Zea systematics: ribosomal ITS evidence

Ribosomal internal transcribed spacer (ITS) sequences were used to evaluate the phylogenetics of Zea and Tripsacum. Maximum likelihood and polymorphism parsimony were used for phylogenetic reconstructions. Zea ITS nucleotide diversity was high compared to other plant species, but approximately equivalent to other maize loci. Coalescence of ITS alleles was rapid relative to other nuclear loci; however, there was still much diversity within populations. Zea and Tripsacum form a clade clearly differentiated from all other Poaceae. Four Zea ITS pseudogenes were identified by phylogenetic position and nucleotide composition. The phylogenetic position of Z. mays ssp. huehuetenangensis was clearly established as basal to the other Z. mays. The ITS phylogeny disfavored a Z. luxurians and Z. diploperennis clade, which conflicted with some previous studies. The introgression of Z. mays alleles into Z. perennis and Z. diploperennis was also established. The ITS data indicated a near contemporary divergence of domesticated maize and its two closest wild relatives.      

The Mycoplasma hominis vaa gene displays a mosaic gene structure

Mycoplasma hominis contains a variable adherence-associated ( vaa ) gene. To classify variants of the vaa genes, we examined 42 M. hominis isolates by PCR, DNA sequencing and immunoblotting. This uncovered the existence of five gene categories. Comparison of the gene types revealed a modular composition of the Vaa proteins. The proteins constituted a conserved N-terminal part followed by a varying number of interchangeable cassettes encoding approximately 110 amino acids with conserved sequence boxes flanking the cassettes. The interchangeable cassettes showed a high mutual homology and a conserved leucine zipper motif. The smallest product contained only one cassette and the largest five. Additionally, two types of stop mutations caused by substitutions resulting in the expression of truncated Vaa proteins were observed. Our results expand the known potential of the Vaa system in generating antigen variation.     

Circumvention of chemotherapy-induced myelosuppression by transfer of themdr1 gene

Drug-induced myelosuppression is a frequent reason for curtailing chemotherapy in cancer patients. Rescue of myelosuppressed patients with autologous marrow transplants is reasonably advanced and permits an increase in the dose of anticancer drugs. Despite this improvement, patients often relapse with drug resistance disease. The human multidrug resistance (mdr1) gene might make it possible to render hemopoietic stem cells resistant to anticancer drugs after transfer of this gene. By introducing resistant stem cells into patients it might be possible to treat these patients repeatedly with otherwise ablative therapy. This review explores the feasibility ofmdr1 gene therapy.Abbreviations MDR multidrug resistance - ABMT autologous bone marrow transplantation - P-gp P-glycoprotein - RCR replication-competent retrovirus     

A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected.     

Antibacterial chalcones--bioisosteric replacement of the 4'-hydroxy group

Hydroxy chalcones, for example, Licochalcone A, has for several years been known to be antibacterial. The low aqueous solubility and the medium antibacterial potency have limited the usefulness of the compounds. We describe the bioisosteric replacement of the essential 4'-hydroxy group in the hydroxy chalcones with bioisosters of varied degrees of acidity resulting in both more potent and more soluble compounds. The more acidic 4'-hydroxy analogues (e.g., 3'-fluoro- or 3',5'-difluoro-) gave almost inactive compounds whereas exchanging the hydroxy group with a carboxy group resulted in a potent compound with a high aqueous solubility. Further optimisation and SAR-analysis resulted in soluble and potent carboxy chalcones [e.g., 3,5-dibromo- and 3,5-di(trifluoromethyl)-].     

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.     

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: Interactions with human β2 integrins and erythrocytes

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α_Lβ₂ (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express β₂ integrin. Cells expressing α_Mβ₂ (CD11b/CD18) or α_Xβ₂ (CD11c/CD18) were killed as efficiently as cells expressing α_Lβ₂. Erythrocytes, which do not express β₂ integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β₂ chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α_Mβ₂ and α_Xβ₂ as novel receptors for LtxA.     

Sex and sex hormones influence the development of albuminuria and renal macrophage infiltration in spontaneously hypertensive rats

There is a sex difference in hypertensive renal injury, with men experiencing greater severity and a more rapid progression of renal disease than women; however, the molecular mechanisms protecting against renal injury in women are unknown. The goal of this study was to determine whether sex hormones modulate blood pressure and the progression of albuminuria during the developmental phase of hypertension in male and female spontaneously hypertensive rats (SHR). Studies were also performed to examine how sex and sex hormones influence two major risk factors for albuminuria, overactivation of the renin-angiotensin system and oxidative stress. Blood pressure was measured by telemetry in gonad-intact and gonadectomized male and female SHR. Microalbumin excretion, measured over time, and macrophage infiltration were used to assess renal health. Male SHR had significantly higher blood pressures than female SHR, and gonadectomy decreased blood pressures in males with no effect in females. Male SHR displayed a gonad-sensitive increase in albuminuria over time, and female SHR had a gonad-sensitive suppression in macrophage infiltration. Female SHR had greater plasma ANG II levels and similar levels of renal cortical ANG II vs. levels shown in males but less AT(1)-receptor protein expression in the renal cortex. Female SHR also had a gonad-sensitive decrease in renal oxidative stress. Therefore, the renal protection afforded to female SHR is associated with lower blood pressure, decreased macrophage infiltration, and decreased levels of oxidative stress.     

Effect of chronic IL-6 infusion on acute pressor responses to vasoconstrictors in mice

Interleukin (IL)-6 has been implicated as a contributing factor in the pathogenesis of hypertension, although the mechanisms involved are unclear. Studies conducted in vitro suggest that IL-6 may have a direct effect on vascular tone and may modulate constrictor responses to agonists. Whether this effect can be observed in vivo is unknown. Therefore, mice were treated with either IL-6 (16 ng/h sc) or vehicle for 14 days, and the acute blood pressure and heart rate responses to endothelin (ET)-1, angiotensin II (ANG II), and phenylephrine (PE) were assessed under isoflurane anesthesia. Blood pressure responses to ET-1 were identical in vehicle- and IL-6-infused mice, both in the presence and the absence of ganglion blockade with chlorisondamine. The fall in heart rate during ET-1 responses was significantly attenuated in IL-6-infused mice with autonomic reflexes intact (vehicle vs. IL-6, P < 0.05 at 1 and 3 nmol/kg of ET-1), but this difference was not observed after ganglionic blockade. Both blood pressure and heart rate responses to ANG II were indistinguishable between IL-6- and vehicle-infused mice, as were responses to PE except for a significant increase in the blood pressure response and decrease in the heart rate response in IL-6-infused mice observed only at the highest dose of PE (300 microg/kg; P < 0.05). These findings show that, despite what might be predicted from studies conducted in vitro, chronic exposure to elevated plasma IL-6 concentrations in itself does not predispose the mouse to enhanced responsiveness to vasoconstrictors in vivo.