Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus

The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The gamma-glutamylcysteine synthetase (gammaecs) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem, with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gammaecs and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

Molecular analysis of the psa permease complex of Streptococcus pneumoniae

The psaBCA locus of Streptococcus pneumoniae encodes a putative ABC Mn2+-permease complex. Downstream of the operon is psaD, which may be co-transcribed and encodes a thiol peroxidase. Previously, there has been discordance concerning the phenotypic impact of mutations in the psa locus, resolution of which has been complicated by differences in mutant construction and the possibility of polar effects. Here, we constructed unmarked, in frame deletion mutants DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaD, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD in S. pneumoniae D39 to examine the role of each gene within the locus in Mn2+ uptake, susceptibility to oxidative stress, virulence, nasopharyngeal colonization and chain morphology. The requirement for Mn2+ for growth and transformation was also investigated for all mutants. Inductively coupled plasma mass spectrometry (ICP-MS) analysis provided the first direct evidence that PsaBCA is indeed a Mn2+ transporter. However, this study did not substantiate previous reports that the locus plays a role in choline-binding protein pro-duction or chain morphology. We also confirmed the importance of the Psa permease in systemic virulence and resistance to superoxide and hydrogen peroxide, as well as demonstrating a role in nasopharyngeal colonization for the first time. Further evi-dence is provided to support the requirement for Mn2+ supplementation for growth and transformation of DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD mutants. However, transformation, as well as growth, of the DeltapsaD mutant was not dependent upon Mn2+ supplementation. We also show that, apart from sensitivity to hydrogen peroxide, the DeltapsaD mutant exhibited essentially similar phenotypes to those of the wild type. Western blot analysis with a PsaD antiserum showed that deleting any of the genes upstream of psaD did not affect its expression. However, we found that deleting psaB resulted in decreased expression of PsaA relative to that in D39, whereas deleting both psaB and psaC resulted in at least wild-type levels of PsaA.

Columnar cell lesions of the breast: The missing link in breast cancer progression? A morphological and molecular analysis

Columnar cell lesions (CCLs) of the breast are a spectrum of lesions that have posed difficulties to pathologists for many years, prompting discussion concerning their biologic and clinical significance. We present a study of CCL in context with hyperplasia of usual type (HUT) and the more advanced lesions ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. A total of 81 lesions from 18 patients were subjected to a comprehensive morphologic review based upon a modified version of Schnitt's classification system for CCL, immunophenotypic analysis (estrogen receptor [ER], progesterone receptor [PgR], Her2/neu, cytokeratin 5/6 [CK5/6], cytokeratin 14 [CK14], E-cadherin, p53) and for the first time, a whole genome molecular analysis by comparative genomic hybridization. Multiple CCLs from 3 patients were studied in particular detail, with topographic information and/or showing a morphologic spectrum of CCL within individual terminal duct lobular units. CCLs were ER an PgR positive, CK5/6 and CK14 negative, exhibit low numbers of genetic alterations and recurrent 16q loss, features that are similar to those of low grade in situ and invasive carcinoma. The molecular genetic profiles closely reflect the degree of proliferation and atypia in CCL, indicating some of these lesions represent both a morphologic and molecular continuum. In addition, overlapping chromosomal alterations between CCL and more advanced lesions within individual terminal duct lobular units suggest a commonality in molecular evolution. These data further support the hypothesis that CCLs are a nonobligate, intermediary step in the development of some forms of low grade in situ and invasive carcinoma. Copyright: © 2005 Lippincott Williams & Wilkins, Inc.

Molecular analysis reveals tighter social regulation of immigration in patrilocal populations than in matrilocal populations

Human social organization can deeply affect levels of genetic diversity. This fact implies that genetic information can be used to study social structures, which is the basis of ethnogenetics. Recently, methods have been developed to extract this information from genetic data gathered from subdivided populations that have gone through recent spatial expansions, which is typical of most human populations. Here, we perform a Bayesian analysis of mitochondrial and Y chromosome diversity in three matrilocal and three patrilocal groups from northern Thailand to infer the number of males and females arriving in these populations each generation and to estimate the age of their range expansion. We find that the number of male immigrants is 8 times smaller in patrilocal populations than in matrilocal populations, whereas women move 2.5 times more in patrilocal populations than in matrilocal populations. In addition to providing genetic quantification of sex-specific dispersal rates in human populations, we show that although men and women are exchanged at a similar rate between matrilocal populations, there are far fewer men than women moving into patrilocal populations. This finding is compatible with the hypothesis that men are strictly controlling male immigration and promoting female immigration in patrilocal populations and that immigration is much less regulated in matrilocal populations.

Imaging, anatomical, and molecular analysis of callosal formation in the developing human fetal brain

A complex set of axonal guidance mechanisms are utilized by axons to locate and innervate their targets. In the developing mouse forebrain, we previously described several midline glial populations as well as various guidance molecules that regulate the formation of the corpus callosum. Since agenesis of the corpus callosum is associated with over 50 different human congenital syndromes, we wanted to investigate whether these same mechanisms also operate during human callosal development. Here we analyze midline glial and commissural development in human fetal brains ranging from 13 to 20 weeks of gestation using both diffusion tensor magnetic resonance imaging and immunohistochemistry. Through our combined radiological and histological studies, we demonstrate the morphological development of multiple forebrain commissures/decussations, including the corpus callosum, anterior commissure, hippocampal commissure, and the optic chiasm. Histological analyses demonstrated that all the midline glial populations previously described in mouse, as well as structures analogous to the subcallosal sling and cingulate pioneering axons, that mediate callosal axon guidance in mouse, are also present during human brain development. Finally, by Northern blot analysis, we have identified that molecules involved in mouse callosal development, including Slit, Robo, Netrin1, DCC, Nfia, Emx1, and GAP-43, are all expressed in human fetal brain. These data suggest that similar mechanisms and molecules required for midline commissure formation operate during both mouse and human brain development. Thus, the mouse is an excellent model system for studying normal and pathological commissural formation in human brain development. (c) 2006 Wiley-Liss, Inc.

Molecular evolution of breast cancer

Molecular analysis of invasive breast cancer and its precursors has furthered our understanding of breast cancer progression. In the past few years, new multi-step pathways of breast cancer progression have been delineated through genotypic-phenotypic correlations. Nuclear grade, more than any other pathological feature, is strongly associated with the number and pattern of molecular genetic abnormalities in breast cancer cells. Thus, there are two distinct major pathways to the evolution of low- and high-grade invasive carcinomas: whilst the former consistently show oestrogen receptor (ER) and progesterone receptor (PgR) positivity and 16q loss, the latter are usually ER/PgR-negative and show Her-2 over-expression/amplification and complex karyotypes. The boundaries between the evolutionary pathways of well-differentiated/low-grade ductal and lobular carcinomas have been blurred, with changes in E-cadherin expression being one of the few distinguishing features between the two. In addition, lesions long thought to be precursors of breast carcinomas, such as hyperplasia of usual type, are currently considered mere risk indicators, whilst columnar cell lesions are now implicated as non-obligate precursors of atypical ductal hyperplasia (ADH) and well-differentiated ductal carcinoma in situ (DCIS). However, only through the combination of comprehensive morphological analysis and cutting-edge molecular tools can this knowledge be translated into clinical practice and patient management. Copyright (C) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley Sons, Ltd.

Gene expression profiles in murine hematopoietic stem cells revisited: Analysis of cDNA libraries reveals high levels of translational and metabolic activities

Gene expression studies from hematopoietic stem cell (HSC) populations purified to variable degrees have defined a set of sternness genes. Unexpectedly, results also hinted toward a HSC chromatin poised in a wide-open state. With the aim of providing a robust tool for further studies into the molecular biology of HSCs, the studies herein describe the construction and comparative molecular analysis of A-phage cDNA libraries from highly purified HSCs that retained their long-term repopulating activities (long-term HSCs [LT-HSCs]) and from short-term repopulating HSCs that were largely depleted of these activities. Microarray analysis of the libraries confirmed the previous results but also revealed an unforeseen preferential expression of translation- and metabolism-associated genes in the LT-HSCs. Therefore, these data indicate that HSCs are quiescent only in regard of proliferative activities but are in a state of readiness to provide the metabolic and translational activities required after induction of proliferation and exit from the HSC pool.