Rapid isolation and characterization of the yeast proteasome regulatory complex.

The 26S proteasome, which catalyzes degradation of ubiquitinated proteins, is composed of the 20S proteasome and the 19S complex. Recently, it has been reported that the 26S complex can be dissociated into the lid complex and the 20S-proteasome-base complex in a mutant yeast and that the lid complex is required for ubiquitin-dependent proteolysis. In the present study, we established methods for rapid isolation of the 19S complex, the lid complex, and the base complex from wild-type yeast.

Nob1p, a new essential protein, associates with the 26S proteasome of growing saccharomyces cerevisiae cells.

Nob1p, which interacts with Nin1p/Rpn12, a subunit of the 19S regulatory particle (RP) of the yeast 26S proteasome, has been identified by two-hybrid screening. NOB1 was found to be an essential gene, encoding a protein of 459 amino acid residues. Nob1p was detected in growing cells but not in cells in the stationary phase.

Extragenic suppressors that rescue defects in the heat stress response of the budding yeast mutant tom1.

The TOM1 gene codes for a so-called HECT protein, a putative ubiquitin ligase, in Saccharomyces cerevisiae. Deletion of the entire gene (tom1-10) or the sequence encoding the HECT domain (tom1-2) causes temperature sensitivity for growth. Here we report the isolation of extragenic, recessive suppressors of tom1-2, which were designated tmr (for tom1 revertant) mutations.

The Pho85 kinase, a member of the yeast cyclin-dependent kinase (Cdk) family, has a regulation mechanism different from Cdks functioning throughout the cell cycle.

Background: The PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85p) which is highly homologous to the Cdc28 kinase (Cdc28p). Although the two kinases share a 51% identity and their functional domains are well conserved, PHO85 fails to replace CDC28. Pho85p forms complexes with G1-cyclin homologues, including Pcl1p, Pcl2p and Pcl9p, and is thought to be involved in the cell-cycle regulation at G1 and the end of M.

Mouse cyclin-dependent kinase (Cdk) 5 is a functional homologue of a yeast Cdk, pho85 kinase.

Mouse cyclin-dependent kinase (Cdk) 5 and yeast Pho85 kinase share similarities in structure as well as in the regulation of their activity. We found that mouse Cdk5 kinase produced in pho85Delta mutant cells could suppress some of pho85Delta mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction. We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins including Pcl1, Pcl2, Pcl6, Pcl9, and Pho80, and that the immunocomplex could phosphorylate Pho4, a native substrate of Pho85 kinase.

Modified expansive open-door laminoplasty in cervical myelopathy.

The authors modified the technique of open-door cervical laminoplasty with a patency maintenance method that relies only on soft tissue reconstruction. Thirty-five patients who underwent this procedure were evaluated at more than 2 years' follow-up. The expanded open-door lamina and excellent alignment on the lateral view of the cervical spine were well maintained after surgery.

Genetic evidence for interaction between components of the yeast 26S proteasome: combination of a mutation in RPN12 (a lid component gene) with mutations in RPT1 (an ATPase gene) causes synthetic lethality.

The 19S regulatory particle of the yeast 26S proteasome consists of six related ATPases (Rpt proteins) and at least 11 non-ATPase proteins (Rpn proteins). RPN12 (formerly NIN1) encodes an Rpn component of the 19S regulatory particle and is essential for growth. To determine which subunit(s) of the 26S proteasome interact(s) with Rpn12, we attempted to screen for mutations that cause synthetic lethality in the presence of the rpn12-1 (formerly nin1-1) mutation.

Cloning and characterization of a gene encoding phosphatidyl inositol-specific phospholipase C from Trypanosoma cruzi.

A gene encoding phosphatidyl inositol-4,5-bisphosphate phospholipase C (PLC) was cloned from the protozoan parasite Trypanosoma cruzi. A partial cDNA encoding putative PLC was obtained by a polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to conserved regions of PLCs. A 2178-bp protein coding region of the T.

Rpn9 is required for efficient assembly of the yeast 26S proteasome.

We have isolated the RPN9 gene by two-hybrid screening with, as bait, RPN10 (formerly SUN1), which encodes a multiubiquitin chain receptor residing in the regulatory particle of the 26S proteasome. Rpn9 is a nonessential subunit of the regulatory particle of the 26S proteasome, but the deletion of this gene results in temperature-sensitive growth. At the restrictive temperature, the Deltarpn9 strain accumulated multiubiquitinated proteins, indicating that the RPN9 function is needed for the 26S proteasome activity at a higher temperature.

Yeast tom1 mutant exhibits pleiotropic defects in nuclear division, maintenance of nuclear structure and nucleocytoplasmic transport at high temperatures.

A tom1-1 mutant was isolated from Saccharomyces cerevisiae. At high temperatures, 60% of the cells were arrested as dumbbell forms with a single large nucleus containing duplicated DNA and a short spindle. Electron-microscopy showed electron-dense structures scattered within the nucleus.

Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component of septin rings at the mother-bud neck in budding yeast.

SMT3 of Saccharomyces cerevisiae is an essential gene encoding a ubiquitin-like protein similar to mammalian SUMO-1. When a tagged Smt3 or human SUMO-1 was expressed from GAL1 promoter, either gene rescued the lethality of the smt3 disruptant. By indirect-immunofluorescent microscopy, the HA-tagged Smt3 was detected mostly in nuclei and also at the mother-bud neck just like septin fibers.

Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70.

The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth. Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S.

Two distinct mechanisms operate in the reactivation of heat-denatured proteins by the mitochondrial Hsp70/Mdj1p/Yge1p chaperone system.

The yeast mitochondrial Hsp70, Ssc1p, functions as a molecular chaperone with its partner proteins, Mdj1p (DnaJ homologue) and Yge1p (GrpE homologue). We have purified a mature form of Ssc1p from yeast mitochondria and those of Mdj1p and Yge1p from Escherichia coli overexpresser cells. With these purified components of the mitochondrial Hsp70 chaperone system, we have succeeded in reconstituting their chaperone functions in the protection of firefly luciferase against thermal damage in vitro.

The PY-motif of Bul1 protein is essential for growth of Saccharomyces cerevisiae under various stress conditions.

The previously identified BUL1 gene was found to encode a protein bound to Rsp5-ubiquitin ligase in budding yeast. We have identified the BUL2 gene as a functional homologue of BUL1. The bul1 bul2 double disruptant was sensitive to various stresses, such as high temperature, salts, and a non-fermentable carbon source.

Measurement of strain distributions within vertebral body sections by texture correlation.

Study Design: A high-resolution strain measurement technique was applied to axially loaded parasagittal sections from thoracic spinal segments.

Objectives: To establish a new experimental technique, develop data analysis procedures, characterize intrasample shear strain distributions, and measure intersample variability within a group of morphologically diverse samples.

Summary Of Background Data: Compression of intact vertebral bodies yields structural stiffness and strength, but not strain patterns within the trabecular bone.

The effect of boundary conditions on experimentally measured trabecular strain in the thoracic spine.

Vertebral bodies are the primary structural entities of the spine, and trabecular bone is the dominant material from which vertebral bodies are composed. Understanding the mechanical characteristics of vertebral trabecular bone, therefore, is of critical importance in the many clinical conditions that affect the spine. Numerous studies have loaded vertebral bodies to investigate the influence of trabecular bone characteristics on deformation and failure patterns, but the methods of load application have been inconsistent.

Growth-dependent change of the 26S proteasome in budding yeast.

The 26S proteasome is assembled from the 20S proteasome and the regulatory subunit complex in an ATP-dependent manner. In the present study, we found that the ATP-dependent activity and the protein amount of the 26S proteasome change during growth of the budding yeast Saccharomyces cerevisiae. Both levels in the stationary phase are higher than those in the exponentially growing phase.

Phosphorylation of sic1, a cyclin-dependent kinase (Cdk) inhibitor, by Cdk including Pho85 kinase is required for its prompt degradation.

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1 for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1 cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Delta cells.

cDNA cloning and functional analysis of p28 (Nas6p) and p40.5 (Nas7p), two novel regulatory subunits of the 26S proteasome.

We employed cDNA cloning to deduce the complete primary structures of p28 and p40.5, two novel subunits of PA700 (also called 19S complex), a 700 kDa multisubunit regulatory complex of the human 26S proteasome. These polypeptides consisted of 226 and 376 amino acids with calculated molecular masses of 24428 Da and 42945 Da, and isoelectric points of 5.

Revision of failed pedicle screws using hydroxyapatite cement. A biomechanical analysis.

Study Design: The biomechanical influence of in situ setting hydroxyapatite cement was examined for use in pedicle screw revision surgery. Pull-out testing of control and pedicle screws augmented with hydroxyapatite cement was performed in human cadaver vertebrae.

Objectives: To determine the immediate effect of using hydroxyapatite cement to augment revision pedicle screws after failure of the primary pedicle screw fixation.

Sro7p, a Saccharomyces cerevisiae counterpart of the tumor suppressor l(2)gl protein, is related to myosins in function.

Yeast SRO7 was identified as a multicopy suppressor of a defect in Rho3p, a small GTPase that maintains cell polarity. Sro7p and Sro77p, a homologue of Sro7p, possess domains homologous to the protein that are encoded by the Drosophila tumor suppressor gene lethal (2) giant larvae [l(2)gl]. sro7Delta sro77Delta mutants showed a partial defect of organization of the polarized actin cytoskeleton and a cold-sensitive growth phenotype.

Functional evaluation using motor scores after cervical spinal cord injuries.

Patient evaluation using Zancolli's classification for cervical cord injuries uses easily understood criteria and accurately defines the state of disability in Frankel grade A or B patients. However, this classification cannot be used in Frankel grade C patients even though they also cannot walk. We compared the Zancolli classification with American Spinal Injury Association (ASIA) motor scores in evaluating self-care in Frankel grade C patients.

Phosphoinositide-specific phospholipase C forms a complex with 14-3-3 proteins and is involved in expression of UV resistance in fission yeast.

The fission yeast plc1+ gene encodes phosphoinositide-specific phospholipase C. The two- hybrid interaction assay with plexA-plc1+ as a bait revealed that Plc1p interacted with the 14-3-3 proteins Rad24p and Rad25p. Formation of a complex containing Plc1p and Rad24p in vivo was confirmed by an immunological method.

Son1p is a component of the 26S proteasome of the yeast Saccharomyces cerevisiae.

A son1 mutant was isolated as a mutant showing synthetic lethality with nin1-1 which is defective in the p31 component of the regulatory subunit of the yeast 26S proteasome. son1delta showed a synthetic effect with sen3delta and sun1delta, both components of the 26S proteasome, and with cdc28-1N. The 26S proteasome was partially purified from the wild type yeast.