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.      

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.      

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     

Involvement of MEKK1/ERK/P21Waf1/Cip1 signal transduction pathway in inhibition of IGF-I-mediated cell growth response by methylglyoxal

The abnormal accumulation of methylglyoxal (MG), a physiological glucose metabolite, is strongly related to the development of diabetic complications by affecting the metabolism and functions of organs and tissues. These disturbances could modify the cell response to hormones and growth factors, including insulin-like growth factor-1 (IGF-I). In this study, we investigated the effect of MG on IGF-I-induced cell proliferation and the mechanism of the effect in two cell lines, a human embryonic kidney cell line (HEK293), and a mouse fibroblast cell line (NIH3T3). MG rendered these cells resistant to the mitogenic action of IGF-I, and this was associated with stronger and prolonged activation of ERK and over-expression of P21(Waf1/Cip1). The synergistic effect of MG with IGF-I in activation of ERK was completely abolished by PD98059 but not by a specific PI3K inhibitor, LY294002, or a specific PKC inhibitor, bisindolylmaleimide. Blocking of Raf-1 activity by expression of a dominant negative form of Raf-1 did not reduce the enhancing effect of MG on IGF-I-induced activation of ERK. However, transfection of a catalytically inactive form of MEKK1 resulted in inactivation of the MG-induced activation of ERK and partial inhibition of the enhanced activation of ERK and over-expression of p21(Waf1/Cip1) induced by co-stimulation of MG and IGF-I. These results suggested that the alteration of intracellular milieu induced by MG through a MEKK1-mediated and PI3K/PKC/Raf-1-independent pathway resulted in the modification of cell response to IGF-I for p21(Waf1/Cip1)-mediated growth arrest, which may be one of the crucial mechanisms for MG to promote the development of chronic clinical complications in diabetes.     

Analysis of cell viability and differential activity of mouse hepatocytes under 3D and 2D culture in agarose gel

Three-dimensional (3D) and two-dimensional (2D) cultures of hepatocytes in various concentrations (0.3–0.7%) of agarose gel revealed that the hepatocytes under 3D cultures in 0.3% agarose gel possess long-term (>3 weeks) viability, significant self-assembly to form tissue like aggregates, low lactate dehydrogenase release and high albumin synthesis. These were in contrast to 2D culture of hepatocytes. Our results suggest that the 3D culture of hepatocytes in agarose gel favors aggregate formation of functionally active cells and would be useful for liver transplantation as well as to analyze hepatocytes biology.     

Adjustment of juvenile tuatara to a cooler,southern climate: operative temperatures,emergence behaviour and growth rate

Translocations for conservation often involve species limited to relict distributions. However, uncertainty can exist regarding the ability of source individuals to acclimatise following a shift to a distant location. We investigated the ability of captive-reared juvenile tuatara (Sphenodon punctatus) of Cook Strait stock (41°S) to adjust to outdoor, predator-protected pens within Orokonui Ecosanctuary (45 °S). We examined potential basking and within burrow temperatures, the influence of temperature on emergence, and growth rates in comparison with other locations. Tuatara at Orokonui reached their preferred temperature when basking over summer, and burrows provided protection from freezing over winter. Emergence was temperature-dependent and essentially ceased during winter. Growth rates of Orokonui-held juveniles were within the range for four other captive-rearing facilities and faster than for wild juveniles from a Cook Strait population. As all Orokonui-held juveniles have survived and grown we conclude that the climate at this southern location is suitable to consider a free-release.