Improving double-ended transition state searches for soft-matter systems.

Transitions between different stable configurations of biomolecules are important in understanding disease mechanisms, structure-function relations, and novel molecular-scale engineering. The corresponding pathways can be characterized efficiently using geometry optimization schemes based on double-ended transition state searches. An interpolation is first constructed between the known states and then refined, yielding a band that contains transition state candidates. Here, we analyze an example where various interpolation schemes lead to bands with a single step transition, but the correct pathway actually proceeds via an intervening, low-energy minimum. We compare a number of different interpolation schemes for this problem. We systematically alter the number of discrete images in the interpolations and the spring constants used in the optimization and test two schemes for adjusting the spring constants and image distribution, resulting in a total of 2760 different connection attempts. Our results confirm that optimized bands are not necessarily a good description of the transition pathways in themselves, and further refinement to actually converge transition states and establish their connectivity is required. We see an improvement in the optimized bands if we employ the adjustment of spring constants with doubly-nudged elastic band and a smaller improvement from the image redistribution. The example we consider is representative of numerous cases we have encountered in a wide variety of molecular and condensed matter systems.

Characterisation of the host inflammatory response to Staphylococcus epidermidis in neonatal whole blood.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens causing late-onset sepsis, and gestational age is the most important risk factor for these infections. OBJECTIVE: To characterise innate immune responses to S epidermidis by assessment of whole blood in vitro cytokine production in a large group of preterm and term infants. RESULTS: The S epidermidis-induced in vitro production of proinflammatory cytokines such as intracytoplasmic interleukin (IL) 6 and tumour necrosis factor alpha in cord blood samples was found to be dependent on gestational age (R = 0.279, 95% CI 0.10 to 0.44, p = 0.002; R = 0.251, 95% CI 0.07 to 0.41, p = 0.005, respectively; n = 121). In contrast, the production of anti-inflammatory cytokines such as IL10 and transforming growth factor beta was not associated with gestational age. When different stimulation strategies were compared, a strong correlation was noted for cytokine responses after lipopolysaccharide and S epidermidis exposure--that is, IL6 (R = 0.431, 95% CI 0.29 to 0.55, p<0.001, n = 161) and IL10 (R = 0.332, 95% CI 0.18 to 0.47, p<0.001, n = 161). In addition, a lower IL6 production was found in supernatants of whole blood cultures infected with a clinically isolated IcaABD-positive (biofilm production) strain compared with a control IcaABD-negative ATCC strain (p = 0.009). CONCLUSIONS: These in vitro data suggest that proinflammatory responses to S epidermidis are dependent on gestational age in preterm infants, whereas the counteracting anti-inflammatory response to S epidermidis may not be directly related to gestational age. Individual host factors may have a role as well as bacterial determinants, such as biofilm production. Further studies are encouraged to investigate the different aspects of innate immune responses to CoNS in vivo.

Retinoic acid-mediated transcription and maturation of SREBP-1c regulates fatty acid synthase via cis-elements responsible for nutritional regulation.

A region of the rat FAS (fatty acid synthase) promoter has been defined as being responsible for RA (retinoic acid) responsiveness. The defined promoter region is devoid of canonical RA-response elements but contains cis-elements binding generalized and specific transcription factors that mediate the dietary response of FAS. Our results are consistent with SREBP-1c (sterol-regulatory-element-binding protein 1c) binding in this region, thus bringing about the RA responsiveness of the rat FAS proximal promoter.

Structure and evolution of the horizontal septum in vertebrates.

Although the horizontal septum (HS) has been identified as playing a role in fish biomechanics and in path finding of cells during zebrafish development, its morphology is poorly known. However, it is generally regarded as an evolutionarily conserved structure. To test this idea, we applied a novel combination of techniques to analyse the HS of 35 species from all major gnathostome clades in which is visualized its collagen fibre architecture. Results show that the HS is a conserved trait only with respect to the presence of caudolateral [= epicentral] and craniolateral [= posterior oblique] collagen fibre tracts, but differs remarkably with respect to the specifications of these tracts. Our data revealed several evolutionary changes within vertebrates. In the gnathostome ancestor, the two tracts are represented by evenly distributed epicentral fibres (ECFs) and posterior oblique fibres (POFs). ECFs are condensed to distinct epicentral tendons (ECTs) in the actinopteran ancestor. POFs independently evolved to distinct posterior oblique tendons (POTs) at least two times within teleosts. Within basal teleostomes, POFs as well as ECFs or ECTs were lost two times independently. POTs were lost at least three times independently within teleosts. This view of a homoplastic HS remains stable regardless of the competing phylogenies used for analysis. Our data make problematic any generalization of biomechanical models on fish swimming that include the HS. They indicate that the pathfinding role of the HS in zebrafish may be extended to gnathostome fishes, but not to agnathans, sarcopterygian fishes and tetrapods.

Transcription factors acting on the promoter of the rat fatty acid synthase gene.

Fatty acid synthase (FAS), one of the main lipogenic enzymes, converts dietary calories into a storage form of energy. The transcription factors, stimulatory proteins 1 and 3 (Sp1 and Sp3), nuclear factor Y (NF-Y), upstream stimulatory factor (USF) and sterol regulatory element binding protein-1 (SREBP-1) have cognate binding sites on the promoter of the FAS gene. It was shown that Sp1 and NF-Y interact co-operatively at the diet-induced DNase I-hypersensitive site at position -500. Adjacent binding sites for NF-Y and Sp1 have also been found between -71 and -52, and -91 and -83. cAMP regulation is mediated via the inverted CAAT element (ICE) at -99 to -92, which binds NF-Y. The FAS insulin-responsive element 3 (FIRE3)-binding site at -71 to -52 is capable of binding NF-Y, USF and SREBP-1, and is required for the sterol response in conjunction with the co-activator NF-Y around -100. Surprisingly, both FIRE3 and ICE are also necessary for the response to retinoic acid that plays a role in development and is an essential component of the diet.

Inhibition of the rat blood-brain barrier choline transporter by manganese chloride.

Choline transport has been characterized by multiple mechanisms including the blood-brain barrier (BBB), and high- and low-affinity systems. Each mechanism has unique locations and characteristics yet retain some similarities. Previous studies have demonstrated cationic competition by monovalent cations at the BBB and cation divalent manganese in the high-affinity system. To evaluate the effects of divalent manganese inhibition as well as other cationic metals at the BBB choline transporter, brain choline uptake was evaluated in the presence of certain metals of interest in Fischer-344 rats using the in situ brain perfusion technique. Brain choline uptake was inhibited in the presence of Cd(2+) (73 +/- 2%) and Mn(2+) (44 +/- 6%), whereas no inhibition was observed with Cu(2+) and Al(3+). Furthermore, it was found that manganese caused a reduction in brain choline uptake and significant regional choline uptake inhibition in the frontal and parietal cortex, the hippocampus and the caudate putamen (45 +/- 3%, 68 +/- 18%, 58 +/- 9% and 46 +/- 15%, respectively). These results suggest that choline uptake into the CNS can be inhibited by divalent cationic metals and monovalent cations. In addition, the choline transporter may be a means by which manganese enters the brain.

The FIRE3-mediated sterol response of the FAS promoter requires NF-Y/CBF as a coactivator.

The transcription of the fatty acid synthase (FAS) gene is regulated by the sterol status of the cell via cleavage of the sterol regulatory element-binding protein (SREBP). When human HepG2 hepatoma cells were cotransfected with an expression plasmid for mature SREBP-1a together with FAS promoter/reporter constructs significant increases in reporter activity were observed. Deletion analysis of the FAS promoter between -151 and -52 relative to the transcription start site pinpoint two cis-elements important in sterol regulation of the FAS gene. One element, FIRE3, between -71 and -52 can bind in vitro translated and transcribed SREBP-1a whereas the other element, the inverted CCAAT element ICE(-97/-92), binds the trimeric transcription factor NF-Y/CBF as shown with rat liver extract and reconstituted, recombinant NF-Y. The results clearly show that the coactivator for SREBP-1a in this cell line is NF-Y. This finding was confirmed by using a dominant negative form of NF-YA, NF-YAm29, which interferes with the effect of ectopically expressed SREBP-1a on FAS reporter activity.

[Acute temporomandibular joint arthritis after Lyme borreliosis]. / Akute Kiefergelenkarthritis nach Lyme-Borreliose.

CASE REPORT: This report is about a rare connection between Lyme disease and an inflammation of the left temporomandibular joint. In this case, an infection was documented in 1998, 5 years after contact with Borrelia burgdorferi. The patient, a 49-year-old female, first came to our department in 1999. She showed the symptoms of a left temporomandibular joint infection. THERAPY: We suggested treatment with ceftriaxone 1 x 2 g/day i.v.

Running-buffer composition influences DNA-protein and protein-protein complexes detected by electrophoretic mobility-shift assay (EMSA).

The gel-shift assay is a rapid, extremely sensitive, technically simple and widely used method for investigating nucleic acid-protein interaction based on the observation that binding of protein to DNA or RNA fragments usually leads to a reduction in the electrophoretic mobility of the fragment in non-denaturing gels. Here we report on the critical role of the running buffer and show that its importance ranks equally with other factors affecting complex formation and stability such as binding buffer, temperature, non-specific competitor or gel concentration and/or composition. We demonstrate differences in the binding patterns obtained with oligonucleotides containing binding sites for the ubiquitously expressed transcription factors Sp1 (stimulatory protein 1), NF-Y (nuclear factor Y) and USF (upstream stimulatory protein), which are dependent on the ionic strength of the running buffer used. Furthermore, we show the influence of glycine concentration on Sp1 binding using recombinant glutathione S-transferase-Sp1 fusion protein expressed in Escherichia coli.

Role of Sp1 and Sp3 in the transcriptional regulation of the fatty acid synthase gene.

Inspection of the 5' region of the sequence of the rat fatty acid synthase (FAS) gene revealed a high GC content between -900 and +500, implying several binding sites for members of the Sp1 family of transcription factors. Using SL2 and H4IIE cells in conjunction with FAS promoter/luciferase constructs either successively deleted or containing defined deletions we characterized six GC boxes--GC-I to GC-VI--located between -557 and -83 and discovered a seventh, GC-VII, in the first intron. In vitro DNAse I-footprinting, electrophoretic mobility shift assays, and the yeast one-hybrid system indicated that Sp1 as well as Sp3 interacts with GC-I to GC-VII. Each of the GC boxes conferred Sp1-dependent transcription on the FAS-Mini promoter and in the case of GC-I, Sp1, and Sp3 exert an additive effect on FAS promoter activity.

Transcriptional repression by Drosophila methyl-CpG-binding proteins.

C methylation at genomic CpG dinucleotides has been implicated in the regulation of a number of genetic activities during vertebrate cell differentiation and embryo development. The methylated CpG could induce chromatin condensation through the recruitment of histone deacetylase (HDAC)-containing complexes by methyl-CpG-binding proteins. These proteins consist of the methylated-DNA binding domain (MBD). Unexpectedly, however, several studies have identified MBD-containing proteins encoded by genes of Drosophila melanogaster, an invertebrate species supposed to be void of detectable m(5)CpG. We now report the genomic structure of a Drosophila gene, dMBD2/3, that codes for two MBD-containing, alternatively spliced, and developmentally regulated isoforms of proteins, dMBD2/3 and dMBD2/3Delta. Interestingly, in vitro binding experiments showed that as was the case for vertebrate MBD proteins, dMBD2/3Delta could preferentially recognize m(5)CpG-containing DNA through its MBD. Furthermore, dMBD2/3Delta as well as one of its orthologs in mouse, MBD2b, could function in human cells as a transcriptional corepressor or repressor. The activities of HDACs appeared to be dispensable for transcriptional repression by dMBD2/3Delta. Finally, dMBD2/3Delta also could repress transcription effectively in transfected Drosophila cells. The surprisingly similar structures and characteristics of the MBD proteins as well as DNA cytosine (C-5) methyltransferase-related proteins in Drosophila and vertebrates suggest interesting scenarios for their roles in eukaryotic cellular functions.

Dependence of rat spot14 promoter activity on NF-Y binding to the inverted CCAAT-element at -100.

Electrophoretic mobility shift assay (EMSA) and in vitro transcription/translation show that NF-Y binds to the inverted CCAAT-element in the promoter of the rodent spot14 gene. The NF-Y-binding sequence has been shown to be responsible for basal activity in H4IIE. Given the similar role found for the inverted CCAAT-element in the promoter of the FAS gene, NF-Y may have an important function in the control of lipogenesis.

Loading of DNA-binding factors to an erythroid enhancer.

The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage- and erythroid lineage-specific expression of the human alpha-like globin genes, the embryonic zeta and the adult alpha2/alpha/1. A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3'-NA, modulates the capability of HS-40 to activate the embryonic zeta-globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecular basis of this model. First, human erythroid K562 cells stably integrated with various HS-40 mutants cis linked to a human alpha-globin promoter-growth hormone hybrid gene were analyzed by genomic footprinting and expression analysis. By the assay, we demonstrate that factors bound at different motifs of HS-40 indeed act in concert to build a fully functional enhanceosome. Thus, modification of factor binding at a single motif could drastically change the configuration and function of the HS-40 enhanceosome. Second, a specific 1-bp, GC-->TA mutation in the 3'-NA motif of HS-40, 3'-NA(II), has been shown previously to cause significant derepression of the embryonic zeta-globin promoter activity in erythroid cells. This derepression was hypothesized to be regulated through competitive binding of different nuclear factors, in particular AP1 and NF-E2, to the 3'-NA motif. By gel mobility shift and transient cotransfection assays, we now show that 3'-NA(II) mutation completely abolishes the binding of small MafK homodimer. Surprisingly, NF-E2 as well as AP1 can still bind to the 3'-NA(II) sequence. The association constants of both NF-E2 and AP1 are similar to their interactions with the wild-type 3'-NA motif. However, the 3'-NA(II) mutation causes an approximately twofold reduction of the binding affinity of NF-E2 factor to the 3'-NA motif. This reduction of affinity could be accounted for by a twofold-higher rate of dissociation of the NF-E2-3'-NA(II) complex. Finally, we show by chromatin immunoprecipitation experiments that only binding of NF-E2, not AP1, could be detected in vivo in K562 cells around the HS-40 region. These data exclude a role for AP1 in the developmental regulation of the human alpha-globin locus via the 3'-NA motif of HS-40 in embryonic/fetal erythroid cells. Furthermore, extrapolation of the in vitro binding studies suggests that factors other than NF-E2, such as the small Maf homodimers, are likely involved in the regulation of the HS-40 function in vivo.

Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1.

The regulation of the rat fatty acid synthase gene by mediators such as diet, hormones, cAMP, sterols or retinoic acid is controlled by three NF-Y binding sites. All three sites have a neighbouring Sp1-binding GC-box. This NF-Y/Sp1 motif is conserved in the FAS promoters of rat, human, goose and chicken. We have previously shown cooperative binding of NF-Y and Sp1 to the promoter region at -500 coincident with a diet-induced DNAse I-hypersensitive site. Here, we show an in-vivo interaction of NF-YA with Sp1 using the yeast two-hybrid system. The interacting domains are located between amino acids 55 and 139 of the NF-Y subunit NF-YA and between amino acids 139 and 344 of Sp1. In addition, we show by co-immunoprecipitation direct interaction of NF-Y subunit NF-YA with Sp1 in extracts of rat hepatoma cells H4IIE. Furthermore, we demonstrate by the GST pull-down assay that NF-YA interacts physically with Sp1 in-vitro in the absence of DNA. Therefore, NF-Y can be added to the list of transcription factors interacting with Sp1.

FIRE3 in the promoter of the rat fatty acid synthase (FAS) gene binds the ubiquitous transcription factors CBF and USF but does not mediate an insulin response in a rat hepatoma cell line.

Several putative insulin-responsive elements (IRE) in the fatty acid synthase (FAS) promoter have been identified and shown to be functional in adipocytes and hepatocytes. Here we report on the insulin-responsiveness in the rat hepatoma cell line H4IIE of four cis-elements in the FAS promoter: the FAS insulin-responsive elements, FIRE2 and FIRE3; the inverted CCAAT element, ICE; and the insulin/glucose-binding element, designated hepatic FIRE element, hFIRE, originally identified in rat hepatocytes. Using electrophoretic mobility shift assay (EMSA) competition experiments together with supershifts and in vitro transcription/translation we show that FIRE3 (-68/-58) binds not only the upstream stimulatory factors USF-1/USF-2 but also the CCAAT-binding factor CBF, also known as the nuclear factor Y, NF-Y. The putative IRE FIRE2, which shows sequence similarity to FIRE3, is located between -267 and -249. Gel retardation experiments indicate that USF-1 and USF-2 also bind to this element, which contains an imperfect E-box motif. Using the same approach we have shown that hFIRE binds the stimulatory proteins Sp1 and Sp3 in addition to CBF. Transient transfection experiments using FAS promoter constructs deleted for FIRE2 and FIRE3 demonstrate that neither of these elements mediates the insulin response of the FAS promoter in the rat hepatoma cell line H4IIE, however, ICE at -103/-87 is responsible for mediating the effect of the insulin antagonist cAMP. The hFIRE element located at -57/-34, in spite of its role in the glucose/insulin response in primary rat hepatocytes, is apparently not involved in the insulin regulation of the rat FAS promoter in H4IIE cells. The fact that the topology of the promoters of the FAS genes in rat, human, goose and chicken is conserved regarding CBF-binding sites and USF-binding sites implies an important role for these ubiquitously expressed transcription factors in the regulation of the FAS promoter.

Cooperative binding of NF-Y and Sp1 at the DNase I-hypersensitive site, fatty acid synthase insulin-responsive element 1, located at -500 in the rat fatty acid synthase promoter.

In vitro DNase I footprint analysis of the rat fatty acid synthase (FAS) promoter from -568 to -468 revealed four protein binding sites: A, B, and C boxes and the FAS insulin-responsive element 1 (FIRE1). As demonstrated by gel mobility shift analysis and supershift experiments, FIRE1, located between -516 and -498, is responsible for binding NF-Y. The C box located downstream of FIRE1 was shown by in vitro footprinting to be a Sp1 binding site, and furthermore, competition with Sp1 also abolished FIRE1 binding. Since the half-life of the Sp1.NF-Y.DNA complex is significantly longer than the half-lives of the Sp1.DNA or NF-Y.DNA complexes, the two transcription factors are deemed to bind cooperatively in the FAS promoter at -500. It is unusual that NF-Y binds at this distance from the start site of transcription. NF-Y binding sites are found in the promoters of at least three other FAS genes, viz. goose, chicken, and man. A second NF-Y binding site is located in the FAS promoter at the more usual position of -103 to -87, and it too has a neighboring Sp1 site. CTF/NF-1 competes for proteins binding to the B box. The A box binds Sp1 and contains a 12/13 match of the inverted repeat sequence responsible for binding the nuclear factor EF-C/RFX-1 in the enhancer regions of hepatitis B virus and the major histocompatibility complex class II antigen promoter. The same relative positions of NF-Y and Sp1 binding sites in the promoters of FAS genes of goose, rat, chicken, and man emphasize the involvement of these transcription factors in the diet and hormonal regulation of FAS.