Drosophila projectin: relatedness to titin and twitchin and correlation with lethal(4) 102 CDa and bent-dominant mutants.

We have investigated projectin, a large protein of insect muscles, in Drosophila melanogaster. The 5.3 kilobases of coding sequence reported here contains Class I and Class II motifs characteristic of titin and twitchin, arranged in a three domain ... [II-I-I] [II-I-I] ... pattern. Two mutants mapped to the location of the projectin gene in the 102C subdivision of chromosome 4, lethal(4) 102 CDa and bent-Dominant, have DNA rearrangements within their projectin gene. The lethal(4) 102 CDa mutant has a 141 nucleotide insertion containing stop codons in all three reading frames within an exon sequence, showing that it cannot synthesize normal projectin. Both bent-Dominant and lethal(4) 102 CDa homozygotes die at the completion of embryogenesis because they are unable to escape the egg vitelline membrane. We propose that this hatching failure is due to muscle weakness caused by projectin defects.     

Arthrin: a new actin-like protein in insect flight muscle

There are one or more proteins of 50,000 to 60,000 Mr in the thin filaments of insect flight muscle. A protein of 55,000 Mr has been isolated from insect fibrillar flight muscle and called arthrin. Despite its higher molecular weight, arthrin is in many ways like actin. The amino acid composition of arthrin was similar to that of actin. There were similarities in the peptides produced by digesting the denatured proteins and mild digestion of polymerized proteins cleaved similar-sized fragments from arthrin and actin. Polymerized arthrin activated the Mg2+ ATPase of myosin to the same extent as actin and the ATPase was regulated by rabbit or Lethocerus troponin and tropomyosin. Arthrin did not itself act as troponin-T. Electron microscopy of negatively stained specimens showed that arthrin and actin filaments were similar in structure and that arthrin could be decorated by rabbit subfragment-1 to form normal-looking arrowheads. Arthrin formed paracrystals at an optimum concentration of MgCl2 (25 mM) that was somewhat lower than the optimum for actin paracrystals. Optical diffraction showed that the structure of the paracrystals was similar to those formed from actin. The mass of arthrin and actin filaments relative to phage fd was measured by scanning transmission electron microscopy; the relative mass of arthrin and actin was 1.33, in agreement with molecular weight estimations. Therefore arthrin has the properties of a heavy form of actin. The proportion of actin, arthrin and troponin-T in Lethocerus myofibrils was six moles of actin to one mole of arthrin and one mole of troponin-T. The function of arthrin is not known.     

Experience using two CT-guided stereotactic biopsy methods

15 patients had intracranial CT-guided stereotactic biopsies. Biopsies were performed either with a Riechert-Mundinger stereotactic frame modified for use in the CT or by using the CT scan to establish the relationship of the intracranial lesion to identifiable bony landmarks, and subsequently performing the biopsy in a standard stereotactic frame. Both systems provided safe and accurate methods for obtaining intracranial tissue.     

Molecular and Genetic Analysis of Rec103, an Early Meiotic Recombination Gene in Yeast

In the yeast Saccharomyces cerevisiae at least 10 genes are required to begin meiotic recombination. A new early recombination gene REC103 is described in this paper. It was initially defined by the rec103-1 mutation found in a selection for mutations overcoming the spore inviability of a rad52 spo13 haploid strain. Mutations in REC103 also rescue rad52 in spo13 diploids. rec103 spo13 strains produce viable spores; these spores show no evidence of meiotic recombination. rec103 SPO13 diploids produce no viable spores, consistent with the loss of recombination. Mutations in REC103 do not affect mitotic recombination, growth, or repair. These phenotypes are identical to those conferred by mutations in several other early meiotic recombination genes (e.g., REC102, REC104, REC114, MEI4, MER2, and SPO11). REC103 maps to chromosome VII between ADE5 and RAD54. Cloning and sequencing of REC103 reveals that REC103 is identical to SKI8, a gene that depresses the expression of yeast double-stranded (``killer') (ds)RNA viruses. REC103/SKI8 is transcribed in mitotic cells and is induced ~15-fold in meiosis. REC103 has 26% amino acid identity to the Schizosaccharomyces pombe rec14(+) gene; mutations in both genes confer similar meiotic phenotypes, suggesting that they may play similar roles in meiotic recombination.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

The anomeric form of D-fructose 1,6-bisphosphate used as substrate in the muscle and yeast aldolase reactions.

From a series of rapid quench kinetic experiments, it has been demonstrated that muscle D-fructose bisphosphate aldolase catalyzes the cleavage of beta-D-fructose 1,6-bisphosphate but not that of the alpha anomer, although the alpha anomer may be tightly bound. Yeast D-fructose bisphosphate aldolase appears to utilize both alpha and beta anomers of the substrate, with yeast apoaldolase catalyzing the interconversion of the alpha and beta forms.     

Glutaminyl‐tRNA Synthetase from Pseudomonas aeruginosa: Characterization,structure, and development as a screening platform

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl‐tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the K_M and k_cat^obs, governing interactions with tRNA, were 1.0 μM and 0.15 s^?1, respectively. The crystal structure of the α₂ form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three‐dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad‐spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild‐type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time‐kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 μg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.