Experimental Indication in Favor of the Introns-Late Theory: The Receptor Tyrosine Kinase Gene from the Sponge Geodia cydonium

We have analyzed the gene that encodes receptor tyrosine kinase (RTK) from the marine sponge Geodia cydonium, which belongs to the most ancient and simple metazoan groups, the Porifera. RTKs are enzymes found only in metazoa. The sponge gene contains two introns in the extracellular part of the protein. However, the rest of the protein (transmembrane and intracellular part), including the tyrosine kinase (TK)-domain, is encoded by a single exon. In contrast, all TK genes, so far known only from higher animals (vertebrates), contain several introns especially in the TK-domain. The TK-domain of G. cydonium shows similarity with numerous members of receptor as well as nonreceptor TKs. Phylogenetic analysis of the sponge TK-domain indicates that this enzyme branched off first from the common tree of metazoan TK proteins. Consequently, we assume that introns, found in the TK-domains of genes from higher animals, were inserted into these genes after splitting off the sponge taxa from other metazoan organisms (over 600 million years ago). Our results support the view that ancient genes were not ``in pieces.' Received: 8 August 1996 / Accepted: 4 November 1996     

Effects of Environmental Parameters on the Formation and Turnover of Acetate by Forest Soils

The capacity to form acetate from endogenous matter was a common property of diverse forest soils when incubated under anaerobic conditions. At 15 to 20(deg)C, acetate synthesis occurred without appreciable delay when forest soils were incubated as buffered suspensions or in microcosms at various percentages of their maximum water holding capacity. Rates for acetate formation with soil suspensions ranged from 35 to 220 (mu)g of acetate per g (dry weight) of soil per 24 h, and maximal acetate concentrations obtained in soil suspensions were two- to threefold greater than those obtained with soil microcosms at the average water holding capacity of the soil. Cellobiose degradation in soil suspensions yielded H(inf2) as a transient product. Under anaerobic conditions, supplemental H(inf2) and CO(inf2) were directed towards the acetogenic synthesis of acetate, and enrichments yielded a syringate-H(inf2)-consuming acetogenic consortium. At in situ temperatures, acetate was a relatively stable anaerobic end product; however, extended incubation periods induced acetoclastic methanogenesis and sulfate reduction. Higher mesophilic and thermophilic temperatures greatly enhanced the capacity of soils to form methane. Although methanogenic and sulfate-reducing activities under in situ-relevant conditions were negligible, these findings nonetheless demonstrated the occurrence of methanogens and sulfate-reducing bacteria in these aerated terrestrial soils. In contrast to the protracted stability of acetate under anaerobic conditions at 15 to 20(deg)C with unsupplemented soils, acetate formed by forest soils was rapidly consumed in the presence of oxygen and nitrate, and substrate-product stoichiometries indicated that acetate turnover was coupled to oxygen-dependent respiration and denitrification. The collective results suggest that acetate formed under anaerobic conditions might constitute a trophic link between anaerobic and aerobic processes in forest soils.     

Anti-Hepatitis A Virus Antibody Response Elicited in Mice by Different Forms of a Synthetic VP1 Peptide

Peptide VP1 (11-25) of the capsid of hepatitis A virus was synthesized by the Fmoc-polyamide solid phase method, and administered to mice in different forms: (1) free, (2) encapsulated in multilamellar liposomes, (3) coupled to keyhole limpet hemocyanin (KHL), and (4) incorporated into a tetrameric branched lysine core. The highest anti-VP1 peptide responses were generated by synthetic peptides entrapped into liposomes and coupled to KLH. No anti-HAV response was generated with the free peptide, while all the other forms induced both anti-HAV and HAV-neutralizing antibodies. Maximum neutralization indices were observed in ascites from mice treated with liposome-entrapped and KLH peptides.     

Aberrant pre-mRNA maturation is caused by LINE insertions into introns of the white gene of Drosophila melanogaster.

Insertional mutagenesis screens have provided thousands of mutant alleles for analysing genes of varied functions in Drosophila melanogaster. We here document mechanisms of insertional mutagenesis by a LINE element, the I factor, by determining the molecular structure of RNAs produced from two alleles of the white gene of D.melanogaster, wIR1 and wIR6. These alleles result from insertion of the I factor into introns of the gene. We show that sequences present within the element direct aberrant splicing and termination events. When the I factor is inserted within the white first intron it may lead to the use of a cryptic 3' splice site which does not contain the dinucleotide AG. This splicing gives rise to a chimeric messenger RNA whose synthesis is controlled differently in tissues where the mutated gene is expressed. When the I factor is inserted within the white last intron it induces synthesis of truncated mRNAs. These results provide, for the first time, mechanisms for I factor insertional mutagenesis. They are discussed in the more general context of RNA processing in Drosophila and the evolution of eukaryotic gene introns.     

Evidence from charcoal analysis for environmental change during the interval late Bronze Age to Roman,at the archaeological site of Castro de Penices,N.W. Portugal

Vegetation and environmental change from late Bronze Age to the Roman period in north-west Portugal is reconstructed on the basis of charcoal analyses. The site was occupied by people of the Castrejo culture, i.e. an Iron Age culture that developed in the north-west Iberian peninsula. The pattern of exploitation of natural wood resources by local populations during this period appears to be similar during the three phases of occupation. The frequencies of light-demanding plant species, mostly Leguminosae, testify to considerable destruction and degeneration of the climax woodlands. The preference of particular wood for specific uses, such as roofing, is discussed and the Holocene history of selected trees within the wider region is considered.     

Fluorescence of eosinophil leucocyte granules induced by 1-hydroxy-3,6,8-pyrenetrisulfonate. Visualization of differences in protein isoelectric points

After treatment of horse, rat and human blood smears with alkaline solutions of 1-hydroxy-3,6,8-pyrenetrisulfonate (HPTS), eosinophil leucocyte granules were the unique cell components which showed a bright green fluorescence. When stained with HPTS at pH 10, the whole granule of horse eosinophils showed high emission which strongly diminished after washing or staining in salt solutions or by using blocking methods for amino groups. Using HPTS at pH 12, the fluorescence reaction of house granules was specifically located in the peripheral region, appearing as fluorescent rings. These microscopic observations, which indicate differences in the isoelectric point of proteins within the eosinophil granule, were also confirmed by HPTS staining of protein blots as model substrates. Spectral analysis of HPTS at pH 10 and 12 showed practically identical absorption and emission spectra with peaks at 450 nm and 510 nm, respectively. Our results indicate that mainly ionic binding occurs between cationic proteins and HPTS in alkaline solution, and that the most cationic proteins (with isoelectric points at pH higher than 12) are located in the peripheral annular region of horse eosinophil granules.     

Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

Pruritus of poison oak dermatitis-other views

The use of the do-not-resuscitate order has become accepted medical practice. To date, however, no study has been done of how often it is used or factors associated with its use. Reports of all deaths of inpatients occurring during calendar year 1981 at San Bernardino County Medical Center were eligible for study. Retrospective review of the 237 cases indicated that a do-not-resuscitate order had been written for 165 (69.6%) of the patients. Comparison of reports of those for whom such an order had been written with those for whom no order had been written indicated that a do-not-resuscitate order was not associated with age, sex, ethnicity or pay status. Indices of mental clarity, however, were associated with orders not to resuscitate; those patients residing in nursing homes, and not alert and oriented on admission were overrepresented in the group given this order. Primary discharge diagnosis was also associated with such an order, as was an increased duration of hospital stay.     

SEM localization of laminin on the basement membrane of the chick corneal epithelium with immunolatex microspheres

Embryonic chick corneal explants were soaked in mild detergent and the anterior corneal epithelium was peeled from its basement membrane, leaving the lamina lucida surface exposed and supported on the subjacent primary stroma. Explants were treated with rabbit anti-laminin IgG, followed by sheep anti-rabbit IgG linked microspheres, and processed for SEM. The lucida surface was heavily decorated with microspheres, whereas controls treated with preimmune rabbit IgG were essentially beadless. Laminin distribution was not regular, appearing denser in some regions than others. However, the connective tissue surface of the basement membrane was never laminin-positive, even after treatment with hyaluronidase. These results suggest the basal lamina of the corneal epithelium is asymmetric, with preferential location of laminin to the lucida surface of the basement membrane.