Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth.

Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms.

Genome-wide analysis of lipoprotein expression in Escherichia coli MG1655.

To gain insight into the cell envelope of Escherichia coli grown under aerobic and anaerobic conditions, lipoproteins were examined by using functional genomics. The mRNA expression levels of each of these genes under three growth conditions--aerobic, anaerobic, and anaerobic with nitrate--were examined by using both Affymetrix GeneChip E. coli antisense genome arrays and real-time PCR (RT-PCR).

Effects of inhibition of myocardial extracellular-responsive kinase and P38 mitogen-activated protein kinase on mechanical function of rat hearts after prolonged hypothermic ischemia.

Background: Mitogen-activated protein kinases (MAPKs), including extracellular-responsive kinase (ERK) and p38 MAPK, are activated by stresses associated with hypothermia-rewarming and ischemia-reperfusion. Their activation in heart is associated with beneficial (preconditioning) and adverse effects (apoptosis and impaired contractility). This study determined whether ERK and p38 MAPK activities are altered by hypothermic ischemia and normothermic reperfusion and the consequences of their inhibition on recovery of myocardial function.

Managing end-of-life care: comparing the experiences of terminally Ill patients in managed care and fee for service.

There have been no published empirical studies comparing the experiences of terminally ill patients in managed care organizations (MCOs) and those in fee for service (FFS). This investigation represents the first empirical study to systematically compare substantive outcomes between populations of terminally ill patients enrolled in MCO and FFS healthcare delivery systems. The investigators interviewed 988 patients whose physicians judged them to be terminally ill and 893 of their caregivers.

Geographic & age variations in invasive cervical cancer in Kentucky, 1995-1999.

The incidence rate of invasive cervical cancer in Kentucky is among the highest in the United States. Data from the Kentucky Cancer Registry and the National Cancer Institute (NCI) show that in 1998, Kentucky's invasive cervical cancer incidence rate was 49% higher than the national rate (11.2 per 100,000 women versus 7.

Mutations in conserved regions 1, 2, and 3 of Raf-1 that activate transforming activity.

To investigate the role of Raf-1 in v-Ha-ras transformation, we have isolated and characterized a number of Raf-1 mutants that display increased transforming activity in Rat2 fibroblasts. A dipeptide deletion (Delta144-145) in the cysteine-rich domain (CRD) of conserved region (CR) 1 increased the interaction between Raf-1 and v-Ha-ras effector loop mutants in the yeast two-hybrid system, supporting the proposal that the CRD serves as a secondary ras-binding domain. Many activating mutations were located in CR2.

History of tobacco use among Kentucky women diagnosed with invasive cervical cancer: 1997-1998.

An association between cigarette smoking and cervical cancer has been demonstrated by numerous epidemiologic studies. However, just because there is an association does not mean that there is an etiologic connection between tobacco use and cervical cancer, although some studies do indicate such a relationship. There are numerous potential explanations, including smoking as a behavior being associated with other behaviors that place women at increased risk of HPV infection.

RasGRP is essential for mouse thymocyte differentiation and TCR signaling.

The Ras signaling pathway plays a critical role in thymopoiesis and T cell activation, but the mechanism of Ras regulation is controversial. At least one mode of Ras regulation in T cells involves the messenger diacylglycerol (DAG). RasGRP, a Ras activator with a DAG-binding C1 domain, is expressed in T cells and thymocytes.

Phorbol esters modulate the Ras exchange factor RasGRP3.

RasGRP represents the prototype of a new class of guanine nucleotide exchange factors that activate small GTPases. The guanyl nucleotide-releasing protein (GRP) family members contain catalytic domains related to CDC25, the Ras exchange factor of Saccharomyces cerevisiae. They also contain a motif resembling a pair of calcium-binding EF-hands and a C1 domain similar to the diacylglycerol interaction domain of protein kinase C.

RasGRP links T-cell receptor signaling to Ras.

Stimulation of the T-cell receptor (TCR) alters a number of intracellular signaling pathways including one that involves protein tyrosine kinases, phospholipase C-gamma1 (PLC-gamma1), diacylglycerol (DAG), and calcium messengers. By a divergent pathway, TCR-stimulated protein tyrosine kinase activity is thought to result independently in recruitment of the Ras activator Sos to the plasma membrane, leading to Ras activation. Here we show that RasGRP, a Ras activator that contains calcium-binding EF hands and a DAG-binding domain, is expressed in T cells.

Hypothermic stress leads to activation of Ras-Erk signaling.

The small GTPase Ras is converted to the active, GTP-bound state during exposure of vertebrate cells to hypothermic stress. This activation occurs more rapidly than can be accounted for by spontaneous nucleotide exchange. Ras-guanyl nucleotide exchange factors and Ras GTPase-activating proteins have significant activity at 0 degrees C in vitro, leading to the hypothesis that normal Ras regulators influence the relative amounts of Ras-GTP and Ras-GDP at low temperatures in vivo.

RasGRP, a Ras activator: mouse and human cDNA sequences and chromosomal positions.

We recently reported the molecular cloning of a novel transforming rat brain cDNA, rbc7, that encodes a Ras activator (Ebinu et al. Science 280, p. 1082, 1998).

RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs.

RasGRP, a guanyl nucleotide-releasing protein for the small guanosine triphosphatase Ras, was characterized. Besides the catalytic domain, RasGRP has an atypical pair of "EF hands" that bind calcium and a diacylglycerol (DAG)-binding domain. RasGRP activated Ras and caused transformation in fibroblasts.

Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells.

v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro.

ras effector loop mutations that dissociate p120GAP and neurofibromin interactions.

ras proteins are positively regulated by nucleotide exchange factors and negatively regulated by GTPase-activating proteins (GAPs). Two GAPs have been found in mammalian cells, p120GAP and neurofibromin, the product of the type 1 neurofibromatosis (NF1) gene. A library of substitutions in the effector loop region of ras in an Escherichia coli plasmid expression system was screened for c-Ha-ras species with altered GAP interactions.

RAS signalling is abnormal in a c-raf1 MEK1 double mutant.

A mutant rat cell clone that suppresses the transformation defects of RAS effector loop substitutions is heterozygous for mutations in c-raf1 and MEK1. The mutant cells can be transformed by many otherwise defective RAS effector mutants, including RAS genes with the effector regions of distantly related GTPases, even though the encoded RAS proteins do not interact with either the mutant or wild-type RAF in Saccharomyces cerevisiae. While the significance of the c-raf1 mutation is unclear, the MEK1 mutation increases MEK1 activity and leads to activation of mitogen-activated protein kinase.

Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa3 and c in Neurospora crassa.

The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa3 and c. Using a sib-selection procedure we have isolated the cyt-12+ allele from a cosmid library of N. crassa genomic DNA.

Isolation and characterization of a cDNA clone coding for the calcium-binding subunit of calcineurin from bovine brain: an identical amino acid sequence to the human protein.

A cDNA clone encoding the calcium-binding subunit of calcineurin, calcineurin B, was isolated from a bovine brain library by immunoscreening. The 841 bp cDNA has a 56 bp 5'-noncoding region, an open reading frame of 510 bp, and a 275 bp 3'-noncoding sequence. The deduced amino acid sequence of bovine calcineurin B differs from the previously reported protein sequence (Aitken et al.

Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction.

The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP.

The murine leukemia inhibition factor gene (Lif) is located on proximal chromosome 11, not chromosome 13.

Lif, the murine gene encoding leukemia inhibition factor (LIF), has been previously localized to proximal Chromosome (Chr) 11. Hilda, the murine gene encoding "human interleukin in DA cells" (HILDA) has been localized to Chr 13. Since these two growth factors are identical, the proposal for two different structural loci is intriguing.

Nucleotide sequence of the Varkud mitochondrial plasmid of Neurospora and synthesis of a hybrid transcript with a 5' leader derived from mitochondrial RNA.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed circular DNAs (3.6 and 3.7 kb, respectively; 1 kb = 10(3) bases or base-pairs), whose characteristics suggest relationships to mitochondrial DNA introns and retrotransposons.