Crucial Role for Neuronal Nitric Oxide Synthase in Early Microcirculatory Derangement and Recipient Survival following Murine Pancreas Transplantation

Objective

Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation.

Background

Tetrahydrobiopterin donor treatment has been shown to specifically attenuate these derangements following pancreas transplantation, and tetrahydrobiopterin-mediated protective effects to rely on its NOS-cofactor activity, rather than on its antioxidant capacity. However, the NOS-isoform mainly involved in this process has still to be defined.

Methods

Using a murine pancreas transplantation model, grafts lacking one of the three NOS-isoforms were compared to grafts from wild-type controls. Donors were treated with either tetrahydrobiopterin or remained untreated. All grafts were subjected to 16 h cold ischemia time and transplanted into wild-type recipients. Following 4 h graft reperfusion, microcirculation was analysed by confocal intravital fluorescence microscopy. Recipient survival was monitored for 50 days.

Results

Transplantation of the pancreas from untreated wild-type donor mice resulted in microcirculatory damage of the transplanted graft and no recipient survived more than 72 h. Transplanting grafts from untreated donor mice lacking either endothelial or inducible NOS led to similar outcomes. In contrast, donor treatment with tetrahydrobiopterin prevented microcirculatory breakdown enabling long-term survival. Sole exception was transplantation of grafts from untreated donor mice lacking neuronal NOS. It resulted in intact microvascular structure and long-term recipient survival, either if donor mice were untreated or treated with tetrahydrobiopterin.

Conclusion

We demonstrate for the first time the crucial involvement of neuronal NOS in early microcirculatory derangements following solid organ transplantation. In this model, protective effects of tetrahydrobiopterin are mediated by targeting this isoform.     

Ascospore septation inDiploschistes (Thelotremataceae, lichenizedAscomycota) and the taxonomic significance of macro- and microcephalic ascospore types

The ascospore septation in 13 species ofDiploschistes was examined. Microcephalic, macrocephalic, as well as irregular ascospore septation were found. In some species and even specimens of some species, different types of spore septation were detected. The taxonomic significance of the character of ascospore septation sequence is questioned, and the distinction of the ordersGraphidales andOstropales, which was primarily based on this character, is rejected.This paper is dedicated to Prof. DrRolf Santesson (Uppsala) on the occasion of his 80th birthday.     

Stem-like cells in human hepatoblastoma.

Hepatoblastoma is a pediatric liver tumor with epithelial components resembling embryonal and fetal liver cells. The existence of teratoid hepatoblastoma suggests the presence of stem cells in hepatoblastoma. The aim of this study was to analyze the expression of stem cell markers in hepatoblastomas. We studied specimens from 10 hepatoblastomas. Five of the hepatoblastomas were of epithelial and five of mixed type. Immunohistochemistry (IHC) for the stem cell markers CD34, Thy1, c-kit, and the hepatic or biliary lineage markers CK-18, OCH, CK-7, and CD56 was performed. Double IHC for stem cell and lineage markers was used to identify putative liver stem cells. The different markers showed distinct distributions on the tumor cells. Cells in atypical ducts were found to express simultaneously stem cell markers and hepatocytic or biliary lineage markers. Other cells in connective tissue showed c-kit expression, but not hepatic or biliary marker expression. The data show the presence of different cell populations bearing stem cell markers in human hepatoblastoma. Ductal cells co-expressing stem cell markers and hepatic lineage markers phenotypically resemble hepatic stem-like cells. These findings support the thesis that stem cells play a role in the histogenesis of hepatoblastoma.     

Inhibition of VEGFR-2 by SU5416 increases neonatally glutamate-induced neuronal damage in the cerebral motor cortex and hippocampus

Vascular endothelial growth factor (VEGF) exerts neuroprotective or proinflammatory effects, depending on what VEGF forms (A–E), receptor types (VEGFR1–3), and intracellular signaling pathways are involved. Neonatal monosodium glutamate (MSG) treatment triggers neuronal death by excitotoxicity, which is commonly involved in different neurological disorders, including neurodegenerative diseases. This study was designed to evaluate the effects of VEGFR-2 inhibition on neuronal damage triggered by excitotoxicity in the cerebral motor cortex (CMC) and hippocampus (Hp) after neonatal MSG treatment. MSG was administered at a dose of 4 g/kg of body weight (b.w.) subcutaneously on postnatal days (PD) 1, 3, 5, and 7, whereas the VEGFR-2 inhibitor SU5416 was administered at a dose of 10 mg/kg b.w. subcutaneously on PD 5 and 7, 30 min before the MSG treatment. Neuronal damage was assessed using hematoxylin and eosin staining, fluoro-Jade staining, and TUNEL assay. Additionally, western blot assays for some proteins of the VEGF-A/VEGFR-2 signaling pathway (VEGF-A, VEGFR-2, PI3K, Akt, and iNOS) were carried out. All assays were performed on PD 6, 8, 10, and 14. Inhibition of VEGFR-2 signaling by SU5416 increases the neuronal damage induced by neonatal MSG treatment in both the CMC and Hp. Moreover, neonatal MSG treatment increased the expression levels of the studied VEGF-A/VEGFR-2 signaling pathway proteins, particularly in the CMC. We conclude that VEGF-A/VEGFR-2 signaling pathway activation could be part of the neuroprotective mechanisms that attempt to compensate for neuronal damage induced by neonatal MSG treatment and possibly also in other conditions involving excitotoxicity.     

Autophagy activity is associated with membranous sodium iodide symporter expression and clinical response to radioiodine therapy in non-medullary thyroid cancer

Although non-medullary thyroid cancer (NMTC) generally has a good prognosis, 30–40% of patients with distant metastases develop resistance to radioactive iodine (RAI) therapy due to tumor dedifferentiation. For these patients, treatment options are limited and prognosis is poor. In the present study, expression and activity of autophagy was assessed in large sets of normal, benign and malignant tissues and was correlated with pathology, SLC5A5/hNIS (solute carrier family 5 member 5) protein expression, and with clinical response to RAI ablation therapy in NMTC patients. Fluorescent immunostaining for the autophagy marker LC3 was performed on 100 benign and 80 malignant thyroid tissues. Semiquantitative scoring was generated for both diffuse LC3-I intensity and number of LC3-II-positive puncta and was correlated with SLC5A5 protein expression and clinical parameters. Degree of diffuse LC3-I intensity and number of LC3-II-positive puncta scoring were not discriminative for benign vs. malignant thyroid lesions. Interestingly, however, in NMTC patients significant associations were observed between diffuse LC3-I intensity and LC3-II-positive puncta scoring on the one hand and clinical response to RAI therapy on the other hand (odds ratio [OR] = 3.13, 95% confidence interval [CI] =1.91–5.12, P = 0.01; OR = 5.68, 95%CI = 3.02–10.05, P = 0.002, respectively). Mechanistically, the number of LC3-II-positive puncta correlated with membranous SLC5A5 expression (OR = 7.71, 95%CI = 4.15–11.75, P<0.001), number of RAI treatments required to reach remission (P = 0.014), cumulative RAI dose (P = 0.026) and with overall remission and recurrence rates (P = 0.031). In conclusion, autophagy activity strongly correlates with clinical response of NMTC patients to RAI therapy, potentially by its capacity to maintain tumor cell differentiation and to preserve functional iodide uptake.     

Lactobacillus intestinalis (ex Hemme 1974) sp. nov., nom. rev., isolated from the intestines of mice and rats

The genetic and phenotypic properties of 10 strains identified as Lactobacillus intestinalis sp. nov. were examined. These strains constitute a distinct species which can be differentiated from all of the previously described homofermentative species in the genus Lactobacillus by their carbohydrate fermentation pattern. The guanine-plus-cytosine contents of their DNAs are 33 to 35 mol%. DNAs from 10 other Lactobacillus species did not exhibit significant levels of relatedness to representative strains of the new species. The name Lactobacillus intestinalis (ex Hemme) sp. nov., nom. rev. is proposed for these isolates, and strain Th4 (= ATCC 49335 = JCM 7548) is the type strain.     

Genome rearrangements in <Emphasis Type="Italic">Escherichia coli</Emphasis> during de novo acquisition of resistance to a single antibiotic or two antibiotics successively

Background

The ability of bacteria to acquire resistance to antibiotics relies to a large extent on their capacity for genome modification. Prokaryotic genomes are highly plastic and can utilize horizontal gene transfer, point mutations, and gene deletions or amplifications to realize genome expansion and rearrangements. The contribution of point mutations to de novo acquisition of antibiotic resistance is well-established. In this study, the internal genome rearrangement of Escherichia coli during to de novo acquisition of antibiotic resistance was investigated using whole-genome sequencing.

Results

Cells were made resistant to one of the four antibiotics and subsequently to one of the three remaining. This way the initial genetic rearrangements could be documented together with the effects of an altered genetic background on subsequent development of resistance. A DNA fragment including ampC was amplified by a factor sometimes exceeding 100 as a result of exposure to amoxicillin. Excision of prophage e14 was observed in many samples with a double exposure history, but not in cells exposed to a single antibiotic, indicating that the activation of the SOS stress response alone, normally the trigger for excision, was not sufficient to cause excision of prophage e14. Partial deletion of clpS and clpA occurred in strains exposed to enrofloxacin and tetracycline. Other deletions were observed in some strains, but not in replicates with the exact same exposure history. Various insertion sequence transpositions correlated with exposure to specific antibiotics.

Conclusions

Many of the genome rearrangements have not been reported before to occur during resistance development. The observed correlation between genome rearrangements and specific antibiotic pressure, as well as their presence in independent replicates indicates that these events do not occur randomly. Taken together, the observed genome rearrangements illustrate the plasticity of the E. coli genome when exposed to antibiotic stress.

     

Molecular dynamics simulation of hydration in myoglobin

This study was carried out to evaluate the stability of the 89 bound water molecules that were observed in the neutron diffraction study of CO myoglobin. The myoglobin structure derived from the neutron analysis was used as the starting point in the molecular dynamics simulation using the software package CHARMM. After solvation of the protein, energy minimization and equilibration of the system, 50 ps of Newtonian dynamics was performed. This data showed that only 4 water molecules are continously bound during the length of this simulation while the other solvent molecules exhibit considerable mobility and are breaking and reforming hydrogen bonds with the protein. At any instant during the simulation, 73 of the hydration sites observed in the neutron structure are occupied by water. © 1995 Wiley-Liss, Inc.