A genetic module regulates the melanization response of Anopheles to Plasmodium

Two modes of refractoriness to Plasmodium, ookinete lysis and melanization, are known in the malaria vector, Anopheles gambiae. Melanization, a potent insect immune response, is manifested in a genetically selected refractory strain and in susceptible mosquitoes that are depleted of specific C-type lectins (CTLs). Here we use a systematic in vivo RNA interference-mediated reverse genetic screen and other recent results to define a melanization-regulating genetic module or network. It encompasses at least 14 genes, including those that encode five Easter-like clip domain serine proteases and four Masquerade-like serine protease homologues of the mosquito CLIPB and CLIPA subfamilies respectively. We show that several but not all CLIPB genes promote Plasmodium melanization, exhibiting partial functional overlap and synergy. We also report that several CLIPA genes have contrasting roles: CLIPA8 is essential for parasite melanization, while three other CLIPAs are novel synergistic inhibitors of this response. Importantly, the roles of certain CLIPAs and CLIPBs are strain specific, indicating that this network may differ between strains. Finally, we provide evidence that in susceptible mosquitoes melanization induced by knockdown of either CTL4 or CLIPA2/CLIPA5 directly kills ookinetes, in contrast to refractory mosquitoes where it merely disposes of dead parasites.     

A test case for structure-based functional assignment: the 1.2 A crystal structure of the yjgF gene product from Escherichia coli

The YER057c/YIL051c/YjgF protein family is a set of 24 full-length homologs, each approximately 130 residues in length, and each with no known function or relationship to proteins of known structure. To determine the function of this family, the structure of one member--the YjgF protein from Escherichia coli--was solved and refined at a resolution of 1.2 A. The YjgF molecule is a homotrimer with exact threefold symmetry. Its tertiary and quaternary structures are related to that of Bacillus subtilis chorismate mutase, although their active sites are completely different. The YjgF protein has an active site curiously similar to protein tyrosine phosphatases, including a covalently modified cysteine, but it is unlikely to be functionally related. The lessons learned from this attempt to deduce function from structure may be useful to future projects in structural genomics.     

Crystal structure of human maspin, a serpin with antitumor properties: reactive center loop of maspin is exposed but constrained

Maspin, a member of the serpin superfamily, has tumor suppressing activity against breast and prostate cancer. Maspin inhibits tumor growth by blocking cell invasion, and its reactive center loop (RCL) is thought to mediate this activity. To understand this function on the molecular level, we have solved the three-dimensional structure of Maspin to 3.1 A resolution. The molecular structure shows the characteristic features of the serpin fold, but the RCL of Maspin is unique in length, composition, and placement. Although the RCL of Maspin is accessible and cleavable by some proteinases, it functions in the uncleaved, constrained conformation observed here. These structural results will contribute to our understanding of the mechanism by which Maspin suppresses tumors.     

Effect of propranolol on cardiac cytokine expression after myocardial infarction in rats

The pro-inflammatory cytokines interleukin (IL)-1 and IL-6 have been shown to be upregulated in the myocardium after injury and after adrenergic receptor stimulation. Together with other cytokines, such as the transforming growth factor (TGF)-, the pro-inflammatory cytokines have been implicated in the initiation of tissue repair and wound healing after myocardial infarction (MI). In the present study, the effect of -adrenergic receptor blockade with propranolol (2 mg/kg·h s.c. by miniosmotic pumps) on cardiac cytokine expression and on wound healing was analyzed in rats from 6–72 h after MI. IL-1 and IL-6 gene expression strongly increased in the infarcted myocardium 6 h after MI and peaked after 12 h, while TGF-, progressively increased from 12 h onwards. Also, TGF-₂ increased after 12 h, peaked after 24 h and declined thereafter, while TGF-, was only elevated after 72 h. Treatment with propranolol had a negative chronotropic effect throughout the observation period of 72 h. It attenuated the initial elevation in LVEDP and increased cardiac output ultimately. Furthermore, propranolol attenuated IL-1 mRNA expression, but had not effect on the other cytokines. Moreover, MMP-9 gelatinolytic activity was markedly attenuated by propranolol indicating a delayed resorption of the necrotic tissue and, possibly, collagen turnover. Replacement by scar tissue, however, was not affected as indicated by normal collagen expression.     

Pigment epithelium-derived factor (PEDF), a serpin with potent anti-angiogenic and neurite outgrowth-promoting properties

Pigment epithelium-derived factor is a member of the serpin superfamily of proteins, but one that lacks inhibitory properties against either serine or cysteine proteinases. Nevertheless it possesses a number of physiological properties that make it a potentially important protein in regulation of angiogenesis, in neuronal cell survival and in protection of neurons from neurotoxic agents. It is also a protein that is highly up-regulated in the G0 phase of early-passage cells compared with rapidly proliferating cells or senescent cells, and so is also linked to both the cell cycle and cell senescence. The determination of a high resolution X-ray crystal structure of native PEDF provides insight into regions of the protein that may be involved in one or more of these functions.     

Endocrine-mediated effects of UV-A irradiation on grass shrimp (Palaemonetes pugio) reproduction

Although much is known regarding photoperiodic effects on crustacean egg production, the effects of ultraviolet (UV) light on reproduction has not been investigated. Likewise, little is known concerning the interaction between UV and xenobiotic exposure on crustacean reproductive cycles. In this study, male and female grass shrimp, Palaemonetes pugio, were exposed to sublethal concentrations of endosulfan (200 ng/l and 400 ng/l ES) under both white fluorescent (WF) and UV-A (315-400 nm) light conditions for 50 days in laboratory bioassays. Female endocrine (vitellogenin, ecdysteroids, and cholesterol), reproductive (percent gravid, clutch size), and embryo (days to hatch, hatching success, and hatching survival) responses were assessed. UV-exposure alone caused a significant (>4-fold) increase in total Palaemonetes pugio female egg production over the course of 50 days. Exposure to ES and UV significantly lowered the percentage of gravid females relative to UV controls, whereas ES-exposed shrimp under WF lighting did not exhibit these trends. Although higher vitellogenin concentrations and lower ecdysteroid titers were correlated with increased female egg production, cholesterol titers only exhibited a dose-dependent change when exposed to ES. Embryos from females exposed to UV had significantly lower ecdysteroid titers and shorter hatching times but there were no differences in embryo vitellogenin concentrations, hatching success, or hatching survival. These results indicate that UV-A exposure has a pronounced effect on grass shrimp (Palaemonetes pugio) reproduction and is likely mediated through 5-hydroxytrptamine (5-HT)-related neuroendocrine pathways. The implications for decapod aquaculture and evaluating chronic contaminant effects are discussed.     

Space flight micro-fungi after 27 years storage in water and in continuous culture

Four species of micro-fungi were selected for study in the National Aeronautics and Space Administration (NASA) Apollo Microbial Ecology Evaluation Device (MEED) mycology experiments. Trichophyton terrestre, Rhodotorula rubra, Saccharomyces cerevisiae and Chaetomium globosum were selected from a series of preflight test fungi for the MEED mycology studies during the 2 years prior to the actual flight (Volz, 1971a, 1972b). Conidia of T. terrestre, ascospores of C. globosum and yeast cells of R. rubra and S. cerevisiae were suspended in sterile distilled water and loaded into wet and dry cuvettes for exposure to specific space flight parameters according to the filters built into the space flight hardware (Volz, 1971b). Living cells were found in the original inocula and phenotype water storage after 27 years. Colony cells were also examined after 27 years of continuous culture.     

A cosmid library specific for human Chromosome regions 6p21.3 and 6q27

We have explored the potential of irradiation-fusion gene transfer (IFGT) hybrids as a source of well-defined human chromosome fragments from which probes can be derived. Extensive characterization of the IFGT hybrid 4J4 with a full panel of markers from Chromosome (Chr) 6 showed that the human DNA content derives largely from 6p21.3 and 6q27. A cosmid library has been constructed from 4J4 DNA, and 370 recombinants containing human DNA have been isolated and overlapping clones ordered into 20 contigs. Regional localization of representative clones from each contig, determined by fluorescent in situ hybridization (FISH), places 13 contigs in 6q27 and 6 in 6p21.3. Preliminary screening of cDNA libraries with selected cosmids has identified two expressed sequences. Since there are a number of medically important genes in both these regions of human Chr 6 with several disease loci linked to the HLA-A region in 6p21.3 and various tumor suppressor genes to 6q27, this library will provide a valuable resource to aid the isolation of candidate genes for these diseases. In addition, unique markers for detailed physical and genetic mapping of these regions of human Chr 6 can be easily obtained.     

Developing manufacturing control software: A survey and critique

The complexity and diversity of manufacturing software and the need to adapt this software to the frequent changes in the production requirements necessitate the use of a systematic approach to developing this software. The software life-cycle model (Royce, 1970) that consists of specifying the requirements of a software system, designing, implementing, testing, and evolving this software can be followed when developing large portions of manufacturing software. However, the presence of hardware devices in these systems and the high costs of acquiring and operating hardware devices further complicate the manufacturing software development process and require that the functionality of this software be extended to incorporate simulation and prototyping. This paper reviews recent methods for planning, scheduling, simulating, and monitoring the operation of manufacturing systems. A synopsis of the approaches to designing and implementing the real-time control software of these systems is presented. It is concluded that current methodologies support, in a very restricted sense, these planning, scheduling, and monitoring activities, and that enhanced performance can be achieved via an integrated approach.     

Phosphate uptake in Saccharomyces cerevisiae Hansen wild type and phenotypes exposed to space flight irradiation.

Rates of phosphate uptake were approximately twice as great for Saccharomyces cerevisiae single-cell phenotypic isolates exposed to space parameters as for the wild-type ground control. Quantitative determination of 32P was performed by liquid scintillation spectrometry utilizing Cerenkov radiation counting techniques.