Reinnervation of experimental superficial wounds in rats

Sensory reinnervation of a superficial skin wound in the rat was studied by labeling sensory axons with anterogradely transported wheat germ agglutinin-horseradish peroxidase. Reinnervation starts after 3 days from the edge of the wound as well as from beneath the wound. About 2 weeks after the production of the wound, some hyperinnervation appears to be present, but after a few additional weeks, the innervation pattern is essentially normal. The results indicate that structural recovery of sensory axons is rapid and probably complete when skin wounds heal with no or minimal scar formation.     

Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization.

"Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.     

Metabolic activation and hepatotoxicity. Toxicity of bromobenzene in hepatocytes isolated from phenobarbital-and diethylmaleate-treated rats.

Hepatocytes freshly isolated from diethylmaleate-treated rats exhibited a markedly decreased concentration of reduced glutathione (GSH) which increased to the level present in hepatocytes from nontreated rats upon incubation in a complete medium. When bromobenzene was present in the medium, however, this increase in GSH concentration upon incubation was reversed and a further decrease occurred that resulted in GSH depletion and cell death. This was prevented by metyrapone, an inhibitor of the cytochrome P-450-linked metabolism of bromobenzene. Bromobenzene metabolism in hepatocytes was accompanied by a fraction of metabolites covalently binding to cellular proteins. The size of this fraction, relative to the amount of total metabolites, was increased in hepatocytes isolated from diethylmaleate-treated rats and in hepatocytes from phenobarbital-treated rats incubated with bromobenzene in the presence of 1,2-epoxy-3,3,3-trichloropropane, an inhibitor of microsomal epoxide hydrase which, however, also acted as a GSH-depleting agent. In addition, the metabolism of bromobenzene by hepatocytes was associated with a marked decrease in various coenzyme levels, including coenzyme A, NAD(H), and NADP(H). Cysteine and cysteamine inhibited the formation of protein-bound metabolites of bromobenzene in microsomes, but did not prevent bromobenzene toxicity in hepatocytes when added at higher concentrations to the incubation medium (containing 0.4 mm cysteine). Methionine, on the other hand, did not cause a significant effect on bromobenzene metabolism in microsomes and prevented toxicity in hepatocytes, presumably by stimulating GSH synthesis and thereby decreasing the amount of reactive metabolites available for interaction with other cellular nucleophiles. It is concluded that, in contrast to hepatocytes with normal levels of GSH, hepatocytes from diethylmaleate-treated rats were sensitive to bromobenzene toxicity under our incubation conditions. In this system, bromobenzene metabolism led to GSH depletion and was associated with a progressive decrease in coenzyme A and nicotinamide nucleotide levels and a moderate increase in the formation of metabolites covalently bound to protein. Methionine was a potent protective agent which probably acted by enhanced GSH synthesis via the formation of cystathionine.     

Getting the right stuff： controlling neural stem cell state and fate in vivo and in vitro with biomaterials

Stem cell therapy holds great promises in medical treatment by, e.g., replacing lost cells, re-constitute healthy cell populations and also in the use of stem cells as vehicles for factor and gene delivery. Embryonic stem cells have rightfully attracted a large interest due to their proven capacity of differentiating into any cell type in the embryo in vivo. Tissue-specific stem ceils are however already in use in medical practice, and recently the first systematic medical trials involving human neural stem cell （NSC） therapy have been launched. There are yet many obstacles to overcome and procedures to improve. To ensure progress in the medical use of stem cells increased basic knowledge of the molecular mechanisms that govern stem cell characteristics is necessary. Here we provide a review of the literature on NSCs in various aspects of cell therapy, with the main focus on the potential of using biomaterials to control NSC characteristics, differentiation, and delivery. We summarize results from studies on the characteristics of endogenous and transplanted NSCs in rodent models of neurological and cancer diseases, and highlight recent advancements in polymer compatibility and applicability in regulating NSC state and fate. We suggest that the development of specially designed polymers, such as hydrogels, is a crucial issue to improve the outcome of stem cell therapy in the central nervous system.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Sensibility and cutaneous reinnervation in free flaps

Sensibility and sensory reinnervation were investigated in 19 free flaps, predominantly located on the lower extremities, between 2 months and 3 years after flap transfer. All patients showed deep pressure sensibility. In 10 of the patients, primarily those examined late after surgery, a heat pain threshold was obtained at about 50 degrees C. None of the patients had superficial sensibility of any other modality. No neurofilament-positive sensory nerve fibers were observed in the dermis or epidermis. In one patient nerve fibers were detected in the subcutaneous tissue. It is concluded that patients will have deep pressure sensibility of the flap area even early after the operation and that most patients will develop a heat pain sensitivity, probably due to subcutaneous reinnervation.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model

Background aimsThere is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion.MethodsAn ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays.ResultsIP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion.ConclusionsIP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.