Synthesis of new symmetrical bis-steroidal pyrazine analogues from diosgenin.

New symmetrical bis-steroidal pyrazine dimers that are cephalostatins/ritterazines analogues have been prepared easily from a cheap, readily available natural steroid (diosgenin). These dimers were obtained by classical, condensation of α-amino ketones in order to construct the pyrazine rings. The three dimers differ in the functionalized diosgenin: (25R)-5α,6ß-dihydroxy-5α-spirosta-3-one, (25R)-4,5α-epoxy-5ß-spirosta-3,6-dione and (25R)-5α-hydroxy-5α-spirosta-3,6-dione respectively.

Acute cellular transplant rejection following laparoscopic adjustable gastric banding in a morbidly obese patient post heart transplantation.

BACKGROUND: Obesity is a worldwide health problem that is often worsened after organ transplantation. As obesity is associated with increased incidence of metabolic syndrome, cardiovascular events and death, it is essential to control weight and avoid weight gain in patients especially following cardiac transplantation. Of the various strategies that are available for weight reduction, bariatric surgery seems to be the most effective in achieving weight loss and in maintaining the reduced body weight. However, this has not been frequently performed in organ-transplant recipients. CASE REPORT: We are reporting a unique case of a bariatric surgery procedure performed in a patient after cardiac transplantation. A 30-year-old African-American man with a history of end-stage heart failure due to idiopathic dilated cardiomyopathy underwent orthotopic cardiac transplantation. Three years after transplantation, the patient underwent laparoscopic adjustable gastric banding surgery for morbid obesity. Two months later, the patient presented with severe heart failure and was diagnosed with acute cellular rejection as evidenced by endomyocardial biopsy results despite continued combined immunosuppressive therapy that had not been changed but with significantly reduced blood levels of calcineurin inhibitors. CONCLUSION: We hypothesize that the altered gastro-intestinal motility and delayed gastric emptying due to laparoscopic adjustable gastric banding may have caused incomplete absorption of the administered immunosuppressant drugs in this particular case, as evidenced by the low tacrolimus level, resulting in acute cellular rejection of the transplanted heart, which has never been described before.

Biochemical basis of Fabry disease with emphasis on mitochondrial function and protein trafficking.

Fabry disease, also known as Anderson-Fabry disease, is an X-linked lysosomal storage disorder. The clinical picture is highly variable and usually milder in females. It is a multisystemic disease involving many organs. Fabry disease is due to a deficiency of alpha-galactosidase A caused by different usually "private" mutations. Enzyme replacement therapy (ERT) has been established, other therapeutic options are at an experimental stage. Classically, mechanical deposition of storage material in blood vessels was believed to lead to decreased blood supply with consecutive organ dysfunction. Recently, however, many secondary biochemical processes have been discussed to be involved in the pathogenesis of Fabry disease. For example, compromised energy metabolism has been found both in vitro and in vivo, altered lipid composition of membranes can lead to abnormalities in trafficking and sorting of rafts-associated proteins. We discuss the role of these secondary phenomena in the pathogenesis of Fabry disease.

Molecular detection of Giardia contamination in water bodies in a zoo.

We used a combined microscopy-molecular approach to determine the occurrence and identities of waterborne Giardia sp. cysts isolated from 18 separate, 10l grab samples collected from a Malaysian zoo. Microscopy revealed that 17 of 18 samples were Giardia cyst positive with concentrations ranging from 1 to 120 cysts/l. Nine (52.9%) of the 17 cyst positive samples produced amplicons of which 7 (77.8%) could be sequenced. Giardia duodenalis assemblage A (6 of 7) and assemblage B (1 of 7), both infectious to humans, were identified at all sampling sites at the zoo. The presence of human infectious cysts raises public health issues, and their occurrence, abundance and sources should be investigated further. In this zoo setting, our data highlight the importance of incorporating environmental sampling (monitoring) in addition to routine faecal examinations to determine veterinary and public health risks, and water monitoring should be considered for inclusion as a separate element in hazard analysis, as it often has a historical (accumulative) connotation.

Reverse genetics of measles virus and resulting multivalent recombinant vaccines: applications of recombinant measles viruses.

An overview is given on the development of technologies to allow reverse genetics of RNA viruses, i.e., the rescue of viruses from cDNA, with emphasis on nonsegmented negative-strand RNA viruses (Mononegavirales), as exemplified for measles virus (MV). Primarily, these technologies allowed site-directed mutagenesis, enabling important insights into a variety of aspects of the biology of these viruses. Concomitantly, foreign coding sequences were inserted to (a) allow localization of virus replication in vivo through marker gene expression, (b) develop candidate multivalent vaccines against measles and other pathogens, and (c) create candidate oncolytic viruses. The vector use of these viruses was experimentally encouraged by the pronounced genetic stability of the recombinants unexpected for RNA viruses, and by the high load of insertable genetic material, in excess of 6 kb. The known assets, such as the small genome size of the vector in comparison to DNA viruses proposed as vectors, the extensive clinical experience of attenuated MV as vaccine with a proven record of high safety and efficacy, and the low production cost per vaccination dose are thus favorably complemented.

Demonstration of lipids in plastic resin-embedded sections of skin material.

Two different fluorescence stains, green: 5-hexadecanoylaminofluorescein, and red: BODIPY(R) 665/676 [(E,E)-3, 5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene, produced good results regarding the demonstration of glycolipids, free fatty acids and triglycerides in mammalian skin material that had been embedded in a water miscible plastic resin (Technovit(R) 7100). In this way, functional aspects of specific structures (epidermal barrier region, sebaceous glands) could be characterized histochemically in the integument of five mammalian species with sparse or dense hair coats.

Intestinal parasites in various animals at a zoo in Malaysia.

A survey was undertaken to investigate the prevalence of intestinal parasites from different groups of mammals housed in a zoological garden in Malaysia. A total of 197 faecal samples were collected randomly from various primates (99), hoofed mammals (70) and feline (28). It was discovered that 89.3% of feline, 54.5% of primates and 45.7% of hoofed mammals were infected with intestinal parasites. Intestinal parasites found in primates were Balantidium coli (19.2%), Cryptosporidium spp. (14.1%), hookworm (10.1%), Trichuris spp. (5.1%), Ascaris (4.0%) and Blastocystis spp. (2.0%). For hoofed mammals, hookworm had the highest prevalence (34.3%) followed by Trichuris spp. and Cryptosporidium spp. (5.7%). Meanwhile, for feline, Toxocara cati was the most prevalent (64.3%), followed by Cryptosporidium spp. (14.3%), Spirometra spp. (7.1%), and hookworm (3.6%). Animals that were infected were all asymptomatic with low parasite load. Routine monitoring of the presence of parasites in animals kept in the zoo is imperative in assisting zoo management in the formulation and implementation of preventive and control measures against the spread of infectious parasitic diseases among animals within the zoo or to humans.

Synthesis of bis-diosgenin pyrazine dimers: new cephalostatin analogs.

A convenient synthesis for bis-diosgenin pyrazine dimers, cephalostatin analogues is reported. These symmetrical dimeric steroid-pyrazines were obtained by the classical condensation of alpha-amino ketones, the most efficient method for pyrazine ring construction.

Insights into post-translational processing of beta-galactosidase in an animal model resembling late infantile human G-gangliosidosis.

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail. For a better understanding of the molecular pathogenesis underlying G(M1)-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of beta-galactosidase. In the canine model the genetic defect results in exclusion or inclusion of exon 15 in the mRNA transcripts and to translation of two mutant precursor proteins. Intracellular localization, processing and enzymatic activity of these mutant proteins were investigated. The obtained results suggested that the beta-galactosidase C-terminus encoded by exons 15 and 16 is necessary for correct C-terminal proteolytic processing and enzyme activity but does not affect the correct routing to the lysosomes. Both mutant protein precursors are enzymatically inactive, but are transported to the lysosomes clearly indicating that the amino acid sequences encoded by exons 15 and 16 are necessary for correct folding and association with protective protein/cathepsin A, whereas the routing to the lysosomes is not influenced. Thus, the investigated canine model is an appropriate animal model for the human late infantile form and represents a versatile system to test gene therapeutic approaches for human and canine G(M1)-gangliosidosis.

Confocal laser scanning analysis of an equine oral mast cell tumor with atypical expression of tyrosine kinase receptor C-KIT.

A 20-year-old female horse showed a nodular, firm, focal ulcerated mast cell tumor at the right dorsobuccal face of the tongue. Histologically, the nonencapsulated tumor consisted of dense, infiltrating aggregates of well-differentiated, Cresyl violet-positive mast cells accompanied by numerous eosinophils. Furthermore, they exhibited a strong, diffuse, intracytoplasmatic immunohistochemical signal for tryptase and a faint membrane-associated and perinuclear signal for tyrosine kinase receptor KIT. Confocal laser scanning microscopy confirmed an aberrant spatial colocalization of KIT in the Golgi apparatus, which may be the result of a defective protein processing within the tumor cells. The tumor was not associated with a poor prognosis.

Pathophysiology in Microvillus inclusion disease.

Microvillus inclusion disease (MID) is a congenital disorder with the clinical signs of watery diarrhea often beginning in the first days of life. The main pathological features of the disease include a villus atrophy and an accumulation of periodic acid-Schiff (PAS)-positive material within the apical cytoplasm of enterocytes on the light microscopy level. Electron microscopic criteria are pathognomonic consisting of an increased amount of secretory granules preferentially in crypt epithelial cells and of the presence of microvillus inclusion bodies (MIBs) which are most frequently found in villus enterocytes. Until now the basic molecular defects have not been disclosed completely. In this review we discuss the actual pathogenetic hypothesis and the therapeutic options besides small bowel transplantation.

[Primary tracheobronchial amyloidosis]. / Pierwotna amyloidoza tchawiczo-oskrzelowa.

The primary tracheo-bronchial amyloidosis is a rare entity with long lasting and progressive course. Precise diagnosis can be established on the basis of pathological features seen in samples derived from the airways, obtained during fibreoptic bronchoscopy or during lung biopsy sometimes. Describing the case of an insidious course of primary tracheobronchial AL type amyloidoisis, that was initially recognised and treated as a chronic obstructive pulmonary disease. The authors represent its clinical course and the diagnostic difficulties. The changes in the respiratory tract, both radiological and endoscopic suggested a tuberculous or proliferative process. They were responsible for a severe increasing dyspnoea, due to bronchial obstruction, with muco-haemoptic expectoration. A forceps resection of the endobronchial lesions enabled to established the right diagnosis and further treatment limited to local procedures.

Early lap-band erosion associated with colonic inflammation: a case report and literature review.

INTRODUCTION: Laparoscopic adjustable gastric banding is an effective and safe surgical modality for the treatment of morbid obesity. Erosion of the band into the stomach has been reported. No reports are available on erosion of the Lap-Band following diverticulitis of the colon. CASE REPORT: A 31-year-old female with a body mass index (BMI) of 52 underwent an uneventful laparoscopic Lap-Band placement. Postoperative contrast study revealed good positioning of the band and no evidence of leakage. The patient's recovery was uneventful except for an elevated temperature of 101.5 degrees F that was attributed to her atelectasis. She had lost 52 lbs. and remained asymptomatic for 3 months. Following this period of successful weight loss, she presented with complaints of abdominal pain for 3 days associated with diarrhea of 7 days' duration. A Gastrografin contrast study showed no evidence of a leak or band slippage but erosion was suspected. Upper endoscopy confirmed erosion of the band into the stomach. Computed tomography (CT) of the abdomen revealed thickening of the sigmoid and descending colon with mesenteric fat stranding consistent with diverticulitis. Laparoscopic removal of the Lap-Band system was performed. CONCLUSION: We postulate that colonic diverticulitis could have been a precipitating factor in the development of band erosion. Intraabdominal sepsis resulting in subacute infection of the Lab-Band system may be the underlying factor.

Cyclization of free radicals at the C-7 position of ethyl indole-2-carboxylate derivatives: an entry to a new class of Duocarmycin Analogues.

Aryl free-radicals generated at the C-7 position of ethyl indole-2-carboxylates bearing N-allyl and propargylic groups triggered intramolecular cyclizations to furnish a new class of Duocarmycin analogues, formal ethyl pyrrolo[3,2,1-ij]quinoline-2- carboxylate derivatives, through the less favorable 6-endo-trig cyclization mode.

Emergent laparoscopic reduction of acute gastric volvulus with anterior gastropexy.

Gastric volvulus is characterized by abnormal rotation of the stomach around an axis made by two fixed portions. Symptoms of gastric volvulus range from anemia and weight loss to severe epigastric or chest pain associated with nonproductive vomiting or upper gastrointestinal bleeding. Ischemia, necrosis, and perforation will occur if this condition remains untreated. We report a case of a 92-year-old patient with acute gastric volvulus treated with laparoscopic reduction and anterior gastropexy. We suggest that the laparoscopic approach to gastric volvulus is safe and feasible and should be considered. High-risk and elderly patients can particularly benefit from minimally invasive access. Anterior gastropexy palliates the symptoms and can be considered a definitive treatment in this patient population.

Autophagocytosis of the apical membrane in microvillus inclusion disease.

BACKGROUND: Microvillus inclusion disease (MID) is a disorder with the clinical signs of intractable diarrhoea in the newborn and infancy. The typical pathological features of the disease are well known whereas the pathophysiology is still unclear. AIM: This study was performed to define possible alterations of the cytoskeleton and exocytic as well as endocytic pathways within enterocytes in MID. PATIENTS: Four patients with MID were studied. Three had a congenital onset of diarrhoea and one patient had a late onset form. METHODS: Thin frozen sections of small bowel biopsies of patients were labelled by antibodies against the cytoskeleton and the brush border enzyme sucrase-isomaltase. The binding sites of the primary antibodies were visualised by immunogold particles in the electron microscope. Biopsies were labelled in organ culture to analyse the biosynthetic and endocytic pathways within the enterocytes. RESULTS: Labelling with antibodies against actin and villin did not differ significantly in control and patient biopsies. Biosynthetic labelling revealed normal intracellular processing and transport of the brush border enzyme sucrase-isomaltase. Secretory granules in crypt epithelial cells were positive for sucrase-isomaltase, differing in its labelling density between patients. Patient biopsies showed microvillus inclusion bodies which endocytosed cationised ferritin within five minutes after uptake as well as ovalbumin after incubation for 10 minutes. These microvillus inclusion bodies correspond to early endosomes because they lack lysosome associated membrane proteins. Late endosomes and lysosomes containing sucrase-isomaltase did not reveal microvillus-like structures. CONCLUSION: Microvillus inclusion bodies in MID originate from autophagocytosis of the apical membrane of enterocytes with engulfing of microvilli.

Characteristics and structural requirements of apical sorting of the rat growth hormone through the O-glycosylated stalk region of intestinal sucrase-isomaltase.

The apical sorting of the small intestinal membrane glycoprotein sucrase-isomaltase (SI) depends on the presence of O-linked glycans and the transmembrane domain. Here, we investigate the role of O-glycans carried by the Ser/Thr-rich stalk region of SI as an apical sorting signal and evaluate the spatial requirements for an efficient recognition of this signal. Several hybrid proteins are generated comprising the unsorted and unglycosylated protein, the rat growth hormone (rGH), fused to either the transmembrane domain of SI (GH-SI(TM)), or the transmembrane and the stalk domains (GH-SI(SR/TM)). Both constructs are randomly distributed over the apical and basolateral membranes of MDCK cells indicating that neither the transmembrane domain nor the O-glycans are sufficient per se for an apical delivery. Only when a polyglycine spacer is inserted between the stalk region of SI and the luminal part of rGH in the GH-SI(Gly/SR/TM) fusion protein does efficient apical sorting of an O-glycosylated protein as well as a time-dependent association with detergent-insoluble lipid microdomains occur. Obviously, the polyglycine spacer facilitates the accessibility of the O-glycans in GH-SI(Gly/SR/TM) to a putative sorting receptor, whereas these glycans are inadequately recognized in GH-SI(SR/TM). We conclude that the O-glycans in the stalk region of SI act as an apical sorting signal within a sorting machinery that comprises at least a carbohydrate-binding protein and fulfills specific spatial requirements provided, for example by a polyglycine spacer in the context of rGH or the P-domain within the SI enzyme complex.

Apical membrane proteins are transported in distinct vesicular carriers.

The function of polarized epithelial cells and neurons is achieved through intracellular sorting mechanisms that recognize classes of proteins in the trans-Golgi network (TGN) and deliver them into separate vesicles for transport to the correct surface domain. Some proteins are delivered to the apical membrane after their association with membrane detergent-insoluble glycophosphatidylinositol/cholesterol (DIG) membrane microdomains [1], while some do not associate with DIGs [2-4]. However, it is not clear if this represents transport by two different pathways or if it can be explained by differences in the affinity of individual proteins for DIGs. Here, we investigate the different trafficking mechanisms of two apically sorted proteins, the DIG-associated sucrase-isomaltase (SI) and lactase-phlorizin hydrolase, which uses a DIG-independent pathway [5]. These proteins were tagged with YFP or CFP, and their trafficking in live cells was visualized using confocal laser microscopy. We demonstrate that each protein is localized to distinct subdomains in the same transport vesicle. A striking triangular pattern of concentration of the DIG-associated SI in subvesicular domains was observed. The original vesicles partition into smaller carriers containing either sucrase-isomaltase or lactase-phlorizin hydrolase, but not both, demonstrating for the first time a post-TGN segregation step and transport of apical proteins in different vesicular carriers.

Molecular basis of aberrant apical protein transport in an intestinal enzyme disorder.

The impaired sorting profile to the apical membrane of human intestinal sucrase-isomaltase is the underlying cause in the pathogenesis of a novel phenotype of intestinal congenital sucrase-isomaltase deficiency. Molecular characterization of this novel phenotype reveals a point mutation in the coding region of the sucrase-isomaltase (SI) gene that results in an amino acid substitution of a glutamine by arginine at residue 117 of the isomaltase subunit. This substitution is located in a domain revealing features of a trefoil motif or a P-domain in immediate vicinity of the heavily O-glycosylated stalk domain. Expression of the mutant SI phenotype in epithelial Madin-Darby canine kidney cells reveals a randomly targeted SI protein to the apical and basolateral membranes confirming an exclusive role of the Q117R mutation in generating this phenotype. Unlike wild type SI, the mutant protein is completely extractable with Triton X-100 despite the presence of O-glycans that serve in the wild type protein as an apical sorting signal and are required for the association of SI with detergent-insoluble lipid microdomains. Obviously the O-glycans are not adequately recognized in the context of the mutant SI, most likely due to altered folding of the P-domain that ultimately affects the access of the O-glycans to a putative sorting element.

Gene transfer into neurons from hippocampal slices: comparison of recombinant Semliki Forest Virus, adenovirus, adeno-associated virus, lentivirus, and measles virus.

Viral vectors are useful for transferring genes into neurons. Here, we characterized recombinant Semliki Forest virus (SFV), adenovirus type 5 (Ad5), adeno-associated virus type 2 (AAV), lentivirus, and measles virus (MV) by their expression of green fluorescent protein (GFP) in rat hippocampal slice cultures. SFV infected more neurons (>90% of all GFP-positive cells) than AAV, lentivirus, and MV (71, 69, and 62%, respectively), whereas no infected neurons were identified with Ad5. AAV-mediated GFP expression was neuron-specific when the platelet-derived growth factor beta-chain promoter rather than cytomegalovirus promoter was used. Transgene expression occurred rapidly but transiently for SFV, increased slowly but remained stable with AAV and lentivirus, and was fast with MV. Resting membrane potential and conductance, action potentials, firing accommodation, and H-current appeared normal in infected CA1 pyramidal cells. Thus, SFV is useful for short-term and AAV and lentivirus for long-term transduction of hippocampal slices, while MV constitutes a novel vector.