Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interface

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus exhibits half-of-the-sites reactivity and negative cooperativity in binding of tyrosine. Protein engineering has been applied to the enzyme to determine whether it can be reversibly dissociated into monomers and if the monomers are active. The target for mutation is the residue Phe-164. The side chain of Phe-164 in one subunit interacts with its symmetry-related partner in the other. Mutation of Phe-164----Asp-164 gives a mutant [TyrTS(Asp-164)] that undergoes dissociation at high pH when the aspartate residues are ionized. The monomer is inactive and does not bind tyrosine. Dissociation is enhanced at low concentrations of enzyme by a mass action effect. Kinetic and binding measurements on TyrTS(Asp-164) with tyrosine and tyrosyl adenylate show that the monomer has very weak affinity for these ligands. Accordingly, dimerization is favored by high concentrations of tyrosine and ATP since the dimeric form has a high affinity for the ligands. The presence of tRNA does not encourage dimer formation, and so it must bind to the monomer. TyrTS(Asp-164) is fully active at pH 6 where dimerization is favored but has low activity at pH 7.8 where dissociation is favored. It should now prove possible to engineer heterodimers that may be used to investigate the subunit interactions further.     

Predictions of knee and ankle moments of force in walking from EMG and kinematic data

A deterministic model was developed and validated to calculate instantaneous ankle and knee moments during walking using processed EMG from representative muscles, instantaneous joint angle as a correlate of muscle length and angular velocity as a correlate of muscle velocity, and having available total instantaneous joint moments for derivation of certain model parameters. A linear regression of the moment on specifically processed EMG, recorded while each subject performed cycled isometric calibration contractions, yielded the constants for a basic moment-EMG relationship. Using the resultant moment for optimization, the predicted moment was proportionally augmented for longer muscle lengths and reduced for shorter lengths. Similarly, the predicted moment was reduced for shortening velocities and increased if the muscle was lengthening. The plots of moments predicted using the full model and those calculated from link segment mechanics followed each other quite closely. The range of root mean square errors were: 3.2-9.5 Nm for the ankle and 4.7-13.0 Nm for the knee.     

Elastic properties of collapsing and expanding trachea

The compliance of the canine trachea under positive and negative transmural pressures was measured in an in-vivo preparation. The average compliance values found in eight animals were 11.7 X 10(-6) (dynes/cm2)-1 at zero transmural pressure and 4.9 and 6.9 X 10(-6) (dynes/cm2)-1 at -20 and +20 X 10(3) dynes/cm2 transmural pressure, respectively. These compliances were significantly lower than those measured by others in excised preparations. Stress relaxation was noted at all pressure levels.     

Anaerobic digestion of high-strength acidic whey in a pH-controlled up-flow fixed film loop reactor

Summary A fixed film loop reactor was developed for the stabilization of undiluted sour whey. Porous clay beads were used to immobilize the population. The fermentation system was self-supporting with the aid of a pH-titrator. Within 2 months; the loading increased automatically to its maximum of 14 kg COD (chemical oxygen demand)/m³ per day. Parallel to this, the bacterial film was formed on the surface of the support material. For a pH of 6.7 the steady state was reached at a hydraulic retention time of 5 days equivalent to a loading of 14 kg COD/m³ per day. An amount of 5.6 m³ biogas was produced per m³ digester content and day and the COD-reduction was 95%. The pH-controlled whey addition led to only minor disturbances when overloading or oxygenation occured and a fast recovery of methanogenesis was observed. The economics of anaerobic whey digestion compared with conventional whey utilization is estimated by a simple cost/benefit calculation.     

Members of novel VH gene families are found in VDJ regions of polyclonally activated B-lymphocytes.

Four potentially productive and two non-productive VDJ gene segments were isolated from the DNA of mouse B-lymphocytes which had been polyclonally activated by bacterial lipopolysaccharide (LPS). Three VDJ regions exhibit VH genes which stem from two novel VH gene families. The complexity of these families is 5-9 genes. One of the non-productive VDJ regions exhibits a D segment which may have been generated by joining of two DSP2 segments. Both non-productive VDJ regions appear to contain rearranged pseudo VH genes. Three potential somatic mutations distributed over two productive VDJ regions are observed.     

First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.     

EcoK selection vectors for shotgun cloning into M13 and deletion mutagenesis.

For shotgun cloning into M13 vectors, a double-stranded cassette of synthetic oligonucleotides containing a SmaI site within the two halves of an EcoK site, has been introduced into the vector M13mp8. Cloning of blunt end DNA into the SmaI site destroys the EcoK site, and recombinants are therefore preferentially selected on transfection into a K strain of E.coli. For deletion mutagenesis using synthetic oligonucleotides, an M13 vector with four copies of the EcoK cassette has been made to facilitate the joining of lacZ or a Factor Xa cleavage site to any protein reading frame.     

Two new monoclonal antibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffin-embedded tissues with follicular center and mantle zone human B lymphocytes and derived tumors

Two monoclonal antibodies (LN-1, LN-2) reactive with B lymphocytes in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by utilizing cell extracts from pokeweed mitogen-stimulated peripheral blood lymphocytes and diffuse histiocytic lymphoma SU-DHL-4 cells, respectively. Both monoclonal antibodies were initially identified by indirect immunofluorescence screening techniques on paraformaldehyde-acetone-fixed cell preparations. Specificity screens with 36 well-characterized human lymphoma and leukemia cell lines showed that both LN-1 and LN-2 stained cell lines of B cell lineage but were unreactive with those of T cell or, with one exception, myeloid derivation. Null cell acute lymphoblastic leukemia cell lines were found to be LN-2+ but LN-1-. The B cell specificity of these reagents was confirmed on 15 lymphoma and 17 leukemia biopsy specimens by using indirect immunofluorescence techniques. Immunoperoxidase staining of sections from B5-fixed, paraffin-embedded human lymphoid tissues showed that LN-1 bound to the cell membrane and cytoplasm of germinal center cells whereas LN-2 stained the nuclear membrane and cytoplasm of germinal center and mantle zone B lymphocytes as well as interfollicular histiocytes and thymic medullary dendritic cells. Both monoclonal antibodies failed to stain cortical thymocytes, lymph node T cells, and peripheral blood T and myeloid cells. Immunoperoxidase staining of 20 nonlymphoid human organs and tissues revealed that LN-1 reacted positively with red blood cell precursors of the bone marrow, ciliated epithelial cells of the bronchus, distal tubular cells of the kidney, and ductal cells from several organs including the breast and prostate. In contrast, LN-2 was unreactive with all human nonlymphoid organs and tissues including the bone marrow. Indirect immunofluorescence staining of a panel of 26 solid tumor cells lines showed that LN-1 was reactive with the majority of epithelium-derived cell lines, glioblastomas, and astrocytomas but was unreactive with neuroblastomas, small cell carcinoma of the lung, and sarcomas. LN-2 was unreactive with 25 of 26 of the solid tumor cell lines by these techniques. Immunobiochemical studies have shown that LN-1 recognizes a cell surface sialoantigen whereas LN-2 is directed against a 35,000 dalton nuclear membrane protein. Because of their high specificity for B cell tumors and their ability to stain B5-fixed, paraffin-embedded tissues, LN-1 and LN-2 are useful reagents for the diagnosis and classification of the human lymphomas and leukemias.     

The 21-acetylation of corticosteroids by Clostridium sporogenes

A strain of Clostridium sporogenes, an anaerobic bacterium, isolated from sewage in New York City synthesizes two constitutive enzymes with action on steroid molecules: (i) an enzyme capable of selectively acetylating the 21-hydroxyl function of certain steroids and (ii) the corresponding esterase. Under our experimental conditions the enzymes have a strict structural requirement for 3-keto-4-ene and C-20-keto or 20 alpha-hydroxyl group and convert their respective substrates to a mixture of free and acetylated products.