Inhibition of Membrane-Active Peptides by Fatty Acid–Peptide Hybrids

Nine fatty acid–peptide hybrid molecules were constructed using the general formula CH₃(CH₂) _n CO-Phe Asp Cys-amide and tested for their ability to inhibit cell lysis induced by the membrane-active peptide melittin. All of these molecules, where n = 4–14, inhibited the action of melittin to some extent, but the longer carbon chains were most effective. Several potential inhibitors were also constructed with conservative substitutions in the peptide portion of the molecule. All were effective to varying degrees. We concluded that in the hexapeptide inhibitor published by Blondelle et al. (1993), the role of the first three residues is only to provide hydrophobic interaction with the melittin and has no particular amino acid sequence specificity. Some of these inhibitors were found to inhibit the lytic activity of a melittin analogue which had only superficial sequence similarity to melittin and also a truncated form of melittin, indicating the generality of the action of the inhibitors.Deceased 5/4/98     

Structure and expression of mouse alpha 1-acid glycoprotein gene-3 (AGP-3).

The genome of Mus domesticus has multiple genes of the alpha 1-acid glycoprotein (AGP). Two cDNA clones were identified corresponding to AGP-1 and AGP-2. Moreover, two alleles of AGP-1 exist in inbred mice. The genomic DNA of the AGP-2 gene has been cloned and studied. Here we report the genomic organization of three M. domesticus AGP genes, the sequence analysis of the AGP-3 genomic DNA, and the expression of the AGP-3 gene. The major structural differences between AGP-2 and AGP-3 genes are located in introns 1 and 5. The low level of AGP-3 mRNA can be detected by the polymerase chain reaction (PCR). The molecular basis of the low level expression of AGP-3 and the possible classification of AGP-3 as a pseudogene are discussed.     

Enzymatic modifications of human plasma fibronectin in relation to opsonizing activity

Plasma fibronectin is one of the largest plasma proteins (Mr approximately 440 000), comprising two approximately equal polypeptide chains which are held together by a disulfide linkage near the C-terminal end of the molecule. The binding of gelatinized latex beads to liver slices as well as the internalization of these particles by macrophages, in the presence of heparin, is greatly enhanced by fibronectin. The question as to whether the entire covalent structure of fibronectin was necessary for opsonizing activity was approached by limited proteolytic degradations of the molecule. Patterns of controlled digestion with trypsin, cathepsin D, Staphylococcus aureus protease, and plasmin all indicate that the minimal unit necessary for retention of opsonic activity is some large (Mr 200 000 and 190 000) single-chain entity. Treatment with plasmin proved to be the most reliable procedure for generating the active split product which could be readily separated from the inactive, disulfide-containing C-terminal fragment. Incorporation of dansylcadaverine into plasma fibronectin (3.5 mol/mol of protein) by fibronoligase (coagulation factor XIIIa) did not affect the opsonic activity of the protein.     

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.     

The Ighmbp2 helicase structure reveals the molecular basis for disease-causing mutations in DMSA1

Mutations in immunoglobulin µ-binding protein 2 (Ighmbp2) cause distal spinal muscular atrophy type 1 (DSMA1), an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. However, despite extensive studies, the mechanism of disease-causing mutations remains elusive. Here we report the crystal structures of the Ighmbp2 helicase core with and without bound RNA. The structures show that the overall fold of Ighmbp2 is very similar to that of Upf1, a key helicase involved in nonsense-mediated mRNA decay. Similar to Upf1, domains 1B and 1C of Ighmbp2 undergo large conformational changes in response to RNA binding, rotating 30° and 10°, respectively. The RNA binding and ATPase activities of Ighmbp2 are further enhanced by the R3H domain, located just downstream of the helicase core. Mapping of the pathogenic mutations of DSMA1 onto the helicase core structure provides a molecular basis for understanding the disease-causing consequences of Ighmbp2 mutations.     

Regulation of G(1) cell-cycle progression by oncogenes and tumor suppressor genes.

Progression of resting quiescent G(0) cells into early G(1) and transition across the restriction point are highly regulated processes. Mutation of proto-oncogenes and tumor suppressor genes regulating these transitions are targeted during oncogenesis. Recent work has underscored the importance of the G(0) to early G(1) transition and metabolism to neoplastic cells.     

Sampling for Explosives-Residues at Fort Greely,Alaska

Fort Greely, Alaska has an extensive complex of weapon training and testing areas located on lands withdrawn from the public domain under the Military Lands Withdrawal Act (PL106-65). The Army has pledged to implement a program to identify possible munitions contamination. Because of the large size (344,165,000 m²) of the high hazard impact areas, characterization of these constituents will be difficult. We used an authoritative sampling design to find locations most likely to contain explosives-residues on three impact areas. We focused our sampling on surface soils and collected multi-increment and discrete samples at locations of known firing events and from areas on the range that had craters, pieces of munitions, targets, or a designation as a firing point. In the two impact areas used primarily by the Army, RDX was the most frequently detected explosive. In the impact area that was also used by the Air Force, TNT was the most frequently detected explosive. Where detected, the explosives concentrations generally were low (<0.05 mg/kg) except in soils near low-order detonations, where the explosive-filler was in contact with the soil surface. These low-order detonations potentially can serve as localized sources for groundwater contamination if positioned in recharge areas.