Specificity of Ubiquitin-Binding Proteins: Recognition of Different Faces of Ubiquitin

To further understand ubiquitin-binding proteins, we have developed a panel of monoubiquitin affinity resins linked through six different positions: residues 6, 11, 29, 48, 63, and 76. Each resin bound a different subset of yeast proteins. MALDI-TOF MS analysis of the eluted proteins identified several of these proteins. Adding excess free ubiquitin competes for the binding of specific ubiquitin-binding proteins. Thus, putative monoubiquitin-binding proteins could be identified by this method. Analysis of yeast protein with this panel of resins demonstrates clear differences in the protein-binding pattern, depending on what ubiquitin residue is coupled to the resin. The results suggested that certain proteins show a preference for binding to different faces of ubiquitin. Sap185, an effector of the Sit4 protein phosphatase, has been identified as a putative ubiquitin-binding protein that binds to a face of ubiquitin other than that containing the hydrophobic patch. The combined affinity chromatography and mass spectrometry approach is a powerful tool for identifying ubiquitin-binding proteins.     

Ethics consultation

Hospices in the U.S. were surveyed in 1990 to find out whether service to blacks and Hispanics was affected by admission criteria and hospice service characteristics of hospices located in or near these populations. Hospice characteristics such as reimbursement patterns, staff interventions, and admission criteria were different depending upon the percent of blacks and/or Hispanics in the hospice service area or actually served by the hospice. Care for Hispanics was more dependent on Medicaid and free care than blacks whose care was financed primarily by Medicare and Medicaid. Hospices identified problems in serving Hispanics as language, reimbursement, and severity-of-illness issues. Hospice admission criteria, especially the primary caregiver requirement, were identified as impeding access for blacks. Hispanics were perceived as presenting the most access and service problems and as the most underserved.     

Tools: An environment for time-shared computing and programming

The Yale Tools environment provides integrated modern computing facilities to a mixed group of programmers and users on time-shared machines. Hundreds of people use the environment every day for systems and applications programming, word-processing and university business. The success of the environment is in its integration and engineering detail. Building such a system without a high-level language and a run-time library would have been impossible.     

Stability of the centred finite difference scheme for the three-dimensional equations of elastodynamics

The displacement equations of motion for elastodynamics in 3 space dimensions are approximated by difference equations using 2nd order centred finite differences in space and time. The stability criterion of the difference scheme without boundary conditions is taken to be that condition on the ratio of step sizes which prevents the growth of an arbitray disturbance, and it requures the ratio of time step size to the space grid size to be less than a certain function of the ratio of the shear wave speed to the compressional wave speed. For this ratio less than 0.5, the condition is more stringent than that obtained by a simple extension from the two-dimensional case.     

Semen quality characteristics of dairy goats

Semen was collected, processed, and frozen from five dairy bucks for 2 successive yr for use in quality classification and evaluation for inclusion in artificial insemination programs. Semen was evaluated for volume, initial, postthaw and 37 degrees C incubated percent progressive motility, percent postthaw 3-h 37 degrees C incubated intact acrosomes, autoagglutination, whey-induced agglutination, and percent primary, secondary, and tertiary abnormalities. Significant high correlations were found between: percent progressive motility and percent intact acrosomes, percent intact acrosomes and percent autoagglutination, and percent autoagglutination and percent whey agglutination. Means of the postthaw quality parameters, percent progressive motility, percent intact acrosomes, and percent primary and secondary abnormalities were used to categorize ejaculates within each incubation time (0 and 2 h). At 0 h, 25 ejaculates were classified as high quality and 11 were low quality. Using 2-h data, 19 ejaculates were classified as high quality and 17 as low. Inclusion of both agglutination parameters in the 2-h data analysis resulted in 13 ejaculates categorized as high and 23 as low quality. Based on assessment with techniques used in bovine artificial insemination programs, semen quality of goat semen could be used to discriminate between acceptable or unacceptable ejaculates. Based on recommended sperm numbers per inseminate and average ejaculate characteristics the low number of marketable units per ejaculate would make incorporation of goats into existing artificial insemination programs prohibitive.     

NDC1: a nuclear periphery component required for yeast spindle pole body duplication

The spindle pole body (SPB) of Saccharomyces cerevisiae serves as the centrosome in this organism, undergoing duplication early in the cell cycle to generate the two poles of the mitotic spindle. The conditional lethal mutation ndc1-1 has previously been shown to cause asymmetric segregation, wherein all the chromosomes go to one pole of the mitotic spindle (Thomas, J. H., and D. Botstein. 1986. Cell. 44:65-76). Examination by electron microscopy of mutant cells subjected to the nonpermissive temperature reveals a defect in SPB duplication. Although duplication is seen to occur, the nascent SPB fails to undergo insertion into the nuclear envelope. The parental SPB remains functional, organizing a monopolar spindle to which all the chromosomes are presumably attached. Order-of-function experiments reveal that the NDC1 function is required in G1 after alpha-factor arrest but before the arrest caused by cdc34. Molecular analysis shows that the NDC1 gene is essential and that it encodes a 656 amino acid protein (74 kD) with six or seven putative transmembrane domains. This evidence for membrane association is further supported by immunofluorescent localization of the NDC1 product to the vicinity of the nuclear envelope. These findings suggest that the NDC1 protein acts within the nuclear envelope to mediate insertion of the nascent SPB.     

NMR study of the transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor complex. Visualization of human TGF-alpha binding determinants through nuclear Overhauser enhancement analysis

The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.     

Pleomorphic xanthoastrocytoma: DNA flow cytometry and outcome analysis of 12 patients

BACKGROUND: Pleomorphic xanthoastrocytoma (PXA) is an astrocytic tumor occurring primarily in childhood and adolescence with some malignant histologic features but a relatively slow clinical course. However, some tumors progress more rapidly and can undergo malignant degeneration. The authors attempted to determine whether various histologic features or tumor cell proliferative indices might help identify lesions at risk for early progression and distinguish PXAs from malignant gliomas. METHODS: In a retrospective study of 12 patients with PXA, the tumor's histologic features and DNA flow cytometric parameters were compared with their clinical course. DNA flow cytometry values for the S- and G2-phase of the PXAs also were compared with control group samples of malignant and low grade astrocytomas. RESULTS: Of the 12 tumors at initial diagnosis, 5 were considered typical PXAs whereas 7 had some atypical features (4 with paucity of reticulin fibers, 1 with focal necrosis, and 2 with both atypical reticulin and focal necrosis). During the follow-up period (range, 3.75-11 years; mean, 6.8 years), 2 patients had recurrences; 1 atypical reticulin PXA progressed to glioblastoma after 6.5 years and the 1 tumor with focal necrosis recurred at 6 months and again at 2 years with typical histologic features. DNA flow cytometry parameters of the typical PXA group were similar to values for malignant astrocytoma and significantly higher than values for control specimens of low grade astrocytomas. There were no distinctive DNA flow cytometric features that could distinguish this last tumor from others with a more benign clinical course. CONCLUSIONS: Measurements of the S-phase and G2-phase obtained from DNA flow cytometry and atypical histologic features cannot reliably identify PXA patients at risk for early progression and overall are significantly higher than values obtained for low grade gliomas. Therefore, frequent (i.e., two to three times per year) postoperative clinical and radiologic examinations are necessary to judge the appropriateness of adjuvant therapy in patients with PXA. The paradox of slow growth but DNA flow cytometry consistent with aggressive malignant lesions may represent a cell-cycle arrest mechanism in these lesions that could be verified in subsequent studies.     

Dominant alleles of Saccharomyces cerevisiae CDC20 reveal its role in promoting anaphase

We identified an allele of Saccharomyces cerevisiae CDC20 that exhibits a spindle-assembly checkpoint defect. Previous studies indicated that loss of CDC20 function caused cell cycle arrest prior to the onset of anaphase. In contrast, CDC20-50 caused inappropriate cell cycle progression through M phase in the absence of mitotic spindle function. This effect of CDC20-50 was dominant over wild type and was eliminated by a second mutation causing loss of function, suggesting that it encodes an overactive form of Cdc20p. Overexpression of CDC20 was found to cause a similar checkpoint defect, causing bypass of the preanaphase arrest produced by either microtubule-depolymerizing compounds or MPS1 overexpression. CDC20 overexpression was also able to overcome the anaphase delay caused by high levels of the anaphase inhibitor Pds1p, but not a mutant form immune to anaphase-promoting complex- (APC-)mediated proteolysis. CDC20 overexpression was unable to promote anaphase in cells deficient in APC function. These findings suggest that Cdc20p is a limiting factor that promotes anaphase entry by antagonizing Pds1p. Cdc20p may promote the APC-dependent proteolytic degradation of Pds1p and other factors that act to inhibit cell cycle progression through mitosis.