Novel hepatic progenitor cell surface markers in the adult rat liver

Hepatic progenitor/oval cells appear in injured livers when hepatocyte proliferation is impaired. These cells can differentiate into hepatocytes and cholangiocytes and could be useful for cell and gene therapy applications. In this work, we studied progenitor/oval cell surface markers in the liver of rats subjected to 2-acetylaminofluorene treatment followed by partial hepatectomy (2-AAF/PH) by using rat genome 230 2.0 Array chips and subsequent RT-PCR, immunofluorescent (IF), immunohistochemical (IHC) and in situ hybridization (ISH) analyses. We also studied expression of the identified novel cell surface markers in fetal rat liver progenitor cells and FAO-1 hepatoma cells. Novel cell surface markers in adult progenitor cells included tight junction proteins, integrins, cadherins, cell adhesion molecules, receptors, membrane channels and other transmembrane proteins. From the panel of 21 cell surface markers, 9 were overexpressed in fetal progenitor cells, 6 in FAO-1 cells and 6 are unique for the adult progenitors (CD133, claudin-7, cadherin 22, mucin-1, ros-1, Gabrp). The specificity of progenitor/oval cell surface markers was confirmed by ISH and double IF analyses. Moreover, study of progenitor cells purified with Ep-CAM antibodies from D-galactosamine injured rat liver, a noncarcinogenic model of progenitor cell activation, verified that progenitor cells expressed these markers. CONCLUSION: We identified novel cell surface markers specific for hepatic progenitor/oval cells, which offers powerful tool for their identification, isolation and studies of their physiology and pathophysiology. Our studies also reveal the mesenchymal/epithelial phenotype of these cells and the existence of species diversity in the hepatic progenitor cell identity.     

In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force

Phagosomes employ lytic enzymes, cationic peptides, and reactive oxygen intermediates to eliminate invading microorganisms. The effectiveness of these microbicidal mechanisms is potentiated by the acidic pH created by H(+)-pumping vacuolar-type ATPases (V-ATPases) on the phagosomal membrane. The degree of phagosomal acidification varies greatly among neutrophils, macrophages, and dendritic cells and can be affected by diseases like cystic fibrosis. The determinants of phagosomal pH are not completely understood, but the permeability to ions that neutralize the electrogenic effect of the V-ATPase has been proposed to play a central role. When counterion conductance is limiting, generation of a large membrane potential will dominate the proton-motive force (pmf), with a proportionally diminished pH gradient. Validation of this notion requires direct measurement of the electrical potential that develops across the phagosomal membrane (Psi(Phi)). We describe a noninvasive procedure to estimate Psi(Phi) in intact cells, based on fluorescence resonance energy transfer. This approach, in combination with measurements of phagosomal pH, enabled us to calculate the pmf across phagosomes of murine macrophages and to analyze the factors that limit acidification. At steady state, Psi(Phi) averaged 27 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A. The comparatively small contribution of the potential to the pmf suggests that proton pumping is not limited by the counterion permeability, a notion that was validated independently by using ionophores. Instead, phagosomal pH stabilizes when the rate of proton pumping, which decreases gradually as the lumen acidifies, is matched by the passive leak of proton equivalents.     

Ancient and continuing Darwinian selection on insulin-like growth factor II in placental fishes

Despite abundant examples of both adaptation at the level of phenotype and Darwinian selection at the level of genes, correlations between these two processes are notoriously difficult to identify. Positive Darwinian selection on genes is most easily discerned in cases of genetic conflict, when antagonistic evolutionary processes such as a Red Queen race drive the rate of nonsynonymous substitution above the neutral mutation rate. Genomic imprinting in mammals is thought to be the product of antagonistic evolution coincident with evolution of the placenta, but imprinted loci lack evidence of positive selection likely because of the ancient origin of viviparity in mammals. To determine whether genetic conflict is a general feature of adaptation to placental reproduction, we performed comparative evolutionary analyses of the insulin-like growth factor II (IGF2) gene in teleost fishes. Our analysis included several members of the order Cyprinodontiformes, in which livebearing and placentation have evolved several times independently. We found that IGF2 is subject to positive Darwinian selection coincident with the evolution of placentation in fishes, with particularly strong selection among lineages that have evolved placentation recently. Positive selection is also detected along ancient lineages of placental livebearing fishes, suggesting that selection on IGF2 function is ongoing in placental species. Our observations provide a rare example of natural selection acting in synchrony at the phenotypic and molecular level. These results also constitute the first direct evidence of parent-offspring conflict driving gene evolution.     

Gastric heterotopia together with squamous metaplasia in the gallbladder

Heterotopic gastric mucosa in the gallbladder is extremely unusual. In this study, we aimed to report a case of gastric heterotopia together with squamous metaplasia in the gallbladder of a 47-year-old female patient who experienced an intensive abdominal pain. He was admitted to the hospital for clinical treatment without any improvement. Ultrasonography showed a stone located in the gallbladder neck and dilatation of intrahepatic bile ducts, both hepatic ducts and common hepatic duct. Laparoscopic cholecystectomy was performed. In the microscopical examination, the epithelium of the gallbladder revealed an unspecified chronic cholecystitis. Besides, at the level of the gallbladder body, a heterotopic gastric mucosa contain chief, parietal and mucosal cells with cystic glands and squamous metaplasia was found. Actually the patient is in long-time follow-up, asymptomatic. We also review 96 other reports of HGM in the gallbladder in the international medical literature from 1934. As heterotopic tissue may promote carcinogenesis of the gallbladder, close attention should be paid to any occurrence of such lesions in this anatomical region. It appears that laparoscopic cholecystectomy may be unavoidable for patients affected by heterotopic gastric mucosa at the present time and care must be taken when a diagnosis is made based on intraoperative frozen sections.     

Reversible Kallmann syndrome: report of the first case with a KAL1 mutation and literature review

Kallmann syndrome (KS) describes the association of isolated hypogonadotropic hypogonadism with hypo/anosmia. A few KS patients may reverse hypogonadism after testosterone withdrawal, a variant known as reversible KS. Herein, we describe the first mutation in KAL1 in a patient with reversible KS and review the literature. The proband was first seen at 22 years complaining of anosmia and lack of puberty. His brother had puberty at 30 years and a maternal granduncle had anosmia and delayed puberty. On physical examination, he was P(2)G(1), testes were 3 ml and bone age was 14 years. During 20 years of irregular testosterone replacement, he developed secondary sexual characteristics and testicular enlargement. At the age of 41 years, after stopping testosterone replacement for 5 months, his testes were 15 ml, serum testosterone, LH, and FSH responses to GnRH were normal, and his wife was pregnant. The molecular study revealed a cytosine insertion in exon 2 of KAL1, generating a frameshift at codon 75 and a premature stop at codon 85. The expected gene product is a truncated peptide with 85 of the 680 [corrected] amino acids present in the wild-type protein. Fourteen cases of reversible KS have been described but the genotype was only studied in a single case showing a heterozygous fibroblast growth factor receptor type 1 (FGFR1) mutation. Considering the low prevalence of mutations in KAL1 or FGFR1 in KS, it is possible that these genotypes are more prevalent in reversible KS than in other KS patients, but additional studies are necessary to confirm this hypothesis.     

Teaching the Medical Interview: Methods and Key Learning Issues in a Faculty Development Course

OBJECTIVE To describe the American Academy on Communication in Healthcare’s (AACH) Faculty Development Course on Teaching the Medical Interview and report a single year’s outcomes. DESIGN We delivered a Faculty Development course on Teaching the Medical Interview whose theme was relationship-centered care to a national and international audience in 1999. Participants completed a retrospective pre-post assessment of their perceived confidence in performing interview, clinical, teaching, and self-awareness skills. PARTICIPANTS AND SETTING A total of 79 participants in the 17th annual AACH national faculty development course at the University of Massachusetts Medical School in June 1999. INTERVENTION A 5-day course utilized the principles of learner-centered learning to teach a national and international cohort of medical school faculty about teaching the medical interview. MEASUREMENTS AND MAIN RESULTS The course fostered individualized, self-directed learning for participants, under the guidance of AACH faculty. Teaching methods included a plenary session, small groups, workshops, and project groups all designed to aid in the achievement of individual learning goals. Course outcomes of retrospective self-assessed confidence in interview, clinical, teaching, self-awareness, and control variables were measured using a 7-point Likert scale. Participants reported improved confidence in interview, clinical, teaching, and self-awareness variables. After controlling for desirability bias as measured by control variables, only teaching and self-awareness mean change scores were statistically significant (p < .001). CONCLUSIONS The AACH Faculty Development course on Teaching the Medical Interview utilized learner-centered teaching methods important to insure learning with experienced course participants. Perceived teaching and self-awareness skills changed the most when compared to other skills. This study was presented in part at the 23rd annual meeting of the Society of General Internal Medicine, Boston, MA, May, 2000.     

Potentially Reduced Exposure Cigarettes Accelerate Atherosclerosis: Evidence for the Role of Nicotine

The tobacco industry markets potentially reduced exposure products (PREPs) as less harmful or addictive alternatives to conventional cigarettes. This study compared the effects of mainstream smoke from Quest, Eclipse, and 2R4F reference cigarettes on the development of atherosclerosis in apolipoprotein E-deficient (apoE −/−) mice. Mice were exposed to smoke from four cigarette types for 12 weeks beginning at age of 12 weeks, and in a separate study for 8 weeks, beginning at age of 8 weeks. In both studies, mice exposed to smoke from high-nicotine, high-tar Quest 1, and 2R4F cigarettes developed greater areas of lipid-rich aortic lesions than did non-smoking controls. Exposure to smoke from the lower-nicotine products, Eclipse, and Quest 3, was associated with smaller lesion areas, but animals exposed to smoke from all of the tested types of cigarette had larger lesions than did control animals not exposed to smoke. Urinary levels of isoprostane F2 alpha VI, increased proportionally to cigarette nicotine yield, whereas induction of pulmonary cytochrome P4501A1 was proportional to tar yield. Lesion area was associated with both nicotine and tar yields, although in multiple regression analysis only nicotine was a significant predictor of lesion area. Smoke exposure did not alter systolic blood pressure (SBP), heart rate (HR), blood cholesterol, or leukocyte count. Taken together, these observations suggest that smoking may accelerate atherosclerosis by increasing oxidative stress mediated at least in part via the actions of nicotine.     

Tyrosine 687 phosphorylated Alzheimer's amyloid precursor protein is retained intracellularly and exhibits a decreased turnover rate

Tyrosine 687 (Y687) of the Alzheimer's amyloid precursor protein (APP) was shown to be phosphorylated in the brains of Alzheimer's disease patients. This residue lies within a typical endocytosis consensus motif commonly found in molecules with receptor functions, strongly suggesting a potential role for APP in signal transduction. Consequently, the work here described addressed how phosphorylation of Y687 may be affecting APP in terms of its proteolytic cleavage and subcellular distribution. Our data show that the APP mutant mimicking constitutive dephosphorylation of Y687 had a faster turnover rate, both in terms of maturation and metabolism, when compared to Wt-APP-GFP and even more so when compared to the mutant mimicking constitutive phosphorylation. Thus, the mutant mimicking constitutively phosphorylated Y687 had a much higher t(1/2) and was significantly retained both in the ER and TGN. Additionally, this mutant was not incorporated into visible vesicular structures, with a concomitant dramatic decrease in Abeta production. Our findings point to the direct phosphorylation of APP on Y687 as an important regulatory mechanism in terms of determining the subcellular localization of APP and modulating its processing via different proteolytic pathways.