Increased number of cardiac adrenergic receptors following local heart irradiation

The effect of local X irradiation on cardiac alpha and beta receptors was studied in Wistar rats. Animals were given local heart irradiation with single doses of 15 or 20 Gy and were examined after a range of latency times of 7 to 400 days. Using the radioactive ligands [3H]CGP-12177 and [3H]prazosin, the maximal binding capacity was determined from saturation experiments. At 7 days after 20 Gy the maximal binding capacity of both alpha and beta receptors was reduced to below the level of untreated control animals. Subsequently it rose continually to a maximum of 160% of the control level for beta receptors and 130% for alpha receptors at 400 days postirradiation. The antagonist affinity as judged from the dissociation constant for [3H]CGP 12177 and [3H]prazosin did not change significantly. A similar effect was observed after 15 Gy. An increase in adrenergic receptors may represent an important pathogenetic link between early morphological and late functional changes in the pathogenesis of radiation-induced heart disease.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

An Integrated Map with Cosmid/PAC Contigs of a 4-Mb Down Syndrome Critical Region

The major phenotypic features of Down syndrome have been correlated with partial trisomies of chromosome 21, allowing us to define the candidate gene region to a 4-Mb segment on the 21q22.2 band. We present here a high-resolution physical map with megabase-sized cosmid/PAC contigs. This ordered clone library has provided unique material for the integration of a variety of mappable objects, including exons, cDNAs, restriction sites, etc. Furthermore, our results have exemplified a strategy for the completion of the chromosome 21 map to sequencing.     

The intrinsic electrophysiological characteristics of fly lobula plate tangential cells: I. Passive membrane properties

The passive membrane properties of the tangential cells in the fly lobula plate (CH, HS, and VS cells, Fig. 1) were determined by combining compartmental modeling and current injection experiments. As a prerequisite, we built a digital base of the cells by 3D-reconstructing individual tangential cells from cobalt-stained material including both CH cells (VCH and DCH cells), all three HS cells (HSN, HSE, and HSS cells) and most members of the VS cell family (Figs. 2, 3). In a first series of experiments, hyperpolarizing and depolarizing currents were injected to determine steady-state I-V curves (Fig. 4). At potentials more negative than resting, a linear relationship holds, whereas at potentials more positive than resting, an outward rectification is observed. Therefore, in all subsequent experiments, when a sinusoidal current of variable frequency was injected, a negative DC current was superimposed to keep the neurons in a hyperpolarized state. The resulting amplitude and phase spectra revealed an average steady-state input resistance of 4 to 5 M and a cut-off frequency between 40 and 80 Hz (Fig. 5). To determine the passive membrane parameters R _m (specific membrane resistance), R _i (specific internal resistivity), and C _m (specific membrane capacitance), the experiments were repeated in computer simulations on compartmental models of the cells (Fig. 6). Good fits between experimental and simulation data were obtained for the following values: R _m = 2.5 kcm², R _i = 60 cm, and C _m = 1.5 F/cm² for CH cells; R _m = 2.0 kcm², R _i = 40 cm, and C _m = 0.9 F/cm² for HS cells; R _m = 2.0 kcm², R _i = 40 cm, and C _m = 0.8 F/cm² for VS cells. An error analysis of the fitting procedure revealed an area of confidence in the R _m -R _i plane within which the R _m -R _i value pairs are still compatible with the experimental data given the statistical fluctuations inherent in the experiments (Figs. 7, 8). We also investigated whether there exist characteristic differences between different members of the same cell class and how much the exact placement of the electrode (within ±100 m along the axon) influences the result of the simulation (Fig. 9). The membrane parameters were further examined by injection of a hyperpolarizing current pulse (Fig. 10). The resulting compartmental models (Fig. 11) based on the passive membrane parameters determined in this way form the basis of forthcoming studies on dendritic integration and signal propagation in the fly tangential cells (Haag et al., 1997; Haag and Borst, 1997).     

Hemoglobin switching in sheep. Synthesis, cloning, and characterization of DNA sequences coding for the beta B, beta C, and gamma-globin mRNAs.

Synthetic double-stranded DNAs (sDNAs) were prepared from sheep globin mRNA templates isolated from reticulocytes producing either hemoglobin B (HbB) (alpha 2 beta B2), HbC (alpha 2 beta C2), or HbF (alpha 2 gamma 2). These DNAs were inserted into the Eco RI site of plasmid pMB9 by the homopolymer tailing method and used to transform Escherichia coli X1776 to tetracycline resistance. Recombinant clones were identified by colony hybridization and further characterized by molecular hybridization and restriction endonuclease analysis. All plasmids analyzed thus far contained either beta- or gamma-globin DNA sequences. Moreover, sDNAs used for cloning yielded restriction endonuclease fragments consistent with the presence of predominantly beta- or gamma-sDNA, indicating that formation of double-stranded alpha-sDNA proceeds much less efficiently under our conditions than the formation of non-alpha-sDNAs. Three recombinant plasmids, pS beta B2, pS beta C69, and pS gamma 56, were selected for detailed study. These were shown to contain, respectively, beta B-, beta C-, and gamma-DNA sequences by molecular hybridization and by protection of the appropriate cDNAs from S1 nuclease digestion. Each contained all of the restriction endonuclease sites defined for the synthetic sDNAs and protected at least 90% of the sequence length of homologous cDNA. Restriction endonuclease maps of the beta B- and beta C-globin genes were identical at all 12 sites that were mapped, whereas four differences were identified in the gamma gene compared to the two others; three of these corresponded to differences in amino acid sequence of the globins. A method was developed to isolate the anti-mRNA strand of the insert for use as a specific molecular hybridization probe analogous to complementary DNA.     

Nucleic Acid Tools for Invasive Fungal Disease Diagnosis

Current Fungal Infection Reports - This review has incorporated the knowledge and experience of the leads of each of the laboratory working parties of the fungal PCR initiative in order to provide...     

The Crown Pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758)

Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species’ unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.