Basic residues in azurocidin/HBP contribute to both heparin binding and antimicrobial activity

Azurocidin/CAP37/HBP is an antimicrobial and chemotactic protein that is part of the innate defenses of human neutrophils. In addition, azurocidin is an inactive serine protease homolog with binding sites for diverse ligands including heparin and the bovine pancreatic trypsin inhibitor (BPTI). The structure of the protein reveals a highly cationic domain concentrated on one side of the molecule and responsible for its strong polarity. To investigate the role of this highly basic region, we produced three recombinant azurocidin mutant proteins that were altered in either one or both of two clusters of 4 basic residues located symmetrically on each side of a central cleft in the cationic domain. Two of the mutant proteins (Loop 3: R5Q, K6Q, R8Q, and R10Q; Loop 4: R61Q, R62Q, R63Q, and R65Q) exhibited little or no change in heparin and BPTI binding or in antimicrobial function. In contrast, the Loop 3/Loop 4 mutant (R5Q, K6Q, R8Q, R10Q, R61Q, R62Q, R63Q, and R65Q) in which all 8 basic residues were replaced showed greatly decreased ability to bind heparin and to kill Escherichia coli and Candida albicans. Thus, we report that the 8 basic residues that were altered in the Loop 3/Loop 4 mutant contribute to the ability of the wild-type azurocidin molecule to bind heparin and to kill E. coli and C. albicans. Because BPTI binding was comparable in wild-type and Loop 3/Loop 4 mutant protein, we conclude that the same 8 basic residues are not involved in the binding of BPTI to azurocidin, supporting the notion that the binding site for BPTI is distinct from the site involved in heparin binding and antimicrobial activity. Finally, we show that removal of all 4 positively charged amino acids in the 20-44 azurocidin sequence (DMC1: R23Q,H24S,H32S,R34Q), a region previously thought to contain an antimicrobial domain, does not affect the activity of the protein against E. coli, Streptococcus faecalis, and C. albicans.     

Erythroid expression of the human alpha-spectrin gene promoter is mediated by GATA-1- and NF-E2-binding proteins

alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.     

Different substrate recognition motifs of human and trypanosome nucleobase transporters. Selective uptake of purine antimetabolites

The therapeutic index of antimetabolites such as purine analogues is in large part determined by the extent to which it is selectively accumulated by the target cell. In the current study we have compared the transport of purine nucleobase analogues by the H2 transporter of bloodstream form Trypanosoma brucei brucei and the equilibrative nucleobase transporter of human erythrocytes. The H2 transporter forms hydrogen bonds with hypoxanthine at positions N3, N7, N(1)H, and N(9)H of the purine ring, with apparent Delta G(0) of 7.7-12.6 kJ/mol. The transporter also appears to H-bond with the amine group of adenine. The human transporter forms hydrogen bonds that form to (6)NH(2) and N1 of adenine. An H-bond is also formed with N3 and the 6-keto and amine groups of guanine but not with the protonated N1, thus explaining the low affinity for hypoxanthine. N7 and N9 do not directly interact with the human transporter in the form of H-bonds, and it is proposed that pi-pi stacking interactions contribute significantly to permeant binding. The potential for selective uptake of antimetabolites by the parasite transporter was demonstrated.     

The binding of Xanthophylls to the bulk light-harvesting complex of photosystem II of higher plants. A specific requirement for carotenoids with a 3-hydroxy-beta-end group

The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single beta-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-trans-neoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3' diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6-C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.     

Temperature influence on embryonic development of Anopheles albitarsis and Anopheles aquasalis

Temperature influence on the embryonic development of Anopheles aquasalis and An. albitarsis was investigated. At 26 degrees C, 75% and 60% of respectively An. aquasalis and An. albitarsis eggs hatched, with one peak of eclosion, between the 2nd and 3rd day after oviposition. At 20 +/- 2 degrees C, around 66-70% of An. aquasalis eggs hatched, with one eclosion peak, on the 5th day. On the other hand, An. albitarsis eclosion at 21+/- 2 degrees C decreased to 10-22%, with two eclosion peaks, on the 4th-5th day and on the 9th-12th day. These data indicate a stronger temperature influence over An.albitarsis than over An. aquasalis embryos.     

An induced Ets repressor complex regulates growth arrest during terminal macrophage differentiation

Defining the molecular mechanisms that coordinately regulate proliferation and differentiation is a central issue in development. Here, we describe a mechanism in which induction of the Ets repressor METS/PE1 links terminal differentiation to cell cycle arrest. Using macrophages as a model, we provide evidence that METS/PE1 blocks Ras-dependent proliferation without inhibiting Ras-dependent expression of cell type-specific genes by selectively replacing Ets activators on the promoters of cell cycle control genes. Antiproliferative effects of METS require its interaction with DP103, a DEAD box-containing protein that assembles a novel corepressor complex. Functional interactions between the METS/DP103 complex and E2F/ pRB family proteins are also necessary for inhibition of cellular proliferation, suggesting a combinatorial code that directs permanent cell cycle exit during terminal differentiation.     

Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log(10) CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log(10) CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log(10) CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log(10) CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.     

Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.     

HNK-1 sulfotransferase null mice express glucuronyl glycoconjugates and show normal cerebellar granule neuron migration in vivo and in vitro

Sulfoglucuronyl carbohydrate (SGC), reactive with antibody against human natural killer cell antigen, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system and has been implicated in cell-cell recognition, neurite outgrowth and neuronal migration during development, through its interaction with SGC-binding protein (SBP) 1. However, sulfotransferase (ST) null mutant mice, which lack SGC, were shown to have normal development with usual gross anatomy of the nervous system and other organs. Failure to observe a severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoproteins of the ST mutant nervous system. In the ST null mice, SGC-containing molecules were absent; instead the precursor glucuronyl carbohydrate (GC)-containing molecules accumulated. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites in the nervous system as the SGC molecules in the wild type. In vitro binding studies showed that, similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP-1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected in the mutant, possibly owing to interaction of SBP-1 with GC molecules. The results suggested that in vivo SBP-1-GC interaction was sufficient to allow normal neurite outgrowth and cell migration in the mutant, giving rise to a wild-type phenotype. However, the role of other compensatory molecules involved in these processes cannot be completely ruled out.