Neurturin protects dopaminergic neurons following medial forebrain bundle axotomy.

To determine whether neurturin (NTN), a recently identified homologue of glial cell line-derived neurotrophic factor (GDNF), is able to preserve tyrosine hydroxylase immunoreactivity (TH-IR) in a rat model of Parkinson's disease, polymer encapsulated cells genetically engineered to release NTN were implanted near the substantia nigra 1 week before a unilateral medial forebrain bundle axotomy. Animals were allowed to survive for 1 week post-axotomy. Upon sacrifice, animals that received a NTN capsule had a significantly higher percentage of TH-IR (lesioned side vs non-lesioned side) than animals that had received a capsule containing non-transfected parent cells. However, in contrast to GDNF, no reduction of turning was observed upon amphetamine rotation with NTN. Nevertheless, these results suggest that NTN might have a therapeutic value for the treatment of Parkinson's disease.

Development of the pattern of photoreceptors in the chick retina.

The various classes of photoreceptor cells found in vertebrate retinae are organized in specific patterns, which are important for visual function. It is not known how these patterns are achieved during development. The chick retina provides an excellent model system in which to investigate this issue, containing cone opsins red, green, blue, and violet, as well as the rod-specific opsin rhodopsin. In this study, whole-mount in situ hybridization has revealed striking differences among opsins in both spatial and temporal aspects of expression. The long-wavelength cone opsins, red and green, were first detected in a small spot within the area centralis at embryonic day 14 (E14). In contrast, the short-wavelength cone opsins, blue and violet, were not detected until 2 d later and showed domains of expression both within the area centralis and in temporal retina. The first rhodopsin transcripts were seen at E15 in inferior retina. When opsin expression was first detected, there were differences in the localization of RNA within the inner segment of cone photoreceptors, suggesting that morphological differentiation preceded the expression of photopigment molecules. Marked differences in the distribution of rods and cones were also found. Within the area centralis, a circular rod-free zone bisected by a narrow rod-sparse region along the nasal-temporal axis was evident as soon as rhodopsin RNA could be detected. Such specialized regions appear to be set aside soon after photoreceptor cells become postmitotic, as evidenced by a spatially restricted pattern of visinin RNA observed at E7. The onset of particular opsins in restricted regions of the retina suggest an underlying pattern related to visual function in the chick.

Isolation and characterization of cDNA clones encoding the Drosophila homolog of the HMG-box SSRP family that recognizes specific DNA structures.

Recently an HMG-box protein denoted SSRP1, for structure-specific recognition protein 1, has been discovered which binds to specific DNA structural elements such as the bent, unwound conformations that occur upon the formation of intrastrand crosslinks by the anticancer drug cisplatin. The SSRP family includes the mouse protein T160, which recognizes recombination signal sequences. In order to delineate functional domains more clearly, a homolog of SSRP1 was cloned from Drosophila melanogaster. This homolog maps to polytene region 60A (1-4) and shares 54% identity with human SSRP1. Comparison of the predicted amino acid sequences among SSRP family members reveals 48% identity, with structural conservation in the carboxy terminus of the HMG box as well as domains of highly charged residues. Interestingly, however, the most highly conserved regions of the protein are in the less well understood amino terminus, strongly suggesting that this portion of the protein is critical for its function.

Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions to DNA caused by binding of the anticancer agent cisplatin.

Human cDNA clones encoding a structure-specific recognition protein, SSRP1, that binds specifically to DNA modified with cisplatin have been isolated and characterized. The SSRP1 gene maps to human chromosome 11q12. The cDNA clones, obtained by using partial-length cDNAs described previously, predict an 81-kDa protein containing several highly charged domains and a stretch of 75 amino acids 47% identical to a portion of the high mobility group (HMG) protein HMG1. This HMG box most likely constitutes the structure recognition element for cisplatin-modified DNA, with the probable recognition motif being the local duplex unwinding and bending toward the major groove that occurs upon formation of intrastrand cis-[Pt(NH3)2]2+ d(GpG) and d(ApG) cross-links. Although the DNA recognition properties of members of the HMG-box family of proteins have been characterized with respect to their sequence specificity, the present work demonstrates that proteins with this domain can recognize particular DNA structures as well. The Pt-DNA SSRP described here is the human homolog of a recently identified mouse protein that binds to recombination signal sequences [Shirakata, M., Hüppi, K., Usuda, S., Okazaki, K., Yoshida, K. & Sakano, H. (1991) Mol. Cell. Biol. 11, 4528-4536]. These sequences have been postulated to form stem-loop structures, further implicating local bends and unwinding in DNA as a recognition target for HMG-box proteins. Expression analysis in a variety of tissues and cisplatin-resistant cell lines and the inability of cisplatin to induce the message in HeLa cells argue against a direct link between SSRP1 mRNA levels and the response of cells to the drug.

Isolation of cDNAs encoding a human protein that binds selectively to DNA modified by the anticancer drug cis-diamminedichloroplatinum(II)

DNA modified by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) was used to identify a factor in mammalian cells that binds to cis-DDP-damaged DNA and hence may play a role in repair. This factor selectively recognizes double-stranded DNA fragments modified by cis-DDP or [Pt(en)Cl2] (en, ethylenediamine). Little or no binding occurs to unmodified double-stranded DNA or to DNA modified with the clinically ineffective compounds trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine). Low levels of binding to single-stranded DNA modified by cis-DDP are observed. The apparent molecular mass of the factor in a variety of mammalian cells is approximately 100 kDa, as determined by modified Western blotting. Two recombinant phage have been isolated from a human B-cell lambda gt11 library by using a cis-DDP-modified DNA restriction fragment as a probe. The two clones have insert sizes of 1.88 and 1.44 kilobases and are aligned at their 5' ends. The polypeptides encoded by the recombinant phage exhibit DNA binding properties similar to those of the cellular factor identified in crude extracts prepared from mammalian cells. Northern analysis with one of the clones revealed an mRNA of 2.8 kilobases that is conserved in humans and rodents. The methods used here should be applicable in studies of other damage-specific DNA binding proteins.