Assembly of the Respiratory Mucin MUC5B: A NEW MODEL FOR A GEL-FORMING MUCIN*

Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D′D3; NT5B) and subdomains (D1, D1-D2, D2-D′-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.     

Properties of the vastus lateralis muscle in relation to age and physiological function in master cyclists aged 55–79 years

In this study, results are reported from the analyses of vastus lateralis muscle biopsy samples obtained from a subset (n = 90) of 125 previously phenotyped, highly active male and female cyclists aged 55–79 years in regard to age. We then subsequently attempted to uncover associations between the findings in muscle and in vivo physiological functions. Muscle fibre type and composition (ATPase histochemistry), size (morphometry), capillary density (immunohistochemistry) and mitochondrial protein content (Western blot) in relation to age were determined in the biopsy specimens. Aside from an age‐related change in capillary density in males (r = ?.299; p = .02), no other parameter measured in the muscle samples showed an association with age. However, in males type I fibres and capillarity (p < .05) were significantly associated with training volume, maximal oxygen uptake, oxygen uptake kinetics and ventilatory threshold. In females, the only association observed was between capillarity and training volume (p < .05). In males, both type II fibre proportion and area (p < .05) were associated with peak power during sprint cycling and with maximal rate of torque development during a maximal voluntary isometric contraction. Mitochondrial protein content was not associated with any cardiorespiratory parameter in either males or females (p > .05). We conclude in this highly active cohort, selected to mitigate most of the effects of inactivity, that there is little evidence of age‐related changes in the properties of VL muscle across the age range studied. By contrast, some of these muscle characteristics were correlated with in vivo physiological indices.     

The digestive gland of Viviparus ater (Mollusca, Gastropoda, Prosobranchia): an ultrastructural and histochemical study

The digestive gland of Viviparus ater was studied using histochemical and ultrastructural methods. Only one cell type was observed in the tubule epithelium of the gland. The cells are involved in an endocytotic process mediated by clathrin-coated vesicles and in the intracellular digestion of food materials (thus they can be regarded as digestive cells). The different stages of digestion and exocytotic extrusion of residual bodies into the tubule lumen were shown by electron microscopy. Very few, small mucocytes are scattered among the digestive cells. Calcium concretions, glycogen-containing cells and endocrine cells are scattered in the area of connective tissue present among the digestive tubules.     

Gelation of fractionated canine submaxillary mucin in a chaotropic solvent

Rheological measurements have been performed on three molecular weight fractions of purified canine submaxillary mucin (CSM) dissolved in the chaotropic solvent 6 M guanidine hydrochloride (GdnHCI). Solutions of the lower molecular weight fractions are viscoelastic sols, and their dynamic moduli can be scaled with respect to molecular weight and concentration according to linear viscoelasticity theory. In contrast, preparations of the highest molecular weight fraction form viscoelastic gels that exhibit an equilibrium shear modulus, G_e^′, which scales with mucin concentration as G_e^′ c³. Amino acid and carbohydrate analyses of all three fractions are similar; thus, the differences in rheological behavior are attributed to molecular weight differences, which affect the degree of coil overlap in solutions of a given concentration. These observations demonstrate conclusively that mucin glycoproteins of high molecular weight form gels under conditions in which the mucin chains physically interpenetrate, even when non-covalent intermolecular interactions are extensively disrupted. A comparison of these results with previous studies of purified submaxillary and tracheobronchial mucins indicates that the carbohydrate side-chain length, in addition to molecular weight, is an important determinant of the observed elastic response and the ability to form physical gels     

Large Specimen Bone Embedment and Cement Line Staining

Undecalcified embedment of large bone specimens is often challenging. A method is presented here that is suitable for methacrylate embedment of sections of canine vertebrae while retaining the ability to localize tartrate-resistant acid phosphatase and alkaline phosphatase activity. Specimens also retained tetracycline labelling, and sectioned preparations were readily stained with routine bone procedures. A modification of the Bodian silver stain, used for examining the nerves and spinal cord in these specimens, provided a useful stain for canaliculi and cement lines in trabecular and cortical bone. This stain is advantageous when both bone and nerve tissue are of interest, as in spinal fusion studies.     

一氧化氮合酶(NOS)等活性变化对肺癌早期诊断的研究——纤支镜活检组织50例的观察

用酶组织化学方法探讨经支气管纤维镜（简称纤支镜）活检组织,对肺癌的早期诊断。包括一氧化氮合酶（ＮＯＳ）、细胞色素氧化酶（ＣＣＯ）、乳酸脱氢酶（ＬＤＨ）、琥珀酸脱氢酶（ＳＤＨ）、酸性磷酸酶（ＡＣＰ）等。酶组织化学反应结果显示为肺癌组病人ＮＯＳ、ＬＤＨ、ＣＣＯ、ＡＣＰ的活性显著增高,分别为强阳性（）,少数呈最强阳性（）;而ＳＯＨ活性为弱阳性（＋）,较正常人组显著下降。可疑组病人ＳＤＨ活性增高呈强阳性（）或最强阳性（）,但ＮＯＳ、ＬＤＨ、ＡＣＰ的活性较肺癌组下降,分别为弱阳性（＋）或中等阳性（）。上述结果提示：ＮＯＳ等活性的变化,可作为提高肺癌早期诊断的重要指标之一。     

兰花蕉种子的解剖学和组织化学研究

兰花蕉种子球形或近球形,具表皮毛,种脊不明显。种子包括假种皮、种皮、外胚乳、内胚乳和胚五部分。假种皮具３～４条粗毛状裂片,包围种子或不定向伸展;裂片最外方为１层表皮细胞和１～３层厚壁细胞,内方为薄壁细胞;表皮细胞和厚壁细胞的壁增厚并木质化;成熟时裂片下部１／２段中空。种皮由外珠被发育而来,但内珠被在种子发育后期才萎缩。种皮分化为外种皮,中种皮与内种皮;外种皮由１层表皮细胞构成,其细胞壁增厚并木质化;中种皮外方为２～３层厚壁细胞,内方由１２～１５层薄壁细胞构成;内种皮由１层径向延长的石细胞构成,其细胞壁网状增厚,胞腔不明显。外胚乳极不显眼,大部分只由１层切向延长的长方形细胞构成,局部为２～１７层细胞;外胚乳细胞主要含许多脂类物质及少量蛋白质颗粒,不含淀粉。内胚乳占据种子很大的体积,由通常径向延长的长方形、长条形或方形薄壁细胞构成;细胞内充满淀粉粒和通常一颗亦有２至多颗菱形或方形蛋白质晶体,脂类物质极少。胚圆柱形,胚根和胚芽不明显。种子珠孔区不分化出珠孔领和孔盖,但具柄,柄的远轴端边缘大部分着生假种皮,着生假种皮一侧柄略膨大。合点区内种皮出现极宽的缺口,缺口间为整体呈弧状长条形的合点区厚壁细胞群。较粗的种脊维管?     

Two-dimensional protein profiles of palisade parenchyma and of spongy parenchyma of Vicia faba leaf

Specific probes such as targeted-enzyme assays have failed to reveal qualitative differences between the metabolism of spongy parenchyma and that of palisade parenchyma cells. To determine whether the results from these definite assays support a general conclusion concerning the overall biochemical similarity of these cells, two-dimensional protein profiles have been constructed for these two types of cells. Protein extracts were prepared from freeze-dried homogeneous tissue samples or from isolated protoplasts of Vicia faba L. cv. Longpod leaves. Proteins separated by micro two-dimensional gel electrophoresis were stained with silver. The relative apparent abundance (based on visual judgment of the area and intensity of staining) was estimated for each spot. Pairwise comparison of nominally 125 stained proteins of each of approximately 75 gel pairs did not reveal a consistent qualitative difference between the profiles. Recognizing that polypeptides escaped detection in our investigation through loss, failure to stain sufficiently, or incomplete resolution, we qualify our conclusion that spongy parenchyma and palisade are similar in their overall protein complement.     

Lectin histochemistry of the human fetal subcommissural organ

The subcommissural organ (SCO) of 7 human fetuses, 3 to 6.5 months old, was investigated by means of: (i) immunocytochemistry employing three different antisera against secretory products extracted from the bovine SCO and Reissner's fiber; (ii) lectin binding using concanavalin A (Con A; affinity: mannose, glucose), wheat-germ agglutinin (WGA; affinity: N-acetyl-glucosamine, sialic acid), and Limax flavus agglutinin (LFA; affinity: sialic acid). Sections of bovine SCO were processed simultaneously and examined for comparative purposes. The human fetal SCO displayed lectin-binding properties identical to those in the SCO of other mammals. Thus, Con A-binding sites were restricted to abundant supranuclear structures that most likely corresponded to the rough endoplasmic reticulum, but were missing from granules located in the apical cytoplasm. The latter secretory material was strongly WGA- and LFA-positive and formed a distinct zone in the most apical portion of the ependymal cells. In contrast, this type of reactivity was missing in the adjacent cells of ependyma proper. In the bovine SCO, LFA-positive granules were also aggregated in an apical layer. The secretory material in the bovine SCO, especially its apical granular component, was strongly immunoreactive with the three antisera used; the human fetal SCO, however, lacked this immunoreactivity. It is postulated that the SCO of human fetuses secretes glycoproteins with a carbohydrate chain similar to--and a protein backbone different from--the secretions elaborated by the SCO of other vertebrate species.     

Identification of separate vasopressin-neurophysin II and oxytocin-neurophysin I containing nerve fibres in the external region of the bovine median eminence

Summary Immuno-enzyme histochemical investigations showed the presence, in the external region of the bovine median eminence, of accumulations of vasopressin-neurophysin II-and oxytocin-neurophysin I-complexes. These two hormone-neurophysin complexes are located in separate fine varicose nerve fibres. The results strongly plead against an important role of tanycytes in the transport of vasopressin, oxytocin and neurophysins from the cerebrospinal fluid to the hypophysial portal blood-vessels.This work was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek.