DNA polymorphisms associated with a new α1-antitrypsin PI Q0 variant (PI Q0riedenburg)

Summary A new PI Q0 variant (PI Q0_riedenburg) is described; it is caused by a complete deletion of the ₁-antitrypsin (₁AT) gene. The deletion gives rise to four new restriction fragment length polymorphisms (RFLPs) detected with a genomic probe of the 5 region of the gene. Analysis of the RFLPs indicates that the deletion starts immediately upstream of exon Ic. The deletion extends into the 3 flanking region of the gene but does not include the ₁AT-related gene (the PIL gene), which is located 12 kb downstream of the ₁AT gene.     

In situ immunocytochemical evidence that a homolog of protein translation elongation factor EF-1α is associated with microtubules in carrot cells

Summary Evidence indicates that elongation factor-1 (EF-1), a ubiquitous and abundant protein factor involved in the first step of peptide elongation, is also associated with the cytoskeleton in a variety of organisms. Although the effects of these associations on EF-1's translational function have not been examined, the associations do appear to result in non-passive effects on the cytoskeleton. A carrot homolog of EF-1, pp 50, has been reported to interact with microtubules in vitro, inducing the formation of microtubule bundles that can be dissociated by Ca²⁺/calmodulin. The characterization of anti-pp 50 antibodies is reported here. Immunocytochemistry, using anti-pp 50 and anti-tubulin antibodies, was used to investigate the co-localization of pp 50 and microtubules in situ. In carrot protoplasts fixed after detergent lysis, at least a fraction of pp 50 appears to be associated with microtubules. Treatment of such protoplasts with amiprophos-methyl (APM) reduced both the presence of microtubules and the co-localizing pp 50-associated fluorescence. In taxol-treated protoplasts, increases in both microtubules and the colocalizing pp 50-associated fluorescence were observed. When carrot protoplasts were fixed prior to detergent extraction, confocal laser scanning microscopy likewise revealed co-localization. Furthermore, what is likely to be a fluorescence resonance energy transfer (FRET) between fluorochromes associated with anti-pp 50 and anti-tubulin reporters was observed, indicating that some pp 50 is intimately associated with microtubules. The in situ cytoarchitectural evidence is consistent with a function previously proposed for pp 50 based on in vitro experiments — that pp 50 is a plant microtubuleassociated protein (MAP) whose function can be modulated by a Ca²⁺/calmodulin signal transduction mechanism in plant cells.Abbreviations APM amiprophos-methyl - BSA bovine serum albumin - EF-1 elongation factor-1-alpha - FRET fluorescence resonance energy transfer - MAP microtubule-associated protein - PBS phosphate buffered saline - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

SUVi and BACH1: a new subfamily of mammalian helicases?

The RecQ family of DNA helicases have been shown to be important for the maintenance of genomic integrity in prokaryotes and eukaryotes. Members of this family are genes responsible for cancer predisposition disorders like Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome. Here, we show the sequence homologies between two recently identified mammalian helicases, namely SUVi and BACH1. These two genes also share strong homologies with other members of the RecQ family. On the basis of published data and sequence analysis we suggest that SUVi/BACH1 may represent a novel subfamily of mammalian helicases, functioning in the processing of lesions induced by different genotoxic agents.     

Accumbal 14-3-3ζ protein downregulation is associated with cocaine-conditioned memory

Cocaine-conditioned memory has been known to cause cocaine craving and relapse, while its underlying mechanisms remain unclear. We explored accumbal protein candidates responsible for a cocaine-conditioned memory, cocaine-induced conditioned place preference (CPP). Two-dimensional gel electrophoresis in conjunction with liquid chromatography mass spectrometry analysis was utilized to identify accumbal protein candidates involved in the retrieval of cocaine-induced CPP. Among the identified candidate proteins, a downregulated 14-3-3ζ protein was chosen and confirmed by Western immunoblotting. A polymer-mediated plasmid DNA delivery system was then used to overexpress 14-3-3 protein in mouse nucleus accumbens before the CPP retrieval tests. Overexpression of accumbal 14-3-3ζ protein was found to diminish conditioned cue/context-mediated cocaine-induced CPP. In contrast, another isoform of 14-3-3 protein, 14-3-3ε protein, did not affect conditioned cue/context-mediated cocaine-induced CPP. Overexpression of accumbal 14-3-3ζ protein did not produce motor activity-impairing effect or alter local dopamine metabolism. Moreover, overexpression of accumbal 14-3-3ζ protein did not affect food-induced CPP. These results, taken together, indicated that overexpressed accumbal 14-3-3ζ protein specifically decreased conditioned cue/context-mediated cocaine memory. Further understanding of the function of accumbal 14-3-3ζ protein may shed light on the treatment of cocaine craving and relapse.     

8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and α-smooth muscle actin polymerization

Reactive oxygen species (ROS) are activators of cell signaling and modify cellular molecules, including DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the prominent lesions in oxidatively damaged DNA, whose accumulation is causally linked to various diseases and aging processes, whereas its etiological relevance is unclear. 8-OxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair (BER) pathway. OGG1 binds free 8-oxoG and this complex functions as an activator of Ras family GTPases. Here we examined whether OGG1-initiated BER is associated with the activation of Rho GTPase and mediates changes in the cytoskeleton. To test this possibility, we induced OGG1-initiated BER in cultured cells and mouse lungs and used molecular approaches such as active Rho pull-down assays, siRNA ablation of gene expression, immune blotting, and microscopic imaging. We found that OGG1 physically interacts with Rho GTPase and, in the presence of 8-oxoG base, increases Rho–GTP levels in cultured cells and lungs, which mediates α-smooth muscle actin (α-SMA) polymerization into stress fibers and increases the level of α-SMA in insoluble cellular/tissue fractions. These changes were absent in cells lacking OGG1. These unexpected data and those showing that 8-oxoG repair is a lifetime process suggest that, via Rho GTPase, OGG1 could be involved in the cytoskeletal changes and organ remodeling observed in various chronic diseases.     

Structural evidence that the small subunit found associated with the TL antigen is β 2-microglobulin

Comparative tryptic peptide mapping and partial amino-terminal primary sequence analysis of the light chain component associated with the TL antigens showed that the small subunit of TL was identical to the ₂m light chain associated with the H-2K or D product of the same strain. Peptide comparison of the ₂m from the Tla products of an A strain X-ray induced leukemia RADA1 (Tla ^a) and of a C57BL/6 strain X-ray induced leukemia ERLD (Tla ^b) showed differences to the extent of 25–35% in their peptides. This is consistent with previous results showing ₂m allelic variations between these mouse strains. The data prove the structural identity of the ₂m molecules from TL and H-2K, D antigens as well as reveal the strain specific polymorphism of the ₂m associated with these products.     

Ethanol-induced increase in Fyn kinase activity in the dorsomedial striatum is associated with subcellular redistribution of protein tyrosine phosphatase α

In vivo exposure of rodents to ethanol leads to a long-lasting increase in Fyn kinase activity in the dorsomedial striatum (DMS). In this study, we set out to identify a molecular mechanism that contributes to the enhancement of Fyn activity in response to ethanol in the DMS. Protein tyrosine phosphatase α (PTPα) positively regulates the activity of Fyn, and we found that repeated systemic administration or binge drinking of ethanol results in an increase in the synaptic localization of PTPα in the DMS, the same site where Fyn resides. We also demonstrate that binge drinking of ethanol leads to an increase in Fyn activity and to the co-localization of Fyn and PTPα in lipid rafts in the DMS. Finally, we show that the level of tyrosine phosphorylated (and thus active) PTPα in the synaptic fractions is increased in response to contingent or non-contingent exposure of rats to ethanol. Together, our results suggest that the redistribution of PTPα in the DMS into compartments where Fyn resides is a potential mechanism by which the activity of the kinase is increased upon ethanol exposure. Such neuroadaptations could be part of a mechanism that leads to the development of excessive ethanol consumption.     

Evidence that human α-thrombin is a monovalent cation-activated enzyme

The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH₃)₄NCl) or choline chloride (HO(CH₂)₂N(CH₃)₃Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p-nitroanilide substrates were measured. Na⁺ enhanced thrombin activity by decreasing the K_m,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average K_A for Na⁺for the four substrates examined was 3.5 × 10^?2m. A comparison of the effects of Na⁺ vs K⁺ on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the K_m,app (0.2 to 04.-fold) and increased thek_cat,app (3.1 to 3.4-fold) except that higher K⁺ concentrations (K_A = 2.8 × 10^?1M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p-tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na⁺ caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δ?_288.5 = +1067). The K_m for Na⁺ was 2.3 × 10^?2m which agrees with the kinetically determined K_A for Na⁺. The results are consistent with Na⁺ binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme.     

Evidence that a polymorphism within the 3′UTR of glutathione peroxidase 4 is functional and is associated with susceptibility to colorectal cancer

Low selenium (Se) status has been associated with increased risk of colorectal cancer (CRC). Se is present as the amino acid selenocysteine in selenoproteins, such as the glutathione peroxidases. Se incorporation requires specific RNA structures in the 3' untranslated region (3'UTR) of the selenoprotein mRNAs. A single nucleotide polymorphism (SNP) occurs at nucleotide 718 (within the 3'UTR) in the glutathione peroxidase 4 gene. In the present study, Caco-2 cells were transfected with constructs in which type 1 iodothyronine deiodinase coding region was linked to the GPx4 3'UTR with either C or T variant at position 718. Higher reporter activity was observed in cells expressing the C variant compared to those expressing the T variant, under either Se-adequate or Se-deficient conditions. In addition, a disease association study was carried out in cohorts of patients with either adenomatous polyps, colorectal adenocarcinomas and in healthy controls. A higher proportion of individuals with CC genotype at the GPx4 T/C 718 SNP was present in the cancer group, but not in the polyp group, compared with the control group (P < 0.05). The present data demonstrate the functionality of the GPx4 T/C 718 SNP and suggest that T genotype is associated with lower risk of CRC.     

Precocious puberty associated with a pineal cyst: is it disinhibition of the hypothalamic-pituitary axis?

Accelerated development of secondary sexual characteristics or sexual precocity is a well-known entity. Most authors recognize two groups of patients, those described as having central precocious puberty (CPP) and those with precocious pseudopuberty. CPP results from premature activation of the hypothalamic-pituitary-gonadal axis and pseudopuberty is caused by lesions that secrete gonadotropin-like substances or hormones. The onset of CPP is usually before age 8 in females and age 9 in males; however, there is contention that the age of onset is much earlier and also differs depending on the patients' race. Previously reported causes of CPP include intracranial neoplasm, infection, trauma, hydrocephalus and Angelman's syndrome. Pineal cysts are usually asymptomatic incidental findings, but have been associated with CPP. We present an interesting case of a patient with CPP and an associated pineal cyst. We review the literature on the pathogenesis of CPP and associated pineal cyst, the neuroendocrine relationship between the pineal gland and puberty and the neurosurgical role in these cases.     

Isolation by zinc-affinity chromatography of the histidine–proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase: Evidence that the formation of a protein–protein complex between the catalytic subunit and the novel component is critical for the stability of the enzyme

The histidine–proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP deaminase under denaturing and reducing conditions specifically binds to a Zn²⁺-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn²⁺ from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95 000 and the N-terminal sequence L-T-P-T-D-X-K-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70 000, characterizing it as a N-glycosylated protein. The separation from AMP deaminase of an 85-kDa component with a blocked N terminus is observed when the enzyme is applied to the Zn-charged column under nondenaturing conditions. On storage under reducing conditions, this component undergoes an 85- to 95-kDa transition yielding a L-T-P-T-D-X-K-T-T-K-P-L N-terminal sequence, suggesting that the shift in the migration on SDS/PAGE as well as the truncation of the protein at its N terminus are promoted by the reduction of a disulfide bond present in freshly isolated HPRG. The separation of HPRG induces a marked reduction in the solubility of AMP deaminase, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme.     

Vaccination against shigellosis: is it the path that is difficult or is it the difficult that is the path?

Following several decades of research, there is not yet a convincing vaccine against shigellosis. It is still difficult, in spite of the breadth of strategies (i.e. live attenuated oral, killed oral, subunit parenteral) to select an optimal option. Two approaches are clearly emerging: (i) live attenuated deletion mutants based on rational selection of genes that are key in the pathogenic process, and (ii) conjugated detoxified polysaccharide parenteral vaccines, or more recently conjugated synthetic carbohydrates. Some of these approaches have already undergone phase I and II clinical trials with promising results, but important issues have also emerged, particularly the discrepancy between colonization and immunogenic potential of live attenuated vaccine candidates depending upon the population concerned (i.e. non endemic vs. endemic areas). Efforts are needed to definitely establish the proof of concept of these approaches, and thus the need for clinical trials which should also soon explore the possibility to associate different serotypes, in response to serotype specific protection against shigellosis. More basic research is also required to improve what we can still consider as first-generation vaccines, and to explore possible new paradigms including the search for cross-protective antigens.     

A new GH13 subfamily represented by the α-amylase from the halophilic archaeon Haloarcula hispanica

Extremophiles - α-Amylase catalyzes the endohydrolysis of α-1,4-glucosidic linkages in starch and related α-glucans. In the CAZy database, most α-amylases have been classified...     

Diagnostic phase antibody response to the human papillomavirus type 16 E2 protein is associated with succesful treatment of genital HPV lesions with systemic interferon α-2b

Background and objective: Systemic interferon α-2b treatment reduces relapses of genital human papillomavirus (HPV) lesions in some but not all females. The aim of the present study was to investigate possible predictive pretreatment factors for the outcome of therapy.Material and methods: HPV DNA status and HPV antibody response were evaluated in 100 randomized patients treated with laser ablation and systemic interferon α-2b or placebo, and followed up to 6 months.Results: Overall, adjuvant therapy with systemic interferon-α did not differ from placebo. However, detectable diagnostic phase levels of serum antibodies to e.g. HPV16 open reading frame (ORF) E2 derived peptide ¹⁴¹EEASVTVVEGQVDYY¹⁵⁵ predicted 10-fold difference in the risk of recurrence of HPV infection following adjuvant interferon α-2b therapy as compared with placebo (odds ratio, OR, 0.5, 95% confidence interval (CI), 0.1–2.3; OR, 4.6, 95% CI 0.5–41, respectively). This trend was statistically significant in the whole study population (2P < 0.05), and in patients with high viral load (2P < 0.01).Conclusions: Evaluation of the E2 antibody responses may help to identify women with genital HPV lesions who respond to systemic interferon α-2b treatment.     

The lectin from the mushroom Pleurotus ostreatus: a phosphatase-activating protein that is closely associated with an α-galactosidase activity: A part of this paper has been presented as a preliminary report at the 17th Interlec. Meeting 1997 in Würzburg,Germany

The lectin from the mushroom Pleurotus ostreatus described earlier [F. Conrad, H. Rüdiger, The lectin from Pleurotus ostreatus: purification, characterization and interaction with a phosphatase, Phytochemistry 36 (1994) 277–283] was further characterized. Determination of the isoelectric point by capillary electrophoresis gave a value of 7.6. The dissociation constant of the lectin-α-lactose-1-phosphate complex determined by capillary electrophoresis is 3 mM. The activation of an endogenous phosphatase by the lectin as found earlier for the pseudosubstrate p-nitrophenylphosphate was confirmed also for naturally occurring substrates as ADP and ATP. We observed that at all purification steps the lectin is accompanied by an α-galactosidase activity. Both activities could neither be resolved by electrophoresis under non-denaturing conditions nor by affinity chromatography. However, carbohydrate binding by the lectin and carbohydrate processing by the enzyme are not due to the same site since: (i) the lectin accepts both α- and β-glycosides whereas the enzyme activity is restricted to the α-anomer; (ii) the interaction with erythrocytes leads to a stable agglutinate, i.e. no ‘clot-dissolving activity’ [C.N. Hankins, J.I. Kindinger, L.M. Shannon, Legume α-galactosidases which have hemagglutinin properties, Plant Physiol. 65 (1980) 618–622] is observed; (iii) the α-galactosidase activity is inhibited by galactose but not by a β-galactoside. Therefore, lectin and enzymatic activities are either properties of two tightly associated proteins, or of just one molecule. The kinetic parameters of the lectin-associated α-galactosidase activity for p-nitrophenyl-α-galactopyranoside are: K_M=2.5 mM, k_cat=66 s⁻¹, and K_I=20 mM for the inhibitor d-galactose.     

Identification of a DNA-binding factor that recognizes an α-coixin promoter and interacts with a Coix Opaque-2 like protein

Transient expression and electrophoretic mobility shift assay were used to investigate the cis elements and the DNA-binding proteins involved in the regulation of expression of a 22 kDa zein-like -coixin gene. A set of unidirectional deletions was generated in a 962 bp fragment of the -coixin promoter that had been previously fused to the reporter gene GUS. The constructs were assayed by transient expression in immature maize endosperm. There was no significant decrease in GUS activity as deletions progressed from –1084 to –238. However, deletion from –238 to –158, which partially deleted the O2c box, resulted in a dramatic decrease in GUS activity emphasizing the importance of the O2 box in the quantitative expression of the gene. The –238 promoter fragment interacted with Coix endosperm nuclear proteins to form 5 DNA-protein complexes, C1–C5, as detected by EMSA. The same retarded complexes were observed when the –158 promoter fragment was used in the binding reactions. Reactions with nuclear extracts isolated from Coix endosperms harvested from 6 to 35 days after pollination revealed that the 5 DNA-protein complexes that interact with the -coixin promoter are differentially assembled during seed development. Deletion analysis carried out on the –238/ATG promoter fragment showed that a 35 bp region from –86 to –51 is essential for the formation of the complexes observed. When nuclear extracts were incubated with an antiserum raised against the maize Opaque-2 protein, the formation of 4 complexes, C1, C3, C4 and C5, was prevented indicating that an Opaque-2 like protein participates in the formation of those complexes. Complex C2 was not affected by the addition of the O2 antibody, suggesting the existence of a novel nuclear factor, CBF1, that binds to the promoter and makes protein-protein associations with other proteins present in Coix endosperm nuclei.