Genomic Basis of Circannual Rhythm in the European Corn Borer Moth

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Immune protection against experimental autoimmune encephalomyelitis: optimal conditions and analysis of mechanism

Protection against experimental autoimmune encephalomyelitis (EAE) was studied in the guinea pig and the Lewis rat. Basic protein of myelin (BPM) injected in incomplete Freund's adjuvant (IFA) gave solid protection against subsequent challenge with normally encephalitogenic doses of BPM in complete Freund's adjuvant (CFA). Protection depended on the amount of BPM in IFA injected and on the duration of the interval between protection and encephalitogenic challenge with BPM in CFA. Notably, protection was long lasting; it remained demonstrable, to some degree for 52 weeks in guinea pig and 32 weeks in rats, these being the longest intervals tested.Protection could not be correlated with serum antibody levels to BPM, and was afforded in the guinea pig by the injection, in IFA, of a synthetic peptide matching residues 112–122 of human BPM; this peptide produced no detectable serum antibody to BPM. Protected guinea pigs had intact cell-mediated immunity to BPM, as measured by inhibition of macrophage migration in vitro. The mechanism of protection may involve the production, following injection of BPM in IFA, of a class of suppressor thymic lymphocytes capable of overriding otherwise encephalitogenic thymic lymphocytes.     

Heterologous expression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction

The diterpene cyclase taxadiene synthase from yew (Taxus) species transforms geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committed step in the biosynthesis of the anti-cancer drug Taxol. Taxadiene synthase is translated as a preprotein bearing an N-terminal targeting sequence for localization to and processing in the plastids. Overexpression of the full-length preprotein in Escherichia coli and purification are compromised by host codon usage, inclusion body formation, and association with host chaperones, and the preprotein is catalytically impaired. Since the transit peptide-mature enzyme cleavage site could not be determined directly, a series of N-terminally truncated enzymes was created by expression of the corresponding cDNAs from a suitable vector, and each was purified and kinetically evaluated. Deletion of up to 79 residues yielded functional protein; however, deletion of 93 or more amino acids resulted in complete elimination of activity, implying a structural or catalytic role for the amino terminus. The pseudomature form of taxadiene synthase having 60 amino acids deleted from the preprotein was found to be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity with kinetics comparable to the native enzyme. In addition to the major product, taxa-4(5),11(12)-diene (94%), this enzyme produces a small amount of the isomeric taxa-4(20), 11(12)-diene ( approximately 5%), and a product tentatively identified as verticillene ( approximately 1%). Isotopically sensitive branching experiments utilizing (4R)-[4-(2)H(1)]geranylgeranyl diphosphate confirmed that the two taxadiene isomers, and a third (taxa-3(4),11(12)-diene), are derived from the same intermediate taxenyl C4-carbocation. These results, along with the failure of the enzyme to utilize 2, 7-cyclogeranylgeranyl diphosphate as an alternate substrate, indicate that the reaction proceeds by initial ionization of the diphosphate ester and macrocyclization to the verticillyl intermediate, followed by a secondary cyclization to the taxenyl cation and deprotonation (i.e., formation of the A-ring prior to B/C-ring closure). Two potential mechanism-based inhibitors were tested with recombinant taxadiene synthase but neither provided time-dependent inactivation nor afforded more than modest competitive inhibition.     

Urinary but Not Brain Isatin Levels Are Reduced in Germ-Free Rats

Germ-free rats excreted considerably smaller amounts of the monoamine oxidase-inhibiting compound isatin than the substantially larger output by conventional animals of the same strain, although concentrations in brain and other tissues were similar in the two groups. Thus, isatin is likely to be elaborated both endogenously in rat tissues and "exogenously" by flora inhabiting the lumen of the alimentary tract.     

Molecular survey of a prevalent mutation, 985A-to-G transition, and identification of five infrequent mutations in the medium-chain Acyl-CoA dehydrogenase (MCAD) gene in 55 patients with MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is an inborn error of fatty-acid oxidation that is characterized by fasting intolerance and recurrent episodes of hypoglycemic coma which can be fatal. Its incidence is one of the highest among genetic metabolic disorders. Using a modified PCR and NcoI digestion method, we have surveyed 46 additional, unrelated MCAD-deficient patients for a prevalent mutation, an 985A-to-G transition (985A----G), that we previously identified in nine MCAD-deficient patients. Among the total of 55 studied, 44 were homozygous and 10 were heterozygous for the 985G allele, whereas one did not carry this mutant allele, indicating that the prevalence of the 985G allele is 89.1%. Furthermore, we identified five other types of mutation: one each in three of the compound heterozygotes and two in the single non-985G patient. An RFLP study of 12 985G-homozygotes showed that all 24 alleles fell into a single haplotype. A questionnaire regarding the ethnic and national origin of their patients was sent to all referring investigators. All 41 patients for whom this information was provided were Caucasians. Of 29 patients whose country of origin was specified, 19 and five were from the British Isles and Germany, respectively. These data suggest that 985A----G may have occurred in a single person in an ancient Germanic tribe.     

Validating Livanow: Molecular Data Agree That Leeches, Branchiobdellidans, and Acanthobdella peledina Form a Monophyletic Group of Oligochaetes

To investigate the phylogenetic relationships of leeches, branchiobdellidans, and acanthobdellidans, whole nuclear 18S rDNA and over 650 bp of mitochondrial cytochrome c oxidase subunit I were acquired from 101 annelids, including 36 leeches, 18 branchiobdellidans, Acanthobdella peledina, as well as 28 oligochaetes and combined with homologous data for 17 polychaete outgroup taxa. Parsimony analysis of the combined aligned dataset supported monophyly of leeches, branchiobdellidans, and acanthobdellidans in 100% of jackknife replicates. Monophyly of the oligochaete order Lumbriculida with Acanthobdellida, Branchiobdellida, and Hirudinea was supported in 84% of jackknife replicates. These results provide support for the hypotheses that leeches and branchiobdellidans are sister groups, that acanthobdellidans are sister to them, and that together with the family Lumbriculidae they all constitute a clade within Oligochaeta. Results support synonymy of the classes Clitellata and the more commonly used Oligochaeta. Leeches branchiobdellidans, and acanthobdellidans should be regarded as orders equal to their closest relatives, the order Lumbriculida.     

Spectral imaging microscopy web sites and data.

The Internet is enabling greater access to spectral imaging publications, spectral graphs, and data than that was available a generation ago. The spectral imaging systems discussed in this issue of Cytometry work because reagent and hardware spectra are reproducible, reusable, and provide input to spectral unmixing and spectral components recognition algorithms. These spectra need to be readily available in order to determine what to purchase, how to use it, and what the output means. We refer to several commercially sponsored and academic spectral web sites and discuss our spectral graphing and data sites. Sites include fluorescent dye graph servers from Invitrogen/Molecular Probes, BD Biosciences, Zeiss/Bio-Rad Cell Sciences, and filter set servers from Chroma Technology and Omega Optical. Several of these sites include data download capabilities. Recently, two microscope manufacturers have published on their web sites transmission curves for select objective lenses-crucial data for anyone doing multiphoton excitation microscopy. Notable among the academic sites, PhotoChemCAD 2.0 has over 200 dyes and a downloadable database/graphing program, and the USC-A Chemistry UV-vis Database displays absorption spectra of many dyes and indicators used in clinical histology and pathology. Our Fluorescent Spectra graphing/calculator site presents dyes, filters, and illumination data from many of these and additional sources. PubSpectra is our free download site which uses Microsoft Excel files as standardized human/machine readable format with over 2,000 biomedical spectra. The principle that data is not subject to copyright provides a framework in which all scientific data should be made freely accessible.     

The chromosomal component of reproductive isolation in the grasshopper Caledia captiva

An analysis of the relative viabilities of recombinant and nonrecombinant chromosomes among the surviving embryos from back-crosses involving the Moreton (M) and Torresian (T) taxa has revealed that these embryos do not contain a representative sample of gametes derived from the F₁ hybrid parent. The significant deviations in the hybrid gametic population arise entirely from intrachromosomal effects with no evidence of any between-chromosome interactions. This is interpreted as clear evidence to show that recombinational repatterning within heterozygous bivalents in the F₁ parent is a significant factor in inducing the observed deviant segregation ratios. Furthermore, by using a population which is chromosomally equivalent to the Torresian but genically similar to the Moreton, it has been shown that over 46% of the F₂ embryonic breakdown arises solely from the effects of chromosomal heterozygosity upon recombination repatterning among (Moreton × Torresian) F₁ hybrids. From these data it is proposed that each chromosome is internally coadapted in the sense that it contains balanced blocks of cis-acting acting loci which can be disrupted by recombinational change. Disruption of the linear association of the genes on structurally different chromosomes by recombination repatterning results in novel intrachromosomal associations which may be functionally inadequate and so lead to arrested embryonic development. It is speculated that an important factor in arresting development may involve interactions between the novel recombinant chromosomes of the gamete and maternal factors laid down in the egg during oogenesis which are responsible for the sequential activation of the genomes of the progeny during development. Thus coadaptation is interpreted in terms of the functional intergration of a chromosome with the products of the genome of the previous generation. The assessment of the relative viabilities of recombinant and nonrecombinant chromosomes has shown that the Torresian nonrecombinant chromosomes possess the highest viabilities in the sequence T^N>M^NT^R = M^R where N and R represent nonrecombinant and recombinant classes. This sequence is relevant to the structure of the hybrid zone between the Torresian and Moreton taxa and explains both its asymmetry and the basis of the observed introgression of Torresian chromosomes into the Moreton taxon and the absence of the reverse movement.     

A preliminary assessment of changes in plant-dwelling insects when threatened plants are translocated

Translocation of threatened species is a tool used increasingly to conserve biodiversity, but the suite of co-dependent species that use the threatened taxa as hosts can be overlooked. We investigate the preliminary impact of translocating three threatened plant species on insect species and the integrity of insect assemblages that depend on these plants as their hosts. We compare the insect assemblages between natural populations of the threatened species, related non-threatened plant species growing wild near the threatened plants, and threatened plants translocated to another site approximately 40?km away. We used host breadth models and a coextinction risk protocol to determine which insect species are potentially host-specific on the threatened plants, and then assessed these insects?? potential presence at the translocation site. We found that insect assemblages on naturally-occurring threatened plants had more individuals, higher species density and higher species richness than assemblages on translocated plants. For one plant species, Leucopogon gnaphalioides, species composition differed significantly between wild and translocated populations (P?<?0.001). Furthermore, four insect species that were host-specific to Banksia brownii and B. montana were not detected on the translocated plants. Instead, translocated plants supported insect assemblages more similar to those of related plant species from the surrounding area. We conclude that threatened plant translocations that involve seed collection and propagation may have limited benefit for individual dependent species or the supported insect assemblage. Additional conservation actions will be required to maintain the diversity of insect assemblages and host-dependent relationships.