Repression of p15INK4b expression by Myc through association with Miz-1.

Deregulated expression of c-myc can induce cell proliferation in established cell lines and in primary mouse embryonic fibroblasts (MEFs), through a combination of both transcriptional activation and repression by Myc. Here we show that a Myc-associated transcription factor, Miz-1, arrests cells in G1 phase and inhibits cyclin D-associated kinase activity. Miz-1 upregulates expression of the cyclin-dependent kinases (CDK) inhibitor p15INK4b by binding to the initiator element of the p15INK4b promoter. Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit transcriptional activation by Miz-1. Expression of Myc in primary cells inhibits the accumulation of p15INK4b that is associated with cellular senescence; conversely, deletion of c-myc in an established cell line activates p15INK4b expression. Alleles of c-myc that are unable to bind to Miz-1 fail to inhibit accumulation of p15INK4b messenger RNA in primary cells and are, as a consequence, deficient in immortalization.

Association of Myc with the zinc-finger protein Miz-1 defines a novel pathway for gene regulation by Myc.

The Myc protein activates transcription as part of a complex with its partner protein, Max. Myc-transformed cells are also characterised by the loss of expression of a number of genes and this repressive effect of Myc on gene expression may not be mediated by the Myc/Max complex. We recently isolated by two-hybrid cloning a novel zinc-finger protein that associates with the carboxy-terminus of Myc. We have termed this protein Miz-1 (Myc-interacting zinc finger protein). Some of the properties of Miz-1 suggest that it may be involved in gene repression by Myc in vivo.

Strep-tag II for one-step affinity purification of active bHLHzip domain of human c-Myc.

The c-Myc protein, the product of the c-myc protooncogene, is a nuclear phosphoprotein with DNA-binding properties when heterodimerized with the Max protein. It contains an amino-terminal transcriptional activation domain and a carboxy-terminal basic helix-loop-helix leucine zipper (bHLHzip) domain that directs heterodimerization and promotes DNA binding. Here, we describe the isolation of the bHLHzip domain of human c-Myc with a technique for efficient single-step purification. Using a C-terminal Strep-tag II affinity peptide and a novel Streptactin-Sepharose matrix, elution is performed under mild conditions by competition with the biotin analog desthiobiotin. No significant influence of the affinity tag on the activity of the bHLHzip domain was observed when the fusion protein was subjected to glutathione S-transferase (GST) pull-down assays for investigating its in vitro-binding properties with GST-Max. The use of the C-terminal Strep-tag II was shown to be more suitable for obtaining pure product fractions than use of the N-terminal GST affinity tag.

Effects of carbon dioxide pneumoperitoneum on cerebral hemodynamics in pigs.

Previous studies have shown that laparoscopic interventions are associated with increases in intracranial pressure. However, the consequences on cerebral blood flow (CBF) are unknown. This study investigates the effects of carbon dioxide (CO2) pneumoperitoneum on CBF in pigs. Ten pigs (weight, 20-26 kg) were anesthetized with 1.4% isoflurane and fentanyl (1 microg/kg per minute). Mechanical ventilation (fraction of inspired oxygen = 0.4) was set to maintain normocapnia (end-tidal CO2 tension = 35 mm Hg). Arterial and central venous catheters were placed for measurement of mean arterial blood pressure and central venous pressure. Bilateral internal carotid artery blood flow was measured using two transient time flow probes placed around both carotid arteries (with ligated external carotid arteries). Cortical and subcortical cerebral blood flow was measured using laser Doppler flowmetry. Sagittal sinus pressure was measured via a superior sagittal sinus catheter. After baseline measurements, the peritoneal cavity of the animals was insufflated with CO2 to achieve an intraabdominal pressure of 12-mm Hg. After 10 minutes of stable CO2, pneumoperitoneum measurements were repeated. Increases in central venous pressure (6.3 +/- 2.1 to 11.1 +/- 3.0 mm Hg) and sagittal sinus pressure (8.0 +/- 2.8 to 11.9 +/- 3.0 mm Hg) were noted during CO2 pneumoperitoneum (P < .05). Bilateral internal carotid artery blood flow (46.0 +/- 7.4 vs 47.7 +/- 7.1 mL/100g per minute), cortical CBF (263 +/- 115 vs 259 +/- 158 tissue perfusion units), and subcortical CBF (131 +/- 145 vs 133 +/- 149 tissue perfusion units) did not change during CO2 pneumoperitoneum. The current data show that CO2 pneumoperitoneum increases sagittal sinus pressure without changing CBF. Increases in sagittal sinus pressure are likely related to decreases in cerebral venous drainage caused by increases in intraabdominal pressure.

The effects of fentanyl and sufentanil on cerebral hemodynamics.

Our study investigated the effects of moderate doses of fentanyl and sufentanil versus high-dose sufentanil on cerebral hemodynamics by using transcranial Doppler ultrasonography (TCD). Thirty American Society of Anesthesiologists (ASA) II and III patients scheduled for elective coronary artery bypass graft (CABG) were studied after Institutional Review Board (IRB) approval and informed consent. The evening before surgery, all patients received oral flurazepam (1 mg/kg), Atropine (0.4 mg/70 kg s.c.) and a combination of droperidol (70 micrograms/kg s.c.) plus fentanyl (1.5 micrograms/kg s.c.) were given as preanesthetic medication 1 h before induction of anesthesia. Anesthesia was induced with either 25 micrograms/kg fentanyl i.v. (group 1, n = 10), 3 micrograms/kg sufentanil i.v. (group 2, n = 10) or 6 micrograms/kg sufentanil i.v. (group 3, n = 10). All patients received 100 micrograms/kg pancuronium i.v. With the induction of respiratory depression, assisted ventilation was performed followed by controlled ventilation to maintain normoxia and normocapnia (FiO2, 1.0). Cerebral blood flow velocity (CBFV, cm/s) was measured continuously in the middle cerebral artery by using a bidirectional 2-MHz TCD system. Monitoring included heart rate (HR, beats/min), direct mean arterial blood pressure (MAP, mm Hg), and PaCO2. Physiologic variables including arterial blood gases were measured at baseline, 5 min, and 10 min after infusion of fentanyl or sufentanil. In all patients, HR, MAP, end-tidal carbon dioxide tension (PetCO2), and PaCO2 were constant over time and did not differ between groups. CBFV did not change with moderate doses of fentanyl (group 1) or sufentanil (group 2). In contrast, infusion of high-dose sufentanil (group 3) was associated with 27 to 30% decreases in CBFV (p < 0.05). Our results suggest that sufentanil decreases CBFV in a dose-related fashion with a threshold effect. Increases in CBFV and CBF seen in previous studies may be related to an increasing PaCO2 when maintenance of normocarbia is based on only real-time capnography with a constant PetCo2 rather than additional arterial blood gas monitoring.

Jugular bulb oxygen saturation and middle cerebral blood flow velocity during cardiopulmonary bypass.

This study investigates changes of jugular bulb oxygen saturation (SjO2) measured by fiberoptic jugular bulb oximetry and changes of intracranial hemodynamics using transcranial Doppler sonography (TCD) during cardiopulmonary bypass (CPB) for coronary artery bypass graft (CABG) in 17 ASA III patients. Anesthesia was maintained with fentanyl, midazolam, and continuous infusion of etomidate. Hypothermic CPB (27 degrees C) was managed according to alpha-stat conditions. SjO2 (%) was measured by a fiberoptic catheter (Opticath F 5.5; Abbott Critical Care Systems) placed in the right jugular bulb via the right internal jugular vein. Mean blood flow velocity (Vmean, cm/s) was measured in the middle cerebral artery using a bidirectional 2-MHz TCD system (Transpect, Medasonics). Data were recorded continuously from the beginning to the end of the CPB. During cooling and hypothermia (27 degrees C); SjO2 and Vmean did not change compared with values at the start of CPB. However, with the beginning of rewarming, Vmean was increased 65% compared with stable hypothermia (27 degrees C). This increase in Vmean was associated with a 25% decrease in SjO2. Maximum desaturation occurred at a 36 degrees C jugular bulb temperature. During cooling and stable hypothermia, global oxygen balance and intracerebral perfusion seemed to be maintained. However, a major alteration in the balance of the cerebral oxygen supply and demand may occur in response to rewarming despite increases in Vmean. Findings suggest inadequate increases in CBF to meet cerebral metabolic demand. Further investigations need to validate these findings with biochemical techniques and neuropsychological tests.

Use of chemostat for selection ofStreptomyces chrysomallus mutants altered in the induction of D-glucose isomerase.

D-glucose isomerase ofStreptomyces chrysomallus PL45 is inducible by D-xylose only. In mutants obtained by means of a selection procedure in a chemostat the isomerase was induced in xylose-free medium containing glucose as carbon source.

Xylose catabolism by a mutant ofStreptomyces chrysomallus altered in the regulation of D-glucose isomerase.

A mutant ofS. chrysomallus with a D-glucose isomerase inducible by D-glucose or its catabolites was characterized. In contrast to the wild-type strain it showed a decreased catabolite repression by D-glucose of D-xylose consumption and a constitutive pentose phosphate pathway as well. A hypothesis concerning altered induction pattern of its D-glucose isomerase is discussed.

Self-monitoring, self-administration of token reinforcement, and goal-setting to improve work rates with retarded clients.

Although many studies have demonstrated the effectiveness of self-regulation strategies with non-retarded populations, relatively few studies have examined their value for retarded workers in vocational settings. A Self-Regulation Package (SRP), which incorporated self-monitoring, self-administration of reinforcement, and goal-setting procedures, was investigated as a strategy for increasing the productivity of sheltered workshop clients. A combined multiple-baseline, multi-element, reversal-to-baseline design was used to evaluate the SRP. As a function of the presence of the SRP, production of the 8 clients increased by an average of 43% (range: 19-60). Social validation procedures revealed that clients preferred to work under the SRP conditions versus baseline conditions. Since many workshops for retarded persons have client/staff ratios which do not readily permit staff to undertake additional duties, the adoption of self-regulation strategies could represent an effective and acceptable means of assessing and improving individual rates of production.

Fabrication and evaluation of a sequence-specific oligonucleotide miniarray for molecular genotyping.

PURPOSE: Molecular genotyping relies on the identification of specific microbial DNA sequences. Accurate genotyping not only requires discrimination between low- and high-risk pathogens for effective diagnosis or disease management but also requires the identity of the specific strain or type of the microbe involved in pathogenesis. The majority of these assays require DNA amplification followed by genome identification either through sequencing or hybridization to specific oligonucleotide probes. We evaluated the use of a DNA microchip assay as a simple and easy-to-use procedure for genotyping. METHODS: Various methodological parameters were optimized for single-base mismatch discrimination on a DNA microarray. The fabrication procedures involved substrate chemistry for immobilization. The effect of various buffers and features associated with oligonucleotide sequences were standardized. The assay was evaluated on a low-density genotyping chip containing the sequences of various (Human Papilloma Virus) HPV subtypes. RESULTS: The specific subtype was identified with high specificity by hybridization in miniaturized condition. CONCLUSIONS: The DNA microchip provides a rapid and cost-effective genotyping procedure for microbial organisms and can be implemented easily in any laboratory.

Arsenical resistance of growth and phosphate control of antibiotic biosynthesis in Streptomyces.

Twenty-six wild-type Streptomyces strains tested for resistance to arsenate, arsenite and antimony(III) could be divided into four groups: those resistant only to arsenite (3) or to arsenate (2) and those resistant (8) or sensitive (13) to both heavy metals. All strains were sensitive to antimony. The structural genes for the ars operon of Escherichia coli were subcloned into various Streptomyces plasmid vectors. The expression of the whole ars operon in streptomycetes may be strain-specific and occurred only from low-copy-number plasmids. The arsC gene product could be expressed from high-copy plasmids and conferred arsenate resistance to both E. coli and Streptomyces species. The ars operon expressed in S. lividans and the arsC gene expressed in S. noursei did not render the synthesis of undecylprodigiosin and nourseothricin, respectively, phosphate-resistant. In addition in wild-type strains of Streptomyces phosphate sensitivity of antibiotic biosynthesis did not show strong correlation with resistance of growth to arsenicals.

Control of the Myc-Max mediated transactivation in yeast by natural promoter elements.

Transcriptional activation studies involving the human oncoprotein and transcription factor Myc and its helix-loop-helix partner protein Max in mammalian cells are critical due to the presence of endogenous Myc and Max proteins. Here we show that co-expression of the human c-myc and max genes from 2micro circle derived high copy number vectors in yeast cells stimulate the transcriptional activation of a LacZ reporter gene fused to the yeast cytochrome-c1 oxidase minimal promoter containing the adenovirus major late promoter element (AMLPE). The exchange of the single Myc binding site in the AMLPE by the two E-box DNA motifs (CACGTG) present in the Myc responsive element of a human Myc target gene (ornithine decarboxylase) in front of a promoter-reporter gene cassette results in a two-fold enhanced beta-galactosidase expression. Low expression of max and high level expression of c-myc at the same time led to a further enhancement of transcriptional activation from this promoter-reporter gene cassette.

Freeze-fracture observations of Corynebacterium glutamicum: the occurrence of an outer membrane-like structure and the influence of temperature on the cytoplasmic membrane.

In order to elucidate temperature-dependent morphological changes in the cell envelope of the Gram-positive L-lysine-overproducing Corynebacterium glutamicum 9366 we carried out freeze-fracture investigations of native cells and studied their fatty acid composition. In addition to the cytoplasmic membrane C. glutamicum possesses at the periphery of the cell an additional fracture plane which is unusual in Gram-positive bacteria and is designated here as outer membrane-like structure. The fracture faces of this layer display a distinguishable appearance in several regions of the cell. Bacteria grown at various temperatures showed changes in the relation of the saturated to unsaturated fatty acids. We demonstrated that the cytoplasmic membrane was affected by these changes in the fatty acid composition.

[Validity of fiber optic cerebral vein oximetry during extracorporeal circulation]. / Validität der fiberoptischen zerebrovenösen Oxymetrie während der extrakorporalen Zirkulation.

INTRODUCTION: This study investigates the accuracy of continuous jugular bulb venous oximetry during different conditions of hypothermic cardiopulmonary bypass (CPB) (27 degrees C) for coronary artery bypass graft. METHODS: 38 ASA III patients were studied following Ethical Care Committee approval and informed consent. Patients were anaesthetized with fentanyl, midazolam, continuous infusion of etomidate and pancuronium. Ventilation was performed with oxygen in air. CPB was managed according to alpha-stat conditions under moderate hypothermia (27 degrees C). SjO2 (%) and jugular bulb temperature (degree C) were measured by a fiberoptic thermodilution catheter (Opticath F 5.5, Abbott Critical Care Systems) placed in the jugular bulb via the internal jugular vein. Appropriate catheter position was x-ray controlled prior to the measurements. The fiberoptic data were compared to co-oximetric data of blood samples after induction of anaesthesia, 2 min following start of CPB, during cooling, stable hypothermia and rewarming of CPB. STATISTICS: Assessing of agreement (Bland/Altman and Bartko). RESULTS: Jugular venous oximetry correlated closely with the co-oximeter determinations after induction of anaesthesia. However, following start of CPB accuracy was decreased. During cooling, stable hypothermia and rewarming oximetric data correlated well with co-oximetry, however, over-estimating the SjO2 by 1.4 to 2%. CONCLUSION: The present data show that continuous jugular bulb venous oximetry is accurate and reliable for continuous SjO2 monitoring during hypothermic CPB for cardiac surgery. Induction of CPB and hemodilution affect accuracy slightly, but changes are well detected. Before clinical intervention SjO2 should be confirmed by laboratory co-oximetry.

ATP levels in phosphate-deregulated and arsenate-resistant mutants of Streptomyces noursei.

Mycelial levels of ATP and glucose-6-phosphate were investigated in mutants of streptothricin-producing S. noursei JA 3880b differing from the wild-type strain in antibiotic formation, in the control by inorganic phosphate of the secondary metabolism, and in the resistance to growth inhibition by toxic arsenate ions. As compared with the ancestral strain, mutants exhibited a lower content of ATP in the mycelium while addition of 0.1 M arsenate to growing cultures provoked only moderate changes in the level of this high-energy metabolite. The results suggest that there exists a correlation between growth resistance to arsenate and insensitivity to phosphate inhibition of the secondary metabolism, on the one hand, and the capacity to produce streptothricin-type antibiotics, on the other.

[Septic encephalopathy]. / Die septische Enzephalopathie.

Impaired mental function, from clouding of consciousness to deep coma is often seen in patients with systemic inflammation. Diagnosis of this syndrome which is called "septic encephalopathy" is dependent on exclusion of other causes. The underlying mechanisms have only been defined in parts. The appearance of cerebral symptoms during an infection increases mortality. Primary symptoms of septic encephalopathy appear early, before other septic organ manifestations become apparent. The most sensible parameter for diagnosis of septic encephalopathy in comatose patients or under sedation is the EEG. It shows general alterations which increase parallelly to the severity of septic encephalopathy. Septic encephalopathy has to be considered a multifactorial event. In an early stage of the development of septic encephalopathy, bacteremia induces overproduction of cytokines and other mediators. This causes metabolic dysregulation with effects on the cerebral protein-, glucose and neurotransmitter metabolism. In addition, cytokines damage the blood-brain-barrier and exert direct cytotoxic effects. This results in histologic detectable neuronal damage. Further effects of the cytokine expression are perivascular edema and hemorrhage. The loss of metabolic regulation of the brain perfusion and local cerebral ischemia additionally contribute to the etiology of septic encephalopathy. A specific therapy is not yet known.

Nucleotide sequence analysis of five putative Streptomyces griseus genes, one of which complements an early function in daunorubicin biosynthesis that is linked to a putative gene cluster involved in TDP-daunosamine formation.

Sequence analysis of the lkmB region of the daunorubicin biosynthetic gene cluster of Streptomyces griseus JA3933 revealed two contiguous open reading frames (ORF) in the same orientation, and three ORFs in the opposite orientation together extending over a 4.6 kb region adjacent to a homologue of the S. peucetius dnrJ gene. ORF1 complemented in trans the lkmB mutation, which seems to affect an early step in daunorubicin biosynthesis. Its deduced product showed no similarity to any known enzyme in the databases. The mutation in ORF1 was localised to a C-T transition at position 1172, leading to the change from a glycine to aspartic acid in the deduced protein. The lack of any homology to known polyketide synthesis enzymes indicates a regulatory role for the product of ORF1, despite the ability of lkmB mutants to further metabolise alkanoic acid. The genes of the oppositely oriented cluster seem to be involved in sugar metabolism. The putative ORF3 protein revealed strong homology to eukaryotic acyl CoA dehydrogenases and might encode an enzyme for the oxidoreduction preceding the introduction of the amino group into daunosamine, and the ORF4 protein is homologous to several epimerases, central enzymes in the formation of the L-2,3,6-trideoxy-3-aminohexoses from TDP-D-glucose. ORF5 seems also to be related to enzymes metabolising nucleotide-activated hexoses.

[Effect of phosphate on the biosynthesis of tentoxin by Alternaria alternata (Fr.) Keissler]. / Einfluss von Phosphat auf die Bildung von Tentoxin durch Alternaria alternata (Fr.) Keissler.

Alternaria alternata (Fr.) Keissler produces a phytotoxic substance tentoxin. The influence of inorganic phosphate on the formation of this secondary metabolite was analyzed. Distinct phases of growth and metabolite formation can be defined. The first phase shows exponential growth, high QO2, protein and nucleic acid values and a rapid uptake of inorganic phosphate from the medium. The second phase shows linear growth and active tentoxin formation takes place. The highest yields of tentoxin are obtained, when inorganic phosphate in the medium was limited. The phosphate level also influences the ATP-pool of the mycelium. The role of ATP as an effector in phosphate mediated control of tentoxin synthesis was discussed.

Mg-chelatase of tobacco: the role of the subunit CHL D in the chelation step of protoporphyrin IX.

The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway. The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H). We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions in vitro and in vivo between the central subunit CHL D and subunits CHL I and CHL H. We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein-protein interaction and enzyme activity. Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity. In addition, it could be demonstrated that CHL D is capable of forming homodimers. Moreover, it interacted with both CHL I and CHL H. Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process.

Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I.

Mg-protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chID, bchH/chIH and bchI/chII encode proteins which are required for in vitro Mg-chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchI have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline-rich region flanked by a highly acid-rich segment. Protein-protein interaction among the three subunits CHL D, H and I was studied using the yeast two-hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg-chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X-Gal-containing agar plates. In vitro Mg2+ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg-chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.