Cumulative mutations of ubiquitin acceptor sites in human immunodeficiency virus type 1 gag cause a late budding defect

The p6 domain of human immunodeficiency virus type 1 (HIV-1) Gag has long been known to be monoubiquitinated. We have previously shown that the MA, CA, and NC domains are also monoubiquitinated at low levels (E. Gottwein and H. G. Krausslich, J. Virol. 79:9134-9144, 2005). While several lines of evidence support a role for ubiquitin in virus release, the relevance of Gag ubiquitination is unclear. To directly address the function of Gag ubiquitination, we constructed Gag variants in which lysine residues in the NC, SP2, and p6 domains were mutated to arginine either in individual domains or in combination. Using these mutants, we showed that in addition to MA, CA, NC, and p6, SP2 is also mono- or di-ubiquitinated at levels comparable to those of the other domains. Replacement of all lysine residues in only one of the domains had minor effects on virus release, while cumulative mutations in NC and SP2 or in NC and p6 resulted in an accumulation of late budding structures, as observed by electron microscopy analysis. Strikingly, replacement of all lysine residues downstream of CA led to a significant reduction in virus release kinetics and a fivefold accumulation of late viral budding structures compared to wild-type levels. These results indicate that ubiquitination of lysine residues in Gag in the vicinity of the viral late domain is important for HIV-1 budding, while no specific lysine residue may be needed and individual domains can functionally substitute. This is consistent with Gag ubiquitination being functionally involved in a transient protein interaction network at the virus budding site.     

Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.     

The power and the limitations of cross-species protein identification by mass spectrometry-driven sequence similarity searches

Mass spectrometry-driven BLAST (MS BLAST) is a database search protocol for identifying unknown proteins by sequence similarity to homologous proteins available in a database. MS BLAST utilizes redundant, degenerate, and partially inaccurate peptide sequence data obtained by de novo interpretation of tandem mass spectra and has become a powerful tool in functional proteomic research. Using computational modeling, we evaluated the potential of MS BLAST for proteome-wide identification of unknown proteins. We determined how the success rate of protein identification depends on the full-length sequence identity between the queried protein and its closest homologue in a database. We also estimated phylogenetic distances between organisms under study and related reference organisms with completely sequenced genomes that allow substantial coverage of unknown proteomes.     

Potential of fecal microbiota for early-stage detection of colorectal cancer

Several bacterial species have been implicated in the development of colorectal carcinoma (CRC), but CRC-associated changes of fecal microbiota and their potential for cancer screening remain to be explored. Here, we used metagenomic sequencing of fecal samples to identify taxonomic markers that distinguished CRC patients from tumor-free controls in a study population of 156 participants. Accuracy of metagenomic CRC detection was similar to the standard fecal occult blood test (FOBT) and when both approaches were combined, sensitivity improved > 45% relative to the FOBT, while maintaining its specificity. Accuracy of metagenomic CRC detection did not differ significantly between early- and late-stage cancer and could be validated in independent patient and control populations (N = 335) from different countries. CRC-associated changes in the fecal microbiome at least partially reflected microbial community composition at the tumor itself, indicating that observed gene pool differences may reveal tumor-related host–microbe interactions. Indeed, we deduced a metabolic shift from fiber degradation in controls to utilization of host carbohydrates and amino acids in CRC patients, accompanied by an increase of lipopolysaccharide metabolism.     

地塞美松中间体的C1,4脱氢和11α-羟基化

地塞美松 (Ｄｅｘａｍｅｔｈａｓｏｎｅ)为高效肾上腺皮质激素药物 ,临床上广泛使用。开拓用梯可吉宁 (Ｔｉｇｏｇｅｎｉｎ)为起始原料生产从化学结构和合成技术上讲较为合理 ,而且适合于资源综合利用[1] 。在合成过程中 ,除了Ａ环需引入Ｃ1,2 和Ｃ4 ,5两个双键 (含Ｃ-3 羟基氧化 )外 ,还需用微生物法在Ｃ-11位引入羟基。国外常采用化学法脱氢 ,然后再用霉菌 11α 羟基化[2 ,3 ] 。本文报道用两类微生物菌种 ,节杆菌 (Ａｒｔｈｒｏｂａｃｔｅｒｓｐ .)ＡＸ86和绿僵菌 (Ｍｅｔａｒｈｉｚｉｕｍｓｐ .)Ｍ 88,混合转化一步完成脱氢和羟基化反应…     

Instantaneously measured traits may detect non-plastic ecophysiological performances in response to drought, explaining distributions of Styrax species in the Cerrado

We analyzed the differences between irrigated and non-irrigated plants of three congeneric Styrax species that present distinct distribution patterns in the physiognomies of the Cerrado vegetation in Brazil. Styrax ferrugineus showed a stomatal conductance (g _s) unresponsive to soil water deficit in potted plants. This may explain the high gas exchange and photochemical efficiency found in this species, which is well adapted to the Cerrado sensu stricto (s. str.), a savanna-type vegetation. S. camporum, which is widely distributed in the Cerrado sensu lato (s. l.) areas, was the only species that exhibited increased intrinsic water use efficiency on the days of maximum water deficit. This result distinguishes S. camporum from S. pohlii, which is a forest species, since the g _s of both species decreased during the days of maximum water stress. In contrast to other studies, we propose that instantaneously measured traits, such as leaf gas exchange rates and chlorophyll fluorescence, may be used to detect non-plastic performances in response to environmental stress, helping explain distinct geographical distributions of congeneric species in the Cerrado vegetation.     

Identification of heterotrimeric G proteins in human sperm tail membranes

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein α-subunits, and A 86, which detects all known pertussis toxin-sensitive α-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein α-subunits failed to detect any specific antigens in enriched tail membranes AS 36, recognizing the ã2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa β₁-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein α-subunits nor G protein β-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive α-subunits. However, membrane preparations of nonpurified human spermatozoa contained α₂ subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein α-subunits; the putative β-subunit was identified as a β₂-subunit. © 1995 Wiley-Liss, Inc.