Caldesmon inhibits active crossbridges in unstimulated vascular smooth muscle: an antisense oligodeoxynucleotide approach.

Caldesmon is a thin-filament-associated protein believed to be important in the regulation of smooth muscle contraction, although the precise mechanism is unknown. We used antisense oligodeoxynucleotides to produce intact swine carotid smooth muscle tissue deficient in h-caldesmon. Caldesmon content was decreased by 78% after 7 days in culture with antisense oligodeoxynucleotides but was unchanged in tissues in the presence of sense oligodeoxynucleotides or vehicle. Antisense oligodeoxynucleotides produced a significant decrease in the caldesmon/actin ratio, but no change was measured in the calponin/actin ratio, suggesting that the effect was specific to caldesmon and not other thin-filament-associated proteins. Basal and KCl-stimulated levels of myosin light chain phosphorylation were not different among tissues from all 3 groups. In contrast, h-caldesmon-deficient tissues produced 62% less KCl-induced force than controls. Unstimulated h-caldesmon-deficient smooth muscle tissues stretched and then released, redeveloped force, demonstrating active crossbridge cycling; strips containing normal h-caldesmon content did not redevelop force on release. We suggest that in resting vascular smooth muscle, active crossbridges are inhibited by caldesmon. Therefore, regulation of smooth muscle includes a thin-filament-based disinhibition component.

Robotic automation of dideoxyribonucleotide sequencing reactions.

We developed a robot to carry out standard Sanger dideoxyribonucleotide sequencing reactions efficiently and with minimal human intervention. A commercial robot was adapted to our design and specifications, and we programmed it to perform up to 240 sequencing reactions in a single unattended run of 7 h. The robot configuration can be easily altered to allow 480 reactions to be performed in an unattended run of 14 h. The special features of our robot include cooled reagent reservoirs and cooled chambers for storage of DNA templates and completed reactions as well as reproducible aspiration of small volumes by using a sensing algorithm. The robot has successfully performed over 3500 DNA reactions in about 30 separate runs in our DNA core facility.

Exclusion of mutations in the gene for type III collagen (COL3A1) as a common cause of intracranial aneurysms or cervical artery dissections: results from sequence analysis of the coding sequences of type III collagen from 55 unrelated patients.

We performed detailed DNA sequencing analysis on type III collagen cDNA from 58 patients with either intracranial artery aneurysms or cervical artery dissections. The 58 patients were of seven different nationalities; among the patients were three pairs of relatives, so that 55 were unrelated, and of these, 29 had at least one blood relative with either an intracranial artery aneurysm or a cervical artery dissection. The age of the patients at the time of diagnosis ranged from 15 to 68 years (mean +/- SD = 40.3 +/- 11.0). The study group consisted of 25 males and 33 females. The analysis covered 3,232 nucleotides of significant (nonredundant) sequences per allele; therefore, we analyzed as many as 355,520 nucleotides. Mutations in the coding sequences for the triple-helical domain of type III collagen were excluded in 40 individuals with intracranial aneurysms and 18 individuals with cervical artery dissections. Direct sequencing of polymerase chain reaction products allowed mutations to be excluded with a high degree of confidence. Mutations that markedly decreased expression from one allele were also excluded in 42 of the 58 individuals, since the presence of both bases at one or more polymorphic sites in the 42 patients showed that two alleles were transcribed. The results indicated that mutations in the gene for type III procollagen (COL3A1) are not a common cause of either intracranial artery aneurysms or cervical artery dissections.

Sequencing of cDNA from 50 unrelated patients reveals that mutations in the triple-helical domain of type III procollagen are an infrequent cause of aortic aneurysms.

Detailed DNA sequencing of the triple-helical domain of type III procollagen was carried out on cDNA prepared from 54 patients with aortic aneurysms. The 43 male and 11 female patients originated from 50 different families and five different nationalities. 43 patients had at least one additional blood relative who had aneurysms. Five overlapping asymmetric PCR products, covering all the coding sequences of the triple-helical domain of type III procollagen, were sequenced with 28 specific sequencing primers. Analysis of the sequencing gels revealed only two nucleotide changes that altered the structure of the protein. One was a substitution of threonine for proline at amino acid position 501 and its functional importance was not clearly established. The other was a substitution of arginine for an obligatory glycine at amino acid position 136. In 40 of the 54 patients, detection of a polymorphism in the mRNA established that both alleles were expressed. The results indicate that mutations in type III procollagen are the cause of only about 2% of aortic aneurysms.

Single base mutation that substitutes glutamic acid for glycine 1021 in the COL3A1 gene and causes Ehlers-Danlos syndrome type IV.

The proposita described here was a 24-year-old woman with an acrogeric form of the Ehlers-Danlos syndrome including a massive dissecting aortic aneurysm. She was found to have a single-base mutation that substituted glutamic acid for glycine at amino acid position 1021 in the triple-helical domain of the type III procollagen. It is the most carboxy-terminal single-base mutation characterized to date in the COL3A1 gene. Analysis of medium and cell layer proteins from proposita's cultured skin fibroblasts showed that the mutant protein was poorly secreted, migrated more slowly on a polyacrylamide gel, and was partially unstable at +25 degrees C to brief digestion with trypsin.

Efficient DNA sequencing on microtiter plates using dried reagents and Bst DNA polymerase.

Sequenase, Taq DNA polymerase and Bst DNA polymerase were tested for sequencing of DNA on microtiter plates using dried down reagents. Several parameters were investigated to expedite the drying process while minimizing damage to the enzyme. Sequenase did not tolerate drying very well, and frequently generated sequences with weak signals and many sites of premature termination. With Taq DNA polymerase it was possible to obtain sequences of good quality. However, there was considerable variation of results between experiments and between batches of microtiter plates. Bst DNA polymerase generated sequences of excellent quality. It was stable for more than a week in dried-down state at -20 degrees C and at least overnight at room temperature. The method described here using Bst DNA polymerase is well suited for laboratory robots and workstations that typically employ 96-well microtiter plates.

An ocular melanoma-associated antigen. Molecular characterization.

Monoclonal antibody (MAb) 8-1H defines a highly conserved ocular melanoma-associated antigen. Based on a potential binding site of MAb8-1, we identified, molecularly cloned, and sequenced the gene, encoding this melanoma-associated antigen from a uveal melanoma copy DNA library. Nucleotide sequence analysis of the melanoma-associated antigen revealed characteristics of a membrane-bound protein of 238 amino acids (approximately 25 kd) with four transmembrane domains. The sequence of the ocular melanoma-associated antigen is identical to the sequence of a recently reported cutaneous melanoma-associated antigen, substantiating our previous studies regarding common antigens between ocular and cutaneous melanoma. In addition, the melanoma-associated antigen shares sequence homology with a number of transmembrane proteins that appear to be involved in growth regulation. The implication of the biological function of this ocular melanoma-associated antigen, its relevance to the malignant phenotype, and clinical applications of the molecular characterization of this melanoma-associated antigen are described.

Simple harmonic motion of tropomyosin: proposed mechanism for length-dependent regulation of muscle active tension.

A simple harmonic theory is proposed to describe the regulatory mechanism of tropomyosin in the activation of muscle contraction. The theory proposes that activation-associated displacement of tropomyosin is inherent to tropomyosin, a consequence of the molecule's large-scale vibrational motion (5-10 A root mean square displacement). In association with thin filament the vibrational motion may become less complex, approaching the ideal case of simple harmonic motion. The degree of activation increases as the amplitude of the simple harmonic motion increases, causing tropomyosin to shorten lengthwise, shiftings its position from the periphery of thin filament (OFF) to the actin groove (ON). The amplitude may be regulated in a rectilinear manner by the thick filament electrostatic force, the thin filament hydrophobic force, and the Ca(2+)-dependent force of the troponin complex. The radial and tangenital components of the resultant force may vary as the muscle is stretched, regulating maximum active tension and Ca2+ sensitivity, respectively. This may represent the molecular basis for Starling's law of the heart. The mechanism may be important for describing the regulatory mechanism of tropomyosin in smooth muscle and nonmuscle cells and may facilitate a clinically relevant understanding of the effects of pH, Mg2+ concentration, ionic strength, and ethanol on the regulation of active tension.

Purification and amino terminal sequencing of human melanoma nerve growth factor receptor.

The nerve growth factor (NGF) receptor, solubilized with Triton X-100 detergent, has been purified from human melanoma cell line A875. Purification to near-homogeneity was achieved by chromatography on wheat germ agglutinin-agarose, followed by immunoaffinity chromatography on Sepharose columns coupled with anti-NGF receptor monoclonal antibody (MAb). The purified receptor, a 75,000-dalton protein, retains the capacity to bind NGF as well as anti-receptor MAbs. Final purification was achieved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of amino acid residues at the amino terminus has been determined. Possible sequence homology between the NGF receptor and several other proteins is discussed. Using the purified receptor as immunogen, new MAbs to the NGF receptor have been produced. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of dorsal root ganglia from monkeys.

Isolation and amino terminal sequencing of a novel melanoma-associated antigen.

The biochemical characteristics are described for a melanoma-associated glycoprotein antigen, whose expression depends on stage of tumor progression and melanocytic differentiation. This antigen, identified using a monoclonal antibody which specifically stains melanoma cells in immunoperoxidase assay of fixed tissue sections, is synthesized as a 30,000-Da precursor and then processed to a 30,000- to 60,000-Da sialylated glycoprotein. Two-dimensional gel electrophoresis of the antigen resolved more than 20 forms, heterogeneous in both charge and molecular weight. The kinetics of post-translational processing, the sensitivity of processing to tunicamycin, and the molecular weight of the oligosaccharide chains indicate that the oligosaccharides are N-linked. Amino acid sequencing of the antigen purified by immunoaffinity chromatography and by high-pressure liquid chromatography or polyacrylamide gel electrophoresis allowed the assignment of the first 20 acids.