Hyperhomocysteinemia leads to adverse cardiac remodeling in hypertensive rats

Hyperhomocysteinemia (Hhe), linked to cardiovascular disease by epidemiological studies, may be an important factor in adverse cardiac remodeling in hypertension. Specifically, convergence of myocardial and vascular alterations promoted by Hhe and hypertension may exacerbate cardiac remodeling and myocardial dysfunction. We studied male spontaneously hypertensive rats fed one of three diets: control, intermediate Hhe inducing, or severe Hhe inducing. After 10 wk of dietary intervention, cardiac function was assessed in vitro, and cardiac and coronary arteriolar remodeling were monitored by histomorphometric, immunohistochemical, and biochemical techniques. Results showed that Hhe induced diastolic dysfunction, as characterized by the diastolic pressure-volume curve, without significant changes in baseline systolic function. Perivascular collagen levels were increased by Hhe, and there was an increase in left ventricular hydroxyproline levels. Myocyte size was not affected. Coronary arteriolar wall thickness increased with Hhe due to smooth muscle hyperplasia. Mast cells increased in parallel with Hhe and collagen accumulation. In summary, 10 wk of Hhe caused coronary arteriolar remodeling, myocardial collagen deposition, and diastolic dysfunction in hypertensive rats.     

Cholangiocytes exhibit dynamic, actin-dependent apical membrane turnover

The present studies of cholangiocytes used complementary histological, biochemical, and electrophysiological methods to identify a dense population of subapical vesicles, quantify the rates of vesicular trafficking, and assess the contribution of the actin cytoskeleton to membrane trafficking. FM 1-43 fluorescence measured significant basal rates of total exocytosis (1.33 +/- 0.16% plasma membrane/min) in isolated cholangiocytes and apical exocytosis in cholangiocyte monolayers. Cell surface area remained unchanged, indicating that there was a concurrent, equivalent rate of endocytosis. FM 1-43 washout studies showed that 36% of the endocytosed membrane was recycled to the plasma membrane. 8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP; cAMP analog) increased exocytosis by 71 +/- 31%, whereas the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; protein kinase A inhibitor) diminished basal exocytosis by 53 +/- 11%. A dense population of 140-nm subapical vesicles arose, in part, from apical membrane endocytosis. Phalloidin staining showed that a subpopulation of the endocytosed vesicles was encapsulated by F-actin. Furthermore, membrane trafficking was inhibited by disrupting the actin cytoskeleton with cytochalasin D (51 +/- 13% of control) or jasplakinolide (58 +/- 9% of control). These studies indicate that there is a high rate of vesicular trafficking at the apical membrane of cholangiocytes and suggest that both cAMP and the actin cytoskeleton contribute importantly to these events.     

The expression of the dodecameric ferritin in Listeria spp. is induced by iron limitation and stationary growth phase

The new discipline of Evolutionary Developmental Biology (Evo-Devo) is facing the fascinating paradox of explaining morphological evolution using conserved pieces or genes to build divergent animals. The cephalochordate amphioxus is the closest living relative to the vertebrates, with a simple, chordate body plan, and a genome directly descended from the ancestor prior to the genome-wide duplications that occurred close to the origin of vertebrates. Amphioxus morphology may have remained relatively invariant since the divergence from the vertebrate lineage, but the amphioxus genome has not escaped evolution. We report the isolation of a second Emx gene (AmphiEmxB) arising from an independent duplication in the amphioxus genome. We also argue that a tandem duplication probably occurred in the Posterior part of the Hox cluster in amphioxus, giving rise to AmphiHox14, and discuss the structure of the chordate and vertebrate ancestral clusters. Also, a tandem duplication of Evx in the amphioxus lineage produced a prototypical Evx gene (AmphiEvxA) and a divergent gene (AmphiEvxB), no longer involved in typical Evx functions. These examples of specific gene duplications in amphioxus, and other previously reported duplications summarized here, emphasize the fact that amphioxus is not the ancestor of the vertebrates but 'only' the closest living relative to the ancestor, with a mix of prototypical and amphioxus-specific features in its genome.     

Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability

The enhanced green fluorescent protein (EGFP) is increasingly used as a reporter gene in viral vectors for a number of applications. To establish a system to study the activity of cis-acting cellular regulatory sequences, we deleted the viral enhancer in EGFP-carrying retroviral vectors and replaced it with cell type-specific elements. In this study, we use this system to demonstrate the activity of the human CD2 lymphoid-specific and the Tie2 endothelial cell type-specific enhancers in cell lines and in primary cells transduced by retroviral vectors. Furthermore, we compare findings obtained with EGFP as the reporter gene to those obtained replacing EGFP with d2EGFP, an unstable variant of EGFP characterized by a much shorter half-life compared to EGFP, and by reduced accumulation in the cells. d2EGFP-carrying vectors were generated at titers which were not different from those generated by the corresponding vectors carrying EGFP. Moreover, the activity of a Moloney murine leukemia virus enhancer could be readily detected following transduction of target cells with either EGFP- or d2EGFP-carrying vectors. However, the activity of the relatively weak CD2 and Tie2 enhancers was exclusively detected using EGFP as the reporter gene.These findings indicate that enhancer replacement is a feasible and promising approach to address the function of cell type-specific regulatory elements in retroviral vectors carrying the EGFP gene.     

Comparative growth analysis of the facultative anaerobes Bacillus subtilis,Bacillus licheniformis,and Escherichia coli

Bacillus subtilis anaerobic respiration and fermentative growth capabilities were compared to two other facultative anaerobes, Bacillus licheniformis and Escherichia coli. In glycerol defined medium, B. subtilis grew with nitrate, but not nitrite or fumarate, while B. licheniformis grew with nitrate or fumarate, but not nitrite. Growth of E. coli occurred in glycerol defined medium with either nitrate, nitrite, or fumarate. In order to grow by fermentation, B. subtilis required both glucose and pyruvate, while B. licheniformis and E. coli were capable of using either glucose or pyruvate.     

Chromatin of Trypanosoma cruzi: in situ analysis revealed its unusual structure and nuclear organization

Chromatin of Trypanosoma cruzi is known to be organized in classical nucleosomal filaments, but surprisingly, these filaments do not fold in visible chromosomes and the nuclear envelope is preserved during cell division. Our hypothesis about the role of chromatin structure in regulating gene expression and, more generally, cell functioning, pressed us to verify if chromatin organization is modulated during the parasite life-cycle. To this end, we analyzed in situ the fine structural organization of T. cruzi chromatin by means of an integrated biophysical approach, using differential scanning calorimetry and fluorescence microscopy. We observed that logarithmic forms exhibit a less condensed chromatin with respect to the stationary ones. Thermal analysis revealed that parasite chromatin is organized in three main levels of condensation, barring from the polynucleosomal filament till to superstructured fibers. Besides, the fluorescence images of nuclei showed a characteristic chromatin distribution, with defined domains localized near to the nuclear envelope. While in stationary parasites, these regions are highly condensed, in logarithmic forms they unfold by extending themselves toward the center of nucleus. These observations suggest that, in comparison with higher eukaryotes, in T. cruzi the nuclear envelope plays an unusual and pivotal role in interphase and in mitosis.     

Higher-accuracy method for measuring minichromosome stability in Saccharomyces cerevisiae

The minichromosome maintenance assay isfrequently used to characterize mutations genetically that affect the initiation of DNA replication or to decipherfunctional components in autonomously replicating sequences. The assay determines minichromosome loss by measuring the percentage of plasmid-containing cells in cultures after a period of growth in nonselective medium. Here we analyze data acquisition errors that contribute to the low accuracy of the routine versions of the assay. We propose modifications that eliminate errors in the acquisition of two variables and significantly improve the accuracy of the assay.     

Human milk oligosaccharides: an enzymatic protection step simplifies the synthesis of 3'- and 6'-O-sialyllactose and their analogues

We describe a chemo-enzymatic synthesis of 3'- and 6'-O-sialyllactose, two trisaccharides occurring in the 'acidic fraction' of the human milk oligosaccharides and endowed with potential antiadhesive activity. The key step is the highly regioselective 6'-O-acylation of benzyllactoside, which gave access to suitably protected lactose building blocks to be used as acceptors in the sialylation reaction. Moreover, the synthesis of the carboxymethyl and sulfo analogues of the title compounds is reported.     

Meiotic chromosome missegregation during apyrene meiosis in the gypsy moth,Lymantria dispar,is preceded by an aberrant prophase I

The gypsy moth, Lymantria dispar, produces two structurally and genetically distinct types of spermatozoa. The eupyrene spermatozoa are genetically haploid and structurally typical. The apyrene spermatozoa are anucleate and structurally different from eupyrene spermatozoa. To understand further the events contributing to meiotic chromosome missegregation in apyrene spermatocytes, we examined the progression of meiosis in these cells with respect to their eupyrene counterparts. Chromosomal bouquet formation and fusion of nucleolar organizing regions are disrupted in apyrene nuclei. In addition, the chromatin of apyrene nuclei is prematurely and extremely condensed compared with that of eupyrene nuclei. An antibody to the conserved synaptonemal complex protein 3 (SCP3) labeled eupyrene pachytene chromosomes, but not apyrene pachytene chromosomes. In addition, apyrene meiotic spindles are missing a subset of microtubules, which likely include kinetochore microtubules. Because the condensation behavior of meiotic chromatin in apyrene spermatocytes deviates from that of eupyrene spermatocytes, we examined the appearance and distribution of the phosphorylated form of histone H3, but no significant differences in histone H3 phosphorylation were found between apyrene and eupyrene spermatocytes. We argue that because a pachytene checkpoint is not initiated in apyrene spermatocytes, this system may provide a way to understand better the underlying biochemical connections between pairing, recombination, synapsis, kinetochore assembly and segregation of chromosomes during meiosis in a higher eukaryote.