Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis

Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3'-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.     

Association of comorbidity between Opisthorchis viverrini infection and diabetes mellitus in the development of cholangiocarcinoma among a high-risk population,northeastern Thailand

BackgroundCholangiocarcinoma (CCA) is a category of lethal hepatobiliary malignancies. Previous studies have found that Opisthorchis viverrini infection and diabetes mellitus (DM) are closely correlated with CCA. However, few studies have discussed the association of CCA with a combination of both O. viverrini infection and DM. This study aimed to assess the correlation of CCA with various combinations of O. viverrini infection and DM among a high-risk population in northeastern Thailand.MethodologyThis study included participants from 20 provinces in northeastern Thailand who had been screened for CCA in the Cholangiocarcinoma Screening and Care Program (CASCAP) between 2013 and 2019. Histories of O. viverrini infection and DM diagnosis were obtained using a health questionnaire. CCA screening used ultrasonography with a definitive diagnosis based on histopathology. Multilevel mixed-effects logistic regression was performed to quantify the association, which is presented as adjusted odds ratios (aOR) and their 95% confidence intervals (CI).Principal findingsOverall, 263,776 participants were included, of whom 32.4% were infected with O. viverrini, 8.2% were diagnosed with DM, and 2.9% had a history of both O. viverrini infection and DM. The overall rate of CCA was 0.36%. Of those infected with O. viverrini, 0.47% had CCA; among those with DM, 0.59% had CCA and among those infected with O. viverrini and had DM, 0.73% had CCA. Compared with participants who were not infected with O. viverrini and were non-DM, the aOR for those infected with O. viverrini and with DM was 2.36 (95% CI: 1.74–3.21; p-value <0.001).ConclusionsThe combination of O. viverrini infection and DM was highly associated with CCA, and these two conditions had a combined effect on this association that was greater than that of either alone. These findings suggest that CCA screening should have a strong focus on people with a combination of O. viverrini infection and DM.     

A PCR-based method, with internal control, for the detection of Banana bunchy top virus in banana

Banana bunchy top disease is a major constraint to banana production in most regions where this crop is grown. The disease is caused by Banana bunchy top virus (BBTV), a multicomponent, single-stranded DNA virus of the family Nanoviridae. We have designed primers to a conserved region of the master replication-associated protein that are useful for the polymerase chain reaction (PCR)-mediated detection of BBTV. In addition, primers to banana genomic sequence are used as an internal control, overcoming the uncertainty (owing to false-negatives) inherent in PCR diagnostics. Together these primer sets are a valuable tool in the effort to control BBTV, particularly in screening micropropagated banana plantlets for the absence of virus before release to farmers.     

Mass spectrometry analysis for the determination of side reactions for cyclic peptides prepared from an Fmoc/t}Bu/Dmab protecting group strategy

The Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group (PSRG) proposed for their annual study that laboratory members prepare cyclo(Tyr-Glu-Ala-Ala-Arg-DPhe-Pro-Glu-Asp-Asn) according to the following synthetic pathway: (i) side-chain anchoring Fmoc-Asp(OH)-ODmab to a Rink amide resin; (ii) linear assembly; (iii) Dmab and Fmoc removal, respectively; (iv) on-resin cyclization with an uronium-based coupling reagent; (v) final cleavage/deprotection with TFA. Based upon this protocol, a variety of side-products were identified:(i) N-terminal guanidine formation; (ii) C-terminal piperidyl amide formation; and (iii) a novel C-terminal benzyl amide-guanidine derivative that formed due to a chemical reaction between the Dmab protecting group and the uronium-based coupling agent. The elemental composition and subsequent structure determination of this unexpected derivative was established by tandem mass spectrometry, i.e. low energy collision-induced dissociation experiments with fragment mass determination within 5 ppm.     

Role of myosin phosphatase isoforms in cGMP-mediated smooth muscle relaxation

In vitro experiments showing the activation of the myosin phosphatase via heterophilic leucine zipper interactions between its targeting subunit (MYPT1) and cGMP-dependent protein kinase I suggested a pathway for smooth muscle relaxation (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). The relationship between MYPT1 isoform expression and smooth muscle responses to cGMP signaling in vivo has not been explored. MYPT1 isoforms that contain or lack a C-terminal leucine zipper are generated in birds and mammals by cassette-type alternative splicing of a 31-nucleotide exon. The avian and mammalian C-terminal isoforms are highly conserved and expressed in a tissue-specific fashion. In the mature chicken the tonic contracting aorta and phasic contracting gizzard exclusively express the leucine zipper positive and negative MYPT1 isoforms, respectively. Expression of the MYPT1 isoforms is also developmentally regulated in the gizzard, which switches from leucine zipper positive to negative isoforms around the time of hatching. This switch coincides with the development in the gizzard of a cGMP-resistant phenotype, i.e. inability to dephosphorylate myosin and relax in response to 8-bromo-cGMP after calcium activation. Furthermore, association of cGMP-dependent protein kinase I with MYPT1 is detected by immunoprecipitation only in the tissue that expresses the leucine zipper positive isoform of MYPT1. These results suggest that the regulated splicing of MYPT1 is an important determinant of smooth muscle phenotypic diversity and the variability in the response of smooth muscles to the calcium desensitizing effect of cGMP signaling.     

Transfer and expression of a hydrocarbon-degrading plasmid pHCL from Pseudomonas putida to marine bacteria

Transfer of a catabolic plasmid from Pseudomonas putida to indigenous marine bacteria and obligate halophilic bacteria was carried out under both in vitro and in situ conditions. The marine recipients, which could not otherwise grow on hydrocarbon substrates, were able to degrade them after the horizontal transfer of the catabolic plasmid from P. putida. Mating conducted on nutrient plates yielded comparatively more transconjugants than in broth mating under laboratory conditions (10⁶ c.f.u./ml). The transconjugants stably maintained the plasmid when they were maintained in seawater amended with selective pressure (antibiotics/Hg (25 g/l) even after 30 days, whereas under non-selective conditions they progressively lost the plasmid after 24 days. The expression of the plasmid in the marine recipients was investigated by gas chromatographic analysis. The overall objective of this study is to evolve a novel strategy for bioremediation of oil spills and the results of the present study suggest that the present approach would offer a better solution for the removal of harmful substances from the environment by avoiding serious interference with the microbial flora of the ecosystem.     

NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICK1 complex

AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity that may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. Here, we show that GluR2, PICK1, NSF, and alpha-/beta-SNAPs form a complex in the presence of ATPgammaS. Similar to SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. Alpha- and beta-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions. Furthermore, we are reporting a non-SNARE substrate for NSF disassembly activity.