Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione.

Reactive oxygen species (ROS) are mediators of lung injury, and glutathione (GSH) is the major nonprotein antioxidant that protects the cell from oxidative stress. We have recently shown that H(2)O(2) induces ceramide-mediated apoptosis in human lung epithelial cells. We hypothesized that ROS-mediated depletion of GSH plays a regulatory role in ceramide generation, and thus in the induction of apoptosis. Our present studies demonstrate that GSH at physiologic concentrations (1 to 10 mM) inhibits ceramide production in a time- and dose-dependent manner in A549 human alveolar epithelial cells. On the other hand, buthionine-sulfoximine-mediated depletion of intracellular GSH induces elevation of ceramide levels and apoptosis. In addition, GSH blocks H(2)O(2)-mediated induction of intracellular ceramide generation and apoptosis. These effects were not mimicked by oxidized GSH (GSSG) or other thiol antioxidants, such as dithiothreitol and 2-mercaptoethanol. Moreover, increase of intracellular H(2)O(2), mediated by inhibition of catalase by aminotriazole, also induces ceramide generation and apoptosis. These effects were blocked by N-acetylcysteine. Our results suggest that GSH depletion may be the link between oxidative stress and ceramide-mediated apoptosis in the lung.

The Drosophila UBC9 homologue lesswright mediates the disjunction of homologues in meiosis I.

BACKGROUND: In Saccharomyces cerevisiae and other organisms, the UBC9 (ubiquitin-conjugating 9) protein modifies the function of many different target proteins through covalent attachment of the ubiquitin-like protein SMT-3/SUMO. RESULTS: Using a second-site suppression screen of a mutation in the nod locus with a variable meiotic phenotype, we have identified mutations in the Drosophila melanogaster UBC9 homologue, encoded by the gene lesswright (lwr). lwr mutations dominantly suppress the nondisjunction and cytological defects of female meiotic mutations that affect spindle formation. The lwr lethal phenotype is rescued by a Drosophila UBC9/lwr transgene. CONCLUSIONS: We suggest that LWR mediates the dissociation of heterochromatic regions of homologues at the end of meiotic prophase I. Our model proposes that when there is less LWR protein, homologues remain together longer, allowing for more normal spindle formation in mutant backgrounds and therefore more accurate meiotic chromosome segregation.

Isolation and analysis of fluP, a gene associated with hyphal growth and sporulation in Aspergillus parasiticus.

Aflatoxins (AF) are polyketide-derived mycotoxins that frequently contaminate food and feed crops, causing health risks to animals and humans. The fluP gene was cloned by screening an Aspergillus parasiticus genomic DNA library with a cDNA probe encoding part of a polyketide synthase (PKS), the 6-methylsalicylic acid synthase (MSAS) from Penicillium patulum. FluP was hypothesized to function as a PKS in AF biosynthesis. The predicted amino acid sequence of FluP demonstrated a high degree of identity to MSAS (55%), moderate identity to another fungal PKS protein encoded by wA from A. nidulans (22%) and low identity (<5%) to fungal fatty acid synthase (FAS) proteins. Disruption of fluP in A. parasiticus resulted in the loss of fluP transcript, a 3- to 4-fold reduction in hyphal growth rate, the appearance of a fluffy, cotton-like hyphal morphology, reduction or elimination of asexual spores and spore-bearing structures, and a twofold reduction in aflatoxin accumulation. Removal of selective pressure on fluP knockout transformants resulted in frequent reversion (10%) to the wild-type genotype and phenotype, establishing a direct link between gene disruption and the associated phenotype. The data suggest that fluP encodes a novel PKS associated with hyphal growth and cell development (sporulation), whose activity indirectly influences aflatoxin accumulation in A. parasiticus.

Detection and analysis of Staphylococcal enterotoxin A in food by Western immunoblotting.

Western blotting has the potential to overcome some of the major problems associated with enzyme-linked immunosorbent assay (ELISA) detection of toxins in food, such as cross-reactivity with unrelated antigens and insensitivity with heat-treated foods, because the Western procedure solubilizes denatured protein and allows characterization of the antigen that reacts with the antibody. A simple Western immunoblotting protocol was developed to identify and measure the level of Staphylococcus aureus enterotoxin A (SEA) in food. Test samples are merely homogenized with no additional solubilization or pretreatment steps. The immunoblots detect SEA at levels as low as 100 pg/ml. Using the simplified sample preparation, both native and heat-denatured SEA were identified in a variety of foods including mushrooms, milk, potato salad and meat products. Our data suggest that SEA is being secreted at mid-log growth in BHI media as well as in mushrooms. These results suggest that Western blotting is a useful tool for determining the presence of SEA in foods because it allows characterization of the antigen reacting with the antibody and can be used for heat-treated foods, thus overcoming some of the limitations of the ELISA test.

Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus.

Staphylococcus aureus causes pathologies ranging from minor skin infections to life-threatening diseases. Pathogenic effects are largely due to production of bacterial toxin, which is regulated by an RNA molecule, RNAIII. The S. aureus protein called RAP (RNAIII activating protein) activates RNAIII, and a peptide called RIP (RNAIII inhibiting peptide), produced by a nonpathogenic bacteria, inhibits RNAIII. Mice vaccinated with RAP or treated with purified or synthetic RIP were protected from S. aureus pathology. Thus, these two molecules may provide useful approaches for the prevention and treatment of diseases caused by S. aureus.

How rolling circle plasmids control their copy number.

Rolling circle DNA replication is inherently continuous and unregulated. This 'go-for-broke' strategy works well for lytic phages but is suicidal for plasmids that must coexist with their host. Plasmids have consequently evolved elaborate copy number control systems that operate at the transcriptional, translational and post-translational levels.

The inactivated plasmid inititator protein RepC/RepC* may have a regulatory role.

During replication of the plasmid pT181, the initiator protein RepC is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. Here we show that during in vitro replication, RepC* is radioactively labeled, suggesting that the source of the RepC* oligodeoxynucleotide is the newly synthesized pT181 DNA. The RepC/RepC* heterodimer retains its ability to bind the pT181 double-strand origin and, therefore, it may act as a competitive inhibitor of the RepC homodimer during replication.

Morewright (mwr), a new meiotic mutant of Drosophila melanogaster affecting nonexchange chromosome segregation.

A new meiotic mutation, morewright (mwr) was identified by screening for new mutations that act as dominant enhancers of the dosage-sensitive Drosophila melanogaster female meiotic mutant, nodDTW.mwr is a recessive meiotic mutant, specifically impairing the segregation of nonexchange chromosomes. Cytological evidence suggests that the meiotic defect in mwr/mwr females is in homologue recognition because the chromosomes appear to be misaligned on an intact spindle. The mwr mutation was recovered during a screen of random P-element insertions on a chromosome with a single insertion located at 50C. The P-element insertion is a recessive female-sterile mutation. While excision of the P element from the mwr-bearing chromosome partially relieves the female sterility, the excisions retain the dominant nodDTW-enhancing activity. The mwr meiotic phenotype maps very close to the female-sterile P insertion. Thus the mwr locus appears to encode a function required for partner recognition in meiosis, although its relationship to the neighboring female-sterile mutation remains to be elucidated.

Modification of the plasmid initiator protein RepC active site during replication.

pT181 is a Staphylococcus aureus rolling circle replicating plasmid whose copy number is controlled by regulating the synthesis and activity of the initiator protein, RepC*. The RepC* dimer is modified during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC. To purify RepC, RepC was expressed in S. aureus as a fusion protein with a polyhistidine tail. The histidine-tagged RepC retains its initiation and topoisomerase activities in vitro. His-tagged RepC/RepC and RepC/RepC* were purified in a two-step procedure. Peptide mapping, mass spectrometric analysis and protein sequencing of purified RepC and RepC* were carried out, and both proteins appeared identical, except that the peptide containing the RepC active site tyrosine used in nicking activity was absent when the purified RepC* sample was analyzed. The absence of the active site in RepC* suggests that this site was modified during replication. The results provide the first direct biochemical evidence that RepC* is a modified form of RepC, and support a model in which RepC replication of pT181 leaves RepC with an oligonucleotide blocking the active site of one of its subunits.

Separation anxiety: the etiology of nondisjunction in flies and people.

Two new studies examine the recombinational history of human chromosomes that nondisjoin at the first meiotic division in females. Our analysis of these studies suggests two possible etiologies of nondisjunction in terms of well-understood properties of chromosome mechanics. For both the X chromosome and for chromosome 21, 60-70% of nondisjoined chromosomes are derived from chiasmate bivalents, many of which display unusual patterns of exchange. The patterns of exchange and nondisjunction observed for human chromosome 21 parallel those exhibited by a mutation in Drosophila that impairs spindle assembly and function. Based on these similarities, we propose that nondisjunction of chromosome 21 in human females results from an age-dependent loss of spindle-forming ability. The recombinational histories of nondisjoining human X chromosomes are quite different from those of chromosome 21, but rather parallel those obtained for spontaneous nondisjunction in Drosophila females. The data for X chromosome disjunction in both species can be explained by a model in which nondisjunction is the consequence of the age-dependent movement of transposable elements. According to this model, nondisjunction is explained as the consequence of the repair of transposon-induced breaks in the DNA. Both models provide reasonable alternatives to biologically implausible explanations such as the 'production line hypothesis'.

A structure-function analysis of NOD, a kinesin-like protein from Drosophila melanogaster.

We have analyzed a collection of 12 mutations in the Drosophila melanogaster nod locus, which encodes a kinesin-like protein involved in female meiotic chromosome segregation. The kinesin-like domain is at the N-terminus of the protein, while the C-terminal portion of the protein is unique. Four of the mutations are missense and affect highly conserved domains of the kinesin-like portion of the predicted protein, and thus demonstrate that the sequence conservation is biologically relevant. Surprisingly, two other mutations, which behave genetically as null alleles, are the result of mutations in the last exon of the nod gene. Thus, these two mutations affect the most C-terminal residues in the unique portion of the predicted protein. Based on these mutations, we suggest that this part of the protein may also be essential for wild-type function. The mutations were induced by either gamma-rays or ethyl methanesulfonate (EMS). All of the gamma-ray induced mutations were small or large chromosomal rearrangements, while all of the EMS mutations were G-->A transitions. These findings are consistent with the biochemical basis of the mode of action of each mutagen.

The lethal(1)TW-6cs mutation of Drosophila melanogaster is a dominant antimorphic allele of nod and is associated with a single base change in the putative ATP-binding domain.

The l(1)TW-6cs mutation is a cold-sensitive recessive lethal mutation in Drosophila melanogaster, that affects both meiotic and mitotic chromosome segregation. We report the isolation of three revertants of this mutation. All three revert both the meiotic and mitotic effects as well as the cold sensitivity, demonstrating that all three phenotypes are due to a single lesion. We further show that these revertants fail to complement an amorphic allele of the nod (no distributive disjunction) locus, which encodes a kinesin-like protein. These experiments demonstrate that l(1)TW-6cs is an antimorphic allele of nod, and we rename it nodDTW. Sequencing of the nod locus on a nodDTW-bearing chromosome reveals a single base change in the putative ATP-binding region of the motor domain of nod. Recessive, loss-of-function mutations at the nod locus specifically disrupt the segregation of nonexchange chromosomes in female meiosis. We demonstrate that, at 23.5 degrees, the meiotic defects in nodDTW/+ females are similar to those observed in nod/nod females; that is, the segregation of nonexchange chromosomes is abnormal. However, in nodDTW/nodDTW females, or in nodDTW/+ females at 18 degrees, we observe a more severe meiotic defect that apparently affects the segregation of both exchange and nonexchange chromosomes. In addition, nodDTW homozygotes and hemizygous males have previously been shown to exhibit mitotic defects including somatic chromosome breakage and loss. We propose that the defective protein encoded by the nodDTW allele interferes with proper chromosome movement during both meiosis and mitosis, perhaps by binding irreversibly to microtubules.

Rex and a suppressor of Rex are repeated neomorphic loci in the Drosophila melanogaster ribosomal DNA.

The Rex locus of Drosophila melanogaster induces a high frequency of mitotic exchange between two separated ribosomal DNA arrays on a single chromosome. The exchanges take place in the progeny of Rex mothers and occur very early, before the third mitotic division. A number of common laboratory stocks have also been found to carry dominant suppressors of Rex (Su(Rex)). Rex was mapped to the X centric heterochromatin, proximal to su(f), by genetic and molecular analysis of two spontaneous recombinants. Using deficiencies and duplications of the heterochromatin, both Rex and one Su(Rex) were shown to behave as neomorphs. Rex-induced exchange in a target chromosome bearing both Rex and Su(Rex) was then used to map these functions to the bb locus itself. Molecular analysis of the recombinants, using length variants of the ribosomal DNA intergenic spacer as genetic markers, mapped Su(Rex) and Rex within the bb locus and demonstrated that both are repeated elements.

Assessment of intraocular lens power on the basis of precataract refractive measurements.

Assessment of intraocular lens (IOL) power may rely, when biometric measurements of the eye are not available, on refraction previous to cataract development. Such an assessment, however, is generally considered unreliable. To improve the predictability of this method, we correlated refraction measurements with predictions of IOL power. In contrast to previous studies which used retrospective assessment of precataract refraction, this study included only noncataractous eyes and correlated the direct refractive measurements obtained with hypothetical IOL power values. These values were calculated by placing axial length and corneal power measurements of the same eyes in the SRK formula. We conclude that the regression formula obtained may improve the clinical judgment required for predicting lens implant power in cataractous eyes, on the basis of precataract refractive measurements.

Lymphoedema due to lymphatic hyperplasia: a case report.

An unusual case of generalised lymphoedema is described. The congenital nature of the lymphatic anomaly and the significance of its lymphographic appearance are discussed.

Congenital and traumatic cataract. The effect on ocular axial length.

Elongation of the eye is one of the drawbacks of implanting intraocular lenses in children. To evaluate possible effects of cataract and aphakia on eye elongation, we measured the axial length in children with unilateral aphakia (15 children with congenital cataract and 27 with traumatic cataract) and in children with bilateral congenital cataract (operated on in 14 cases and not operated on in eight cases). In all cases of unilateral aphakia, the aphakic eye was consistently longer than the normal fellow eye. Excessive eye elongation was related to corresponding reduction in visual acuity. The presumed rate of elongation was quantitatively expressed using the time elapsed since surgery and the age of the child. In bilateral congenital cataract, the axial length measured in aphakic eyes that were operated on was similar to that in eyes that were not operated on. We suggest that unilateral cataract or aphakia is associated with excessive eye elongation of affected eyes. Eye elongation seems to be related to amblyopia and poor vision rather than to aphakia.