Selection of phage antibodies by binding affinity. Mimicking affinity maturation.

We describe a process, based on display of antibodies on the surface of filamentous bacteriophage, for selecting antibodies either by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen. For affinity selection, phage are mixed with small amounts of soluble biotinylated antigen (less than 1 microgram) such that the antigen is in excess over phage but with the concentration of antigen lower than the dissociation constant (Kd) of the antibody. Those phage bound to antigen are then selected using streptavidin-coated paramagnetic beads. The process can distinguish between antibodies with closely related affinities. For off-rate selection, antibodies are preloaded with biotinylated antigen and diluted into excess unlabelled antigen for variable times prior to capture on streptavidin-coated paramagnetic beads. To mimic the affinity maturation process of the immune system, we introduced random mutations into the antibody genes in vitro using an error-prone polymerase, and used affinity selection to isolate mutants with improved affinity. Starting with a small library (40,000 clones) of mutants (average 1.7 base changes per VH gene) of the mouse antibody B1.8, and using several rounds of affinity selection, we isolated a mutant with a fourfold improved affinity to the hapten 4-hydroxy-5-iodo-3-nitrophenacetyl-(NIP)-caproic acid (mutant Kd = 9.4(+/- 0.3) nM compared with B1.8 Kd = 41.9(+/- 1.6) nm). The relative increase in affinity of the mutant is comparable to the increase seen in the anti-4-hydroxy-3-nitrophenylacetyl/NIP-caproic acid murine secondary immune response.     

By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.

By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection. Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage. Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues. The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA). Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range. The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3. However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin. Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro.     

Comparison of 4-hydroxynzoate decarboxylase and phenol carboxylase activities in cell-free extracts of a defined, 4-hydroxybenzoate and phenol-degrading anaerobic consortium

Summary Two 4-hydroxybenzoate decarboxylase activities and a phenol carboxylase activity were found in cell-free extracts of a defined, 4-hydroxybenzoate- or phenol-grown consortium. Both decarboxylase activities were loosely membrane-associated and required K⁺ but a different pH and ion strength. Loss of activity of both decarboxylases by EDTA could be compensated by Zn²⁺ ions. The K _m values for 4-hydroxybenzoate and K⁺ of the decarboxylase activities with pH optima at 6.4 or 7.8 were 0.02 and 2.5 or 0.004 and 0.5 mm, respectively. 3,4-Dihydroxybenzoate, 3,4,5-tridydroxybenzoate, 3,5-dimethoxy-4-hydroxybenzoate and 3-chloro-4-hydroxybenzoate were also decarboxylated by both enzyme activities. The phenol carboxylase was a soluble enzyme with its pH optimum at 6.5. It required K⁺, Rb⁺ or NH _inf4 ^sup+ as monovalent, Zn²⁺, Mg²⁺, Mn²⁺ or Ni²⁺ as divalent cations and catalysed the carboxylation of phenol if 2,4-,2,3,4- or 2,4,6-hydroxybezoates were absent. The three enzyme activities were not influenced by Avidin and thus were probably not biotin-dependent enzymes. Offprint requests to: J. Winter     

Identification of CpG islands in a physical map encompassing the Friedreich's ataxia locus

The Friedreich's ataxia locus has been previously assigned to chromosome 9q 13-21.1 by the demonstration of tight linkage to two anonymous DNA markers. MCT112 (Z greater than 80, theta = 0) and DR47 (Z greater than 50, theta = 0). The absence of recombination between these three loci has prevented the resolution of gene/probe order in this region, impeding strategies for gene isolation. We report physical mapping over a 4-Mb genomic interval, linking the markers MCT112 and DR47 on a common 460-kb NotI fragment and identifying 11 CpG islands in the 1.7-Mb interval most likely to contain the Friedreich's ataxia locus. Four of these islands were detected only by analysis of three YAC clones spanning a 700-kb interval including the MCT112/DR47 cluster. Without clear evidence of the precise location of the disease locus from recombination events, each of these regions must be considered as specifying a potential "candidate" sequence for the mutated gene.     

Relation of birth weight and childhood respiratory infection to adult lung function and death from chronic obstructive airways disease.

OBJECTIVE--To examine whether birth weight, infant weight, and childhood respiratory infection are associated with adult lung function and death from chronic obstructive airways disease. DESIGN--Follow up study of men born during 1911-30 whose birth weights, weights at 1 year, and childhood illnesses were recorded at the time by health visitors. SETTING--Hertfordshire, England. SUBJECTS--5718 men born in the county during 1911-30 and a subgroup of 825 men born in the county during 1920-30 and still living there. MAIN OUTCOME MEASURES--Death from chronic obstructive airways disease, mean forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), and respiratory symptoms. RESULTS--55 men died of chronic obstructive airways disease. Death rates fell with increasing birth weight and weight at 1 year. Mean FEV1 at age 59 to 70 years, adjusted for height and age, rose by 0.06 litre (95% confidence interval 0.02 to 0.09) with each pound (450 g) increase in birth weight, independently of smoking habit and social class. Bronchitis or pneumonia in infancy was associated with a 0.17 litre (0.02 to 0.32) reduction in adult FEV1 and with an increased odds ratio of wheezing and persistent sputum production in adult life independently of birth weight, smoking habit, and social class. Whooping cough in infancy was associated with a 0.22 litre (0.02 to 0.42) reduction in adult FEV1. CONCLUSIONS--Lower birth weight was associated with worse adult lung function. Intrauterine influences which retard fetal weight gain may irrecoverably constrain the growth of the airways. Bronchitis, pneumonia, or whooping cough in infancy further reduced adult lung function. They also retarded infant weight gain. Consistent with this, death from chronic obstructive airways disease in adult life was associated with lower birth weight and weight at 1 year. Promoting lung growth in fetuses and infants and reducing the incidence of lower respiratory tract infection in infancy may reduce the incidence of chronic obstructive airways disease in the next generation.     

Fetal and infant growth and impaired glucose tolerance at age 64.

OBJECTIVE--To discover whether reduced fetal and infant growth is associated with non-insulin dependent diabetes and impaired glucose tolerance in adult life. DESIGN--Follow up study of men born during 1920-30 whose birth weights and weights at 1 year were known. SETTING--Hertfordshire, England. SUBJECTS--468 men born in east Hertfordshire and still living there. MAIN OUTCOME MEASURES--Fasting plasma glucose, insulin, proinsulin, and 32-33 split pro-insulin concentrations and plasma glucose and insulin concentrations 30 and 120 minutes after a 75 g glucose drink. RESULTS--93 men had impaired glucose tolerance or hitherto undiagnosed diabetes. They had had a lower mean birth weight and a lower weight at 1 year. The proportion of men with impaired glucose tolerance fell progressively from 26% (6/23) among those who had weighted 18 lb (8.16 kg) or less at 1 year to 13% (3/24) among those who had weighed 27 lb (12.25 kg) or more. Corresponding figures for diabetes were 17% (4/23) and nil (0/24). Plasma glucose concentrations at 30 and 120 minutes fell with increasing birth weight and weight at 1 year. Plasma 32-33 split proinsulin concentration fell with increasing weight at 1 year. All these trends were significant and independent of current body mass. Blood pressure was inversely related to birth weight and strongly related to plasma glucose and 32-33 split proinsulin concentrations. CONCLUSIONS--Reduced growth in early life is strongly linked with impaired glucose tolerance and non-insulin dependent diabetes. Reduced early growth is also related to a raised plasma concentration of 32-33 split proinsulin, which is interpreted as a sign of beta cell dysfunction. Reduced intrauterine growth is linked with high blood pressure, which may explain the association between hypertension and impaired glucose tolerance.     

Multiple coordinate controls contribute to a balanced expression of ribulose-1,5-bisphosphate carboxylase/oxygenase subunits in rye leaves

In the leaves of rye (Secale cereale L.), control mechanisms acting at multiple molecular levels contribute to a coordinate expression of the subunit polypeptides of ribulose-1,5-bisphosphate carboxylase. The relevance and hierarchy of the different control steps were evaluated by comparing the time courses of changes in levels of translatable mRNA, rates of in vivo amino acid incorporation, and the turnover of subunit polypeptides after selective interference with translation at either cytoplasmic 80S ribosomes, or at the 70S ribosomes of the chloroplast, by compartment-specific inhibitors, or by the use of 70S-ribosome-deficient leaves. The latter were generated by growing the plants at a non-permissive elevated temperature of 32 degrees C. The rates of synthesis of the two ribulose-1,5-bisphosphate carboxylase subunits were most rapidly adapted to each other by translational controls. Within 0.5-2.5 h after selective inhibition of the synthesis of either subunit, that of the other subunit made in the unaffected compartment also declined by more than 90% without any marked change in its mRNA. After prolonged inhibition (24 h) of either cytoplasmic or chloroplast protein synthesis, the levels of mRNAs for both subunits were greatly diminished. In rye, the mRNA levels for both subunits changed under all experimental conditions tested in a closely parallel manner and appeared to be always maintained in a balanced, fairly constant ratio by strong coordinate controls. Even 70S-ribosome-deficient leaves contained mRNAs for both the small and the large subunits, although only in small amounts. The mRNAs for both subunits were also markedly further decreased in 70S-ribosome-deficient leaves after application of an inhibitor of cytoplasmic translation. MDMP [2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide], suggesting that the suppression of the large subunit mRNA in the plastids was not mediated through feedback effects of accumulating unassembled large subunits. Coordinate controls at both the mRNA and the translational level require a bidirectional exchange of regulatory signals between chloroplast and cytoplasm. However, these controls were not absolutely restrictive and allowed low rates of uncoupled synthesis of either large or small subunits. Large subunits made in the presence of MDMP were stable over 24 h. However, unassembled small subunits synthesized in 70S-ribosome-deficient leaves were degraded with a half-time of 10.5 h, in contrast to their behavior after integration into the holoprotein in normal leaves, where no turnover was detected. The proteolytic removal of surplus free small subunits is regarded as a final post-translational fine-tuning step to establish a balanced subunit stoichiometry in leaves.     

Assignment of the Charcot-Marie-Tooth neuropathy type 1 (CMT 1a) gene to 17p11.2-p12.

Charcot-Marie-Tooth disease type 1a (CMT 1a) is an autosomal dominant peripheral neuropathy linked to the DNA markers D17S58 and D17S71, located in the pericentromeric region of the chromosome 17p arm. We analyzed an extended 5-generation Belgian family, multiply affected with CMT 1a, for linkage with eight chromosome 17 markers. The results indicated that the CMT 1a mutation is localized in the chromosomal region 17p11.2-p12 between the marker D17S71 and the gene for myosin heavy polypeptide 2 of adult skeletal muscle.     

Oxidation of benzaldehydes to benzoic acid derivatives by three Desulfovibrio strains.

Desulfovibrio vulgaris Marburg, "Desulfovibrio simplex" XVI, and Desulfovibrio sp. strain MP47 used benzaldehydes such as vanillin, 3,4,5-trimethoxybenzaldehyde, protocatechualdehyde, syringaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, and 2-methoxybenzaldehyde as electron donors for sulfate reduction and carbon dioxide and/or components of yeast extract as carbon sources for cell synthesis. The aldehydes were oxidized to their corresponding benzoic acids. The three sulfate reducers oxidized up to 7 mM vanillin and up to 4 mM p-anisaldehyde. Higher concentrations of vanillin or p-anisaldehyde were toxic. In addition, pyridoxal hydrochloride and o-vanillin served as electron donors for sulfate reduction. Salicylaldehyde, pyridine-2-aldehyde, pyridine-4-aldehyde, and 4-hydroxy-3-methoxybenzylalcohol were not oxidized. No molecular hydrogen was detected in the gas phase. The oxidized aldehydes were not further degraded.