Improved limits on nu(e) emission from mu+ decay.

We investigated mu(+) decays at rest produced at the ISIS beam stop target. Lepton flavor (LF) conservation has been tested by searching for nu(e) via the detection reaction p(nu(e),e(+))n. No nu(e) signal from LF violating mu(+) decays was identified. We extract upper limits of the branching ratio (BR) for the LF violating decay mu(+)-->e(+)+nu(e)+nu(-) compared to the standard model (SM) mu(+)-->e(+)+nu(e)+nu(mu) decay: BR<0.9(1.7) x 10(-3) (90% C.L.) depending on the spectral distribution of nu(e) characterized by the Michel parameter rho=0.75(0.0). These results improve earlier limits by one order of magnitude and restrict extensions of the SM in which nu(e) emission from mu(+) decay is allowed with considerable strength. The decay mu(+)-->e(+)+nu(e)+nu(mu) often proposed as a potential source for the nu(e) signal observed in the LSND experiment can be excluded.

N-terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo.

Most tumor cells depend upon activation of the ribonucleoprotein enzyme telomerase for telomere maintenance and continual proliferation. The catalytic activity of this enzyme can be reconstituted in vitro with the RNA (hTR) and catalytic (hTERT) subunits. However, catalytic activity alone is insufficient for the full in vivo function of the enzyme. In addition, the enzyme must localize to the nucleus, recognize chromosome ends, and orchestrate telomere elongation in a highly regulated fashion. To identify domains of hTERT involved in these biological functions, we introduced a panel of 90 N-terminal hTERT substitution mutants into telomerase-negative cells and assayed the resulting cells for catalytic activity and, as a marker of in vivo function, for cellular proliferation. We found four domains to be essential for in vitro and in vivo enzyme activity, two of which were required for hTR binding. These domains map to regions defined by sequence alignments and mutational analysis in yeast, indicating that the N terminus has also been functionally conserved throughout evolution. Additionally, we discovered a novel domain, DAT, that "dissociates activities of telomerase," where mutations left the enzyme catalytically active, but was unable to function in vivo. Since mutations in this domain had no measurable effect on hTERT homomultimerization, hTR binding, or nuclear targeting, we propose that this domain is involved in other aspects of in vivo telomere elongation. The discovery of these domains provides the first step in dissecting the biological functions of human telomerase, with the ultimate goal of targeting this enzyme for the treatment of human cancers.

Comprehensive evaluation of community-based diabetic patients: effect of feedback to patients and their physicians: a randomized controlled trial.

OBJECTIVE: To demonstrate improvements in diabetes care stimulated by comprehensive evaluation of community-based diabetic patients with feedback to the patients and their physicians. RESEARCH DESIGN AND METHODS: A comprehensive evaluation of community-based diabetic patients with annotated reporting of results to both patients and their physicians (universal intervention) was followed by random assignment of 50% of patients to individual counseling (randomized intervention). In four communities, two large and two small, 55 type 1 and 376 type 2 diabetic patients were recruited, evaluated, and reassessed at 1 year. Outcome measures were HbA1c, serum cholesterol, and systolic and diastolic blood pressure. RESULTS: There were significant improvements in all outcome measures for type 2 diabetic patients randomized to individual counseling (P = 0.03; follow-up rate 84%) and significant improvements in all outcome measures for all high-risk type 2 patients (highest P value = 0.004; follow-up rate 85%). CONCLUSIONS: Comprehensive evaluation of diabetic patients at the community level with annotated reporting of results to the patients and their physicians was associated with improvement of mean HbA1c, cholesterol, and systolic and diastolic blood pressure, particularly in patients in high-risk status for these outcome variables. Individual counseling of 50% of patients, randomly selected, enhanced these results.

Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density.

Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.

Evaluation of an activated patient diabetes education newsletter.

This study evaluated a monthly, activated patient newsletter sent to over 7000 patients in Michigan with diabetes. The newsletter provided concise and action-oriented information about diabetes care. Patients who had signed up to receive the newsletter during the first 4 months of the project (1863) were surveyed to determine how many patients found the newsletter helpful; 80% (1498) of the patients replied. Patients who found the newsletter most helpful were older; had lower incomes, and reported more complications, less understanding of diabetes, and being in poorer overall health. They also were more likely to have non-insulin-dependent diabetes mellitus (NIDDM) than insulin-dependent diabetes mellitus (IDDM). We concluded that the activated patient newsletter is a useful public health/patient education intervention for persons with diabetes. Such a newsletter should be part of a coordinated system of ongoing patient care, education, screening, and social and psychological support.

Influence of stimulation parameters on auditory stimulus processing in schizophrenia and major depression: an auditory evoked potential study.

The influence of stimulation frequency and stimulus intensity on the auditory evoked potential components N1 and P2 was investigated in schizophrenic and major depressive patients. The findings in the patients were compared with those in normal controls. At a high stimulation frequency the amplitude of N1 was enhanced in both schizophrenic and major depressive patients; the latency of N1 increased only in the schizophrenic patients. These changes may be related to impairments of auditory input control and processing in these diseases. In the schizophrenic patients, P2 latency was prolonged under treatment with high-potency neuroleptic drugs.

Ultrastructural localization of rRNA in HeLa cells, rat liver cells and Xenopus laevis oocytes by means of the monoclonal antibody--protein a--gold technique.

The postembedding localization of rRNA was investigated in ultrathin sections of HeLa cells, rat liver and Xenopus laevis oocytes by means of the monoclonal antibody to rRNA and protein A-gold technique. The incidence of gold particles was highest in nucleoli and cytoplasmic areas containing ribosomes. The chromosomes were labelled less than the surrounding cytoplasm in mitotic HeLa cells. In nucleoli of HeLa cells and rat hepatocytes, the labelling of areas containing ribonucleoprotein components was greater than the labelling of fibrillar centres. In segregated nucleoli of X. laevis oocytes, the labelling of the granular region substantially exceeded that of the fibrillar regions. The incidence of nucleoplasmic gold particles in interphasic HeLa cells was found to be slightly increased in the vicinity of nucleoli. The labelling of clusters of interchromatin granules in rat hepatocytes was not significantly different from that of the rest of the nucleophasmic interchromatin spaces.

Actin and RNA are components of the chromatoid bodies in spermatids of the rat.

Chromatoid bodies present in spermatocytes and spermatids of the rat show directed movements around spermatid nuclei during differentiation. This transient organelle contains RNA and establishes contact to intranuclear material and to the acrosomal complex. In order to determine possible components of motility and to verify the presence of RNA, we used a recently developed low-temperature embedding resin combined with protein A-gold and enzyme-gold techniques for studies at the ultrastructural level. All chromatoid bodies analyzed display high concentrations of gold particles over the electron-dense regions when labeled with antiactin. In contrast, RNase-gold particles were localized mainly in the electron-translucent areas. Corresponding controls were always negative. The results suggest a relationship between the impressive motility of the chromatoid body and actin present in the organelle. In addition, specific localization of RNA supports earlier findings that consider the chromatoid body an essential element for differentiation during spermiogenesis.

Immunocytochemical localization of cytoskeletal proteins and histone 2B in isolated membrane-depleted nuclei, metaphase chromatin, and whole Chinese hamster ovary cells.

Immunocytochemical techniques employing protein A-gold labeling were used to locate actin, tubulin, and histone 2B in thin sections of embedded, isolated membrane-depleted nuclei, metaphase chromosomes, and whole Chinese hamster ovary (CHO) cells. Actin and tubulin were detected in significant amounts in the cytoplasm and the nucleus of whole cells. The isolated membrane-depleted nuclei and metaphase chromosomes showed levels of actin and tubulin labeling either comparable to or sometimes lower than the levels of labeling in whole interphase cells. The results suggest that actin and tubulin are normal components of nuclei throughout the cell cycle. A mouse monoclonal antibody to histone 2B gave relatively weak labeling of nuclei and chromosomes in whole cells but intense labeling of isolated nucleoids and chromosomes. An increased concentration of antibody at the periphery of interphase nucleoids was noted.

Dehydration and embedding temperatures affect the antigenic specificity of tubulin and immunolabeling by the protein A-colloidal gold technique.

Qualitative and quantitative tests were performed to determine whether the temperature at which dehydration and embedding occur affects the antigenic specificity of tubulin and the protein A-gold (pAg) immunolabeling technique. The analysis indicates that low temperature (-35 degrees C) treatment increased the specificity and density of pAg labeled anti-tubulin antibodies to Leishmania tropica subpellicular microtubules as compared to samples prepared at 0 degrees C or 20 degrees C.

Long-term prognosis and course of schizo-affective, schizophreniform, and cycloid psychoses.

From the Bonn study including 502 patients followed up an average 22.4 years after onset of schizophrenic disease diagnosed according to the criteria of K. Schneider, we selected 113 cases of schizo-affective, schizophreniform, and cycloid psychoses in accordance to the definitions given by Kasanin, Retterstøl, Angst, and Leonhard. These psychoses have a better prognosis than the whole sample: characteristic residues are seen more rarely, complete remissions and noncharacteristic residues more frequently. This group of psychoses differs from the whole sample in the hereditary taint, too: the morbidity risk with affective psychoses and with schizophrenic psychoses in first- and second-degree relatives is higher than in the total sample of the Bonn study. In spite of the better prognosis and other differences described in the paper, we believe that these results do not justify the classification of schizo-affective and related disorders as an independent disease group. Between these different subtypes of schizophrenia only a differential typology and not a differential diagnostic is possible.

Comparative studies on the structural organization of membrane-depleted nuclei and metaphase chromosomes.

Interphase membrane-depleted nuclei and metaphase chromosomes were prepared in parallel with a nonionic detergent lysis procedure at low ionic strength. By flow microfluorometry we showed for the first time that cell lysates contain all stages of the cell cycle in the same proportions as the starting cell population. Morphologically intact membrane-depleted nuclei and metaphase chromosomes were isolated as non-aggregated structures on sucrose gradients. When analysed in the electron microscope, membrane-depleted nuclei that had been treated with 2M NaCl appeared as residual structures containing the pore complex-lamina layer attached to a halo of DNA filaments. In contrast, no distinct high salt-resistant structure was found with metaphase chromosomes. They formed a highly fragile network which disintegrated easily into small complexes connected with DNA filaments. High salt-resistant DNA-protein complexes were purified by Metrizamide density gradient centrifugation. The main difference in the protein composition of interphase and metaphase residual complexes was the presence in interphase of a protein triplet in the 60-75 kilodalton molecular weight range and its absence in metaphase. This protein triplet most likely corresponds to the lamins A, B, and C of the nuclear lamina. The combined results suggest that the main difference in the structural organization of interphase nuclei and metaphase chromosomes is the presence or absence of the pore complex-lamina layer.

Ultrastructural localization of DNA in tissue culture cell nuclei by means of enzyme-gold and autoimmune sera-protein A-gold techniques.

The post-embedding localization of DNA was investigated in cell nuclei by means of DNase I-gold and autoimmune sera-protein A-gold techniques. Using the former technique, gold particles were found mainly over euchromatin, nucleoli exhibit occasionally high labeling. In the latter technique, the use of the serum binding dsDNA confined the label mainly to condensed chromatin.

Analysis of ring-shaped nucleoli in serially sectioned human lymphocytes.

Serial section analysis has demonstrated that ring-shaped nucleoli of mature human lymphocytes are spherical structures consisting of a peripheral ribonucleoprotein shell that surrounds one large fibrillar center. The shell exhibits usually one or, less frequently, two openings. The fibrillar center is in contact with the nucleoplasm and perinucleolar condensed chromatin, which frequently appears as a pedicle-like structure. Several chromocenters are associated with the ring-shaped nucleolus.

Biochemical and structural analyses of microtubules in the pellicular membrane of Leishmania tropica.

The structure of the major protein of the pellicular membrane of Leishmania tropica was investigated. This protein is composed of two polypeptides, of ca. 50,000 d molecular weight, that were found to cross-react immunologically with the alpha and beta subunits of pig brain tubulin. The polypeptides and pig brain tubulin subunits were partially digested with S. aureus V8 protease, and the peptides obtained analysis by SDS-polyacrylamide gel electrophoresis. A comparison of the patterns showed that the beta subunits of Leishmania and pig tubulin have very similar primary structures, while the alpha subunits have evolved divergently. These experiments demonstrate that the major polypeptides found in the pellicular membrane of L. tropica are alpha and beta subunits of tubulin. Immunoelectron microscopy indicates that the tubulin is located in the microtubules associated with the pellicular membrane of Leishmania. Arrays of microtubules were prepared by nonionic detergent treatment of the cells and observed by electron microscopy after negative staining. Optical diffraction reveals a 5 nm spacing between protofilaments in the microtubule and a 4 nm axial periodicity corresponding to the tubulin subunits. The pitch of the shallow left-hand three-start helix is 12 degrees. A distance of 47 nm separates each microtubule from the next. These data show that the dimensions and supramolecular organization of the tubulin subunits in the microtubules are identical in the pellicular membrane of L. tropica and in mammalian brain.