S1P lyase inhibition prevents lung injury following high pressure-controlled mechanical ventilation in aging mice.

Ventilator-induced Lung Injury (VILI) is characterized by hypoxia, inflammatory cytokine influx, loss of alveolar barrier integrity, and decreased lung compliance. Aging influences lung structure and function and is a predictive factor in the severity of VILI; however, the mechanisms of aging that influence the progression or increased susceptibility remain unknown. Aging impacts immune system function and may increase inflammation in healthy individuals. Recent studies suggest that the bioactive sphingolipid mediator sphingosine-1-phosphate (S1P) and the enzyme that degrades it S1P lyase (SPL) may be involved in lung pathologies including acute lung injury. It is unknown whether aging influences S1P and SPL expression that have been implicated in lung inflammation, injury, and cell apoptosis. We hypothesized that aging and injurious mechanical ventilation synergistically impair S1P levels and enhance S1P lyase (SPL) expression that amplifies alveolar barrier damage and diminishes pulmonary function. Young (2-3 mo) and old (20-25 mo) C57BL/6 mice were mechanically ventilated for 2 h using pressure-controlled mechanical ventilation (PCMV) at 45 cmH2O and 35 cmH2O, respectively. We assessed the impact of aging and PCMV on several indications of acute lung injury, immune cell recruitment, S1P levels and SPL activity. Furthermore, we evaluated the protective effects of inhibiting SPL by tetrahydroxybutylimidazol (THI) administration on the negative outcomes associated with aging and mechanical injury. PCMV exacerbated lung injury in old mice and increased neutrophil influx that was further exacerbated due to aging. SPL expression increased in the young and old ventilated mice and the old nonventilated group. THI treatment reduced several of the indicators of lung injury and resulted in elevated S1P levels in lung tissue and plasma from mice that were injured from mechanical ventilation. CD80 and CD206 activation markers of alveolar and interstitial macrophages were also influenced by THI. SPL inhibition may be a viable therapeutic approach for patients requiring mechanical ventilation by preventing or regulating the exaggerated inflammatory response and reducing lung injury.

TET-mediated hydroxymethylcytosine at the Pparγ locus is required for initiation of adipogenic differentiation.

BACKGROUND/OBJECTIVES: Adipose tissue is one of the main organs regulating energy homeostasis via energy storage as well as endocrine function. The adipocyte cell number is largely determined by adipogenesis. While the molecular mechanism of adipogenesis has been extensively studied, its role in dynamic DNA methylation plasticity remains unclear. Recently, it has been shown that Tet methylcytosine dioxygenase (TET) is catalytically capable of oxidizing DNA 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) toward a complete removal of the methylated cytosine. We investigate whether expression of the Tet genes and production of hydroxymethylcytosine are required for preadipocyte differentiation. SUBJECTS/METHODS: Murine 3T3-L1 preadipocytes were used to evaluate the role of Tet1 and Tet2 genes during adipogenesis. Changes in adipogenic ability and in epigenetic status were analyzed, with and without interfering Tet1 and Tet2 expression using small interfering RNA (siRNA). The adipogenesis was evaluated by Oil-Red-O staining and induced expression of adipogenic genes using quantitative polymerase chain reaction (qPCR). Levels of 5-hmC and 5-mC were measured by MassARRAY, immunoprecipitation and GC mass spectrometry at specific loci as well as globally. RESULTS: Both Tet1 and Tet2 genes were upregulated in a time-dependent manner, accompanied by increased expression of hallmark adipogenic genes such as Pparγ and Fabp4 (P<0.05). The TET upregulation led to reduced DNA methylation and elevated hydroxymethylcytosine, both globally and specifically at the Pparγ locus (P<0.05 and P<0.01, respectively). Knockdown of Tet1 and Tet2 blocked adipogenesis (P<0.01) by repression of Pparγ expression (P<0.05). In particular, Tet2 knockdown repressed conversion of 5-mC to 5-hmC at the Pparγ locus (P<0.01). Moreover, vitamin C treatment enhanced adipogenesis (P<0.05), while fumarate treatment inhibited it (P<0.01) by modulating TET activities. CONCLUSIONS: TET proteins, particularly TET2, were required for adipogenesis by modulating DNA methylation at the Pparγ locus, subsequently by inducing Pparγ gene expression.

IL-10 production by CLL cells is enhanced in the anergic IGHV mutated subset and associates with reduced DNA methylation of the IL10 locus.

Chronic lymphocytic leukemias (CLLs) with unmutated (U-CLL) or mutated (M-CLL) IGHV have variable features of immunosuppression, possibly influenced by those CLL cells activated to produce interleukin 10 (IL-10). The two subsets differ in their levels of anergy, defined by low surface immunoglobulin M levels/signaling capacity, and in their DNA methylation profile, particularly variable in M-CLL. We have now found that levels of IL-10 produced by activated CLL cells were highly variable. Levels were higher in M-CLL than in U-CLL and correlated with anergy. DNA methylation analysis of IL10 locus revealed two previously uncharacterized 'variably methylated regions' (CLL-VMRs1/2) in the gene body, but similarly low methylation in the promoter of both U-CLL and M-CLL. CLL-VMR1/2 methylation was lower in M-CLL than in U-CLL and inversely correlated with IL-10 induction. A functional signal transducer and activator of transcription 3 (STAT3) binding site in CLL-VMR2 was confirmed by proximity ligation and luciferase assays, whereas inhibition of SYK-mediated STAT3 activation resulted in suppression of IL10. The data suggest epigenetic control of IL-10 production. Higher tumor load may compensate the reduced IL-10 production in U-CLL, accounting for clinical immunosuppression in both subsets. The observation that SYK inhibition also suppresses IL-10 provides a potential new rationale for therapeutic targeting and immunological rescue by SYK inhibitors in CLL.

Epigenetics in radiation-induced fibrosis.

Radiotherapy is a major cancer treatment option but dose-limiting side effects such as late-onset fibrosis in the irradiated tissue severely impair quality of life in cancer survivors. Efforts to explain radiation-induced fibrosis, for example, by genetic variation remained largely inconclusive. Recently published molecular analyses on radiation response and fibrogenesis showed a prominent role of epigenetic gene regulation. This review summarizes the current knowledge on epigenetic modifications in fibrotic disease and radiation response, and it points out the important role for epigenetic mechanisms such as DNA methylation, microRNAs and histone modifications in the development of this disease. The synopsis illustrates the complexity of radiation-induced fibrosis and reveals the need for investigations to further unravel its molecular mechanisms. Importantly, epigenetic changes are long-term determinants of gene expression and can therefore support those mechanisms that induce and perpetuate fibrogenesis even in the absence of the initial damaging stimulus. Future work must comprise the interconnection of acute radiation response and long-lasting epigenetic effects in order to assess their role in late-onset radiation fibrosis. An improved understanding of the underlying biology is fundamental to better comprehend the origin of this disease and to improve both preventive and therapeutic strategies.

Carnitine status in early-treated children, adolescents and young adults with phenylketonuria on low phenylalanine diets.

BACKGROUND: In patients with phenylketonuria (PKU), the carnitine status may be impaired for metabolic or dietary reasons, including low carnitine intake, a deficient synthesis and acylcarnitine production from phenylalanine (Phe) metabolites. METHODS: Free carnitine and acylcarnitine status was assessed in 30 PKU patients, aged 0.5-36 years, mean age 13.8 years. Our cohort was divided into 2 groups according to the preparations of Phe-free amino acids (AA) prescribed, with or without carnitine supplementation. Daily Phe intake, dosage of AA mixtures and body weight were recorded along with measurements of acylcarnitines in blood spots (by tandem mass spectrometry) and serum AA. Control data were obtained from 50 healthy volunteers (aged 0.2-39 years, mean age 14.2. years). Statistical analysis comprised the t test, ANOVA and Pearson's correlation. RESULTS: PKU patients had lower free carnitine (C0) concentrations than controls (25.82 +/- 7.38 vs. 31.28 +/- 6.17 micromol/l; p < 0.001) and lower octanoyl- and decanoylcarnitine. Mean C0 and acylcarnitine concentrations did not differ between PKU patients taking the various protein substitutes with or without carnitine; mean C0 levels in PKU patients receiving AA enriched with carnitine were still lower compared with controls (p < 0.05). CONCLUSIONS: Actual dietary regimens can not completely normalize the carnitine status; therefore, carnitine levels should be given careful consideration in subjects with PKU.

Effects of various dietary amino acid preparations for phenylketonuric patients on the metabolic profiles along with postprandial insulin and ghrelin responses.

AIM: We investigated the metabolic profiles along with insulin and ghrelin responses following ingestion of various amino acid (AA) substitutes commonly used in the treatment of phenylketonuria to study the effects of added macronutrients. METHODS: Twenty healthy and 6 phenylketonuric adults ingested AA mixtures with or without carbohydrates and fat (Anamix, Easiphen, or p-am 3; 0.35 g AA/kg body weight); milk powder shakes were used for control purposes. Serum AA, glucose, urea, insulin, and ghrelin were measured over 5 h. RESULTS: Peak AA concentrations were achieved at around 60 min postprandially for supplemented AA powders and control shakes, significantly later than for pure AA. Of interest, the mean Phe/Tyr ratio declined by 40-50% in phenylketonuric patients following intake of Easiphen, Anamix, or p-am 3. The insulin peaks, up to 500% as compared with baseline, occurred at 30 min and were approximately 100% higher after intake of AA plus macronutrients. Glucose and urea remained constant. Ghrelin showed a nadir at 60 min, followed by a rise leading to a 30% increase of initial concentrations for pure AA as compared with more constant levels for preparations with macronutrients. CONCLUSION: An oral AA bolus together with macronutrients retards hyperaminoacidemia, displays a higher insulin secretion, normoglycemia, and more stable ghrelin concentrations, whereas the pure AA tested here exerted weaker anabolic effects.

[Teleradiology and mammography screening: evaluation of a network with dedicated workstations for reporting]. / Teleradiologie im Mammographie-Screening: Evaluation eines Testnetzes mit dedizierten Befundungsstationen.

PURPOSE: Evaluation of a system that supports a workflow for breast cancer screening by mammography. The time of installation, system reliability and workstation operation were evaluated. MATERIALS AND METHODS: The evaluated system (Image Diagnost, Munich, Germany) contains 2 diagnostic mammography workstations, a centralized server, and 2 Dicom shuttles for exchanging images via a physically existing network structure. Temporary archiving is possible. A mask designed for the needs of mammography screening facilitates assignment of BIRADS categories. The system automatically compares the categories assigned by a first and second reviewer and decides whether a consensus conference should be held. In the event that a conference is needed, the reviews are transmitted to the mammography expert responsible for the screening program and the consensus conference. Images are transferred via ISDN, Germany's National Research and Education Network (in the following DFN) and a central server between 2 sites which are approx. 100 km apart. We evaluated the duration of installation, the reliability of the system, and the usability of the workstation. Since we used curative mammography for evaluating the system, the patient age was noted for comparison. RESULTS: The system was installed in five days. Once installed, the system functioned without any major breakdowns. Mammography units of 2 manufacturers were able to be connected to the system without difficulty. Mammographies of 151 patients were exchanged between the sites and evaluated by 2 radiologists. 57 % of the patients were in the screening age (50-69 years). 9 exams were classified BIRADS 4a, 2 were 4b and 3 were BIRADS 5. The evaluations were technically perfect in 146/151 cases; hanging protocols had to be altered manually in 6 cases; the window/level had to be manually adjusted in 26/151 cases. Magnification was sufficient in all cases. The system exchanges examinations extremely quickly; up to 100 mammography exams may be evaluated in 1 hr. CONCLUSION: The system supports the workflow given by the German Mammography Screening program both locally and in a network.

[No detectable nephrotoxic side effect using a dimer, non-ionic contrast media in cerebral perfusion computed tomography in case of suspected brain ischemia]. / Keine nachweisbare Nephrotoxizität eines dimeren nichtionischen Röntgenkontrastmittels bei der notfallmässigen kraniellen Perfusions-Computertomographie.

PURPOSE: In suspected brain ischemia, the perfusion cerebral computed tomography (cCT) should be performed with the lowest amount of contrast media to avoid a contrast media induced nephropathy (CIN) even if the patient already is in renal failure. We were interested to find the best parameters for this examination. MATERIAL AND METHODS: From February 2000 to March 2003, 138 patients (58 females, 80 males, mean age 66.8 years) underwent cCT-perfusion immediately after the admission to our stroke unit. Of these patients, 62% (n = 86) had normal renal function and 38% (n = 52) renal failure (up to 381 micromol/l basic serum creatinine). We varied volume (20-80 ml), flow (5 vs. 7.2 ml/s) and concentration (270 vs. 320 ml/mg iodine) of a dimer, non-ionic contrast media (Visipaque) to establish 5 groups. So we got patients receiving 6 g, 12 g, 16 g, 19 g and 25 g of iodine. After generating the perfusion maps, two radiologists reviewed the quality of the maps and scored it (1-5). We measured the serum creatinine before contrast application and at follow up cCT (days 3 and 7). RESULTS: The quality of the maps increases with increasing amount of iodine. However, the diagnostic result was not significantly better using more than about 16 g of iodine (e. g., 60 ml--7.2 ml/s--270 mg/ml) in cCT-perfusion studies. Only one patient had a pathologic increase in serum creatinine (day 1: 93; day 4: 146 micromol/l) but died at day 5 because of massive co-morbidity and septic pneumonia. No CIN occurred even in the patient group with pre-existent renal failure. CONCLUSIONS: About 60 ml contrast media and a moderate flow rate of about 7 ml/s ensure good results in perfusion-cCT, even if the patients have poor blood circulation or arteriosclerosis. The use of a dimer, non-ionic contrast media (range of 6-25 g iodine) seems to minimize the risk of CIN in the daily routine.

[Laser-induced thermotherapy (LITT) of tumors of the liver in central location: results and complications]. / Laserinduzierte Thermotherapie (LITT) von Lebertumoren in zentraler Lokalisation: Ergebnisse und Komplikationen.

PURPOSE: To investigate whether laser-induced thermotherapy (LITT) for tumors of the liver in central location is a sufficient and safe therapeutic option. MATERIAL AND METHODS: According to predefined criteria, 23 of 136 patients were chosen to be treated with LITT because of malignant liver tumors. At the time of the first LITT, the patients had 28 central tumors (27 metastases, one HCC), which were treated in 34 sessions with 64 laser applications and had a clinical and imaging follow-up every 3 months. RESULTS: The primary effectiveness rate was 74.1% and the secondary effectiveness rate 82.1%. The mortality rate was 0%. Major complications occurred in one patient (hemorrhagic pleural effusion), while minor complications occurred in 10 patients. During the median follow-up of 20 months (range 3-57 months), local tumor progression developed in 22% of the tumors. The effectiveness rate was 78.6%, 71.4% and 64.3% after 3, 6 and 9 months. The median survival was 46.0 months (95% confidence interval: 28.6-47.1 months). CONCLUSION: In our patients, complications and ablation rate of laser-induced thermotherapy for central liver tumors do not differ from those in peripheral location as described in the literature.

[Percutaneous laser-induced thermotherapy of lung metastases: experience gained during 4 years]. / Perkutane laserinduzierte Thermotherapie von Lungenmetastasen: Erfahrungen nach 4-jähriger Anwendung.

We perform laser ablation of lung metastases for four years. In the following we report on technical success, ablation results and follow-up. 30 patients with a total of 42 lung metastases were treated. Patients were aged between 42 and 74 years. Primary tumors were mostly colorectal carcinomas, head and neck and uro-genital tumors. Technical success was achieved in 40, complete ablation in 14 metastases (33%); however, in 8 metastases follow-up was too short for a valid evaluation (4-8 weeks). Further analysis may improve the overall results: metastases located in the central parts of the lungs were more easily treated than those located peripherally, best results were achieved in metastases not larger than 3 cm, percutaneous ablation of lung metastases has potential as an additional therapeutic option if surgery is not possible.

Endogenous retroviral syncytin: compilation of experimental research on syncytin and its possible role in normal and disturbed human placentogenesis.

Placental syncytin was first described in the year 2000 as a fusogenic glycoprotein originally derived from a human endogenous retroviral envelope gene. Although the presence of stable integrated retroviral elements within the human genome has been known for many years, their biological significance is still obscure and has usually been designated as irrelevant or even harmful. Syncytin, however, demonstrates tissue-specific expression and distinctive receptor interaction during trophoblast cell differentiation and syncytium formation. These findings indicate an involvement of syncytin in the development of the human placenta. Disturbances in placental architecture leading to severe placental dysfunction, such as pre-eclampsia, may therefore be discussed as a consequence of an altered syncytin system. We evaluate the hypothesis that syncytin is essential for human placenta formation and may also have played an important role in human placental evolution.

Does excessive occlusal load affect osseointegration? An experimental study in the dog.

AIM: The purpose of this study was to evaluate the effect of excessive occlusal load following placement of titanium implants in the presence of healthy peri-implant mucosal tissues. MATERIALS AND METHODS: Mandibular bilateral recipient sites in six Labrador dogs were established by extracting premolars and molars. After 3 months, two TPS (titanium plasma sprayed) implants and two SLA (sandblasted, large grit, acid etched) implants were placed on each side of the mandible in each dog. Three implants were lost in the initial healing phase, leaving 45 implants for evaluation. Following 6 months of healing, gold crowns were placed on implants on the test side of the mandible. The crowns were in supra-occlusal contact with the opposing teeth in order to create excessive occlusal load. Implants on the control side were not loaded. Plaque control was performed throughout the experimental period. Clinical measurements and standardised radiographs were obtained at baseline and 1, 3 and 8 months after loading. At 8 months, the dogs were killed and histologic analyses were performed. RESULTS: At 8 months, all implants were osseointegrated. The mean probing depth was 2.5+/-0.3 and 2.6+/-0.3 mm at unloaded and loaded implants, respectively. Radiographically, the mean distance from the implant shoulder to the marginal bone level was 3.6+/-0.4 mm in the control group and 3.7+/-0.2 mm in the test group. Control and test groups were compared using paired non-parametric analyses. There were no statistically significant changes for any of the parameters from baseline to 8 months in the loaded and unloaded implants. Histologic evaluation showed a mean mineralised bone-to-implant contact of 73% in the control implants and 74% in the test implants, with no statistically significant difference between test and control implants. CONCLUSION: In the presence of peri-implant mucosal health, a period of 8 months of excessive occlusal load on titanium implants did not result in loss of osseointegration or marginal bone loss when compared with non-loaded implants.

[Percutaneous thermoablation of lung metastases. Indication, performance, initial results, and imaging findings]. / Perkutane Laserablation von Lungenmetastasen. Indikation, technische Durchführung, erste Ergebnisse und Bildbefunde.

Not unlike thermoablation of liver metastases, thermoablation of metastases to the lungs is gaining clinical interest. Radiofrequency ablation and laser-induced interstitial thermotherapy are both used clinically. Initially it was suspected that percutaneous treatment of lung metastases would result in a rate of pneumothoraces and tissue reactions which would not be clinically acceptable. However, this did not prove true. Fear of pneumothoraces however did lead to the desire for an applicator with a maximally reduced diameter. While clinical results are not yet available, technical success rates of laser-induced interstitial thermotherapy of lung metastases are promising. The percentage of pneumothoraces does not differ significantly from that seen in diagnostic procedures. Large metastases may be treated by simultaneous use of multiple applicators or by repositioning of an applicator (pullback technique). Surgical experience is still guiding us in deciding which primaries' metastases may be successfully treated percutaneously. The literature indicates that lung metastases from colorectal primaries are especially suited. Radiotherapy is only an alternative method in cases of lung metastases if they cause symptoms (such as pain because of thorax infiltration or difficulty in breathing because of bronchial lesion). Due to the risk of radiation-induced pneumonia general radiotherapy is not to be recommended. The possibility of stereotactic ray treatment is being considered, but because breathing shifts the metastases it is not yet feasible. Therefore, percutaneous thermoablation could be used as a minimally invasive, rather riskless therapeutic option for a relatively high percentage of inoperable lung metastases.

[Laser-induced thermotherapy (LITT) of lung metastases: description of a miniaturized applicator, optimization, and initial treatment of patients]. / Laser-induzierte Thermotherapie (LITT) von Lungenmetastasen: Beschreibung eines miniaturisierten Applikators, Optimierung und erste Patientenbehandlungen.

PURPOSE: A thin-caliber applicator system was developed for introducing a laser fiber under CT guidance into lung metastases with only minimal complications. MATERIALS AND METHODS: A space-saving 5.5 French Teflon cannula with a titanium trocar and connectors for a laser light guide (2 or 3 cm Dornier Diffusor-Tip H-6111-T2 or H-6111-T3 coupled to a Dornier Medilas Fibertom 5100 laser, wavelength of 1064 nm) and a perfusion line for physiologic saline solution were developed. After puncture the laser Diffusor-Tip remains in the cannula and is cooled during its tissue passage by slowly flowing saline solution. The miniaturized applicator system (Monocath) was calibrated in nonperfused bovine liver for maximum energy supply and necessary flow of the cooling saline solution in reference to a commercially available 9 French laser catheter with an 11.5 French inducer sheath (Power-Applicator). The new applicator system was used for treating lung metastases in 10 patients over a period of 21 months. RESULTS: The size of heat coagulation in bovine liver was 24 +/- 2 ml using the miniaturized system with application of 15 W for 20 min and a saline flow of 0.75 ml/min, in comparison to a size of 29 +/- 7 ml for the commercial applicator (30 W, 20 min, 60 ml/min). All metastases could be safely approached with the miniaturized applicator, except for two metastatic lesions at the lung base in two patients. A minor pneumothorax developed in three patients and intrapulmonary bleeding in two. Contrast-enhanced CT demonstrated necrosis of the treated metastatic areas in 6 patients. Follow-up of three patients after 5, 6, and 8 months showed complete tumor regression with minimal scarring in one patient. CONCLUSION: The miniaturized applicator system enables the introduction of a laser fiber into pulmonary metastases with only minor complications. Complete ablation seems to be achievable in suitable patients with the applied laser energy and a slow cooling fluid flow rate.

Spring, a novel RING finger protein that regulates synaptic vesicle exocytosis.

The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca(2+)-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation.

The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome.

We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.

Bacterial replication initiator DnaA. Rules for DnaA binding and roles of DnaA in origin unwinding and helicase loading.

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

The interaction domains of the DnaA and DnaB replication proteins of Escherichia coli.

The initiation of chromosome replication in Escherichia coli requires the recruitment of the replicative helicase DnaB from the DnaBC complex to the unwound region within the replication origin oriC, supported by the oriC-bound initiator protein DnaA. We defined physical contacts between DnaA and DnaB that involve residues 24-86 and 130-148 of DnaA and residues 154-210 and 1-156 of DnaB respectively. We propose that contacts between DnaA and DnaB occur via two interaction sites on each of the proteins. Interaction domain 24-86 of DnaA overlaps with its N-terminal homo-oligomerization domain (residues 1-86). Interaction domain 154-210 of DnaB overlaps or is contiguous with the domains known to interact with plasmid initiator proteins. Loading of the DnaBC helicase in vivo can only be performed by DnaA derivatives containing (in addition to residues 24-86 and the DNA-binding domain 4) a structurally intact domain 3. Nucleotide binding by domain 3 is, however, not required. The parts of DnaA required for replication of pSC101 were clearly different from those used for helicase loading. Domains 1 and 4 of DnaA, but not domain 3, were found to be involved in the maintenance of plasmid pSC101.

Transcriptional regulation of gene expression of sec6, a component of mammalian exocyst complex at the synapse.

Sec6, an essential component of the mammalian brain exocyst complex, is believed to function in synapse formation and synaptic plasticity. During neuronal development, the expression of the Sec6 gene correlates temporally with neurite outgrowth and synaptogenesis. To understand the mechanisms that regulate the Sec6 gene expression, we have cloned and characterized the 5'-terminal region of the murine Sec6 gene. We have shown that the 5'-untranslated region of the murine Sec6 gene is encoded by two exons that are separated by a 1560-bp intron. Primer extension analysis demonstrates that Sec6 gene transcription is initiated from a unique site. The Sec6 promoter is embedded in a CpG island and lacks canonical TATA or CAAT boxes. Sequence analysis of the 5'-flanking region and the first intron reveals the presence of a number of binding sites for transcription factors AP-1, AP-2, AP-4, ATF, C/EBPbeta, GATA-1, Oct 1, SP1, STAT, and NRSF. Transfection experiments using Sec6-luciferase fusion genes demonstrate that the 5'-flanking sequence functions as a strong promoter in neuronal but not in nonneuronal cells. Deletion analysis reveals the presence of a core promoter between nucleotide position -139 and +53, and two enhancer and four silencer elements within the 5'-flanking region and the first intron sequence. These results indicate that neuronal expression of the Sec6 gene involves a relatively specific core promoter and interplay between multiple positive and negative regulatory elements.