Identification and simulation of new non-random statistical properties common to different populations of eukaryotic non-coding genes

The autocorrelation function analysing the occurrence probability of the i-motif YRY(N)iYRY in genes allows the identification of mainly two periodicities modulo 2, 3 and the preferential occurrence of the motif YRY(N)6YRY (R = purine = adenine or guanine, Y = pyrimidine = cytosine or thymine, N = R or Y). These non-random genetic statistical properties can be simulated by an independent mixing of the three oligonucleotides YRYRYR, YRYYRY and YRY(N)6 (Arquès & Michel, 1990b). The problem investigated in this study is whether new properties can be identified in genes with other autocorrelation functions and also simulated with an oligonucleotide mixing model. The two autocorrelation functions analysing the occurrence probability of the i-motifs RRR(N)iRRR and YYY(N)iYYY simultaneously identify three new non-random genetic statistical properties: a short linear decrease, local maxima for i identical to 3[6] (i = 3, 9, etc) and a large exponential decrease. Furthermore, these properties are common to three different populations of eukaryotic non-coding genes: 5' regions, introns and 3' regions (see section 2). These three non-random properties can also be simulated by an independent mixing of the four oligonucleotides R8, Y8, RRRYRYRRR, YYYRYRYYY and large alternating R/Y series. The short linear decrease is a result of R8 and Y8, the local maxima for i identical to 3[6], of RRRYRYRRR and YYYRYRYYY, and the large exponential decrease, of large alternating R/Y series (section 3). The biological meaning of these results and their relation to the previous oligonucleotide mixing model are presented in the Discussion.     

Localization of calretinin mRNA in rat and guinea pig inner ear by in situ hybridization using radioactive and non-radioactive probes

The localization of calretinin mRNA was studied in the rat and guinea pig inner ear by in situ hybridization, and compared to the distribution of the protein previously examined by immunocytochemistry. Radioactive and non-radioactive in situ hybridization (ISH) were performed using oligonucleotide probes labelled with 35S or digoxigenin. Radioactive ISH was more sensitive than non-radioactive ISH. In cochlear and vestibular ganglia, calretinin mRNA was localized in subpopulations of neurons with patterns of distribution similar to those shown by immunocytochemistry. By contrast, the observations in the sensory epithelia differed with the two techniques, ISH revealing less positive structures than immunocytochemistry. Rat inner hair cells and guinea pig inner hair cells, Hensen's cells and Deiters cells, which had been described strongly immunoreactive, appeared positive with radioactive but not with non-radioactive ISH. On the other hand, rat vestibular type II hair cells and guinea pig interdental cells of the spiral limbus which were faintly immunoreactive were not positive with both ISH techniques.     

Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis

In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin-induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death.     

Three-year prospective adherence to three breast cancer screening modalities

PURPOSES: To determine the long-term risk/benefit ratio of phacoemulsification and intraocular lens (IOL) implantation combined with trabeculotomy to manage eyes with pseudoexfoliation syndrome and co-existing cataract. SETTING: Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine; Kurihara Eye Clinic; Departments of Ophthalmology, Tenri Hospital, Kumamoto University, and Matsue Red Hospital; Nagata Eye Clinic, Japan. METHODS: This multicenter retrospective study comprised 49 eyes of 36 patients with pseudoexfoliation syndrome and co-existing cataract who had the combined procedure for uncontrolled intraocular pressure (IOP) (> 21 mm Hg) even on antiglaucoma medication. RESULTS: After a mean follow-up of 20.0 months +/- 13.2 (SD), IOP in all 49 eyes was well controlled (< or = 21 mm Hg). Mean IOP at the final examination was 14.6 +/- 2.6 mm Hg on a mean of 0.9 +/- 0.8 glaucoma medications. Complications included an IOP spike in 11 eyes and fibrin exudation in 1 eye. CONCLUSION: Phacoemulsification and IOL implantation combined with trabeculotomy was an effective treatment for patients with pseudoexfoliation syndrome and cataract.     

p53 protein accumulation and response to adjuvant chemotherapy in premenopausal women with node-negative early breast cancer

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Mouse prolactin receptor gene: genomic organization reveals alternative promoter usage and generation of isoforms via alternative 3'-exon splicing

In rodents, the prolactin receptor is expressed as multiple isoforms with identical extracellular and membrane-proximal region sequences but with different 3' sequences, encoding different cytoplasmic regions, and different 5' untranslated region (UTR) sequences. These divergent sequences could be the result of multiple prolactin receptor genes or of a single gene which displays alternative promoter usage and 3'-exon splicing. To investigate the molecular basis for these observations, we have cloned and determined the organization of the mouse prolactin receptor gene. Genomic DNA cloning allowed the arrangement of promoters 1A, 1B, and 1C to be determined. 5'-RACE-PCR from mouse liver identified two novel 5' prolactin receptor sequences, indicating that the gene has at least five different promoters, four of which are active in liver. The remaining nonvariable 5' UTR is encoded by a separate exon (exon 2), while a further 11 coding exons follow, the last 4 of which are alternatively spliced to produce the four isoforms of the receptor. Functional units were found to be exon specific. Thus, the multiple prolactin receptor isoforms are the product of a single gene of >120 kb which displays multiple promoter usage and 3'-exon splicing.