GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons.

A potent neurotrophic factor that enhances survival of midbrain dopaminergic neurons was purified and cloned. Glial cell line-derived neurotrophic factor (GDNF) is a glycosylated, disulfide-bonded homodimer that is a distantly related member of the transforming growth factor-beta superfamily. In embryonic midbrain cultures, recombinant human GDNF promoted the survival and morphological differentiation of dopaminergic neurons and increased their high-affinity dopamine uptake. These effects were relatively specific; GDNF did not increase total neuron or astrocyte numbers nor did it increase transmitter uptake by gamma-aminobutyric-containing and serotonergic neurons. GDNF may have utility in the treatment of Parkinson's disease, which is marked by progressive degeneration of midbrain dopaminergic neurons.

Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin.

Administration of extracellular hemoglobin-based oxygen carriers often induces mild increases in blood pressure. In order to test whether nitric oxide (NO) scavenging is responsible for the hypertensive effect, we constructed and tested a set of recombinant hemoglobins that vary in rates of reaction with NO. The results suggest that the rapid reactions of oxy- and deoxyhemoglobin with nitric oxide are the fundamental cause of the hypertension. The magnitude of the blood-pressure effect correlates directly with the in vitro rate of NO oxidation. Hemoglobins with decreased NO-scavenging activity may be more suitable for certain therapeutic applications than those that cause depletion of nitric oxide.

Optimization of heterologous protein production in Escherichia coli.

Escherichia coli has long been the primary prokaryotic host for the synthesis of heterologous proteins. Recent advances have been made in the expression of complex proteins as soluble, functional molecules, complete with prosthetic groups, disulfide bonds, and quaternary structure. The development of alternative promoter and induction strategies has improved the options available for manipulating the expression conditions, which are frequently critical to soluble yield.

Genetic engineering of polysaccharide structure: production of variants of xanthan gum in Xanthomonas campestris.

Xanthan gum is an extracellular heteropolysaccharide produced by the bacterium Xanthomonas campestris. Xanthan has wide commercial application as a viscosifier of aqueous solutions. Previously, through genetic engineering, a set of mutants defective in the xanthan biosynthetic pathway has been obtained. Certain mutants were shown to synthesize and polymerize structural variants of the xanthan repeating unit and thus produce "variant xanthans". Initial studies of solution viscosities of these polymers, presented here, indicate that the variants have rheological properties similar to, but not identical with, xanthan. These results indicate that acetylation and pyruvylation can affect the viscometric properties of xanthan. Specifically, the presence of pyruvate increases viscosity, whereas acetate decreases viscosity. In addition, the elimination of sugar residues from xanthan side chains also has a major effect on viscosity. Compared to wild-type xanthan, polymer lacking the terminal mannose (polytetramer) is a poor viscosifier. In contrast, polymer lacking both the terminal mannose and glucuronic acid (polytrimer) is a superior viscosifier, on a weight basis. There is a negative effect of acetylation on the viscosity of polytetramer xanthan, but there is seemingly no effect of acetylation on polytrimer xanthan viscosity. The further study of these materials should provide insight into the relationship between xanthan structure and rheological behavior.

Genetic studies on capsid-length determination in bacteriophage T4. I. Isolation and partial characterization of second-site revertants of a gene 23 mutation affecting capsid length.

The T4 mutation ptg19-80 affects the mechanism of capsid-length determination. It is located in gene 23, which encodes the major structural protein of the capsid. The mutation results in the production of abnormal-length capsids in high frequencies. This paper describes the isolation and partial characterization of second-site revertants of ptg19-80. In the course of their analysis, it was discovered that ptg19-80 is itself a double mutation consisting of a gene 23 mutation (ptg19-80c), which causes the morphogenetic defect, and a suppressor mutation located near the lysozyme gene. Phenotypic characterization of nine pseudo-wild-type revertants of this double-mutation revealed that these revertants all produced lower frequencies of abnormal capsids than did ptg19-80. Seven of these revertants were shown to contain two suppressor mutations, one mapping in or near gene 22 and done mapping in or near gene 24. Both mutations were required for suppression. These suppressors displayed no discernible phenotype in the absence of ptg19-80c.

Genetic studies on capsid-length determination in bacteriophage T4. II. Genetic evidence that specific protein-protein interactions are involved.

A bacteriophage T4 mutation (ptg19-80c) located in gene 23, which encodes the major structural protein of the T4 capsid, results in the production of capsids of abnormal length. Mutations outside gene 23 which partially suppress ptg19-80c have been described in the accompanying paper (D. H. Doherty, J. Virol. 43:641-654, 1982). Characterization of these suppressors was extended. A complementation test suggested that the suppressors were in genes 22 and 24. These genes coded for the major component of the morphogenetic core of the capsid precursor and the vertex protein of the capsid, respectively. The suppressor mutations were found to have no obvious phenotype in the absence of ptg19-80c. Suppression was shown to be allele specific: other ptg mutations at different sites in gene 23 were not suppressed by the suppressors of ptg19-80c. These results indicated that specific interactions among the three proteins gp22, gp23, and gp24 may play a role in the regulation of T4 capsid-length determination. Current models for capsid-length determination are considered in the light of these results.

Bacterial and phage mutations that reveal helix-unwinding activities required for bacteriophage T4 DNA replication.

An Escherichia coli strain with a mutation in the optA gene restricts the growth of bacteriophage T4 strains partially defective in gene 43 (DNA polymerase) or missing gene dda (DNA-dependent ATPase). The mutations in the dda gene inactivate a DNA-dependent ATPase that has been shown to have DNA helicase activity in vitro. We show that the restriction of phage growth after infection of the optA bacterium is the result of a block in DNA replication. We infer that the block arises from a defect in DNA unwinding.

On the role of the single-stranded DNA binding protein of bacteriophage T4 in DNA metabolism. I. Isolation and genetic characterization of new mutations in gene 32 of bacteriophage T4.

The product of gene 32 of bacteriophage T4 is a single-stranded DNA binding protein involved in T4 DNA replication, recombination and repair. Functionally differentiated regions of the gene 32 protein have been described by protein chemistry. As a preliminary step in a genetic dissection of these functional domains, we have isolated a large number of missense mutants of gene 32. Mutant isolation was facilitated by directed mutagenesis and a mutant bacterial host which is unusually restrictive for missense mutations in gene 32. We have isolated over 100 mutants and identified 22 mutational sites. A physical map of these sites has been constructed and has shown that mutations are clustered within gene 32. The possible functional significance of this clustering is considered.

Determination of the amount of homology required for recombination in bacteriophage T4.

Homology is an important feature of recombination. We have used the rll cistrons of bacteriophage T4 to determine the extent of homology required for recombination. We varied the amount of homologous DNA available for recombination in both marker rescue experiments and deletion-by-deletion crosses. Our results suggest that the primary pathway for recombination in T4 requires 50 bp of homology. Our finding that recombination is detectable when fewer than 50 bp of homology are available suggests that there is a second, less efficient pathway of recombination in T4. This pathway may be used during the formation of deletions.

Mechanism of NO-induced oxidation of myoglobin and hemoglobin.

Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. The consumption of NO by extracellular hemoglobin and subsequent vasoconstriction have been suggested to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue. In order to determine the mechanism of this key reaction, we have measured the kinetics of NO-induced oxidation of a variety of different recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rates depend linearly on [NO] but show no dependence on [O2]. The bimolecular rate constants for NO-induced oxidation of MbO2 and HbO2 are large (k.ox,NO = 30-50 microM-1 s-1 for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb and Hb. Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitric oxide with the iron atom to produce Fe(2+)-N=O or with Fe(2+)-O-O delta- to produce Fe(3+)-OH2 and nitrate. Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the distal pocket which normally hinder NO entry into the protein. Decreasing the volume of the distal pocket by replacing Leu(B10) and Val(E11) with aromatic amino acids markedly inhibits NO-induced oxidation of MbO2. The latter results provide a protein engineering strategy for reducing hypertensive events caused by extracellular hemoglobin-based O2 carriers. This approach has been explored by examining the effects of Phe(B10) and Phe(E11) substitutions on the rates of NO-induced oxidation of the alpha and beta subunits in recombinant human hemoglobin.