Organization of assembly factors Cbp3p and Cbp4p and their effect on bc 1 complex assembly in Saccharomyces cerevisiae

The bc₁ complex (complex III) of Saccharomyces cerevisae is composed of ten subunits that are assembled in the inner mitochondrial membrane. Cbp3p and Cbp4p are two mitochondrial proteins which are postulated to act as chaperones in bc₁ complex formation. Here, we show by blue native PAGE that cbp3 and cbp4 mutants are disturbed in complex III assembly and accumulate intermediate-sized forms of the complex. Moreover, deletion of CBP3 interferes with the formation of complex III/IV supracomplexes. Our studies show that Cbp3p and Cbp4p interact and are present in high-molecular-weight complexes, some of which might represent intermediates of complex III assembly. Overexpression of Cbp4p cannot substitute for the function of Cbp3p, but high-level expression of Cbp3p can partially compensate for the lack of Cbp4p. The finding that mitochondria of cbp3 and cbp4 mutants exhibit a wild-type lipid composition favors the idea that Cbp3p and Cbp4p are specific assembly factors for complex III rather than components of the mitochondrial lipid metabolism.     

Interactions of the RAD7 and RAD23 excision repair genes of Saccharomyces cerevisiae with DNA repair genes in different epistasis groups

Summary The RAD7 and RAD23 genes of S. cerevisiae affect the efficiency of excision repair of UV-damaged DNA. We have examined the UV survival of strains carrying the rad7 or rad23 deletion mutation in combination with deletion mutations in genes affecting different DNA repair pathways. As expected, the rad7 and rad23 mutations interact epistatically with the excision repair defective rad1 mutation, and synergistically with the rad6 and rad52 mutations that affect the postreplication repair and recombinational repair pathways, respectively. However, the rad7rad6 and the rad23rad6 mutants exhibit the same level of UV sensitivity as the radlrad6 mutant. This observation is of interest since, in contrast to the rad7 or the rad23 mutations, the rad1 mutant is very UV sensitive and highly excision defective. This observation suggests that RAD6 and RAD7 and RAD23 genes compete for the same substrate during DNA repair.     

Separating the direct effect of hypoxia from the indirect effect of changes in cardiac output on the maximum pressure difference across the tricuspid valve in healthy humans

In healthy humans, changes in cardiac output are commonly accommodated with minimal change in pulmonary artery pressure. Conversely, exposure to hypoxia is associated with substantial increases in pulmonary artery pressure. In this study we used non-invasive measurement of an index of pulmonary artery pressure, the maximum systolic pressure difference across the tricuspid valve (P_max), to examine the pulmonary vascular response to changes in blood flow during both air breathing and hypoxia. We used Doppler echocardiography in 33 resting healthy humans breathing air over 6–24 h to measure spontaneous diurnal variations in P_max and cardiac output. Cardiac output varied by up to ~2.5 l/min; P_max varied little with cardiac output [0.61±0.74 (SD) mmHg min l^–1]. Eight of the volunteers were also exposed to eucapnic hypoxia (end-tidal ) for 8 h. In this group P_max rose progressively from 21 mmHg to 37 mmHg over 8 h. By comparing diurnal variations in P_max during air breathing with changes in P_max during hypoxia in the same eight individuals, we concluded that only approximately 5% of the changes in P_max during hypoxia could be attributed to concurrent changes in cardiac output. The low sensitivity of P_max to changes in cardiac output makes it a useful index of hypoxic pulmonary vasoconstriction in healthy humans.     

Neutrophil elastase α1-proteinase inhibitor complexes in pleural effusions

Summary Polymorphonuclear (PMN) granulocyte derived neutrophil elastase (NE) is rapidly antagonized by ₁-proteinase inhibitor (₁ PI) in vivo. To determine the clinical value of elastase ₁-proteinase inhibitor complexes (E-₁ PI) in pleural effusions, fluid samples of 99 patients were examined. Fifty-six had malignant effusions, 30 had nonmalignant exudates (pleural protein above 3 g/dl) mainly of inflammatory origin, and 13 patients had low protein transudates (below 3 g/dl) due to congestive heart failure. Nonmalignant exudates showed significantly higher (P<0.001) concentrations of E-₁ PI compared with malignant effusions or low protein transudates (P<0.001). Malignant exudates secondary to lung cancer were characterized by higher (P<0.001) median pleural E-₁ PI concentrations compared to malignant exudates due to primarily extrathoracic malignancies. Total pleural leukocyte counts and pleural neutrophil counts were performed in 68 effusions. By this means no clear-cut differentiation between malignant and nonmalignant exudates seems possible except for marked empyema. In conclusion, E-₁ PI complexes in pleural fluid may better reflect the stage of inflammation of pleural effusions rather than mere pleural leukocyte counts. Low levels of E-₁ PI complexes (<75 ng/ml) in pleural exudates with protein values above 3 g/dl are characteristic of malignant exudates. Determination of E-₁ PI in pleural exudates may serve as a sensitive marker of inflammation and useful adjunct to pleural cytology in aspects of differential diagnosis of pleural effusions.Abbreviations E-₁ PI elastase ₁ proteinase inhibitor complexes - NE neutrophil elastase - PMN polymorphonuclear     

Symmetry, broken symmetry, and handedness in bimanual coordination dynamics

Summary The symmetrical dynamics of 11 rhythmic bimanual coordination may be specified by an order parameter equation involving the relative phase between rhythmic components, and an interlimb coupling which determines the relative attractiveness of in-phase and anti-phase patterns. Symmetry breaking of these dynamics can occur via the difference in the natural frequencies, , of the left and right rhythmic components, or by the intrinsic asymmetrical dynamics of the body. The latter is captured by additional terms that render the symmetrical coupling slightly anisotropic. A major prediction resulting from this step is that although =0, as the frequency of coordination is increased, the asymmetrical coupling will increase and the symmetrical coupling will decrease. This results in a greater left-limb bias in left-handers and right-limb bias in right-handers. This increased handedness prediction was confirmed in an experiment in which 20 left-handed and 20 right-handed individuals performed 1 1 coordination with hand-held rigid pendulums. Manipulations of left and right pendulum lengths controlled , and the coupled frequency was determined by a metronome. Also confirmed was the prediction that the small shift in equilibria from in-phase and anti-phase due to the intrinsic asymmetry should be amplified in left-handers when > 0 and in right-handers when < 0. Further, the bias in left-handers was more consistent than the bias in right-handers, and a subgroup of right-handers was identified who performed similarly to left-handers. The coordination dynamics of functional asymmetry provides insights into the elementary synergy between the limbs, the dynamical mechanism that modulates it, and the nature of the asymmetry in left-handed and right-handed individuals.     

Transient Exposure of Human Bronchial Epithelial Cells to Cytokines Leads to Persistent Increased Expression of ICAM-1

Effects of several cytokines on kinetics of Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) expression were studied on a bronchial epithelial cell line (BEAS-2B). VCAM-1 was neither constitutively expressed on BEAS-2B cells nor induced by Interferon-gamma (IFN-), Tumor Necrosis Factor-alpha (TNF-), Interleukin-1beta (IL-1), IFN-, IL-4, IL-6, IL-8 or Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). ICAM-1 was constitutively expressed on BEAS-2B cells. IFN- and TNF- upregulated ICAM-1 expression on these cells. The functional importance of IFN- plus TNF- upregulation of ICAM-1 expression on BEAS-2B cells was demonstrated by neutrophil-BEAS-2B cell adhesion assays. Cytokines are rapidly released and cleared in animals. Therefore, transient cytokine(s) exposure might occur on the bronchial mucosa. Brief incubation of BEAS-2B cells with IFN- plus TNF- led initial upregulation of ICAM-1 expression followed by a protracted downregulation. Our findings stress the importance of studying the mechanism(s) controlling the persistent increased expression of ICAM-1 after brief cytokine(s) exposure.     

Structural and functional mitochondrial abnormalities associated with high levels of partially deleted mitochondrial DNAs in somatic cell hybrids

Kearns-Sayre syndrome (KSS) is a progressive and ultimately fatal human encephalomyopathy that is associated with large-scale deletions of mitochondrial DNA (mtDNA). To gain new insights into the developmental pathobiology of this disease, we studied the maintenance and expression of deleted mtDNAs (-mtDNAs) in somatic cell hybrids generated by fusion of HeLacot cells with a KSS fibroblast clone containing both wild-type and -mtDNAs. We observed that -mtDNAs were preferentially maintained over the KSS wild-type mtDNAs (wt-mtDNAs) in almost all isolated hybrid clones. Mitochondrial metabolism was not compromised in hybrids containing as much as 70–79% -mtDNAs. Two clones containing more than 99% -mtDNA were severely deficient in oxidative phosphorylation and exhibited abnormal, enlarged mitochondria. These clones had undetectable levels of mtDNA-encoded polypeptides, but contained normal amounts of a nuclear DNA-encoded mitochondrial protein. The data suggest a nonrandom pattern of mtDNA segregation in the triplasmic hybrids and a correlation among -mtDNA, structural mitochondrial abnormalities, and mitochondrial dysfunction.     

Processing of binaural stimuli by cat superior olivary complex neurons

Summary A method was developed to record stereotactically from the cat Superior Olivary Complex (SOC) using glass micropipettes. Sound stimulation was given through a closed system that permitted independent variation of interaural time (time) and intensity (int) differences. The most common binaural units found (n = 34) were ipsilateral excitatory, contralateral inhibitory (EI¹), cells of the Lateral Superior Olive (LSO). Some Medial Superior Olive (MSO) cells and presumed MSO ascending afferents were found but, as noted by other authors, we found it difficult to obtain single unit recordings from this nucleus. The LSO EI cells were mostly sensitive to higher frequencies and showed Peristimulus Time Histograms (PSTHs) consisting of a sharp On response followed by a plateau when stimulated with Best Frequency (BF) tone bursts or noise bursts. This On response was sensitive to time and int such that ipsilateral time lead or intensity increase resulted in a stronger response. The response reached a minimum around zero time or int. No sharp peaks or dips were seen in the physiological range needed for localization, instead the response increased with increasing ipsilateral lead or intensity to the maximum values tested (2048 s time, 30 dB int). In the physiological range the time and int response were complementary (both increasing response as ipsilaterality was increased). Provided enough sound energy in the unit's sensitive region was present, the same time curves were produced when BF tone bursts, masked tone bursts, sharp onset tone bursts or noise bursts were used. Changing the time of the carrier of the tone burst alone had no effect (except for one cell with a BF of 560 Hz), only the relative time of arrival of the stimulus envelope seemed to be important. In contrast to these LSO EI cells MSO-type units showed EI or EE predominantly low frequency phase-locked responses. When stimulated with interaurally phase shifted (pha) BF tones the unit response was a cyclic function of pha. Some cells (all that were tested, n = 6 including the 560 Hz LSO EI cell) showed these cyclic responses when stimulated with noise bursts or non-BF tones. However, these characteristic delays were not necessarily in the physiological range, i.e. we could find no evidence that these units were responding to time/pha values corresponding to a particular sound source direction. In both LSO and MSO it seems that integration of information higher in the CNS from a population of these cells is necessary for unambiguous coding of sound source direction. The time intensity trading ratios measured in two MSO type cells (11 and 26 /dB) were clearly different to those measured in LSO EI cells (n = 6, 99–550 s/dB). These ratios correspond approximately to those of the psychophysical time and int images measured by Hafter and Jeffress (1968).Supported by the Deutsche Forschungsgemeinschaft (SFB 45)     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Differential Regulation of IL-8 by IL-1β and TNFα in Hyaline Membrane Disease

Mechanisms that regulate cytokine-mediated inflammation in the lungs of preterm infants, including factors which regulate production of the chemokine IL-8, remain poorly defined. Sequential bronchoalveolar lavage samples were obtained from preterm newborns with hyaline membrane disease over a 28-day period. Bronchoalveolar lavage cell cytokine relationships were evaluated and the differential regulation of IL-8 by IL-1 and TNF was studied in a short-term culture system. In vivo, IL-8 and IL-l protein levels correlated closely with each other and with macrophage counts. In cell culture, exogenous anti-IL-1 antibody led to a 40% maximum inhibition (approximately) of IL-8 production by lipopolysaccharide stimulated lung inflammatory cells. Comparable amounts of exogenous anti-TNF antibodies achieved a 15% maximum inhibition (approximately) of IL-8 production. Anti-IL-1 and anti-TNF antibodies in combination did not inhibit IL-8 production beyond that achieved by anti-IL-l antibody alone. These results, in preterm newborns, support the concept of lung inflammation mediated in part by a macrophage, IL-1, and IL-8 cell cytokine pathway. The results also suggest that factors other than IL-1 and TNF regulate IL-8 expression in the lungs of preterm infants.     

Efficacy of the two-microelectrode voltage clamp technique in crayfish muscle

Crayfish muscle fibres of different dimensions were voltage clamped and white noise current was injected into the fibres at various distances from the voltage clamp current electrode. The clamp current was measured and power spectral densities were calculated. This method revealed the efficacy of the voltage clamp in these fibres. In large fibres (l=1.8–2.0 mm; =100–180m) a space clamp was achieved only for a band width f=40Hz. At a distance of 100m from the clamp electrodes f was 250–500Hz. In fibres of medium size (l=1.0–1.3mm; =60–120m) f was about 80Hz and about 800 Hz at a distance of 100m. In experiments with very small muscle fibres (l=400–600m; =30–50m) f was more than 500Hz. The improvement of the space clamp for the smaller muscle fibres resulted mainly from the reduced total membrane capacity,c _m, of these fibres. The limitations of the space clamp could be derived from the impedance properties of the fibres. The band width of the space clamp correlated with the band width for which the square of the absolute impedance, |Z _p|², of the muscle fibre could be described by a simple RC-model. This correlation was demonstrated in a model circuit.Power density spectra of membrane current fluctuations were measured also. To optimize the resolution of these measurements the contribution of instrumental noise was minimized. The effects of instrumental noise are discussed.This investigation was supported by the Deutsche Forschungs-gemeinschaft     

A Matrix Associated Region Localizes the Human SOCS-1 Gene to Chromosome 16p13.13

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3 end of the human PRM1 PRM2 TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3 of the spermatid-expressed PRM1 PRM2 TNP2 domain at position 16p13.13.     

Indices for the role played by regional flow resistance in blood pressure reflex

Summary An index, = (r/r)/(R/R), is proposed for assessment of the relative degree of participation of a regional flow resistance in a blood pressure reflex, wherer andR are regional and total peripheral resistances respectively and 's refer to their reflexive changes. For actual computation, =I (iP – Pi)/i(IP – PI), whereI=cardiac output,i=regional flow rate,P=mean arterial pressure. When the change in cardiac output is neglegible, the above equation is simplified as =1 – Pi/iP. Another index, = g/G = (Pi – iP)/(PI – IP), is introduced for the degree of contribution of a region to a reflex, whereg andG are regional and total conductances.i = 1. Some examples of application of the indices are presented.Supported in part by a grant from the Ministry of Education of Japan.     

Synchronous appearance of fibronectin,integrin α5β1, vinculin and actin in epithelial cells and fibroblasts during rat tracheal wound healing

The distribution of integrin 51 (51) and associated components during wound healing was investigated in the rat trachea following mechanical injury. Under anesthesia, the ventral surface of the trachea was scratched, and tissue specimens were obtained from 6 h to 3 weeks after injury and studied using light and electron microscopy and immunohistochemistry. 51, vinculin and actin in regenerating epithelial cells and extracellular fibronectin appear virtually simultaneously after injury (from 12 h to 7 days) as do 51, vinculin and -smooth muscle actin in fibroblasts and cellular fibronectin in granulation tissue (from 3 to 10 days). Immunoelectron microscopy 2 days after injury showed that 51 and vinculin were localized on the basal and lateral surfaces of regenerating epithelial cells and fibroblast surfaces, and fibronectin was localized just under the regenerating epithelial cells, around collagen fibrils and sporadically around fibroblasts. Bromodeoxyuridine labeling showed that the appearance of these components was associated with the period of cell proliferation. The appearances of fibronectin, 51, vinculin and actin in regenerating epithelial cells and fibroblasts during tracheal wound healing are well coordinated. During the initial cell migration phase, plasma fibronectin may stimulate cell migration before cellular fibronectin is produced in situ, and regenerating epithelial cells appear to begin to migrate into the wound before cell proliferation starts.     

Ultrasonography gives directly but noninvasively elastic characteristic of human tendon in vivo

To obtain an insight into tendon elasticity during human movement, a real-time ultrasonography was applied to the contracting tibialis anterior muscle. The insertion point of fascicles onto the aponeurosis was clearly visualized, and its position relative to a fixed marker on the skin moved proximally (1) according to the increasing dorsiflexion force (F) with a fixed ankle joint. Notably, the 1 – F relationship in the tendon was found to be quadratic in nature (F = c1²; c=1.48 2.24, r=0.985 0.992, n=9) as has been reported in the isolated tendon, although the F – 1 curves were slightly underestimated in comparison with the stiffness constant estimated from tendon architecture. This underestimation might be caused by changes in the height of the foot arch with the application of force.     

Comparison of the eIF-2α Homologous Proteins of Seven Ranaviruses (Iridoviridae)

The -subunit of the eukaryotic initiation factor 2 (eIF-2) is a key component of the translation machinery of the cell. In response to cellular stress such as viral infections, eIF-2 is phosphorylated by double-stranded RNA-dependent protein kinase (PKR) leading to the inhibition of cellular protein synthesis. The importance of eIF-2 as a regulatory mechanism for protein synthesis is illustrated by the wide variety of strategies employed by viruses to down-regulate PKR. Thus, Vaccinia virus encodes K3L protein, which resembles eIF-2 and acts as a pseudo-substrate inhibitor of PKR. Nucleotide sequencing of the genome of epizootic haematopoietic necrosis virus (EHNV), a member of the genus ranavirus of Iridoviridae, has revealed an eIF-2 equivalent gene. We have cloned and sequenced eIF-2 genes of several iridoviruses of fishes and frogs. The eIF-2 open reading frames and deduced proteins of the iridoviruses investigated exhibit a high degree of homology of both nucleotide and amino acid sequences. At the N-terminus, the iridoviral eIF-2 shows significant homology to the N-termini of cellular initiation factor 2- of various species, to full-length poxviral eIF-2 proteins, and to the S1 domain of ribosomal proteins. Comparison of amino acid sequences of corresponding iridoviral proteins with eIF-2 homologous proteins of poxviruses and eukaryotes has revealed a high conservation of motifs. A phylogenetic analysis of eukaryotic eIF-2 and poxvirus and iridovirus eIF-2 sequences has demonstrated the relationship of these iridoviruses. In order to investigate the role of the eIF-2 equivalent, respective genes have been expressed in prokaryotic and eukaryotic (insect, fish and chicken cell) systems. The iridoviral eIF-2 protein has a molecular weight of 31 kDa and is cytoplasmic. The cellular and viral protein synthesis of iridoviruses is probably regulated by a mechanism similar to that of Vaccinia virus. Frog-virus 3, the type species of the genus ranavirus of Iridoviridae, has a unique translational efficiency and, moreover, down-regulates the cellular protein synthesis of infected cells.     

Poly ADP Ribose-Polymerase Inhibitors Prevent the Upregulation of ICAM-1 and E-selectin in Response to Th1 Cytokine Stimulation

We studied the role of poly-ADP-ribose polymerase (PARP) in the mobilization of ICAM-1, VCAM-1, and E-selectin by TNF- and IL-1 in cultured human endothelial cells. Enzyme linked immunosorbent analysis (ELISA) was used to assess if ICAM-1, VCAM-1, and E-selectin were expressed at the cell surface, and if PARP inhibition (using the selective PARP inhibitor GPI 6150) blocked the induced expression. Endothelial cell adhesion molecule expression was evaluated at 4 and at 24 h after cytokine stimulation. At 4 h ICAM-1 and E-selectin, but not VACM-1, were stimulated by both IL-1 and TNF-. Blocking PARP via GPI 6150 only affected TNF- induced E-selectin expression at 4 hours. ICAM-1, VCAM-1, and E-selectin expression were all stimulated by both IL-1 and TNF- in the 24 h assays. PARP inhibition with GPI 6150 blocked the IL-1 mediated stimulation of both ICAM-1 and E-selectin expression, and blocked TNF- stimulation of ICAM-1 expression at 24 h. These experiments suggest that specific PARP inhibition may provide a novel method of controlling leukocyte dependent inflammation through the reduction of ICAM-1 and E-selectin expression in endothelial cells in response to cytokines.     

The effect of an α-ketoglutarate-pyridoxine complex on human maximal aerobic and anaerobic performance

Summary The administration of 30 mg/kg of body weight of an -ketoglutarate-pyridoxine complex (-KG compl; stoichiometric ratio -KG: pyridoxine 46.35 to 53.65) to trained non-athletic individuals increases max by 6% (p<0.005). The kinetics of the on- and off-responses at the onset and offset of a rectangular work load is not affected by the drug. Peak blood lactate concentration [La_b] following two supramaximal running work loads lasting 60 s and 132±4 s, respectively is significantly (p<0.05 and p<0.005) less after the -KG compl treatment (La_b=–1.1 and –2.7 mmol·1^–1, respectively) than in a control group. The half time (t1/2) of La disappearance from blood during recovery is unaffected by the -KG compl treatment (19.7 min vs 19.5 min). The increase in max and the corresponding decrease of [La_b] are not found after separate administration of either of the components of the complex. It is concluded that -KG complex stimulates aerobic metabolism, probably prompting mitochondrial reabsorption of -KG, which activates the malate-oxalacetate shuttle and the generation of high energy phosphates at the substrate level.     

Psychophysics of psilocybin and 48-148-148-1

Visual, tactile and intermodal sensory magnitude estimations were performed by college students prior to and after the oral administration of either 160 g/kg psilocybin or 30 mg ⁹-THC. Although the drugs induce inversely related changes in the exponent of the psychophysical power function (R=kS ⁿ ), the straight line relationship between log S (Stimulus) and log R (Response) is altered by neither psilocybin nor ⁹-THC. The remarkable consistency of magnitude estimations in the face of a changing exponent and constant may be interpreted as a demonstration of state boundness (i.e., performance that is consistently changed and characteristic of a particular state of consciousness).     

Altered expression of the steroid bioconverting pathway in pAN 7-1 transformants of Cochliobolus lunatus

Summary The filamentous fungus C. lunatus converts progesterone mainly to its 11-hydroxy derivative. C. lunatus transformed with the plasmid pAN 7-1, which contains the E. coli hph gene expressed under the control of the A. nidulans gpd and trpC expression signals, lacks this activity, but exhibits acetyl side chain degradation of progesterone through the reaction scheme progesterone20-hydroxy-progesterone ⁴-androstene-3,17-dione testolactone+testosterone. The main partof this metabolic pathway is not expressed in the non-transformed strain. It was determined that the site-specific integration of the plasmid into the genome directly influences the expression of genes involved in the bioconversion of steroids.