Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNF-synthesis attenuates MPTP toxicity in mouse striatum

The impact of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in the pathology of Parkinson's disease (PD) and in MPTP neurotoxicity remains unclear. Here, male TNF-alpha (-/-) deficient mice and C57bL/6 mice were treated with MPTP (4 x 15 mg/kg, 24 h intervals) and in one series, thalidomide was administered to inhibit TNF-alpha synthesis. Real-time RT-PCR revealed that the striatal mRNA levels of TNF-alpha, of the astrocytic marker glial fibrillary acidic protein (GFAP) and of the marker for activated microglia, macrophage antigen complex-1 (MAC-1), were significantly enhanced after MPTP administration. Thalidomide (50 mg/kg, p.o.) partly protected against the MPTP-induced dopamine (DA) depletion, and TNF-alpha (-/-) mice showed a significant attenuation of striatal DA and DA metabolite loss as well as striatal tyrosine hydroxylase (TH) fiber density, but no difference in nigral TH and DA transporter immunoreactivity. TNF-alpha deficient mice suffered a lower mortality (10%) compared to the high mortality (75%) seen in wild-type mice after acute MPTP treatment (4 x 20 mg/kg, 2 h interval). HPLC measurement of MPP(+) levels revealed no differences in TNF-alpha (-/-), wild-type and thalidomide treated mice. This study demonstrates that TNF-alpha is involved in MPTP toxicity and that inhibition of TNF-alpha response may be a promising target for extending beyond symptomatic treatment and developing anti-parkinsonian drugs for the treatment of the inflammatory processes in PD.     

Vertebrate-like βγ-crystallins in the ocular lenses of a copepod

The diverse crystallins are water-soluble proteins that are responsible for the optical properties of cellular lenses of animal eyes. While all vertebrate lenses contain physiological stress-related - and -crystallins, some also contain taxon-specific, often enzyme-related crystallins. To date, the - and -crystallins have been found only in vertebrate lenses. Here we report lenses from an invertebrate, the pontellid copepod Anomalocera ornata, accumulate -crystallin family members as judged by immunocytochemistry, western immunoblotting and microsequencing. Our data provide the first example of -crystallin members in an invertebrate lens, establishing that the use of this protein family as lens crystallins is not confined to vertebrates.     

Species-specific lens activation of the thymidine kinase promoter by a single copy of the mouse alpha A-CRYBP1 site and loss of tissue specificity by multimerization.

One copy of the mouse alpha A-crystallin gene alpha A-CRYBP1 site activated the thymidine kinase (tk) promoter in a mouse lens epithelial cell line but not in primary chicken lens cells; multiple copies further activated the tk promoter and extended expression to fibroblasts, B cells, and chicken lens cultures. The loss of lens specificity by multimerization may place selective constraints on the number of alpha A-CRYBP1 sites in the alpha A-crystallin promoter.     

Characterization of squid crystallin genes. Comparison with mammalian glutathione S-transferase genes.

Previous experiments have indicated that the crystallins of the squid lens (S-crystallins) are evolutionarily related to glutathione S-transferases (GST) (EC 2.5.1.18). Here we confirm by peptide sequencing that the crystallins of the lens of the squid Ommastrephes sloani pacificus comprise a family of GST-like proteins. Squid lens extracts showed 400 times less GST activity than those of liver using 1-chloro-2,4-dinitrobenzene as a substrate, suggesting that the abundant GST-like crystallins lack enzymatic activity. Four different cDNAs (pSL20-1, pSL18, pSL11, and pSL4) showed 20-25% similarity in homologous regions with mammalian GST polypeptides. pSL20-1, pSL18, and pSL4 each encode an S-crystallin with a unique internal peptide that is unrelated to mammalian GSTs or any other sequence in GenBank. The S-crystallin family is encoded in a minimum of 9-10 genes, and the exon-intron structures of at least two of these (SL20-1 and SL11) are similar to those of the mammalian GST genes. The SL20-1 gene has six exons, with the its unique internal peptide encoded precisely in exon 4; the SL11 gene lacks a unique internal peptide and has five exons. Experiments using bacterial chloramphenicol acetyltransferase as a reporter gene showed that at least 84 and 111 base pairs of 5'-flanking sequence are needed for function of the SL20-1 and SL11 promoters, respectively, in a transfected rabbit lens epithelial cell line (N/N1003A). Within these regions each has a putative TATA box and an upstream AP-1 site overlapping with antioxidant responsive-like elements, which are regulatory elements in the rat GST Ya and quinone reductase genes responsive to oxidative stress.     

The chicken delta-crystallin gene family. Two genes of similar structure in close chromosomal approximation

delta-Crystallin is a major protein product of the differentiated chicken lens. We have isolated two, non-allelic delta-crystallin genes using a recombinant bacteriophage/chicken genomic DNA library. There appear to be only these two delta-crystallin genes in the haploid chicken genome. Southern hybridization and R-loop analyses indicate that the two genes are oriented on the chromosome with similar 5'-3' polarity. delta 1, arbitrarily designated as the directionally 5' of the two genes, is 6.7 kilobases in length, while delta 2 is 9.2 kilobases. The two delta-crystallin genes are about 4.2 kilobases apart. Structurally, both genes are arranged in a similar and characteristic pattern of 17 exons/16 introns, as judged by electron microscopy. The delta-crystallin gene locus represents a simple model for the study of structural co-evolution and/or functional co-expression of two related genes within a developmentally modulated region of the genome.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic δ-crystallin

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens.The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of δ-crystallin, since δ-crystallin peptides could be identified in tryptic pepetide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable δ-crystallin peptides, although it is possible that modified δ-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added δ-[³⁵S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of δ-crystallin in lens membrane preparations.