Biological and physical factors influencing the successful engraftment of a cultured human skin substitute

Skin tissue may be engineered in a variety of ways. Our cultured skin substitute (Graftskin, living skin equivalent or G-LSE), Apligraftrade mark, is an organotypic culture of skin, containing both a "dermis" and "epidermis." The epidermis is an important functional component of skin, responsible for biologic wound closure. The epidermis possesses a stratum corneum which develops with time in culture. The stratum corneum provides barrier function properties and gives the LSE improved strength and handling characteristics. Clinical experience indicated that the stratum corneum might play an important role in improving the clinical utility of the LSE. Handling and physical characteristics improved with time in culture. We examined the LSE at different stages of epidermal maturation for barrier function and ability to persist as a graft. LSE grafted onto athymic mice before significant development of barrier function did not withstand bandage removal at 7 days postgraft. LSE grafted after barrier function had been established in vitro were able to withstand bandage removal at day 7. Corneum lipid composition and structure are critical components for barrier function. Media modifications were used in an attempt to improve the fatty acid composition of the stratum corneum. The barrier developed more rapidly and was improved in a serum-free, lipid-supplemented condition. Lipid lamellar structure was improved with 10% of the stratum corneum exhibiting broad-narrow-broad lipid lamellar arrangements similar to human skin. Fatty acid metabolism was not appreciably altered. Barrier function in vitro was 4- to 10-fold more permeable than human skin. Epidermal differentiation does not compromise engraftment or the wound healing ability of the epidermis. The stratum corneum provides features beneficial for engraftment and clinical use. (c) 1996 John Wiley & Sons, Inc.     

Evidence That the Pathway of Dimethylsulfoniopropionate Biosynthesis Begins in the Cytosol and Ends in the Chloroplast

In the flowering plant Wollastonia biflora (L.) DC. the first step in 3-dimethylsulfoniopropionate (DMSP) synthesis is conversion of methionine to S-methylmethionine (SMM) and the last is oxidation of 3-dimethylsulfoniopropionaldehyde (DMSP-ald) (F. James, L. Paquet, S.A. Sparace, D.A. Gage, A.D. Hanson [1995] Plant Physiol 108: 1439-1448). DMSP-ald was shown to undergo rapid, spontaneous decomposition to dimethylsulfide and acrolein. However, it was stable enough (half-life [greater than or equal to] 1 h) in tertiary amine buffers to use as a substrate for enzyme assays. A dehydrogenase catalyzing DMSP-ald oxidation was detected in extracts of W. biflora mesophyll protoplasts. This enzyme had a high affinity for DMSP-ald (Km = 1.5 [mu]M), was subject to substrate inhibition, preferred NAD to NADP, and was immunologically related to plant betaine aldehyde dehydrogenases. After fractionation of protoplast lysates, [greater than or equal to]90% of DMSP-ald dehydrogenase activity was recovered from the chloroplast stromal fraction, whereas the enzyme that mediates SMM synthesis, S-adenosylmethionine:methionine S-methyltransferase, was found exclusively in the cytosolic fraction. Immunohistochemical analysis confirmed that the S-methyltransferase was cytosolic. Intact W. biflora chloroplasts were able to metabolize supplied [35S]SMM to [35S]DMSP. These findings indicate that SMM is made in the cytosol, imported into the chloroplast, and there converted successively to DMSP-ald and DMSP.     

Sucrose Synthase Localization during Initiation of Seed Development and Trichome Differentiation in Cotton Ovules

Sucrose synthase in cotton (Gossypium hirsutum L.) ovules was immunolocalized to clarify the relationship between this enzyme and (a) sucrose import/utilization during initiation of seed development, (b) trichome differentiation, and (c) cell-wall biosynthesis in these rapidly elongating "fibers." Analyses focused on the period immediately before and after trichome initiation (at pollination). Internal tissues most heavily immunolabeled were the developing nucellus, adjacent integument (inner surface), and the vascular region. Little sucrose synthase was associated with the outermost epidermis on the day preceding pollination. However, 1 d later, immunolabel appeared specifically in those epidermal cells at the earliest visible phase of trichome differentiation. The day following pollination, these cells had elongated 3- to 5-fold and showed a further enhancement of sucrose synthase immunolabel. Levels of sucrose synthase mRNA also increased during this period, regardless of whether pollination per se had occurred. Timing of onset for the cell-specific localization of sucrose synthase in young seeds and trichome initials indicates a close association between this enzyme and sucrose import at a cellular level, as well as a potentially integral role in cell-wall biosynthesis.     

Effect of endurance exercise training on muscle glycogen supercompensation in rats

Nakatani, Akira, Dong-Ho Han, Polly A. Hansen, Lorraine A. Nolte, Helen H. Host, Robert C. Hickner, and John O. Holloszy. Effect of endurance exercise training on muscle glycogensupercompensation in rats. J. Appl.Physiol. 82(2): 711-715, 1997.The purpose of this study was to test the hypothesis that the rate and extent ofglycogen supercompensation in skeletal muscle are increased byendurance exercise training. Rats were trained by using a 5-wk-long swimming program in which the duration of swimming was gradually increased to 6 h/day over 3 wk and then maintained at 6 h/day for anadditional 2 wk. Glycogen repletion was measured in trained anduntrained rats after a glycogen-depleting bout of exercise. The ratswere given a rodent chow diet plus 5% sucrose in their drinking waterad libitum during the recovery period. There were remarkabledifferences in both the rates of glycogen accumulation and the glycogenconcentrations attained in the two groups. The concentration ofglycogen in epitrochlearis muscle averaged 13.1 ± 0.9 mg/g wet wtin the untrained group and 31.7 ± 2.7 mg/g in the trained group(P < 0.001) 24 h after the exercise.This difference could not be explained by a training effect on glycogensynthase. The training induced ~50% increases in muscle GLUT-4glucose transporter protein and in hexokinase activity inepitrochlearis muscles. We conclude that endurance exercise trainingresults in increases in both the rate and magnitude of muscle glycogensupercompensation in rats.

     

MiRNA-21 Expression Decreases from Primary Tumors to Liver Metastases in Colorectal Carcinoma

Objective

Metastasis is the major cause of death in colorectal cancer patients. Expression of certain miRNAs in the primary tumors has been shown to be associated with progression of colorectal cancer and the initiation of metastasis. In this study, we compared miRNA expression in primary colorectal cancer and corresponding liver metastases in order to get an idea of the oncogenic importance of the miRNAs in established metastases.

Methods

We analyzed the expression of miRNA-21, miRNA-31 and miRNA-373 in corresponding formalin-fixed paraffin-embedded (FFPE) tissue samples of primary colorectal cancer, liver metastasis and healthy tissues of 29 patients by quantitative real-time PCR.

Results

All three miRNAs were significantly up-regulated in the primary tumor tissues as compared to healthy colon mucosa of the respective patients (p < 0.01). MiRNA-21 and miRNA-31 were also higher expressed in liver metastases as compared to healthy liver tissues (p < 0.01). No significant difference of expression of miRNA-31 and miRNA-373 was observed between primary tumors and metastases. Of note, miRNA-21 expression was significantly reduced in liver metastases as compared to the primary colorectal tumors (p < 0.01).

Conclusion

In the context of previous studies demonstrating increased miRNA-21 expression in metastatic primary tumors, our findings raise the question whether miRNA-21 might be involved in the initiation but not in the perpetuation and growth of metastases.     

Ancestral population reconstitution from isofemale lines as a tool for experimental evolution

Experimental evolution is a powerful tool to study adaptation under controlled conditions. Laboratory natural selection experiments mimic adaptation in the wild with better‐adapted genotypes having more offspring. Because the selected traits are frequently not known, adaptation is typically measured as fitness increase by comparing evolved populations against an unselected reference population maintained in a laboratory environment. With adaptation to the laboratory conditions and genetic drift, however, it is not clear to what extent such comparisons provide unbiased estimates of adaptation. Alternatively, ancestral variation could be preserved in isofemale lines that can be combined to reconstitute the ancestral population. Here, we assess the impact of selection on alleles segregating in newly established Drosophila isofemale lines. We reconstituted two populations from isofemale lines and compared them to two original ancestral populations (AP) founded from the same lines shortly after collection. No significant allele frequency changes could be detected between both AP and simulations showed that drift had a low impact compared to Pool‐Seq‐associated sampling effects. We conclude that laboratory selection on segregating variation in isofemale lines is too weak to have detectable effects, which validates ancestral population reconstitution from isofemale lines as an unbiased approach for measuring adaptation in evolved populations.     

The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats

Background and Purpose

Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach.

Methods

For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation.

Results

Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis.

Conclusions

With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.