Reconstructive surgery for immunosuppressed organ-transplant recipients

Prolonged vascularized organ allograft survival and an improved quality of life are now possible for many transplant recipients. These advances are due largely to greater understanding of the immune response, the development of potent immunosuppressive agents (cyclosporin A), and improved surgical techniques. Thus more of these patients may require surgical procedures related or unrelated to their original operation, and the plastic surgeon, among other specialists, should be aware of the special problems of the immunocompromised transplant recipient who needs to undergo reconstructive surgery. We report our experience with 15 kidney, heart, and liver transplant recipients who required reconstructive surgery for a variety of conditions. The combined team approach by reconstructive and transplant surgeons is described, as well as the perioperative drug protocol and the special problems that immunosuppressed transplant recipients present. We conclude that these patients can successfully undergo major reconstructive procedures as long as the plastic surgeon not only performs technically flawless surgery, but also familiarizes himself or herself with the special problems of the immunosuppressed host, including the ever-present risk of sepsis and delayed and impaired wound healing, the potential for acute Addisonian crisis, and the possibility of multiple complicating comorbid conditions.     

THE RELATION BETWEEN THE INTRACELLULAR RIBONUCLEIC ACID DISTRIBUTION AND AMINO ACID INCORPORATION IN THE LIVER OF THE DEVELOPING CHICK EMBRYO

The RNA-P and DNA-P content of the nucleus and the RNA-P content of the whole cell of the livers of 8- to 20-day chick embryos and of adult fowls have been determined. The DNA-P content of the liver nuclei was slightly higher in the 8- and 10-day embryo than in all the other stages examined. A significant decrease in the RNA content of the cell occurred during embryonic development. The RNA content of the adult cell was the same as that of the 14- to 16-day embryo. The proportion of the cellular RNA contributed by the nucleus also decreased during development. In respect to both nuclear RNA content and distribution of RNA between nucleus and cytoplasm, the adult resembled the 8- to 12-day embryo. Examination of the fine structure of the cell showed that, as development progressed, free ribosomes decreased in number and the rough membranes increased. Slices of 8-, 14-, and 20-day embryonic livers and of adult livers were incubated with ¹⁴C-leucine, and the amount of labeled amino acid incorporated into whole tissue protein and into the proteins of the subcellular fractions was measured. Embryonic liver incorporated ¹⁴C-leucine 15 to 30 times more rapidly than adult liver. The microsomal protein was always more highly labelled than the protein in any other subcellular fraction; however, in the 8-day embryonic and the adult liver the proportion of total counts found in the nuclear fraction was considerably higher than in the 14- or 20-day embryonic liver. The significance of an apparent correlation between the proportion of the cell's RNA contributed by the nucleus and the proportion of total counts in the nuclear fraction is discussed.     

HH2A,an immortalized bovine mammary epithelial cell line,expresses the gene encoding mammary derived growth inhibitor (MDGI)

Summary We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression.     

Intracellular concentrations of phenylalanine,tyrosine and α-aminobutyric acid in 13 homozybotes and 19 heterozygotes for phenylketonuria (PKU) compared with 26 normals

Summary Intracellular concentrations of phenylalanine, tyrosine, -aminobutyric acid, and seven other aminoacids (glycine, alanine, valine, cystine, methionine, isoleucine, leucine) were measured in lymphocytes of 13 homozygotes and 19 heterozygotes for phenylketonuria and in lymphocytes of 26 normals. Intracellular concentrations for phenylalanine, tyrosine, and -aminobutyric acid were significantly higher in homo- and heterozygotes than in normals (P<0.001; P<0.01). For the other seven aminoacids there were no or only questionable differences. Between homo-and heterozygotes there was no difference in any of the aminoacids. The intracellular phenylalanine: tyrosine ratio was essentially the same in all three groups of individuals. There was no correlation between intracellular phenylalanine above or below 10nmol/10⁶ cells and IQ in heterozygotes. The same is true for phenylalanine: tyrosine ratio greater or smaller than 1. In homozygotes there was no correlation between intracellular phenylalanine and age—to which DQ/IQ is correlated. There was no significant difference in intracellular phenylalanine between homozygotes with blood levels above and below 908 mol/l (15 mg/100 ml) at the time of blood sampling and no correlation between intra- and extracellular phenylalanine concentrations.Among the 26 normals there were only two with intracellular phenylalanine above 10 nmol/10⁶ cells, both showing phenylalanine loading test curves suggestive of heterozygosity.The results are discussed and important functions of the cell wall are proposed. The formation of an abnormal unknown intracellular metabolite being the real noxious agent could explain the incomparably different degrees of brain dysfunction in individuals with equal though elevated intracellular phenylalanine concentrations, i.e., homozygotes and heterozygotes for PKU.     

Strain-dependent expression of metabolic proteins in the mouse hippocampus

Individual mouse strains differ significantly in terms of behavior and cognitive function. Strain-specific variation of metabolic protein levels in the hippocampus among various commonly used mouse strains, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry [high capacity ion trap (HCT)] has been chosen to address this question by determining strain-dependent levels of metabolic proteins in hippocampal tissue of four inbred and one outbred mouse strain. Statistical analysis of protein spots on 2-DE gels of the individual strains (n = 10) revealed significant strain-dependent differences in densities of 39 spots. Subsequent HCT analysis led to the identification of 22 different metabolic proteins presenting with differential protein levels among the five mouse strains investigated. Among those are proteins concerned with the metabolism of amino acid, nucleic acid, carbohydrate and energy. Moreover, proteins known to play a pivotal role in the processes of learning and memory, such as calcium/calmodulin-dependent protein kinase type II alpha chain, were found to present with significant inter-strain variability, which is also in agreement with our previous reports. Strain-specific protein levels of metabolic proteins in the mouse hippocampus may provide some insight into the molecular underpinnings and genetic determination of strain-dependent neuronal function. Furthermore, data presented herein emphasize the significance of the genetic background for the analysis of metabolic pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments.     

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.     

Global Metabolic Responses to Salt Stress in Fifteen Species

Cells constantly adapt to unpredictably changing extracellular solute concentrations. A cornerstone of the cellular osmotic stress response is the metabolic supply of energy and building blocks to mount appropriate defenses. Yet, the extent to which osmotic stress impinges on the metabolic network remains largely unknown. Moreover, it is mostly unclear which, if any, of the metabolic responses to osmotic stress are conserved among diverse organisms or confined to particular groups of species. Here we investigate the global metabolic responses of twelve bacteria, two yeasts and two human cell lines exposed to sustained hyperosmotic salt stress by measuring semiquantitative levels of hundreds of cellular metabolites using nontargeted metabolomics. Beyond the accumulation of osmoprotectants, we observed significant changes of numerous metabolites in all species. Global metabolic responses were predominantly species-specific, yet individual metabolites were characteristically affected depending on species’ taxonomy, natural habitat, envelope structure or salt tolerance. Exploiting the breadth of our dataset, the correlation of individual metabolite response magnitudes across all species implicated lower glycolysis, tricarboxylic acid cycle, branched-chain amino acid metabolism and heme biosynthesis to be generally important for salt tolerance. Thus, our findings place the global metabolic salt stress response into a phylogenetic context and provide insights into the cellular phenotype associated with salt tolerance.     

Alpha-actinin-4 is required for normal podocyte adhesion

Mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant human kidney disease. Mice deficient in alpha-actinin-4 develop a recessive phenotype characterized by kidney failure, proteinuria, glomerulosclerosis, and retraction of glomerular podocyte foot processes. However, the mechanism by which alpha-actinin-4 deficiency leads to glomerular disease has not been defined. Here, we examined the effect of alpha-actinin-4 deficiency on the adhesive properties of podocytes in vivo and in a cell culture system. In alpha-actinin-4-deficient mice, we observed a decrease in the number of podocytes per glomerulus compared with wild-type mice as well as the presence of podocyte markers in the urine. Podocyte cell lines generated from alpha-actinin-4-deficient mice were less adherent than wild-type cells to glomerular basement membrane (GBM) components collagen IV and laminin 10 and 11. We also observed markedly reduced adhesion of alpha-actinin-4-deficient podocytes under increasing shear stresses. This adhesion deficit was restored by transfecting cells with alpha-actinin-4-GFP. We tested the strength of the integrin receptor-mediated linkages to the cytoskeleton by applying force to microbeads bound to integrin using magnetic pulling cytometry. Beads bound to alpha-actinin-4-deficient podocytes showed greater displacement in response to an applied force than those bound to wild-type cells. Consistent with integrin-dependent alpha-actinin-4-mediated adhesion, phosphorylation of beta1-integrins on alpha-actinin-4-deficient podocytes is reduced. We rescued the phosphorylation deficit by transfecting alpha-actinin-4 into alpha-actinin-4-deficient podocytes. These results suggest that alpha-actinin-4 interacts with integrins and strengthens the podocyte-GBM interaction thereby stabilizing glomerular architecture and preventing disease.     

The maturation of the inner membrane of foetal rat liver mitochondria.

A new method was devised for the isolation of foetal and neonatal rat lvier mitochondria, giving higher yields than conventional methods. 2. During development from the perinatal period to the mature adult, the ratio of cytochrome oxidase/succinate-cytochrome c reductase changes. 3. The inner mitochondrial membrane of foetal liver mitochondria possesses virtually no osmotic activity; the permeability to sucrose decreases with increasing developmental age. 4. Foetal rat liver mitochondria possess only marginal respiratory control and do not maintain Ca2+-induced respiration; they also swell in respiratory-control medium in the absence of substrate. ATP enhances respiratory control and prevents swelling, adenylyl imidodiphosphate, ATP+atractyloside enhance the R.C.I. (respiratory control index), Ca2+-induced respiratory control and prevent swelling, whereas GTP and low concentrations of ADP have none of these actions. It is concluded that the effect of ATP depends on steric interaction with the inner mitochondrial membrane. 5. When 1-day pre-partum foetuses are obtained by Caesarean section and maintained in a Humidicrib for 90 min, mitochondrial maturation is "triggered", so that their R.C.I. is enhanced and no ATP is required to support Ca2+-dependent respiratory control or to inhibit mitochondrial swelling. 6. It is concluded that foetal rat liver mitochondria in utero do not respire, although they are capable of oxidative phosphorylation in spite of their low R.C.I. The different environmental conditions which the neonatal rat encounters ex utero enable the hepatic mitochondria to produce ATP, which interacts with the inner mitochondrial membrane to enhance oxidative phosphorylation by an autocatalytic mechanism.