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.

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.

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.

A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans.

A novel human gene, SARM, encodes the orthologue of a Drosophila protein (CG7915) and contains a unique combination of the sterile alpha (SAM) and the HEAT/Armadillo motifs. The SARM gene was identified on chromosome 17q11, between markers D17S783 and D17S841 on BAC clone AC002094, which also included a HERV repeat and keratin-18-like, MAC30, TNFAIP1, HSPC017, and vitronectin genes in addition to three unknown genes. The mouse SARM gene was located on a mouse chromosome 11 BAC clone (AC002324). The SARM gene is 1.8 kb centromeric to the vitronectin gene, and the two genes share a promoter region that directs a high level of liver-specific expression of both the SARM and the vitronectin genes. In addition to the liver, the SARM gene was highly expressed in the kidney. A 0.4-kb antisense transcript was coordinately expressed with the SARM gene in the kidney and liver, while in the brain and malignant cell lines, it appeared independent of SARM gene transcription. The SARM gene encodes a protein of 690 amino acids. Based on amino acid sequence homology, we have identified a SAM motif within this derived protein. Structure modeling and protein folding recognition studies confirmed the presence of alpha-alpha right-handed superhelix-like folds consistent with the structure of the Armadillo and HEAT repeats of the beta-catenin and importin protein families. Both motifs are known to be involved in protein-protein interactions promoting the formation of diverse protein complexes. We have identified the same conserved SAM/Armadillo motif combination in the mouse, Drosophila, and Caenorhabditis elegans SARM proteins.

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.

Genomic organization of DHR38 gene in Drosophila: presence of Alu-like repeat in a translated exon and expression during embryonic development.

The recombinant lambda clone 4-2 containing the genomic region of the Drosophila hormone receptor 38 (DHR 38) gene, homologous to mammalian neuronal growth factor I-B (NGFI-B), was isolated by radioactive labelled oligonucleotide hybridization. The nucleotide sequence of the genomic clone revealed three exons that encode the functional domains of the protein. The N-terminal exon1 had no homology at the amino acid level with NGFI-B, the mammalian homologue. A glutamine-rich region, probably involved in transcriptional activation, was observed at the C-terminal part of this exon. A similar motif is also present upstream in another reading frame of the same strand. Both motifs are preceded by a repetitive non-anucleotide sequence containing an AluI site, resembling a duplicated human Alu-sequence. A monomeric version of this sequence, coding similarly for an oligoglutamine peptide, occurs at a surprisingly high frequency in other regulatory genes in Drosophila. In contrast to mammalian Alu sequences, this sequence is found almost exclusively in the coding regions of Drosophila genes, but not in the non-coding parts of the genes. The DNA-binding domain with two zinc-fingers, and at least part of the ligand-binding peptide, is coded by the largest middle exon2 in DHR38 and exhibits up to 100% homology in short peptide motifs to its mammalian counterpart, where these domains are split into exons 3, 4, 5, and 6. However, the length, information content, stop codon, and splice site are conserved in the last exons in both fly and rat. In situ hybridization to 0-24 h wholemount embryos showed strong expression of DHR38 in neurogenic regions and in the intestinal tract during embryogenesis, suggesting a spatial and temporal control of transcription, partially analogous to the central nervous system-specific expression of NGFI-B in mammals.

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.

Developmental and cell cycle regulation of alfalfa nucMs1, a plant homolog of the yeast Nsr1 and mammalian nucleolin.

We report here the isolation and characterization of the nucMs1 alfalfa cDNA, whose predicted amino acid sequence structurally resembles the yeast Nsr1 protein and animal nucleolins. These proteins consist of an N-terminal acidic domain, centrally located RNA recognition motifs (RRMs), and a C-terminal glycine- and arginine-rich domain. In comparison with animal nucleolins that contain four RRMs, NucMs1 more closely resembles the yeast Nsr1 protein, which contains only two RRMs. A NucMs1 C-terminal peptide antibody specifically recognized a 95-kD nucleolar protein in alfalfa cells that changed its localization in a cell cycle-dependent manner. The nucMs1 transcript and p95nucMs1 protein levels correlated with cell proliferation, and nucMs1 gene expression was found to be induced in the G1 phase upon mitogenic stimulation of G0-arrested leaf cells. In situ hybridization analysis of different alfalfa organs during various developmental stages showed that nucMs1 gene expression is highest in root meristematic cells, but it is also found in other meristematic cells of the plant body. nucMs1 expression is tightly linked to cell proliferation but does not depend on a particular cell cycle phase. No nucMs1 expression was observed in cells that had exited the cell cycle and were undergoing differentiation or polar growth, indicating that nucMs1 may not be necessary for processes other than cell proliferation.

DNA-based immunization with chimeric vectors for the induction of immune responses against the hepatitis C virus nucleocapsid.

Vectors expressing the first 58 amino acids of the hepatitis C virus (HCV) nucleocapsid alone or as a fusion protein with the middle (pre-S2 and S) or major (S) surface antigens of hepatitis B virus (HBV) were constructed. Intramuscular immunization of BALB/c mice with the chimeric constructs in the form of naked DNA elicited humoral responses to antigens from both viruses within 2 to 6 weeks postinjection. No anti-HCV responses were obtained in mice immunized with the vector expressing the HCV sequence in the nonfusion context. Sera from chimera-injected mice specifically recognized both HCV capsid and HBV surface antigens in enzyme-linked immunosorbent assay and immunoblot testing. Anti-HCV serum titers formed plateaus of approximately 1:3,000; these remained stable until the end of the study (18 weeks postinfection). Anti-HBV immune responses were found to be lower in the chimera-injected animals (< 200 mIU/ml) than in those immunized with the native HBV vector (> 2,000 mIU/ml). This is the first report of the use of DNA-based immunization for the generation of immune responses to an HCV protein. In addition, these findings show that it is possible to elicit responses to viral epitopes from two distinct viruses via DNA immunization with chimeric vectors.

Characterization and mapping of a B-cell immunogenic domain in hepatitis C virus E2 glycoprotein using a yeast peptide library.

To identify conserved humoral antigenic determinants within the hepatitis C virus (HCV) envelope protein E2, we expressed a peptide library containing random short fragments of the HCV envelope in yeast. Clones were identified using a monospecific rabbit antibody to a region downstream of the E2 hypervariable region. The clones define the limits of two original antigenic domains: a major one (aa 493-576) and a minor one (aa 535-606). The major antigenic domain maps in a region that displays a high degree of homology within a (HCV) subtype (92-97.6% identity). Yeast-encoded determinants were characterized by Western blot analysis, N-glycosidase F digestion, and using a panel of synthetic peptides. The data suggest that the major antigenic domain contains at least two determinants, one of them mimicked by an 18-mer peptide (aa 514-531). ELISA and competitive inhibition assays demonstrated that: (1) the determinants appear subtype 1a-specific, (2) seroprevalence of antibody to the determinants is rather low (20.6%), and (3) donors show a heterologous response to the different determinants. Antibody response to the E2 determinants was studied in HCV-infected chimpanzees and post-transfusion-associated NANB hepatitis cases. The antibody response was found during chronic infection and may not be effective for virus clearance.

Rescue of a 7502-nucleotide (49.3% of full-length) synthetic analog of respiratory syncytial virus genomic RNA.

A cDNA was constructed to encode an internally-truncated version of negative-sense genomic RNA (vRNA) of respiratory syncytial virus (RSV) containing (in 3' to 5' order) the 3' vRNA leader region, the complement of the open reading frame for bacterial chloramphenicol acetyl transferase (CAT) under the control of RSV gene-start and gene-end signals, an intergenic nucleotide, the complete L gene including the gene-start and gene-end signals, and the 5' vRNA trailer region. The encoded vRNA analog (RSV-CAT-L) would be 7502 nucleotides (nt) in length, 49.3% of the complete 15,222-nt parental vRNA. RSV-CAT-L vRNA was synthesized in vitro from HgaI-digested cDNA and transfected into RSV-infected cells. The vRNA was "rescued" such that it was expressed to yield CAT and was packaged into particles that could be passaged to express CAT in fresh cells. The efficiency of rescue was greatly improved by a single nucleotide substitution in the leader region that had been found to increase the efficiency of rescue and passage of the previously described 935-nt RSV-CAT vRNA. Compared to the 8-fold smaller RSV-CAT vRNA, and adjusted to molar equivalence of transfected RNA, RSV-CAT-L vRNA was 119- to 347-fold less efficient in expressing CAT upon transfection, whereas RSV-CAT-L vRNA containing the above-mentioned nucleotide substitution was 15.8-fold less efficient.

Alfalfa cyclins: differential expression during the cell cycle and in plant organs.

Cell division in eukaryotes is mediated by the action of the mitosis promoting factor, which is composed of the CDC2 protein kinase and one of the various mitotic cyclins. We have recently isolated a cdc2 gene from alfalfa. Here, we report the isolation of two cyclin genes, cycMs1 and cycMs2, from alfalfa. The cycMs2 gene shows highest similarity to type B cyclins. In contrast, the predicted amino acid sequence of the cycMs1 gene shows similar homology scores to cyclins of all types (25 to 35%). Both genes are expressed in dividing suspension cultured cells but cease to be expressed when the cells enter stationary phase. In synchronized alfalfa suspension cultured cells, the mRNAs of cycMs1 and cycMs2 show maximal expression in the G2 and M phases. Transcripts of cycMs2 are found only in late G2 and M phase cells, an expression pattern typical for cyclin B genes, whereas cycMs1 appears with the onset of G2. This pattern indicates that alfalfa cycMs1 and cycMs2 belong to different classes of cyclins. In young leaves, expression of both genes is high, whereas in mature leaves no transcripts can be detected, indicating that the two cyclin genes are true cell division markers at the mRNA level. In other organs, a more complex expression pattern of the two cyclin genes was found.

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.

Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene.

The viral genomic RNA (vRNA) of human respiratory syncytial virus is a nonsegmented negative strand that is not infectious alone. To develop methods for complementing synthetic vRNA with viral proteins, a cDNA was constructed to encode a vRNA in which all of the viral protein-coding sequences were removed and replaced with a negative-sense copy of the bacterial chloramphenicol acetyltransferase gene. Upon transfection into respiratory syncytial virus-infected cells, the synthetic vRNA was "rescued" such that it was amplified, expressed, and packaged into infectious virions. A heterologous paramyxovirus, parainfluenza virus 3, was inactive in rescue. Further internal deletions mapped the cis-acting viral sequences required for rescue to two segments totaling 105 nucleotides (nt) derived from the two vRNA ends. Rescue was unaffected by replacement of the 44-nt 3'-terminal leader region with a 50-nt sequence that is complementary to the 5' terminus and represents the 3' end of the positive-sense replicative intermediate RNA. This 5'-end complement was related to the parental leader region only near the 3' terminus (91% or 73% identical for the first 11 or 22 nt, respectively). The addition of 11 heterologous nt to the 3' end of the parental leader region ablated rescue, suggesting that the 3'-proximal conserved domain is required and cannot function from an internal site. However, deletion of the 3'-terminal 3 nt, or a double transition at positions 4 and 5, had no effect on rescue. Thus, the 3'-terminal 5 nt, although conserved between 3' ends of the negative- and positive-sense RNAs, do not appear to be essential.

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.

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.

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.

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.

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.