An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichia coli.

Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (kcatNADH/KMNADH)/(kcatNADPH/KMNADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli, opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB.

Improving the nutritional value of Golden Rice through increased pro-vitamin A content.

"Golden Rice" is a variety of rice engineered to produce beta-carotene (pro-vitamin A) to help combat vitamin A deficiency, and it has been predicted that its contribution to alleviating vitamin A deficiency would be substantially improved through even higher beta-carotene content. We hypothesized that the daffodil gene encoding phytoene synthase (psy), one of the two genes used to develop Golden Rice, was the limiting step in beta-carotene accumulation. Through systematic testing of other plant psys, we identified a psy from maize that substantially increased carotenoid accumulation in a model plant system. We went on to develop "Golden Rice 2" introducing this psy in combination with the Erwinia uredovora carotene desaturase (crtI) used to generate the original Golden Rice. We observed an increase in total carotenoids of up to 23-fold (maximum 37 microg/g) compared to the original Golden Rice and a preferential accumulation of beta-carotene.

Radioimmunoassay studies of human apolipoprotein E.

This report describes the development and first applications of a sensitive and specific double antibody radioimmunoassay for human apoplipoprotein E (apoE). ApoE was purified from the very low density lipoproteins of hypertriglyceridemic patients by heparin-agarose affinity chromatography, DEAE-cellulose chromatography, and preparative polyacrylamide gel electrophoresis. The purified apoprotein had an amino acid composition characteristic of apoE and resulted in the production of monospecific antisera when injected into rabbits. The radioimmunoassay, which was carried out in the presence of 5 mM sodium decyl sulfate, had a working range of 0.8-12 ng. The withinassay coefficient of variation was 9% and the coefficient of variation for systematic between-assay variability was 3%. Prior delipidation of samples with organic solvents did not alter their immunoreactivity. In 26 normal volunteers, the mean plasma apoE concentration was 36 +/- 13 microgram/ml. Hyperlipidemic patients (n = 68) had higher mean apoE levels. A single patient with type III hyperlipoproteinemia had a plasma apoE level of 664 microgram/ml. The plasma apoE level was independently related to plasma cholesterol and triglyceride levels in a population of 108 normal and nonchylomicronemic hyperlipidemic patients. The multiple correlation coefficient for this relationship was 0.73. Thus, variation in plasma cholesterol and triglyceride concentrations described 53% of the variation in apoE concentrations in this population. The lipoprotein distribution of apoE was investigated by agarose column chromatography and ultracentrifugation of plasma. Agarose column chromatography demonstrated that all or nearly all plasma apoE is associated with lipoproteins. In plasma from normal volunteers and hypercholesterolemic patients, apoE was found in two discrete lipoprotein classes: very low density lipoproteins and a set of lipoprotein particles with size and density characteristics similar to HDL2. In hypertriglyceridemic patients, nearly all apoE was associated with the triglyceride-rich lipoproteins.

The St. Lawrence Island famine and epidemic, 1878­80: a Yupik narrative in cultural and historical context.

A collaborative study of the Smithsonian Institution's ethnology collections has inspired the narration of Alaska Native oral traditions, including Yupik Elder Estelle Oozevaseuk's re-telling (in 2001) of the story of Kukulek village and the St. Lawrence Island famine and epidemic of 1878­80. The loss of at least 1,000 lives and all but two of the island's villages was a devastating event that is well documented in historical sources and archaeology, as well as multiple Yupik accounts. Yupiget have transmitted memories of extreme weather, bad hunting conditions, and a wave of fatal contagion that swept the island. The Kukulek narrative, with origins traceable to the late nineteenth century, provides a spiritual perspective on the disaster's underlying cause, found in the Kukulek people's disrespect toward the animal beings that sustained them. This paper explores the cultural and historical contexts of this narrative, and contrasts it with Western perspectives.

Ventricular arrhythmias in rehabilitated and nonrehabilitated post-myocardial infarction patients with left ventricular dysfunction.

The incidence of ventricular arrhythmias in rehabilitated post-myocardial infarction (MI) patients with left ventricular dysfunction included in a long-term rehabilitation program was assessed and compared with that in similar patients who were not in such a program. Thirty-eight post-MI patients (2 to 19 years after the acute event) with ejection fraction < 40% were investigated by 48-hour Holter monitoring. They were divided into the following 3 groups: group I, 11 patients who underwent arm training for 60 months; group II, 11 patients who underwent calisthenics for 36 months; and group III, 16 patients who were not in any rehabilitation program; the age of the patients was 61 +/- 7, 61 +/- 6 and 61 +/- 9 years, respectively, (p = not significant). Ejection fraction at rest was 31 +/- 9 for group I, 29 +/- 7 for group II, and 29 +/- 7 for group III (p = not significant). There were no significant differences concerning the location of MI, and antiarrhythmic treatment received by patients from all groups. At the conclusion of 48-hour Holter monitoring, 2 blood samples were obtained for assessment of norepinephrine (at rest and after postural change). Quality of life was determined by a detailed questionnaire, including questions concerning social activity, life satisfaction and sexual function. After 36 and 60 months, an improvement in hemodynamic condition of patients in group I was noted. Quality of life was higher in the rehabilitated patients, with enhanced emotional stability, satisfaction with work and social life, and a high percentage of return to work (82 vs 40%).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo.

A clone containing a 6.4 kb Borrelia burgdorferi chromosomal DNA insert reacted only with sera from patients with Lyme disease and not with any normal human or rabbit sera. Restriction enzyme analysis indicated that this DNA fragment was located on the B. burgdorferi chromosomal map between rpoB and p22A; its direction of transcription was towards p22A. Sequence analysis suggests that LA006 encodes six proteins: three previously described immunodominant lipoproteins of the 39 kDa Bmp protein family, BmpA, BmpB and BmpC; a 51 kDa MgtE magnesium transporter protein; a 16 kDa protein kinase C inhibitor; and a 56 kDa protein with similarity to an uncharacterized Escherichia coli chromosomal open reading frame.

Cloning and DNA sequence analysis of bmpC, a gene encoding a potential membrane lipoprotein of Borrelia burgdorferi.

Immunoscreening of a lambda gt11 genomic library of Borrelia burgdorferi expressed in Escherichia coli permitted detection of a clone containing a partial sequence of a B. burgdorferi gene encoding a protein with significant homology to TmpC of Treponema pallidum. Subsequent cloning and DNA sequence analysis revealed an open reading frame encoding a protein with 353 amino acid residues. The open reading frame is preceded by putative promoter sequences and a ribosome binding site, and is initiated with a TTG. The putative protein shares 26% identity with TmpC, contains a signal peptidase II sequence, and is also homologous to the gene products of the recently described bmpA and bmpB of B. burgdorferi. This gene has been designated bmpC. Additional sequencing and restriction analysis indicate that it is located at approximately 400 kbp on the chromosomal map of B. burgdorferi, immediately upstream of bmpA and bmpB.

Prolonged PR interval associated with an abnormal frontal plane QRS axis as an electrocardiographic criterion of left ventricular function.

The aim of this study was to investigate whether, in patients who had had one or more myocardial infarctions, the electrocardiographic appearances of prolonged PR interval associated with an abnormal frontal plane QRS axis are correlated with the value of left ventricular ejection fraction. Seventy-three consecutive patients all of whom had had at least one myocardial infarction, were divided into 3 groups: Group A, 22 patients having on their electrocardiogram a prolonged PR interval and an abnormal frontal plane QRS axis; Group B, 15 patients having an abnormal QRS axis as a solitary conduction disturbance; and Group C, 36 patients without conduction abnormalities. The ejection fraction, determined in all patients by gated blood pool imaging, was found to be 30% +/- 8 (mean +/- 1 SD) in Group A, 53% +/- 21 in Group B, and 52% +/- 14 in Group C (P less than 0.001). An ejection fraction of less than 40% was found in 91% of patients in Group A, in 33.33% in Group B, and in 8.33% in Group C (P less than 0.01). Multiple segment ventricular wall contraction abnormalities were found in 50, 6.66, and 11.11%, respectively (P less than 0.001). It can be presumed, therefore, that in patients after one or more myocardial infarctions, this association of conduction abnormalities signifies a severe impairment of ventricular contractility and may identify a subgroup with a high prevalence of severely depressed ejection fraction.

Production and characterization of monoclonal antibodies to psittacine beak and feather disease virus.

Monoclonal antibodies specific for the virus that causes psittacine beak and feather disease (PBFD) were produced by fusing spleen cells from mice immunized with purified concentrated PBFD virus with mouse myeloma cell line Sp2/0. The resulting hybridomas were tested for reactivity against whole purified virus by an enzyme-linked immunosorbent assay (ELISA) system. Four clones, designated 15H8, 8E3, 11G12, and 2C3, were subcloned by limiting dilution. Isotyping indicated that clone 15H8 was secreting IgG, whereas the remaining clones secreted IgM. The secreted immunoglobulins were characterized by reactivity against purified PBFD virus using immunoblotting procedures, by immunohistochemical staining of virus-induced lesions in infected tissues, and by inhibition of PBFD virus agglutination of cockatoo erythrocytes. Antibodies secreted by clones 15H8 and 8E3 had the strongest activity against purified whole virus. Only immunoglobulin secreted by the clone 15H8 could be used to detect viral antigen in infected tissues. None of the monoclonal antibodies had hemagglutination-inhibition activity.

Holter monitoring for dizziness and syncope in old age.

Among 509 patients referred to our Institute for Holter monitoring, between 1st September, 1982-30th October, 1983, 28 patients aged 65-90 (mean 76) were referred for dizziness and syncope. There were 17 men and 11 women. Seven patients had a M.I. in their past, 4 angina pectoris, 5 hypertension, 4 aortic stenosis or aortic insufficiency or both, hemodynamically significant, one had mitral valve prolapse (MVP) and one transient ischemic attacks (TIA). In our series 16 out of 28 patients received digoxin and antiarrhythmic drugs (quinidine, propranolol, procainamide, Neo-gilurythmal, amiodarone), 2 of them digoxin and quinidine in full doses and one digoxin and amiodarone. Other drugs administered to our patients included Aldomin, Isordil, Lasix, aminophylin, cromoglycate etc. In 10 patients (35.7%) we found complex ventricular arrhythmias (7 with M.I., 3 patients of 4 with significant aortic valve lesion, 2 patients of 2 with left anterior hemiblock (LAH), 1 patient with MVP, 1 patient with TIA). In another 5 patients (17.8%) we found atrial fibrillation, fast rhythm (2 with chronic obstructive lung disease, 2 with hypertension and 1 in post M.I.) which explained their symptomatology. From our data we conclude that the pluripathology found in old age as well as the multimedication administered, cause a plurietiology of syncope, arrhythmias playing an important role in its determination, in this particular age group.

Mutual regression: altered states in the psychoanalytic situation.

Regression is one of the most controversial topics in the psychoanalytic literature, and disagreements continue regarding a number of propositions central to the issue. On the one hand are those who maintain a traditional view that analysis facilitates regression, through both its setting and its method. On the other are critics from diverse theoretical schools, Freudian, Kleinian, and interpersonal, who have argued that the setting does not, and that the analyst should not, induce or encourage regression. This paper reexamines the fundamentals of regression and relates the concept to the notion of altered states of consciousness. From a relational position, it is argued that patient and analyst mutually regulate regressive states in each other, and that this is an important aspect of the analytic process. One of the analyst's roles is to facilitate the patient's shift into the state best suited for the task of the moment and, often, to improve the patient's ability to internally and interpersonally regulate and move between various states of consciousness. The analyst's commitment to state control as a mutual endeavor helps safeguard the patient's autonomy.

Free association and changing models of mind.

The method of free association, like most major psychoanalytic concepts, was developed in and has been conceptually tied to a one-person, intrapsychic, drive psychology. Psychoanalysts who have retained the classical, drive-structure metapsychology have continued to emphasize the free association method, while those who have shifted to a relational, two-person, interactional or interpersonal point of view have often minimized or abandoned the free association method. This article explores this unfortunate dichotomy and examines the ways in which the free association method, stripped of its unnecessary tie to drive theory and reconceptualized within the context of a two-person psychology, remains a valuable clinical method. Free association, as a method, can be useful to psychoanalysts of all theoretical orientations as a reminder of Freud's most important discoveries, that is, of unconscious mental life, psychic determinism, psychic continuity, and meaning, as well as of the value and priority of careful, restrained, and disciplined listening to patients and of taking our lead from them. The method of free association, with the corresponding assumption of the analyst's evenly hovering attention, provides a fundamental context within which our personalities and theoretical biases, instead of being disruptions or impositions, become active and analyzable aspects of the analytic process.

Functional analysis of SPINDLY in gibberellin signaling in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-delta17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-delta17 phenotype but does not reduce rga-delta17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification.

Stressful life events and Rorschach content.

This study investigated the construct validity of Elizur's (1949) Rorschach Content Test anxiety score. The study compared the Rorschach protocols of subjects experiencing high and low life-stress conditions. The Rorschach test was administered to 56 college students, half men and half women, who were selected from a larger group on the basis of their extreme scores on a measure of stressful life events, the College Schedule of Recent Experience (Anderson, 1972). Subjects in the high stress condition responded to the Rorschach with significantly (p less than .05) more anxious content than did subjects in the low stress condition. The findings of the study add support to the construct validity of Elizur's anxiety score. Implications for future research are discussed.