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.

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.

Cross-hybridization between the avian myeloblastosis oncogene and eukaryotic 28S ribosomal RNA.

In Northern blots, avian myeloblastosis (myb) oncogene probes (genomic or cDNA) cross-hybridize to the 28S rRNA band mimicking a myb-specific transcript. A misinterpretation of the hybridization data can be avoided by using an oligodeoxyribonucleotide probe.

Expression of the breast cancer associated gene pS2 and the pancreatic spasmolytic polypeptide gene (hSP) in diffuse type of stomach carcinoma.

Expression of the pancreatic spasmolytic peptide (hSP) gene and pS2 (a gene isolated from oestrogen-induced breast carcinoma cells) were analysed in 36 samples of human stomach carcinoma. 17 tumours were investigated at the RNA level (by northern blots) as well as at the gene product level (by immunochemistry). Since pS2 had been shown to be expressed in normal stomach mucosa its activity in carcinoma samples was expected. Surprisingly, strong pS2 immunoreactivity was noted in the diffuse carcinoma type, whereas the intestinal type displayed weak reactivity. The tumour samples showing strong immunostaining expressed the regular 0.6 kb pS2 RNA band and weak staining was paralleled by aberrant transcripts. Additionally, only in tumour samples with regular pS2 transcription was the typical 0.7 kb hSP RNA band seen; samples with aberrant pS2 bands did not express hSP at all. This is the first demonstration of hSP gene activity in a human tumour.

The cellular myb oncogene is amplified, rearranged and activated in human glioblastoma cell lines.

The human c-myb gene which encodes a DNA binding protein and which is rarely amplified in neoplastic cells was found to be altered in four human glioblastoma cell lines. It exists in multiple copies in 2 out of 4 cases studied. The degree of amplification as determined by densitometry was about 10-fold, a rearrangement within the coding region and an enhanced gene activity of c-myb were noted. The observation of c-myb oncogene amplification and activity in glioblastoma cell lines presents the first report of this effect in human brain tumor cells.

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.

Differential activity of two oncogenes from chromosome #7 in human glioblastoma cell lines.

Human glioblastoma cell lines are known to develop polysomy of cytogenetically intact chromosomes #7 and overexpression of the erbB oncogene (7p12-p14) at a level even higher than is to be expected from the number of #7 chromosomes. The met oncogene, however, which is also located on chromosome #7 (7q31-q32), was shown not to be overexpressed in a panel of 7-polysomic glioblastoma cell lines overexpressing erbB. Molecular analysis of the cell line HeRo gave proof that there is no detectable amplification or rearrangement of the erbB gene which could be responsible for its overexpression. These findings favor the assumption of differential regulation of the met and erbB oncogenes, e.g. by means of insufficient activity of a trans-acting erbB suppressor gene possibly located on a chromosome with a low copy number.

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.

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.

Assignment of the gene for human spasmolytic protein (hSP/SML1) to chromosome 21.

A human cDNA corresponding to the porcine pancreatic spasmolytic protein (PSP) was isolated, and the recombinant clone was originally termed hSP for human spasmolytic protein. Later, the term SML1 for spasmolysin was suggested for the human gene. This protein shows a remarkable sequence homology to pS2, a protein coded by an estrogen-induced gene isolated from the breast carcinoma cell line MCF-7. Although, at the DNA level, the gene sequences pS2 and hSP/SML1 display insufficient homology for cross-hybridization, their expression in tumor cells occurs with remarkable coordination. The human pS2 gene sequence has been assigned to chromosome 21, and we have therefore attempted to map the hSP/SML1 gene by using cDNA and Southern blotting of genomic DNAs from a panel of human-rodent somatic cell hybrids carrying different complements of human chromosomes. Interestingly, the hSP/SML1 gene is also localized on chromosome 21.

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.

Generating highly labeled oligonucleotides for DNA-protein interaction.

We developed a new strategy to prepare double-stranded oligonucleotides containing recognition sites for specific binding proteins to examine DNA-protein interactions in various assays (gel mobility shift, UV-crosslinking, and affinity chromatography). The advantages of our procedures are as follows. Only one strand needs to be synthesized using a commercial oligonucleotide synthesizer. The probes can be labeled to a high specific activity and the exact position of labeling can be chosen, which is necessary for UV-crosslinking studies. Furthermore, multimeric binding sites for efficient DNA affinity chromatography can easily be generated. It is also possible to precisely place modified bases without the need for chemical precursors. Using this protocol, more detailed information about the binding protein factors and their behavior in interaction with recognition sites can be obtained.

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.

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 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.

Association of the human spasmolytic polypeptide and an estrogen-induced breast cancer protein (pS2) with human pancreatic carcinoma.

The human pS2 gene, isolated from the breast carcinoma cell line MCF-7 and shown to be under estrogen transcriptional control in a subclass of breast cancer cells was reported to be secreted in normal stomach surface epithelial cells, whereas additional gastrointestinal tissues like pancreas and colon do not secrete pS2 at all. In porcine pancreas, a spasmolytic polypeptide (sharing domains of homology with pS2) was observed; a corresponding human gene (hSP) was shown to be active in normal stomach mucosa. hSP and pS2 gene activity in normal and neoplastic pancreas tissues was then compared. Whereas both genes are inactive in normal pancreatic cells, activation of the pS2 sequence in a primary pancreatic carcinoma cell culture and in 23 tumor tissues was noted when investigated by immunostaining. In all cases when pS2 showed a regular 0.6 kb transcript, hSP displayed a transcript of 0.7 kb. Six of these tumors showed a reduced pS2 immunoreactivity and, at the same time, aberrant pS2 mRNA bands and a complete shut-down of the hSP gene were noted. In one case, whereas normal pancreas remained negative, the corresponding tumor and its metastasis displayed regular transcripts of pS2 and hSP. This remarkably high correlation suggests that pS2 and hSP expression in the pancreatic tumors, but not in their corresponding healthy tissue is significantly linked to molecular steps leading to tumorigenesis.

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.