Portal hypertension and liver lesions in chronically alcohol drinking rats prevented and reversed by stable gastric pentadecapeptide BPC 157 (PL-10, PLD-116), and propranolol, but not ranitidine.

Liver lesions and portal hypertension in rats, following chronic alcohol administration, are a particular target for therapy. Portal hypertension (mm Hg) assessed directly into the portal vein, and liver lesions induced by 7.28 g/kg b.w. of alcohol given in drinking water for 3 months, were counteracted by a stable gastric pentadecapeptide BPC 157, GEPPPGKPADDAGLV, M.W. 1419, known to have a beneficial effect in a variety of models of gastrointestinal or liver lesions (10 microg or 10 ng/kg b.w. i.p. or i.g.) and propranolol (10 mg/kg b.w. i.g.), but not ranitidine (10 mg/kg b.w. i.g.) or saline (5 ml/kg b.w. i.p./i.g.; control). The medication (once daily) was throughout either the whole 3 months period (1) or the last month only (2) (last application 24 h before sacrifice). In the background of 7.28 g/kg/daily alcohol regimen similar lesions values were assessed in control rats following alcohol consumption, after 2 or 3 months of drinking. Both prophylactic and therapeutic effects were shown. After a period of 2 or 3 months, in all control saline [intragastrically (i.g.) or intraperitoneally (i.p.)] treated rats, the applied alcohol regimen consistently induced a significant rise of portal blood pressure values over values noted in healthy rats. In rats that received gastric pentadecapeptide BPC 157 or propranolol the otherwise raised portal pressure was reduced to the values noted in healthy rats. Besides, a raised surface area (microm(2)) and increased circumference (microm) of hepatocyte or hepatocyte nucleus [HE staining, measured using PC-compatible program ISSA (VAMS, Zagreb, Croatia)] and an advanced steatosis [scored (0-4), Oil Red staining] (on 100 randomly assigned hepatocytes per each liver), an increased liver weight, all together parallel a raised portal pressure in controls. Some of them were completely eliminated (not different from healthy rats, i.e. portal pressure, the circumference and area of hepatocytes, liver weight), while others were markedly attenuated (values less than in drinking controls, still higher than in healthy rats, i.e. circumference and area of hepatocytes nucleus). On the other hand, ranitidine application attenuated only steatosis development. In summary, despite continuous chronic alcohol drinking, pentadecapeptide BPC 157, and propranolol may prevent portal hypertension as well as reverse already established portal hypertension along with related liver disturbances.     

Lung lesions and anti-ulcer agents beneficial effect: anti-ulcer agents pentadecapeptide BPC 157, ranitidine, omeprazole and atropine ameliorate lung lesion in rats.

Anti-ulcer agents may likely attenuate lesions outside the gastrointestinal tract, since they had protected gastrectomized rats (a "direct cytoprotective effect"). Therefore, their therapeutic potential in lung/stomach lesions were shown. Rats received an intratracheal (i.t.) HCl instillation [1.5 ml/kg HCl (pH 1.75)] (lung lesion), and an intragastric (i.g.) instillation of 96% ethanol (gastric lesion; 1 ml/rat, 24 h after i.t. HCl instillation), then sacrificed 1 h after ethanol. Basically, in lung-injured rats, the subsequent ethanol-gastric lesion was markedly aggravated. This aggravation, however, in turn, did not affect the severity of the lung lesions in the further period, at least for 1 h of observation. Taking intratracheal HCl-instillation as time 0, a gastric pentadecapeptide, GEPPPGKPADDAGLV, M.W.1419, coded BPC 157 (10 microg, 10 ng, 10 pg), ranitidine (10 mg), atropine (10 mg), omeprazole (10 mg), were given [/kg, intraperitoneally (i.p.)] (i) once, only prophylactically [as a pre-treatment (at -1h)], or as a co-treatment [at 0)], or only therapeutically (at +18h or +24 h); (ii) repeatedly, combining prophylactic/therapeutic regimens [(-1 h)+(+24 h)] or [(0)+(+24 h)], or therapeutic/therapeutic regimens [(+18 h)+(+24 h)]. For all agents, combining their prophylactic and salutary regimens (at -1 h/+24 h, or at 0/+24 h) attenuated lung lesions; even if effect had been not seen already with a single application, it became prominent after repeated treatment. In single application studies, relative to controls, a co-treatment (except to omeprazole), a pre-treatment (at -1 h) (pentadecapeptide BPC 157 and atropine, but not ranitidine and omeprazole) regularly attenuated, while therapeutically, atropine (at +18 h), pentadecapeptide BPC 157 highest dose and omeprazole (at +24 h), reversed the otherwise more severe lung lesions.     

The effect of a novel pentadecapeptide BPC 157 on development of tolerance and physical dependence following repeated administration of diazepam

A novel gastric pentadecapeptide BPC 157 with different beneficial activities and anticonvulsant effect interacting with GABAergic system could improve diazepam efficacy coadministered (10 microg/kg, 10 ng/kg i.p.) with diazepam (5.0 mg/kg i.p.) twice daily for 10 days, since diazepam chronic medication would otherwise predispose for diazepam- tolerance/withdrawal development (shorter latency to convulsion after convulsant). In diazepam chronically treated mice, it attenuated diazepam tolerance (provoked by later acute administration of diazepam together with convulsant) and postponed physical dependence/withdrawal effects (provoked by later administration of isoniazid). In tolerance assay, at 42 h after the end of conditioning regimen, shorter preconvulsive latencies than in healthy (non-diazepam conditioned) mice following isoniazid (800 mg/kg i.p.) (as hallmark of tolerance) were observed if diazepam (5.0 mg/kg i.p.) was again given acutely to mice previously conditioned with diazepam alone (use of picrotoxin 3.0 mg/kg i.p., as convulsant, with acute application of diazepam in previously diazepam conditioned mice did not lead to tolerance hallmark). This was completely avoided in diazepam+BPC 157 10 microg or diazepam+BPC 157 10 ng chronically treated animals. In physical dependence assay (isoniazid challenge assessed at 6, 14, 42 and 72 h after conditioning medication), when compared to diazepam non-conditioned healthy mice, in diazepam conditioned mice residual anticonvulsive activity was not present already at the earliest post-conditioning interval (i.e., not different latency to isoniazid-convulsions), whereas shorter preconvulsive latencies (as physical dependence/withdrawal hallmark) were noted in diazepam conditioned mice following isoniazid challenge at 42 h and at 72 h after end of conditioning treatment. In diazepam+BPC 157 10 microg- conditioned mice, a residual anticonvulsive activity (i.e., longer latency to isoniazid convulsion) was noted at 6 h post-conditioning, whereas shorter preconvulsive latencies appeared only at 72 h-post-conditioning period. In conclusion, taken together these data (lack of tolerance development (tolerance studies), prolonged residual anticonvulsive activity, and postponed physical dependence/withdrawal hallmark in diazepam+BPC 157 chronically treated mice) with common benzodiazepines tolerance/withdrawal knowledge, it could be speculated that BPC 157 acts favoring the natural homeostasis of the GABA receptor complex as well as enhancing the GABAergic transmission, and having a mechanism at least partly different from those involved in diazepam tolerance/withdrawal, it may be likely used in further therapy of diazepam tolerance and withdrawal.     

Characterisation of a 19-year-old "long-term survivor" with Edwards syndrome

Trisomy 18 is the second most frequent autosomal aneuploidy affecting about 1 in 8,000 new-borns. Similar to trisomy 13 more than 90% of the patients die within the first year. Main causes of death are failure of vital organ function, in most cases of brain, heart, kidney, and gut, sometimes combined with severe infections. The degree to which essential organs are affected at birth and the clinical course differ considerably. Unknown genetic factors and various environmental effects are most likely involved. A less severe course of Edwards syndrome can be caused by a partial trisomy due to a deletion of the extra chromosome 18 or somatic mosaicism with a trisomic and a normal cell-line in the patient. In this report conventional chromosome analysis, FISH, and QF-PCR have been performed on a 19-year-old female patient with trisomy 18 to investigate a large number of cells including non-mitotic cells from various different tissues. This study supports evidence for an apparently pure form of trisomy 18 in this "long-living" patient with Edwards syndrome.     

Mild phenotype due to inverse duplication 4p16.3 - P15.3 including the Wolf-Hirschhorn critical region

Duplication of distal 4p results in a recognizable clinical phenotype. We report here on a 3 year old girl with a de novo inverse duplication of the chromosome segment 4p16.3-p15.3. The symptoms in this patient are milder than those of previously described patients with 4p duplication syndrome and include a deep hairline, deep-set eyes, short pug nose, full cheeks, simian crease, clinodactily of the fifth digit, no speech development and a moderate psychomotor retardation. Fluorescence in situ hybridization (FISH) using a chromosome 4 painting probe confirmed that the extra material is of chromosome 4 origin. Further analysis with the Wolf-Hirschhorn critical region probe demonstrated the duplication of this region. The lysosomal hydrolase alpha-L-iduronidase (IDUA) gene which is mutated in mucopolysaccaridosis type I (MPS I) and mapped to 4p16.3 might be responsible for some of the MPS like facial features. A phenotype-genotype correlation analysis in combination with literature review was undertaken to allow a further delineation of partial trisomy 4p syndromes.     

Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks

The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications.     

The effect of pentadecapeptide BPC 157, H2-blockers, omeprazole and sucralfate on new vessels and new granulation tissue formation

A clear protection of the gastrointestinal tract and an evident anti-inflammatory effect were shown for a novel stomach pentadecapeptide BPC 157 (i.p./i.g.) in comparison with several reference standards in various ulcer models along with a protection of endothelium and particular interaction with the NO-system. Thus, we evaluated whether this pentadecapeptide along with other gastroprotective agents could affect angiogenesis and the healing process in vivo using a procedure initially described by Szabo and co-workers. In each rat, two sterile sponges (1 x 1 x 0.25 cm; V = 0.25 mL) with the same quantities of BPC 157 (10 ng x mL(-1), 10 microg x mL(-1), 50 microg x kg(-1)) or reference agents (cimetidine: 10, 100, 500 mg x mL(-1); ranitidine: 2.5, 25, 250 mg x mL(-1); famotidine: 10, 50, 100 mg x mL(-1); omeprazole: 10, 50, 100 mg x mL(-1); sucralfate: 1, 5, 10 mg x mL(-1) were implanted subcutaneously in the lumbar region. The sponges were removed after 3 or 7 d, fixed in formalin, and processed for histologic and histochemical evaluation and morphometry assessment. Compared with the control values, the number of newly formed endothelial spaces inside newly formed granulation tissue was markedly increased in all animals treated with BPC 157, cimetidine, ranitidine, famotidine, sucralfate and omeprazole, a consistent finding noted after either 3 or 7 d. Compared with control values, markedly more granulation tissue was noted in the rats in the groups of animals treated with BPC 157 (50 microg) and in the rats treated with sucralfate in all dosages used, euthanized after 3 d. In all groups treated with H2-blockers however, similar values to those of controls were noted. Thus, it could be concluded that an evident angiogenic property was consistently noted for the novel pentadecapeptide BPC 157, H2-blockers (cimetidine, famotidine and ranitidine) and omeprazole, besides the well known angiogenic effect of sucralfate. Furthermore, unlike H2-blockers and omeprazole, BPC 157 stimulates the formation of granulation tissue, suggesting a particular activity, similar to that previously noted for sucralfate.     

Metabolic engineering of yeast: the perils of auxotrophic hosts

Auxotrophic mutants may have physiological alterations and sensitivities which are not generally recognized. Such features are shown here by observations that final cell densities attained by several leucine-auxotrophic Saccharomyces cerevisiae strains depend differently on the initial leucine concentration in the medium. Furthermore, complementing such auxotrophic strains with the plasmid-based LEU2 selection marker resulted in different final cell densities than chromosomal expression of LEU2 in the otherwise isogenic, prototrophic strains. These results warn that auxotrophic host-related physiological influences overlay any metabolic effect of a cloned gene expressed in such a host, clearly complicating interpretation of the effect of that gene's product in scientific or metabolic engineering research.     

Caveolae: biochemical analysis

Caveolae appear in a multitude of processes encompassing growth regulation and trafficking. We demonstrate the abundant presence of ESA/reggie-1/flotillin-2, ATP synthase beta subunit and annexin V in endothelial caveolae by immunopurification of caveolae from vascular endothelial membrane. Five proteins are abundant in a caveolin-1 protein complex, analyzed by sucrose gradient velocity sedimentation following octyl-beta-D-glucopyranoside extraction. Caveolin-1 alpha interacts with caveolin-1beta, caveolin-2, actin, the microsomal form of NADH cytochrome B5 reductase and ESA/reggie-1/flotillin-2 as shown by co-immunoprecipitation. We propose the concept that ATP biosynthesis in caveolae regulates mechanosignaling and is induced by membrane depolarization and a proton gradient. Pressure stimuli and metabolic changes may trigger gene regulation in endothelial cells, involving a nuclear conformer of caveolin-1, shown here with an epitope-specific caveolin-1 antibody, and immediate response of ion channel activity, regulated by ESA/reggie-1/flotillin-2.