Healing of burn wounds in transgenic mice overexpressing transforming growth factor-beta 1 in the epidermis.

Transforming growth factor-beta (TGF-beta) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-beta in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-beta on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-beta in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-beta. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-beta 1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-beta 1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO(2) laser. Using this model, we demonstrated activation of latent TGF-beta after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-beta1 gene into keratinocytes markedly increases the release and activation of TGF-beta after burn injury. Elevated local TGF-beta significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls, P < 0.05) and homozygous (25% versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.

Hydrophobic adsorption of aromatic compounds on polyurethane foam as a carbon source for Pseudomonas growth.

The use of polyurethane foam appears to be efficient to extract hydrophobic pollutants from aqueous media. Their adsorption is the result of spontaneous hydrophobic interactions with the foam, The rate of adsorption is a function of the diffusion of the molecules into the foam as well as their hydrophilic/lipophilic balance. A mixture of different molecules modifies the adsorption capacities of each type of molecule on the foam, probably resulting from stacking phenomena between the molecules. The Pseudomonas species can grow in the presence of the polyurethane foam and be adsorbed on it. Moreover, a strain of Pseudomonas pseudoalcaligenes tested in this study can use adsorbed biphenyl as the sole carbon source. Polyurethane foam therefore shows favorable characteristics for being chosen as a method of concentrating aromatic compounds and optimizing the rate of degradation of these molecules by bacteria.

Induction of a stable morphological change in Propionibacterium freudenreichii.

When cells of Propionibacterium freudenreichii were incubated under fasting conditions and then plated in the presence of an inhibitor of protein synthesis, a variable but significant (greater than 10(-2) fraction of the population changed their morphology from rod to sphere, with a considerable thickening of the cell wall. This change was accompanied by metabolic and antibiotic-resistance modifications, including the synthesis of at least one new enzyme (alpha-glucosidase), and by the simultaneous appearance of several new species of DNA, presumably plasmids. The round cells grew faster than the parent strain and maintained their morphology indefinitely when propagated on complex medium containing glucose as the main carbon source. However, when glucose was omitted, cells returned to the rod form and regained their previous characteristics, including the absence of detectable plasmids.

Alteration of active transport after bacteriophage T5 infection.

Bacteriophage T5 absorption immediately followed by injection of the first-step-transfer DNA segment produces alterations in the bacterial membrane which reduce the uptake of amino acids and of o-nitrophenyl-beta-D-galactopyranoside. Concomitantly, intracellular ATP is hydrolyzed. The extent of inhibition of uptake and of ATP hydrolysis is cooperatively increased with the multiplicity of infection. Inhibition of active transport at a high multiplicity of infection (greater than 3) is also observed after the second step of DNA injection. In contrast, at low multiplicities of infection, phage proteins are able to enhance amino acid uptake. Infection of su- bacteria with amber mutants in the first-step-transfer DNA suggests that protein A1 is implicated in this enhancement.

Analysis of the coliphage T5 DNA ejection process with free and liposome-associated TonA protein.

Outer membrane protein TonA, the receptor for coliphage T5, has been partially purified and incorporated into the phospholipid bilayer of liposomes. Adsorption of the phage to its receptor in either a free or liposome-associated form is fast and sufficient to trigger the ejection of encapsidated DNA. In both in vitro systems the exit of DNA from the phage capsid is a very slow process. Ejected DNA can partially accumulate inside the liposome aqueous compartment, but the transfer from the phage head to the liposome internal space is never complete, perhaps because the liposome volume is too small. The presence of polyamines or divalent cations (magnesium) or both in the incubation medium diminished the extent of DNA ejection, possibly by stabilizing DNA inside the head. DNA movement was slowed as the temperature was decreased from 37 to 18 degrees C. Furthermore, incubation at 4 degrees C totally prevented this DNA movement, even if a large part of the DNA had already exited the capsid.

Rescue of first-step-transfer amber mutants by "second-step-transfer-blocked" bacteriophage T5 on an su- strain.

T5 st0 phages irreversibly blocked in the injection of their second-step-transfer DNA can produce active A1 and A2 proteins which complement first-step-transfer amber mutants infecting an su(-) strain.

Evidence for heterogeneity in populations of T5 bacteriophage. II. Some particles are unable to inject their second-step-transfer DNA.

A new class of bacteriophage was characterized in purified T5 stocks. Regardless of the host cell, these phages were irreversibly blocked at the first-step-transfer stage under conditions in which whole DNA injection normally takes place. However, they expressed their first-step-transfer functions. These observations confirmed the previously established heterogeneity of T5 bacteriophage populations and provided a new way to define a phage function necessary to release the blocking of T5 DNA injection at the first-step-transfer stage.

Single-stranded bacteriophage T5 stO DNA as template for in vitro fMet-tRNA binding to ribosomes and protein synthesis.

Good evidence is provided that fMet-tRNA binding and aminoacid incorporation, when single-stranded DNA is used instead of mRNA in an E. coli cell-free system, are strictly dependent on the magnesium concentration. Ten sites homologous to the initiation sites of translation can be detected on denatured T5 stO DNA when using ribosomes and initiation factors from uninfected E. coli F. In S-30 extracts, at high magnesium concentrations and in the presence of neomycin, initiation of the translation of denatured T5 stO DNA begins anywhere on the molecule, and yet high molecular weight polypeptides are synthesized. The template potentiality of the denatured T5 stO DNA decreased when using ribosomes plus initiation factors and crude extracts from T5 stO-infected bacteria. By in vitro formation of initiation complexes sites analogous to initiation sites of translation were localized on T5 stO DNA molecules using single-stranded fragments separated by sedimentation in alkaline sucrose gradient.

In vitro synthesis of large peptide molecules using glucosylated single-stranded bacteriophage T4D DNA template.

Denatured Bacteriophage T4D DNA is able to stimulate aminoacid incorporation into TCA-precipitable material in an in vitro protein synthesis system according to base DNA sequences. Newly synthesized polypeptides remain associated with ribosomes and have a molecular weight in range of 15,000 to 45,000 Daltons.

Evidence for heterogeneity in populations of T5 bacteriophage.

Each T5 stock contains a population of particular phages that, just after adsorption onto the host bacteria. release their entire chromosome outside the bacterial membrane in a place where it is sensitive to bacterial enzymes. This release takes place before the sensitization step to deprivation of calcium and before the transfer of the first-step DNA fragment. Secondarily, this released DNA is degraded by bacterial enzymes, mainly by the endonuclease I; the products of degradation are spontaneously released in the surrounding medium. Thus, in each T5 phage stock it seems that there is a minor population that is deficient for the mechanism of controlled DNA injection into the bacteria.

Penetration into host cells of naked, partially injected (post-FST) DNA of bacteriophage T5.

The experiments presented here suggest that the naked post-FST DNA, attached to the bacteria after a centrifugation at 6,000 x g of Escherichia coli-T5 complexes arrested at the FST stage, can be injected into the host cell if protein synthesis is allowed. This results from the observation that the production of infectious centers by this naked DNA is sensitive to DNase or moderate shearing treatments. The process of injection seems to be the same as that for DNA normally rolled in an adsorbed capsid.