Acetopyruvate hydrolase production by Pseudomonas putida O1--optimization of batch and fed-batch fermentations

The main objective of this work was the optimization of the production of the beta-ketolase, acetopyruvate hydrolase, from Pseudomonas putida O1. Orcinol was used as an inducer for enzyme production. The growth medium was optimized in two steps. In the first step, screening for optimal glucose concentration was performed. In the second step, a central composite design was used to optimize carbon and nitrogen sources in the medium. After this optimization procedure, a medium was obtained which produced seven times more biomass than the initial medium. Acetopyruvate hydrolase enzyme production was optimized by determining the optimal time of feed and amount of orcinol, using statistical methods. In a subsequent step, the maximal orcinol-degradation rate was determined. The results obtained were used to find an optimal feeding profile for enzyme production. By using the optimized fed-batch process, acetopyruvate hydrolase activity was enhanced from 10 units l(-1)to 400 units l(-1), in comparison with previously reported fermentation experiments. Productivity could even be increased by a factor of 75, to a value of 20 units l(-1 )h(-1).     

Growth factors in cartilage tissue engineering

Tissue engineering of cartilage consists of two steps. Firstly, the cells from a small biopsy of patient's own tissue have to be multiplied. During this multiplication process they lose their cartilage phenotype. In the second step, these cells have to be stimulated to re-express their cartilage phenotype and produce cartilage matrix. Growth factors can be used to improve cell multiplication, redifferentiation and production of matrix. The choice of growth factors should be made for each phase of the tissue engineering process separately, taking into account cell phenotype and the presence of extracellular matrix. This paper demonstrates some examples of the use of growth factors to increase the amount, the quality and the assembly of the matrix components produced for cartilage tissue engineering. In addition it shows that the "culture history" (e.g., addition of growth factors during cell multiplication or preculture period in a 3-dimensional environment) of the cells influences the effect of growth factor addition. The data demonstrate the potency as well as the limitations of the use of growth factors in cartilage tissue engineering.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Epinephrine and glucagon counteract inhibition of protein synthesis induced by D-galactosamine in isolated mouse hepatocytes.

10 mM D-galactosamine enhibited protein synthesis (1 h incubation time) by 67% in isolated mouse liver cells. Counteracting uridylate deficiency induced by D-galactosamine by preventive administration of 20 mM uridine did not decrease the extent of protein synthesis inhibition. 20 mM D-galactose reverted the inhibition of protein synthesis by D-galactosamine. 10(-5) M epinephrine and 10(-7) M glucagon decreased the incorporation of D-galactosamine into glycogen to 38% and 26% of the control value, respectively, after a 35 min incubation and reduced the inhibition of protein synthesis by D-galactosamine effectively. Experimental evidence supports the view that aminoglycogen formed after D-galactosamine treatment is responsible for the inhibition of protein synthesis.     

Estimation of heterozygosity for single-probe multilocus DNA fingerprints

In spite of the increasing application of DNA fingerprinting to natural populations and to the genetic identification of humans, explicit methods for estimation of basic population genetic parameters from DNA fingerprinting data have not been developed. Contributing to this omission is the inability to determine, for multilocus fingerprinting probes, relatively important genetic information, such as the number of loci, the number of alleles, and the distribution of these alleles into specific loci. One of the most useful genetic parameters that could be derived from such data would be the average heterozygosity, which has traditionally been employed to measure the level of genetic variation within populations and to compare genetic variation among different loci. We derive here explicit formulas for both the estimation of average heterozygosity at multiple hypervariable loci and a maximum value for this estimate. These estimates are based upon the DNA restriction-pattern matrices that are typical for fingerprinting studies of humans and natural populations. For several empirical data sets from our laboratory, estimates of average and maximal heterozygosity are shown to be relatively close to each other. Furthermore, variances of these statistics based on simulation studies are relatively small. These observations, as well as consideration of the effect of missing alleles and alternate numbers of loci, suggest that the average heterozygosity can be accurately estimated using phenotypic DNA fingerprint patterns, because this parameter is relatively insensitive to the lack of certain genetic information.      

Isolation of capsid protein dimers from the tick-borne encephalitis flavivirus and in vitro assembly of capsid-like particles

Flaviviruses have a spherical capsid that is composed of multiple copies of a single capsid protein and, in contrast to the viral envelope, apparently does not have an icosahedral structure. So far, attempts to isolate distinct particulate capsids and soluble forms of the capsid protein from purified virions as well as to assemble capsid-like particles in vitro have been largely unsuccessful. Here we describe the isolation of nucleocapsids from tick-borne encephalitis (TBE) virus and their disintegration into a capsid protein dimer by high-salt treatment. Purified capsid protein dimers could be assembled in vitro into capsid-like particles when combined with in vitro transcribed viral RNA. Particulate structures could also be obtained when single-stranded DNA oligonucleotides were used. These data suggest that the dimeric capsid protein functions as a basic building block in the assembly process of flaviviruses.     

Characterization of sulfate transport in the hepatic endoplasmic reticulum

The transport of sulfate ion across the endoplasmic reticulum membrane was investigated using rapid filtration and light scattering assays. We found a protein-mediated, bi-directional, low-affinity, and high-capacity, facilitative sulfate transport in rat liver microsomes, which could be inhibited by the prototypical anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. It was resistant to various phosphate transport inhibitors and was not influenced by high concentration of phosphate or pyrophosphate, which is contradictory to involvement of phosphate transporters. It was sensitive to S3483 that has been reported to inhibit the glucose 6-phosphate transporter (G6PT), but the weak competition between sulfate and glucose 6-phosphate did not confirm the participation of this transporter. Moreover, the comparison of the activity and S3483 sensitivity of sulfate transport in microsomes prepared from G6PT-overexpressing or wild type COS-7 cells did not show any significant difference. Our results indicate that sulfate fluxes in the endoplasmic reticulum are mediated by a novel, S3483-sensitive transport pathway(s).     

Mutational evidence for an internal fusion peptide in flavivirus envelope protein E

The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus promotes cell entry by inducing fusion of the viral membrane with an intracellular membrane after uptake by endocytosis. This protein differs from other well-studied viral and cellular fusion proteins because of its distinct molecular architecture and apparent lack of involvement of coiled coils in the low-pH-induced structural transitions that lead to fusion. A highly conserved loop (the cd loop), which resides at the distal tip of each subunit and is mostly buried in the subunit interface of the native E homodimer at neutral pH, has been hypothesized to function as an internal fusion peptide at low pH, but this has not yet been shown experimentally. It was predicted by examination of the X-ray crystal structure of the TBE virus E protein (F. A. Rey et al., Nature 375:291-298, 1995) that mutations at a specific residue within this loop (Leu 107) would not cause the native structure to be disrupted. We therefore introduced amino acid substitutions at this position and, using recombinant subviral particles, investigated the effects of these changes on fusion and related properties. Replacement of Leu with hydrophilic amino acids strongly impaired (Thr) or abolished (Asp) fusion activity, whereas a Phe mutant still retained a significant degree of fusion activity. Liposome coflotation experiments showed that the fusion-negative Asp mutant did not form a stable interaction with membranes at low pH, although it was still capable of undergoing the structural rearrangements required for fusion. These data support the hypothesis that the cd loop may be directly involved in interactions with target membranes during fusion.