Donor Preconditioning After the Onset of Brain Death With Dopamine Derivate n-Octanoyl Dopamine Improves Early Posttransplant Graft Function in the Rat.

Heart transplantation is the therapy of choice for end-stage heart failure. However, hemodynamic instability, which has been demonstrated in brain-dead donors (BDD), could also affect the posttransplant graft function. We tested the hypothesis that treatment of the BDD with the dopamine derivate n-octanoyl-dopamine (NOD) improves donor cardiac and graft function after transplantation. Donor rats were given a continuous intravenous infusion of either NOD (0.882 mg/kg/h, BDD+NOD, n = 6) or a physiological saline vehicle (BDD, n = 9) for 5 h after the induction of brain death by inflation of a subdural balloon catheter. Controls were sham-operated (n = 9). In BDD, decreased left-ventricular contractility (ejection fraction; maximum rate of rise of left-ventricular pressure; preload recruitable stroke work), relaxation (maximum rate of fall of left-ventricular pressure; Tau), and increased end-diastolic stiffness were significantly improved after the NOD treatment. Following the transplantation, the NOD-treatment of BDD improved impaired systolic function and ventricular relaxation. Additionally, after transplantation increased interleukin-6, tumor necrosis factor TNF-α, NF-kappaB-p65, and nuclear factor (NF)-kappaB-p105 gene expression, and increased caspase-3, TNF-α and NF-kappaB protein expression could be significantly downregulated by the NOD treatment compared to BDD. BDD postconditioning with NOD through downregulation of the pro-apoptotic factor caspase-3, pro-inflammatory cytokines, and NF-kappaB may protect the heart against the myocardial injuries associated with brain death and ischemia/reperfusion.

Quality of isolated pig islets is improved using perfluorohexyloctane for pancreas storage in a split lobe model.

Pancreas transportation between donor center and islet production facility is frequently associated with prolonged ischemia impairing islet isolation and transplantation outcomes. It is foreseeable that shipment of pig pancreases from distant centralized biosecure breeding facilities to institutes that have a long-term experience in porcine islet isolation is essentially required in future clinical islet xenotransplantation. Previously, we demonstrated that perfluorohexyloctan (F6H8) is significantly more efficient to protect rat and human pancreata from ischemically induced damage compared to perfluorodecalin (PFD). To evaluate the effect of F6H8 on long-term stored pig pancreases in a prospective study, we utilized the split lobe model to minimize donor variability. Retrieved pancreases were dissected into the connecting and splenic lobe, intraductally flushed with UW solution and immersed alternately in either preoxygenated F6H8 or PFD for 8-10 h. Prior to pancreas digestion, the intrapancreatic pO2 and the ratio of ATP-to-inorganic phosphate was compared utilizing 31P-NMR spectroscopy. Isolated islets were cultured for 2-3 days at 37°C and subjected to quality assessment. Pancreatic lobes stored in preoxygenated F6H8 had a significantly higher intrapancreatic pO2 compared to pancreata in oxygen-precharged PFD (10.11 ± 3.87 vs. 1.64 ± 1.13 mmHg, p < 0.05). This correlated with a higher ATP-to-inorganic phosphate ratio (0.30 ± 0.04 vs. 0.14 ± 0.01). No effect was observed concerning yield and purity of freshly isolated islets. Nevertheless, a significantly improved glucose-stimulated insulin response, increased viability and postculture survival (57.2 ± 5.7 vs. 39.3 ± 6.4%, p < 0.01) was measured in islets isolated from F6H8-preserved pancreata. The present data suggest that F6H8 does not increase islet yield but improves quality of pig islets isolated after prolonged cold ischemia.

Aerosolized semifluorinated alkanes as excipients are suitable for inhalative drug delivery--a pilot study.

Semifluorinated alkanes (SFAs) have been described as potential excipients for pulmonary drug delivery, but proof of their efficacy is still lacking. We tested whether SFA formulations with the test drug ibuprofen can be nebulised and evaluated their pharmacokinetics. Physico-chemical properties of five different ibuprofen formulations were evaluated: an aqueous solution (H2O), two different SFAs (perfluorohexyloctane (F6H8), perfluorobutylpentane (F4H5)) with and without ethanol (SFA/EtOH). Nebulisation was performed with a jet catheter system. Inhalative characteristics were evaluated by laser diffraction. A confirmative animal study with an inhalative single-dose (6 mg/kg) of ibuprofen with each formulation was performed in anaesthetised healthy rabbits. Plasma samples at defined time points and lung tissue harvested after the 6-h study period were analyzed by HPLC-MS/MS. Pharmacokinetics were calculated using a non-compartment model. All formulations were nebulisable. No differences in aerodynamic diameters (MMAD) were detected between SFA and SFA/EtOH. The ibuprofen plasma concentration-time curve (AUC) was highest with F4H5/EtOH. In contrast, F6H8/EtOH had the highest deposition of ibuprofen into lung tissue but the lowest AUC. All tested SFA and SFA/EtOH formulations are suitable for inhalation. F4H5/EtOH formulations might be used for rapid systemic availability of drugs. F6H8/EtOH showed intrapulmonary deposition of the test drug.

Semifluorinated alkanes--a new class of excipients suitable for pulmonary drug delivery.

INTRODUCTION: Semifluorinated alkanes (SFAs) are considered as diblock molecules with fluorocarbon and hydrocarbon segments. Unlike Perfluorocarbons (PFCs), SFAs have the potential to dissolve several lipophilic or water-insoluble substances. This makes them possibly suitable as new excipients for inhalative liquid drug carrier systems. PURPOSE: The aim of the study was to compare physico-chemical properties of different SFAs and then to test their respective effects in healthy rabbit lungs after nebulisation. METHODS: Physico-chemical properties of four different SFAs, i.e. Perfluorobutylpentane (F4H5), Perfluorohexylhexane (F6H6), Perfluorohexyloctane (F6H8) and Perfluorohexyldodecane (F6H12) were measured. Based on these results, aerosol characteristics of two potential candidates suitable as excipients for pulmonary drug delivery, i.e. F6H8 and F4H5, were determined by laser light diffraction. Tracheotomised and ventilated New Zealand White rabbits were nebulised with either a high- or a low dose of SFAs (F6H8(low/high) and F4H5(low/high)) or saline (NaCl). Ventilated healthy animals served as controls (Sham). Arterial blood gases, lung mechanics, heart rate and blood pressure were recorded prior to nebulisation and in 30 min intervals during the 6-h study period. RESULTS: Out of the four SFAs studied initially, no satisfactory behaviour as a solvent has to be expected because of low lipophilicity for F6H6. Output rate during aerosolisation was very low for F6H12. F6H8 and F4H5 presented comparable aerosolisation characteristics and lipophilicity and were therefore tested in the in vivo model. Aerosol therapy, either SFAs or saline, impaired paO2/FiO2 ratio, dynamic lung compliance and respiratory mechanics in all groups, except for F4H5(low) group which behaved like the control group (Sham). F4H5(low) had no adverse effects on gas exchange or pulmonary mechanics. CONCLUSIONS: Perfluorobutylpentane (F4H5) in a low-dose application may be suitable as a new inhalable excipient in SFA-based pulmonary drug delivery systems for lipophilic or water-insoluble substances.

A cleaning solution for silicone intraocular lenses: "sticky silicone oil".

AIM: The aim of the study was to compare the efficacy of perfluorobutylpentane (F4H5) and perfluorohexyloctane (F6H8) in dissolving silicone oil from the surface of silicone intraocular lenses (IOL). METHODS: Droplets of stained silicone oil were applied to an object slide either lying flat or tilted by 30 degrees . Mixing with H(2)O, F4H5 or F6H8 was documented by a digital camera. Droplets of silicone oil were applied to silicone lenses and washed off by repeated rinsing with F4H5 or F6H8. The silicone lenses of 11 patients with silicone oil remnants on the posterior IOL surface were rinsed intraoperatively with F4H5 during removal surgery. RESULTS: Only F4H5 was able to mix with silicone oil and to remove it form the surface of a glass object slides. Rinsing with 25 mul F4H5 reduced the amount of silicone oil 1000 mPas or 5000 mPas attached on a silicone lens to 15% and 28%, respectively. A hanging droplet of silicone oil 5000 beneath a silicone lens was completely removed from below by F4H5. In all patients sufficient IOL cleaning was possible using F4H5. There was no significant postoperative inflammation in the vitreous or anterior chamber. CONCLUSION: Polydimethylsiloxanes dissolve effectively in F4H5 due to its lipophilic chemical structure. A much smaller volume of F4H5 than F6H8 is able to remove silicone oil from silicone lenses completely. Intraocular use of F4H5 is safe, and initial clinical data underlines its effectiveness as a cleaning agent after contact of silicone lenses with silicone oil.

CAPN 8: isolation of a new mouse calpain-isoenzyme.

Recent molecular biological approaches indicate that calpain, also named CANP for calcium-activated neutral protease and originally characterized as an intracellular cytoplasmatic nonlysosomal cysteine protease that requires calcium ions for activity, constitutes a large superfamily consisting of ubiquitous and tissue specific homologues, which are widely distributed in cells of various organisms from human to fungus. Due to the increasing number of substrates along with the involvement of calpain isoenzymes in mammalian diseases, especially in malignancies, members of the calpain superfamily seem to be important biomodulators in physiological as well as pathological cell function. Here we report the characterisation of a new calpain, named CAPN 8 with a different C-terminal domain, implicating a putative new regulatory mechanism. Northern blot analysis revealed an ubiquitous expression with different RNA levels in all tissues examined. Highest levels were found in brain, kidney, and digestive tract, suggesting a specific regulatory function of CAPN 8 in these tissues.

Expression of calpain I messenger RNA in human renal cell carcinoma: correlation with lymph node metastasis and histological type.

Calpain, also named CANP (for calcium-activated neutral protease), is an intracellular cytoplasmatic non-lysosomal cysteine endopeptidase that requires calcium ions for activity. Many substrates of the calpain isoenzymes, such as the transcription factors c-Fos and c-Jun, the tumor supressor protein p53, protein kinase C, pp60c-src and the adhesion molecule integrin, have been implicated in the pathogenesis of different human tumors, suggesting an important role of the calpains in malignant diseases. We now report differential expression of the calpain I gene (CL I) in a variety of tumors, extending our study to a larger series of renal cell carcinomas. Using Northern-blot analysis, we studied calpain I expression in 30 renal cell carcinomas as compared with matched healthy tissues. Tumor samples were classified according to their histological type: 21 clear cell carcinomas, 4 chromophobe carcinomas, 3 papillary carcinomas and 2 oncocytomas. In renal tumor samples, calpain I gene mRNA was expressed at highly variable levels, significantly depending on the different histological types. Moreover, there was a correlation of higher calpain I expression with increased malignancy: within the clear cell carcinoma subset, tumor samples with advanced nodal status (N1 and N2) showed a significantly higher calpain I expression than tumors without metastasis to regional lymph nodes. Our data suggest an important role of calpain isoenzymes in carcinogenesis and tumor progression.

Glyceraldehyde-3-phosphate dehydrogenase and Nm23-H1/nucleoside diphosphate kinase A. Two old enzymes combine for the novel Nm23 protein phosphotransferase function.

We have recently discovered an alternative function of the putative metastasis suppressor protein Nm23, which is identical to nucleoside diphosphate kinase, as a protein phosphotransferase in vitro. While purified native Nm23 protein did not phosphorylate other proteins, we could purify a Nm23-associated protein that activates the protein phosphotransferase function; it was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isoenzyme. Co-expression and purification of (His)6-tagged GAPDH in combination with either Nm23-H1 or Nm23-H2 in baculovirus-infected Sf9 cells showed that only Nm23-H1, but not Nm23-H2, forms a stable complex with GAPDH. Protein phosphotransferase activity was confirmed for the recombinant GAPDH.Nm23-H1 complex but not for either of the enzymes alone, nor was this activity observed after simple mixing of the purified proteins in vitro. The molecular mass of the highly purified recombinant GAPDH.Nm23-H1 complex suggests that a dimer of GAPDH interacts with a dimer of Nm23-H1. In contrast to the complex with GAPDH, co-expression of Nm23-H1 with antioxidant protein (MER-5) or creatine kinase did not activate the protein phosphotransferase function, indicating that this activation may specifically require GAPDH as a binding partner.

NM23-H1 and NM23-H2 gene expression in human renal tumors.

The two nm23 genes, nm23-H1 and nm23-H2, are implicated in the metastatic process and tumor progression in some human tumors. Until now no data exist about nm23 expression in the different types of human renal tumors. To investigate if the nm23 genes play a central role in the progression of renal tumors, we have examined nm23-H1 and nm23-H2 gene expression using Northern-blot analysis and immunohistochemistry. We analysed clear cell type RCC, chromophilic RCC, chromophobic RCC, collecting duct type RCC and renal oncocytomas. Our results indicate that the nm23 genes do not play a central role in the prognosis of renal cell carcinoma in the analysed tumors.

Isolation and characterization of new microsatellites at the nm23-H1 and nm23-H2 gene loci and application for loss of heterozygosity (LOH) analysis.

The nm23-H1 gene has been suggested to be a metastasis suppressor gene. Studies about the events of loss of heterozygosity (LOH) at the nm23 locus and its correlation to metastasis are controversially discussed. To optimize detection of LOH at the nm23 locus, we screened two P1 clones for additional microsatellites. Tumor and normal DNA from 37 colorectal, 16 gastric, and 8 germ cancer patients were examined for LOH. We found two new CA repeats, one 5' to nm23-H1 and another 3' to nm23-H2. Using these nm23 locus-specific CA repeats and five other chromosome 17 loci (D17S1522, D17S1566, D17S855, D17S515, and TP53), allele loss was observed in 4/32 (12.5%) patients with colon cancer, 2/14 (14.3%) with gastric cancer, and 1/7 (14%) with germ cancer. No isolated LOH of the nm23 region was observed.

A second trefoil protein, ITF/hP1.B, is transcribed in human breast cancer.

Trefoil proteins form a specific group of stable secreted polypeptides. They are expressed in a lot of human cancers and during inflammatory processes of the gastrointestinal tract. Recently a new human trefoil protein, ITF/hP1.B, was isolated. Until now no studies of the activity of this gene in human solid tumors exist. In our examination we show for the first time that this gene is transcribed in human breast cancer. In contrast to another trefoil protein, pS2, the expression of ITF/hP1.B is not under control of estrogen in the human breast cancer cell line MCF-7. We suggest that the gene activity of ITF/hP1.B in addition to pS2 expression may be an improved prognostic marker in human breast cancer.

A novel serine/threonine-specific protein phosphotransferase activity of Nm23/nucleoside-diphosphate kinase.

Two human nm23 genes have been identified, designated nm23-H1 and nm23-H2, which encode the 88% identical nucleoside-diphosphate kinase (NDPK) A and NDPK B polypeptides, respectively. The nm23-H1 gene product has been shown to play a functional role in the suppression of tumor metastasis. The Nm23 proteins/NDPK are highly conserved throughout evolution and are implicated in controlling cellular differentiation and development in various species, while the underlying mechanisms remain undefined. Neither the NDPK activity nor the DNA-binding activity, identified recently for NDPK B, can satisfactory explain the regulatory functions of Nm23. The present study provides evidence that purified Nm23 proteins are capable of transferring a phosphate group to other proteins when non-denaturing amounts of urea are present. This novel Nm23/NDPK activity was found to be specific for serine and threonine residues, and the transphosphorylation of substrate proteins occurred stoichiometrically. Because of the absence of a substrate turn-over, the novel function was termed protein phosphotransferase activity instead of protein kinase activity. It is demonstrated that urea stimulates the interaction of NDPK with other proteins. Identical phosphoprotein patterns were obtained using purified NDPK preparations from human, Drosophila, yeast and Dictyostelium in the presence of urea. Partially purified NDPK from human erythrocytes produced a similar phosphorylation pattern independent of urea addition and also acted stoichiometrically. In this preparation, a protein phosphotransferase activity of Nm23 species may possibly be generated and/or stabilized by the interaction with copurified proteins. Using different mutants of Dictyostelium NDPK it was shown that the protein phosphotransferase activity depends on the same active site as the NDPK activity. A phosphotransfer mechanism analogous to that of protein-histidine kinases is proposed, involving a high-energy phosphohistidine intermediate. Furthermore, the novel Nm23 function is compared with an apparently similar protein phosphotransferase activity which was observed previously with partially purified NDPK from different plant species.

Presence of regulatory sequences within intron 4 of human and murine c-myb genes.

The molecular mechanisms that modulate c-myb mRNA transcription in hematopoietic cells appear to involve intron regulatory sequences. We have characterized the fourth of ten introns from both human and murine c-myb genes in regard to nucleotide sequence and specific protein binding. For this approach complete genomic c-myb intron 4 fragments were isolated from mouse and human DNA using PCR amplification with flanking exon-primers derived from the mouse gene. Comparison of the obtained sequences revealed strong homology between the two species. Using crude nuclear protein extracts from mouse and human myb expressing cells (70Z/3B; Molt4) and gel shift experiments we found specific protein interaction for both introns and to determine the protein binding site in detail, we performed DNase I footprinting. Our results indicate that the binding factor is absent in control cell lines without c-myb transcriptional activity, suggesting a possible positive regulatory function of the DNA-protein complex. To confirm these findings we introduced the human c-myb intron 4 DNA sequence into the EcoRI site of the pCAT-Promoter plasmid and transfected Molt4 cells with this chimeric construct. The transient expression studies revealed that intron 4 sequences possess enhancer activity. Thus, we have demonstrated that intron 4 sequences can be important for the regulation of c-myb proto-oncogene expression.

Expression pattern of breast-cancer-associated protein pS2/BCEI in colorectal tumors.

Recently, several carcinomas of the gastrointestinal tract were tested for pS2/BCEI activity, a gene isolated from breast-cancer cells and coding for a small secreted peptide. In the latter tumors, its activity is under estrogen control; surprisingly, it was also found expressed in carcinomas of the stomach, biliary tract and pancreas. We have now investigated the expression of this gene in 64 colorectal carcinomas, 31 adenomas and 13 polyps in comparison with their matrix tissues by applying molecular (RNA analysis) and immunohistochemical (pS2 antibody) techniques. Positive pS2 immunostaining (ranging from focal to strong immunoreaction) was noted in 89% of human colon cancers, while 11% remained negative. Furthermore, all 40 transitional mucosae were strongly positive, whereas normal mucosa was negative. Of hyperplastic polyps, 68.2% displayed a significant immunoreaction, and 80.6% of adenomas were focally positive. Finally, 6 out of 16 cases showed significant pS2 transcription in Northern blot analysis. These data clearly indicate that the breast-cancer-associated pS2 protein also plays an as yet undetermined role in the tumorigenesis of human colorectal carcinomas.

Isolation and characterization of the human genomic locus coding for the putative metastasis control gene nm23-H1.

Nm23-H1 gene expression is inversely correlated with tumor metastatic potential in certain tumors, including melanomas, breast carcinomas, and hepatocellular carcinomas. Using nm23-H1 c-DNA primer and genomic polymerase chain reaction (PCR) amplification, we purified three PCR fragments (one of 4kb and two of 2 kb) covering the whole human genomic locus of the gene (8.460bp). We recombined the PCR products into pUC18 and produced a restriction map to perform subcloning. Complete sequencing of genomic PCR fragments, including the whole coding region of nm23-H1, revealed that the gene consists of five exons and four introns spanning 8.5kb. A sequence homology analysis between human nm23-H1 and the homolog gene of the rat (NDP-K beta) shows that exon-intron boundaries are well conserved between these two species.

Retrospective analysis of prognostic significance of the estrogen-inducible pS2 gene in male breast carcinoma.

BACKGROUND: The estrogen-inducible pS2 gene, originally isolated from a breast cancer cell line, is correlated with hormone-dependent female breast tumors and its expression is associated with longer overall and disease-free survival. METHODS: The authors have investigated 38 samples of carcinomas of the male breast for pS2 expression by using a monoclonal antibody. The immunostaining was compared with clinical data, in particular, to the progesterone receptor status, to assess a possible prognostic value of this parameter. RESULTS: Although most cases (27 of 38) were immunopositive (i.e., above the 5th percentile of immunoreactive cells), no correlation with tumor grade and survival was notable. CONCLUSIONS: Therefore, on the contrary to the situation in female breast cancer, pS2 activity failed to constitute a new prognostic parameter in male breast carcinomas.

High levels of nm23-H1 and nm23-H2 messenger RNA in human squamous-cell lung carcinoma are associated with poor differentiation and advanced tumor stages.

Expression of the candidate metastasis-suppressor gene nm23-H1 has been shown to correlate inversely with metastatic potential in some human tumors, but not in all. Until now, few studies have been carried out on the activity of the homologous nm23-H2 gene in human cancer. No nm23 transcription studies exist for human lung cancer so far. To determine whether the nm23 genes could have a metastasis-suppressor function in non-small-cell lung carcinoma (NSCLC), pulmonary sarcoma and carcinoids, we analysed both nm23-HI and nm23-H2 mRNA levels in 37 tumor samples obtained from patients who underwent potentially curative resection between 1986 and 1990, and in 4 metastatic tumors obtained from autopsy. As compared to corresponding healthy lung parenchyma, both nm23-HI and nm23-H2 transcript levels were elevated in 37 of 41 tumors. The increases in nm23 mRNA expression were stronger in advanced stages of squamous-cell carcinoma, large-cell carcinoma, sarcoma and carcinoids than in early stages of the respective tumor types. Within stages I and II of squamous-cell carcinoma, significantly higher nm23 mRNA levels were found in poorly differentiated tumors than in moderately differentiated ones. Moreover, an inverse correlation between nm23 expression and disease-free survival of the patients was observed. In conclusion, our results indicate that the increased nm23 expression in the analysed tumors is not consistent with the proposed metastasis-suppressor function, but the 2 nm23 genes nevertheless may be implicated in the mechanism of tumor progression.

Influence of steroid hormones on pS2/BCEI gene expression in xenografted colon tumors.

The biological function of the hormone inducible human pS2/BCEI gene, cloned from the breast cancer cell line MCF-7, still remains unknown. Our aim was to determine the in vivo influence of steroid hormones on pS2 expression and tumour growth in colorectal tumours. We transplanted aliquots of human colon tumours into male and female nude mice and studied tumour growth and expression of the pS2/BCEI gene by immunostaining and mRNA analysis. Our results show that the sex of the host and therefore the hormonal background does not play a dominant role in pS2 expression and growth of the xenografts.