The defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes.

A recombinant Anopheles gambiae defensin peptide was used to define the antimicrobial activity spectrum against bacteria, filamentous fungi and yeast. Results showed that most of the Gram-positive bacterial species tested were sensitive to the recombinant peptide in a range of concentrations from 0.1 to 0.75 microM. No activity was detected against Gram-negative bacteria, with the exception of some E. coli strains. Growth inhibitory activity was detected against some species of filamentous fungi. Defensin was not active against yeast. The kinetics of bactericidal and fungicidal effects were determined for Micrococcus luteus and Neurospora crassa, respectively. Differential mass spectrometry analysis was used to demonstrate induction of defensin in the hemolymph of bacteria-infected adult female mosquitoes. Native peptide levels were quantitated in both hemolymph and midgut tissues. The polytene chromosome position of the defensin locus was mapped by in situ hybridization.

Image analysis combined with visual cytology in the early detection of recurrent bladder carcinoma.

BACKGROUND: Early detection of recurrent transitional cell carcinoma of the bladder (TCC) is important to permit early treatment, which produces maximal preservation of the bladder and maximum survival. METHODS: This retrospective cohort study attempted to determine the period of time over which urinary DNA image analysis combined with visual cytology is useful in the early detection of recurrent TCC of the bladder. The authors believe this study is unique in that it measured the effectiveness of this test (image analysis plus visual cytology combined) at varying times before clinical diagnosis of recurrence was made. The cohort was comprised of 175 urologic patients from urologic practices across the U.S. Data, collected between January 1991 and February 1994, included cystoscopy, biopsy, DNA image analysis, and visual cytologic reports. RESULTS: Sixty patients in the cohort were found to have active TCC whereas 115 patients had a history of, but no active, disease during the follow-up period. As expected, the sensitivity and specificity of DNA image analysis in combination with visual cytology, and DNA image analysis alone, were greatest when urinary samples were obtained close to the time of diagnosis. In general, the longer the interval from the combined tests to the time of diagnosis, the lower the sensitivity. The combined tests had predictive value up to 3 months prior to clinical diagnosis when any detectable cytologic abnormality was considered positive. At the optimal cutoff points as determined from receiver operating characteristic curves, sensitivity increased when DNA image analysis was supplemented with visual cytology. CONCLUSIONS: The combination of DNA image analysis and visual cytology provides a better method for the early detection of recurrent TCC than DNA image analysis alone. This test potentially may be useful in providing information regarding bladder tumor recurrence up to 3 months prior to clinical evidence of disease.

Plasmodium activates the innate immune response of Anopheles gambiae mosquitoes.

Innate immune-related gene expression in the major disease vector mosquito Anopheles gambiae has been analyzed following infection by the malaria parasite, Plasmodium berghei. Substantially increased levels of mRNAs encoding the antibacterial peptide defensin and a putative Gram-negative bacteria-binding protein (GNBP) are observed 20-30 h after ingestion of an infected blood-meal, at a time which indicates that this induction is a response to parasite invasion of the midgut epithelium. The induction is dependent upon the ingestion of infective, sexual-stage parasites, and is not due to opportunistic co-penetration of resident gut micro-organisms into the hemocoel. The response is activated following infection both locally (in the midgut) and systemically (in remaining tissues, presumably fat body and/or hemocytes). The observation that Plasmodium can trigger a molecularly defined immune response in the vector constitutes an important advance in our understanding of parasite-vector interactions that are potentially involved in malaria transmission, and extends knowledge of the innate immune system of insects to encompass responses to protozoan parasites.

Inducible immune factors of the vector mosquito Anopheles gambiae: biochemical purification of a defensin antibacterial peptide and molecular cloning of preprodefensin cDNA.

Larvae of the mosquito vector of human malaria, Anopheles gambiae, were inoculated with bacteria and extracts were biochemically fractionated by reverse-phase HPLC. Multiple induced polypeptides and antibacterial activities were observed following bacterial infection, including a member of the insect defensin family of antibacterial proteins. A cDNA encoding An. gambiae preprodefensin was isolated using PCR primers based on phylogenetically conserved sequences. The mature peptide is highly conserved, but the signal and propeptide segments are not, relative to corresponding defensin sequences of other insects. Defensin expression is induced in response to bacterial infection, in both adult and larval stages. In contrast, pupae express defensin mRNA constitutively. Defensin expression may prove a valuable molecular marker to monitor the An. gambiae host response to infection by parasitic protozoa of medical importance.

Morphogenesis in Drosophila requires nonmuscle myosin heavy chain function.

We provide the first link between a known molecular motor and morphogenesis, the fundamental process of cell shape changes and movements that characterizes development throughout phylogeny. By reverse genetics, we generate mutations in the Drosophila conventional nonmuscle myosin (myosin II) heavy chain gene and show that this gene is essential. We demonstrate that these mutations are allelic to previously identified, recessive, embryonic-lethal zipper mutations and thereby identify nonmuscle myosin heavy chain as the zipper gene product. Embryos that lack functional myosin display defects in dorsal closure, head involution, and axon patterning. Analysis of cell morphology and myosin localization during dorsal closure in wild-type and homozygous mutant embryos demonstrates a key role for myosin in the maintenance of cell shape and suggests a model for the involvement of myosin in cell sheet movement during development. Our experiments, in conjunction with the observation that cytokinesis also requires myosin, suggest that the processes of cell shape change in morphogenesis and cell division are intimately and mechanistically related.

Use of Neurospora spore killer strains to obtain centromere linkage data without dissecting asci.

Use of a centromere-linked Spore killer gene Sk reduces manyfold the labor involved in obtaining tetrad data that would otherwise require ordered dissection of intact linear eight-spored asci. Heterozygous crosses are made for Spore killer (SkK X SkS) and for markers to be tested. In such crosses only SkK ascospores survive. The four viable (SkK) and four aborted (SkS) ascospores of each ascus are ejected from the perithecium as a physically disordered group. The four surviving SkK ascospores of individual asci are germinated and scored. SkK segregates from SkS at the first meiotic division. If both marker alleles are represented in the surviving products, they must therefore have segregated from one another at the second division. Four-spore (Fsp) genes have been used to eliminate one postmeiotic nuclear division, so that only two ascospores per ascus need to be scored. The Spore killer method has been useful for mapping closely linked genes in centromere regions, for identifying genes that are far out on chromosome arms, for obtaining information on meiotic crossing-over, and for comparing linkages in different species.