Bioactivation of free-fat transfers: a potential new approach to improving graft survival.

A new approach to free-fat autotransplantation resorption was evaluated experimentally in a rat animal model. Bioactive fat grafts were created by the addition of basic fibroblast growth factor delivered by dextran beads to the grafts and compared with free fat alone, free fat plus beads, and free fat plus beads and a control solution in the same animal. The grafts were assessed by weight and histology at 1 and 12 months postoperatively in 40 animals. A graded response in weight retention was observed at 1 and 12 months, with the growth factor-treated grafts exhibiting near complete weight maintenance after 1 year. All other bead-containing grafts had an intermediate response, with free fat alone averaging more than one-half graft weight loss after 1 year. Histologically, the bead-containing grafts had good fibroblastic ingrowth, but extensive intercellular collagen formation and the occurrence of small-sized adipocytes among the larger adipocytes were seen only in the growth factor-treated grafts. These findings indicate that graft manipulations that affect the preadipocyte cells of the graft or fibroblastic components of the recipient site, either through polypeptide stimulation or surface charge attraction, may offer a viable approach to postoperative fat-graft volume maintenance.     

A new approach to microhomogenization

A versatile microhomogenizer is described which is capable of handling sample volumes ranging from 1 μl to several millititers. The sample and homogenization buffer are sealed within a segment of flexible plastic tubing. The sample is disrupted by rubbing or rolling a metal rod back and forth over the length of tubing. Centrifugation of the homogenate is performed in the same sealed tubing segment. This method can be used to homogenize single cells.     

Skin graft survival after external beam irradiation.

Difficulties with skin graft ulceration after radiation therapy for cancer have led many to question the suitability of this method of soft-tissue coverage and its cost-effectiveness. The objective of this study was, therefore, to assess skin-graft integrity subjected to postoperative external beam irradiation in a rat model. The model consisted of a rectangular full-thickness skin graft raised and reapplied to its original bed on the dorsum of each rat. Five groups of adult male Sprague-Dawley rats (n = 8 per group) were established. Group A was the control group and was not given postoperative irradiation. Groups B, C, D, and E received postoperative unfractionated cobalt60 irradiation 4 weeks after grafting for a total dose of 15, 20, 25, or 30 Gy, respectively. Weekly skin-graft evaluation was performed for the 4 weeks after irradiation (8 weeks after surgery) by measuring areas of graft loss using computerized planimetry. After the animals were killed, histologic samples were obtained from normal unirradiated skin and from both intact and ulcerated skin-graft sites. Graft loss after irradiation of < or = 20 Gy was similar to that of the unirradiated controls. Occurring as early as 1 week after treatment, a two-fold increase in graft ulceration was observed with doses of > or = 25 Gy (p = 0.0007). Only partial healing of ulcerations was noted by the fourth week after treatment. Histologic changes associated with the irradiation of skin grafts using doses of 25 Gy or higher included hyaline degeneration, fibrinoid necrosis, telangiectasia, and edema. Granulation tissue predominated as a mechanism of healing in areas of graft ulceration. The intensity of inflammatory cell infiltrate did not correlate with radiation dose. The authors concluded that postoperative, unfractionated irradiation can induce skin-graft loss at doses of 25 Gy or higher. Fractionated irradiation or longer intervals between grafting and irradiation may increase skin-graft tolerance; however, further studies are warranted.     

Skin pigmentation,biogeographical ancestry and admixture mapping

Ancestry informative markers (AIMs) are genetic loci showing alleles with large frequency differences between populations. AIMs can be used to estimate biogeographical ancestry at the level of the population, subgroup (e.g. cases and controls) and individual. Ancestry estimates at both the subgroup and individual level can be directly instructive regarding the genetics of the phenotypes that differ qualitatively or in frequency between populations. These estimates can provide a compelling foundation for the use of admixture mapping (AM) methods to identify the genes underlying these traits. We present details of a panel of 34 AIMs and demonstrate how such studies can proceed, by using skin pigmentation as a model phenotype. We have genotyped these markers in two population samples with primarily African ancestry, viz. African Americans from Washington D.C. and an African Caribbean sample from Britain, and in a sample of European Americans from Pennsylvania. In the two African population samples, we observed significant correlations between estimates of individual ancestry and skin pigmentation as measured by reflectometry (R(2)=0.21, P<0.0001 for the African-American sample and R(2)=0.16, P<0.0001 for the British African-Caribbean sample). These correlations confirm the validity of the ancestry estimates and also indicate the high level of population structure related to admixture, a level that characterizes these populations and that is detectable by using other tests to identify genetic structure. We have also applied two methods of admixture mapping to test for the effects of three candidate genes (TYR, OCA2, MC1R) on pigmentation. We show that TYR and OCA2 have measurable effects on skin pigmentation differences between the west African and west European parental populations. This work indicates that it is possible to estimate the individual ancestry of a person based on DNA analysis with a reasonable number of well-defined genetic markers. The implications and applications of ancestry estimates in biomedical research are discussed.     

A new approach to immunoFET operation

A new method is presented for the detection of an immunological reaction taking place in a membrane, which covers the gate area of an ISFET. By stepwise changing the electrolyte concentration of the sample solution, a transient diffusion of ions through the membrane-protein layer occurs, resulting in a transient membrane potential, which is measured by the ISFET. The diffusion rate is determined by the immobile charge density in the amphoteric protein layer, which changes upon formation of antibody-antigen complexes. No membrane potential is induced at zero fixed charge density as occurs at a protein characteristic pH. Isoelectric points of embedded proteins can be determined by detecting the zero potential response. Up to now, the authors have studied the membrane adsorption of lysozyme, human serum albumin (HSA) and the immune reaction of HSA with the antibody anti-human serum albumin (alpha HSA). The influence of protein parameters on the amplitude of the transient can be described with an empirical equation. Assuming Langmuir behaviour, the protein concentration in the solution can well be correlated with the concentration in the membrane. This new detection method is unique concerning direct measurements of charge densities and isoelectric points of amphoteric macromolecules adsorbed in the membrane. The simple procedure of one incubation stage followed by one detection stage, without separate washing and labelling techniques, gives direct information about specific charge properties of the macromolecules to be studied.     

A new approach to biogeographical subdivision

The authors propose a new approach permitting subdivision of a water area into sectors, each including species with a similar biogeographical status. This approach relies on computer methods for construction of maps presenting the spatial distribution of continuous fields. The method is illustrated by data on the nekton from the north-western Sea of Japan. Different modifications of the method and prospects of its further development are discussed.     

A new approach to microtubule depolymerization

Microtubule thermal denaturation is treated in the framework of the Ising model. The partition function is calculated with a new iterative procedure which allows us to calculate values of the denaturation curves and enthalpy changes in excellent agreement with the experimental data, We also obtain the excess heat capacity due to polymerization, OCp.     

A new approach to the construction of potentiometric immunosensors.

An electrochemical immunosensor based on a new detection principle was developed. Laccase, which is able to catalyse the electroreduction of oxygen via the direct (mediatorless) mechanism was used as an enzyme label. The new detection method does not require the presence of an electrochemically active mediator, and the reaction substrates are atmospheric oxygen and electrons, the latter being taken by the active site of the enzyme label directly from the electrode. The formation of the complex between laccase-labelled antibody and antigen on the electrode surface resulted in a considerable (more than 300 mV) shift of the electrode potential. The rate of the increase of the electrode potential was inversely proportional to the concentration of the free antigen in the sample. The non-specific adsorption of conjugate and other proteins on the electrode could be eliminated by using a polyethylenimine-based polymer on the electrode surface. Insulin was used as a model analyte. The sensitivity limit for this antigen was approximately 3 micrograms ml-1.     

A new approach to overcome potassium-mediated inhibition of triplex formation.

G,A-containing purine oligonucleotides of various lengths form extremely stable and specific triplexes with the purine-pyrimidine stretch of the vpx gene [Svinarchuk,F., Monnot,M., Merle,A., Malvy,C. and Fermandjian,S. (1995) Nucleic Acids Res., 22, 3742--3747]. The potential application of triple-helix-forming oligonucleotides (TFO) in gene-targeted therapy has prompted us to study triplex formation mimicking potassium concentrations and temperatures in cells. Triplex formation was tested by dimethyl sulphate (DMS) footprinting, gel-retardation, UV melting studies and electron microscopy. In the presence of 10 mM MgCl2, KCl concentrations up to 150 mM significantly lowered both efficiency (triplex : initial duplex) and rate constants of triplex formation. The KCl effect was more pronounced for 11mer and 20mer TFOs than for 14mer TFO. Since the dissociation half-life for the 11mer TFO decreases from 420 min in the absence of monovalent cations to 40 min in the presence of 150 mM KCI, we suggest that the negative effect could be explained by a decrease in triplex stability. In contrast, for the 20mer TFO no dissociation of the triplex was observed during 24 h of incubation either in the absence of monovalent cations or in the presence of 150 mM KCl. We suppose that in the case of the 20mer TFO the negative effect of KCI on triplex formation is probably due to the self-association of the oligonucleotide in competitive structures such as parallel duplexes and/or tetraplexes. This negative effect may be overcome by the prior formation of a short duplex either on the 3'- or 5'-end of the 20mer TFO. We refer to these partial duplexes as 'zipper' TFOs. It was demonstrated that a 'zipper' TFO can form a triplex over the full length of the target, thus unzipping the short complementary strand. The minimal single-stranded part of the 'zipper' oligonucleotide which is sufficient to initiate triplex formation can be as short as three nucleotides at the 3'-end and six nucleotides at the 5'-end. We suggest that this type of structure may prove useful for in vivo applications.