Towards the molecular biology of cell adhesion in Drosophila

The characterization of extracellular matrix molecules and their putative receptors is rapidly evolving in Drosophila. Where corresponding vertebrate and Drosophila extracellular proteins have been identified they are very similar with respect to their structural properties, suggesting a high degree of conservation during evolution. By contrast, indications for components homologous to vertebrate cell-cell adhesion molecules are still very sparse. Studies on the regulation of the Drosophila genes encoding cell adhesion molecules that are involved in general basic functions during morphogenesis, together with a knowledge of the function of the genes responsible for pattern formation, should lead towards a more complete understanding of the organism's developmental program.     

The role of dendritic cells in the initiation of immune responses to contact sensitizers. I. In vivo exposure to antigen

Twenty-four hours after skin painting mice with picryl chloride (PIC) there was a four- to fivefold increase in the numbers of dendritic cells (DC) isolated from the lymph nodes. These DC initiated primary proliferative and cytotoxic responses when added to cultures of normal syngeneic lymph node cells. The proliferative response was enhanced when the donors of the responding lymph node cells were sensitized with the same antigen. Contact sensitivity developed in syngeneic mice injected into the footpads with 30,000-50,000 DC from lymph nodes of mice painted with picryl chloride 1 day previously. Thus, 1 day after skin painting mice, there were dendritic cells in the draining lymph nodes which were able both to initiate primary stimulation of lymphocytes in vitro and to sensitize recipient mice to give specific delayed hypersensitivity reactions.     

Epithelial differentiation in the absence of extracellular matrix

Summary To investigate the regulation of epithelial differentiation, normal human epidermal keratinocytes were cultured floating on the surface of culture medium without attachment to a solid substrate. Keratinocytes spread out on the surface of the medium, proliferated and differentiated either into several flat lacy sheets 1 to 3 cells thick (on medium containing 0.15 mM calcium) or formed one single aggregate of cells from 5 to 15 cells in thickness on medium containing 1.15 mM calcium. The cell aggregates demonstrated a pattern of ordered epithelial differentiation. Levels of progressive differentiation resembling the structure of normal human epidermis were identified by light microscopy, immunohistochemistry, and electron microscopy. Differentiation proceeded from cells at the air side toward cells at the medium side with basal appearing cells on the air side and keratinocytes expressing filaggrin and involucrin on the side toward the medium. These results demonstrate that organized epithelial differentiation can occur in the absence of extracellular matrix. In contrast with other air-liquid interface cultures, epithelial differentiation in the absence of extracellular matrix progresses from air towards medium.     

The relationship between consumption of tyrosine and phenylalanine as precursors of catecholamine at breakfast and the circadian typology and mental health in Japanese infants aged 2 to 5 years

Background

This study aims to examine the relationship between tyrosine and phenylalanine intake at breakfast as precursors of dopamine, and scores on the Torsvall-Åkerstedt Diurnal Type Scale and of mental health in Japanese infants aged 2 to 5 years.

Results

An integrated questionnaire was administered to parents of 1,367 infants attending one of ten nursery schools governed by Kochi City or a kindergarten affiliated with the Faculty of Education at Kochi University (775 answers for analysis: 56.7%) in May and June 2008. Questionnaires included the Torsvall-Åkerstedt Diurnal Type Scale and questions on sleep habits (onset, offset, quality, quantity, and so on), meal habits (content and regularity of timing), and mental health (depressive states). Amount of tyrosine and phenylalanine intake was calculated based on a breakfast content questionnaire and data on the components of amino acids in foods. Infants who ingested more than 800 mg of tyrosine or phenylalanine at breakfast per meal were more morning-type than those who ingested less than 800 mg (ANOVA: P= 0.005). However, this relationship disappeared in the ANCOVA analysis (with the covariance of tryptophan intake, P= 0.894). Infants who ingested more than 800 mg of the two amino acids at breakfast showed significantly higher mental health scores (lower frequency of depressive states) than those who ingested less than 800 mg (ANOVA: P = 0.004). This relationship remained significant when ANCOVA analysis was performed with the covariance of tryptophan (ANCOVA: P= 0.017).

Conclusions

These results suggest that tyrosine and phenylalanine ingested at breakfast are not related with circadian phase, but are relate with mental health in infants.     

Targeted deletions created in yeast vectors by recombinational excision

We have developed a simple method for creating defined deletions in yeast vectors by utilizing the ability of Saccharomyces cerevisiae to perform homologous recombination. Two complementary single-stranded oligonucleotides are designed so that the 5' and 3' halves of the resulting double-stranded oligonucleotide are homologous to the 5' and 3' side of a desired deletion junction, respectively. The sequence to be deleted is cleaved by restriction endonuclease digestion, followed by co-transformation of the linearized plasmid and the oligonucleotide into yeast. By homologous recombination in vivo, a subset of the plasmids will recircularize and simultaneously acquire the deletion as defined by the oligonucleotide.     

ATPase and DNA helicase activities of the Saccharomyces cerevisiae anti-recombinase Srs2

Saccharomyces cerevisiae SRS2 encodes an ATP-dependent DNA helicase that is needed for DNA damage checkpoint responses and that modulates the efficiency of homologous recombination. Interestingly, strains simultaneously mutated for SRS2 and a variety of DNA repair genes show low viability that can be overcome by inactivating homologous recombination, thus implicating inappropriate recombination as the cause of growth impairment in these mutants. Here, we report on our biochemical characterization of the ATPase and DNA helicase activities of Srs2. ATP hydrolysis by Srs2 occurs efficiently only in the presence of DNA, with ssDNA being considerably more effective than dsDNA in this regard. Using homopolymeric substrates, the minimal DNA length for activating ATP hydrolysis is found to be 5 nucleotides, but a length of 10 nucleotides is needed for maximal activation. In its helicase action, Srs2 prefers substrates with a 3' ss overhang, and approximately 10 bases of 3' overhanging DNA is needed for efficient targeting of Srs2 to the substrate. Even though a 3' overhang serves to target Srs2, under optimized conditions blunt-end DNA substrates are also dissociated by this protein. The ability of Srs2 to unwind helicase substrates with a long duplex region is enhanced by the inclusion of the single-strand DNA-binding factor replication protein A.     

Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.     

Interaction with Rad51 is indispensable for recombination mediator function of Rad52

In the yeast Saccharomyces cerevisiae, the RAD52 gene is indispensable for homologous recombination and DNA repair. Rad52 protein binds DNA, anneals complementary ssDNA strands, and self-associates to form multimeric complexes. Moreover, Rad52 physically interacts with the Rad51 recombinase and serves as a mediator in the Rad51-catalyzed DNA strand exchange reaction. Here, we examine the functional significance of the Rad51/Rad52 interaction. Through a series of deletions, we have identified residues 409-420 of Rad52 as being indispensable and likely sufficient for its interaction with Rad51. We have constructed a four-amino acid deletion mutation within this region of Rad52 to ablate its interaction with Rad51. We show that the rad52delta409-412 mutant protein is defective in the mediator function in vitro even though none of the other Rad52 activities, namely, DNA binding, ssDNA annealing, and protein oligomerization, are affected. We also show that the sensitivity of the rad52delta409-412 mutant to ionizing radiation can be complemented by overexpression of Rad51. These results thus demonstrate the significance of the Rad51-Rad52 interaction in homologous recombination.     

Chromosome location, genomic organization of the porcine COL10A1 gene and model structure of the NC1 domain

The porcine COL10A1 gene, encoding the alpha1(X) chain of type X collagen, has been sequenced. The gene structure is evolutionarily conserved, consisting of three exons and two introns spanning 7100 bp. Linkage mapping localized the gene to chromosome 1, which is in agreement with human-pig homology maps. Furthermore, protein structure comparison of the functionally important carboxyl domain between species revealed that amino acid changes were few and mainly situated in loop regions.