Lipofilling (fat grafting) in the secondary prevention of ischial tuberosity and pelvic pressure ulcers.

STUDY DESIGN: Retrospective study. OBJECTIVES: Lipofilling was proposed to adult chronic spinal cord injury patients with history of ischial tuberosity pressure ulcers surgery, at risk of recurrence of pressure ulcers due to unsatisfactory adipose tissue thickness. SETTING: Fondation Hopale, Berck, France. METHODS: The three staged (Coleman) procedure for fat grafting consisted of water-jet assisted liposuction (Harvest-Jet), decantation, and reinjection of the autologous fat in three-dimensional plan. RESULTS: Ten consecutive patients (eight paraplegics and two tetraplegics) benefited from bilateral ischial lipofilling, with additional lipofilling of the sacrum (three patients) and the trochanters (two patients). All patients attended the seating clinic with pressure mapping before and after lipofilling for appropriate cushion prescription. Mean follow-up time was 16 months (4-24 months). Complications were limited to stage I (two patients) and stage II (one patient) pressure ulcers, due to negligence. Excellent results with minimal fat wasting (between 10% and 25%) underneath the ischial tuberosity were seen in eight patients, with significant improvement of adipose tissue thickness. Total fat waste was seen in two patients, one of whom following dramatic weight loss. Results were always good in the sacrum and trochanteric areas. Lipofilling allowed longer sitting times (three patients), improved skin quality (nine patients), improved quality of life (six patients), 'a better feeling of positioning in their wheelchair' (four patients), and decrease in pelvic pain (three patients). DISCUSSION AND CONCLUSION: Pelvic lipofilling is safe, cost-effective, and can now be considered in the primary prevention of pelvic pressure ulcers in patients with insufficient adipose tissue.

Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility.

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.

The similarities of bar and pat gene products make them equally applicable for plant engineers.

The bar and pat genes, isolated from different Streptomyces species, both encode a phosphinothricin acetyltransferase (PAT) and are widely applied in plant genetic engineering. The genes were expressed in Escherichia coli and the corresponding proteins were purified and used for functional and structural comparison. Both proteins are homodimers regardless of whether they are expressed in microorganisms or in plants. They have comparable molecular weights and show immuno-cross-reactivity to their respective polyclonal antisera. The enzymes have a similar substrate affinity towards L-phosphinothricin and do not acetylate any of the other L-amino acids tested. In model digestion experiments using simulated human gastric fluids, their enzymatic activity is decreased within seconds, accompanied by a rapid and complete breakdown of both proteins. These data demonstrate the structural and functional equivalence of the PAT proteins, which is also reflected in the comparable performance of transgenic plants carrying the bar or pat gene.

Quantitative analysis of the contribution of Glu46 and Asn98 to the guanosine specificity of ribonuclease T1.

In the crystal structure of the ribonuclease T1 (RNase T1; EC 3.1.27.3)-2'-GMP complex the hydrogen-bonding potential of the guanine base is saturated [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368]. The oxygens of the Glu46 carboxylate and the Asn98 main-chain carbonyl act as hydrogen-bond acceptors for the N(1)H-C(2)-N(2)H2 part of the base. We measured the transesterification kinetics of wild-type and Glu46Ala RNase T1 using the GpU, IpU, and XpU series of analogous substrates. We found that the N(1)H---Glu46 O epsilon 1, the N(2)H---Glu46 O epsilon 2, and the N(2)H---Asn98 O hydrogen bonds have an apparent contribution of 2.7, 1.1, and 1.2 kcal/mol to the interaction energy of the enzyme and the transition state of the substrate. Wild-type RNase T1 discriminates guanine from nonionized xanthine (a guanine analogue in which the exocyclic amino group is replaced by an oxygen) by about 4.4 kcal/mol. Loss of the specific hydrogen bonds with the exocyclic amino group of the guanine base accounts for 2.4 kcal/mol of this discrimination energy; 2.0 kcal/mol is due to unfavorable non-H-bonded oxygen-oxygen contacts in the enzyme-xanthine complex. A pH dependence study shows that the deprotonated form of xanthine (i.e., the 6-keto-2-enolate anion; pKa = 5.4) is far less preferred, if not excluded, as substrate by wild-type RNase T1; this may be attributed to an electrostatic repulsion of the negatively charged xanthine by the Glu46 carboxylate group.

Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.

Purification of recombinant glycosylated human gamma interferon expressed in transformed Chinese hamster ovary cells.

Human IFN-gamma was produced in cultures of a Chinese hamster ovary (CHO) cell line transformed with a combination of plasmids encoding HuIFN-gamma cDNA and mouse DHFR cDNA and subsequently selected for growth in the presence of methotrexate. Confluent monolayers of these cells constitutively secrete HuIFN-gamma into the medium reaching a concentration of 2-5 micrograms/ml; the supernatant of the monolayer could be harvested daily for a period of more than 10 days. IFN-gamma was purified by passing the filtered CHO cell culture medium directly through a phosphocellulose column followed by elution and adsorption on a Con A-Sepharose column. Further concentration on an AMICON PM 10 filter and removal of high mw contaminating proteins with DEAE-Sephacel resulted in a IFN-gamma preparation of more than 99% purity (specific activity of about 10(8) International units per mg of protein). Each liter of CHO conditioned culture medium yielded 1-2 mg pure HuIFN-gamma. Its molecular weight, as determined by gel filtration, is about 50 kD and corresponds to a dimer structure. SDS-polyacrylamide gel electrophoresis indicated the presence of a 21 kD and a 25 kD polypeptide as compared with 17 kD for unglycosylated, bacterially made HuIFN-gamma and consistent with the two glycosylated forms of HuIFN-gamma produced in mitogen-stimulated human lymphocyte cultures.

Addition by ATP: RNA adenylyltransferase from Escherichia coli of 3'-linked oligo(A) to bacteriophage Qbeta RNA and its effect on RNA replication.

An oligo(A) or poly(A) segment was added in a stepwise fashion to the 3'-end of bacteriophage Qbeta-RNA with the aid of ATP : RNA adenylyltransferase from Escherichia coli. Nearly all RNA molecules, present in the reaction mixture, could be polyadenylated. For tail lengths not exceeding 200 nucleotide residues, the physical properties of Qbeta-RNA-poly(A) were found to be only slightly different from those of the original RNA. The polyadenylated RNA was purifed by affinity chromatography. The properties of Qbeta-RNA with oligo(A) tails of different average lengths were investigated in the in vitro replication reaction. Almost complete abolishment of template activity, even by short oligo(A) stretches, was found. Furthermore, polyadenylated Qbeta-RNA inhibited the normal replication reaction of Qbeta-RNA by removal of host factor HFI, in the same way as does free poly(A).