Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.     

Fission yeast <Emphasis Type="Italic">ucp3</Emphasis> gene encodes a putative Arf6 GTPase-activating protein

In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as ‘New End Take Off’ (NETO). We previously found that Arf6, a member (class III) of the ADP-ribosylation factor GTPase (Arf) family, is necessary for NETO in fission yeast. Here we report the characterization of a S. pombe gene, ucp3, encoding a putative Arf GTPase-activating protein (GAP) for Arf6. The Ucp3 contains Arf GAP domain, and has a high similarity to Gts1, which was identified as a GAP for Arf3 (class III Arf) in Saccharomyces cerevisiae. Overexpression of ucp3 inhibited growth from new end possibly by disturbing the GDP/GTP-cycling of Arf6. Gene disruption of ucp3 revealed that Ucp3 is essential for cell viability. Ucp3 uniformly localizes to the cell periphery. And its localization is not dependent on microtubules, actin cytoskeletons, Arf6 and Syt22 (guanine nucleotide exchange factor for Arf6). We hypothesize that Ucp3 functions as a GAP for Arf6. Moreover, Ucp3 might have another function important for cell viability.     

Clearly different mechanisms of enhancement of short-lived Nef-mediated viral infectivity between SIV and HIV-1

Background

One of the major functions of Nef is in the enhancement of the infectivity of the human and simian immunodeficiency viruses (HIV and SIV, respectively). However, the detailed mechanism of the enhancement of viral infectivity by Nef remains unclear. Additionally, studies of mechanisms by which Nef enhances the infectivity of SIV are not as intensive as those of HIV-1.

Methods

We generated short-lived Nef constructed by fusing Nef to a proteasome-mediated protein degradation sequence to characterize the Nef role in viral infectivity.

Results

The apparent expression level of the short-lived Nef was found to be extremely lower than that of the wild-type Nef. Moreover, the expression level of the short-lived Nef increased with the treatment with a proteasome inhibitor. The infectivity of HIV-1 with the short-lived Nef was significantly lower than that with the wild-type Nef. On the other hand, the short-lived Nef enhanced the infectivity of SIV_mac239, an ability observed to be interestingly equivalent to that of the wild-type Nef. The short-lived Nef was not detected in SIV_mac239, but the wild-type Nef was, suggesting that the incorporation of Nef into SIV_mac239 is not important for the enhancement of SIV_mac239 infectivity.

Conclusions

Altogether, the findings suggest that the mechanisms of infectivity enhancement by Nef are different between HIV-1 and SIV_mac239. Lastly, we propose the following hypothesis: even when the expression level of a protein is extremely low, the protein may still be sufficiently functional.

     

Rax1, a protein required for the establishment of the bipolar budding pattern in yeast

In Saccharomyces cerevisiae, cell type determines two distinct spatial budding patterns. Haploid cells exhibit an axial pattern, whereas diploid cells exhibit a bipolar pattern. Axl1, a member of the insulin-degrading enzyme (IDE) family, is the key morphological determinant for the haploid axial pattern. Here we identified a novel gene, RAX1, specifically required for the bipolar budding pattern. Loss of RAX1 alters the bipolar pattern of axl1 haploids resulting in reversion to the axial pattern, and also alters the bipolar patterns of bud3 and bud4 haploids. However, bud10 rax1 haploids exhibit a random budding pattern, suggesting Bud10 acts as the key proximal landmark in axial budding. Rax1 is required for the localization of Bud8, the distal bipolar budding landmark. Interestingly, Rax1 contains a C-terminal domain possessing some similarity to insulin-related peptides. Our results suggest that Rax1 is necessary for the establishment of the bipolar budding landmark.     

The nucleotide and deduced amino acid sequences of a cDNA encoding a new species of pregnancy-specific beta 1-glycoprotein (PS beta G)

We screened a cDNA library of a human placenta with cDNA for nonspecific cross-reacting antigen, a member of the carcinoembryonic antigen gene family. One of the positive clones, PS34, was found to encode a 426 amino acid protein belonging to pregnancy-specific beta 1-glycoprotein (PS beta G). The mature PS34 protein consisted of domains, N, A1, A2, B2 and C. The domain-N of PS34 showed sequence similarities of 79.8-83.5% to those of the PS beta G members so far reported, indicating PS34 is a new member of PS beta G and also of the carcinoembryonic antigen gene family.     

Synthesis, structure, and hypochromism of (2,9)(6,9)purinophane

The title compound 1 was synthesized via 8 steps from phthalimide derivative 2. The molecular structure of 1 was determined by X-ray analysis and the relationship between the hypochromism and the stacking mode of two purine rings was discussed.     

Identification of novel proteins in isolated polyphosphate vacuoles in the primitive red alga Cyanidioschyzon merolae

Plant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate‐rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF‐MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density‐gradient ultracentrifugation. The vacuole‐containing fraction was subjected to SDS–PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non‐lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o‐methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin‐tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features.     

The minimal eukaryotic ribosomal DNA units in the primitive red alga Cyanidioschyzon merolae.

Cyanidioschyzon merolae is a small unicellular red alga that is considered to belong to one of the most deeply branched taxa in the plant kingdom. Its genome size is estimated to be 16.5 Mbp, one of the smallest among free-living eukaryotes. In the nucleus containing this small genome, one nucleolus is clearly observed, but the molecular basis for the intranuclear structure including ribosomal DNA organization is still unclear. We constructed a bacterial artificial chromosome library for C. merolae 10D composed of two subsets with different insert size distributions. The two subsets have average insert sizes of 97 and 48 kb, representing 10.0- and 6.9-fold genome-equivalent coverage of the haploid genome, respectively. For application to whole-genome shotgun sequencing, the termini of each clone were sequenced as sequence-tagged connectors and mapped on the contigs assigned to chromosomes. Screening for rRNA genes by conventional colony hybridization with high-density filter blots and subsequent sequencing revealed that the C. merolae genome contained the smallest number of ribosomal DNA units among all the eukaryotes examined to date. They consist of only 3 single units of rRNA genes distributed on separate chromosomal loci, representing an implication for concerted evolution. Based on these results, the origin and evolution of the nucleolus are discussed.     

Synthesis and SAR of novel parenteral anti-pseudomonal cephalosporins: discovery of FR264205

We describe herein the synthesis and biological evaluation of a series of novel cephalosporins with potent activity against Pseudomonas aeruginosa. Introduction of various amino groups to the 4-position of a 3-amino-2-methylpyrazole cephalosporin 3-side chain resulted in enhanced MIC values against multiple Pseudomonas aeruginosa strains and ultimately led to the discovery of FR264205 (15) with excellent anti-bacterial activity and weak convulsion effect by direct intracerebroventricular injection assay.