Molecular evolution of mammalian lactate dehydrogenase-A genes and pseudogenes: association of a mouse processed pseudogene with a B1 repetitive sequence

A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a B1 repetitive element was isolated, and a nucleotide sequence of approximately 3 kb was determined. The pseudogene and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of the LDH-A genes and processed pseudogenes from mouse, rat, and human were compared, and a phylogenetic tree was constructed. The rate and pattern of nucleotide substitutions in the LDH-A pseudogenes are similar to previously reported results (Li et al. 1984). The average rate of nucleotide substitutions in the LDH-A pseudogenes is 4.3 X 10(- 9)/site/year. The substitutions of C----T and G----A are most frequent, and A----G substitutions are relatively high. The rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which is not significantly higher than the average rate of 4.7 X 10(-9) for 35 mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88 X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to be highly conserved in evolution.      

3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

Herceptin failure is a major clinical problem in breast cancer. A subset of breast cancer patients with high HER-2/neu levels eventually experience metastatic disease progression when treated with Herceptin as a single agent. Mechanistic details of development of this aggressive disease are not clear. Therefore, there is a dire need to better understand the mechanisms by which drug resistance develops and to design new combined treatments that benefit patients with aggressive breast cancer and have minimal toxicity. We hypothesized that 3, 3′-diindolylmethane (DIM), a non-toxic agent can be combined with Herceptin to treat breast cancers with high levels of HER-2/neu. Here, we evaluated the effects of Herceptin alone and in combination with DIM on cell viability, apoptosis and clonogenic assays in SKBR3 (HER-2/neu-expressing) and MDA-MB-468 (HER-2/neu negative) breast cancer cells. We found that DIM could enhance the effectiveness of Herceptin by significantly reducing cell viability, which was associated with apoptosis-induction and significant inhibition of colony formation, compared with single agent treatment. These results were consistent with the down-regulation of Akt and NF-kB p65. Mechanistic investigations revealed a significant upregulation of miR-200 and reduction of FoxM1 expression in DIM and Herceptin-treated breast cancer cells. We, therefore, transfected cells with pre-miR-200 or silenced FoxM1 in these cells for understanding the molecular mechanism involved. These results provide experimental evidence, for the first time, that DIM plus Herceptin therapy could be translated to the clinic as a therapeutic modality to improve treatment outcome of patients with breast cancer, particularly for the patients whose tumors express high levels of HER-2/neu.     

Does Auxin Binding Protein 1 Control Both Cell Division and Cell Expansion?

The Auxin-Binding Protein 1 (ABP1) was identified over 30 years ago thanks to it''s high affinity for active auxins. ABP1 plays an essential role in plant life yet to this day, its function remains ‘enigmatic.’ A recent study by our laboratory shows that ABP1 is critical for regulation of the cell cycle, acting both in G₁ and at the G₂/M transition. We showed that ABP1 is likely to mediate the permissive auxin signal for entry into the cell cycle. These data were obtained by studying a conditional functional knock-out of ABP1 generated by cellular immunization in the model tobacco cell line, Bright Yellow 2.Key Words: auxin responses, auxin-binding protein 1, immunomodulation, cellular immunisation     

A niche for Wolbachia

Wolbachia are endosymbionts of arthropods and filarial nematodes. Arthropods infected with these endobacteria display altered reproductive phenotypes, including cytoplasmic incompatibility and sex-ratio distortion. In nematodes, the endobacteria are essential for embryogenesis and worm survival. Wolbachia are transmitted vertically from mother to progeny, and Frydman et al. recently showed that, after transfer to uninfected Drosophila, Wolbachia rapidly accumulate in the somatic stem cell niche. From this location, the endobacteria might enter the developing oocytes and infect the progeny.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.