Comparison of llama VH sequences from conventional and heavy chain antibodies

Forty different PCR clones encoding a llama variable heavy chain domain were analysed. The majority of these clones are derived from heavy-chain antibody cDNA in which the entire CH1 exon is absent. It appears from the amino acid within the VHH framework 1 and 3 that all the llama clones belong to the VH III family. However, the individual llama VHH sequences differ more substantially from each other than expected for members of the same family. Several remarkable amino acid substitutions in the framework 2 hinder the proper association of the VL. However, they lay the foundation for the secretion from the endoplasmic reticulum and good solubility behaviour of llama H2 antibodies. The repertoire of the llama VHHs may be extensive due to the presence of a long CDR3-loop, often constrained by a disulfide bridge and the occurrence of H1 and H2 loop conformations not yet encountered in mice or human VHs. The variability plot of the amino acids in the VHH shows that the first hypervariable region coincides with the structural H1 loop in contrast to the situation found in mice and man where the CDR1 and H1 are slightly offset. We propose that the amino acids of the llama H1 loop participate actively in the antigen binding. All these observations are characteristic for the llama VHHs of the homodimeric heavy-chain H2 antibodies, but are not maintained in the llama clones from conventional heterotetrameric H2L2 immunoglobulins.     

Characterization of unconventional MYO6, the human homologue of the gene responsible for deafness in Snell's waltzer mice

Deafness is the most common form of sensory impairment in humans. Mutations in unconventional myosins have been found to cause deafness in humans and mice. The mouse recessive deafness mutation, Snell's waltzer, contains an intragenic deletion in an unconventional myosin, myosin VI (locus designation, Myo6). The requirement for Myo6 for proper hearing in mice makes this gene an excellent candidate for a human deafness disorder. Here we report the cloning and characterization of the human unconventional myosin VI (locus designation, MYO6) cDNA. The MYO6 gene maps to human chromosome 6q13. The isolation of the human gene makes it now possible to determine if mutations in MYO6 contribute to the pathogenesis of deafness in the human population.     

A novel small conductance Ca2+-activated K+ channel blocker from Oxyuranus scutellatus taipan venom. Re-evaluation of taicatoxin as a selective Ca2+ channel probe

Taicatoxin, isolated from the venom of the Australian taipan snake Oxyuranus scutellatus, has been previously regarded as a specific blocker of high threshold Ca2+ channels in heart. Here we show that taicatoxin (in contrast to a range of other Ca2+ channel blockers) interacts with apamin-sensitive, small conductance, Ca2+-activated potassium channels on both chromaffin cells and in the brain. Taicatoxin displays high affinity recognition of 125I-apamin acceptor-binding sites, present on rat synaptosomal membranes (Ki = 1.45 +/- 0.22 nM) and also specifically blocks affinity-labeling of a 33-kDa 125I-apamin-binding polypeptide on rat brain membranes. Taicatoxin (50 nM) completely blocks apamin-sensitive after-hyperpolarizing slow tail K+ currents generated in rat chromaffin cells (mean block 97 +/- 3%, n = 12) while only partially reducing total voltage-dependent Ca2+ currents (mean block 12 +/- 4%, n = 6). In view of these findings, the use of taicatoxin as a specific ligand for Ca2+ channels should now be reconsidered.     

Corneal scarring in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study: baseline prevalence and repeatability of detection

Fibroblast growth factor-16 (FGF-16) is the most recent member of the FGF family to be cloned. Since the biologic activity of rat FGF-16 (rFGF-16) was unknown, and this protein has no apparent signal sequence, we transformed its entire cDNA into Escherichia coli for high-level expression and further characterization of this novel protein. An attempt was made to purify the expressed protein from the supernatant of mechanically lysed cells using sequential cation-exchange chromatography. This resulted in a gradual loss of the protein as precipitate throughout the purification process. In addition to precipitation during purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the partially purified materials showed a cluster of protein bands around 20k to 29k. Sequence analysis of the major bands indicated that two N-terminal truncations had occurred, during E. coli fermentation, purification, or both. The largest truncation resulted in the removal of the 34 N-terminal amino acids, including the initiation codon methionine. We cloned d34 rFGF-16, expressed the gene in E. coli, and developed a purification process for this form. Even with this truncated form, precipitation was a problem. We were largely able to overcome this problem, however, by including EDTA throughout the purification process. We have characterized the structure of purified d34 rFGF-16 extensively using circular dichroism, Fourier transform infrared spectroscopy, and sedimentation velocity analysis. These studies revealed that the protein has a distinct tertiary structure, consists primarily of beta-strands, has a weak tendency to self-associate, and is fairly extended. We then performed biologic assays which showed that d34 rFGF-16 induces oligodendrocyte proliferation in vitro, and induces hepatocellular proliferation and increased liver weight in vivo. In summary, FGF-16, a novel FGF family member, has both unique structural and biological properties.     

Prodrugs for targeting hypoxic tissues: regiospecific elimination of aspirin from reduced indolequinones

We report the cases of three patients with anorexia nervosa (AN) who each recovered rapidly after experiencing a life-threatening episode with severe thrombocytopenia. All three cases were the typical restricting-type of AN, occurring in adolescence. They refused to be admitted to a hospital until their general condition had been severely deteriorated. Their lowest platelet counts were 2.9, 4.6, and 2.3 x 10(4)/mm3, respectively. Apparent hemorrhagic tendencies, such as purpura, gingival and nasal bleeding, and gastrointestinal bleeding were observed. The bone marrow examination showed apparent hypoplasia in two patients. No evidence of disseminated intravascular coagulation or autoantibody to platelets was detected. The platelet counts recovered rapidly by water and nutritional supplementation. The recovery from the AN itself was excellent in all three patients without specific psychotherapy.     

Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels

A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched in isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method.     

Erythema migrans-like rash illness at a camp in North Carolina: a new tick-borne disease?

BACKGROUND: Borrelia burgdorferi, the causative agent of Lyme disease, has never been isolated from a patient thought to have acquired Lyme disease in any southeastern state. OBJECTIVE: To investigate 14 cases of an erythema migrans (EM)-like rash illness that occurred during 2 summers at an outdoor camp in central North Carolina in an effort to determine the etiologic, epidemiological, and clinical aspects of this illness. METHODS: Using active surveillance, we identified cases of clinically diagnosed EM in residents and staff of the camp. We collected clinical and demographic information; history of exposure to ticks; acute and convalescent serum antibodies to B. burgdorferi, Rickettsia rickettsii, and Ehrlichia chaffeensis; and cultures for spirochetes from biopsy specimens of skin lesions. Serum samples from a group of residents and staff who did not develop rashes were tested for the same antibodies. We speciated ticks removed from people and collected from vegetation. RESULTS: We identified 14 cases of EM-like rash illness during the 2 summers. Of the 14 case-patients, 10 had associated mild systemic symptoms and 1 had documented fever. All 14 case-patients had removed attached ticks, and 8 remembered having removed a tick from the site where the rash developed a median of 12 days earlier (range, 2-21 days). One tick removed from the site where a rash later developed was identified as Amblyomma americanum, the Lone Star tick; 97% of ticks collected from vegetation and 95% of ticks removed from people were A. americanum. No spirochetes were isolated from skin biopsy specimens. Paired serum samples from 13 case-patients did not show diagnostic antibody responses to B. burgdorferi or other tick-borne pathogens. CONCLUSIONS: This investigation suggests the existence of a new tick-associated rash illness. We suspect that the disease agent is carried by A. americanum ticks. In the southern United States, EM-like rash illness should no longer be considered definitive evidence of early Lyme disease.