HGF/SF Increases Number of Skin Melanocytes but Does Not Alter Quality or Quantity of Follicular Melanogenesis

Melanins are an important factor determining the vulnerability of mammalian skin to UV radiation and thus to UV-induced skin cancers. Transgenic mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF) have extra-follicular dermal melanocytes, notably in the papillary upper dermis, and are susceptible to UV-induced melanoma. Pigmented HGF/SF neonatal mice are more susceptible than albino HGF/SF animals to UVA -induced melanoma, indicating an involvement of melanin in melanoma formation. This raises the question of the effect of transgenic HGF/SF on melanization. We developed a methodology to accurately quantitate both the production of melanin and the efficiency of melanogenesis in normal, and HGF/SF transgenic mice in vivo. Skin and hair shafts of 5 day old and adult (3 week old) C57BL/6-HGF/SF and corresponding C57BL/6 wild type mice were investigated by electron paramagnetic resonance spectroscopy (EPR) to quantitate melanin, by transmission electron microscopy (TEM) for the presence of melanosomes, and by standard histology and by Western blotting and zymography to determine the expression and activity of melanogenesis-related proteins. Eumelanin but no phaeomelanin was detected in transgenic C57BL/6-HGF and C57BL/6 wild type mice. Transgenic HGF/SF overexpression did not change the type of melanin produced in the skin or hair, did not affect the terminal content of melanin production in standard samples of hair and did not influence hair cycle/morphogenesis-related changes in skin thickness. No melanocytes were found in the epidermis and no melanosomes were found in epidermal keratinocytes. HGF/SF transgenic mice thus lack the epidermal melanin UV-protection found in constitutively dark human skin. We conclude that melanocytes in the HGF/SF transgenic mouse, particularly in the papillary dermis, are vulnerable to UVA which interacts with eumelanin but not phaeomelanin to induce melanoma.     

Biological invasion by a benthivorous fish reduced the cover and species richness of aquatic plants in most lakes of a large North American ecoregion

Biological invasions are projected to be the main driver of biodiversity and ecosystem function loss in lakes in the 21st century. However, the extent of these future losses is difficult to quantify because most invasions are recent and confounded by other stressors. In this study, we quantified the outcome of a century‐old invasion, the introduction of common carp to North America, to illustrate potential consequences of introducing non‐native ecosystem engineers to lakes worldwide. We used the decline in aquatic plant richness and cover as an index of ecological impact across three ecoregions: Great Plains, Eastern Temperate Forests and Northern Forests. Using whole‐lake manipulations, we demonstrated that both submersed plant cover and richness declined exponentially as carp biomass increased such that plant cover was reduced to <10% and species richness was halved in lakes in which carp biomass exceeded 190 kg ha^?1. Using catch rates amassed from 2000+ lakes, we showed that carp exceeded this biomass level in 70.6% of Great Plains lakes and 23.3% of Eastern Temperate Forests lakes, but 0% of Northern Forests lakes. Using model selection analysis, we showed that carp was a key driver of plant species richness along with Secchi depth, lake area and human development of lake watersheds. Model parameters showed that carp reduced species richness to a similar degree across lakes of various Secchi depths and surface areas. In regions dominated by carp (e.g., Great Plains), carp had a stronger impact on plant richness than human watershed development. Overall, our analysis shows that the introduction of common carp played a key role in driving a severe reduction in plant cover and richness in a majority of Great Plains lakes and a large portion of Eastern Temperate Forests lakes in North America.     

Epithelial Mesenchymal Transition and Pancreatic Tumor Initiating CD44+/EpCAM+ Cells Are Inhibited by γ-Secretase Inhibitor IX

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high rate of metastasis. Recent studies have indicated that the Notch signalling pathway is important in PDAC initiation and maintenance, although the specific cell biological roles of the pathway remain to be established. Here we sought to examine this question in established pancreatic cancer cell lines using the γ-secretase inhibitor IX (GSI IX) to inactivate Notch. Based on the known roles of Notch in development and stem cell biology, we focused on effects on epithelial mesenchymal transition (EMT) and on pancreatic tumor initiating CD44+/EpCAM+ cells. We analyzed the effect of the GSI IX on growth and epithelial plasticity of human pancreatic cancer cell lines, and on the tumorigenicity of pancreatic tumor initiating CD44+/EpCAM+ cells. Notably, apoptosis was induced after GSI IX treatment and EMT markers were selectively targeted. Furthermore, under GSI IX treatment, decline in the growth of pancreatic tumor initiating CD44+/EpCAM+ cells was observed in vitro and in a xenograft mouse model. This study demonstrates a central role of Notch signalling pathway in pancreatic cancer pathogenesis and identifies an effective approach to inhibit selectively EMT and suppress tumorigenesis by eliminating pancreatic tumor initiating CD44+/EpCAM+ cells.     

Homoplasmic MELAS A3243G mtDNA mutation in a colon cancer sample

We have analyzed mtDNA variation in various cancer samples, comparing them with normal tissue controls, and identified mutations and polymorphisms, both known and novel, in mitochondrial tRNA, rRNA and protein genes. Most remarkably, in a colon cancer sample we have found the A3243G mutation in the homoplasmic state. This mutation is known to cause severe mitochondrial dysfunction and, until now, has not been found in cancer cells, nor in the homoplasmic state in living subjects. The mutation was absent from normal tissue, suggesting that mtDNA mutation and resulting respiratory deficiency played a role in carcinogenesis.     

Kinetics of increased generation of (.)NO in endotoxaemic rats as measured by EPR

Ferrous-diethyldithiocarbamate (Fe(DETC)(2)) chelate is a lipophilic spin trap developed for (.)NO detection by electron paramagnetic resonance (EPR) spectroscopy. Using this spin trap we investigated the kinetics of (.)NO production in endotoxaemia in rats induced by lipopolysaccharide (Escherichia coli, 10 mg/kg). The NO-Fe(DETC)(2) complex was found to give a characteristic EPR signal, and the amplitude of the 3rd (high-field) component of its hyperfine splitting was used to monitor the level of (.)NO. We found that in blood, kidney, liver, heart and lung (.)NO production starts to increase as early as 2 h after LPS injection, reaches the maximum 6 h after LPS injection and then returns to basal level within further 12-18 h. Interestingly, in the eye bulb the maximum of (.)NO production was detected 12 h after LPS, and the signal was still pronounced 24 h after LPS. In brief, the highly lipophilic exogenous spin trap, Fe(DETC)(2) is well suited for assessment of (.)NO production in endotoxaemia. We demonstrated that the kinetics of increased production of (.)NO in endotoxaemic organs, with the notable exception of the eye, do not follow the known pattern of NOS-2 induction under those conditions. Accordingly, only in early endotoxaemia a high level of (.)NO is detected, while in late endotoxaemia (.)NO detectability is diminished most probably due to concomitant oxidant stress.     

Enhancement of epigastric skin flap survival by adenovirus-mediated VEGF gene therapy

A novel approach to treat ischemic tissues by using gene therapy has recently been introduced on the basis of the angiogenic potential of certain growth factors. The authors investigated the effect of adenovirus-mediated gene therapy with vascular endothelial growth factor (VEGF) delivered into the subdermal space to treat compromised skin flaps. For this purpose, the epigastric skin flap model in rats, based solely on the right inferior epigastric vessels, was used. Thirty male Sprague-Dawley rats were divided into five groups of six rats each. Viral transfection with 108 plaque-forming units was performed 2 days before the epigastric flap elevation. Rats received subdermal injections of adenovirus encoding VEGF (Ad-VEGF) or green fluorescent protein (Ad-GFP) as treatment control. Another set of animals (n = 6) received no injections and were designated as control. To determine whether site of injection had an impact on flap viability, injections were given into the predicted local ischemic area (Ad-VEGF local, n = 6; Ad-GFP local, n = 6) and into the midline of the flap (Ad-VEGF midline, n = 6; Ad-GFP midline, n = 6). A flap measuring 8 x 8 cm was outlined on the abdominal skin extending from the xiphoid process proximally and the pubic region distally, to the anterior axillary lines bilaterally. Then, the epigastric flap was elevated as an island on the right inferior epigastric vessels and sutured back to its bed. Flap viability was evaluated at 7 and 14 days after the first operation. The epigastric flaps were scanned to the computer and areas of hypoxic and/or necrotic zones relative to total flap surface area were measured and expressed as percentages by using Image Pro Plus software. Specimens were taken for histologic evaluation at day 14 before the animals were killed. Combined area of necrotic and hypoxic zones as well as necrotic zone were decreased to 9.7 +/- 1.4 percent and 1.4 +/- 0.9 percent in Ad-VEGF local, and 11.8 +/- 1.9 percent and 3.5 +/- 1.64 percent in Ad-VEGF midline compared with the control and Ad-GFP treatment groups (control, 23 +/- 3.6 percent and 20.1 +/- 3.3 percent; Ad-GFP local, 24.8 +/- 4.8 percent and 16.2 +/- 5.9 percent; and Ad-GFP midline, 23.4 +/- 6.9 percent and 19.5 +/- 7.7 percent; p < 0.05). Histologic evaluation by light microscopy failed to demonstrate any quantitative difference in vascularity of skin flaps between the treatment groups. In this study, the authors demonstrated that adenovirus-mediated gene therapy using VEGF enhanced epigastric skin flap survival, as confirmed by the significant reduction in combined area of necrotic and hypoxic zones of the flap. Compared with the control, both local and midline subdermal injections of Ad-VEGF showed improvement in overall flap survival by 57.9 and 48.7 percent, respectively. The results of this study raise the possibility of using adenovirus-mediated therapeutic angiogenesis for safer flap surgery in high-risk patients.     

Type II diabetes mellitus,congestive heart failure,and zinc metabolism

Zinc status was assessed in patients with type II diabetes mellitus and congestive heart failure (CHF). Three groups of patients were enrolled into the study: Group 1: 15 patients with type II diabetes mellitus and CHF; Group 2: 20 patients with isolated type II diabetes mellitus; and Group 3: nine patients with isolated CHF. Twenty-four-hour urine was measured for creatinine, protein, and zinc, and blood was drawn for creatinine, proteins, liver enzymes, hemoglobin A₁c, and zinc. Insulin treatment and hemoglobin A₁c were comparable in the diabetic patients of groups 1 and 2, but group 1 was also treated with captopril and diuretics like the CHF patients of group 3. Plasma zinc levels were statistically similar in all three groups, but urinary zinc excretion (μmol/24 h) and urinary zinc: creatinine (μmol/mmol) ratio were significantly higher in the type II diabetics and CHF group (27.2±1.5; 1.69±0.6, respectively) compared to the diabetic patients alone (19.4±0.76; 0.97±0.3, respectively) and the CHF patients (9.7±0.3; 0.62±0.3, respectively). Patients with type II diabetes mellitus and CHF were treated with higher doses of captopril than the CHF patients (56.25±24 mg vs 18.8±11 mgP<0.05). Thus, patients with type II diabetes mellitus and CHF excrete larger amounts of zinc, which may eventually lead to zinc deficiency.     

Stem cells and pancreatic differentiation in vitro

Cell therapy using pancreatic islets would be a promising therapy to treat diabetes. But, because of the limited supply of human donor islets, other cellular sources have to be considered. Stem cells characterized by extensive proliferation and differentiation capacity may be a valuable source for the in vitro generation of islets. Insulin-producing cells derived from embryonic stem (ES) cells have been shown to reverse experimentally induced diabetes in animal models. However, the oncogenic properties of ES cells are critical in the context of clinical applications and efficient cell-lineage selection systems need to be established. Future studies have to demonstrate whether somatic stem cells residing in adult tissues, such as bone marrow, pancreatic ducts, intestine or liver may provide alternatives to generate functional pancreatic endocrine cells.     

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-alphaGal antibody

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6' OH of Galbeta(1 --> 4)Glcbeta-OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). UDP-[6-(3)H]Gal studies indicated that alpha1,3-galactosyltransferase recognized the C-6' modified Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and alpha1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15-22. Galalpha(1 --> 3)Galbeta(1 --> 4)Glcbeta-OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6" modified derivatives (23-30). An ELISA bioassay was performed utilizing human anti-alphaGal antibodies to study the binding affinity of the derivatized epitopes (6, 15-30). Modifications made at the C-6' position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6" position resulted in significant or complete abrogation of recognition. The results indicate that the C-6' OH of the alphaGal trisaccharide epitope is not mandatory for antibody recognition.