Prevention of collagen-induced arthritis in mice transgenic for the complement inhibitor complement receptor 1-related gene/protein y

The objective of these studies was to examine collagen-induced arthritis (CIA) in C57BL/6 mice transgenic for the rodent complement regulatory protein complement receptor 1-related gene/protein y (Crry) (Crry-Tg), a C3 convertase inhibitor. The scores for clinical disease activity and for histological damage in the joints were both significantly decreased in Crry-Tg mice in comparison to wild-type (WT) littermates. The production of both IgG1 and IgG2a anti-collagen Abs was reduced in the Crry-Tg mice, although spleen cell proliferation in response to collagen type II was not altered. The production of IFN-gamma, TNF-alpha, and IL-1beta by LPS-stimulated spleen cells was decreased, and IL-10 was increased, in cells from Crry-Tg mice in comparison to WT. The steady-state mRNA levels for IFN-gamma, TNF-alpha, and IL-1beta were all decreased in the joints of Crry-Tg mice in comparison to WT. The synovium from Crry-Tg mice without CIA contained the mRNA for the Crry transgene, by RT-PCR, and the synovium from transgenic mice with CIA exhibited little deposition of C3 protein by immunohistological analysis. These results suggest that suppression of CIA in Crry-Tg mice may be due to enhanced synthesis of Crry locally in the joint with decreased production of proinflammatory cytokines.     

Functional evolution in the ancestral lineage of vertebrates or when genomic complexity was wagging its morphological tail

Early vertebrate evolution is characterized by a significant increase of organismal complexity over a relatively short time span. We present quantitative evidence for a high rate of increase in morphological complexity during early vertebrate evolution. Possible molecular evolutionary mechanisms that underlie this increase in complexity fall into a small number of categories, one of which is gene duplication and subsequent structural or regulatory neofunctionalization. We discuss analyses of two gene families whose regulatory and structural evolution shed light on the connection between gene duplication and increases in organismal complexity.     

Arbutin synthase, a novel member of the NRD1beta glycosyltransferase family, is a unique multifunctional enzyme converting various natural products and xenobiotics

Plant glucosyltransferases (GTs) play a crucial role in natural product biosynthesis and metabolization of xenobiotics. We expressed the arbutin synthase (AS) cDNA from Rauvolfia serpentina cell suspension cultures in Escherichia coli with a 6 x His tag and purified the active enzyme to homogeneity. The recombinant enzyme had a temperature optimum of 50 degrees C and showed two different pH optima (4.5 and 6.8 or 7.5, depending on the buffer). Out of 74 natural and synthetic phenols and two cinnamyl alcohols tested as substrates for the AS, 45 were accepted, covering a broad range of structural features. Converting rates comparable to hydroquinone were not achieved. In contrast to this broad acceptor substrate specificity, only pyrimidine nucleotide activated glucose was tolerated as a donor substrate. Nucleotide and amino acid sequence analysis revealed AS to be a new member of the NRD1beta family of glycosyl transferases and placed the enzyme into the group of plant secondary product GTs. Arbutin synthase is therefore the first example of a broad spectrum multifunctional glucosyltransferase.     

Muscle contraction increases lactate transport while reducing sarcolemmal MCT4, but not MCT1

Rates of lactate uptake into giant sarcolemmal vesicles were determined in vesicles collected from rat muscles at rest and immediately after 10 min of intense muscle contraction. This contraction period reduced muscle glycogen rapidly by 37-82% in all muscles examined (P < 0.05) except the soleus muscle (no change P > 0.05). At an external lactate concentration of 1 mM lactate, uptake into giant sarcolemmal vesicles was not altered (P > 0.05), whereas at an external lactate concentration of 20 mM, the rate of lactate uptake was increased by 64% (P < 0.05). Concomitantly, the plasma membrane content of monocarboxylate transporter (MCT)1 was reduced slightly (-10%, P < 0.05), and the plasma membrane content of MCT4 was reduced further (-25%, P < 0.05). In additional studies, the 10-min contraction period increased the plasma membrane GLUT4 (P < 0.05) while again reducing MCT4 (-20%, P < 0.05) but not MCT1 (P > 0.05). These studies have shown that intense muscle contraction can increase the initial rates of lactate uptake, but only when the external lactate concentrations are high (20 mM). We speculate that muscle contraction increases the intrinsic activity of the plasma membrane MCTs, because the increase in lactate uptake occurred while plasma membrane MCT4 was decreased and plasma membrane MCT1 was reduced only minimally, or not at all.     

Estimating the fraction of invariable codons with a capture-recapture method

Summary A codon-based approach to estimating the number of variable sites in a protein is presented. When first and second positions of codons are assumed to be replacement positions, a capture-recapture model can be used to estimate the number of variable codons from every pair of homologous and aligned sequences. The capture-recapture estimate is compared to a maximum likelihood estimate of the number of variable codons and to previous approaches that estimate the number of variable sites (not codons) in a sequence. Computer simulations are presented that show under which circumstances the capture-recapture estimate can be used to correct biases in distance matrices. Analysis of published sequences of two genes, calmodulin and serum albumin, shows that distance corrections that employ a capture-recapture estimate of the number of variable sites may be considerably different from corrections that assume that the number of variable sites is equal to the total number of positions in the sequence. Offprint requests to: A. Sidow     

Insulin stimulates fatty acid transport by regulating expression of FAT/CD36 but not FABPpm

Because insulin has been shown to stimulate long-chain fatty acid (LCFA) esterification in skeletal muscle and cardiac myocytes, we investigated whether insulin increased the rate of LCFA transport by altering the expression and the subcellular distribution of the fatty acid transporters FAT/CD36 and FABPpm. In cardiac myocytes, insulin very rapidly increased the expression of FAT/CD36 protein in a time- and dose-dependent manner. During a 2-h period, insulin (10 nM) increased cardiac myocyte FAT/CD36 protein by 25% after 60 min and attained a maximum after 90-120 min (+40-50%). There was a dose-dependent relationship between insulin (10(-12) to 10(-7) M) and FAT/CD36 expression. The half-maximal increase in FAT/CD36 protein occurred at 0.5 x 10(-9) M insulin, and the maximal increase occurred at 10(-9) to 10(-8) M insulin (+40-50%). There were similar insulin-induced increments in FAT/CD36 protein in cardiac myocytes (+43%) and in Langendorff-perfused hearts (+32%). In contrast to FAT/CD36, insulin did not alter the expression of FABPpm protein in either cardiac myocytes or the perfused heart. By use of specific inhibitors of insulin-signaling pathways, it was shown that insulin-induced expression of FAT/CD36 occurred via the PI 3-kinase/Akt insulin-signaling pathway. Subcellular fractionation of cardiac myocytes revealed that insulin not only increased the expression of FAT/CD36, but this hormone also targeted some of the FAT/CD36 to the plasma membrane while concomitantly lowering the intracellular depot of FAT/CD36. At the functional level, the insulin-induced increase in FAT/CD36 protein resulted in an increased rate of palmitate transport into giant vesicles (+34%), which paralleled the increase in plasmalemmal FAT/CD36 (+29%). The present studies have shown that insulin regulates protein expression of FAT/CD36, but not FABPpm, via the PI 3-kinase/Akt insulin-signaling pathway.     

Denervation provokes greater reductions in insulin-stimulated glucose transport in muscle than severe diabetes

We have examined the independent and combined effects of insulin insufficiency (streptozotocin (STZ)-induced diabetes, 85 mg/kg i.p.) and reduced muscle activity (denervation) (7 days) on basal, insulin-stimulated and contraction-stimulated glucose transport in rat muscles (soleus, red and white gastrocnemius). There were four treatments: control, denervated, diabetic, and denervated + diabetic muscles. Contraction-stimulated glucose transport was lowered (~ 50%) (p < 0.05) to the same extent in all experimental groups. In contrast, there was a much smaller reduction insulin-stimulated glucose transport in muscles from diabetic animals (18-24% reduction, p < 0.05) than in denervated muscles (40-60% reduction, p < 0.05) and in denervated + diabetic muscles (40-60% reduction, p < 0.05). GLUT-4 mRNA reduction was greatest in denervated + diabetic muscles (~ -75%, p < 0.05). GLUT-4 protein was decreased (p < 0.05) to a similar extent in all three experimental conditions (~ -30-40%). In conclusion, (1) muscle inactivity (denervation) and STZ-induced diabetes had similar effects on reducing contraction-stimulated glucose transport, but (2) muscle inactivity (denervation), rather than severe diabetes, produced a 2-fold greater impairment in skeletal muscle insulin-stimulated glucose transport.     

Impact of peptides on the recognition of HLA class I molecules by human HLA antibodies

MHC class I molecules expressed on cell surfaces are composed of H chain, beta2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.     

Functional evaluation of Dent’s disease-causing mutations: implications for ClC-5 channel trafficking and internalization

ClC-5 is a member of the ClC family of voltage-gated chloride channels. Loss-of-function mutations of its corresponding gene (CLCN5) cause Dents disease, an X-linked kidney disorder, characterized by low-molecular weight proteinuria, hypercalciuria, nephrocalcinosis/nephrolithiasis, and progressive renal failure. Here, we examined the effect of different mutations on function and cellular trafficking of the recombinant protein. Mutant CLCN5 cDNAs were generated by site directed mutagenesis for two premature stop codon variants (R347X and M517IfsX528), and several missense mutations (C221R, L324R, G462 V, and R516 W). We also tested L521R (instead of L521RfsX526 observed) and mutants G506E and R648X (previously reported by others). After heterologous expression in Xenopus oocytes, ClC-5 channel activity and surface expression were determined by two-electrode voltage-clamp analysis and ClC-5 surface ELISA, respectively. Except for the R516 W and R648X variants, none of the mutated proteins induced functional chloride currents or reached the plasma membrane. This is readily understandable for the truncation mutations. Yet, the tested missense mutations are distributed over different transmembrane regions, implying that correct channel structure and orientation in the membrane is not only a prerequisite for proper ClC-5 function but also for Golgi exit. Interestingly, the R648X mutant although functionally compromised, displayed a significant increase in surface expression. This finding might be explained by the deletion of a ClC-5 carboxy-terminal PY-like internalization signal, which in turn impairs channel removal from the membrane. Our observations further imply that recruitment of ClC-5 to alternative routes (plasma membrane or early endosomes) in the trans-Golgi network is mediated via different signal sequences.Electronic Supplementary Material Supplementary material is available for this article at .