 0.81. EBCT-GFR (ml/min) correlated with inulin clearance as GFREBCT= 11.7 + 1.02 ∗ GFRinulin, r = 0.80. During vasodilation, RBF and GFR increased, whereas ITC decreased along the nephron. In renal artery stenosis, single-kidney GFR decreased linearly with the degree of stenosis, and ITC increased along the nephron, indicating increased fluid reabsorption. EBCT-RBF, GFR, and ITC were similar among repeated measurements. This approach might be invaluable for simultaneous quantification of regional hemodynamics and function in the intact kidneys, in a manner potentially applicable to humans.

Noninvasive breath tests for gastric emptying are important techniques for understanding the changes in gastric motility that occur in disease or in response to drugs. Mice are often used as an animal model; however, the gamma variate model currently used for data analysis does not always fit the data appropriately. The aim of this study was to determine appropriate mathematical models to better fit mouse gastric emptying data including when two peaks are present in the gastric emptying curve. We fitted 175 gastric emptying data sets with two standard models (gamma variate and power exponential), with a gamma variate model that includes stretched exponential and with a proposed two-component model. The appropriateness of the fit was assessed by the Akaike Information Criterion. We found that extension of the gamma variate model to include a stretched exponential improves the fit, which allows for a better estimation of T1/2and Tlag. When two distinct peaks in gastric emptying are present, a two-component model is required for the most appropriate fit. We conclude that use of a stretched exponential gamma variate model and when appropriate a two-component model will result in a better estimate of physiologically relevant parameters when analyzing mouse gastric emptying data.

Plasma membrane calcium pumps differentially affect the pattern of calcium signals.

Abstract Killer Ig-like receptors (KIRs) are expressed on CD4+CD28null T cells, a highly oligoclonal subset of T cells that is expanded in patients with rheumatoid arthritis. It is unclear at what stage of development these T cells acquire KIR expression. To determine whether KIR expression is a consequence of clonal expansion and replicative senescence, multiple CD4+CD28null T cell clones expressing the in vivo dominant TCR β-chain sequences were identified in three patients and analyzed for their KIR gene expression pattern. Based on sharing of TCR sequences, the clones were grouped into five clone families. The repertoire of KIRs was diverse, even within each clone family; however, the gene expression was not random. Three particular receptors, KIR2DS2, KIR2DL2, and KIR3DL2, had significant differences in gene expression frequencies between the clone families. These data suggest that KIRs are successively acquired after TCR rearrangement, with each clone family developing a dominant expression pattern. The patterns did not segregate with the individual from whom the clones were derived, indicating that peripheral selection in the host environment was not a major shaping force. Several models were examined using a computer algorithm that was designed to simulate the expression of KIRs at various times during T cell proliferation. The computer simulations favored a model in which KIR gene expression is inducible for a limited time during the initial stages of clonal expansion.

Endothelial cell proliferation and migration is essential to angiogenesis. Typically, proliferation and chemotaxis of endothelial cells is driven by growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF activates phospholipases (PLCs) – specifically PLCγ1 – that are important for tubulogenesis, differentiation and DNA synthesis. However, we show here that VEGF, specifically through VEGFR2, induces phosphorylation of two serine residues on PLCβ3, and this was confirmed in an ex vivo embryoid body model. Knockdown of PLCβ3 in HUVEC cells affects IP3 production, actin reorganization, migration and proliferation; whereas migration is inhibited, proliferation is enhanced. Our data suggest that enhanced proliferation is precipitated by an accelerated cell cycle, and decreased migration by an inability to activate CDC42. Given that PLCβ3 is typically known as an effector of heterotrimeric G-proteins, our data demonstrate a unique crosstalk between the G-protein and receptor tyrosine kinase (RTK) axes and reveal a novel molecular mechanism of VEGF signaling and, thus, angiogenesis.

Choline acetyltransferase (ChAT; EC 2.3.1.6 ) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist in Caenorhabditis elegans and Drosophila , but no CHAT mutations have been observed in humans to date. Here we report that mutations in CHAT cause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and to CHAT as one of the candidate genes. Direct sequencing of CHAT reveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, V506L, and R560H) have significantly impaired catalytic efficiencies.