A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma.

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.     

Thermal denaturation and loss of viability in Escherichia coli and Bacillus stearothermophilus

When Escherichia coli was heated at 10°C/min in a differential scanning calorimeter, the onset of irreversible thermal denaturation occurred at 51°C, about 5°C above the maximum growth temperature. The temperature at which death rate was maximal (63°C) coincided with the thermogram peak caused by denaturation of the 30S ribosomal subunit. The maximum death rate in vegetative cells of Bacillus stearothermophilus occurred at the higher temperature of 71°C which also coincided with the leading edge of the main thermogram peak.     

Stabilization of proteins immobilized on Sepharose from leakage by glutaraldehyde crosslinking

A technique has been developed to prevent leakage of proteins immobilized on Sepharose without destroying their biological functions. This involves the use of glutaraldehyde at concentrations ranging from 0.015 to 0.25% ($\text{v}\text{v}$) to crosslink proteins, which had been coupled to Sepharose by conventional methods. Glutaraldehyde crosslinking decreases immuno-globulin G leakage from Sepharose-immunoadsorbents to undetectable levels without noticeably affecting antigen-binding activity and reduces leakage of lactoperoxidase from solid-phase lactoperoxidase with only a moderate reduction of enzymatic activity.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

An examination of the epiphytic nature of Gambierdiscus toxicus, a dinoflagellate involved in ciguatera fish poisoning

Twenty-four specimen of macroalgae were collected in nearshore waters of the island of Hawaii, identified, and maintained to examine how the epiphytic relationship between Gambierdiscus toxicus (isolate BIG12) varied among the macroalgal species. Gambierdiscus cells were introduced to Petri dishes containing 100 g samples of each macroalgal host, which were examined at two, 16, 24, and every 24–72 h thereafter, over a 29-day period. Gambierdiscus proliferated in the presence of some host species (e.g., Galaxaura marginata and Jania sp.), but grew little in the presence of other species (e.g., Portieria hornemannii). Gambierdiscus exhibited high survival rates (>99%) in the presence of Chaetomorpha sp., but died before the end of the experiment (after 21 days) with other host species (e.g., Dictyota and Microdictyon spp.). Gambierdiscus avoided contact with P. hornemannii, but averaged up to 30% attachment with other host species. The numbers of Gambierdiscus cells belonging to one of three classes (alive and attached; alive and unattached; and dead) were determined for each time point. The 24 algal hosts were grouped according to their commonalities relative to these three classes using a Bray-Curtis similarity index, similarity profile (SIMPROF) permutation tests, and Multi-Dimensional Scaling (MDS) analysis (PRIMER 6). The resultant six groupings were used to construct different Gambierdiscus growth profiles for the different algal hosts. Group A is characterized by a preponderance of unattached cells and high mortality rates. Groups B, C, E, and F also displayed high proportions of unattached cells, but mortality either occurred later (Groups B and C) or rates were lower (Groups E and F). Group D had the highest proportion of attached cells. Group E contained three out of the four chlorophyte species, while Group F contained the majority of the rhodophytes. Over 50% of the species in Group F are considered to be palatable, whereas Groups A, B, and C are composed of species that exhibit chemical defenses against herbivory. The results of this study coupled with previous findings indicate that Gambierdiscus is not an obligate epiphyte; it can be free-swimming and found in the plankton. The conditions that lead to changes between epiphytic and planktonic stages need to be better studied in order to determine how they affect Gambierdiscus growth and physiology, connectivity and dispersion mechanisms, and toxin movement up into the foodweb.     

The design and discovery of novel amide CCR5 antagonists

The synthesis of a range of novel amine-containing structures and their primary potency as inhibitors of HIV-1 fusion via blocking of the CCR5 receptor is described. The development of the medicinal chemistry strategy and SAR’s which led to the identification of the piperidine amide compounds 33 and 36 as excellent leads for further evaluation is described, along with key physicochemical data which highlighted their lead potential.     

Physiological Heterothallism and Sexuality in Euascomycetes: A Partial History

Copyright 1998 Academic Press.