ABSTRACT
PURPOSE: To identify and analyze differentially genes expressed between lens epithelia dissected from age-related cataractous and noncataractous human lenses. METHODS: RNAs from 50 pooled cataractous and 25 pooled noncataractous epithelia were compared by reverse transcription-polymerase chain reaction differential display (RT-PCR-DD). Two differentially displayed bands were chosen for further study. These were reamplified, cloned, and sequenced. Expression of these genes was further evaluated in pooled and individual epithelia by RT-PCR with gene-specific primers. RESULTS: Significant differences in gene expression were detected between the cataractous and the noncataractous epithelia. Three mRNAs displayed higher levels and 12 mRNAs displayed lower levels of expression in the cataractous samples compared with that in the noncataractous samples. Of the mRNAs expressed at higher levels, one was identified as metallothionein IIa (METII). Of the mRNAs with decreased expression, one was identified as protein phosphatase 2A regulatory subunit (P2A-RS). Overexpression of METII and underexpression of P2A-RS were confirmed in pooled and individual epithelia. CONCLUSIONS: These results provide evidence that age-related cataract is associated with alterations in the expression of multiple epithelial genes including METII and P2A-RS. METII is a detoxification protein induced by oxidative stress, and P2A-RS is a mitotic suppressor involved in cell-cycle control. These results implicate these proteins and their associated functions in the maintenance of lens transparency.
Subject(s)
Cataract/genetics , Crystallins/genetics , Gene Expression , Lens, Crystalline/metabolism , Metallothionein/genetics , Phosphoprotein Phosphatases/genetics , Adult , Aged , Aged, 80 and over , Base Sequence , Cataract/metabolism , Crystallins/metabolism , DNA Primers/chemistry , Electrophoresis, Agar Gel , Epithelium/metabolism , Female , Humans , Male , Metallothionein/metabolism , Middle Aged , Molecular Sequence Data , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 2 , RNA/isolation & purification , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence AlignmentABSTRACT
The intra-acrosomal human sperm protein SP-10 was previously designated a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. In the present study, a monoclonal antibody to SP-10 (MHS-10) was employed on Western blots to identify immunoreactive SP-10 in sperm extracts from baboon (Papio cyanocephalus anubis) and two macaques (Macaca mulatta and Macaca fascicularis). In each of these primates, the MHS-10 monoclonal antibody recognized a polymorphic pattern of immunoreactive peptides similar to that in humans. Immunoreactive SP-10 was also demonstrated in pig sperm. Using purified preparations of the previously described intra-acrosomal molecules acrosin and sperminogen in the pig, we observed that the MHS-10 monoclonal antibody did not react with these proteins, indicating SP-10 is distinct from these known acrosomal components. Sperm from several common species including the rabbit, bull, rat, guinea pig and cat did not immunoreact with the MHS-10 monoclonal antibody. By use of a radioactive probe spanning 628 nucleotides of the open reading frame for SP-10 on Northern blots of poly A + RNA obtained from testes of Macaca fascicularis, Papio papio, and Papio cyanocephalus anubis, a 1.35-kb mRNA of identical size to the mRNA from human testes was identified. These results indicate that baboons, macaques, and pigs may be appropriate models for testing an SP-10-based contraceptive vaccine.
Subject(s)
Acrosome/immunology , Antigens/immunology , Macaca fascicularis/immunology , Macaca mulatta/immunology , Macaca/immunology , Papio/immunology , Spermatozoa/immunology , Swine/immunology , Acrosome/ultrastructure , Animals , Blotting, Northern , Blotting, Western , Contraception, Immunologic/methods , Contraceptive Agents, Male/immunology , Cross Reactions , Humans , Male , Species Specificity , Spermatozoa/ultrastructure , Vaccines/immunologyABSTRACT
Theory and prototype (at wavelength lambda = 5 mm) partial coherence scattering data for optical applications in diagnostic measurements on two component suspensions or gases are presented. Results are given for equal volume mixtures of two sizes of moving randomly distributed large spheres for all realizable values of the fractional volume w (the fraction of the slab region container filled by scattering material). The relative index of refraction of the spheres was about 1.017, and their diameters were 6.52lambda and 3.33lambda (so that we used about eight times as many small spheres as large ones for each value of w). The spheres were of lightweight Styrofoam, and their motion arose from turbulent air streams (flowing through grids that form the top and bottom of a Styrofoam container), and the data were obtained in real time by processing the instantaneous phase quadrature components of the scattered field with an electronic analog computer. We give results for the forward scattered coherent phase, for the coherent, incoherent, and total intensities, and for the covariant magnitude and phase which (together with the incoherent intensity) provide the variances and covariance of the instantaneous phase quadrature components. We also consider certain reduced data records (from which the major effects of scatterer size and material have been eliminated) to indicate the dependence of the scattering on the fractional volume and to facilitate comparison with earlier data for distributions of identical spheres.
