Regulation of heme-controlled eukaryotic polypeptide chain initiation factor 2 alpha-subunit kinase of reticulocyte lysates.

We have obtained highly purified preparations of the heme-controlled eukaryotic initiation factor 2 alpha-subunit (eIF-2 alpha) kinase (HCI) from rabbit reticulocyte lysates containing five different polypeptides. One of these is a 87-kDa (p87) phosphopeptide which appears to show an autokinase activity. The controlled digestion with trypsin of HCI preparations leads to the suggestion that phosphorylation of p87 is not needed for kinase activity and, furthermore, that another 89-kDa polypeptide could be the kinase catalytic subunit. In agreement with this, monoclonal antibodies directed against p87 do not interfere with eIF-2 alpha kinase activity. Moreover, the anti-p87 antibodies and those directed against the mammalian 90-kDa heat shock protein recognize the same p87 polypeptide from rabbit reticulocyte lysates. Upon incubation of the HCI preparation with hemin (5-10 microM), the eIF-2 alpha kinase is converted into an inactive form and appears to become associated with related peptides forming high molecular weight complexes which can be reversibly activated by 2-mercaptoethanol. The maintenance of the integrity of the porphyrin ring is absolutely required for kinase inactivation and although the presence of metal ion is not essential, the iron and cobalt metalloporphyrins are more effective than protoporphyrin IX. The formation of the inactive form of HCI by hemin is prevented by either N-ethylmaleimide, monoclonal antibodies directed against p87, or phosphorylation of p87. The data strongly suggest that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive dimer HCI.p87 via disulfide bonds and direct binding of hemin. A model of HCI regulation is discussed.     

Ceratitis capitata brain adenylate cyclase and its membrane environment

Adenylate cyclase activation by GTP and octopamine as well as basal activity (in the presence of Mg2+) have been studied as a function of membrane structure in plasma membranes from brain of the dipterous Ceratitis capitata. Benzyl alcohol and lidocaine, but not phenobarbital, inhibited the three activities to the same extent. Triton X-100-solubilized adenylate cyclase was also inhibited by benzyl alcohol and lidocaine, but not by phenobarbital. Results could be explained by an effect on the catalytic unit lipid environment, which would be maintained after solubilization, counteracting the effect of these drugs to facilitate lateral diffusion and coupling of adenylate cyclase components in the lipid bilayer. The observation that the insect adenylate cyclase is relatively insensitive to changes in bulk bilayer fluidity is strengthened by the absence of effect of phenobarbital on enzyme activities. Indeed, this compound was as active as lidocaine or benzyl alcohol in increasing bulk membrane fluidity. The response of C. capitata adenylate cyclase to changes in membrane fluidity is different from that recorded in mammalian systems. This may be functionally important and result from the fact that insects are not warm-blooded.     

Differences in synovial fluid cytokine levels but not in synovial tissue cell infiltrate between anti-citrullinated peptide/protein antibody-positive and –negative rheumatoid arthritis patients

Introduction

Comparative data on synovial cell infiltrate and cytokine levels in anti citrullinated peptide/protein antibody (ACPA)-positive and ACPA negative rheumatoid arthritis (RA) patients are scarce. Our aim was to analyze synovial cell infiltrate and synovial fluid (SF) levels of cytokines in patients with RA according to the presence or absence of ACPA in serum.

Methods

A cross-sectional study in a single center including consecutive RA patients was performed. Patients were defined as ''ACPA negative'' if serum was negative to two different ACPAs [second generation commercial anti-cyclic citrullinated peptide antibodies (CCP2) and chimeric fibrin/filaggrin citrullinated antibodies]. Parallel synovial tissue (ST) biopsies and SF were obtained by knee arthroscopy. Synovial cell infiltrate and endothelial cells were analyzed by immunohistochemistry and SF levels of Th1, Th2, Th17 and pro-inflammatory cytokines by Quantibody(R) Human Array.

Results

A total of 83 patients underwent arthroscopy, with a mean age of 55.9 ± 12 years, and mean disease duration of 45 months (interquartile range, IQR 10.8 to 122). 62% were female and 77% were ACPA positive. No significant differences were found in clinical variables, acute phase reactants, synovial cell infiltrate or lymphoid neogenesis (LN) between ACPA positive and negative patients. However ACPA positive patients had significantly higher levels of IL-1β, IL-10, IL-17 F and CC chemokine ligand 20 (CCL-20) than ACPA negative patients.

Conclusions

In our cohort of patients with RA no significant differences were found in synovial cell infiltrate or synovial LN according to ACPA status. However, ACPA positive patients had higher levels of T-cell derived and pro-inflammatory cytokines than ACPA negative patients. As systemic and local inflammation was similar in the two groups, these findings support a distinct synovial physiopathology.     

Population genetics structure of glyphosate‐resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance

Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate‐resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K‐means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) encoding gene from glyphosate‐resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target‐site resistance mechanism.     

Chagas disease in Mexico

Mexico - the northernmost country of Latin America - has long been thought to have an unusually low prevalence of Chagas disease compared with other Latin American countries. This has seemed unusual because of the large number of vector species and subspecies reported from the country, and the social and ecological conditions that seem to parallel those in other countries where Chagas disease is recognized as a major public health priority. This review seeks to clarify the question, and suggests that the epidemiological, parasitological, and entomological pattern of Chagas disease in Mexico may also parallel that of other endemic regions, but has been masked by poor awareness of the disease both at local and institutional levels.     

Central wedge nymphectomy with a 90-degree Z-plasty for aesthetic reduction of the labia minora

The labia minora or nymphae of the vulva are two cutaneous-mucosal refolds located between the labia majora, the internal aspect of which is separated by the interlabial cleft. The enlargement of the labia minora may be attributable to several factors, most commonly congenital. Although some women require surgical reduction for functional reasons, most seek reduction of their labia minora because of psychological concerns. The authors describe a modified plastic surgery procedure for functional and aesthetic reduction of the labia minora.     

Characterization of extradiol dioxygenases from a polychlorinated biphenyl-degrading strain that possess higher specificities for chlorinated metabolites

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBDLB400; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2',6'-dichloro-2,3-dihydroxybiphenyl (2',6'-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBDP6-I and DHBDP6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBDLB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBDP6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3'-Cl DHB approximately DHB approximately 4'-Cl DHB > 2'-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBDP6-I were among the best for DHBDP6-III (4-Cl DHB > 5-Cl DHB approximately 6-Cl DHB approximately 3'-Cl DHB > DHB > 2'-Cl DHB approximately 4'-Cl DHB; DHBDP6-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBDP6-I and 2'-Cl DHB for DHBDP6-III. As observed in DHBDLB400, chloro substituents influenced the reactivity of the dioxygenases with O2. For example, the apparent specificities of DHBDP6-I and DHBDP6-III for O2 in the presence of 2'-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBDP6-I and DHBDP6-III shared the relative inability of DHBDLB400 to cleave 2',6'-diCl DHB (apparent catalytic constants of 0.088 +/- 0.004 and 0.069 +/- 0.002 s(-1), respectively). However, these isozymes had remarkably different apparent K(m) values for this compound (0.007 +/- 0.001, 0.14 +/- 0.01, and 3.9 +/- 0.4 micro M for DHBDLB400, DHBDP6-I, and DHBDP6-III, respectively). The markedly different reactivities of DHBDP6-I and DHBDP6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.     

Heavy metals and arsenic uptake by wild vegetation in the Guadiamar river area after the toxic spill of the Aznalcóllar mine

On 25 April 1998, approximately 4.5 hm(3) of pyritic sludge, containing 5000 mg of As kg(-1) among other pollutants, was spilled into the Agrio and Guadiamar rivers and the surrounding agricultural areas (Aznalcóllar, Seville, Southern Spain). Many trace metals such as Pb, Cu, Zn, Cd, Tl, Sb and As reached the Do?ana National Park, the largest wetland area in Europe, affecting soils, different plant and animal species. In order to recuperate the affected lands by employing plants capable of accumulating high levels of contaminants in shoots, periodical field surveys have been made to identify the metal-tolerant species that are spontaneously growing in the polluted soils, and are able to uptake one or various of the contaminants. Among the 99 different plant species studied, Anchusa azurea, Beta vulgaris, Chamaemelum fuscatum, Convolvulus arvensis, Cynodon dactylon, Diplotaxis virgata, Erodium aethiopicum, Lavatera cretica, Malva nicaeensis, Silybum marianum and, above all, Amaranthus blitoides highlight as the most promising to be used in the remediation of the affected area.