Marangoni flow: an additional mechanism in boiling heat transfer.

Experimental results indicate that extensive liquid circulation can be established by surface tension gradients near bubbles attached to heated surfaces. This circulation can contribute significantly to the high rate of heat transfer observed under boiling conditions.

Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae.

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.

CSE1 and CSE2, two new genes required for accurate mitotic chromosome segregation in Saccharomyces cerevisiae.

By monitoring the mitotic transmission of a marked chromosome bearing a defective centromere, we have identified conditional alleles of two genes involved in chromosome segregation (cse). Mutations in CSE1 and CSE2 have a greater effect on the segregation of chromosomes carrying mutant centromeres than on the segregation of chromosomes with wild-type centromeres. In addition, the cse mutations cause predominantly nondisjunction rather than loss events but do not cause a detectable increase in mitotic recombination. At the restrictive temperature, cse1 and cse2 mutants accumulate large-budded cells, with a significant fraction exhibiting aberrant binucleate morphologies. We cloned the CSE1 and CSE2 genes by complementation of the cold-sensitive phenotypes. Physical and genetic mapping data indicate that CSE1 is linked to HAP2 on the left arm of chromosome VII and CSE2 is adjacent to PRP2 on chromosome XIV. CSE1 is essential and encodes a novel 109-kDa protein. CSE2 encodes a 17-kDa protein with a putative basic-region leucine zipper motif. Disruption of CSE2 causes chromosome missegregation, conditional lethality, and slow growth at the permissive temperature.

Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae.

Mutations in the ESP1 gene of Saccharomyces cerevisiae disrupt normal cell-cycle control and cause many cells in a mutant population to accumulate extra spindle pole bodies. To determine the stage at which the esp1 gene product becomes essential for normal cell-cycle progression, synchronous cultures of ESP1 mutant cells were exposed to the nonpermissive temperature for various periods of time. The mutant cells retained viability until the onset of mitosis, when their viability dropped markedly. Examination of these cells by fluorescence and electron microscopy showed the first detectable defect to be a structural failure in the spindle. Additionally, flow cytometric analysis of DNA content demonstrated that massive chromosome missegregation accompanied this failure of spindle function. Cytokinesis occurred despite the aberrant nuclear division, which often resulted in segregation of both spindle poles to the same cell. At later times, the missegregated spindle pole bodies entered a new cycle of duplication, thereby leading to the accumulation of extra spindle pole bodies within a single nucleus. The DNA sequence predicts a protein product similar to those of two other genes that are also required for nuclear division: the cut1 gene of Schizosaccharomyces pombe and the bimB gene of Aspergillus nidulans.

Ligands for the receptor tyrosine kinases hek and elk: isolation of cDNAs encoding a family of proteins.

Hek is a member of the eph subfamily of receptor tyrosine kinases whose members include elk, hek2, sek, eph and eck among others. Using a soluble form of hek consisting of the extracellular region of the receptor fused to the Fc domain of human IgG1 and an expression cloning strategy, we have isolated two different but related cDNAs from the human T-lymphoma line HSB-2 that encode ligands for hek. The cDNAs encode proteins of 238 and 201 amino acids (44% amino acid identity) that are anchored to the membrane by glycosylphosphatidylinositol (GPI)-linkage. The proteins encoded by these cDNAs are bound by hek with affinity constants of 2 x 10(8) M-1. These proteins also bind the elk tyrosine kinase receptor. These cDNAs are related to other cDNAs that we have recently isolated from a human placental library that encode ligands for both hek and elk and define an emerging family of ligands for eph-related kinases (LERKs).

Nucleocytoplasmic shuttling of the thyroid hormone receptor alpha.

The thyroid hormone receptor alpha (TR alpha) exhibits a dual role as an activator or repressor of gene transcription in response to thyroid hormone (T(3)). Our studies show that TR alpha, formerly thought to reside solely in the nucleus tightly bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. The finding that TR alpha shuttles reveals an additional checkpoint in receptor control of gene expression. Using Xenopus oocyte microinjection assays, we show that there are two coexisting mechanisms for nuclear entry of TR alpha. First, nuclear import of TR alpha (molecular mass 46 kDa) was not sensitive to general inhibitors of signal-mediated transport, indicating that TR alpha can enter the oocyte nucleus by passive diffusion. Second, when TR alpha was tagged with glutathione-S:-transferase, import of the fusion protein (molecular mass 73 kDa) was completely blocked by these inhibitors, demonstrating that an alternative, signal-mediated import pathway exists for TR alpha. Nuclear retention of TR alpha in oocytes is enhanced in the presence of T(3), suggesting that more intranuclear binding sites are available for the ligand-bound receptor. Using mammalian cells, we show that shuttling of green fluorescent protein (GFP)-tagged and untagged TR alpha is inhibited in both chilled and energy-depleted cells, suggesting that there is an energy-requiring step in the nuclear retention/export process. Nuclear export of TR alpha is not blocked by leptomycin B, a specific inhibitor of the export receptor CRM1, indicating that TR alpha does not require the CRM1 pathway to exit the nucleus. Dominant negative mutants of TR with defects in DNA binding and transactivation accumulate in the cytoplasm at steady state, illustrating that even single amino acid changes in functional domains may alter the subcellular distribution of TR. In contrast to TR alpha, nuclear export of its oncogenic homolog v-ErbA is sensitive to leptomycin B, suggesting that the oncoprotein follows a CRM1-mediated export pathway. Acquisition of altered nuclear export capabilities may contribute to the oncogenic properties of v-ErbA.

Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants.

Pichia pastoris is a yeast capable of expressing large amounts of some proteins. When expression vectors are introduced into P. pastoris, individual transformants typically express widely varying amounts of protein. Because clones expressing the highest level of protein occur infrequently during the transformation process, finding them can be very labor-intensive. We developed an immunological based filter screening method that rapidly detects transformants secreting large amounts of a heterologous protein. We have applied this method to the expression of a soluble trimeric form of CD40L, a molecule that regulates B-cell responses. Using this method, we identified transformants with one to 13 copies of the CD40L expression cassette. Maximum expression was obtained with clones containing eight or more copies of the expression cassette, and a clone with eight copies was selected for further analysis. High cell density fermentation of this clone using a mixed glycerol:methanol feed yielded 255 mg CD40L per liter of supernatant.

Designing proteins that work using recombinant technologies.

Therapeutic proteins have been engineered for a variety of purposes including reduced antigenicity, longer half-life, simplified process development, and increased affinity. Fusion proteins bring together functions from two different molecules creating therapeutics with completely novel activities. Protein engineering technologies have relied on rational design, directed evolution, DNA shuffling, RNA-peptide fusion, phage and ribosomal display methods to select out candidate protein forms with the desired therapeutic properties. Engineered site-specific pegylation and glycosylation strategies have improved circulation half-life, reduced immunogenicity and increased protein therapeutic stability. In this review we describe how protein engineering techniques have been used to select out, improve stability and clinical efficacy of protein therapeutics.

Measuring the fidelity of implementation of a mental health program model.

A fidelity index of program implementation for assertive community treatment (ACT) was developed. In Study 1, 20 experts rated the importance of 73 elements proposed as critical ACT ingredients, also indicating ideal model specifications for elements. Agreement among experts on ratings of importance was high (intraclass r = .98). In Study 2, a 17-item subset of the expert-identified critical ingredients was used to construct a fidelity index with three subscales: Staffing, Organization, and Service. Internal consistencies ranged from .50 to .72, with a .81 reliability for the total scale. Fidelity was linearly related to program "generations," suggesting "program drift." In 18 ACT programs, fidelity also was associated with measures of reduction in days in psychiatric hospitals. The correlation was significant for the total scale and for the Organization and Staffing subscales but not for the Service subscale.

A multisite study of client outcomes in assertive community treatment.

OBJECTIVE: This study examined outcomes of clients admitted to assertive community treatment programs simultaneously implemented at six sites in northeastern Indiana. METHODS: A total of 212 clients at risk for psychiatric rehospitalization were assessed at baseline and at six-month intervals for 18 months after admission to assertive community treatment programs. Data on rehospitalization, quality of life, and level of functioning were compared using t tests. Progressive improvement was also examined by linear trend analysis. RESULTS: Frequency of psychiatric hospitalization was reduced by one-third and the number of inpatient days by 50 percent after admission to the program. Improvements were progressive, with continued reductions over the 18-month period. Progressive improvements also occurred in quality of life as measured by both client and staff ratings. Case managers rated clients as having improved family and social support, increased self-reliance and independence, and improved daily living skills. Clients reported significantly more legal problems, which may have been an artifact of increased monitoring during treatment. A key element of the programs' success was the position of clinical coordinator, important functions of which are described. CONCLUSIONS: Results of this study provide support for wide-scale dissemination of assertive community treatment as an effective form of community care for persons with serious mental illness.

Prospective screening of substance dependence: the advantages of directness.

This study evaluated the ability of several subtle and direct alcoholism screening scales to identify DSM-III-R-defined substance dependence in a university setting. Study participants were university students (N = 495) who completed the substance use disorder modules of the Diagnostic Interview Schedule along with the Michigan Alcoholism Screening Test (MAST), the Substance Abuse Subtle Screening Inventory (SASSI), and five direct screening questions concerning the consequences of alcohol and drug use. Results indicated that the SASSI had no appreciable ability to identify the 57 study participants who met criteria for a substance use disorder. The MAST also demonstrated limited predictive ability, and results suggested that this is because many MAST items assess the more advanced features of alcoholism, features less common in the substance abuse found among university students. The five direct screening questions were modestly useful, and it is argued that there are advantages to directness when screening for substance use disorders.

Closing of a state hospital: an overview and framework for a case study.

This article introduces the trends in deinstitutionalization, the limitations of previous research, and the design and research questions of the Central State Hospital (CSH) closing studies. Previously, the central engine of deinstitutionalization has been the downsizing, and not the closing, of facilities to decrease available beds. Only 14 state hospitals closed between 1970 and 1990. However, since 1990, 40 hospitals have closed. Moreover, beginning in 1993, for the first time since deinstitutionalization began, funding for state psychiatric facilities was less than for community-based services. Previous research on both the downsizing and closing of hospitals has focused predominantly on relatively short-term clinical and social outcomes of patients. The current study is a multidisciplinary, longitudinal, multiple-stakeholder study of the closing of a state-run, long-term care facility in Indiana. The articles that follow focus on the clinical, psychological, social, and attitudinal outcomes for patients, workers, families, and the public following the closing of CSH.

The closing of Central State Hospital: long-term outcomes for persons with severe mental illness.

This study examined the clinical/community functioning of long-stay patients following closing of a large state psychiatric hospital. Two overlapping samples were followed: (1) the tracking project collected information on patient location, treatment provision, legal contacts, and level of functioning (LOF) and followed all discharged patients and (2) the research study subsample, drawn from the final group of discharged patients, gathered information on quality of life (QOL), LOF, and general physical and mental health. At follow-up, patients were functioning equal to or better than prior to discharge. There were consistent improvements in QOL (especially safety and occupational satisfaction) and LOF (especially housing and income/benefits). Fewer than 27% of patients discharged into the community were rehospitalized, and fewer than 4% were either in jail or homeless after 24 months. The study demonstrates that even persons who have been hospitalized for extremely long periods can do well in the community.

The effects of hospital closure on mental health workers: an overview of employment, mental and physical health, and attitudinal outcomes.

This article examines the physical, psychological, and attitudinal impact of the closure of Central State Hospital (CSH) on its former employees. Eighty-five former CSH employees were interviewed at two points in time, preclosure and postclosure. Data on the psychological and physical health and employment attitudes of workers were collected prior to and eight months after organizational closure. Over time, workers had more positive attitudes about the hospital closure as well as reporting less depression, less work stress, and use of more coping strategies at postclosure. However, at post-closure, they also reported increased work conflict, lower income, and a more pessimistic outlook toward their future. Implications for hospital closure are discussed.

An estimate and comparison of MMPI and MMPI-2 concurrent validity. Predicting DSM-III-R diagnoses among college students.

The ability of the MMPI and MMPI-2 to identify persons who were either free or not free from DSM-III-R-defined psychopathology was assessed and compared. University students completed either the MMPI (N = 388) or the MMPI-2 (N = 302) along with a computerized version of the Diagnostic Interview Schedule, which was scored according to the criteria of the DSM-III-R. MMPI profiles were categorized with several different rules as being within normal limits or not. DSM-III-R status served as the criterion variable, and 189 (27%) study participants met criteria for a current axis I disorder. Although MMPI profiles were more elevated than MMPI-2 profiles, the proportion of profiles categorized as either normal or abnormal did not differ. Both the MMPI and MMPI-2 demonstrated a statistically reliable degree of relation with the broadly applied DSM-III-R standard of current disorder or not. Predictive relationships were modest. The variance in DSM-III-R-measured psychopathology accounted for by MMPI or MMPI-2 categorizations averaged gamma 2 = .12. Contrary to hypotheses, our results did not demonstrate improved MMPI-2 discrimination.

Familial resemblance of schizotypic traits.

The goal of this study was to examine the familial resemblance of schizotypic traits, and to explore whether different schizotypic traits are familially related to one another. Several of the Chapmans' psychosis proneness scales were administered to a group of college students and their families. Evidence of at least some familial resemblance was found for both physical anhedonia and perceptual aberration, though it was stronger for anhedonia. Anhedonia and perceptual aberration appeared to be familially influenced independently of one another. Finally, the results indicated that offspring resembled their mothers more than they resembled their fathers on both anhedonia and perceptual aberration.

Stimulation of human Jurkat cells by monoclonal antibody crosslinking of transfected-Ly-6A.2 (TAP) molecules.

Monoclonal antibody crosslinking of phosphatidylinositol-anchored Ly-6A.2 molecules on the surface of murine T lymphocytes leads to cell activation and secretion of IL-2. To examine the potential activity of these molecules in human T cells we transfected the Ly-6A.2 gene into Jurkat cells. Transfection of Jurkat cells with genomic Ly-6A.2 sequences results in low levels of Ly-6A.2 on the cell surface. However, linking the Ly-6A.2 sequences to the enhancer from the human CD2 gene results in greatly increased expression of Ly-6A.2. These molecules are anchored to the membrane via a phosphatidylinositol linkage. Crosslinking of Ly-6A.2 molecules with soluble mAb stimulates the transfected Jurkat cells to produce IL-2. This stimulation is abrogated by treatment with phosphatidylinositol-specific phospholipase C. The transfected human T cells displayed the same unusual crosslinking requirements for stimulation with anti-Ly-6A.2 mAbs as previously observed for murine T cells. Crosslinking of Ly-6A.2 with soluble antibodies is stimulatory, whereas immobilized antibodies are inactive. The crosslinking requirements for antiCD3 mAb stimulation display a reciprocal pattern. These data demonstrate that the Ly-6A.2 pathway for T cell activation is conserved between human and murine T cells.