A new device to measure the structural properties of the femur-anterior cruciate ligament-tibia complex

Previous studies of biomechanical properties of femur-anterior cruciate ligament-tibia complex (FATC) utilized a wide variety of testing methodologies, particularly with respect to ligament orientation relative to loading direction. A new device was designed and built to test the anterior-posterior displacement of the intact porcine knee at 30 and 90 deg of flexion, as well as the tensile properties of the FATC at any loading direction and flexion angle. Tensile tests were performed with the knees at 30 and 90 deg of flexion with the loading direction along either the axis of the tibia (tibial axis) or the axis of the anterior cruciate ligament (ligament axis). The results showed that the stiffness, ultimate load and energy absorbed were all significantly increased when the FATC was tested along the ligament axis. This study demonstrates the importance of alignment in the evaluation of the biomechanical characteristics of the femur-ACL-tibia complex.     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Microbial hydroxylation of quinoline in contaminated groundwater: evidence for incorporation of the oxygen atom of water

Studies conducted in an aquifer contaminated by creosote suggest that quinoline is converted to 2(1H)quinolinone by an indigenous consortium of microorganisms. Laboratory microbial experiments using H218O indicate that water is the source of the oxygen atom for this hydroxylation reaction under aerobic and anaerobic conditions.     

Mapping of functional and antigenic domains of the alpha 4 protein of herpes simplex virus 1.

Monoclonal antibodies to alpha 4, the major regulatory protein of herpes simplex virus 1, have been shown to differ in their effects on the binding of the protein to its DNA-binding site in the promoter-regulatory domain of an alpha gene. To map the epitopes, we expressed truncated genes in transient expression systems. All 10 monoclonal antibodies tested reacted with the N-terminal 288-amino-acid polypeptide. To map the epitopes more precisely, 29 15-mer oligopeptides, overlapping by five amino acids at each end, were synthesized and reacted with the monoclonal antibodies. The nine reactive monoclonal antibodies were mapped to seven sites. Of the two monoclonal antibodies which blocked the binding of alpha 4 to DNA, one (H950) reacted with oligopeptide no. 3 near the N terminal of the protein, whereas the second (H942) reacted with oligopeptide no. 23 near the C terminus of the 288-amino-acid polypeptide. In further tests, oligopeptide no. 19 was found to compete with two host proteins, designated as alpha H1 and alpha H2-alpha H3, for binding to DNA as well as to retard DNA in a band shift assay, whereas oligopeptides no. 26, 27, and 28 enhanced the binding of alpha 4 to DNA. Moreover, oligopeptide no. 27 was also found to retard DNA in a band shift assay. Polypeptide no. 19 competed with alpha 4 for binding to DNA, whereas no. 27 neither enhanced nor competed with the binding of the host polypeptide alpha H1 to its binding site in the promoter-regulatory domain of an alpha gene, but did enhance the binding of the alpha H2-alpha H3 protein to its binding site. In contrast to these results, the truncated alpha 4 polypeptide, 825 amino acids long, bound to the viral DNA, whereas a shorter, 519-amino-acid-long, truncated polypeptide did not. The 825-amino-acid polypeptide was previously shown to induce in transient expression of a late (gamma 2) viral gene.     

Primary structure of a Plasmodium falciparum malaria antigen located at the merozoite surface and within the parasitophorous vacuole

DNA encoding an antigen of 101,000 apparent molecular weight from the human malaria parasite Plasmodium falciparum was cloned and sequenced. Genomic DNA from the Camp strain covering the complete coding region along with cDNA from the FCR3 strain covering 81% of the coding region were obtained. The cloned DNA specified a full-length protein of 743 amino acids which included two tandemly repeated regions, one near the amino terminus containing eight hexapeptide repeats of sequence TVNDEDED, and the second near the carboxyl terminus containing primarily KE and KEE repeats. The latter repeated region is encoded by a 174-base stretch of mRNA containing only a single pyrimidine. Except for a putative leader sequence located at the amino terminus of the protein, the protein is hydrophilic and highly charged with a calculated isoelectric point of 5.6. Sequences from the Camp and FCR3 strains are very close and are also nearly identical to the partial cDNA sequence of the acidic basic repeated antigen (ABRA) protein from the FC27 strain (Stahl, H.D., Bianco, A.E., Crewther, R.F., Anders, R.F., Kyne, A.P., Coppel, R. L., Mitchell, G.F., Kemp, D.J., and Brown, G.V. (1986) Mol. Biol. Med. 3, 351-368). ABRA was previously shown to be located at the merozoite surface and in the parasitophorous vacuole. Because of its location and because it becomes complexed to merozoites when schizonts rupture in the presence of immune serum, ABRA is a candidate component of a malaria vaccine.     

Characterization of a cell surface-expressed disulfide-linked dimer involved in murine T cell activation

We have produced a hamster mAb, H1.2F3, which was derived by immunization with a murine TCR-gamma delta + epidermal T cell line. H1.2F3 immunoprecipitates a cell surface-expressed disulfide-linked dimer that has a m.w. of 85,000 under non-reducing conditions and consists of subunits of 35,000 to 39,000 m.w. This dimer is distinct from the CD3-associated TCR-gamma delta complex (CD3/TCR), inasmuch as H1.2F3 does not co-precipitate or co-modulate with the CD3/TCR complex and recognizes an Ag with a single-peptide backbone of 22,000 m.w. after N-Glycanase treatment. H1.2F3 is weakly reactive with a small percentage of cells from unfractionated thymus, spleen, or lymph node, but reactivity with both T and B lymphocytes is markedly enhanced by a brief period of stimulation with Con A or PMA in vitro. This enhancement requires de novo protein synthesis. Enhanced expression of the H1.2F3 Ag can also be induced in vivo by injection of Con A or anti-CD3. H1.2F3 is a potent stimulator of T, but not B, cell proliferation in the presence of PMA and FcR-bearing accessory cells. These functional and biochemical studies strongly suggest that the Ag recognized by H1.2F3 is the murine homologue of the human CD28 Ag recognized by mAb 9.3.     

UK-73,093: A non-peptide neurotensin receptor antagonist

UK-73,093 was identified in a screening program as a compound able to displace [³H]-neurotensin from its bovine brain receptor. We describe the discovery of this compound, species differences in receptor affinity and its characterization as a functional neurotensin antogonist in vitro and in vivo.     

Genetic and molecular analysis of spontaneous respiratory deficient (res-) mutants of Escherichia coli K-12

Respiratory deficient (res-) mutants of E. coli are slow growing microcolonial, anaerobic, catalase and benzidine negative strains whose broad phenotypic alteration may result from pleiotropic mutations in genes of the hemin biosynthetic pathway. They are easily recovered from platings of sensitive cells on concentrations of gentamicin higher than the minimal inhibitory concentration. These mutants show a dramatic change in their biochemical diagnostic profile resulting primarily from deficiencies in the active transport mechanisms of the cell. Using well-marked F- and Hfr strains, 157 mutants were analyzed from 3 different parent strains; all but 2 resulted from mutations in 3 loci of the hemin biosynthetic pathway. Of these a marked skew to hemB- mutations was seen, with more than 80% mapping there. The possibility that this hot spot resulted from transpositional activity was tested by Southern hybridization of EcoRI digests of the chromosomal DNA, using as a probe, a 2.8-kb fragment containing the hemB gene. The WT and other hemB+ control strains contained a 14.6-kb fragment. Of 18 hemB strains tested, 14 showed deletion and insertion mutations which fell into four classes based on the variation in the size of the fragment or on the absence of hybridization. The latter resulted from complete deletion of the hemB gene. An increase in fragment size from 1.5-kb to 3.4-kb was observed in some of the strains.     

Specific chemical modifications of link protein and their effect on binding to hyaluronate and cartilage proteoglycan

Specific chemical modifications of amino acid residues were performed on purified, native link protein from bovine articular cartilage. The effects of these on link protein's interactions with hyaluronate and bovine articular cartilage proteoglycan were assayed by gel chromatography. Interaction with hyaluronate was significantly perturbed by modification of lysine, arginine, tyrosine and aspartic/glutamic acid residues, but not histidine and tryptophan residues. No free, accessible sulphydryl group was found on native link protein. The requirement for unmodified lysine and arginine residues resembles that of the hyaluronate-binding site of pig laryngeal cartilage proteoglycan (Hardingham, T.E., Ewins, R.J.F. and Muir, H. (1976) Biochem. J. 157, 127-143). In contrast, proteoglycan binding was only significantly perturbed by the loss of arginine residues. This resistance may reflect hydrophobicity of the binding site or masking of the site from chemical modification by link protein self-association. Amidation of carboxyl groups, which destroyed hyaluronate binding but left proteoglycan binding intact, provides a means of generating a monofunctional link protein molecule of potential use in proteoglycan aggregation studies.