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PubMed2000

Transgenic mouse models for lung cancer.

Transgenic technology allows the ability to target regulatory genes to the lungs in a cell-specific fashion. Using this technology, we have generated a model to investigate the phenotypic consequences of targeting oncogenes to particular cell types in the lungs and are developing a second model for the regulated expression of oncogenes in the lung. The transgenic model involves the constitutive expression of simian virus 40 large T antigen in the Clara cells of mouse lungs. This model has been used to investigate changes in expression of cell cycle regulatory genes in the Clara cells during the transformation process, as well as the expression of the transcription factors regulating the expression of Clara cell differentiation markers. The second model we are developing investigates the regulated expression of the genes in the lungs. This system is based on the establishment of two types of transgenic lines. The regulator line consists of a chimeric transcriptional factor placed under the control of a lung-specific SPC (surfactant protein C) promoter. This chimeric regulator is composed of a transcription activation domain, the GAL4 DNA-binding domain, and a truncated progesterone receptor that is responsive to RU 486, but not to endogenous progesterone. The second transgenic mouse line contains the silent target transgene under the control of a minimal promoter with upstream activating sequences (UAS) that are recognized by the regulator transgene. Upon breeding these two lines, the resulting bitransgenic mice can then be induced to express the target transgene only with the administration of RU 486. Two generations of regulators have been evaluated on their ability to regulate the expression of a growth hormone reporter gene. This system demonstrated the inducible expression of the reporter genes in the distal airways of the lungs.

Adenocarcinoma↗

PubMed1988

Poly(A) shortening and degradation of the 3' A+U-rich sequences of human c-myc mRNA in a cell-free system.

The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A) shortening, which generated a pool of oligoadenylated mRNA molecules. In contrast, the poly(A) of a stable mRNA, gamma globin, was not excised, even after prolonged incubation. The second step, degradation of oligoadenylated c-myc mRNA, generated decay products whose 3' termini were located within the A+U-rich portion of the 3' untranslated region. These products disappeared soon after they were formed, consistent with rapid degradation of the 3' region. In contrast, the 5' region, corresponding approximately to c-myc exon 1, was stable in vitro. The data indicate a sequential degradation pathway in which 3' region cleavages occur only after most or all of the poly(A) is removed. To account for rapid deadenylation, we suggest that the c-myc poly(A)-poly(A)-binding protein complex is readily dissociated, generating a protein-depleted poly(A) tract that is no longer resistant to nucleases.

Adenine↗

PubMed1993

Tumor-specific lysis of human renal cell carcinomas by tumor-infiltrating lymphocytes. I. HLA-A2-restricted recognition of autologous and allogeneic tumor lines.

Metastatic renal cell carcinoma (RCC), like melanoma, belongs to the small group of human tumors in which partial or complete remission has been observed in some patients after treatment with various forms of immunotherapy. In contrast to melanoma, CTL showing MHC-restricted lysis of RCC have not been easily found among tumor-infiltrating lymphocytes (TIL). This has led to the suggestion by some that responses to immunotherapy are mediated predominantly by non-MHC-restricted effector cells. We have characterized an MHC-restricted, CD8+ CTL line obtained from an uncloned TIL population of a primary RCC using a low concentration of rIL-2; in fact, these CTL represented a majority of the short-term cultured TIL population. The CTL lysed autologous tumor cells but not normal kidney cells or target cells sensitive to non-MHC-restricted effector cells. In contrast, lymphokine-activated killer (LAK) cells grown in a high concentration of rIL-2 from the patient's PBL lysed autologous tumor and normal kidney cells in addition to several allogeneic tumors. The TIL could be expanded optimally using an autologous tumor line retrovirally transduced with the human cDNA encoding IL-2. TIL were 20-fold more potent than LAK cells in eliminating the IL-2 expressing tumor cells in vitro. The cultured TIL utilized a restricted number of V alpha gene families, suggesting that they may recognize only a limited number of MHC-peptide complexes presented by autologous tumor cells. HLA-A2 was identified as an MHC restriction molecule for presentation of one tumor-derived peptide to these CTL. Only some allogeneic HLA-A2 RCC tumors were lysed. Sequencing of the second and third exons of the HLA-A2 alleles of these cells revealed that both heterogeneity in MHC and peptide availability influenced CTL recognition. These studies demonstrate that some RCC express common antigenic determinants that can be recognized by MHC-restricted CTL and open the possibility of defining the nature of RCC-derived peptides, which, combined with HLA-A2, can generate specific immune responses.

Antibodies, Monoclonal↗

PubMed2000

Use of a repetitive DNA probe to type clinical and environmental isolates of Aspergillus flavus from a cluster of cutaneous infections in a neonatal intensive care unit.

Aspergillus flavus is second to A. fumigatus as a cause of invasive aspergillosis, but no standard method exists for molecular typing of strains from human sources. A repetitive DNA sequence cloned from A. flavus and subcloned into a pUC19 vector, pAF28, was used to type 18 isolates from diverse clinical, environmental, and geographic sources. The restriction fragment length polymorphisms generated with EcoRI- or PstI-digested genomic DNA and probed with digoxigenin-labeled pAF28 revealed complete concordance between patterns. Eighteen distinct fingerprints were observed. The probe was used to investigate two cases of cutaneous A. flavus infection in low-birth-weight infants in a neonatal intensive care unit (NICU). Both infants were transported by the same ambulance and crew to the NICU on the same day. A. flavus strains of the same genotype were isolated from both infants, from a roll of tape used to fasten their umbilical catheters, from a canvas bag used to store the tape in the ambulance, and from the tape tray in the ambulance isolette. These cases highlight the need to consider exposures in critically ill neonates that might occur during their transport to the NICU and for stringent infection control practices. The hybridization profiles of strains from a second cluster of invasive A. flavus infections in two pediatric hematology-oncology patients revealed a genotype common to strains from a definite case patient and a health care worker. A probable case patient was infected with a strain with a genotype different from that of the strain from the definite case patient but highly related to that of an environmental isolate. The high degree of discrimination and reproducibility obtained with the pAF28 probe underscores its utility for typing clinical and environmental isolates of A. flavus.

Ambulances↗

PubMed1996

Genomic location and nucleotide sequence of a porcine adenovirus penton base gene.

The putative penton base gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and sequenced. The genomic location of the PAV3 penton base was deduced by probing a Southern blot with a polymerase chain reaction generated product containing the human adenovirus type 2 (HAV2) penton base gene. Sequencing revealed an open reading frame (ORF) of 1527 nucleotides coding for a polypeptide of 509 amino acids. However, cDNA analysis indicated an acceptor splice site one nucleotide upstream of the second ATG in the ORF. This produced an ORF of 1452 nucleotides coding for a polypeptide of 484 amino acids with a calculated molecular weight of 54.5 kDa. Comparison with the HAV2 penton base amino acid sequence revealed the putative PAV3 penton base homologue to be 87 amino acids shorter with an overall amino acid homology of approximately 65%. Comparison with the penton base proteins of other HAV types revealed a region between amino acid positions 283 and 379 with no similarity.

Adenoviridae↗

PubMed1996

Construction of adenoviral and retroviral vectors coexpressing the genes encoding the hepatitis B surface antigen and B7-1 protein.

Recombinant retroviral (re-Rv) and adenoviral (re-Ad) vectors for delivery of two foreign genes were constructed, using the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) which mediates initiation of cap-independent translation. The first gene encoded the hepatitis B surface antigen (HBsAg) and the second encoded human or murine B7-1 molecule, a cell surface protein which is a costimulator for T cell activation. The EMCV IRES sequence was placed between the first and second coding sequences to form a dicistronic DNA fragment. In Rv vectors, the dicistronic fragment was inserted between the 5' long terminal repeat (LTR) and an internal promoter for the neomycin (neo) gene, so that the transcription initiated from the 5' LTR would generate a dicistronic mRNA for the HBsAg and B7-1 molecules. For Ad vectors, the dicistronic fragment was inserted between a cytomegalovirus promoter and a polyA signal to form a transcription cassette. This transcription cassette was inserted into the early region 1 of Ad5 genome to form a replication-defective re-Ad vector, or into early region 3 to form replication-competent vectors. Human cell line A549 infected with the re-Rv vectors or with the re-Ad vectors synthesized and secreted HBsAg at comparable levels, while the B7-1 molecules were detected at the surface of the infected cells, indicating both foreign genes carried by the Rv and Ad vectors were expressed efficiently.

Adenoviridae↗

PubMed1999

Rapid detection of Hepatitis E virus RNA by reverse transcription-polymerase chain reaction using universal oligonucleotide primers.

A rapid reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of Hepatitis E virus (HEV) RNA in serum is described. Total nucleic acids are extracted from a small volume of human serum and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR employing degenerate HEV consensus primers. These primers are designed to sequences conserved between the Burma, Mexico, and US HEV strains, generating amplicons within each of the three open reading frames. Reactions are analyzed by agarose gel electrophoresis and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated as positive. This protocol allows for the rapid and sensitive detection of HEV infection in human serum.

DNA Primers↗

PubMed1998

Simultaneous measurement of perfusion and oxygenation changes using a multiple gradient-echo sequence: application to human muscle study.

We have developed a magnetic resonance imaging (MRI) technique based on a multiple gradient-echo sequence designed to probe perfusion and oxygenation simultaneously within skeletal muscle. Processing of the images acquired at successive echo times (TEs) generates two functional maps: one of the signal intensity (SI) extrapolated to zero echo time, which is sensitive to perfusion; and a second one of R2*, which reflects oxygenation. An advantage of the processing procedure lies in the selection of tissue of interest through the profile of T2* decay, leading to automatic rejection of pixels containing small vessels. This allows a more specific assessment of tissue perfusion and oxygenation. This technique was demonstrated successfully during post-ischemic reactive hyperemia in human calf. A perfusion peak of 123 mL x 100 g(-)1 x min(-1) was measured immediately after ischemia, whereas R2* value showed an 11.5% decrease at the same time, essentially reflecting blood oxygenation changes. Differences in the time courses of reperfusion and re-oxygenation were observed, oxygenation presenting a slower recovery. The mechanisms responsible for such a differential dynamic response are discussed.

Adult↗

PubMed2005

Total synthesis of polycavernoside A, a lethal toxin of the red alga Polycavernosa tsudai.

[structure: see text] Two approaches to the synthesis of the aglycon 120 of polycavernoside A (1) were developed, only one of which was completed. The successful "second-generation" route assembled the aglycon seco acids 102 and 106 via Nozaki-Hiyama-Kishi coupling of aldehyde 70, prepared from methyl (S)-3-hydroxy-2-methylpropionate (72) and (S)-pantolactone (73), with vinyl bromide 71. The latter was obtained from a sequence which commenced from the silyl ether 24 of 3-hydroxypropionaldehyde and entailed cyclization of (Z)-zeta-hydroxy-alpha,beta-unsaturated ester 82. Regioselective Yamaguchi lactonization of trihydroxycarboxylic acids 102 and 106 and subsequent functional-group adjustments led to macrolactone 120, to which the fucopyranosylxylopyranoside moiety was attached. Stille coupling of the glycosidated aglycon 128 with dienylstannane 129 furnished polycavernoside A in a synthesis for which the longest linear sequence was 25 steps. The overall yield to lactone 120 was 4.7%.

4-Butyrolactone↗

PubMed2006

An integrated digital microfluidic chip for multiplexed proteomic sample preparation and analysis by MALDI-MS.

To realize multiplexed sample preparation on a digital microfluidic chip for high-throughput Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), several fluidic functions need to be integrated. These include the generation of multiple droplets from a reservoir and parallel in-line sample purification. In this paper, we develop two critical new functions in handling protein solutions and standard proteomic reagents with electrowetting-on-dielectric (EWOD) actuation, leading to an integrated chip for multiplexed sample preparation for MALDI-MS. The first is a voltage sequence designed to generate a series of droplets from each of the three reservoirs--proteomic sample, rinsing fluid, and MALDI reagents. It is the first time that proteomic reagents have been dispensed using EWOD in an air (as opposed to oil) environment. The second is a box-in-box electrode pattern developed to allow droplet passing over dried sample spots, making the process of in-line sample purification robust for parallel processing. As a result, parallel processing of multiple sample droplets is demonstrated on the integrated EWOD-MALDI-MS chip, an important step towards high-throughput MALDI-MS. The MS results, collected directly from the integrated devices, are of good quality, suggesting that the tedious process of sample preparation can be automated on-chip for MALDI-MS applications as well as other high-throughput proteomics applications.

Angiotensin II↗

PubMed1998

The instantaneous measurement of multiple Doppler spectra in the investigation of ovarian masses.

The use of Doppler indices of tumor vascularity as markers for malignancy has been a subject of debate, with inconsistencies in specificity and sensitivity as well as in the diagnostic cut-off values. Part of the discrepancy might be explained by the limited number of vessels selected within the tumor for Doppler evaluation. A typical sample of three or four vessels may not be sufficiently large for correct representation of the vasculature of the tumor, which may contain many vessels. The existing conventional Doppler (color and spectral) techniques clearly have limitations. We present here a novel Doppler modality, namely two-dimensional spectral Doppler imaging (SDI), which allows the acquisition of many Doppler spectra within a few seconds. The operator selects a region of interest within a color Doppler image. The Doppler sequence is initiated and the entire selected region is automatically scanned for about 20 s until all spectral Doppler data have been acquired and processed. The system computer generates a color-coded map of the desired Doppler indices overlaid on the gray-scale image. The system also displays a cumulative histogram or a table of the requested Doppler index from all the sections in the tissue. The whole process is automatically performed by the system computer, without any need for operator intervention. The system provides the examiner with a 'fishing net' for Doppler indices, instead of the 'fishing hook' used in current techniques.

Female↗

PubMed1986

Two DNA recognition domains of the specificity polypeptides of a family of type I restriction enzymes.

The hsd genes of Salmonella typhimurium and Salmonella potsdam encode related type I restriction and modification systems designated SB and SP, respectively; the polypeptide encoded by the hsdS gene dictates the DNA sequence recognized. The hsdS genes of the SB and SP systems have a conserved sequence of around 100 base pairs flanked by two nonhomologous (variable) regions of around 500 base pairs. Recombination between the hsdS genes of SB and SP generated a system (SQ) with a different recognition specificity. We have localized the position of the crossover in the central conserved region by analysis of nucleotide sequences. Concomitant with the generation of a new combination of flanking variable regions is the recombination of minor differences in the central conserved region. A polypeptide domain encoded on the 5' side of the crossover dictates recognition of the trinucleotide component of the target sequence, and a second domain, encoded on the 3' side of the crossover, similarly governs recognition of the tetra- or penta-nucleotide component. Our analysis implicates at least parts of the variable regions in the determination of the specificity of interaction between protein and DNA. Furthermore, the trinucleotide components of the recognition sequences of S. typhimurium and Escherichia coli K-12 are identical, and the 5' segments of their hsdS genes are strikingly homologous rather than variable.

Amino Acid Sequence↗

PubMed1997

A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism.

Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms. The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself. We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl- and carboxylmethylcysteine modifications. The formation and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl1) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor. Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-dependent but length-specific proteolytic activity. Third, a-factor and AFRP use distinct cellular machinery for their secretion. Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion. Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor. Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.

ATP-Binding Cassette Transporters↗

PubMed1986

Sequences: toward a common denominator of family therapy.

This paper presents a conceptual framework that is designed to help clinicians and researchers organize their observations of the complex network of interconnected sequences of behavior and thinking that constitute family interactions. This framework distinguishes four classes of recursive sequences, each of which is defined by its period, i.e., the amount of time it takes for a sequence to complete one cycle. The shortest sequences (S1) are those face-to-face interaction patterns that range in length from seconds to an hour. S2 sequences are played out over one day to one week, often depending on the routines of the family. S3 sequences range from several weeks to a year, and S4 sequences are those patterns of interaction and thought that repeat from generation to generation. In addition to outlining this framework, the paper contends that in many families with a problem, pertinent sequences from each class may be related to the problem and to each other in a cybernetic manner. Suggestions are offered for identifying pertinent sequences from each class and for selecting interventions that best address the targeted sequence. We have found that this conceptual framework has enabled us to use techniques from many different schools of family therapy while avoiding the conceptual contradictions inherent in previous attempts to integrate approaches.

Adolescent↗

PubMed2006

Use of intersimple sequence repeats markers to develop strain-specific SCAR markers for Lentinula edodes.

Although Lentinula edodes is the second most important cultivated mushroom worldwide, most industrially cultivated strains have been identified only through traditional phenotypic analysis. Here, we report for the first time the use of sequence characterized amplified region (SCAR) markers for strain differentiation. SCAR markers were created by first generating and sequencing single intersimple sequence repeats fragments, and then designing primers based on these sequences to amplify strain-specific fragments of a certain size. One SCAR primer pair, ISL450F/R7 (amplifying a band of c. 450 bp), was designed to identify one strain of L. edodes (strain No. 7). The SCAR primer pair was then used to correctly amplify the single unique fragment from DNA samples taken from a total of 85 strains representing three separate species. Our data provide the foundation for a precise and rapid PCR-based strain-diagnostic system for L. edodes.

Base Sequence↗

PubMed1999

A simple RT-PCR-based strategy for screening connexin identity.

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

Animals↗

PubMed2006

[Randomized study comparing piroxicam analgesia and tramadol analgesia during outpatient electromagnetic extracorporeal lithotripsy].

OBJECTIVE: To analyse the efficacy of two types of analgesia protocols during outpatient electromagnetic extracorporeal lithotripsy (ESWL) with a STORZ Modulith SLX second generation lithotriptor. MATERIAL AND METHODS: One hundred and seventy one patients were prospectively randomized to 2 groups. Group 1 (n-82) received 40 mg of Piroxicam IM, and group 2 (n=89) received 100 mg Tramadol IV Lithotripsy was performed in 2 sequences, T1 and T2, during which quantitative evaluation of pain (VE) was performed according to a VAS scale and qualitative evaluation of pain (QE) was performed according to the McGill questionnaire. Endpoints included the mean VE and QE pain scores at T1 and T2, the maximum power tolerated at T2, the postoperative pain at the 6th, 12th and 24th hours after ESWL, the pain score according to the power and according to the frequency and adverse effects. Comparisons were performed by analyses of variance (significant for p < 0.05). RESULTS: No significant difference was observed between the two groups for stone dimensions, duration and number of ESWL shots (p > 0.05) and significant difference was observedfor pain with the two drugs used. Pain was more severe as frequency and power increased. Only one intervention was stopped because of pain. Data analysis suggests that tramadol induces more adverse effects. CONCLUSION: Piroxicam and tramadol are two analgesics commonly used in clinical practice and both are suitable for the treatment of pain during outpatient extracorporeal lithotripsy.

Ambulatory Care↗

PubMed1990

Cartilage proteoglycan aggregate is degraded more extensively by cathepsin L than by cathepsin B.

The degradative actions of cathepsins L and B on human articular-cartilage proteoglycan aggregates were examined. Cathepsin L was found to be much more extensive than cathepsin B in degrading proteoglycan aggregates. It released products with size similar to that of single chondroitin sulphate chains, and a series of degraded link-protein fragments in the digestion mixtures. These proteolytically modified link-protein components (Mr 25,000 and 33,000) have similar Mr values to those of fragments observed in adult human cartilage. In contrast, cathepsin B exhibited a much more limited degradation on both proteoglycan subunits and link-protein components. Both cathepsins L and B generate multiple but distinct cleavage sites on human link proteins, and the hydrolysed bonds have been identified in the region between residues 18 and 29. Protein sequencing analysis of these modified link-protein components also provided evidence for the location of a second N-linked glycosylation site at residue 41 in human link proteins, in addition to that previously described at residue 6 on a proportion of the link proteins. Furthermore, it allows us to report the sequence of human link protein up to residue 65.

Amino Acid Sequence↗