A Ca-stimulated ATPase activity in rabbit neutrophil membranes

An ATPase activity specifically stimulated by micromolar Ca²⁺ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca²⁺-ATPase activity with regard to neutrophil function.     

Replacement of the native crayfish Astacus astacus by the introduced species Pacifastacus leniusculus in a small, enclosed Finnish lake: a 30-year study

The native noble crayfish Astacus astacus L., and the introduced North American signal crayfish Pacifastacus leniusculus Dana, co‐occur in Slickolampi, a small lake in southern Finland. That both species have lived side‐by‐side for 30 yr without any signs of crayfish plague Aphanomyces astaci, indicates that the P. leniusculus population must be plague‐free. According to annual trap catches and population size estimates, A. astacus was clearly dominant in the 1970s and most of the 1980s. At the end of the 1980s, however, there was a shift in the relative abundances of the two species, and in the 1990s, P.leniusculus became dominant. As the 1990s drew to a close, it accounted for >98% of total catches. Originating from a minor stocking (only 900 2nd stage juveniles) P. leniusculus has not augmented the existing fauna in this lake but has almost completely replaced A. astacus. Both species seem to a great extent to prefer the same types of biotope but P. leniusculus is distinctly more demanding and was encountered less often than A. astacus on gently‐sloping soft shores. The proportion of A. astacus with chelae injuries (16 yr, mean 17.3%) was nearly twice that of P. leniusculus (9.3%), suggesting that agonistic interspecific encounters do occur and that P. leniusculus is much more competitive. However, the consistent weakening of A. astacus, even at sites with only a low density of P. leniusculus, indicates that the elimination of A. astacus is not adequately explained by competitive exclusion. We suggest that its disappearance is governed by a combination of several interacting mechanisms, of which harvest (≥100 mm specimens of both species) and interspecific competition with P.leniusculus were initially the main reasons for the decline in the population. The ultimate reason for the collapse of A. astacus seems to have been the almost complete cessation of successful reproduction, presumably due to reproductive interference between the two species. Interspecific mating results in females laying sterile eggs. Although both species suffer from the ensuing loss of recruitment, the consequences are less serious for P. leniusculus, which has a higher capacity for population increase than A. astacus: the smaller the proportion of A. astacus, the greater the role played by reproductive interference as a replacement mechanism.     

Interferon-induced Mx proteins form oligomers and contain a putative leucine zipper.

Interferons induce a number of different proteins that mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. At least three different proteins mediate the antiviral response, and one of them, Mx protein, specifically inhibits the replication of influenza virus and (vesicular stomatitis virus). Mouse and rat Mx1 proteins are nuclear, whereas other presently known Mx proteins are cytoplasmic. The cellular functions of Mx proteins are unknown, but all of them contain a consensus GTP binding site. Very little information is available on the structure and characteristics of the mouse Mx1 protein itself. For biochemical characterization, we expressed mouse Mx1 protein in a baculovirus system and purified it to homogeneity. The purified protein as well as the authentic murine cellular Mx1 protein exists in dimers and trimers in the presence of dissociating solvents, whereas in physiological buffers they form aggregates. Cross-linking experiments done on Mx-expressing cells from various species revealed that mouse, rat, and human Mx proteins exist predominantly in trimers. Amino acid sequence analysis shows that all known Mx proteins have conserved leucine repeats typical for a leucine zipper at their COOH-terminal end. In vitro translation of chimeric catechol O-methyltransferase-Mx1 gene constructs revealed that the leucine zipper domain of Mx1 protein is responsible for the oligomerization. The COOH terminus also functions as a nuclear localization signal. Microinjection of purified oligomers into the cell cytoplasm resulted in a fast accumulation of the protein in the resulted in a fast accumulation of the protein in the nucleus. Immunoelectron microscopy revealed that nuclear murine Mx1 protein exists in distinct, electron-dense structures separate from nuclear membrane, and chromatin, or nucleolus. These observations reveal that a COOH-terminal leucine zipper domain is an important structural element of all Mx proteins. Its relevance to the biology and functions of Mx proteins is presently not known.     

Biotechnology: impact on biological warfare and biodefense

Advances in biological research likely will permit development of a new class of advanced biological warfare (ABW) agents engineered to elicit novel effects. In addition, biotechnology will have applications supporting ABW weaponization, dissemination, and delivery. Such new agents and delivery systems would provide a variety of new use options, expanding the BW paradigm. Although ABW agents will not replace threats posed by traditional biological agents such as Bacillus anthracis (anthrax) and Variola (smallpox), they will necessitate novel approaches to counterproliferation, detection, medical countermeasures, and attribution.     

Matrix assembly induction and cell migration and invasion inhibition by a 13-amino acid fibronectin peptide

Fibronectin (FN) is an extracellular matrix (ECM) protein involved in tumor growth and metastasis. Five human FN cDNA segments encoding for FN fragments, all starting with the II1 repeat and ending with different C-terminal extensions, have been stably expressed in chick embryo fibroblasts (CEF). These FN cDNAs induce the formation of an organized ECM in CEF as long as they retain a sequence coding for a 13-amino acid stretch (FN13), with collagen binding activity, localized between type II2 and I7 repeats. An FN13 synthetic peptide induces in control CEF the assembly of an FN-ECM comparable with that observed in CEF-expressing FN fragments. The activity of FN13 is specific for its amino acid sequence, although the cysteine present in the 6th position can be substituted with a polar serine without affecting the induction of a fibrillar FN-ECM. A less fibrillar matrix is induced by FN13-modified peptides in which the cysteine is methylated or substituted by a non-polar alanine. FN13 induces the assembly of an FN-ECM also in Rous sarcoma virus-transformed CEF lacking the ECM and in hepatoma (SK-Hep1) and fibrosarcoma (HT-1080) human cell lines. FN13 also promotes the adhesion of CEF and Rous sarcoma virus-CEF at levels comparable with those obtained with purified intact FN. Finally, FN13 inhibits the migratory and invasive properties of tumorigenic cells, whereas intact FN favors their migration. All FN13-modified peptides show similar effects, although with reduced efficiency. None of these activities is supported by a scrambled peptide. These data suggest a possible role of FN13 in tumor growth and metastasis inhibition and its possible use as anti-tumorigenic agent.     

Oxidative Inhibition of Protein Phosphatase 2A Activity: Role of Catalytic Subunit Disulfides

A molecular basis for the inhibition of brain protein phosphatase 2A (PP2A) activity by oxidative stress was examined in a high-speed supernatant (HSS) fraction from rat cerebral cortex. PP2A activity was subject to substantial disulfide reducing agent-reversible inhibition in the HSS fraction. Results of gel electrophoresis support the conclusions that inhibition of PP2A activity was associated with the both the disulfide cross-linking of the catalytic subunit (PP2A_C) of the enzyme to other brain proteins and with the formation of an apparent novel intramolecular disulfide bond in PP2A_C. Additional findings that the vicinal dithiol cross-linking reagent phenylarsine oxide (PAO) produced a potent dithiothreitol-reversible inhibition of PP2A activity suggest that the cross-linking of PP2A_C vicinal thiols to form an intramolecular disulfide bond may be sufficient to inhibit PP2A activity under oxidative stress. We propose that the dithiol–disulfide equilibrium of a vicinal thiol pair of PP2A_C may confer redox sensitivity on cellular PP2A.     

Chemical characterization of the two forms of epidermal growth factor in murine saliva

Extracts of murine salivary glands contain two molecular forms of epidermal growth factor, EGF I and EGF II (Petrides, P.E., Levine, A.E., Shooter, E.M. in: Peptides: - Synthesis, Structure and Function (Rich, D.H., Gross, E.eds.) p. 781 (1981]. We have identified both molecules not only in salivary gland extracts but also in saliva using only reverse phase liquid chromatography methodology. EGF I and II were isolated from submaxillary gland extracts in a ratio of 3:1 regardless of whether the classical isolation procedure or our rapid RPLC based technique was used. Both molecular forms were present in the same ratio in saliva of mice of both sexes when salivation was induced by epinephrine treatment of the animals. As judged by amino acid analysis and N-terminal sequencing salivary EGF I corresponds to the 53 amino acid sequence of murine EGF and EGF II is Des-ASN-EGF. The observation that EGF and Des-ASN-EGF are consecreted into saliva upon epinephrine stimulation implies a physiological role of EGF II which may be related to the high molecular weight EGF-complex.