HS5 of the human beta-globin locus control region: a developmental stage-specific border in erythroid cells

Elements with insulator/border activity have been characterized most extensively in Drosophila melanogaster. In vertebrates, the first example of such an element was provided by a hypersensitive site of the chicken beta-globin locus, cHS4. It has been proposed that the homologous site in humans, HS5, functions as a border of the human beta-globin locus. Here, we have characterized HS5 of the human beta-globin locus control region. We have examined its tissue-specificity and assessed its insulating properties in transgenic mice using a lacZ reporter assay. Most importantly, we have tested its enhancer blocking activity in the context of the full beta-globin locus. Our results show that HS5 is erythroid-specific rather than ubiquitous in human tissues. Furthermore, HS5 does not fulfil the criteria of a general in vivo insulator in the transgene protection assay. Finally, a HS5 conditional deletion from the complete locus demonstrates that HS5 has no discernable activity in adult erythroid cells. Surprisingly, HS5 functions as an enhancer blocker in embryonic erythroid cells. We conclude that HS5 is a developmental stage-specific border in erythroid cells.     

PLD pathway involved in carbachol-induced Cl- secretion: possible role of TNF-alpha

In a previous study, it was found thatexposure to tumor necrosis factor- (TNF-) potentiated theelectrophysiological response to carbachol in a time-dependent andcycloheximide-sensitive manner. It was deduced that the potentiationcould be due to protein kinase C activity because of increased1,2-diacylglycerol. It was also observed that propranolol coulddecrease the electrophysiological response to carbachol (Oprins JC,Meijer HP, and Groot JA. Am J Physiol Cell Physiol 278:C463-C472, 2000). The aim of the present study was to investigatewhether the phospholipase D (PLD) pathway plays a role in the carbacholresponse and the potentiating effect of TNF-. Thetransphosphatidylation reaction in the presence of the primary alcohol1-butanol [leading to stable phosphatidylbutanol (Pbut) formation]was used to measure activity of PLD. The phosphatidic acid (PA) levelswere also measured. Muscarinic stimulation resulted in an increasedformation of Pbut and PA. TNF- decreased levels of PA.

     

Selective sparing of goblet cells and paneth cells in the intestine of methotrexate-treated rats

Proliferation, differentiation, and cell death were studied in small intestinal and colonic epithelia of rats after treatment with methotrexate. Days 1-2 after treatment were characterized by decreased proliferation, increased apoptosis, and decreased numbers and depths of small intestinal crypts in a proximal-to-distal decreasing gradient along the small intestine. The remaining crypt epithelium appeared flattened, except for Paneth cells, in which lysozyme protein and mRNA expression was increased. Regeneration through increased proliferation during days 3-4 coincided with villus atrophy, showing decreased numbers of villus enterocytes and decreased expression of the enterocyte-specific genes sucrase-isomaltase and carbamoyl phosphate synthase I. Remarkably, goblet cells were spared at villus tips and remained functional, displaying Muc2 and trefoil factor 3 expression. On days 8-10, all parameters had returned to normal in the whole small intestine. No methotrexate-induced changes were seen in epithelial morphology, proliferation, apoptosis, Muc2, and TFF3 immunostaining in the colon. The observed small intestinal sparing of Paneth cells and goblet cells following exposure to methotrexate is likely to contribute to epithelial defense during increased vulnerability of the intestinal epithelium.     

Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.     

Membrane assembly of the bacteriophage Pf3 major coat protein

The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly alpha-helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacetyl)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane. A nearly identical result was seen previously for the membrane-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an "L-shaped" membrane-bound model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.     

Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley

A number of novel alkaliphilic organotrophic bacteria have been isolated from several saline and alkaline East African soda lakes. The new isolates grow at pH values between 7.0 and 11.0, with pH optima for growth between 9.0 and 10.0. Growth occurs at total salts concentration between 0% and 20% (w/v) with optimum at 0%–7% (w/v). Phylogenetic analyses based on 16S rDNA sequence comparison indicate that these isolates are related (>96% similarity) to members of the Halomonadaceae within the γ-3 subdivision of the Proteobacteria. These analyses indicate that existing species within the Halomonadaceae fell within three main groups, one group comprising the type species of Halomonas, Halomonas elongata, and a number of other known species including one soda lake isolate. A second group constituting most of the remaining known species of Halomonas and related Chromohalobacter spp. includes 3 soda lake isolates with high DNA–DNA homologies. The third group included Halomonas halodenitrificans, Halomonas desiderata, Halomonas cupida, and 13 soda lake isolates. Phenotypic comparisons indicated that the majority of soda lake strains shared similar morphological, phenotypic, and chemotaxonomic properties to known strains of Halomonas but grew under alkaline conditions. The 3 soda lake isolates with high DNA–DNA homologies were, however, significantly different in antibiotic sensitivity pattern and in the utilization of several substrates, were unable to reduce nitrite, and showed low DNA–DNA homologies with known halomonads in the same group. We propose that these isolates comprise a new species of the genus Halomonas that we name Halomonas magadii sp. nov. The type strain is strain 21 MI (NCIMB 13595). Received: July 20, 1999 / Accepted: October 29, 1999     

Muscle contraction history: modified Hill versus an exponential decay model

In recent years, it has been recognised that improvements to classic models of muscle mechanical behaviour are often necessary for properly modelling co-ordinated multi-joint actions. In this respect, the purpose of the present study was to improve on modelling stretch-induced force enhancement and shortening-induced force depression of muscle contraction. For this purpose, two models were used: a modified Hill model and a model based loosely on mechano-chemistry of the cross-bridge cycle (exponential decay model). The models were compared with a classic Hill model and experimental data. Parameter values were based, as much as possible, on experimental findings in the literature, and tested with new experiments on the gastrocnemius of the rat. Both models describe many features of slow-ramp movements well during short contractions (300–500 ms), but long-duration behaviour is described only partly. The exponential decay model does not incorporate a force–velocity curve. Therefore, its good performance indicates that the status of the classic force–velocity characteristic may have to be reconsidered. Like movement-induced force depression and enhancement, it seems a particular manifestation of time-dependent force behaviour of muscle, rather than a fundamental property of muscle (like the length–tension curve). It is argued that a combination of the exponential decay model (or other models based on the mechano-chemistry of contraction) and structurally based models may be fruitful in explaining this time-dependent contraction behaviour. Furthermore, not in the least because of its relative simplicity, the exponential decay model may prove more suitable for modelling multi-joint movements than the Hill model. Received: 19 March 1999 / Accepted in revised form: 9 June 2000     

Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected cell lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of alpha-synuclein is regulated by phosphorylation/dephosphorylation.     

Identification and functional characterization of human soluble epoxide hydrolase genetic polymorphisms

Human soluble epoxide hydrolase (sEH), an enzyme directing the functional disposition of a variety of endogenous and xenobiotic-derived chemical epoxides, was characterized at the genomic level for interindividual variation capable of impacting function. RNA was isolated from 25 human liver samples and used to generate full-length copies of soluble epoxide hydrolase cDNA. The resulting cDNAs were polymerase chain reaction amplified, sequenced, and eight variant loci were identified. The coding region contained five silent single nucleotide polymorphisms (SNPs) and two variant loci resulting in altered protein sequence. An amino acid substitution was identified at residue 287 in exon 8, where the more common arginine was replaced by glutamine. A second variant locus was identified in exon 13 where an arginine residue was inserted following serine 402 resulting in the sequence, arginine 403-404, instead of the more common, arginine 403. This amino acid insertion was confirmed by analyzing genomic DNA from individuals harboring the polymorphic allele. Slot blot hybridization analyses of the liver samples indicated that sEH mRNA steady-state expression varied approximately 10-fold. Transient transfection experiments with CHO and COS-7 cells were used to demonstrate that the two new alleles possess catalytic activity using trans-stilbene oxide as a model substrate. Although the activity of the glutamine 287 variant was similar to the sEH wild type allele, proteins containing the arginine insertion exhibited strikingly lower activity. Allelic forms of human sEH, with markedly different enzymatic profiles, may have important physiological implications with respect to the disposition of epoxides formed from the oxidation of fatty acids, such as arachidonic acid-derived intermediates, as well in the regulation of toxicity due to xenobiotic epoxide exposures.     

Bioreactor-scale production and one-step purification of Epstein-Barr nuclear antigen 1 expressed in baculovirus-infected insect cells

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated malignancies and is essential for EBV-genome maintenance. Antibodies to EBNA1 are abundantly detected in serum of most EBV carriers but EBNA1 escapes recognition by effector T-lymphocytes. To further study the functional and immunological characteristics of EBNA1 it is important to have sufficient quantities of purified EBNA1 available. This paper describes a simple, reproducible method for the production and purification of EBV-encoded EBNA1 expressed in insect cells (bEBNA1). For quantification of EBNA1 expression levels in cell lines and for monitoring bEBNA1 purification and overall yields we developed a quantitative and EBNA1-specific capture ELISA. We observed that EBV-positive cell lines express EBNA1 at different levels, with the B cell lymphoblastoid cell line X50/7 having the highest production. However, much larger quantities (380-fold) were obtained by expressing bEBNA1 in recombinant-baculovirus-infected Sf9 insect cells. Scaling-up experiments revealed that bEBNA1 expression kinetics and protein stability are identical in 1-liter stirred bioreactors when compared to expression in stationary culture flasks. Optimal expression was reached after 72 h following inoculation at 1 pfu/cell, when insect cell viability was about 50%. For purification the nuclear fraction containing most of the bEBNA1 (>95%) was isolated. Solubilized bEBNA1 was purified by a one-step oriP DNA-Sepharose affinity purification procedure, using biotinylated PCR-amplified family of repeats (FR)-domain products immobilized onto streptavidin agarose. A >200-fold specific enrichment was reached and yields of bEBNA1 with an estimated purity of >95%.