Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.     

The primary structure of the alpha subunit of human elongation factor 1. Structural aspects of guanine-nucleotide-binding sites

The primary structure of the alpha subunit of elongation factor 1 (EF-1 alpha) from human MOLT 4 cells was determined by cDNA sequencing. The data show that the conservation of the amino acid sequence is more than 80% when compared with yeast and Artemia EF-1 alpha. An inventory of amino acid sequences around the guanine-nucleotide-binding site in elongation factor Tu from Escherichia coli and homologous amino acid sequences in G proteins, initiation and elongation factors and proteins from the RAS family shows two regions containing conserved sequence elements. Region I has the sequence apolar-Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Yaa-Xaa-Gly-LYs-Thr(Ser)- -Xaa-Xaa-Xaa-Xaa-X-apolar. Except for RAS proteins, Yaa is always an acidic amino acid residue. Region II is characterized by the invariant sequence apolar-apolar-Xaa-Xaa-Asn-Lys-Xaa-Asp. In order to facilitate sequence comparison we have used a graphic display, which is based on the hydrophilicity values of individual amino acids in a sequence.     

Wet cleaving of cells: A method to introduce macromolecules into the cytoplasm : Application for immunolocalization of cytosol-exposed antigens

A new method, called "wet cleaving," has been introduced to allow direct exposure of cytoplasm to externally supplied macromolecules by mechanical rupture of the plasma membrane. Monolayers of adherent cells or poly-L-lysine-attached suspension cells are overlayed with nitrocellulose sheets. By subsequent removal of the sheets, cells are cleaved, thereby exposing the cytoplasm. The method allows bulk quantities of cells to be cleaved in an efficient manner. Cleavage, although imposing some mechanical stress on the cells, leaves most if not all organelles morphologically intact, as shown by electron microscopy. Mechanically ruptured cells are well suited for use in immunocytochemical studies, as is demonstrated with the immunofluorescence localization of vinculin in chicken embryo fibroblasts.     

Aspergillus fumigatus chsE: A Gene Related to CHS3 of Saccharomyces cerevisiae and Important for Hyphal Growth and Conidiophore Development but Not Pathogenicity

The Aspergillus fumigatus chsE (AfchsE) gene was isolated from an A. fumigatus DNA library on the basis of hybridization to a segment of Saccharomyces cerevisiae CHS3 (ScCHS3). The amino acid sequence derived from AfchsE is 28% identical with ScCHS3 and 80% identical with the product of Aspergillus nidulans chsD (AnchsD). A mutant strain constructed by disruption of AfchsE has reduced levels of mycelial chitin, periodic swellings along the length of hyphae, and a block in conidiation that can be partially restored by growth in osmotic stabilizer. This phenotype is different from that reported for an AnchsD mutant, in which germinating conidia and hyphal tips undergo lysis and the colonial growth rate is significantly reduced. Despite the defects associated with the AfchsE- strain, its virulence was not significantly reduced when compared with the wild-type parental strain in a mouse model of pulmonary aspergillosis.     

Suppression of food intake and body weight gain by naloxone in rats

The effect of acute and chronic administration of naloxone on food acquisition and weight gain in rats was studied in 3 experiments. One injection of a sparingly-soluble salt of naloxone in slow-release vehicle markedly lowered mean food intake over that of control rats injected with the vehicle only. Mean body weight of the naloxone-injected rats was significantly lower than that of the control group for one week.Repeated evening injections (2000 h) of naloxone hydrochloride in saline tended to reduce the night-time feeding below control levels throughout the 10-day period of naloxone administration. Food intake was significantly lower in the 4- and 8-h periods after the first injection of naloxone than that on the preceding saline control night. The initial decreases were offset by increased day-time feeding so that total daily food intake was not significantly altered over the 10 days. When saline was substituted for naloxone, food intake increased.Rats given naloxone following 24 h of fasting consumed significantly less food and gained less weight during 4 h of access to food compared to those receiving saline. After a 48-h fast naloxone-treated rats also gained significantly less body weight than those given saline, but the reduction in food intake was not statistically significant. These results suggest the possibility that endorphins may have a modulating effect on feeding activity.     

Disintegration of adhesion plaques in chicken embryo fibroblasts upon Rous sarcoma virus-induced transformation: different dissociation rates for talin and vinculin

The localization of talin and vinculin in chicken embryo fibroblasts (CEF) during transformation was studied by immunoelectron microscopy. CEF cells were infected with a temperature-sensitive mutant of Rous sarcoma virus. After 16 h at 42 degrees C, transformation was induced by incubation at 37 degrees C for different intervals up to 3 h. Cells were cleaved by "wet cleaving" as reported previously by us (R. Brands and C.A. Feltkamp, 1988, Exp. Cell Res. 176, 309) and labeled with affinity-purified polyclonal antibodies to talin or vinculin, or monoclonal anti-vinculin. We observed a rapid reduction of vinculin in adhesion plaques within 15 min and a much slower dissociation of talin. This was found using single-labeling procedures and also within the same cell using double labeling. Seemingly intact microfilament bundles were observed associated with adhesion plaques that contained relatively little vinculin. These observations show that an early event in src-induced transformation is the release of vinculin from adhesion plaques. Furthermore, since adhesion plaques with attached filament bundles can exist at least transiently with very little or no vinculin present, it seems likely that vinculin is not, or not the only protein, linking actin filaments to adhesion plaques.     

Sister chromatid cohesion role for CDC28-CDK in Saccharomyces cerevisiae

High-fidelity chromosome segregation requires that the sister chromatids produced during S phase also become paired during S phase. Ctf7p (Eco1p) is required to establish sister chromatid pairing specifically during DNA replication. However, Ctf7p also becomes active during G(2)/M in response to DNA damage. Ctf7p is a phosphoprotein and an in vitro target of Cdc28p cyclin-dependent kinase (CDK), suggesting one possible mechanism for regulating the essential function of Ctf7p. Here, we report a novel synthetic lethal interaction between ctf7 and cdc28. However, neither elevated CDC28 levels nor CDC28 Cak1p-bypass alleles rescue ctf7 cell phenotypes. Moreover, cells expressing Ctf7p mutated at all full- and partial-consensus CDK-phosphorylation sites exhibit robust cell growth. These and other results reveal that Ctf7p regulation is more complicated than previously envisioned and suggest that CDK acts in sister chromatid cohesion parallel to Ctf7p reactions.     

Function of a plant stress-induced gene,HVA22. Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic

Expression of the barley (Hordeum vulgare) HVA22 gene is induced by environmental stresses, such as dehydration, salinity, and extreme temperatures, and by a plant stress hormone, abscisic acid. Genes sharing high level of sequence similarities with HVA22 exist in diverse eukaryotic organisms, including animals, plants, and fungi, but not in any prokaryotic organisms. The yeast (Saccharomyces cerevisiae) HVA22 homolog, Yop1p, has been shown to interact with the GTPase-interacting protein, Yip1p. Deletion of YOP1 led to only a modest reduction of the stationary phase titer at 37C. A synthetic enhancement mutant screen was performed in the yop1 deletion background to identify genes interacting with YOP1. The open reading frame YOR165W (renamed SEY1 for synthetic enhancement of YOP1) was identified as a YOP1-dependent complementation gene. The yeast SEY1 is a homolog of the Arabidopsis RHD3 gene whose mutations cause the accumulation of transport vesicles near the tips of defective root hairs. The yeast double mutant of yop1 and sey1 is defective in vesicular traffic as evidenced by the accumulation of transport vesicles and the decrease in invertase secretion. Based on these observations, we suggest that Yop1p/HVA22 regulates vesicular traffic in stressed cells either to facilitate membrane turnover, or to decrease unnecessary secretion.     

Identification and characterization of the first class of potent bacterial acetyl-CoA carboxylase inhibitors with antibacterial activity

The multisubunit acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis, is broadly conserved among bacteria. Its rate-limiting role in formation of fatty acids makes this enzyme an attractive target for the design of novel broad-spectrum antibacterials. However, no potent inhibitors have been discovered so far. This report describes the identification and characterization of highly potent bacterial acetyl-CoA carboxylase inhibitors with antibacterial activity for the first time. We demonstrate that pseudopeptide pyrrolidine dione antibiotics such as moiramide B inhibit the Escherichia coli enzyme at nanomolar concentrations. Moiramide B targets the carboxyltransferase reaction of this enzyme with a competitive inhibition pattern versus malonyl-CoA (K(i) value = 5 nm). Inhibition at nanomolar concentrations of the pyrrolidine diones is also demonstrated using recombinantly expressed carboxyltransferases from other bacterial species (Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa). We isolated pyrrolidine dione-resistant strains of E. coli, S. aureus, and Bacillus subtilis, which contain mutations within the carboxyltransferase subunits AccA or AccD. We demonstrate that such mutations confer resistance to pyrrolidine diones. Inhibition values (IC(50)) of >100 microm regarding an eukaryotic acetyl-CoA carboxylase from rat liver indicate high selectivity of pyrrolidine diones for the bacterial multisubunit enzyme. The natural product moiramide B and synthetic analogues show broad-spectrum antibacterial activity. The knowledge of the target and the availability of facile assays using carboxyltransferases from different pathogens will enable evaluation of the antibacterial potential of the pyrrolidine diones as a promising antibacterial compound class acting via a novel mode of action.     

High Level Expression of Tissue-Nonspecific Alkaline Phosphatase in the Milk of Transgenic Rabbits

Alkaline phosphatase is a promising therapeutic agent in the Gram-negative bacterial lipopolysaccharide (LPS) mediated acute and chronic diseases. Contrary to other alkaline phosphatase isozymes, purified tissue-nonspecific alkaline phosphatase (TNAP) is not available in large quantities from tissue sources, which would enable to analyse its efficacy in animal sepsis models. Two transgenic rabbit lines were created by pronuclear microinjection with the whey acidic protein promoter-humanTNAP minigene (WAP-hTNAP). Lactating females of both lines produced biologically active human TNAP. As indicated by fractionation of milk samples the recombinant alkaline phosphatase was associated with the membrane of milk fat globules. Alkaline phosphatase enzymatic activity was two orders of magnitude higher compared to normal human serum levels. The demonstration that this TNAP is physiologically active would provide the clue to use transgenic animals as bioreactor for bulk production of the human tissue-nonspecific alkaline phosphatase in milk. This may be a valuable and possibly viable option with important implication in attenuating LPS mediated inflammatory responses.