Cryofractographic study of intercellular junctions in the populations of agar-cultivated Bordetella pertussis

The characteristic feature of replicas obtained from the freeze-fractures of B. pertussis unfixed cultures developing on casein charcoal agar for 1-7 days is the associative growth of highly polymorphic cells, ensured by the ramified system of intercellular connections (IC) formed by the derivatives of the outer layers of the cell wall. This proves that the associative location of bacterial cells, linked by numerous IC, in the preparation is not the artefact appearing in the process of their chemical fixation. In replicas obtained from the freeze-fractures of B. pertussis cultures, previously fixed with glutaraldehyde, osmic acid and uranyl acetate, oval cells with the cytoplasm having a relatively homogeneous structure and with the smoothed-out three-layer cell wall prevail. As a rule, IC are limited to the sites of direct contacts between individual cells.     

BHK-21-derived cell lines that produce basic fibroblast growth factor, but not parental BHK-21 cells, initiate neuronal differentiation of neural crest progenitors.

We present evidence that basic fibroblast growth factor (bFGF)-producing cells stimulate primary differentiation of neurons from neural crest progenitors. Baby hamster kidney (BHK-21) cells were stably cotransfected with plasmid pSV2/neo, which contains the gene conferring resistance to the neomycin analog G418 and expression vectors containing the human bFGF cDNA. Various clones, which differed in their bFGF production levels, were isolated. Homogeneous neural crest cells were cultured on monolayers of bFGF-producing, BHK-21-derived cell lines. While the parental BHK-21 cells, which do not produce detectable bFGF, had poor neurogenic ability, the various bFGF-producing clones promoted a 1.5- to 4-fold increase in neuronal cell number compared to the parental cells. This increase was correlated with the levels of bFGF produced by the different transfected clones, which ranged between 2.3 and 140 ng/mg protein. In contrast, no stimulation of neuronal differentiation was observed when neural crest cells were grown on monolayers of parental BHK cells transfected with plasmid pSV2/neo alone, or on a parental BHK-derived clone, which secretes high amounts of recombinant vascular endothelial growth factor (VEGF). Furthermore, the neuron-promoting ability of bFGF-producing cells could be mimicked by addition of exogenous bFGF to neural crest cells grown on the parental BHK line. A similar treatment of neural crest cells grown on laminin substrata, instead of BHK cells, resulted in increased survival of non-neuronal cells, but not of neurons (see also Kalcheim, C. 1989, Dev. Biol. 134, 1-10). Taken together, these results suggest that bFGF stimulates neuronal differentiation of neural crest cells by a cell-mediated signalling mechanism.     

The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, inSaccharomyces cerevisiae, theCDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence ofCDC40 (455 amino acids) contains four copies of a β-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in thecdc40-1 allele) results in normal vegetative growth at 23°C, and cell cycle arrest at 36°C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36°C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.     

Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^−/− mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.     

Irreconciliation as practice: resisting impunity and closure in Argentina

In Argentina, irreconciliation is created through everyday practices of vigilance against closure and collective struggles against impunity. In this essay, I show how over several decades since the fall of the dictatorial regime (1976-83), human rights activists and laypeople have devised ways to keep the past alive while attending to injustices through embodied collective engagements with the country's history and its legacies. By examining large protests, the everyday experiences of impunity, and a filmic exploration of kinship bonds and their entanglement with civilian complicity in the repression, the essay illustrates the ways in which irreconciliation is materialized and enacted as a form of social reconstruction many years after state terrorism.     

A new topological method to measure protein structure similarity

A method for the quantitative evaluation of structural similarity between protein pairs is developed that makes use of a Delaunay-based topological mapping. The result of the mapping is a three-dimensional array which is representative of the global structural topology and whose elements can be used to construe an integral scoring scheme. This scoring scheme was tested for its dependence on the protein length difference in a pairwise comparison, its ability to provide a reasonable means for structural similarity comparison within a family of structural neighbors of similar length, and its sensitivity to the differences in protein conformation. It is shown that such a topological evaluation of similarity is capable of providing insight into these points of interest. Protein structure comparison using the method is computationally efficient and the topological scores, although providing different information about protein similarity, correlate well with the distance root-mean-square deviation values calculated by rigid-body structural alignment.     

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

The involvement of IL-6 and IL-8 in acute invasive gastroenteritis of children

The involvement of the proinflammatory cytokines, interleukin 8 (IL-8) and 6 (IL-6), was studied during the first 72 h of acute invasive gastroenteritis. Study population included 33 infants and young children aged six months to six years and seven age-matched controls. As a group, patients with acute invasive gastroenteritis had an increased serum level of IL-8 and IL-6 as compared with healthy controls (p < 0.002 and p < 0.001, respectively). Subjects were then divided into two groups based on stool cultures (proven and non-proven bacterial cultures). Patients with bacterial-proven acute invasive gastroenteritis tended to have increased IL-8 serum concentrations (p < 0.07) as compared with those with non-proven bacterial etiologies and IL-6 levels were only detected in subjects with positive bacterial cultures (p < 0.05). When dividing each sub-group into early and late blood drawing with respect to disease onset, no statistical differences were found in each group but subjects with bacterial-proven etiologies had significant higher IL-6 levels as compared with non-proven etiologies at the two time points (p < 0.019 and p < 0.015, respectively).In conclusion, the proinflammatory cytokines, IL-6 and IL-8, are involved in acute invasive gastroenteritis. The difference in IL-6, and to a lesser degree IL-8, between proven and non-proven bacterial etiologies, needs further investigation.     

Unique misinsertion specificity of poliota may decrease the mutagenic potential of deaminated cytosines

DNA polymerase iota (poliota) is a distributive error-prone enzyme that can incorporate nucleotides opposite a variety of DNA lesions. Further elongation is, however, either substantially inhibited or completely abolished. Here, we provide evidence that poliota can facilitate the efficient bypass of uracil and its derivatives as well as oxidized cytosine and guanine residues. The fidelity of translesion replication depends upon the lesion encountered. Correct nucleotides were inserted preferentially opposite 7,8-dihydro-8-oxoguanine (8-oxoG) and 5-hydroxycytosine (5-OHC). However, when bypassing uracil, 5-hydroxyuracil (5-OHU) or 5,6-dihydrouracil (5,6-DHU), poliota inserted T and G with a 4- to 26-fold preference over the Watson-Crick base, A. While the T:U, T:5-OHU and T:5,6-DHU mispairs were extended poorly, the G:U, G:5-OHU and G:5,6-DHU mispairs were extended with equal or greater efficiency than the correctly paired primer termini. Thus, poliota-dependent misinsertion of G opposite uracil and its derivatives may actually provide a mechanism whereby mammalian cells can decrease the mutagenic potential of lesions formed via the deamination of cytosine.     

Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication

Dpo4 and Dbh are Y-family polymerases that originate from two closely related strains of Sulfolobaceae. Quite surprisingly, however, the two polymerases exhibit different enzymatic properties in vitro. For example, Dpo4 can replicate past a variety of DNA lesions, yet Dbh does so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is prone to make base pair substitutions, whereas Dbh predominantly makes single-base deletions. Overall, the two proteins are 54% identical, but the greatest divergence is found in their respective little finger (LF) domains, which are only 41% identical. To investigate the role of the LF domain in the fidelity and lesion-bypassing abilities of Y-family polymerases, we have generated chimeras of Dpo4 and Dbh in which their LF domains have been interchanged. Interestingly, by replacing the LF domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. Our studies indicate that the unique but variable LF domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member.