Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.     

Use of the free dorsalis pedis flap in head and neck repairs.

Many defects of the head and neck can be readily repaired with a free dorsalis pedis flap, and we report success with these flaps in 9 of 12 cases. A precise knowledge of the anatomy of the arterial supply of the flap is necessary. Preoperative arteriography is recommended if the dorsalis pedis artery is not easily palpable, or if an anomalous distribution of the artery along the dorsum of the foot is sus pected. However, the transfer of the flap should be delayed for two weeks after preoperative arteriography is performed. The one-stage soft tissue reconstruction with a free dorsalis pedis flap has been associated with minimal morbidity and good acceptance by patients. A delay procedure for the flap seems to enhance the chances of complete survival which is so necessary in the repair of intraoral and pharyngeal defects. Careful attention to details and close monitoring of the flap will minimize morbidity. In case of an early failure of a flap, a secondary reconstruction by a different flap can be done in the first 48 to 72 hours. Early postoperative radiotherapy has been well tolerated over these free flaps.     

The accumulation of drugs within large unilamellar vesicles exhibiting a proton gradient: a survey

We have shown previously that transmembrane proton gradients can be used to efficiently accumulate biogenic amines [M.B. Bally et al. (1988) Chem. Phys. Lipids 47, 97-107] and doxorubicin [L.D. Mayer, M.B. Bally and P.R. Cullis (1986) Biochim. Biophys. Acta 857, 123-126] to high concentrations within liposomes. To determine the generality of this loading procedure, representative drugs from a variety of different classes (antineoplastics, local anaesthetics, antihistamines, etc.) were examined as to their ability to redistribute in response to a proton gradient. While the majority of drugs examined, all of which are weak bases, were accumulated by large unilamellar vesicles exhibiting a pH gradient (interior acid) the extent of uptake varied considerably between different pharmaceuticals. These differences are discussed in the context of various factors which will likely influence drug accumulation including its membrane/water partition coefficient and its solubility in the intravesicular medium.     

Effect of temperature and host plant on the development of the blackfaced leafhopper

Population dynamics of the blackfaced leafhopper, Graminella nigrifrons (Forbes) (Homoptera: Cicadellidae), was studied at five temperatures (18, 21, 24, 27, & 30°C) in the laboratory on seedling maize (Zea mays L.), oats (Avena sativa L.), and the perennial johnsongrass (Sorghum halepense (L.) Pers.). Effects of temperature and host plant on egg to adult mean development time, adult size and weight, and fecundity were determined. Leafhoppers on all three hosts developed fastest at the highest temperature tested (21.3 days), and slowest at the lowest temperature tested (73.2 days). The duration from first to last adult eclosion was shortest at 30°C, (11.5 days) and longest at 18°C (43 days). The sex ratio of males to females did not differ from 1:1, but males developed an average of 1.2 days faster than females on all three hosts. Mean percent development/day ranged from 1.4% at 18°C to 4.7% at 30°C. The relationship of this development rate and temperature was determined using both a linear model and a variable parameter biophysical model. Based on these models, the developmental threshold is estimated at 12–15°C. The lowest temperature yielded larger and heavier adults (312 g, dry weight) than did the highest temperature (225 g). Fewer leafhoppers developed on the perennial than the annuals at 30°C and fewer on the annuals than the perennial at 18°C. Our results suggest that early in the season johnsongrass and perhaps other perennials are the superior developmental hosts for this leafhopper, whereas in midsummer when temperatures are highest, annuals are the better hosts.

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Résumé La dynamique des populations (durée de développement de l'uf à l'adulte, poids et taille des adultes, fécondité) de G. nigrifrons Forbes (Homop. Cicadellidae) a été étudiée au laboratoire à 5 températures sur plantules de maïs (Zea mays L.), avoine (Avena sativa L.) et sorgho vivace (Shorgum halepense (L.) Pers.). Sur les 3 plantes, les mâles se développent en moyenne 1,2 j plus vite que les femelles. Les relations entre vitesse de développement et température ont été déterminées en utilisant à la fois un modèle linéaire et le modèle biophysique à 2 paramètres de Sharpe & DeMichele (1977).Les températures plus basses donnent des adultes des 2 sexes plus gros et plus lourds. Moins de G. nigrifrons se sont développés sur la graminée vivace que sur les 2 graminées annuelles à la température la plus élevée (30°C), tandis qu'à la température la plus basse (18°C) moins de cicadelles se sont développées sur les graminées annuelles. La température semble jouer un rôle significatif en déterminant l'adéquation des plantes comme hôtes convenant au développement de G. nigrifrons. Le potentiel de ponte de cette cicadelle avait été sous-estimé par les étudies précédentes.

     

Identification of novel Drosophila meiotic genes recovered in a P-element screen.

The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes alphaTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.     

Acetaldehyde (CH3CHO) production in rodent lung after exposure to metal-rich particles

Epidemiological reports demonstrate an association between increased human morbidity and mortality with exposure to air pollution particulate matter (PM). Metal-catalyzed oxidative stress has been postulated to contribute to lung injury in response to PM exposure. We studied the effects of residual oil fly ash (ROFA), a component of ambient air PM, on the formation of lung carbonyls that are indicators of lipid peroxidation. Rats were instilled intratracheally with ROFA (62.5–1000 μg) and underwent lung lavage. Lavage fluid carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and measured by high performance liquid chromatography with UV detection. Dose-dependent increases in a peak that eluted with the same retention time as the acetaldehyde (CH₃CHO) derivative was observed in rats treated with ROFA 15 min after instillation (up to 25-fold greater than saline treated controls). The identification of CH₃CHO was confirmed using gas chromatography-mass spectroscopy. ROFA-induced increases in other lavage fluid carbonyls were not seen. Increased CH₃CHO in lavage fluid was observed as late as 8 h later. No increase in CH₃CHO was observed in plasma from ROFA-treated rats. An increased formation of CH₃CHO was observed in a human airway epithelial cell line incubated with ROFA suggesting a pulmonary source of CH₃CHO production. Instillation of solutions of metals (iron, vanadium, nickel) contained in ROFA, or instillation of another ROFA-type particle containing primarily iron, also induced a specific increase in CH₃CHO. These data support the hypothesis that metals were involved in the increased CH₃CHO formation. Thus metals on PM may mediate lung responses through induction of lipid peroxidation and carbonyl formation.     

Aspartate facilitates mitochondrial function,growth arrest and survival during doxorubicin exposure

Genomic screens of doxorubicin toxicity in S. cerevisiae have identified numerous mutants in amino acid and carbon metabolism which express increased doxorubicin sensitivity. This work examines the effect of amino acid metabolism on doxorubicin toxicity. S. cerevisiae were treated with doxorubicin in combination with a variety of amino acid supplements. Strains of S. cerevisiae with mutations in pathways utilizing aspartate and other metabolites were examined for sensitivity to doxorubicin. S. cerevisiae cultures exposed to doxorubicin in minimal media showed significantly more toxicity than cultures exposed in rich media. Supplementing minimal media with aspartate, glutamate or alanine reduced doxorubicin toxicity. Cell cycle response was assessed by examining the budding pattern of treated cells. Cultures exposed to doxorubicin in minimal media arrested growth with no apparent cell cycle progression. Aspartate supplementation allowed cultures exposed to doxorubicin in minimal media to arrest after one division with a budding pattern and survival comparable to cultures exposed in rich media. Aspartate provides less protection from doxorubicin in cells mutant in either mitochondrial citrate synthase (CIT1) or NADH oxidase (NDI1), suggesting aspartate reduces doxorubicin toxicity by facilitating mitochondrial function. These data suggest glycolysis becomes less active and mitochondrial respiration more active following doxorubicin exposure.     

Aging and death in an organism that reproduces by morphologically symmetric division

In macroscopic organisms, aging is often obvious; in single-celled organisms, where there is the greatest potential to identify the molecular mechanisms involved, identifying and quantifying aging is harder. The primary results in this area have come from organisms that share the traits of a visibly asymmetric division and an identifiable juvenile phase. As reproductive aging must require a differential distribution of aged and young components between parent and offspring, it has been postulated that organisms without these traits do not age, thus exhibiting functional immortality. Through automated time-lapse microscopy, we followed repeated cycles of reproduction by individual cells of the model organism Escherichia coli, which reproduces without a juvenile phase and with an apparently symmetric division. We show that the cell that inherits the old pole exhibits a diminished growth rate, decreased offspring production, and an increased incidence of death. We conclude that the two supposedly identical cells produced during cell division are functionally asymmetric; the old pole cell should be considered an aging parent repeatedly producing rejuvenated offspring. These results suggest that no life strategy is immune to the effects of aging, and therefore immortality may be either too costly or mechanistically impossible in natural organisms.     

Association of the cystic fibrosis transmembrane regulator with CAL: structural features and molecular dynamics

The association of the cystic fibrosis transmembrane regulator (CFTR) with two PDZ-containing molecular scaffolds (CAL and EBP50) plays an important role in CFTR trafficking and membrane maintenance. The CFTR-molecular scaffold interaction is mediated by the association of the C-terminus of the transmembrane regulator with the PDZ domains. Here, we characterize the structure and dynamics of the PDZ of CAL and the complex formed with CFTR employing high-resolution NMR. On the basis of NMR relaxation data, the alpha2 helix as well as the beta2-beta3 loop of CAL PDZ domain undergoes rapid dynamics. Molecular dynamics simulations suggest a concerted motion between the alpha2 helix and the beta1-beta2 and beta2-beta3 loops, elements which define the binding pocket, suggesting that dynamics may play a role in PDZ-ligand specificity. The C-terminus of CFTR binds to CAL with the final four residues (-D(-)(3)-T-R-L(0)) within the canonical PDZ-binding motif, between the beta2 strand and the alpha2 helix. The R(-)(1) and D(-)(3) side chains make a number of contacts with the PDZ domain; many of these interactions differ from those in the CFTR-EBP50 complex, suggesting sites that can be targeted in the development of PDZ-selective inhibitors that may help modulate CFTR function.     

Accelerated prediction of recombinant protein production in Saccharomyces cerevisiae by using rapid monitoring techniques

The use of a stopped-flow analyser for the monitoring of the production of a secreted recombinant protein, a wild-type cutinase, from S. cerevisiae CEN.PK111-32D pUR7320 is described. Induction is through use of a galactose promoter, and the monitoring facility is used to record the formation of the cutinase and cell density with optical density measurements. A range of induction conditions was studied with a view to using the monitoring to predict the likely level of cutinase formation. Results achieved within 4 to 5 h of induction were of sufficient quality to allow the use of simple modelling relating cutinase formation and cell production to predict likely final specific activities of the product. The utility of such monitoring and prediction is discussed with regards to improved process confidence and definition during fermentation production.