bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA interstrand cross-links of trans-diamminedichloroplatinum(II) are preferentially formed between guanine and complementary cytosine residues.
Authors: Brabec V , Leng M
Published: May 2006

Bases in the opposite strands of DNA cross-linked by clinically ineffective trans-diamminedichloroplatinum(II) (trans-[Pt(NH3)2Cl2]) have been identified by means of three experimental approaches. These include HPLC analysis of enzymatic digests of synthetic oligonucleotide duplexes containing the interstrand cross-link, footprinting experiments on the interstrand cross-linked oligonucleotide duplexes, and termination of the duplex transcription on trans-[Pt(NH3)2Cl2]-treated fragments of plasmid DNA. The results reveal that deoxyguanine and complementary deoxycytosine residues are preferential binding sites of trans-[Pt(NH3)2Cl2] in the interstrand adducts. The interstrand cross-linking reaction was studied by means of gel electrophoresis for the cis and trans isomers. The rate of formation of interstrand cross-links was lower for the trans isomer; however, trans-[Pt(NH3)2Cl2] formed about twice the amount of interstrand cross-links as compared with the cis isomer after 48 hr. The present results are suggested to be relevant to differences in clinical activity of the two platinum(II) isomers.
The beta subunit of the mitochondrial processing peptidase from rat liver: cloning and sequencing of a cDNA and comparison with a proposed family of metallopeptidases.
Authors: Paces V , Rosenberg L E , Fenton W A , Kalousek F
Published: May 2006

Most nuclearly encoded mitochondrial proteins are synthesized with amino-terminal leader peptides that are removed by the mitochondrial processing peptidase (MPP) after translocation. Earlier we reported cloning and sequencing of a cDNA for the larger subunit (MPP alpha subunit) of this enzyme from rat liver mitochondria. We have now completed the cloning and sequencing of a cDNA encoding the smaller subunit of the enzyme (MPP beta subunit) from the same source. The cDNA consists of 1570 bp: 17 bp of 5'-untranslated sequence, 1467 bp of coding sequence, and 86 bp of 3'-untranslated sequence. The predicted protein consists of 489 amino acid residues, including a 45-amino acid leader peptide at the amino terminus and a 444-amino acid mature protein. The amino acid sequences of four tryptic peptides derived from purified MPP beta subunit precisely match those predicted by the cDNA sequence, as does the predicted mature amino terminus. The amino-terminal sequence is typical of a mitochondrial leader peptide, with eight positively charged arginine residues and a single negatively charged aspartate residue. When the amino acid sequence of rat MPP beta subunit is compared with sequences in the protein data bases, significant homology is found with the protease-enhancing protein of Neurospora crassa, the smaller subunit of MPP from Saccharomyces cerevisiae, and the core I protein of bovine ubiquinol:cytochrome c reductase. Lower homology is found with other members of a recently proposed class of endoproteases, which includes human insulinase and protease III from Escherichia coli.
Differently spliced cDNAs of human leukocyte tyrosine kinase receptor tyrosine kinase predict receptor proteins with and without a tyrosine kinase domain and a soluble receptor protein.
Authors: Toyoshima H , Kozutsumi H , Maru Y , Hagiwara K , Furuya A , Mioh H , Hanai N , Takaku F , Yazaki Y , Hirai H
Published: May 2006

Leukocyte tyrosine kinase (LTK) is a tyrosine kinase that has been suggested to be specific for hematopoietic cells and neuronal cells and reported as an unusual membrane protein lacking an extracellular domain. Here we report the cloning of a human LTK cDNA clone containing the complete open reading frame of a putative receptor tyrosine kinase protein. The extracellular domain of the receptor protein is larger than previously predicted. Furthermore, we have cloned a set of cDNAs representing differently spliced human LTK mRNAs. These cDNAs predict a truncated receptor protein lacking the tyrosine kinase domain and a soluble receptor protein that has neither a transmembrane nor a tyrosine kinase domain. Our results suggest that the LTK gene produces not only the putative receptor tyrosine kinase for unknown ligand but also multiple protein products that may have different functions.
A motility in the eukaryotic flagellum unrelated to flagellar beating.
Authors: Kozminski K G , Johnson K A , Forscher P , Rosenbaum J L
Published: May 2006

We report a motility in the flagella of the green alga Chlamydomonas that is unrelated to dynein-based flagellar beating. This motility, referred to as intraflagellar transport, was observed as the rapid bidirectional movement of granule-like particles along the length of the flagella. Intraflagellar transport could be experimentally separated from other, previously reported, nonbeat flagellar motilities. EM of flagella showed groups of nonvesicular, lollipop-shaped structures positioned between the outer doublet microtubules and the flagellar membrane. Movement of these complexes along the length of the flagella may be responsible for intraflagellar transport.
Generation of osteoclast-inductive and osteoclastogenic cell lines from the H-2KbtsA58 transgenic mouse.
Authors: Chambers T J , Owens J M , Hattersley G , Jat P S , Noble M D
Published: May 2006

The development of osteoclastic cell lines would greatly facilitate analysis of the cellular and molecular biology of bone resorption. Several cell lines have previously been reported to be capable of osteoclastic differentiation. However, such cell lines form at best only occasional excavations, suggesting that osteoclastic differentiation is either incomplete or that osteoclasts represent a very small proportion of the cells present. We have used the recently developed H-2KbtsA58 transgenic mouse, in which the interferon-inducible major mouse histocompatibility complex H-2Kb promoter drives the temperature-sensitive (ts) immortalizing gene of simian virus 40 (tsA58), to develop cell lines from bone marrow with high efficiency. Bone marrow cells were incubated with gamma interferon at 33 degrees C, then cloned, and expanded. The cell lines were characterized at 39.5 degrees C in the absence of gamma interferon. First, stromal cell lines were established that induced osteclast formation (resorption of bone slices) when cocultured with hemopoietic spleen cells. Some of the stromal cell lines so generated were able to resorb approximately 30 mm2/cm2 of bone surface. We then established cell lines of hemopoietic origin, several of which possess osteoclastic potential. When these osteoclast-precursor cell lines were cocultured with stromal cell lines, extensive bone resorption was observed. Osteoclast formation did not occur if the precursor cell lines were incubated on bone slices without stromal cells; osteoclast formation was also dependent upon the presence of 1 alpha,25-dihydroxyvitamin D3. These cell lines represent a model for osteoclast formation and a valuable resource for identification of the mechanisms and factors that regulate osteoclast differentiation and function.
Noninvolvement of the long terminal repeat of transposable element 17.6 in insecticide resistance in Drosophila.
Authors: Delpuech J M , Aquadro C F , Roush R T
Published: May 2006

Waters and colleagues recently suggested [Waters, L. C., Zelhof, A. C., Shaw, B. J. & Ch'ang, L.-Y. (1992) Proc. Natl. Acad. Sci. USA 89, 4855-4859] that an insertion of a long terminal repeat of transposable element 17.6 into the 3' untranslated region of a P450 gene leads to susceptibility to the insecticide DDT in Drosophila melanogaster. We tested this hypothesis by screening lines from around the world and found that the presence or absence of a long terminal repeat was uncorrelated with resistance in 31 strains of D. melanogaster and Drosophila simulans. Thus we must reject the hypothesis that the insertion of a long terminal repeat leads to DDT susceptibility in Drosophila.
Optimizing transmembrane domain helicity accelerates insulin receptor internalization and lateral mobility.
Authors: Goncalves E , Yamada K , Thatte H S , Backer J M , Golan D E , Kahn C R , Shoelson S E
Published: May 2006

Transmembrane (TM) domains of integral membrane proteins are generally thought to be helical. However, a Gly-Pro sequence within the TM domain of the insulin receptor is predicted to act as a helix breaker. CD analyses of model TM peptides in a lipid-like environment show that substitution of Gly and Pro by Ala enhances helicity. On this basis, Gly933 and Pro934 within the TM domain of the intact human insulin receptor were mutated to Ala (G-->A, P-->A, GP-->AA) to assess effects of altered helicity on receptor functions. Mutated and wild-type receptors, expressed stably in cultured CHO cells at equivalent levels, were properly assembled, biosynthetically processed, and exhibited similar affinities for insulin. Receptor autophosphorylation and substrate kinase activity in intact cells and soluble receptor preparations were indistinguishable. In contrast, insulin-stimulated receptor internalization was accelerated 2-fold for the GP-->AA mutant, compared to a wild-type control or the G-->A and P-->A mutants. Insulin degradation, which occurs during receptor endocytosis and recycling, was similarly elevated in cells transfected with GP-->AA mutant receptors. Fluorescence photobleaching recovery measurements showed that the lateral mobility of GP-->AA mutant receptors was also increased 2- to 3-fold. These results suggest that lateral mobility directly influences rates of insulin-mediated receptor endocytosis and that rates of endocytosis and lateral mobility are retarded by a kinked TM domain in the wild-type receptor. Invariance of Gly-Pro within insulin receptor TM domain sequences suggests a physiologic advantage for submaximal rates of receptor internalization.
Caldesmon and a 20-kDa actin-binding fragment of caldesmon inhibit tension development in skinned gizzard muscle fiber bundles.
Authors: Pfitzer G , Zeugner C , Troschka M , Chalovich J M
Published: May 2006

Caldesmon is known to inhibit actin-activated myosin ATPase activity in solution, to inhibit force production when added to skeletal muscle fibers, and to alter actin movement in the in vitro cell motility assay. It is less clear that caldesmon can inhibit contraction in smooth muscle cells in which caldesmon is abundant. We now show that caldesmon and its 20-kDa actin-binding fragment are able to inhibit force in chemically skinned gizzard fiber bundles, which are activated by a constitutively active myosin light-chain kinase in the presence and absence of okadaic acid. This inhibitory effect is reversed by high concentrations of Ca2+ and calmodulin. Therefore, caldesmon may act by increasing the level of myosin phosphorylation required to obtain full activation. Our results also suggest that caldesmon does not act to maintain force in smooth muscle by cross-linking myosin with actin since competition of binding of caldesmon with myosin does not cause a reduction in tension.
Hydroxyl radical footprint analysis of human immunodeficiency virus reverse transcriptase-template.primer complexes.
Authors: Metzger W , Hermann T , Schatz O , Le Grice S F , Heumann H
Published: May 2006

Human immunodeficiency virus type 1 reverse transcriptase protects sugar moieties of a model template.primer DNA in a region from positions +3 to -15 from hydroxyl radical attack. A protected region of equivalent size migrates in concert with the translocating enzyme, as shown by hydroxyl radical footprints of replication complexes after primer extension by 4, 10, and 19 nt. The pattern of these footprints suggests that the DNA template.primer is in the A conformation when complexed with reverse transcriptase. Enhanced accessibility of the DNA template strand around position -15 to hydroxyl radicals indicates a conformational change in the template induced by the C-terminal RNase H-containing domain of p66 reverse transcriptase.
Specific mitochondrial proteins in pollen: presence of an additional ATP synthase beta subunit.
Authors: De Paepe R , Forchioni A , Chétrit P , Vedel F
Published: May 2006

A protocol was designed to obtain a pure fraction of pollen mitochondria from the diploid species Nicotiana sylvestris, the female parent of the allotetraploid Nicotiana tabacum. Most organelles were morphologically intact and able to perform in organello mitochondrial (mt) protein synthesis. As revealed by two-dimensional protein electrophoresis, numerous quantitative differences exist between leaf and pollen mt proteins. Moreover, additional mt polypeptides, named R (for reproductive), encoded by either nuclear or mitochondrial genes, are found in pollen. The most abundant R polypeptide, R1 (M(r) 53,000, pI 5.6), is nuclearly encoded, is membrane bound, and cross-reacts with an antibody directed against the beta subunit of the mt ATP synthase (ATPase). N-terminal microsequence analysis showed that the two ATPase beta subunits present in leaves (beta 1 and beta 2) and the R1 pollen-specific subunit are encoded by distinct genes. A similar additional ATPase beta subunit was observed in pollen mitochondria from Petunia, suggesting that this polypeptide is of general importance for male gametophytic development in Solanaceaes.
Identification of an ethylene-responsive region in the promoter of a fruit ripening gene.
Authors: Montgomery J , Goldman S , Deikman J , Margossian L , Fischer R L
Published: May 2006

Transcription of the E4 gene is controlled by an increase in ethylene concentration during tomato fruit ripening. To investigate the molecular basis for ethylene regulation, we have examined the E4 promoter to identify cis elements and trans-acting factors that are involved in E4 gene expression. In transgenic tomato plants a chimeric gene construct containing a 1.4-kilobase E4 promoter fused to a beta-glucuronidase reporter gene is rapidly induced by ethylene in ripening fruit. Deletion of E4 promoter sequences to 193 base pairs reduces the level of GUS activity but does not affect ethylene induction. Transient expression of E4 promoter-luciferase chimeric gene constructs containing various deletions, introduced into tomato fruit pericarp by particle bombardment, indicates that a positive ethylene-responsive region is localized between nucleotides -161 and -85 relative to the transcription start site. DNase I footprint analysis shows that a nuclear factor in unripe fruit interacts specifically with sequences in this element, from -142 to -110, which are required for the ethylene response. The DNase I footprint of this factor is reduced in ethylene-treated unripe fruit and undetectable in ripe fruit. Based on the correlation of a nuclear factor binding site with promoter sequences required for ethylene induction, we propose that this in vitro DNA-binding activity may represent a factor that is involved in ethylene-regulated E4 gene expression.
Accelerated evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes.
Authors: Nakashima K , Ogawa T , Oda N , Hattori M , Sakaki Y , Kihara H , Ohno M
Published: May 2006

Six Trimeresurus flavoviridis (Habu snake) venom gland phospholipase A2 (PLA2) isozyme genes were found to consist of four exons and three introns and to encode proteins of 138 amino acid residues, including the signal sequence of 16 amino acid residues. Comparison of the nucleotide sequences showed that the introns are much more homologous than the protein-coding regions of exons except for the signal peptide-coding region of the first exon. The numbers of nucleotide substitutions per site (KN) for introns are approximately one-fourth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions, indicating that the introns are unusually conserved. The absence of an apparent functional role for the introns suggests that the protein-coding regions, except for the signal peptide-coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per nonsynonymous site (KA) are close to or larger than KS values for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions or exons. This is compatible with the presence of PLA2 species with diverse physiological activities in the venom.
Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila.
Authors: Ferreira P A , Shortridge R D , Pak W L
Published: May 2006

The Drosophila norpA gene encodes a phospholipase C involved in phototransduction. However, phospholipase C apparently is not directly involved in phototransduction in vertebrate photoreceptors, although light-activated phospholipase C activity has been reported in vertebrate rod outer segments. Conserved regions of norpA cDNA were used to isolate bovine cDNAs that would encode four alternative forms of phospholipase C of the beta class that are highly homologous to the norpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the norpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the norpA protein but not in other phospholipases C. Results suggest that these proteins together with the norpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the norpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina.
Loss of expression of transforming growth factor beta in skin and skin tumors is associated with hyperproliferation and a high risk for malignant conversion.
Authors: Glick A B , Kulkarni A B , Tennenbaum T , Hennings H , Flanders K C , O'Reilly M , Sporn M B , Karlsson S , Yuspa S H
Published: May 2006

Mouse skin carcinomas arise from a small subpopulation of benign papillomas with an increased risk of malignant conversion. These papillomas arise with limited stimulation by tumor promoters, appear rapidly, and do not regress, suggesting that they differ in growth properties from the majority of benign tumors. The transforming growth factor beta (TGF-beta) proteins are expressed in the epidermis and are growth inhibitors for mouse keratinocytes in vitro; altered TGF-beta expression could influence the growth properties of high-risk papillomas. Normal epidermis, tumor promoter-treated epidermis, and skin papillomas at low risk for malignant conversion express TGF-beta 1 in the basal cell compartment and TGF-beta 2 in the suprabasal strata. In low-risk tumors, 90% of the proliferating cells are confined to the basal compartment. In contrast, the majority of high-risk papillomas are devoid of both TGF-beta 1 and TGF-beta 2 as soon as they arise; these tumors have up to 40% of the proliferating cells in the suprabasal layers. Squamous cell carcinomas are also devoid of TGF-beta, suggesting that they arise from the TGF-beta-deficient high-risk papillomas. In some high-risk papillomas, TGF-beta 1 loss can occur first and correlates with basal cell hyperproliferation, while TGF-beta 2 loss correlates with suprabasal hyperproliferation. Similarly, TGF-beta 1-null transgenic mice, which express wild-type levels of TGF-beta 2 in epidermis but no TGF-beta 1 in the basal layer, have a hyperproliferative basal cell layer without suprabasal proliferation. In tumors, loss of TGF-beta is controlled at the posttranscriptional level and is associated with expression of keratin 13, a documented marker of malignant progression. These results show that TGF-beta expression and function are compartmentalized in epidermis and epidermal tumors and that loss of TGF-beta is an early, biologically relevant risk factor for malignant progression.
Mechanism of resistance of human immunodeficiency virus type 1 to 2',3'-dideoxyinosine.
Authors: Martin J L , Wilson J E , Haynes R L , Furman P A
Published: May 2006

A molecular clone containing the wild-type reverse transcriptase (RT) coding region of human immunodeficiency virus type 1 (HIV-1) was constructed, and site-directed mutagenesis was used to introduce mutations--Leu74-->Val (L74V), T215Y, and the combination L74V/T215Y--into the RT coding region. The proteins were purified by immunoaffinity chromatography. Assays were performed with mutant and wild-type RT to determine substrate and inhibitor specificity. All three mutant enzymes catalyzed the incorporation of substrate 2'-deoxynucleoside 5'-triphosphates (dNTPs) as efficiently as wild-type HIV-1 RT. Small changes were observed in the Km values for dNTPs with all three mutant enzymes, while more significant changes were noted in sensitivity to nucleoside 5'-triphosphate analogues that inhibit the enzyme activity. Results suggest that altered substrate recognition by the HIV-1 RT is involved in the mechanism of resistance.
Opsins from the lateral eyes and ocelli of the horseshoe crab, Limulus polyphemus.
Authors: Smith W C , Price D A , Greenberg R M , Battelle B A
Published: May 2006

cDNA clones encoding opsins from the lateral eyes and median ocelli of the horseshoe crab, Limulus polyphemus, were isolated from cDNA libraries. The opsin cDNAs obtained from the lateral eye and ocellar libraries code for deduced proteins with 376 amino acids. The two cDNAs are 96% identical at the nucleic acid level, differing primarily at the 3' untranslated region, and are apparently the products of two separate genes. The deduced opsin proteins are 99% identical to each other, differing at only 5 amino acids. The opsins encoded by these cDNAs are most likely the protein moiety of the visible-wavelength rhodopsins in this animal. In the lateral eye, expression of the opsin gene is restricted to the photoreceptor cells of the ommatidia. Comparisons with opsins of other species show that the Limulus opsin proteins are most similar (53% identity) to the opsin from the R1-6 photoreceptors of flies.
Human immunodeficiency virus type 1 transactivator protein, tat, stimulates transcriptional read-through of distal terminator sequences in vitro.
Authors: Graeble M A , Churcher M J , Lowe A D , Gait M J , Karn J
Published: May 2006

The human immunodeficiency virus type 1 transactivator protein, tat, specifically stimulates transcription from the viral long terminal repeat. We used cell-free transcription systems to test whether tat can stimulate transcriptional read-through of an artificial terminator sequence (e.g., a stable RNA stem-loop structure followed by a tract of nine uridine residues) placed downstream of the viral long terminal repeat. In the absence of tat, RNA polymerases are prematurely released from the template at the terminator sequence. Recombinant tat protein purified from Escherichia coli increased the synthesis of full-length transcripts approximately 25-fold and decreased the amount of transcripts ending at the terminator sequence. The reaction is strictly dependent upon the presence of a functional transactivation-responsive region (TAR) sequence. Mutations in the tat binding site on TAR RNA and mutations in the TAR RNA loop block transactivation in vivo. Neither type of mutation is able to respond to tat in vitro. These results strongly suggest that after transcription through the TAR region, tat modifies the transcription complex to increase its elongation capacity.
Kinetics of human immunodeficiency virus type 1 (HIV-1) DNA and RNA synthesis during primary HIV-1 infection.
Authors: Graziosi C , Pantaleo G , Butini L , Demarest J F , Saag M S , Shaw G M , Fauci A S
Published: May 2006

HIV-1 replication and viral burden in peripheral blood mononuclear cells (PBMC) have been reported to be high in primary infection but generally very low during the prolonged period of clinical latency. It is uncertain precisely when this transition occurs during the HIV-1 infection and what the relationship is between the changes in HIV-1 replication versus the clearance of infected cells in the overall control of viral replication. In the present study, the kinetics of viral burden (i.e., frequency of HIV-1-infected cells) and replication during primary and early-chronic infection were analyzed in PBMC of four acutely infected individuals. High frequencies of HIV-1-infected cells and high levels of virus replication were observed in PBMC after primary HIV-1 infection. Down-regulation of virus replication in PBMC was observed in all four patients coincident with the emergence of HIV-1-specific immune responses. Other parameters of virus replication, such as circulating plasma p24 antigen and plasma viremia showed similar kinetics. In contrast, a significant decline in viral burden in PBMC was observed in only one of four patients. These results indicate that the down-regulation in the levels of virus replication associated with the clinical transition from acute to chronic infection does not necessarily reflect a reduction in viral burden, thus suggesting the involvement of additional factors. Identification of these factors will be important in elucidating the host mechanisms involved in the early control of HIV-1 infection and disease.
"Diabodies": small bivalent and bispecific antibody fragments.
Authors: Holliger P , Prospero T , Winter G
Published: May 2006

Bivalent and bispecific antibodies and their fragments have immense potential for practical application. Here we describe the design of small antibody fragments with two antigen-binding sites. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. As indicated by a computer graphic model of the dimers, the two pairs of domains can pack together with the antigen-binding sites pointing in opposite directions. The dimeric antibody fragments, or "diabodies," can be designed for bivalent or bispecific interactions. Starting from the monoclonal antibodies NQ11.7.22 (NQ11) and D1.3 directed against the hapten phenyloxazolone and hen egg lysozyme, respectively, we built bivalent fragments (VHNQ11-VLNQ11)2 and (VHD1.3-VLD1.3)2 and bispecific fragments VHNQ11-VLD1.3 and VHD1.3-VLNQ11. The fragments were expressed by secretion from bacteria and shown to bind specifically to the hapten and/or antigen. Those with 5- and 15-residue linkers had similar binding affinities to the parent antibodies, but a fragment with the VH domain joined directly to the VL domain was found to have slower dissociation kinetics and an improved affinity for hapten. Diabodies offer a ready means of constructing small bivalent and bispecific antibody fragments in bacteria.
Yersinia enterocolitica invasin: a primary role in the initiation of infection.
Authors: Pepe J C , Miller V L
Published: May 2006

The ability to invade the intestinal epithelium of mammals is an essential virulence determinant of Yersinia enterocolitica. The chromosomally encoded Y. enterocolitica 8081v invasion gene, inv, was disrupted to assess its role in pathogenesis. The inv mutant (JP273v) was approximately 80-fold less invasive than wild type for cultured epithelial cells. When mice were infected intragastrically, up to 10(7) fewer JP273v were recovered from Peyer's patches early (6-18 hr) after infection compared with wild type. Analysis of the course of infection revealed that the inv mutant had distinct differences relative to wild type in the distribution of visible infectious foci and in tissue colonization; however, the mutant and wild-type strains had similar LD50 values for both orally and intraperitoneally infected mice. The invasion defect of the inv mutant was fully complemented in vitro and in vivo by introduction of the wild-type inv gene in trans. The inv gene product, invasin, appears to play a vital role in promoting entry during the initial stage of infection. During the subsequent establishment of a systemic infection, invasin may be of secondary importance, since the Y. enterocolitica inv mutant was as proficient as wild type at causing a fatal infection in mice. Based on these data, we discuss the role of invasin in a naturally occurring Y. enterocolitica infection.
A general two-metal-ion mechanism for catalytic RNA.
Authors: Steitz T A , Steitz J A
Published: May 2006

A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA. The mechanism postulates that chemical catalysis is facilitated by two divalent metal ions 3.9 A apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I. One metal ion activates the attacking water or sugar hydroxyl, while the other coordinates and stabilizes the oxyanion leaving group. Both ions act as Lewis acids and stabilize the expected pentacovalent transition state. The symmetry of a two-metal-ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P. The role of the RNA is to position the two catalytic metal ions and properly orient the substrates via three specific binding sites.
Six S100 genes are clustered on human chromosome 1q21: identification of two genes coding for the two previously unreported calcium-binding proteins S100D and S100E.
Authors: Engelkamp D , Schäfer B W , Mattei M G , Erne P , Heizmann C W
Published: May 2006

The human genome contains large regions that are highly structured. Sequence-related members of multigene families are often found in a clustered organization. Here we describe a previously unrecognized gene cluster composed of genes coding for calcium-binding proteins of the S100 family. The linkage of six S100 genes was established by pulsed-field gel electrophoresis, and a contiguous DNA sequence of 15 kilobases containing the full coding region of four different S100 genes was characterized. This is the tightest mammalian gene cluster discovered so far to our knowledge. Two additional S100 genes are located within the cluster, both of which exhibit unique structural features when compared with other S100 genes. The product of S100E is cysteine-rich, whereas that of S100D contains a long hydrophobic N-terminal tail. The gene cluster was assigned to chromosome 1q21, one of the bands showing rearrangements in neoplasms at high frequency. The deregulated expression of some S100 genes in the cluster during tumor progression suggests that chromosomal abnormalities may influence the expression of S100 genes in late stages of cancer, particularly in association with the formation of metastases.
DNA topoisomerase I is essential in Drosophila melanogaster.
Authors: Lee M P , Brown S D , Chen A , Hsieh T S
Published: May 2006

Both biochemical and genetic experiments suggest that the type I DNA topoisomerase may participate in DNA replication, recombination, transcription, and other aspects of DNA metabolism. Despite its apparent importance, genetic studies in unicellular organisms including eubacteria and yeasts indicate that topoisomerase I is not essential for viability. We have previously isolated the cDNA clone encoding DNA topoisomerase I from Drosophila melanogaster. We report here the cytogenetic mapping of top1 to the X chromosome at 13C1 and isolation of top1 genomic DNA. Using P-element mutagenesis, we have isolated a mutant deficient in Drosophila topoisomerase I functions. Genetic studies of this mutant show that topoisomerase I is essential for the growth and development of the fruit fly, a multicellular organism. The biological functions of topoisomerase I are inferred from our analysis of the regulation of topoisomerase I expression during Drosophila development.
Cloning and functional comparison of kappa and delta opioid receptors from mouse brain.
Authors: Yasuda K , Raynor K , Kong H , Breder C D , Takeda J , Reisine T , Bell G I
Published: May 2006

While trying to identify new members of the somatostatin receptor family of G protein-coupled receptors, we isolated cDNAs from a mouse brain library encoding two related receptor-like proteins, designated msl-1 and msl-2, of 380 and 372 amino acids, respectively. There was 61% identity and 71% similarity between the sequences of msl-1 and msl-2. Among members of the G protein-coupled receptor superfamily, the sequences of both msl-1 and msl-1 were most closely related to those of the somatostatin receptors (SSTRs), having approximately 35% identity with the sequence of SSTR1. Transient expression in COS-1 cells showed that msl-1 and msl-2 did not bind somatostatin. Rather they bound opioids selectively and with high affinity and had the pharmacological properties of kappa and delta opioid receptors, respectively. Indeed, the sequence of msl-2 was identical to that of a delta opioid receptor recently cloned by other workers. Functional characterization of kappa/msl-1 and delta/msl-2 opioid receptors showed that they were coupled to G proteins and mediated opioid receptor class-specific agonist inhibition of forskolin-stimulated cAMP formation. RNA blotting studies and in situ hybridization histochemistry showed that kappa opioid receptor mRNA was expressed at high levels in brain in the neocortex, hippocampus, amygdala, medial habenula, hypothalamus (arcuate and paraventricular nuclei), locus ceruleus, and parabrachial nucleus, suggesting that this receptor may play a role in arousal and regulation of autonomic and neuroendocrine functions.
Sequence analysis of the beta-N-acetylhexosaminidase gene of Vibrio vulnificus: evidence for a common evolutionary origin of hexosaminidases.
Authors: Somerville C C , Colwell R R
Published: May 2006

DNA cloned from the marine bacterium Vibrio vulnificus into Escherichia coli HB101 can hydrolyze chitin oligomer analogs in the recipient. The nucleotide sequence of the cloned DNA was determined and a single long open reading frame of 2541 base pairs (initiation codon through termination codon) was found. The nucleotide sequence predicts a gene product of 847 amino acids and a molecular mass of 94.3 kDa. In vitro transcription and translation analyses indicated a single protein of 94 kDa encoded by the cloned DNA. The gene product hydrolyzes methylumbelliferyl beta-D conjugates of chitotriose, chitobiose, N-acetylglucosamine, and N-acetylgalactosamine and has, therefore, been termed a beta-N-acetylhexosaminidase. The predicted protein shares a high degree of sequence similarity with the chitobiase of Vibrio harveyi and limited similarity with the alpha chain of human beta-hexosaminidase. Cluster analyses suggest a common evolutionary ancestor for all known hexosaminidase enzymes, with no detectable relationship to known chitinases.
On how a myosin tryptophan may be perturbed.
Authors: Bivin D B , Kubota S , Pearlstein R , Morales M F
Published: May 2006

A well-known indication that a nucleotide has bound to myosin is the enhancement of the fluorescence of a specific tryptophan in the "subfragment 1" segment of the protein. Empirically the effect has been enormously useful in myosin enzymology. But beyond an early suggestion that it arises from a purine-tryptophan charge-transfer complex, the mechanism of the effect has not been considered. Here we consider the alternative that it arises from an ionizable group (either another residue or the phosphate of the nucleotide) whose proximity to the tryptophan is altered by substrate binding. We study this possibility by studying the interaction of an ionizable residue and tryptophan when both are incorporated in a diketopiperazine structure. The geometry of the situation is inferred from molecular mechanics simulations. Unexpectedly, the best explanation seems to be that the field of the imposed charge, acting across space, affects events in the excited state of the indole.
Even in culture, oligodendrocytes myelinate solely axons.
Authors: Lubetzki C , Demerens C , Anglade P , Villarroya H , Frankfurter A , Lee V M , Zalc B
Published: May 2006

Cerebral hemispheres from mouse embryos at 15 days of gestation were dissociated and maintained in culture for several weeks in a medium which permitted homochronic and homotypic oligodendrocytes and neurons to interact in the presence of other central nervous system cells. After 13-14 days in culture a few oligodendrocytes changed from highly branched, "sun-like," nonmyelinating cells to sparcely branched myelinating cells. The number of fibers myelinated per oligodendrocyte ranged from 1 to 10, similar to that described previously in vivo in the corpus callosum. When an oligodendrocyte began to myelinate, it immediately myelinated a maximum number of fibers, suggesting that the number of axons to be myelinated by the oligodendrocyte was predetermined. When only one fiber was in the vicinity of a myelinating oligodendrocyte, whorls of myelin-like figures were seen at the tip of oligodendrocyte processes that had not reached an axon. Myelinated fibers were unambiguously identified as axons both by immunostaining and by electron microscopy. Myelin was not observed around astrocyte processes or around dendrites. The exclusive myelination of axons suggests the existence of a specific axonal recognition signal which attracts oligodendrocyte processes.
Ethanol selectively blocks a noninactivating K+ current expressed in Xenopus oocytes.
Authors: Covarrubias M , Rubin E
Published: May 2006

There is presently a debate regarding the relative merits of lipid-based and protein-based theories of anesthesia and the action of ethanol in the central nervous system. Voltage-sensitive K+ channels play a key role as regulators of neuronal electrical activity and are potential targets of ethanol and other anesthetic agents. We investigated the action of low concentrations of ethanol on four structurally homologous cloned K+ channels expressed in Xenopus oocytes. We report that only the Drosophila Shaw2 channel, which does not inactivate upon prolonged depolarization, is rapidly and reversibly blocked by ethanol in a concentration-dependent manner (17-170 mM). The concentration dependence of the blockade can be explained by assuming a bimolecular interaction between ethanol and the channel. We also found that Shaw2 K+ channels were selectively blocked by halothane (1 mM). Our results support the "protein hypothesis" of ethanol and anesthetic action. These findings open ways to elucidate directly the molecular mechanism of interaction between general anesthetics and a voltage-sensitive K+ channel.
hP1.B, a human P-domain peptide homologous with rat intestinal trefoil factor, is expressed also in the ulcer-associated cell lineage and the uterus.
Authors: Hauser F , Poulsom R , Chinery R , Rogers L A , Hanby A M , Wright N A , Hoffmann W
Published: May 2006

The six-cysteine P-domain motif forms the basic repeat unit of a growing family of mucin-associated peptides. A precursor for a human secretory polypeptide has been discovered by molecular cloning and deduced to have a single P-domain, termed hP1.B. The pre-pro-peptide has 67% amino acid identity with rat intestinal trefoil factor. We find, using the techniques of RNA analysis and in situ hybridization, that this P-domain peptide is expressed in the human gastrointestinal tract, where a number of pathological conditions affect its expression, and surprisingly find it is expressed in the uterus also. In the intestine, hP1.B is expressed by goblet cells, but in Crohn disease this peptide is synthesized and secreted additionally by the ulcer-associated cell lineage that is known to secrete two other trefoil peptides, pS2 and spasmolytic polypeptide (hSP). In the stomach, hP1.B mRNA is relatively scarce but is more abundant in foci of intestinal metaplasia and near to ulceration. Mucin-rich epithelial cells in hyperplastic polyps of the colon also express this peptide. The discovery of this P-domain peptide and its expression in association with mucins support the hypothesis that P-domains with mucins may subserve related functions in the maintenance and repair of mucosal function.
Structure of a human rhinovirus-bivalently bound antibody complex: implications for viral neutralization and antibody flexibility.
Authors: Smith T J , Olson N H , Cheng R H , Chase E S , Baker T S
Published: May 2006

The structure of a neutralizing immunoglobulin (monoclonal antibody mAb17-IA), bound to human rhinovirus 14 (HRV14), has been determined by cryo-electron microscopy and image reconstruction. The antibody bound bivalently across icosahedral twofold axes of the virus, and there were no detectable conformational changes in the capsid. Thus, bivalently bound IgGs do not appear to cause gross deformations in the capsid. Differences between the electron density of the constant domains of the bound Fab fragment and IgG structures suggested that conformational changes occur about elbow axes upon bivalent attachment as was previously predicted. No significant density was observed for the Fc fragment, which adds further evidence for a high degree of mobility about the hinge region.
Habitat-dependent hybrid parentage and differential introgression between neighboringly sympatric Daphnia species.
Authors: Taylor D J , Hebert P D
Published: May 2006

Allozyme, morphological, and PCR-generated mtDNA markers were used to analyze hybrid parentage and introgression between the neighboringly sympatric crustaceans Daphnia galeata mendotae and Daphnia rosea. mtDNA analysis of D. galeata mendotae from 15 lakes and of D. rosea from 8 lakes revealed species-specific fragment patterns resulting from TaqI and RsaI digestion. No individuals of one parent species possessed the typical mtDNA of the other parent species, suggesting that mtDNA introgression is rare or nonexistent. Hybrids from 18 lakes possessed either the mtDNA patterns of D. rosea or of D. galeata mendotae, indicating that reciprocal hybridization occurs. The mtDNA genome of the dominant parent species in a lake was overrepresented in the hybrids, suggesting that hybridization most often involves females of the common species and males of the rare species. Such a pattern is consistent with the differing importance of density to the environmental induction of males and sexual eggs in Daphnia. For the assessment of nuclear gene flow, eight sympatric populations of each parental taxon and seven allopatric populations of D. galeata were analyzed for allozyme variation at nine polymorphic loci. Our results provided evidence for asymmetric interspecific gene flow involving alleles at six loci that are unlikely to be convergent or symplesiomorphic. This reticulate evolution accounted for much of the genetic divergence between European and North American populations of D. galeata.
Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo.
Authors: Missiakas D , Georgopoulos C , Raina S
Published: May 2006

We have identified and characterized the Escherichia coli gene dsbB, whose product is required for disulfide bond formation of periplasmic proteins, by using two different approaches: (i) screening of a multicopy plasmid library for clones which protect E. coli from the lethal effects of dithiothreitol (DTT), and (ii) screening of insertion libraries of E. coli for DTT-sensitive mutants. Mapping and characterization of mutations conferring a DTT-sensitive phenotype also identified the dsbA, trxA, and trxB genes, whose products are involved in different oxidation-reduction pathways. Null mutations in dsbB conferred pleiotropic phenotypes such as sensitivity to benzylpenicillin and inability to support plaque formation of filamentous phages, and they were shown to severely affect disulfide bond oxidation of secreted proteins such as OmpA and beta-lactamase. These phenotypes resemble the phenotype of bacteria carrying either a null mutation in the dsbA gene or the double mutation dsbA dsbB. Sequencing and expression of the dsbB gene revealed that it encodes a 20-kDa protein predicted to possess an "exchangeable" disulfide bond in -Cys-Val-Leu-Cys-. The dsbB gene maps at 26.5 min on the genetic map of the E. coli chromosome, and its transcription is directed from two promoters, neither of which resembles the canonical E sigma 70-recognized promoter.
Reversal of muscle differentiation during urodele limb regeneration.
Authors: Lo D C , Allen F , Brockes J P
Published: May 2006

Recent studies suggest that maintenance of the differentiated state requires continuous regulation. Limb regeneration in urodele amphibians provides a context in which to address this issue, as limb regeneration may involve the dedifferentiation of multinucleate myotubes to yield mononucleate blastemal cells, which then proliferate and contribute to regenerate tissues. To evaluate this possibility, cultured newt limb myotubes were selectively microinjected with the lineage tracer rhodamine-dextran and introduced into regenerating limbs. In culture, such labeled myotubes were stable after 6-8 weeks, and transfer of the tracer to mononucleate cells was not observed. In contrast, after implantation of labeled myotubes under the wound epidermis of limb blastemas, strongly labeled mononucleate cells were observed after 1 week. These cells could be double-labeled with the cytoplasmic lineage tracer and [3H]thymidine that had been incorporated into the nuclei of implanted myotubes. The number of labeled mononucleate cells increased significantly by 2-3 weeks after implantation, indicating that these cells proliferated. Although the fate of these cells at later times was uncertain, we provide evidence consistent with their subsequent differentiation. These results demonstrate reversal in the mononucleate-to-multinucleate transition of vertebrate myogenesis.
Nonreciprocal homologous recombination between Agrobacterium transferred DNA and a plant chromosomal locus.
Authors: Offringa R , Franke-van Dijk M E , De Groot M J , van den Elzen P J , Hooykaas P J
Published: May 2006

Previously, we demonstrated the occurrence of gene targeting in tobacco cells after Agrobacterium-mediated transformation. In these experiments a defective kanamycin resistance (Kmr) gene residing at a chromosomal location was restored via homologous recombination with an incoming transferred DNA (T-DNA) repair construct (pSDM101) containing a different defective Kmr gene. In this article we describe gene targeting experiments with the same target line, but using an improved repair construct, pSDM321. In one of the Kmr calli obtained after transformation with pSDM321 (line A) the product of homologous recombination was detected using PCR. Further molecular analysis revealed that the defective Kmr gene present on the incoming T-DNA had been restored via homologous recombination with the target locus. The target locus was left unchanged and the corrected T-DNA was found to be inserted on the same chromosome but not close to the target locus. This paper presents molecular evidence in plants for the conversion of an introduced DNA molecule (in this case, T-DNA) by a homologous chromosomal locus.