Structures of the Arm-type Binding Domains of HPI and HAI7 Integrases

The structures of the N-terminal domains of two integrases of closely related but not identical asn tDNA-associated genomic islands, Yersinia HPI (high pathogenicity island; encoding siderophore yersiniabactin biosynthesis and transport) and an Erwinia carotovora genomic island with yet unknown function, HAI7, have been resolved. Both integrases utilize a novel four-stranded β-sheet DNA-binding motif, in contrast to the known proteins that bind their DNA targets by means of three-stranded β-sheets. Moreover, the β-sheets in Int_HPI and Int_HAI7 are longer than those in other integrases, and the structured helical N terminus is positioned perpendicularly to the large C-terminal helix. These differences strongly support the proposal that the integrases of the genomic islands make up a distinct evolutionary branch of the site-specific recombinases that utilize a unique DNA-binding mechanism.     

不同退化红砂荒漠草地的水分分配格局

研究了内蒙古阿拉善盟不同过牧退化红砂草地的土壤 植物 大气系统的水分分配格局、不同退化草地和主要植物种的水分利用效率 .2 0 0 1年降雨量 12 4 .3mm ,其中试验期 119.4mm .1m深土壤水分结果表明 ,10～ 4 0cm土层受蒸散影响最大 ;由于主要共存种红砂和无芒隐子草根系分布和蒸腾强度不同等 ,含水量在 10～ 2 0cm土层以中度退化区显著低于其它样区 (P <0 .0 5 ) ,而 2 0～ 4 0cm土层以轻度退化区较低 .样地年均蒸发量为 30 .6mm ,红砂种群的年均蒸腾量为 11.9mm .随着草地退化加剧 ,裸地的蒸发量和退化指示种匍根骆驼蓬种群的蒸腾量增加 ,而红砂种群的蒸腾量降低 .与较轻度退化区比 ,中度和重度退化区的水分利用率分别下降了 14 .6 %和 4 6 .1% ,红砂水分利用率分别下降了 37.8%和 73.8% .     

The oxygen delivery response to acute hypoxia during incremental knee extension exercise differs in active and trained males

Background

It is well known that hypoxic exercise in healthy individuals increases limb blood flow, leg oxygen extraction and limb vascular conductance during knee extension exercise. However, the effect of hypoxia on cardiac output, and total vascular conductance is less clear. Furthermore, the oxygen delivery response to hypoxic exercise in well trained individuals is not well known. Therefore our aim was to determine the cardiac output (Doppler echocardiography), vascular conductance, limb blood flow (Doppler echocardiography) and muscle oxygenation response during hypoxic knee extension in normally active and endurance-trained males.

Methods

Ten normally active and nine endurance-trained males (VO_2max = 46.1 and 65.5 mL/kg/min, respectively) performed 2 leg knee extension at 25, 50, 75 and 100% of their maximum intensity in both normoxic and hypoxic conditions (FIO₂ = 15%; randomized order). Results were analyzed with a 2-way mixed model ANOVA (group × intensity).

Results

The main finding was that in normally active individuals hypoxic sub-maximal exercise (25 – 75% of maximum intensity) brought about a 3 fold increase in limb blood flow but decreased stroke volume compared to normoxia. In the trained group there were no significant changes in stroke volume, cardiac output and limb blood flow at sub-maximal intensities (compared to normoxia). During maximal intensity hypoxic exercise limb blood flow increased approximately 300 mL/min compared to maximal intensity normoxic exercise.

Conclusion

Cardiorespiratory fitness likely influences the oxygen delivery response to hypoxic exercise both at a systemic and limb level. The increase in limb blood flow during maximal exercise in hypoxia (both active and trained individuals) suggests a hypoxic stimulus that is not present in normoxic conditions.

     

Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2

Leech carboxypeptidase inhibitor (LCI) is a novel protein inhibitor present in the medicinal leech Hirudo medicinalis. The structures of LCI free and bound to carboxypeptidase A2 (CPA2)have been determined by NMR and X-ray crystallography, respectively. The LCI structure defines a new protein motif that comprises a five-stranded antiparallel beta-sheet and one short alpha-helix. This structure is preserved in the complex with human CPA2 in the X-ray structure, where the contact regions between the inhibitor and the protease are defined. The C-terminal tail of LCI becomes rigid upon binding the protease as shown in the NMR relaxation studies, and it interacts with the carboxypeptidase in a substrate-like manner. The homology between the C-terminal tails of LCI and the potato carboxypeptidase inhibitor represents a striking example of convergent evolution dictated by the target protease. These new structures are of biotechnological interest since they could elucidate the control mechanism of metallo-carboxypeptidases and could be used as lead compounds for the search of fibrinolytic drugs.     

Role of murine cytomegalovirus US22 gene family members in replication in macrophages

The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs suggested a common ancestry and related functions for these gene products. Two members of this family, m140 and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22 members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43 mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an interaction at the protein level. The M36-dependent macrophage growth phenotype could be explained by the antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family members have diverged through evolution to serve a variety of functions for the virus.     

Efforts towards the design of 'teflon' proteins: in vivo translation with trifluorinated leucine and methionine analogues

In vivo incorporation of monofluorinated noncanonical amino acids into recombinant proteins has been well-established for decades. Proteins fluorinated in this way proved to be useful tools for many practical applications. In contrast, trifluorinated amino acids have been incorporated in only a few peptides and relatively small proteins by using expression systems in living cells. A novel class of proteins with a fluorous core can be envisaged only if full replacement of the core-building hydrophobic and aliphatic amino acids such as leucine or methionine with the related analogues trifluoromethionine and trifluoroleucine would be feasible. However, our systematic efforts to introduce these amino acids in larger proteins (over 10 Da) that contain different structural motifs clearly show that only partial substitutions are possible. The reasons are high toxicity of these substances and difficulties to accommodate them into the compact cores of natural proteins without adverse effects on their structural integrity. Therefore, engineering of such three dimensional 'Teflon'-like structures would require, besides an expansion of the amino acid repertoire of the genetic code, a de novo protein design as well.     

Structure of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum

Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51-226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an alpha helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1-226), CAP(255-464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.     

The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (T _m) between 177 and 179°C, enthalpies of fusion (ΔH _f) of 20 cal/g and glass transition temperatures (T _g) of 3–4°C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the T _m values were 161°C, ΔH _f values were 7cal/g and T _g values were −3°C. Journal of Industrial Microbiology & Biotechnology (2002) 28, 147–153 DOI: 10.1038/sj/jim/7000231 Received 30 July 2001/ Accepted in revised form 04 November 2001