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.      

Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo

To investigate the extent to which in vivo mutation spectra might reflect the intrinsic specificities of active mutators, genetic and biochemical assays were used to analyse the DNA target specificities of cytidine deaminases of the APOBEC family. The results reveal the critical importance of nucleotides immediately 5' of the targeted C for the specificity of all three enzymes studied (AID, APOBEC1 and APOBEC3G). At position -1, APOBEC1 showed a marked preference for dT, AID for dA/dG and APOBEC3G a strong preference for dC. Furthermore, AID and APOBEC3G showed distinct dependence on the nucleotide at position -2 with dA/dT being favoured by AID and dC by APOBEC3G. Most if not all activity of the recombinant deaminases on free dC could be attributed to low-level contamination by host enzymes. The target preference of APOBEC3G supports it being a major but possibly not sole contributor to HIV hypermutation without making it a dominant contribution to general HIV sequence variation. The specificity of AID as deduced from the genetic assay (which relies on inactivation of sacB of Bacillus subtilis) agrees well with that deduced by Pham et al. using an in vitro assay although we postulate that major intrinsic mutational hotspots in immunoglobulin V genes in vivo might reflect favoured sites of AID action being generated by proximal DNA targets located on opposite DNA strands. The target specificity of AID also accords with the spectrum of mutations observed in B lymphoma-associated oncogenes. The possibility of deaminase involvement in non-lymphoid human tumours is hinted at by tissue-specific differences in the spectra of dC transitions in tumour-suppressor genes. Thus, the patterns of hypermutation in antibodies and retroviruses owe much to the intrinsic sequence preferences of the AID/APOBEC family of DNA deaminases: analogous biases might also contribute to the spectra of cancer-associated mutation.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Security Analysis and Improvement of ‘a More Secure Anonymous User Authentication Scheme for the Integrated EPR Information System’

Over the past few years, secure and privacy-preserving user authentication scheme has become an integral part of the applications of the healthcare systems. Recently, Wen has designed an improved user authentication system over the Lee et al.’s scheme for integrated electronic patient record (EPR) information system, which has been analyzed in this study. We have found that Wen’s scheme still has the following inefficiencies: (1) the correctness of identity and password are not verified during the login and password change phases; (2) it is vulnerable to impersonation attack and privileged-insider attack; (3) it is designed without the revocation of lost/stolen smart card; (4) the explicit key confirmation and the no key control properties are absent, and (5) user cannot update his/her password without the help of server and secure channel. Then we aimed to propose an enhanced two-factor user authentication system based on the intractable assumption of the quadratic residue problem (QRP) in the multiplicative group. Our scheme bears more securities and functionalities than other schemes found in the literature.     

AID Enzymatic Activity Is Inversely Proportional to the Size of Cytosine C5 Orbital Cloud

Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID's enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H > F > methyl > hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.     

AID is essential for immunoglobulin V gene conversion in a cultured B cell line

Following productive V gene rearrangement, the functional immunoglobulin genes in the B lymphocytes of man and mouse are subjected to two further types of genetic modification. Class-switch recombination, a region-specific but largely nonhomologous recombination process, leads to a change in constant region of the expressed antibody. Somatic hypermutation introduces multiple single nucleotide substitutions in and around the rearranged V gene segments and underpins affinity maturation. However, in chicken and rabbits (but not man or mouse), an additional mechanism, gene conversion, is a major contributor to V gene diversification. It has been demonstrated recently that both switch recombination and hypermutation are ablated in mice and humans lacking AID, a B cell-specific protein of unknown molecular activity. Here we show that disruption of AID in the DT40 chicken B cell lymphoma leads to a failure to perform immunoglobulin V gene conversion. Thus, AID is required for all three immunoglobulin gene modification programs (gene conversion, hypermutation, and switch recombination) and acts in the initiation or execution of these processes rather than in bringing the B cell to an appropriate stage of differentiation.     

A lipopolysaccharide mutant of Bradyrhizobium japonicum that uncouples plant from bacterial differentiation.

The Tn5-containing fragment from a non-nodulating mutant of Bradyrhizobium japonicum, strain ML142, was introduced into B. japonicum strain 61A101c by marker exchange to construct strain JS314. Strain JS314 failed to nodulate several soybean varieties tested. However, on a few varieties nodulelike structures were induced to a frequency of 54% of the plants inoculated. The ultrastructure of these nodules was studied in detail by light and electron microscopy. The nodules were devoid of internal bacteria, possessed central vascular tissue (unlike the lateral vascular tissue of a normal nodule), and exhibited localized cell death of epidermal cells. Study of the cell surface polysaccharides of strain JS314 revealed that the exopolysaccharide of this strain was identical to that of the wild type. However, the lipopolysaccharide (LPS) of strain JS314 showed gross differences from that isolated from the wild-type strain. Specifically, the LPS of strain JS314 appeared to lack the high molecular weight LPS I form, strongly suggesting that the LPS lacks the O-chain. Glycosyl-composition analysis showed that the LPS of mutant JS314 lacked 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, fucose, and quinovosamine. These results indicate that LPS I in B. japonicum is essential for bacterial infection of soybean, but is not required to initiate plant cortical cell division, an early plant response to infection.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.