Search

Use of zebrafish as a model organism in biomedical research has led to the generation of many genetically modified mutant lines to investigate various aspects of developmental and cellular processes. However, the broader effects of the underlying mutations on social and motor behavior remain poorly examined. Here, we compared the dynamics of social interactions in the Tüpfel long-fin nacre mutant line, which lacks skin pigmentation, to wild-type zebrafish; and we determined whether status-dependent differences in escape and swimming behavior existed within each strain. We show that despite similarities in aggressive activity, Tüpfel long-fin nacre pairs exhibit unstable social relationships characterized by frequent reversals in social dominance compared to wild-type pairs. The lack of strong dominance relationships in Tüpfel long-fin nacre pairs correlates with weak territoriality and overlapping spatial distribution of dominants and subordinates. Conversely, wild-type dominants displayed strong territoriality that severely limited the movement of subordinates. Additionally, the sensitivity of the startle escape response was significantly higher in wild-type subordinates compared to dominants. However, status-related differences in sensitivity of escape response in Tüpfel long-fin nacre pairs were absent. Finally, we present evidence suggesting that these differences could be a consequence of a disruption of proper visual social signals. We show that in wild-type pairs dominants are more conspicuous, and that in wild-type and Tüpfel long-fin nacre pairings wild-type fish are more likely to dominate Tüpfel long-fin nacres. Our results serve as a cautionary note in research design when morphologically engineered zebrafish for color differences are utilized in the study of social behavior and central nervous system function. [ABSTRACT FROM AUTHOR]

• In mice, LHANts neurons are positioned to coordinate feeding and drinking. • During the light cycle, activating LHANts neurons biases fluid over food intake. • In the dark cycle, activating LHANts neurons mediates aphagia but not drinking. • Activating LHANts neurons projecting to the LPO promotes drinking but not aphagia. The lateral hypothalamic area (LHA) is essential for ingestive behavior but has primarily been studied in modulating feeding, with comparatively scant attention on drinking. This is partly because most LHA neurons simultaneously promote feeding and drinking, suggesting that ingestive behaviors track together. A notable exception are LHA neurons expressing neurotensin (LHANts neurons): activating these neurons promotes water intake but modestly restrains feeding. Here we investigated the connectivity of LHANts neurons, their necessity and sufficiency for drinking and feeding, and how timing and resource availability influence their modulation of these behaviors. LHANts neurons project broadly throughout the brain, including to the lateral preoptic area (LPO), a brain region implicated in modulating drinking behavior. LHANts neurons also receive inputs from brain regions implicated in sensing hydration and energy status. While activation of LHANts neurons is not required to maintain homeostatic water or food intake, it selectively promotes drinking during the light cycle, when ingestive drive is low. Activating LHANts neurons during this period also increases willingness to work for water or palatable fluids, regardless of their caloric content. By contrast, LHANts neuronal activation during the dark cycle does not promote drinking, but suppresses feeding during this time. Finally, we demonstrate that the activation of the LHANts → LPO projection is sufficient to mediate drinking behavior, but does not suppress feeding as observed after generally activating all LHANts neurons. Overall, our work suggests how and when LHANts neurons oppositely modulate ingestive behaviors [ABSTRACT FROM AUTHOR]

Abstract: Mammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase. Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition. Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle. Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles. Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells. Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity. These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells. [Copyright &y& Elsevier]

Abstract: Retroviral infection triggers the cytoplasmic translocation of two Crm1-dependent shuttle factors, namely the Ini1 (integrase interactor 1, hSNF5) and the promyelocytic leukemia (PML) protein. Blocking nuclear export of shuttle factors by leptomycin B increases the efficiency of retroviral integration, suggesting that some may mediate antiviral activity. While PML was shown to counteract proviral establishment, it remained unclear whether Ini1, a protein implicated in various processes during human immunodeficiency virus replication, has the same potential. Employing RNA interference-mediated knock-down of Ini1, we show here that the simultaneous accumulation of both proteins in the cytoplasm likely reflects two non-interdependent phenomena. Furthermore, Ini1 does not interfere with retroviral integration, as cells lacking Ini1 show no increased infection susceptibility. [Copyright &y& Elsevier]

Abstract: Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants, because there are only two efficient resistance markers available for allelic exchange experiments. We have established a system based on the Flp recombinase of the yeast Saccharomyces cerevisiae for use in the nonpathogenic model organism Mycobacterium smegmatis. This system consists of a hygromycin resistance cassette flanked by two Flp recognition targets (FRT) in direct orientation and a curable plasmid for expression of the flp gene. The FRT-hyg-FRT cassette was used on a suicide plasmid and on a conditionally replicating plasmid to delete two of the four known porin genes of M. smegmatis, mspA and mspC, respectively, by homologous recombination. The hyg gene was specifically removed from the chromosome of both mutants upon expression of the flp gene. Based on the marker-less mspC mutant strain, a double knock-out mutant lacking also mspA was obtained using the same strategy. Thus, by a fast and efficient two-step procedure, each of the porin genes was replaced by a single FRT site, which can be further used for site-specific integration. These results show that the Flp/FRT system is a suitable genetic tool for constructing unmarked mutations and for the analysis of redundant genes by consecutive gene deletions in M. smegmatis. [Copyright &y& Elsevier]

The neuropeptide galanin exhibits anticonvulsant effects in experimental epilepsy. Two galanin receptor subtypes, GalR1 and GalR2, are present in the brain. We examined the role of GalR1 in seizures by studying the susceptibility of GalR1 knockout (KO) mice to status epilepticus (SE) and accompanying neuronal injury.SE was induced in GalR1 KO and wild type (WT) mice by Li-pilocarpine, 60 min electrical perforant path stimulation (PPS), or systemic kainic acid (KA). Seizures were analyzed using Harmonie software. Cell injury was examined by FluoroJade B- and terminal deoxynucleotidyl transferase-mediated uridine triphosphatenick end labeling; neurogenesis was studied using bromodeoxyuridine labeling.Compared with WT littermates, GalR1 KO showed more severe seizures, more profound injury to the CA1 pyramidal cell layer, as well as injury to hilar interneurons and dentate granule cells upon Li-pilocarpine administration. PPS led to more severe seizures in KO, as compared with WT mice. No difference in the extent of neuronal degeneration was observed between the mice of two genotypes in CA1 pyramidal cell layer; however, in contrast to WT, GalR1 KO developed mild injury to hilar interneurons on the side of PPS. KA-induced seizures did not differ between GalR1 KO and WT animals, and led to no injury to the hippocampus in either of experimental group.No differences were found between KO and WT mice in both basal and seizure-induced neuronal progenitor proliferation in all seizure types. Li-pilocarpine led to more extensive glia proliferation in GalR1 KO than in WT, and in both mouse types in two other SE models.In conclusion, GalR1 mediate galanin protection from seizures and seizure-induced hippocampal injury in Li-pilocarpine and PPS models of limbic SE, but not under conditions of KA-induced seizures. The results justify the development and use of GalR1 agonists in the treatment of certain forms of epilepsy. [Copyright &y& Elsevier]

Apurinic/apyrimidinic endonuclease (AP endo, HAP1) recognizes abasic sites in ds DNA and makes a single nick in the backbone 5′ to the abasic site. In this report we examine the roles of three conserved tyrosine residues in close proximity to the active site. We show that Tyr128 and Tyr269, which interact upstream and downstream of the abasic site, respectively, are involved in recognition and binding of abasic site-containing double stranded DNA. However, the two residues are not equivalent, as their effects are differentiated by changes in salt concentration. In sharp contrast, Tyr171 is directly involved in catalysis as well as binding. Y171F, Y171H, and Y171A all show decreased catalytic efficiencies 25,000–50,000-fold from the WT enzyme. Both imidazole and basic pH markedly stimulate the WT enzyme. Imidazole stimulates Tyr171 mutant enzymes when tyrosine is also present but basic pH eliminates remaining mutant activity. These results underscore the importance of tyrosines in AP endo catalysis. They render the current hypotheses regarding enzyme action unlikely and allow us to consider the possibility that the phenolate of Tyr171 is the nucleophile that attacks the scissile phosphate. [Copyright &y& Elsevier]

Myogenic differentiation is characterized by permanent and irreversible cell cycle withdrawal and increased resistance to apoptosis. These functions correlate with changes in expression and activity of several cyclin-dependent kinase inhibitors, including p18, p21, and p27. In this study, we examined the requirements for p18, p21, and p27 in initiating growth arrest in multinucleated myotubes under differentiation conditions and in maintaining terminal arrest upon restimulation of differentiated myotubes with mitogenic signals. Under differentiation conditions, only p27−/− or p18−/−p27−/− myotubes are capable of reentering the cell cycle and synthesizing DNA at a very low frequency. Escape from cell cycle arrest was significantly greater in p18−/−p27−/− myotubes than in p27−/− myotubes. Stimulation of differentiated cultures with a mitogen-rich growth medium enhances p18−/−p27−/− myotube proliferation to encompass approximately half of the nuclei. p18−/−p21−/− and p21−/−p27−/− myotubes remain terminally arrested. Nuclei within individual restimulated p18−/−p27−/− myotubes can be found in all phases of the cell cycle, and a myotube can be multiphasic without any obvious deleterious effects. Increasing the time of differentiation or serum stimulation of p18−/−p27−/− myotubes neither increases the proliferation index of the myotube nuclei, nor does it alter the percentage of nuclei in each of the cell cycle phases. During the first 24 h of serum stimulation, the p18−/−p27−/− myotube nuclei that escape G0 arrest will rearrest in either S or G2 phase, without either mitosis or endoreplication. Apoptosis is increased in restimulated p18−/−p27−/− myotube nuclei, but is not specific for any cell cycle phase. These results suggest a collaborative role for p18 and p27 in initiating and maintaining G0 arrest during myogenic differentiation. While p18 and p27 appear to be essential in initiating G0 arrest in a proportion of postmitotic myotube nuclei, there must be another cell cycle inhibitor protein that functions with p18 and p27 in maintaining terminal arrest. We propose that the combined rate-limiting expressions of p18, p27, and this other inhibitor determine whether the myotube nuclei will remain postmitotic, or reenter the cell cycle, and if the nuclei escape G0 arrest, in which phase of the cell cycle the nuclei will ultimately rearrest. [Copyright &y& Elsevier]

Both the ferrous and ferric forms of wild-type neuroglobin are found to be hexacoordinated with axial ligation of the F8-His and E7-His. Rapidly growing Escherichia coli cell cultures with low O2 concentration generate nitric oxide (NO). Combined electron paramagnetic resonance (EPR) and optical measurements show that wild-type human recombinant neuroglobin, overexpressed in such E. coli cells, still favors the F8His-Fe2+-E7His conformation, whereby only a small fraction of the protein binds NO. Upon mutation of the E7-His to Leu and Gln, the competition with the distal histidine disappears and the nitrosyl ferrous form is readily observed. At low temperature, the EPR spectra of the NO-ligated Ngb proteins consist of contributions from two geometrically different NO-heme conformations. In combination with EPR data of vertebrate hemoglobins and myoglobins, the temperature dependence of the EPR spectra of the NO adducts of ferrous hNgb and its E7-mutants proves a strong stabilization of one isomer by the E7-histidine in wt hNgb. It is shown that this is not related to the polarity of histidine, but to its specific binding characteristics. [Copyright &y& Elsevier]

The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the α-helical surface loop, the so-called ‘lid’ of HLL comprised of residues 86–93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the ‘open’ conformation in triacetin, with the α-helical surface loop moving so as to expose the active site. (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue. [Copyright &y& Elsevier]

Op18 (Oncoprotein 18, Stathmin) is an important mitotic regulator that is highly expressed in many cancers including the metastatic prostate carcinoma cell line PC-3-M. Recent studies indicate that antisense-mediated down-regulation of Op18 can inhibit cellular proliferation. However, the transcriptional mechanisms responsible for its normal regulation and for its high level of expression in proliferating cells remain poorly understood. In the study presented here, we have characterized transcription factor binding sites that together account for nearly 80% of the Op18 expression in PC-3-M cells. The 5′ flanking region of the Op18 gene contains four putative E2F sites located at −700 (site 1), −28 (site 2), −19 (site 3), and +720 (site 4) relative to the transcriptional start site. E2F has been implicated in both the c-Jun-mediated up-regulation and the doxorubicin-induced repression of Op18 expression. We have used promoter–reporter assays and mobility shift assays to functionally examine each of these E2F sites. Mutagenesis studies indicate that all sites contribute to the basal expression of Op18. Mutagenesis of either site 1 or 4 reduced the reporter activity by 40%, mutagenesis of site 2 reduced reporter activity by 20%, and mutations in site 3 had no effect on reporter activity. Combinatorial mutagenesis indicates that site 1 and 4 function independently, whereas site 2 functions synergistically with either site 3 or 4. Mobility shift assays indicate that all elements bind factors in the nuclear extracts of PC-3-M cells. Characterization of the sites show that site 1, though a positive element, is not E2F specific; sites 2 and 3 may contain an overlapping binding site for E2F and NF1; and site 4, which resides in intron 1, is the only site shown to interact exclusively with E2F. These studies suggest that the overexpression of Op18 in PC-3-M cells is mediated predominately through the E2F family of transcription factors. [Copyright &y& Elsevier]

Estrogen receptor-alpha (ER-α) is a nuclear transcriptional factor that is part of the nuclear receptor superfamily. In this study, we isolated and identified a new LXXLL-containing protein that interacts with the ER-α via a yeast two-hybrid assay. We have termed this protein estrogen receptor repressor-10 (ERR-10). The ERR-10 cDNA is predicted to encode a polypeptide of 94 amino acids, with a molecular mass of about 10 kDa. Although the ERR-10 mRNA transcript is expressed in a wide range of normal human tissues, higher expression levels are found in endocrinal tissues relative to other tissues. We have demonstrated, through immunoprecipitation, Western blot and GST pull-down assays, that ERR-10 associates with ER-α. Moreover, ERR-10 decreased 17β-estrodial-induced activation of ER-α transcriptional activity in transient transfection assays of mammalian cells. The ERR-10 N-terminus, which resembles two LXXLL motifs, is essential for ER-α binding and repression activity. Estrogen modulation of estrogen-responsive gene expression was markedly blocked by ERR-10. These results suggest that ERR-10 is a novel mediator in ER transcriptional activation. [Copyright &y& Elsevier]

Knockout of the translation inhibitor 4E-BP1 induces an overexpression of uncoupling protein-1 (UCP1) [Nature Medicine 7 (2001) 1128]. A possible inverse control of UCP1 and 4E-BP1 expressions in mouse brown adipose tissue was investigated. Cold-exposure, which increases the expression of UCP1, decreased that of 4E-BP1 mRNA in wild type but not in β1/β2/β3-adrenoceptor knockout mice. Administration of the β3-adrenoceptor agonist CL 316 246 decreased 4E-BP1 mRNA by 75% and protein by 41% after 6 and 48 h, respectively. Our data are the first report of a regulation by the β3-adrenoceptor of 4E-BP1 expression. They support a role of the latter in adaptive thermogenesis. [Copyright &y& Elsevier]

We sought to investigate the role of fibroblast growth factor (FGF)-1 during acute myocardial ischemia and reperfusion.The FGFs display cardioprotective effects during ischemia and reperfusion.We investigated FGF-1–induced cardioprotection during ischemia and reperfusion and the intracellular signaling pathways responsible for these effects in an ex vivo murine setup of myocardial ischemia and reperfusion.Cardiac-specific human FGF-1 overexpression was associated with enhanced post-ischemic hemodynamic recovery and decreased lactate dehydrogenase release during reperfusion. Inhibition of the FGF receptor, protein kinase C (PKC), and tyrosine kinase (TK) resulted in blockade of FGF-1-induced protective effects on cardiac functional recovery and cell death.The overexpression of FGF-1 induces cardioprotection through a pathway that involves the FGF receptor, PKC, and TK. [Copyright &y& Elsevier]

Ycf53 is a hypothetical chloroplast open reading frame with similarity to the Arabidopsis nuclear gene GUN4. In plants, GUN4 is involved in tetrapyrrole biosynthesis. We demonstrate that one of the two Synechocystis sp. PCC 6803 ycf53 genes with similarity to GUN4 functions in chlorophyll (Chl) biosynthesis as well: cyanobacterial gun4 mutant cells exhibit lower Chl contents, accumulate protoporphyrin IX and show less activity not only of Mg chelatase but also of Fe chelatase. The possible role of Gun4 for the Mg as well as Fe porphyrin biosynthesis branches in Synechocystis sp. PCC 6803 is discussed. [Copyright &y& Elsevier]

RNA–protein interactions are fundamental for different aspects of molecular biology such as gene expression, assembly of biomolecular complexes or macromolecular transport. The 3a movement protein (MP) of a plant virus, Cucumber mosaic virus (CMV), forms ribonucleoprotein (RNP) complexes with viral RNA, capable of trafficking from cell-to-cell throughout the infected plant only in the presence of the CMV capsid protein (CP). However, deletion of the C-terminal 33 amino acid residues of the CMV MP (in the mutant designated 3aΔC33 MP) resulted in CP-independent cell-to-cell movement. The biological differences in the behaviour of CMV wild type (wt) 3a MP and 3aΔC33 MP could have been a consequence of differences in the RNA-binding properties of the two MPs detected previously using biochemical assays on ensembles of molecules. To investigate the physical mechanisms of MP–RNA interactions at a single molecule level, we applied atomic force microscopy to measure for the first time unbinding forces between these individual binding partners. Minimal unbinding forces determined for individual interaction of the CMV RNA molecule with the CMV wt or truncated MPs were estimated to be ∼45 pN and ∼90 pN, respectively, suggesting that the distinct differences in the strength of MP–RNA interactions for the wt MP and truncated MP are attributable to the molecular binding mechanism. We also demonstrated that molecules of both CMV 3a MP and 3aΔC33 MP were capable of self-interaction with minimal unbinding forces of ∼50 pN and ∼70 pN, respectively, providing a physical basis for the cooperative mechanism of the RNA binding. The significance of intermolecular force measurements for understanding the structural and functional aspects of viral RNP formation and trafficking is discussed. [Copyright &y& Elsevier]

We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K+ channels. Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2. In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K+ channel SK1 and SK3 like. Directed by the results of the toxin receptor on the ShakerK+ channel, we changed single amino acids of the SK2 K+ channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3. Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK+ channel to improve Shaker K+ channel CTX sensitivity. Interestingly SK2 V342G became CTX sensitive with a Kd of 19 nM and was also KTX sensitive Kd=97 nM. SK2 S344E (Kd CTX=105 nM,Kd KTX=144 nM) and G348D (Kd CTX=31 nM,Kd KTX=89 nM) became also CTX and KTX sensitive with a lower affinity. The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity (Kd=6 nM,Kd=48 nM, and Kd=12 nM). Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K+ channel pore is very similar to those of voltage-gated K+ channels such as the Shaker K+ channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel. From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore. [Copyright &y& Elsevier]

Here, we identify ADP-ribosylation factor (ARF)-like 7 (ARL7) as the only ARF- and ARL-family member whose mRNA-expression is induced by liver X-receptor/retinoid X-receptor agonists or cholesterol loading in human macrophages. Moreover, subcellular distribution of mutant and wild type ARL7-enhanced green fluorescent protein (EGFP) supports that ARL7 may be involved in a vesicular transport step between a perinuclear compartment and the plasma membrane. Therefore, we investigated the effect of ARL7 over-expression on the cholesterol secretory pathway. We found that expression of wild type and dominant active ARL7-EGFP stimulated the rate of apolipoprotein AI-specific cholesterol efflux 1.7- and 2.8-fold. In contrast, expression of the dominant negative form of ARL7-EGFP led to ∼50% inhibition of cholesterol efflux. This data is consistent with a model in which ARL7 is involved in transport between a perinuclear compartment and the plasma membrane apparently linked to the ABCA1-mediated cholesterol secretion pathway. [Copyright &y& Elsevier]

Toxoplasma gondii (T. gondii)-derived heat shock protein 70 (T.g.HSP70) has been identified as a virulent molecule expressing only in T. gondii tachyzoites during lethal acute infection. Therefore, it is of importance to determine the expression of T.g.HSP70 mRNA in a quantitative manner for analysis of virulence of T. gondii in tissues. We have constructed a competitor T.g.HSP70 and have successfully established a quantitative competitive-reverse transcriptase–polymerase chain reaction (QC-RT-PCR) targeting T.g.HSP70 gene. By using the established QC-RT-PCR method, we have demonstrated that the copy number of T.g.HSP70 mRNA per T. gondii tachyzoite was highest in the lung among the organs examined in interferon-γ knockout (GKO) mice. [Copyright &y& Elsevier]

Presenilins (PS1 and PS2) are supposed to be unusual aspartic proteases and components of the γ-secretase complex regulating cleavage of type I proteins. Multiple mutations in PS1 are a major cause of familial early-onset Alzheimer’s disease (AD). We and others recently identified PS-related families of proteins (IMPAS/PSH/signal peptide peptidases (SPP)). The functions of these proteins are yet to be determined. We found that intramembrane protease-associated or intramembrane protease aspartic protein Impas 1 (IMP1)/SPP induces intramembranous cleavage of PS1 holoprotein in cultured cells coexpressing these proteins. Mutations in evolutionary invariant sites in hIMP1 or specific γ-secretase inhibitors abolish the hIMP1-mediated endoproteolysis of PS1. In contrast, neither AD-like mutations in hIMP1 nor in PS1 substrate abridge the PS1 cleavage. The data suggest that IMP1 is a bi-aspartic polytopic protease capable of cleaving transmembrane precursor proteins. These data, to our knowledge, are a first observation that a multipass transmembrane protein or the integral protease per se may be a primary substrate for an intramembranous proteolysis. [Copyright &y& Elsevier]

Adenocarcinoma of the small intestine (ACSI) is a rare condition with few studies addressing follow-up and prognosis. Tumors of 35 patients with curative resection of an ACSI were retrospectively analyzed by immunohistochemistry: p53, hMLH1, hMSH2 and hMSH6 and microsatellite instability (MSI): BAT-26, BAX, TGF-β RII. With a median follow up of 6.1 years, the median cancer-specific survival (CSS) was 36.2 months. Patients who were highly instable (MSI-H) (n=10) had a CSS of 49.6 months in contrast to patients with stable tumors (23.2 months) (P=0.010). Additionally, a low tumor stage according to UICC and MSI-H were shown to be independent factors (P=0.005 and P<0.001) for an increased survival in multivariate analysis. Therefore, it is suggested that analysis of the MSI status might prove useful in discerning prognosis within cancers of the same stage. [Copyright &y& Elsevier]

Epithelial–mesenchymal transitions (EMTs) are an important mechanism for reorganizing germ layers and tissues during embryonic development. They have both a morphogenic function in shaping the embryo and a patterning function in bringing about new juxtapositions of tissues, which allow further inductive patterning events to occur [Genesis 28 (2000) 23]. Whereas the mechanics of EMT in cultured cells is relatively well understood [reviewed in Biochem. Pharmacol. 60 (2000) 1091; Cell 105 (2001) 425; Bioessays 23 (2001) 912], surprisingly little is known about EMTs during embryonic development [reviewed in Acta Anat. 154 (1995) 8], and nowhere is the entire process well characterized within a single species. Embryonic (developmental) EMTs have properties that are not seen or are not obvious in culture systems or cancer cells. Developmental EMTs are part of a specific differentiative path and occur at a particular time and place. In some types of embryos, a relatively intact epithelium must be maintained while some of its cells de-epithelialize during EMT. In most cases de-epithelialization (loss of apical junctions) must occur in an orderly, patterned fashion in order that the proper morphogenesis results. Interestingly, we find that de-epithelialization is not always necessarily tightly coupled to the expression of mesenchymal phenotypes.Developmental EMTs are multi-step processes, though the interdependence and obligate order of the steps is not clear. The particulars of the process vary between tissues, species, and specific embryonic context. We will focus on ‘primary’ developmental EMTs, which are those occurring in the initial epiblast or embryonic epithelium. ‘Secondary’ developmental EMT events are those occurring in epithelial tissues that have reassembled within the embryo from mesenchymal cells. We will review and compare a number of primary EMT events from across the metazoans, and point out some of the many open questions that remain in this field. [Copyright &y& Elsevier]

A psaFJ-null mutant of Synechocystis sp. strain PCC 6803 was characterised. As opposed to similar mutants in chloroplasts of green algae, electron transfer from plastocyanin to photosystem 1 was not affected. Instead, a restraint in full chain photosynthetic electron transfer was correlated to malfunction of photosystem 1 at its stromal side. Our hypothesis is that absence of PsaF causes oxidative stress, which triggers the induction of the ‘iron stress inducible’ operon isiAB. Products are the IsiA chlorophyll-binding protein (CP43′) and the isiB gene product flavodoxin. Supporting evidence was obtained by similar isiAB induction in wild type cells artificially exposed to oxidative stress. [Copyright &y& Elsevier]

The molecular basis of potassium uptake in cyanobacteria has not been elucidated. However, genes known from other bacteria to encode potassium transporters can be identified in the genome of Synechocystis sp. strain PCC 6803. Mutants defective in kdpA and ntpJ were generated and characterized to address the role of the Kdp and KtrAB systems in this strain. KtrAB is crucial for K+ uptake, as the ΔntpJ mutant shows slowed growth, slowed potassium uptake kinetics, and increased salt sensitivity. The ΔkdpA mutant has the same phenotype as the wild type even at limiting potassium, but a ΔkdpAΔntpJ double mutant is not viable, indicating a role of Kdp for potassium uptake when the Ktr system is not functioning. [Copyright &y& Elsevier]

We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3′ proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3′ rapid amplification of cDNA ends (3′-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3′ ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3′ sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants. [Copyright &y& Elsevier]

Our understanding of estrogen signaling has undergone a true paradigm shift over recent years, following the discovery in 1995 of a second estrogen receptor, estrogen receptor β (ERβ). In many contexts ERβ appears to antagonize the actions of ERα (yin/yang relationship) although there also exist genes that are specifically regulated by one of the two receptors. Studies of ERβ knockout mice have shown that ERβ exerts important functions in the ovary, central nervous system, mammary gland, prostate gland, hematopoiesis, immune system, vessels and bone. The use of ERβ-specific ligands against certain forms of cancer represents one of the many pharmaceutical possibilities that have been created thanks to the discovery of ERβ. [Copyright &y& Elsevier]

Background: Tick borne encephalitis virus (TBEV), is a human flavivirus causing tick borne encephalitis (TBE), a viral infection of the central nervous system endemic in Europe and Asia. Objectives: To develop a reverse transcription polymerase chain reaction (RT-PCR) assay based on quantitative real-time RT-PCR technology (TaqMan) for detection and quantification of TBEV RNA. The test includes an internal control (IC) to avoid false negative results. Study design: The system was established and validated using wild-type (WT) non-infectious synthetic RNA representing a fragment of the 3′ non-coding region of the TBEV genome. In addition, synthetic RNA differing from the WT synthetic RNA by a unique probe binding region was used as IC to monitor the overall efficiency of the RT-PCR. Results: The analytical sensitivity of the assay was at least ten copies of the TBEV synthetic transcript in presence of 50 copies of the IC. Successful amplification was obtained for different strains within the TBEV complex (Hypr, Hochosterwitz, Laibach, Elsass=Alsace, ZZ9, Wladiwostok). Among 14 serum and 21 cerebrospinal fluid (CSF) samples obtained from 28 patients with clinical suspicion of TBEV 1 CSF sample tested positive for TBEV RNA. In addition, no TBEV RNA could be detected in blood samples obtained from three vaccinated people 1 and 3 days post-vaccination. Thus indicating that a positive result is unlikely to be caused by recent vaccination. Conclusions: A quantitative, highly sensitive and specific real-time RT-PCR assay has been developed for the detection of TBEV RNA. Inclusion of an IC is important to monitor the possible occurrence of false-negative results caused by the presence of inhibitory factors. This assay should be an important asset for the routine laboratory detection of TBEV RNA. [Copyright &y& Elsevier]

We have developed a method called ‘gene gorging’ to make precise mutations in the Escherichia coli genome at frequencies high enough (1–15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants. [Copyright &y& Elsevier]

Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29–33 and 39–43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor. [Copyright &y& Elsevier]

The forkhead box f1 (Foxf1) transcription factor is expressed in septum transversum mesenchyme and splanchnic (visceral) mesoderm, which give rise to mesenchymal cells of gut-derived organs such as liver, gall bladder, lung, stomach, and intestine. Foxf1 −/− embryos die in utero and haploinsufficiency of the Foxf1 gene resulted in a variety of developmental abnormalities of the lung, gall bladder, esophagus, and trachea and caused defects in liver and lung repair. In this study, we used Foxf1 +/− mouse embryos containing the β-Galactosidase (β-Gal) gene knocked into the Foxf1 locus to examine whether Foxf1 is expressed in the developing brain and head region. In addition to previously reported Foxf1 expression patterns, β-Gal staining was also found in the mesenchyme of developing pituitary gland, eye, pancreas, heart, notochord, genital tubercle, Meckel''s cartilage, and cartilage primordia of nasal septum and vertebral bodies. In adult Foxf1 +/− mice, β-Gal staining was detected in anterior and posterior pituitary gland, astrocytes of the cerebellum and cerebral cortex, lens and retina of the eye, tracheal cartilage, parathyroid, cortical layer of thymus, capsule of the spleen, coronary arteries, and pancreatic blood vessels. [Copyright &y& Elsevier]

A novel gap junction forming protein, mouse connexin-30.2 (Cx30.2) contains 278 amino acid residues, and is 79% identical to human Cx31.9 (GJA11). Northern analysis showed that Cx30.2 is ubiquitously expressed, most prominently in testis. Polyclonal antibodies against Cx30.2 detected a 30 kDa protein in cells overexpressing Cx30.2, and in mouse testis. Immunofluorescence showed that Cx30.2 was expressed in vascular smooth muscle, but also in cell types where Cx31.9 was not detected. These data demonstrate that Cx30.2 is a bona fide gene, and suggest that it is the orthologue of Cx31.9, but that it may have additional roles compared with Cx31.9. [Copyright &y& Elsevier]

Human host cell factor-1 (HCF-1) is a large, 2035-residue nuclear protein that interacts with cellular and viral transcription factors. It contains an N-terminal kelch domain, C-terminal fibronectin type III (FnIII) domain, and a central region including tandem repeats which act as cleavage sites. A second human HCF-1 related gene encodes a protein with a high degree of homology in both the N-terminal kelch domain and C-terminal FnIII domain, but lacks the central portion and as a result is considerably smaller at 792 residues. A unique HCF orthologue has been found in Caenorhabditis elegans which is structurally more related to HCF-2 than HCF-1. Here we report the cloning and expression of the single Drosophila melanogaster host cell factor orthologue (dHCF). The dHCF is 1500 residues in size, intermediate between HCF-1 and HCF-2 and contains an N-terminal kelch domain, and C-terminal FnIII domain both of which show a very high degree of identity, and a central region of some 700 residues with more limited homology. Despite containing a central region no repeat-related motifs were apparent. The dHCF is expressed as a single unprocessed polypeptide consistent with the lack of the internal HCF-1 processing sites, and exhibits a predominantly nuclear localization. We show that this nuclear localization is dependent on a bipartite nuclear localization signal at the C-terminus of the protein, which contains a long spacer of 20 amino acids between two basic clusters. Finally, we also show that dHCF is unable to rescue the tsBN67 cell cycle arrest phenotype. These results indicate that dHCF is an orthologue of HCF-1, although both proteins might not be functionally exchangeable. [Copyright &y& Elsevier]

In this study we have modified the neuroinvasiveness of pseudorabies virus strain Bartha, a commonly utilized trans-synaptic tract-tracer. In addition, we sought to facilitate detection of cellular mRNAs in neurons infected with the virus. In order to modify spreading characteristics, we inserted the lacZ or the GFP (green fluorescent protein) genes into the genomic loci containing the putative latency-associated transcript promoter (PLAT2), resulting in the disruption of the promoter function. Following rat kidney injection, mutant viruses labeled central autonomic neurons in a slower and much more restricted manner than the parent Bartha strain. Since both reporter genes were controlled by the human cytomegalovirus immediate early (IE) 1 promoter, they exhibited IE expression kinetics. This property proved to be important for the co-detection of reporter proteins with neuronal mRNAs, readily detected at early but not at late stage of infection, as shown in tyrosine-hydroxylase expressing A5 catecholaminergic neurons and in serotonin transporter expressing raphe magnus neurons. [Copyright &y& Elsevier]

Both the β-d-(+) and β-l-(−)-enantiomers of 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (D4FC) are clinically relevant compounds because of their potent anti-HIV and anti-HBV activities. Cross-resistance to l-D4FC with HBV containing a mutation in the conserved polymerase YMDD region has been observed. In order to better understand the effects of stereochemistry on planar 5-fluorinated cytidine analogs and to gain insight into resistance caused by YMDD mutations in HIV-1 reverse transcriptase (RT), a combination of transient kinetic studies and computer modeling were employed. In contrast to studies with the (+) and (−) isomers of 3TC-TP and FTC-TP, it was found that wild type RT had a high enantiomeric selectivity between the d-(+) and l-(−) isomers of D4FC-TP. While no resistance was conferred by the methionine 184 to valine mutation to d-D4FC-TP, l-D4FC-TP was incorporated 50- to 70-fold less efficiently. The kinetic parameters of incorporation in the presence of l-D4FC-TP by RTWT and the mechanism of resistance by RTM184V were found to be distinct from those seen with the corresponding l-isomers containing an oxathiolane ring: (−)-3TC-TP and (−)-FTC-TP. Molecular modeling suggests that l- and d-D4FC-TP are positioned in the active site favorably for incorporation by RTWT and that l-D4FC-TP, but not d-D4FC-TP, is sterically hindered by the addition of a β branched amino acid at position 184 of RTM184V. [Copyright &y& Elsevier]

Neuronal nicotinic acetylcholine receptors (nAChR), composed of 12 subunits (α2–α10, β2–β4), are expressed in autonomic ganglia, playing a central role in autonomic transmission. The repertoire of nicotinic subunits in autonomic ganglia includes α3, α5, α7, β2 and β4 subunits. In the last 10 years, heterologous expression studies have revealed much about the nature of neuronal nAChRs. However, there is only limited understanding of subunit actions in autonomic system. Functional deletions of subunit by gene knockout in animals could overcome these limitations. We review recent studies on nAChRs on autonomic ganglia for physiological and pharmacological properties and potential locations of the subunits. [Copyright &y& Elsevier]

Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator in tissue repair. In non-healing human wounds plasmin cleaves and inactivates VEGF165. In the present study, we generated recombinant VEGF165 mutants resistant to plasmin proteolysis. Substitution of Arg110 with Ala110 or Gln110, and Ala111 with Pro111 yielded plasmin-resistant and biologically active VEGF165 mutants. In addition, substitution of Ala111 with Pro111 resulted in a substantial degree of stabilization when incubated in wound fluid obtained from non-healing wounds. These results suggest that the plasmin cleavage site Arg110/Ala111 and the carboxyl-terminal domain play an important role in the mitogenic activity of VEGF165. [Copyright &y& Elsevier]

Dystrophin, the protein which is absent or non-functional in Duchenne muscular dystrophy, consists of four main domains: an N-terminal actin binding domain, a rod shaped domain of spectrin-like repeats, a cysteine-rich domain and a unique C-terminal domain. In muscle, dystrophin forms a linkage between the cytoskeletal actin and a group of membrane proteins (dystrophin associated proteins). The N-terminal domain binds to the cytoskeletal actin and the association with the dystrophin associated proteins is mediated mainly by the cysteine-rich and C-terminal domains of dystrophin. The dystrophin gene also encodes two isoforms of non-muscle dystrophins and a number of smaller products consisting of the two C-terminal domains with different extensions into the spectrin-like repeat domain. Dp71, which consist of the C-terminal and the cysteine-rich domains of dystrophin, is the major product of the gene in all non-muscle tissues tested so far, but it is absent in differentiated skeletal muscle. In an attempt to understand the functions of Dp71, we produced transgenic mice over-expressing this protein in several tissues. The highest levels of exogeneous Dp71 were detected in skeletal muscle, in association with the sarcolemma. This resulted in muscle damage similar to that found in mice which lack dystrophin. The data indicates that Dp71 competes with dystrophin for the binding to the dystrophin associated proteins. Since Dp71 lacks the actin binding domain, it cannot form the essential linkage between the dystrophin associated proteins complex and the cytoskeleton. [Copyright &y& Elsevier]

The putative cell-lysis gene lys of Lactobacillus plantarum G1e phage ϕg1e encodes for a 442 amino-acids protein Lys. The N-terminal region (about 80 amino acids) of Lys consists of two discrete regions (the signal-peptide-like domain and the DE domain containing putative active sites of endolysin). To elucidate functions of the regions of Lys, mutational (random, site-directed, and/or fusion) analysis was performed. The plasmid pNdEHL, expressing the wild type Lys protein under promoter of lacZ′ gene in Escherichia coli, was constructed. Two molecular species (44 kDa; referred to as pre-Lys, and 42 kDa; mature-Lys) from the protein extract of XL1-Blue/pNdEHL were detected on a sodium dodecyl sulfate gel and zymogram with L. plantarum G1e cells. Based on the N-terminal amino acid sequences, the two molecules were determined as; pre-Lys (the amino acid position deduced from lys gene, 1–7) MKLKNKL, mature-Lys (27–33) QTLSSQS. The mature Lys was hardly detected in the cells treated with sodium azide.These results suggested that the N-terminal 26 amino acids region of Lys precursor form is possibly processed posttranslationally, by a SecA-dependent manner at least in E. coli.Analysis of the point mutants (pLD36A, pLE39A, pLE55A, pLE67A and pLD71A), indicated that the acidic residues (aspartic acids at position 36, 71 and glutamic acids at position 39, 55) of N-terminal region and the serine at the position 48 of ϕg1e Lys are essential for the lytic activity. [Copyright &y& Elsevier]

Thrombopoietin (Tpo) and its receptor, c-mpl, are expressed in murine embryonic stem (ES) cells. ES cells are maintained in a pluripotent state by leukemia inhibitory factor (LIF) via activation of the Janus kinase (Jak)–STAT3 signaling pathway. Tpo, like LIF, activates STAT3. We report that Tpo increases the number of undifferentiated colonies derived from wild type or Shp-2 mutant (Shp-2Δ46–110) ES cells. Tpo plus LIF acted synergistically on the Shp-2Δ46–110 ES cells to maintain undifferentiated colonies but no evidence of synergism via Jak–STAT3 activation was detected. Collectively, these data suggest that Tpo can play a role in preventing ES cell differentiation via Jak–STAT3 activation and perhaps via novel pathways that are enhanced in the absence of functional Shp-2. [Copyright &y& Elsevier]

Cultured murine F9 teratocarcinoma stem cells resemble pluripotent stem cells of the inner cell mass of the mouse blastocyst and, depending upon their treatment, can be induced to differentiate along the primitive endoderm, the parietal endoderm (PE), or the visceral endoderm (VE) pathway. The Rex-1 gene encodes a zinc finger family transcription factor which is expressed at high levels in undifferentiated F9 stem cells, embryonic stem cells, and in other types of stem cells. To examine whether the Rex-1 protein plays a role in F9 cell differentiation, homologous recombination was employed to generate F9 cell lines which lack both alleles of Rex-1. F9 wild type cells in monolayer culture require both retinoic acid and cyclic AMP analogs to differentiate into PE, whereas the F9 Rex-1−/− cells differentiate into PE, as assessed by several molecular markers, including thrombomodulin and laminin B1, in the presence of RA alone. The F9 Rex-1−/− cells do not completely differentiate into VE after RA treatment in aggregate culture; they do not express alpha-fetoprotein, a definitive marker of VE differentiation. These results indicate that the Rex-1 transcription factor regulates the differentiation of F9 stem cells along several distinct cell lineages found in the early embryo. [Copyright &y& Elsevier]

The dopamine D2 receptor (D2) is implicated in drug addiction, learning and memory. Two isoforms of the D2 receptor, termed D2L (long form) and D2S (short form), have been identified. We previously generated mice lacking D2L (D2L−/−), but expressing functional D2S. In this study, we investigated the role of D2L in the positive and negative reinforcing properties of abused drugs and electrical stimuli, using D2L−/− mice as a model system. Mice were trained in three associative learning tasks: conditioned place preference to morphine and cocaine, conditioned place aversion to naloxone-precipitated morphine withdrawal, and active avoidance. D2L−/− mice, like wild type mice, developed a place preference to cocaine. In contrast to wild type mice, D2L−/− mice did not develop a place preference to morphine, nor did they attain a place aversion to morphine withdrawal. D2L−/− mice also failed to acquire avoidance behavior in response to electrical stimuli. There were no significant differences between D2L−/− and wild type mice in μ-opioid receptor density, morphine-induced locomotor stimulation and morphine withdrawal symptoms.These results suggest that D2L may have a greater impact than D2S on the rewarding aspects of morphine, and the aversive properties of morphine withdrawal and electrical stimulus. These findings also suggest that the presence of D2L is critical in the acquisition (learning) and/or retention (memory) of context–stimulus associations in certain situations. On the other hand, D2L is not essential for the rewarding aspects of cocaine and for the development of morphine dependence. Thus, these studies reveal distinct functional roles of D2L and/or D2S in drug addiction and avoidance learning, which may lead to a better understanding of the neurobiological basis underlying these behaviors. [Copyright &y& Elsevier]

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18 kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic α-helices (H1–H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C–apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C–apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene “excimer” fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene–A8C–apoLp-III/DMPC or pyrene–A138C–apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene–A8C/A138C–apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle. [Copyright &y& Elsevier]

Application of delivery systems in cancer therapy is restricted as a result of the lack of cell specificity of the respective vectors. Recently, a vector system based on virus-like particles (VLPs) of modified polyoma-VP1 was described which were able to bind specifically a tumor-specific antibody fragment, thus directing the vector system towards tumor cells. The functional gene transfer using the VP1 variant VP1-E8C, coupled with the antibody fragment of the tumor-specific antibody B3 is described in this paper. The specific targeting of the antigen expressing cells was highly efficient as determined by fluorescence microscopy. However, only a low percentage of these cells showed a functional gene transfer. This discrepancy could be accounted for by a rather low capacity of the virus like particles to transport DNA and the mechanism of their internalization by the target cells, which led to a lysosomal degradation of the particles. These limitations could be surmounted partially in cell culture experiments, and the principles suitable for applying this vector system in vivo are discussed. [Copyright &y& Elsevier]

A long-hypocotyl phytochrome B deficient mutant (lh) and its isogenic wild type (WT) of cucumber (Cucumis sativus L.) were used to study interacting effects between thermo- and photomorphogenetic stem elongation in long- and short-term experiments. The plants were grown under a 12 h high light intensity period and day/night temperature (DT/NT) of 25/19 °C (positive DIF, difference between DT and NT) or 19/25 °C (negative DIF) in combination with 30 min end-of-day (EOD)-red (R) or far red (FR) light. Thirty minutes EOD-FR, compared to EOD-R, significantly increased the length of stem, hypocotyl and internodes, and the number of visible internodes in WT. Positive DIF enhanced stem elongation compared to negative DIF under an EOD-R treatment, while the effect of negative DIF was strongly reduced by EOD-FR. For instance, an exposure to EOD-FR increased the plant height 68 and 41% under negative and positive DIF, respectively. In contrast, the lh mutant showed little difference in stem elongation in response to EOD-R and EOD-FR treatments, and small difference between positive and negative DIF treatments. These results indicate that DIF interacts significantly with EOD light quality on stem elongation, and that phytochrome B may play a key role in the interaction between thermo- and photomorphogenetic responses. A study on the kinetics of hypocotyl elongation showed that DIF interacted with EOD treatments in manipulating hypocotyl elongation rate (HER). In the photoperiod, positive DIF resulted in greater average HER than zero DIF and negative DIF, and the effect of DIF was stronger under EOD-R than EOD-FR. In the dark period, negative DIF showed larger HER compared to positive and zero DIF, and the effect was enhanced by EOD-FR compared to EOD-R. An EOD-FR increased HER both in photoperiod and dark period, in comparison to an EOD-R. Further, under EOD-R, negative DIF postponed a distinct peak of HER early in the dark period by approximately 4 h compared to positive DIF. A sharp peak was only found in the photoperiod under positive DIF. Under EOD-FR, a sharp peak of HER appeared at the end of the dark period or early in the photoperiod under negative DIF. This may indicate that the phytochrome is involved in thermoperiodic control of the kinetics of hypocotyl elongation. [Copyright &y& Elsevier]

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme’s Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme. [Copyright &y& Elsevier]

Mouse Emx2 homeobox gene is a very good dorsal marker for the developing cerebral cortex, as it is mainly expressed in this area from the very beginning of corticogenesis. Its cortical expression includes proliferating neuroblasts of the neuroepithelium, or ventricular zone, and the postmitotic Cajal-Retzius cells, known to control neuronal radial migration. Analysis of the phenotype of Emx2 null embryos has shown that this transcription factor plays important roles in neuroblast proliferation, migration and differentiation, as well as in the development of the diencephalon, where it has been shown to cooperate with Otx2. Moreover, the graded distribution of EMX2 homeoprotein along the antero-posterior and medial-lateral cortical axis, is responsible for the patterning of the forebrain, in particular for the specification process that defines cortical territories and area identity during neocortical development. Emx2 participates to this process as Pax6 and COUP-TF1. Finally Emx2 is very interesting from the evolutionary point of view, as it has been shown to share a high degree of homology in its sequence and function with Vax1, another homeobox gene regulating basal forebrain development. This homology traces back to Emx and Vax gene families, which are strongly related, as they are thought to derive from a common ancestor gene. [Copyright &y& Elsevier]

Human immunodeficiency virus type-1 envelope glycoprotein (gp120env) binding to cell surface CD4 receptor triggers a broad range of intracellular effects leading to T cell activation and cell cycle entry. Among these effects we and others previously reported on the nuclear translocation of the nuclear factor-κB (NF-κB) transcription factor. The present work further investigates the signal transduction pathways involved in gp120env-induced NF-κB activation. We demonstrate that gp120env–CD4 interaction stimulates the hyperphosphorylation of IκB-α inhibitory protein. Conversely, overexpression of a dominant-negative IκB-α transgene mutated at S32 and S36 residues, abolishes gp120env-induced NF-κB activation. IκB kinases (IKKs) activity was found to be selectively enhanced following CD4 engagement with gp120env and to mediate the phosphorylation of IκB-α while co-transfection experiments using dominant-negative forms of IKKs inhibited gp120env-induced NF-κB activation. Taken together, these results confirm that IKKs complex play a key role in gp120env-induced NF-κB activation. [Copyright &y& Elsevier]

Cardiac troponin I, the inhibitory subunit of the heterotrimeric cardiac troponin (cTn) complex is phosphorylated by protein kinase A at two serine residues located in its heart-specific N-terminal extension. This flexible arm interacts at different sites within cTn dependent on its phosphorylation degree. Bisphosphorylation is known to induce conformational changes within cTnI which finally lead to a reduction of the calcium affinity of cTnC. However, as we show here, the bisphosphorylated cTnI arm does not interact with cTnC, but with cTnT and/or cTnI. [Copyright &y& Elsevier]