Biomedical subjects
Publications and source records attributed to C J Lobb.
Based on a quantum analysis of two capacitively coupled current-biased Josephson junctions, we propose two fundamental two-qubit quantum logic gates. Each of these gates, when supplemented by single-qubit operations, is sufficient for universal quantum computation. Numerical solutions of the time-dependent Schrödinger equation demonstrate that these operations can be performed with good fidelity.
We present spectroscopic evidence for the creation of entangled macroscopic quantum states in two current-biased Josephson-junction qubits coupled by a capacitor. The individual junction bias currents are used to control the interaction between the qubits by tuning the energy level spacings of the junctions in and out of resonance with each other. Microwave spectroscopy in the 4 to 6 gigahertzrange at 20 millikelvin reveals energy levels that agree well with theoretical results for entangled states. The single qubits are spatially separate, and the entangled states extend over the 0.7-millimeter distance between the two qubits.
We show that dc voltage versus current measurements of a YBa(2)Cu(3)O(7-delta) film in a magnetic field can be collapsed onto scaling functions proposed by Fisher et al. [Phys. Rev. B 43, 130 (1991)] as is widely reported in the literature. We find, however, that good data collapse is achieved for a wide range of critical exponents and temperatures. These results strongly suggest that agreement with scaling alone does not prove the existence of a phase transition. We propose a criterion to determine if the data collapse is valid, and thus if a phase transition occurs. To our knowledge, none of the data reported in the literature meet our criterion.
Circular DNA, derived from lymphocytes of juvenile channel catfish, was used to construct lambda libraries that were screened to identify the products of immunoglobulin DH-JH excision events. Clones were characterized that contained DH to JH recombination signal joints. The signal joints represented 23-bp recombination signal sequences (RSS) identical to germline JH segments that were adjacent to DH 12-bp RSS elements. DH flanking regions within the clones were used to probe a genomic library. Three germline DH gene segments containing 11-19 bp coding regions flanked by 12-bp RSS elements with conserved heptamers and nonamers were identified. The DH locus is closely linked to the JH locus, and Southern blots indicate that the DH segments represent different single member gene families. Analysis of H chain cDNA shows that each germline DH segment was expressed in functional VDJ recombination events involving different JH segments and members of different VH families. Several aspects of CDR3 junctional diversity were evident, including deletion of coding region nucleotides, N- and P-region nucleotide additions, alternate DH reading frame utilization, and point mutations. Coding region motifs of catfish DH segments are phylogenetically conserved in some DH segments of higher vertebrates. These studies indicate that the structure, genomic organization, and recombination patterns of DH segments typically associated with higher vertebrates evolved early in vertebrate phylogeny at the level of the bony fish.
Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a full-length zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47-52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R(43), a 5' splice variant that lacked 17 amino acids in the amino-terminal extracellular domain, showed cAMP accumulation when challenged with [Tyr(34)]hPTH(1-34)-amide (hPTH) (EC(50), 1.64 +/- 0. 95 nM) and [Ile(5),Trp(23),Tyr(36)]hPTHrP-(1-36)-amide ([Ile(5), Trp(23)]hPTHrP) (EC(50), 46.8 +/- 12.1 nM) but not when stimulated with [Tyr(36)]hPTHrP-(1-36)-amide (hPTHrP), [Trp(23), Tyr(36)]hPTHrP-(1-36)-amide ([Trp(23)]hPTHrP), or [Ala(29),Glu(30), Ala(34),Glu(35),Tyr(36)]fugufish PTHrP-(1-36)amide (fuguPTHrP). FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands.
The structure, organization, and partial sequence of a 25-kb genomic region containing a second cluster of H chain gene segments in the channel catfish (Ictalurus punctatus) has been determined. Multiple VH gene segments, representing different VH families, are located upstream of a germline-joined VDJ. The VDJ segment has a split leader sequence and a single open reading consistent with that expressed in members of the VH1 family. Downstream of the germline-joined VDJ is a single JH segment and two pseudogene exons structurally similar to the Cmu1 and Cmu2 exons of the functional gene. Both pseudogene exons are multiply crippled with RNA splice sites destroyed, and open reading frames are interrupted by termination codons, insertions, and/or deletions. Sequence alignment of a 10.8-kb region within the second H chain cluster with the genomic sequence of the nine JH segments and the functional Cmu within the first H chain gene cluster indicates that the second H chain gene cluster probably arose by a massive duplication event. The JH region of the VDJ, the coding and flanking regions of the single JH segment, and the pseudogene Cmu exons were readily aligned with homologous segments in the first gene cluster. This duplication event may have extended to include the upstream VH segments. A member of the Tc1 mariner family of transposable elements is located downstream of the pseudogene Cmu2, which suggests that the transposition may have contributed to the evolution of the duplicated Cmu.
Earlier studies distinguished two classes of catfish light (L) chain (designated F and G). The cDNA structure and genomic organization of G L chain gene clusters has also been characterized previously. In this study, full length cDNA encoding F L chain was derived using PCR strategies based on the determined amino-terminal protein sequence. The encoded V region is readily delineated into framework regions (FR) and complementarity-determining regions (CDR). Multiple sequence alignments indicate that the F V(L) is closely related to kappa gene families. The F C(L) cannot be generally classified but it is structurally distinct from the C(L) regions of G: the amino acid sequence similarity is <35%. cDNA sequences representing processed sterile F transcripts of different loci were identified. Each sequence begins within the J(L) recombination signal sequence and extends downstream through the I(L)-C(L) segments. Genomic blots hybridized with C(L) probes indicate that there are at least 50 different C(L) segments. Based upon V(L) hybridization studies, different families of V(L) segments appear to be associated with closely related F C(L) segments. In characterized genomic clones, F gene segments are arranged in closely linked clusters with single copies of V(L), J(L), and C(L) segments within each cluster. The V(L) segments are located in opposite transcriptional polarity relative to the J(L) and C(L) segments, which indicates that V(L) segments rearrange by inversion. These combined studies establish that two structurally distinct classes of L chains are present in teleost fish and that both of the L chain classes evolved within a common organizational pattern of clustered segmental genes.
OBJECTIVE: To evaluate a new enzyme-linked immunosorbent assay (ELISA) for amphotericin B in serum samples. Results are compared with those obtained by HPLC and bioassay. DESIGN: Comparison of results obtained by ELISA, HPLC, and bioassay. METHODS: We developed a new ELISA using a polyclonal rabbit antibody to measure serum amphotericin B concentrations. Blinded samples of amphotericin B in concentrations of 0.15-78 micrograms/mL were prepared in human serum and assayed simultaneously by the ELISA, HPLC, and bioassay. The results of each assay were derived from standard curves and evaluated by using the Table Curve 2D computer program. These data were compared by using correlation analysis with evaluation of Pearson's correlation coefficient by Student's t-test. RESULTS: ELISA and bioassay compared favorably at amphotericin B concentrations of 0.3-20 micrograms/mL with a correlation coefficient of r = 0.993, while ELISA and HPLC compared with a correlation coefficient of r = 0.944. The average coefficient of variation over the range 0.3-20.0 micrograms/mL was 28% +/- 7% for HPLC, 26% +/- 9% for ELISA, and 13% +/- 4% for bioassay. Comparison of all three assays revealed the highest correlation with the ELISA assay (r = 0.998) for the range of concentrations (0.3-20 micrograms/mL) routinely achieved. Samples containing concentrations in excess of 20 micrograms/mL could be diluted. Desiccation for concentrations less than 0.3 microgram/mL was not tested. CONCLUSION: The determination of serum amphotericin B concentrations by ELISA gave results similar to those obtained by a bioassay and HPLC technique. Although variability appears greater with ELISA, the ease of performing yjis assay expedites the evaluation of amphotericin B concentrations from lipid formulations without interference from coadministered antibacterials of azole antifungals.
A VH gene segment that could not be assigned to any of the six known VH gene families of the channel catfish was identified in a genomic clone containing VH gene segments. This gene segment (designated VH7.1) exhibited the structural features characteristic of vertebrate VH genes, specifically potential upstream regulatory sequences, a leader sequence split by an intron, a reading frame that could be readily divided into framework and complementarity determining regions, and a 3' recombination signal sequence. Two regions of nucleotide deletions coupled with degeneracy in the nonamer sequence indicate that this VH gene segment is a pseudogene. Genomic DNA restricted with different enzymes and hybridized under stringent conditions with probes derived from VH7.1 showed that 8-10 bands were present in Southern blots. Reverse transcriptase PCR approaches were used to determine if any of these related sequences were expressed. Sequence analysis of cloned PCR products indicates that different VH gene segments exhibiting > 80% similarity to germline VH7.1 are expressed. Multiple sequence alignments showed that the expressed cVH7a cDNA sequence shared less than 60% nucleotide similarity with representative cDNA sequences from the other known catfish VH gene families. These combined results thus fulfil the criteria for the definition of a new family of catfish VH gene segments. This newly defined, small VH family is designated VH7.
Our purpose was to develop and characterize an enzyme-linked immunosorbent assay (ELISA) which could measure the concentration of amphotericin B in serum. Amphotericin B was assayed by competition ELISA. Multiwell ELISA plates coated with amphotericin B (1.0 micrograms/ml) conjugated to bovine serum albumin were used to test replicates of serum samples spiked with amphotericin B. Purified rabbit polyclonal antibody against amphotericin B (1.4 micrograms/ml) was added subsequent to the instillation of samples spiked with unknown amounts of amphotericin B. Experiments were performed to test the sensitivity, specificity, precision, and accuracy of the assay. The ability to measure lipid-associated amphotericin B was also evaluated in preliminary studies. Analysis of reference samples containing amphotericin B yielded a traditional sigmoidal curve. The limits of detection were 0.15 to 156 micrograms/ml. The sensitivity of the assay was affected by light and temperature exposure. Assay specificity was altered only by the presence of nystatin, a polyene antifungal agent similar to amphotericin B. Intrarun (coefficient of variation = 3.0%) and interrun (coefficient of variation = 12.8%) coefficients of variation were calculated and were comparable to those in similar assays. The assay's correlation coefficient (r = 0.907) demonstrated a statistically significant correlation between the optical density of the sample and the concentration of drug in the sample. The amphotericin B ELISA's ease, precision, and overall accuracy suggest that this assay could be used for assessments of serum amphotericin B concentrations. Multiple research questions concerning the role of serum amphotericin B concentrations in toxicity and efficacy have gone unanswered because of the labor-intensive nature of the assays which have been available to date. The ability to easily and rapidly measure 40 duplicate samples containing amphotericin B should also prove to be a distinct advantage for clinical research or reference laboratories in addressing these questions.
Nucleated erythrocytes of lower vertebrates provide a source of genomic DNA that can be used in pulsed-field gel electrophoresis (PFGE) studies. Difficulties reported in the preparation of chicken erythrocyte DNA for analysis by PFGE suggest that the presence of hemoglobin iron may result in iron-mediated DNA degradation. We report here modifications to the procedures established for isolation of high molecular weight DNA from mammalian cells. By increasing the volume of buffers and extending the incubation periods to allow for the removal of hemoglobin iron, we have successfully prepared channel catfish erythrocyte DNA suitable for analysis by PFGE.
To determine the structure and organization of germline VH gene segments in the channel catfish, genomic lambda libraries were screened with cDNA probes representing different catfish VH gene families. Thirty-six VH positive genomic clones were isolated and four of these were characterized by restriction mapping and Southern blot analysis with probes specific for each known VH gene family. The four clones, representing about 65 kb of DNA, contained 21 VH segments. The average distance between segments was about 3 kb and gene segments representing different VH gene families were interspersed with each other. Dot-blot hybridization analysis of all 36 genomic clones (average insert size 16-18 kb) indicated that the average clone contained gene segments representing four different VH families. In addition, these analyses indicated that VH segments representing each VH family could be found closely linked to gene segments representing each of the other VH families. Genomic restriction fragments containing a VH segment of each gene family were sequenced. These analyses showed that the general structure of VH segments is conserved in catfish. These structural features include the presence of a leader sequence split by a short intron, an uninterrupted open reading frame encoding readily identified framework and complementarity determining regions, and a downstream recombination signal sequence represented by a consensus heptamer, a 22-24 bp spacer, and an A-rich nonamer. Upstream of the VH segments was an octamer sequence. These analyses indicate that the organization and structure of VH segments typically associated with VH loci of higher vertebrates evolved early in phylogeny at the level of the bony fishes.
Channel catfish L chain cDNA was obtained through a PCR strategy and used to isolate multiple L chain clones from cDNA and genomic libraries. Sequence analysis of full-length cDNA indicates that the V region is preceded by a leader peptide, and represented by framework and CDR regions. Both VL and CL domains contain the invariant cysteines and tryptophans as well as other phylogenetically conserved L chain residues. The sequence similarity of the catfish L chain with higher vertebrate kappa- and lambda-chains, however, does not readily allow the catfish L chain to be classified. Eight cDNA clones isolated from a cDNA library were shown to represent different processed derivatives of sterile L chain transcripts. These transcripts share a similar upstream sequence region and extend downstream to include a CL or alternatively a JL segment in partial germ-line configuration that has been spliced into a CL. Sequence comparisons indicate that these transcripts represent the product of different L chain loci. Genomic Southern blot analyses with VL and CL probes indicate that there are at least 30 VL segments and at least 15 CL segments. The analysis of 17 genomic L chain clones showed that each hybridized with VL-, JL-, and CL-specific probes. Characterization of the gene segments in three of these clones indicates a previously undescribed pattern of segmental gene organization. Gene segments are found in clusters with VL, JL, and CL segments in each cluster. Within a cluster VL segments reside upstream of single copies of closely linked JL and CL segments. The proximity of VL segments downstream from JL-CL segments suggests that individual clusters may be closely linked. The VL segments are located in opposite transcriptional polarity relative to the JL and CL gene segments, which indicates that VL segments are likely rearranged to JL-CL segments by inversion rather than deletion events.
The JH locus of the channel catfish has been characterized to determine the organization and structural diversity of JH segments. These analyses indicate that there are a total of nine JH segments tightly clustered within a region spanning about 2.2 kb. The JH locus is closely linked to the CH 1 domain of the expressed catfish H chain; the distance between the CH proximal JH segment (JH9) and the CH 1 domain is about 1.8 kb. Each JH segment has an upstream recombination sequence, which includes a T-rich nonamer, a 22- to 24-bp spacer, and a phylogenetically conserved heptamer. Each JH segment also has an open reading frame that encodes the conserved framework region 4 tryptophan (Trp-103) and terminates with a RNA donor splice site. The catfish JH locus contains an internal repetitive sequence region characterized by a short (183-188 bp) repeat that occurs sequentially five times. Strong sequence homology as well as the unified length of the repeated sequences indicate that JH segments JH3-JH7 probably arose as the result of a series of homologous but unequal crossover events. Sequence alignments of the duplicated JH segments indicates that there is diversity within the 5-11 nucleotides located immediately downstream from the heptamer, an observation which indicates that closely related JH segments can serve to enhance CDR3 diversity in the expressed H chain. Comparisons of the genomic JH sequences with different cDNA clones indicate that each JH segment is probably functional and that junctional diversity serves an important role in the generation of CDR3 diversity. In addition, single base differences observed in comparisons of JH-encoded regions indicate that there is probably somatic mutation or allelic variation of genomic JH segments. These studies suggest that the characteristic structure and organizational pattern of JH segments in higher vertebrates may have evolved early in vertebrate phylogeny at the level of the bony fish.
Genomic DNA from 18 phylogenetically diverse species of bony fish was hybridized with probes specific for the channel catfish immunoglobulin heavy chain constant (CH) gene, as well as with immunoglobulin heavy chain variable (VH) probes specific for five channel catfish VH gene families. The results showed that CH probes strongly hybridized only to genomic fragments from other catfish species. In contrast, restricted DNA from most other species hybridized with at least two channel catfish VH probes. In those species whose DNA hybridized with multiple VH probes, the restriction pattern of hybridizing fragments was probe-dependent. These studies suggest that (1) the CH gene defined in channel catfish appears to share similarity only with CH genes in other catfish species, (2) families of VH genes appear to have diverged in early phylogenetic lineages of teleosts, and (3) VH genes similar to those defined in catfish appear to be widely represented in phylogenetically diverse species of teleosts.
Several studies have shown that isolates of Edwardsiella ictaluri obtained from infected channel catfish in the southeastern United States harbor two cryptic plasmids, designated pCL1 (5.7 kb) and pCL2 (4.9 kb). These isolates appear to be serologically homogeneous. To extend these studies, we focused our analyses on two isolates of nonictalurid origin. Plasmid analyses of a danio isolate showed that it harbored plasmids which were similar if not identical to pCL1 and pCL2. This strain was also serologically indistinguishable from those isolated from channel catfish. In contrast, a green knife fish (GNF) isolate harbored four plasmids with relative mobilities of 6.0, 5.7, 4.1, and 3.1 kb. Southern blot analyses indicated that only the 5.7- and 4.1-kb plasmids strongly hybridized under high-stringency conditions to probes specific for pCL1 and pCL2, respectively. The GNF isolate showed minimal reactivity when reacted with polyclonal antiserum prepared against a channel catfish isolate. However, polyclonal antiserum to the GNF isolate strongly reacted with the GNF isolate in both surface fluorescence and agglutination reactions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of cell lysates showed that the protein banding patterns of the strains compared were similar. However, Western blots of proteinase K-digested cell extracts showed that O antigen of the GNF isolate was antigenically distinct from the O antigen of the other isolates. These studies indicate that there are different serotypes of E. ictaluri and suggest that plasmid and serological analyses of future isolates of E. ictaluri can be used to determine whether structurally distinct strains are emerging in major channel catfish aquaculture areas.
A channel catfish genomic lambda library was screened with CH and JH probes which were derived from our earlier sequence analyses on different full-length heavy chain cDNA clones. One clone, designated C7, contained a genomic insert of about 18 kb and hybridized with specific probes for each of the four domains of the known C region gene as well as with different oligonucleotides specific for JH gene segments. Southern blot hybridization analysis identified a cluster of JH gene segments which are closely linked to the CH gene. Sequence analysis of the CH-proximal JH element, located about 1.9 kb upstream from the CH1 domain, showed that this element contains 5'-recombination signals typical of JH elements defined in higher vertebrates, i.e. a nonamer, a 24 bp spacer, and a heptamer. The coding region of this JH element was identical to that contained in the variable region sequence of a cDNA clone previously reported. Sequence analysis of the catfish JH-CH intron suggests that several sequences are present which appear similar to important transcriptional regulatory elements found within JH-CH introns of higher vertebrates. These features include sequences similar to higher vertebrate enhancer elements and regulatory octamers. An additional feature reminiscent of some higher vertebrate heavy chain switch regions is a repetitive sequence area composed of tandemly repeated simple sequences. Lastly, several restriction length polymorphisms were identified and mapped within a 1 kb region located immediately upstream from the JH cluster. This finding suggests that polymorphisms within the IgH locus should be useful in the analyses of channel catfish populations. These combined studies provide further evidence that the genomic organization of heavy chain genes in bony fish shares common organizational features with those known from higher vertebrates.
The V regions of channel catfish H chain cDNA clones have been analyzed. Based upon sequence relationships and hybridization analyses, five different groups of VH genes are identified whose definition is consistent with that of five different VH families. Genomic Southern blots indicate that as many as 100 different germ-line VH genes are likely represented by these families. The sequence diversity between identified members of these different families is similar in magnitude to the divergence represented between members of different human or mouse VH families. The FR regions are the most conserved regions when members of different catfish VH families are compared; specific amino acid positions appear to be highly conserved in phylogeny. Equally important is that diversity is represented in complementarity-determining regions CDR1 and CDR2 in members of the different families as well as in members of the same VH family. These results suggest that an extensive repertoire of VH genes can contribute to antibody diversity in this lower vertebrate. Sequence comparisons indicate that one of the catfish VH families shares considerable structural similarity to several higher vertebrate VH gene families--a relationship which suggests that this VH family may be ancestral to some VH gene families of higher vertebrates. Characteristic of the genomic organization of higher vertebrate H chains, catfish appear to have different VH families wherein a VH gene likely undergoes functional recombination with putative DH gene segments and one of apparently several different JH segments. The recombined V region is expressed with the same C region gene. These combined results suggest that bony fishes are the earliest known phylogenetic representatives to have evolved extensive V region gene families.