[Geographic environment-related research advances on children's myopia: intraocular and environmental exposure factors and analytical methods].

Myopia has become a global phenomenon, transitioning into a significant public health issue of worldwide reach. The escalating prevalence of myopia may lead to an increase in the incidence of related complications, potentially resulting in irreversible vision damage for individuals. This not only causes considerable economic strain on societies but also poses a serious threat to vital sectors like national defense. This review outlines various external and internal exposure factors related to childhood myopia. It places particular focus on the analysis of the interaction between geographical environmental factors and internal exposure factors, and examines the limitations of applying traditional methods in studying the relationship between childhood myopia and geographical environmental factors. The paper also introduces two spatial regression methodologies based on frequency estimation and Bayesian estimation, summarizing their feasibility and merits when applied in the study of external exposure elements related to childhood myopia. Finally, it provides a fresh perspective on regional childhood myopia prevention strategies that are conscious of geographical environmental factors.

Biochemical genetic analysis of indanocine resistance in human leukemia.

Indanocine is a potent tubulin-binding drug that is cytotoxic to multidrug-resistant cancer cell lines. We demonstrated that indanocine specifically induces apoptosis in malignant B cells from patients with chronic lymphocytic leukemia. To address the exact biochemical basis for indanocine toxicity, an indanocine-resistant clone was selected from mutagenized CEM human lymphoblastoid cells. The resistant cells displayed a stable indanocine-resistant phenotype for at least 9 months in drug-free culture. The cloned cells are cross-resistant to colchicine and vinblastine, but not to paclitaxel, and do not have increased expression of the multidrug-resistant p170 glycoprotein. In both parental cells and cell extracts, indanocine treatment caused tubulin depolymerization. In contrast, the tubulin in the resistant clone did not depolymerize under identical conditions. Both extract mixing and cell fusion experiments suggested that a stable structural change in microtubules, rather than a soluble factor, was responsible for indanocine resistance. Sequence analysis of parental and resistant cells revealed a single point mutation in the M40 isotype of beta-tubulin at nucleotide 1050 (G-->T, Lys(350)-->Asn) in the indanocine-resistant clone, in a region close to the putative colchicine binding site.

Identification of a preinitiation step in DNA replication that is independent of origin recognition complex and cdc6, but dependent on cdk2.

Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and minichromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation complexes. Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA replication fails to initiate. We also find that after assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication. Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity.

A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle.

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2-cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2-cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2-cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2-cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2-cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2-cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.

Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling.

In Drosophila imaginal discs, the spatially restricted activities of the achaete (ac) and scute (sc) proteins, which are transcriptional activators of the basic-helix-loop-helix class, define proneural clusters (PNCs) of potential sensory organ precursor (SOP) cells. Here, we report the identification of several genes that are direct downstream targets of ac-sc activation, as judged by the following criteria. The genes are expressed in the PNCs of the wing imaginal disc in an ac-sc-dependent manner; the proximal promoter regions of all of these genes contain one or two high-affinity ac-sc binding sites, which define the novel consensus GCAGGTG(T/G)NNNYY; where tested, these binding sites are required in vivo for PNC expression of promoter-reporter fusion genes. Interestingly, these ac-sc target genes, including Bearded, Enhancer of split m7, Enhancer of split m8, and scabrous, are all known or believed to function in the selection of a single SOP from each PNC, a process mediated by inhibitory cell-cell interactions. Thus, one of the earliest steps in adult peripheral neurogenesis is the direct activation by proneural proteins of genes involved in restricting the expression of the SOP cell fate.