Probing the topological exciton condensate via Coulomb drag.

The onset of exciton condensation in a topological insulator thin film was recently predicted. We calculate the critical temperature for this transition, taking into account screening effects. Furthermore, we show that the proximity to this transition can be probed by measuring the Coulomb drag resistivity between the surfaces of the thin film as a function of temperature. This resistivity shows an upturn upon approaching the exciton-condensed state.

A novel fibrotic disorder associated with increased dermal fibroblast proliferation and downregulation of genes of the microfibrillar network.

Clinical evaluation of a young woman with subcutaneous fibrotic nodules, progressive distal joint contractures and marfanoid stature revealed a previously unrecognized fibrotic disorder characterized by several unique phenotypic features and some features overlapping with known disorders. Mutational analysis of the FBN1 and FBN2 genes excluded Marfan syndrome and congenital contractural arachnodactyly. MMP2 gene sequence analysis excluded multicentric osteolysis, nodulosis and arthropathy. The lack of mutations within the MAGP2 gene also excluded an MAGP2-associated disorder. In order to establish the mechanistic basis for the severe skin pathology noted in this patient, we performed transcriptional profiling of dermal fibroblasts, and candidate gene expression studies in conjunction with immunocytochemistry and cell-based and functional assays. Data from these experiments have further excluded any previously recognized fibrotic disorder and identified a unique pattern of gene expression in this patient consistent with progressive fibrosis. The pathogenic mechanisms included persistent proliferation of dermal fibroblasts in coexistence with a disarray of the microfibrillar network. Collagen accumulation, moreover, could be linked to extensive crosslinking resulting from increased activities of lysyl oxidases (LOX and LOXL), and lack of remodelling due to deficiencies in collagenolytic matrix metalloproteinases. The disorder may represent a novel syndrome in which transforming growth factor-ß1-independent dermal fibrosis, unlike known microfibrillar disorders caused by single gene deficiencies, associates with a disarray of the microfibrillar network.

Sequence homology of nucleic acids from human breast cancer cells and complementary DNA's from murine mammary tumor virus and Mason-Pfizer monkey virus.

Simultaneous presence of murine mammary tumor virus- and Mason-Pfizer monkey virus-specific sequences has been detected in nucleic acids isolated from some human breast tumors and from MCF-7 cells, a well-characterized human breast cancer cell line. Carefully characterized long complementary DNA transcripts were used in the molecular hybridization experiments. From the data that are presently available, it would appear that when homology is detected with one of the mammary tumor probes the other also generally shows shows homology. Among all the complementary DNA-RNA hybrids only three, all murine mammary tumor virus hybrids, show Tm values close to 80 degrees. The rest of the hybrids are low melting with shallow slopes for their Crt curves, indicating partial and imperfect hybrids in the majority of cases. Low levels of weak hybrid formation are also detectable with the tumor DNA's. The present experiments cannot ascertain whether the hybridizing sequences from Mason-Pfizer monkey virus and murine mammary tumor virus code for specific viral functions in their natural hosts. Annealing experiments using gene specific cDNA's would be required for fully characterizing these sequences.

Structural and functional diversity of lysyl oxidase and the LOX-like proteins.

Lysyl oxidase (LOX) and four lysyl oxidase-like proteins, LOXL, LOXL2, LOXL3 and LOXL4, each contain a copper binding site, conserved lysyl and tyrosyl residues that may contribute to quinone co-factor formation, and a cytokine receptor-like domain. Each protein differs mainly in their N-terminal sequence, which may confer individual functions. Processing of the LOX proteins by BMP-1 and possibly other mechanisms may result in multiple functional forms. Splicing, reported for LOXL3, may also generate additional variants with unique functions. Each LOX, with its individual, developmentally regulated tissue and cell-specific expression and localization, results in a complex structural and functional variation for the LOX amine oxidases. The presence of only two LOX-like proteins in Drosophila, each with distinct spatial and temporal expression, allows for the assignment of individual function to one of these amine oxidases. Comparative expression analysis of each LOX protein is presented to help determine their functional significance.

Genetic and molecular analysis in the 70CD region of the third chromosome of Drosophila melanogaster.

A collection of lethal and semi-lethal P-element insertions in the 70CD region of chromosome 3 of Drosophila melanogaster was used to investigate genes and gene arrangements by a combination of genetic, cytological, functional and molecular methods. The 12 lethal insertions studied fall into seven complementation groups of six genes. Lethal phases, expression patterns and other phenotypic aspects of these genes were determined. The genes and additional available sequences were placed on cloned genomic DNA fragments and arranged in an EcoRI map of 150kb that covers approximately the bands 70C7-8 to 70D1. Determination of deficiency breakpoints links the genetic, physical and molecular data. The sequences adjacent to seven independent P-element insertions were established after plasmid rescue or polymerase chain reaction. Similarity searches allowed the assignment of the P-element insertions to known mutations, expressed sequence tags, sequence tagged sites, or homologous genes of other species. Among these were identified a putative transacylase, a putative cell cycle gene, and the gene responsible for the dominant Polycomb-suppressor phenotype of devenir. The genomic sequence of the l(3)70Ca/b gene reveals a novel heat shock protein (hsc70Cb). l(3)70Da was identified as a member of the CDC48/PEX1 ATPase family and its coding sequence was determined.

Molecular cloning and sequence analysis of the human parainfluenza 3 virus genes encoding the surface glycoproteins, F and HN.

The sequence of the genes encoding the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of the human parainfluenza 3 virus was determined by molecular cloning. The genes were cloned by primer extension using genomic 50 S RNA as the template. A series of four overlapping clones was generated from the 3' end of the fusion gene which extended across the gene end and intergenic boundaries of the F-HN and HN-L genes. The F gene extends 1851 nucleotides (inclusive of the putative transcription initiation and polyadenylation signals) and encodes a protein consisting of 539 amino acids (mol wt 60,067). This protein contains four potential sites for N-linked glycosylation in the F1 subunit polypeptide and none in the F2 subunit polypeptide. The lack of a potential site of glycosylation in F2 makes this protein unique compared to other reported paramyxoviral F proteins. The HN gene extends 1888 nucleotides and encodes a protein consisting of 572 amino acids (mol wt 64,255). This protein contains four potential sites for N-linked glycosylation.

A simple method for increased recovery of purified paramyxovirus virions.

A simple method involving vigorous agitation of infected cell monolayers prior to collection of culture medium is described to greatly increase the recovery of purified virions of measles virus, respiratory syncytial virus and human parainfluenza virus. Vigorous agitation of the flasks containing monolayers of infected cells increased the recovery of purified virions by at least 3- to 10-fold as judged by the intensity of [35S]methionine labeled viral proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). These protein profiles also indicated that these virions were as clean as those purified from gently collected medium. Analysis of titers of infectious virus recovered from medium of agitated and non-agitated flasks showed similar increases. These results suggest that the cell associated nature of these viruses may be at least partly due to either partially budded virions or mature virions sticking to the cell membrane, since these both might be expected to be freed from the cell by mechanical shearing.

Endonuclease-free, protoplast-forming enzyme preparation and its application in fungal transformation.

An endonuclease-free, protoplast-forming enzyme complex was prepared from the "snail enzyme." The purified preparation has high protoplast-forming activity comparable to the crude enzyme complex without destroying circular plasmid DNA. Furthermore, a higher transformation rate was achieved by the application of the endonuclease-free enzyme complex in both yeast and filamentous fungal vector-host systems.

Techniques of treatment of peritoneal endometriosis: the cavitational ultrasonic surgical aspirator.

The efficacy of medical therapy in endometriosis-associated infertility has been called into question. For decades, surgery has been used in the treatment of endometriosis. However, before the 1960s it consisted of either excision or hysterectomy and bilateral adnexectomy. Although controversy exists about whether laparotomy or operative laparoscopy is the more efTective therapeutic approach for endometriosis, the efficacy of surgery in reducing implants, relieving dysmenorrhea and pelvic pain, and improving fertility has been well-established.

Impact of human immunodeficiency virus type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding and antiviral potency of enfuvirtide in vitro.

Enfuvirtide (ENF), a novel human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, has potent antiviral activity against HIV-1 both in vitro and in vivo. Resistance to ENF observed after in vitro passaging was associated with changes in a three-amino-acid (aa) motif, GIV, at positions 36 to 38 of gp41. Patients with ongoing viral replication while receiving ENF during clinical trials acquired substitutions within gp41 aa 36 to 45 in the first heptad repeat (HR-1) of gp41 in both population-based plasma virus sequences and proviral DNA sequences from isolates showing reduced susceptibilities to ENF. To investigate their impact on ENF susceptibility, substitutions were introduced into a modified pNL4-3 strain by site-directed mutagenesis, and the susceptibilities of mutant viruses and patient-derived isolates to ENF were tested. In general, susceptibility decreases for single substitutions were lower than those for double substitutions, and the levels of ENF resistance seen for clinical isolates were higher than those observed for the site-directed mutant viruses. The mechanism of ENF resistance was explored for a subset of the substitutions by expressing them in the context of a maltose binding protein chimera containing a portion of the gp41 ectodomain and measuring their binding affinity to fluorescein-labeled ENF. Changes in binding affinity for the mutant gp41 fusion proteins correlated with the ENF susceptibilities of viruses containing the same substitutions. The combined results support the key role of gp41 aa 36 to 45 in the development of resistance to ENF and illustrate that additional envelope regions contribute to the ENF susceptibility of fusion inhibitor-naïve viruses and resistance to ENF.

Indication for deletion of two introns in the oxi-3 gene of a respiratory-competent Saccharomyces cerevisiae strain.

Physical mapping of the mitochondrial DNA of the wild-type Saccharomyces cerevisiae strain RXII revealed that most of the restriction sites as well as the location of the apocytochrome b gene were identical in comparison with the known maps of the mitochondrial genome in other Saccharomyces cerevisiae strains. In the middle of the SalI linearized map of the RXII mitochondrial DNA, a deletion was detected which resulted in the loss of two EcoRI and one BamHI restriction sites. The corresponding region, however, exists in most other laboratory strains of Saccharomyces mapped so far. This region overlaps the introns aI2 and aI3 surrounding exon A3 sequences of the subunit 1 of the cytochrome oxidase gene. The nucleotide sequence of the subunit 1 gene showed that the BamHI site was located close to the aI3-A4 intron-exon junction and the distal EcoRI site close to the aI2-A2 boundary. I therefore conclude that these two introns are deleted in the mitochondrial genome of strain RXII. The exon A3 must have been conserved since this strain was respiratory competent. This result, while being a good example of the morphological diversity of a genome with the same function, may contribute to an understanding of the role of introns in the mitochondrial split genes in yeast.

Isolation of a DNA sequence stimulating recombination in yeast.

A series of DNA sequences was rescued from the yeast Saccharomyces cerevisiae transformed by a gene library and selected for the cdc35ts+, TRP1+ phenotype. These sequences did not complement the cdc35ts mutation, and were found in various amounts and orientations in degraded plasmids. A similar phenomenon was demonstrated when the HIS3 gene was cloned into one of them: a highly deleted plasmid was rescued from complemented homozygous diploid yeast cells, in which the HIS3+/his3- character was inherited at a 2:2 ratio. These results suggest that the insert sequences rescued from the cdc35ts transformants stimulate vigorous non-reciprocal recombination events by the transfer of HIS3 gene or the TRP-ARS fragment. This event was detected in the transformation of cdc35 or his3- hosts and was followed by the re-isolation of the degraded plasmid molecules.

Purification and electron microscopy of a large plasmid of Rhizobium meliloti 41.

A large plasmid DNA molecule was purified from Rhizobium meliloti 41 by CsCl-ethidium bromide density gradient centrifugation. Electron microscopic and agarose gel electrophoretic data suggest that addition of alkali effectively removes the chromosomal DNA, the plasmid DNA can be precipitated from the cleared lysate and no gradient centrifugation is needed for plasmid purification.

Domains of the Saccharomyces cerevisiae CDC25 gene controlling mitosis and meiosis.

The cell division cycle gene CDC25 was replaced by various disrupted and deleted mutant copies. Mutants disrupted at a central position of the gene, or lacking 532 residues within the amino-terminal half of the gene product grow normally in glucose, but not in acetate media, and they fail to sporulate as homozygous diploids. Disruptions or deletions within the carboxy-terminal half are lethal, except for the deletion of the 38 carboxy-terminal residues, which are required for sporulation but not for growth in glucose or acetate media. It is concluded that distinct domains of the CDC25 gene product are involved in the control of mitosis and/or meiosis.

Detection of a 2 mu derivative yeast plasmid with altered properties.

The Saccharomyces cerevisiae (Sacch. cerevisiae) strain RXII, like many others, harbours plasmid DNAs. and one category of them is homologous to the 2 mu plasmid of yeast. DNA-DNA hybridization experiments indicated altered structures of this species as regards the number and distribution of the restriction sites. The efforts made to clone either the whole plasmid in pBR328 or its fragments in pBR322 vectors remained unsuccessful, since deleted plasmids were isolated without insert DNA, and even the loss of vector sequences was observed. The data suggest, that the 2 mu derivative plasmid in strain RXII represent an unique category of this extrachromosomal genetic element.

Molecular cloning and sequence analysis of the human parainfluenza 3 virus gene encoding the L protein.

The sequence of the gene encoding the L protein of the human parainfluenza 3 virus was determined by direct dideoxy sequence analysis of the genomic 50 S RNA and confirmed by molecular cloning and sequence analysis of recombinant clones. A series of three overlapping clones was generated by primer extension using genomic 50 S RNA as the template. These clones originate within the 5' end of the hemagglutinin-neuraminidase gene, span the entire L gene, and extend into the extracistronic 5' end of the viral RNA. The L gene extends 6755 nucleotides (inclusive of the putative transcription initiation and polyadenylation signal sequences) and encodes a protein consisting of 2233 amino acids (MW 255,812). There are 44 nucleotides downstream of the putative polyadenylation signal sequence which may represent a negative-strand leader. The complementary sequence of the extracistronic region is nearly identical to the 3' end of the viral RNA. Thirty-three of the first thirty-nine nucleotides of the 3' ends of the plus and minus strands are conserved. Comparison of amino acid sequence homology with other paramyxoviral L proteins indicates a high degree of sequence conservation with Sendai virus (62%) and Newcastle disease virus (28%). In addition, four smaller regions were identified which shared extensive homology with the L protein of vesicular stomatitis virus, a member of the Rhabdoviridae family.

Restoration of the yeast LEU2 gene by transcriptionally controlled recombination between tandem repeats.

The LEU2 gene of a his3 strain was inactivated by inserting the HIS3 gene between two overlapping inactive leu2 gene fragments, and mitotic stability of the resulting leu2:HIS3::leu2 sequence was measured under leucine repression and derepression. Both inactive leu2 regions were transcribed under derepressing conditions (growth in low leucine), and the LEU2 gene was completely restored by illegitimate recombination between the overlapping tandem repeats, leading to the loss of the intervening HIS3 gene. In contrast, only the downstream leu2 fragment was transcribed upon leucine repression, and the HIS3 insert in the leu2 region remained intact. The reciprocal experiment (inactivation of the HIS3 gene by inserting the marker gene LEU2) revealed a moderate rate of HIS3 restoration and LEU2 excision, reflecting transcriptional activity of the HIS3 region intermediate between that of LEU2 transcription in the repressed and derepressed state.

Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.