Cell dynamics during cocoon secretion in the aquatic leech, Theromyzon tessulatum (Annelida: Clitellata: Glossiphoniidae)

One distinguishing feature of clitellate annelids is the presence of specialized segments comprising the clitellum, whose primary function is to secrete a cocoon. Using histological analyses, we have documented cell types (I-V) and cellular processes associated with cocoon secretion in the aquatic leech, Theromyzon tessulatum. Our data indicate that the bulk of the cocoon's biomass arises from precursor cells of a single type that hypertrophy and proliferate ∼1 week prior to egg laying, and then differentiate into either of two cell types (i.e., Type II or Type III) depending on their position within the clitellum. Type II cells are concentrated along the lateral edges and venter of the clitellum and secrete alcian blue-staining granules that form opercula (i.e., glue-like material that seals both cocoon ends), while Type III cells populate the dorsal midline and secrete azocarmine-staining granules that build the cocoon wall. Both cell types occupy spaces between deep muscle layers and extend long-neck tubules to the surface epithelium as they fill with granules a few days prior to egg laying. Other cell types appear to make minor contributions to the cocoon (e.g., Type I, Type IV) or have supporting or signaling roles (e.g., Type V). Our observations suggest that post-translational modification (i.e., glycosylation) of the same core protein(s) distinguishes the granules of Type II/III cells, and that the default state of the Type II/III precursor may be evolutionarily linked to secretory cells in basal polychaetes.     

Particulate phase cigarette smoke increases MnSOD, NQO1, and CINC-1 in rat lungs

Loss of antioxidant/oxidant homeostasis perpetuates inflammation in the lungs and may contribute to the development of COPD and lung cancer. Cigarette smoke (CS) is a primary source of airway oxidative stress and recruits inflammatory cells into smokers' lungs. However, whether these consequences are attributable to a specific or the collective fraction of CS is unknown. We investigated whether the particulate or the gas phase of CS would alter expression of the antioxidant enzymes MnSOD and NQO1 or CINC-1. Sprague Dawley rats were exposed to sham (n = 10) or the particulate phase (PP; n = 10) or gas phase (n = 10) of a Kentucky reference cigarette (1R4F) for 2 h/d for 28 d, after which animals were sacrificed and the lower left lobe of the lung was removed. Immunoblots for SOD and NQO1 revealed that lungs exposed to PP had higher MnSOD/actin and NQO1/actin ratios than either sham-or gas phase-treated animals. In contrast, CuZnSOD remained unchanged. In PP-exposed animals, CINC-1 was 3-fold higher than in sham-exposed animals. The increases in MnSOD and NQO1 protein were associated with increases in total SOD, NQO1, and MPO activities. These data provide evidence that the PP of CS alters oxidant/antioxidant homeostasis in the lungs and participates in the pathogenesis of CS-induced lung diseases such as COPD and cancer.     

5''-3'' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.

The application of T7 and lambda exonuclease to phosphorothioate-based oligonucleotide-directed mutagenesis was investigated. Oligonucleotide primers designed to introduce single or double base mismatches, an insertion or a deletion (each of 16 bases) were annealed to M13 phage derivatives. Double stranded closed circular DNA (RF IV) containing phosphorothioate internucleotidic linkages in the (-)strand was prepared enzymatically from these templates. A nick was introduced into the (+)strand of the hetroduplex DNA. This nicked DNA (RF II) was subjected to treatment with T7 or lambda exonuclease. Both of these enzymes were able to degrade almost all of the viral (+)strand when presented with DNA containing one or two base mismatches. Repolymerisation of the DNA after the gapping reaction, followed by transfection into E. coli cells gave mutational efficiencies of up to 95%. In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.     

Further characterization of the covalent linking reaction of alpha 2-macroglobulin.

It is shown that non-proteolytic proteins can become covalently linked to alpha 2M (alpha 2-macroglobulin) during its reaction with proteinases, and that this probably occurs by the mechanism that leads to the covalent linking of proteinases described previously [Salvesen & Barrett (1980) Biochem. J. 187, 695-701]. The covalent linking of trypsin was at least partly dependent on the presence of unblocked lysine side chains on the protein. The covalent linking of proteinases was inhibited by nucleophiles of low Mr, and these compounds were themselves linked to alpha 2M in a molar ratio approaching one per quarter subunit. Peptide "mapping" indicated that the site of proteinase-mediated incorporation of the amines was the same as that at which methylamine is incorporated in the absence of a proteinase. The nucleophile-reactive site revealed in alpha 2M after reaction with a proteinase was shown to decay with a t1/2 of 112 s, at pH 7.5. After the reaction with a proteinase or with methylamine, a free thiol group was detectable on each subunit of alpha 2M. We propose that the site for incorporation of methylamine in each subunit is a thiol ester, which in S-alpha 2M (the electrophoretically "slow" form) is sterically shielded from reaction with large nucleophiles, but is revealed as a highly reactive group, free from steric hindrance, after the proteolytic cleavage. We have designated the activated species of the molecule "alpha 2M".     

Reduced VLDL clearance in Apoe−/−Npc1−/− mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels

Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe^−/−Npc1^−/− mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe^−/−Npc1^−/− liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe^−/−Npc1^−/− liver was unexpected. However, several other LXR target genes also increased in Apoe^−/−Npc1^−/− liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe^−/− mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe^−/−Npc1^−/− mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL.     

Serum 25-hydroxyvitamin d(3) and d(2) and non-clinical psychotic experiences in childhood

Objective

Non-clinical psychotic experiences are common and distressing. It has been hypothesized that early life vitamin D deficiency may be a risk factor for psychosis-related outcomes, but it is not known if circulating concentrations of 25-hydroxyvitamin D (25(OH)D) during childhood are associated with psychosis-related outcomes or whether the two different forms of 25(OH)D, (25(OH)D₃ and 25(OH)D₂, have similar associations with psychosis-related outcomes.

Methods

We investigated the association between serum 25(OH)D₃ and 25(OH)D₂ concentrations and psychotic experiences in a prospective birth cohort study. Serum 25(OH)D₃ and 25(OH)D₂ concentrations were measured at mean age 9.8 years and psychotic experiences assessed at mean age 12.8 years by a psychologist (N = 3182).

Results

Higher 25(OH)D₃ concentrations were associated with lower risk of definite psychotic experiences (adjusted odds ratio: OR (95% confidence interval: CI) 0.85 (0.75–0.95)). Higher concentrations of 25(OH)D₂ were associated with higher risk of suspected and definite psychotic experiences (adjusted odds ratio: OR (95% confidence interval: CI) 1.26 (1.11, 1.43)). Higher 25(OD)D₂ concentrations were also weakly associated with definite psychotic experiences (adjusted OR (95% CI) 1.17 (0.96, 1.43), though with wide confidence intervals including the null value.

Conclusions

Our findings of an inverse association of 25(OH)D₃ with definite psychotic experiences is consistent with the hypothesis that vitamin D may protect against psychosis-related outcomes.