Functional analysis of tryptophans alpha 62 and beta 120 on HLA-DM.

In the endocytic pathway of antigen-presenting cells, HLA-DM catalyzes the exchange between class II-associated invariant chain peptide (CLIP) and antigenic peptides onto major histocompatibility complex class II molecules. At low pH of lysosomal compartments, both HLA-DM and HLA-DR undergo conformational changes, and it was recently postulated that two partially exposed tryptophans on HLA-DM might be involved in the interaction between the two molecules. To define contact regions on HLA-DM, we have conducted site-directed mutagenesis on those two hydrophobic residues. The HLA-DM alphaW62A,betaW120A (DM(W62A/W120A)) double mutant was expressed in HLA-DR(+) HeLa cells expressing invariant chain, and the activity of this DM molecule was assessed. Flow cytometry analysis of cell surface DR-CLIP complexes revealed that DM(W62A/W120A) removes CLIP as efficiently as its wild-type counterpart. DM(W62A/W120A) was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations alphaW62A and betaW120A on HLA-DM did not affect the association with HLA-DO. The complex egresses the endoplasmic reticulum and accumulates in endocytic vesicles. Moreover, DO and DM(W62A/)W120A were co-immunoprecipitated at pH 7. We conclude that the alpha62 and beta120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.     

Genetic and physical mapping of a novel region close to the fragile X site on the human X chromosome

We report the isolation and characterization of a novel DNA marker (1A1) in Xqter in the region of the fragile X. Genetic studies in families segregating for the fragile X syndrome suggest that 1A1 lies between the disease mutation and the distal locus, DXS52. Studies in normal and fragile X families show that 1A1 is tightly linked to DXS52 (Zmax = 17.20; theta max = 0.03) and F8 (Zmax = 7.01; theta max = 0.08). Multipoint mapping of families supports the order Xcen-DXS105-FRAXA-1A1-DXS52-(F8, DXS115)-Xqter. Pulsed-field gel electrophoresis (PFGE) studies demonstrate that 1A1 defines a new region of at least 2 Mb of DNA not physically linked to DXS52 or F8, thus extending the physical map of Xq27-qter to over 4 Mb. Complex partial digestion PFGE patterns, probably due to differing degrees of methylation, are observed with 1A1 in unrelated normal and fragile-X-positive individuals, whereas other distal markers give uniform digestion profiles. Physical data suggest that 1A1 lies in a region less CpG rich than other distal markers in Xq27-qter.     

Transforming growth factor-alpha in the mammalian brain. Immunohistochemical detection in neurons and characterization of its mRNA

In this communication, we demonstrate that adult mammalian brain neurons express transforming growth factor-alpha (TGF-alpha). We used the anti-TGF-alpha monoclonal antibody, MF9, to immunohistochemically localize TGF-alpha in human and rat brain. We found specific immunoreactivity in neurons throughout the brain which was not a result of cross-reactivity of MF9 with the neuropeptide, synenkephalin. Northern blot analysis of bovine and rat brain RNA using human and rat TGF-alpha cDNA probes, respectively, revealed a single 4.8-kilobase pair mRNA with approximately equal abundance in the bovine brainstem, cerebellum, hypothalamus, and cerebral cortex. Fetal rat brain had about 2-fold more TGF-alpha mRNA than did adult rat. The brain TGF-alpha cDNA was cloned from a human neonatal brainstem library. Four identical clones were isolated after screening 10(6) recombinant lambda gt11 phage. The sequence of the 894-base pair cDNA was virtually identical with the cDNA isolated from a human renal cell carcinoma. A single alanine codon was deleted in the brain cDNA at an exon-exon junction. The alanine deletion is within the amino-terminal region of the TGF-alpha precursor that is thought to be removed by proteolytic processing of the precursor to the mature growth factor. These studies indicate that the normal mammalian brain neurons express TGF-alpha.     

A 33-yr follow-up of peak oxygen uptake and related variables of former physical education students

Åstrand, Per-Olof, Ulf Bergh, and ÅsaKilbom. A 33-yr follow-up of peak oxygen uptake and relatedvariables of former physical education students. J. Appl. Physiol. 82(6): 1844-1852, 1997.In 1949, 27 female and 26 male physical education students were studied at amean age of 22 and 25 yr, respectively. They were restudied in 1970 and1982. Measurements included oxygen uptake, heart rate, and pulmonaryventilation during submaximal and maximal exercise on a cycle ergometerand treadmill. After 21 yr, peak aerobic power was significantlyreduced, from 2.90 to 2.18 l/min and from 4.09 to 3.28 l/min for womenand men, respectively. After another 12 yr, the 1970 maxima were notreduced further. From 1949 to 1982 there was a decrease in peak heartrate from 196 to 177 beats/min in women and from 190 to 175 beats/minin men (P < 0.05). Highest pulmonaryventilation did not change significantly. At an oxygen uptake of 1.5 l/min, the heart rate was the same in 1949 as in 1982. In conclusion,the physical fitness level of the subjects was well above average forthese ages. From 1970 to 1982 there was no decline in the average peakaerobic power, a finding possibly related to increased habitualphysical activity.

     

Distribution of soluble epoxide hydrolase and of cytochrome P450 2C8, 2C9, and 2J2 in human tissues.

Soluble epoxide hydrolase (sEH) hydrolyzes a wide variety of endogenous and exogenous epoxides. Many of these epoxides are believed to be formed by cytochrome P450 epoxygenases. Here we report the distribution of sEH and cytochrome P450 epoxygenases 2C8, 2C9, and 2J2 by immunohistochemistry. A large number of different tissues from different organs were evaluated using high-throughput tissue microarrays. sEH was found in the liver, kidney, and in many other organs, including adrenals, pancreatic islets, pituitary gland, lymphoid tissues, muscles, certain vascular smooth muscles, and epithelial cells in the skin, prostatic ducts, and the gastrointestinal tract. Immunolabeling for sEH was highly specific for particular tissues and individual cell types. CYP2C9 was also found in almost all of these organs and tissues, suggesting that 2C9 and sEH are very similar in their tissue-specific patterns of expression. CYP2C8 and 2J2 were also widely distributed in human tissues but were less frequently associated with sEH. The results suggest potentially distinct pathways of endogenous fatty acid epoxide production and hydrolysis in a variety of human tissues.     

DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation

Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to alpha-synuclein, a protein implicated in PD pathogenesis.