Methanol Fixation Research Articles

Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa.

This study evaluates the use of two fluorescein-labelled (FITC) plant lectins, Pisum sativum (edible pea) agglutinin (PSA) and Arachis hypogaea (peanut) agglutinin (PNA), in order to determine the most accurate and reliable method to experimentally detect and assess the acrosome reaction in mouse spermatozoa. PNA-FITC labelling was restricted to the acrosome and was not influenced by the fixation procedure; either absolute methanol or paraformaldehyde. In contrast, PSA-FITC not only labelled the acrosome, but also the whole head and the flagellum. This aspect was especially marked after methanol fixation. The cytoplasmic droplet, when present, was also stained by PSA-FITC. Incubation with the calcium ionophore ionomycin induced a concentration and time-dependent increase in the number of acrosome reactions. Compared to spotted preparations, smear samples exhibited a high proportion of spermatozoa with damaged acrosome. In conclusion, PNA-FITC labelling was more accurate than PSA-FITC labelling to detect the acrosome of mouse spermatozoa. The fixation method (methanol vs. paraformaldehyde) had no influence on the staining pattern of PNA-FITC labelling, but spotted preparations are recommended to avoid mechanical damage to the acrosome. Ionophore challenge confirmed the existence of a calcium-dependent acrosome reaction in mouse spermatozoa and validated the use of PNA-FITC to quantify this physiological process. The present study illustrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome reaction.

Immunohistochemical determination of the appropriate anti-hTERT antibodies for in situ detection of telomerase activity in frozen sections of head and neck squamous cell carcinomas and tumor margin tissues

In previous studies we demonstrated telomerase activity in frozen tissue from head and neck squamous cell carcinoma (HNSCC) and their tumor-free tumor margins. In the present study frozen sections from the same tissues were examined for in situ presence of hTERT. In preliminary investigations we established that the most suitable method of tissue preparation was fixation in acetone and methanol followed by steaming and visualization by APAAP. Most of the assays involved eleven anti-hTERT antibodies and were supplemented with the inclusion of antibodies Ki-67, anti-nucleolin and CD45. hTERT expression was investigated in the tissues of 61 patients with HNSCC and 37 patients without tumor. Semi-quantitative immunoreactive scores were correlated with telomerase activity. We examined the prognostic significance of hTERT expression with Kaplan-Meier curves and tested the immunological specificity of the antibodies by immunoabsorption with two hTERT peptides and a nucleolin peptide. Nuclear staining of satisfactory distribution and intensity was achieved in seven anti-hTERT antibodies both in the carcinomas and in the squamous epithelia of the tumor resection margins and in the control tissues. Proof of hTERT did not differ from telomerase activity. The telomerase activity demonstrated in tumor-free resection margins and in control tissues did, however, correlate with lymphocytic-monocytic infiltration (CD45 expression). This telomerase activity might be related to nuclear hTERT expression in the squamous epithelium, given that the hTERT score values in the connective tissue tended to be negative. The prognostic significance of hTERT expression demonstrated on paraffin sections from different tumor localizations was not confirmed for the frozen sections of patients with HNSCC. The hTERT specificity of the monoclonal NCL-L-hTERT, whose use as an antibody against hTERT has been questioned, was re-examined with immunohistochemical methods, but the intensity of its immunoabsorption with the nucleolin peptide did not exceed that observed in the other anti-hTERT antibodies.

DNA elution from buccal cells stored on Whatman FTA Classic Cards using a modified methanol fixation method

We describe here a method for DNA elution from buccal cells and whole blood both collected onto Whatman FTA technology, using methanol fixation followed by an elution PCR program. Extracted DNA is comparable in quality to published Whatman FTA protocols, as judged by PCR-based genotyping. Elution of DNA from the dried sample is a known rate-limiting step in the published Whatman FTA protocol; this method enables the use of each 3-mm punch of sample for several PCR reactions instead of the standard, one PCR reaction per sample punch. This optimized protocol therefore extends the usefulness and cost effectiveness of each buccal swab sample collected, when used for nucleic acid PCR and genotyping.

High-Density Gene Expression Analysis of Tumor-Associated Macrophages from Mouse Mammary Tumors

Clinical and experimental evidence indicates that tumor-associated macrophages (TAMs) promote malignant progression. In breast cancer, TAMs enhance tumor angiogenesis, tumor cell invasion, matrix remodeling, and immune suppression against the tumor. In this study, we examined late-stage mammary tumors from a transgenic mouse model of breast cancer. We used flow cytometry under conditions that minimized gene expression changes to isolate a rigorously defined TAM population previously shown to be associated with invasive carcinoma cells. The gene expression signature of this population was compared with a similar population derived from spleens of non-tumor-bearing mice using high-density oligonucleotide arrays. Using stringent selection criteria, transcript abundance of 460 genes was shown to be differentially regulated between the two populations. Bioinformatic analyses of known functions of these genes indicated that formerly ascribed TAM functions, including suppression of immune activation and matrix remodeling, as well as multiple mediators of tumor angiogenesis, were elevated in TAMs. Further bioinformatic analyses confirmed that a pure and valid TAM gene expression signature in mouse tumors could be used to assess expression of TAMs in human breast cancer. The data derived from these more physiologically relevant autochthonous tumors compared with previous studies in tumor xenografts suggest tactics by which TAMs may regulate tumor angiogenesis and thus provide a basis for exploring other transcriptional mediators of TAM trophic functions within the tumor microenvironment.

Immunofluorescence imaging of the influenza virus M1 protein is dependent on the fixation method

The distribution of the matrix (M1) protein of influenza virus in infected cells was examined using immunostaining. The fixation method influenced strongly the immunofluorescence pattern of the M1 protein. The M1 protein was distributed uniformly in both the cytoplasm and in nuclei when cells that had been infected with virus were fixed with paraformaldehyde. In cells that had been fixed with methanol, however, nuclear dots of the M1 protein were clearly visible. The dots were evident at 8h post-inoculation. Up to 6h post-inoculation, only a diffuse distribution of the M1 protein was observed. The dots were co-localized with promyelocytic leukemia (PML) protein, a major component of nuclear domain 10 (ND10), also called PML oncogenic domains (PODs) or PML-nuclear bodies (NBs). These results indicate that the nuclear dots of the M1 protein in cells that had been fixed with methanol are not artifacts of the fixation method. Furthermore, methanol fixation is preferred for localization of the influenza M1 protein in nuclei using immunostaining.

An Evaluation of Pfmdr1 Mutations in Chloroquine Resistance: Effect of Host Factor

Background: Pfcrt and Pfmdr1 mutations have been reported to be associated with chloroquine resistance, but these reports have been inconsistent. Red cell factors, sickle cell trait, ABO blood group antigens and glucose-6-phosphate dehydrogenase (G6PD) deficiency are known to confer some degree of resistance against malaria parasite infection but their interplay with CQ resistance is yet unknown. This study therefore aims at evaluating the correlation of CQ resistance with Pfmdr1 mutation and certain host factors in humans. Methods: One hundred and twenty patients (1–15yrs) with acute uncomplicated falciparum malaria were enrolled into the study. They were administered standard doses of CQ and followed up for parasitological and clinical response for 14 days. Thick and thin blood films as well as filter paper blood samples were obtained from patients before enrolment and during follow up. Blood samples were collected in EDTA bottles for assessment of haematological parameters. DNA was extracted from filter paper using the methanol fixation method and used for Pfmdr1 and msp1 analyses by polymerase chain reaction. Results: Seventy patients were followed up to day 14.53% of the patients were cured, with a 47% failure rate to CQ. Although Pfmdr1Y86 was detected in majority of the patients (19/33) with clinical failure, this was not statistically significant (p > 0.05). 88% of the clinical failures was due to recrudescence and this was highly significant with treatment outcome (p = 0.000). No significant association was found between Pfmdr1 alleles and red cell factors. However, individuals with HbAA were found to be 2.3 times more likely to develop resistance to CQ treatment compared to those having HbAS. Conclusion: In conclusion, the prevalence of CQ resistance and Pfmdr1Y86 mutation was high in this malaria endemic setting. There was no clear influence of ABO, G6PD deficiency and Hb genotype on CQ resistance and Pfmdr1 mutation.

The effect of cell fixation on the discrimination of normal and leukemia cells with laser tweezers Raman spectroscopy

Laser tweezers Raman spectroscopy (LTRS) was used to characterize the effect of different chemical fixation procedures on the Raman spectra of normal and leukemia cells. Individual unfixed, paraformaldehyde-fixed, and methanol-fixed normal and transformed lymphocytes from three different cell lines were analyzed with LTRS. When compared to the spectra of unfixed cells, the fixed cell spectra show clear, reproducible changes in the intensity of specific Raman markers commonly assigned to DNA, RNA, protein, and lipid vibrations (e.g. 785, 1230, 1305, 1660 cm(-1)) in mammalian cells, many of which are important markers that have been used to discriminate between normal and cancer lymphocytes. Statistical analyses of the Raman data and classification using principal component analysis and linear discriminant analysis indicate that methanol fixation induces a greater change in the Raman spectra than paraformaldehyde. In addition, we demonstrate that the spectral changes as a result of the fixation process have an adverse effect on the accurate Raman discrimination of the normal and cancer cells. The spectral artifacts created by the use of fixatives indicate that the method of cell preparation is an important parameter to consider when applying Raman spectroscopy to characterize, image, or differentiate between different fixed cell samples to avoid potential misinterpretation of the data.

The human granulocyte nucleus: Unusual nuclear envelope and heterochromatin composition

The human blood granulocyte (neutrophil) is adapted to find and destroy infectious agents. The nucleus of the human neutrophil has a segmented appearance, consisting of a linear or branched array of three or four lobes. Adequate levels of lamin B receptor (LBR) are necessary for differentiation of the lobulated nucleus. The levels of other components of the nuclear envelope may also be important for nuclear shape determination. In the present study, immunostaining and immunoblotting procedures explored the levels of various components of the nuclear envelope and heterochromatin, comparing freshly isolated human neutrophils with granulocytic forms of HL-60 cells, a tissue culture model system. In comparison to granulocytic HL-60 cells, blood neutrophil nuclear envelopes contain low-to-negligible amounts of LBR, lamins A/C, B1 and B2, LAP2 β and emerin. Surprisingly, a “mitotic” chromosome marker, H3(S10)phos, is elevated in neutrophil nuclei, compared to granulocytic HL-60 cells. Furthermore, neutrophil nuclei appear to be more fragile to methanol fixation, than observed with granulocytic HL-60 cells. Thus, the human neutrophil nucleus appears to be highly specialized, possessing a paucity of nuclear envelope-stabilizing proteins. In consequence, the neutrophil nucleus appears to be very malleable, supporting rapid migration through tight tissue spaces.

A novel histological technique for distinguishing between epithelial cells in forensic casework

There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods – Dane's, Csaba's and Ayoub-Shklar – were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange–pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.

Intersectin-1s Regulates the Mitochondrial Apoptotic Pathway in Endothelial Cells

Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X(L) overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.

Liprinα1 Degradation by Calcium/Calmodulin-Dependent Protein Kinase II Regulates LAR Receptor Tyrosine Phosphatase Distribution and Dendrite Development

Neural activity regulates dendrite and synapse development, but the underlying molecular mechanisms are unclear. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an important sensor of synaptic activity, and the scaffold protein liprinalpha1 is involved in pre- and postsynaptic maturation. Here we show that synaptic activity can suppress liprinalpha1 protein level by two pathways: CaMKII-mediated degradation and the ubiquitin-proteasome system. In hippocampal neurons, liprinalpha1 mutants that are immune to CaMKII degradation impair dendrite arborization, reduce spine and synapse number, and inhibit dendritic targeting of receptor tyrosine phosphatase LAR, which is important for dendrite development. Thus, regulated degradation of liprinalpha1 is important for proper LAR receptor distribution, and could provide a mechanism for localized control of dendrite and synapse morphogenesis by activity and CaMKII.

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing

Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB. The optimized optical clearing procedure of ethanol fixation followed by methyl salicylate clearing preserved the fluorescence of constitutive RFP in whole xenograft tumour specimens, about 1 cc in dimension, indicating successful extension from cell plating experiments to whole tissue samples. Finally, the feasibility of imaging the 3D distribution of viable tumour cells (as indicated by the RFP emission) is demonstrated by optical-ECT imaging of cleared xenograft tumours using an in-house system.For more information on this article, see medicalphysicsweb.org

Requirement of Vimentin Filament Assembly for β3-Adrenergic Receptor Activation of ERK MAP Kinase and Lipolysis

Catecholamine stimulation of beta-adrenergic receptors (betaAR) in adipocytes activates the cAMP-dependent protein kinase to promote liberation of fatty acids as a fuel source. The adipocyte beta3AR also activates extracellular signal-regulated kinases (ERK)-1 and -2 through direct recruitment and activation of Src kinase. This pathway together with cAMP-dependent protein kinase contributes to maximal beta3AR-stimulated lipolysis. In a search for other molecules that might associate with beta3AR upon agonist stimulation, we identified vimentin using a proteomics approach. Immunoprecipitation of beta3AR from adipocytes in the absence or presence of the beta3AR agonist CL316,243, followed by Western blotting for vimentin confirmed this specific interaction. Since vimentin has also been identified on lipid droplets, the functional consequences of blocking the expression or structural integrity of vimentin intermediate filaments on beta3AR regulation of ERK activation and lipolysis was assessed. Following disruption of intermediate filaments with beta,beta'-iminodipropionitrile, as confirmed by confocal microscopy, beta3AR-stimulated ERK activation was blocked, and lipolysis was reduced by more than 40%. Independently, depletion of vimentin by small hairpin RNA (shRNA) completely inhibited beta3AR-mediated ERK activation and significantly reduced lipolysis. By contrast, disruption of actin-containing microfilaments by cytochalasin D or microtubules by nocodazole had no effect on either lipolysis or ERK activation. These results indicate that vimentin plays an essential role in the signal transduction pathway from beta3AR to the activation ERK and its contribution to lipolysis.

Conformation State-sensitive Antibodies to G-protein-coupled Receptors

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of micro-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of mu receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize micro receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native mu receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G(alpha)(i)-coupled (delta-opioid, CB1 cannabinoid, alpha(2A)-adrenergic) as well as G(alpha)(s)-(beta(2)-adrenergic) and G(alpha)(q)-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.

Trypanosoma evansi in three dogs in Iran

Three crossbreed dogs were referred to the Small Animal Hospital of Tehran University. Corneal opacity was obvious in two of these cases. The animals were pyretic (T: 40–41°C) but other vital signs were normal. Blood samples were collected from the cephalic veins of the dogs using EDTA. Complete blood cell count showed a nonregenerative anemia with low packed cell volume, Hb, RBC, mean corpuscular volume, mean corpuscular Hb and mean corpuscular Hb concentration. Thin smears were made and stained by Giemsa method after fixation in methanol and then examined under a light microscope (×100). Trypanosoma evansi detected with 17–36 μm length (mean = 25 μm). The kinetoplasts of the parasites were subterminal, the undulating membranes were well-developed and there was a substantial free flagellum. Serum analysis showed hyperproteinemia and normal values of alanine aminotransferase, aspartate aminotransferase and total bilirubin. Serum analysis showed a polyclonal gammopathy and a beta–gamma bridge in the electrophoresis of serum samples of two dogs. After confirming the disease, treatment started by diminazen aceturate (Berenil, Intervet).

P.166 Salivary urea,creatinine,ascorbic and oxalic acid in CRF patients

To gain preliminary data on the location of a 195 kD cell peripheral protein of epidermal keratinocytes recognized by monoclonal antibody AE11, immunofluorescence staining was carried out on cultured human epidermal cells. B11 antidesmosomal polyclonal antibody and AE3 antikeratin monoclonal antibody staining were used as comparative reference controls. Desmosomal antigens span the cell membrane and keratins constitute a family of cytoplasmic proteins. The cultured cells were systematically used either unfixed, fixed with 4% paraformaldehyde, or fixed with methanol at 4°C. The former two preparations were designed to expose only the surface antigens, whereas the latter method permeated the membrane to allow cytoplasmic staining. Differences in staining patterns were observed in the basal layer as compared with the upper layers with all three antibodies. B11 antidesinosomal antibody staining was consistent in pattern after either paraformaldehyde or methanol fixation. In addition to the punctate staining along adjacent cell surfaces as shown in the basal layer, the surface planes of the upper cells exhibited parallel arrays of linear streaks, demonstrating the distribution of desmosomal proteins. Antikeratin staining by AE3 showed the typical filamentous staining in basal cells. However, a homogeneous patchy staining of suprabasal cells was observed. The presence of punctate surface staining using antikeratin antibody on paraformaldehyde fixed cells suggests leakage of keratin to the cell surface. AE11 showed stronger staining in the top cells on methanol treated cultures and a punctate surface staining of the cells fixed with paraformaldehyde. These observations provide useful preliminary information in localizing peripheral proteins in epidermal keratinocytes.

Cox-2 Is Regulated by Toll-Like Receptor-4 (TLR4) Signaling: Role in Proliferation and Apoptosis in the Intestine

We recently showed that mice deficient in Toll-like receptor 4 (TLR4) or its adapter molecule MyD88 have increased signs of colitis compared with wild-type (WT) mice after dextran sodium sulfate (DSS)-induced injury. We wished to test the hypothesis that cyclooxygenase 2 (Cox-2)-derived prostaglandin E2 (PGE2) is important in TLR4-related mucosal repair. Cox-2 expression was analyzed by real-time polymerase chain reaction, immunohistochemistry, Western blotting, and luciferase reporter constructs. Small interfering RNA was used to inhibit expression of MyD88. TLR4-/- or WT mice were given 2.5% DSS for 7 days. Proliferation and apoptosis were assessed using bromodeoxyuridine staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays, respectively. PGE2 was given orally to DSS-treated mice. Intestinal epithelial cell lines up-regulated Cox-2 expression in a TLR4- and MyD88-dependent fashion. Lipopolysaccharide-mediated stimulation of PGE2 production was blocked by a selective Cox-2 inhibitor or small interfering RNA against MyD88. After DSS injury, Cox-2 expression increased only in WT mice. TLR4-/- mice have significantly reduced proliferation and increased apoptosis after DSS injury compared with WT mice. PGE2 supplementation of TLR4-/- mice resulted in improvement in clinical signs of colitis and restoration of proliferation and apoptosis to WT values. The mechanism for improved epithelial repair may be through PGE2-dependent activation of the epidermal growth factor receptor. We describe an important link between TLR4 signaling and Cox-2 expression in the gut. TLR4 and MyD88 signaling are required for optimal proliferation and protection against apoptosis in the injured intestine. Although TLR4 signaling is beneficial in the short term, chronic signaling through TLR4 may lower the threshold for colitis-associated cancer.

Keratin 20 Serine 13 Phosphorylation Is a Stress and Intestinal Goblet Cell Marker

Keratin polypeptide 20 (K20) is an intermediate filament protein with preferential expression in epithelia of the stomach, intestine, uterus, and bladder and in Merkel cells of the skin. K20 expression is used as a marker to distinguish metastatic tumor origin, but nothing is known regarding its regulation and function. We studied K20 phosphorylation as a first step toward understanding its physiologic role. K20 phosphorylation occurs preferentially on serine, with a high stoichiometry as compared with keratin polypeptides 18 and 19. Mass spectrometry analysis predicted that either K20 Ser(13) or Ser(14) was a likely phosphorylation site, and Ser(13) was confirmed as the phospho-moiety using mutation and transfection analysis and generation of an anti-K20-phospho-Ser(13) antibody. K20 Ser(13) phosphorylation increases after protein kinase C activation, and Ser(13)-to-Ala mutation interferes with keratin filament reorganization in transfected cells. In physiological contexts, K20 degradation and associated Ser(13) hyperphosphorylation occur during apoptosis, and chemically induced mouse colitis also promotes Ser(13) phosphorylation. Among mouse small intestinal enterocytes, K20 Ser(13) is preferentially phosphorylated in goblet cells and undergoes dramatic hyperphosphorylation after starvation and mucin secretion. Therefore, K20 Ser(13) is a highly dynamic protein kinase C-related phosphorylation site that is induced during apoptosis and tissue injury. K20 Ser(13) phosphorylation also serves as a unique marker of small intestinal goblet cells.

Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation

The reiterated nature of histone genes has hampered genetic approach to dissect the role of histones in chromatin dynamics. We here report isolation of three temperature-sensitive (ts) Schizosaccharomyces pombe strains, containing amino-acid substitutions in the sole histone H2B gene (htb1+). The mutation sites reside in the highly conserved, non-helical residues of H2B, which are implicated in DNA-protein or protein-protein interactions in the nucleosome. In the allele of htb1-72, the substitution (G52D) occurs at the DNA binding loop L1, causing disruption of the gene silencing in heterochromatic regions and lagging chromosomes in anaphase. In another allele htb1-223 (P102L) locating in the junction between alpha3 and alphaC, the mutant residue is in contact with H2A and other histones, leading to structural aberrations in the central centromere chromatin and unequal chromosome segregation in anaphase. The third allele htb1-442 (E34K) near alpha1 displayed little defect. Evidence is provided that monoubiquitinated H2B is greatly unstable in P102L mutant, possibly owing to proteasome-independent destruction or enhanced deubiquitination. Histone H2B thus plays an important role in centromere/kinetochore formation.

24-norUrsodeoxycholic Acid Is Superior to Ursodeoxycholic Acid in the Treatment of Sclerosing Cholangitis in Mdr2 (Abcb4) Knockout Mice

Current therapy for primary sclerosing cholangitis is of limited efficacy. Multidrug resistance gene 2 knockout mice (Mdr2(-/-)) represent a well-characterized model for sclerosing cholangitis. Experiments were performed to test in such mice the therapeutic effects of 24-norUrsodeoxycholic acid, a C(23) homologue of ursodeoxycholic acid with 1 fewer methylene group in its side chain. Mdr2(-/-) mice were fed a diet containing 24-norUrsodeoxycholic acid (0.5% wt/wt) or ursodeoxycholic acid (0.5% wt/wt) as a clinical comparator for 4 weeks; controls received standard chow. Effects on serum liver tests, liver histology, markers of inflammation and fibrosis, and bile acid transport and metabolism were compared. 24-norUrsodeoxycholic acid metabolism was studied in serum, liver, bile, and urine. 24-norUrsodeoxycholic acid markedly improved liver tests and liver histology and significantly reduced hydroxyproline content and the number of infiltrating neutrophils and proliferating hepatocytes and cholangiocytes. 24-norUrsodeoxycholic acid underwent extensive phase I/II metabolism (hydroxylation, sulfation, and glucuronidation), thereby increasing the hydrophilicity of biliary bile acid secretion. There was a coordinated induction of bile acid detoxifying enzymes (Cyp2b10, Cyp3a11, and Sult2a1) and efflux pumps (Mrp3 and Mrp4). Ursodeoxycholic acid, in contrast, increased alanine transaminase and alkaline phosphatase levels, had no significant effects on hydroxyproline content, and induced biliary transporters and detoxification enzymes to a much smaller extent than 24-norUrsodeoxycholic acid. 24-norUrsodeoxycholic acid ameliorates sclerosing cholangitis in Mdr2(-/-) mice. Its therapeutic mechanisms involve (1) increasing the hydrophilicity of biliary bile acids, (2) stimulating bile flow with flushing of injured bile ducts, and (3) inducing detoxification and elimination routes for bile acids.