An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle

The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl₂ (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.     

Structure and function of vertebrate CMP-sialic acid synthetases

Activation of sugars into nucleotide sugars is critical for their entry into biosynthetic pathways. In eukaryotic cells, the activation of the acidic nine-carbon sugar sialic acid to CMP-sialic acid takes place in the cell nucleus, whereas all other nucleotide sugars are made in the cytoplasm. Molecular cloning of vertebrate CMP-sialic acid synthetases confirmed the nuclear localization and introduced new molecular tools for directly exploring the functional mechanisms of the enzymes, as well as the physiological relevance of their nuclear transport. Although major advances have been made in understanding structure-function relationships and defining elements involved in the nuclear transport, the riddle surrounding the physiological relevance of nuclear localization awaits resolution.     

Molecular characterization of the diurnal/circadian expression of the chlorophyll a/b-binding proteins in leaves of tomato and other dicotyledonous and monocotyledonous plant species

Diurnal oscillations of steady-state mRNA levels encoding the chlorophyll a/b-binding proteins were monitored inLycopersicon esculentum, Glycine max, Phaseolus vulgaris, P. aureus, P. coccineus, Pisum sativum, Sinapis alba, Hordeum vulgare, Triticum aestivum andZea mays. In these plant speciescab mRNA accumulation increases and decreases periodically indicating i) that the expression of the genes for chlorophyll a/b-binding proteins (cab genes) is controlled by a circadian rhythm, and ii) that the rhythm is widely distributed among monocotyledonous and dicotyledonous plant species. A detailed characterization of the pattern ofcab mRNA expression in tomato leaves shows that the amplitude of the oscillation is dependent on i) the developmental stage of the leaves, ii) the circadian phase and duration of light and iii) the circadian phase and duration of darkness. In addition to the chlorophyll a/b-binding proteins, genes coding for other cellular functions were examined for cyclic variations of their mRNA levels. The analysis includes genes involved in i) carbon metabolism (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, alpha amylase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase), ii) photosynthesis (large and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, Q_B-binding protein, reaction-center protein of photosystem I) and iii) other physiological or morphological reactions (e.g. ubiquitin, actin). However, no periodic fluctuation pattern was detected for the mRNA levels of these genes in tomato and maize leaves.     

rctB mutations that increase copy number of Vibrio cholerae oriCII in Escherichia coli

RctB serves as the initiator protein for replication from oriCII, the origin of replication of Vibrio cholerae chromosome II. RctB is conserved between members of Vibrionaceae but shows no homology to known replication initiator proteins and has no recognizable sequence motifs. We used an oriCII based minichromosome to isolate copy-up mutants in Escherichia coli. Three point mutations rctB(R269H), rctB(L439H) and rctB(Y381N) and one IS10 insertion in the 3'-end of the rctB gene were obtained. We determined the maximal C-terminal deletion that still gave rise to a functional RctB protein to be 165 amino acids. All rctB mutations led to decreased RctB-RctB interaction indicating that the monomer is the active form of the initiator protein. All mutations also showed various defects in rctB autoregulation. Loss of the C-terminal part of RctB led to overinitiation by reducing binding of RctB to both rctA and inc regions that normally serve to limit initiation from oriCII. Overproduction of RctB(R269H) and RctB(L439H) led to a rapid increase in oriCII copy number. This suggests that the initiator function of the two mutant proteins is increased relative to the wild-type.     

Nocturnal vision and landmark orientation in a tropical halictid bee

BACKGROUND: Some bees and wasps have evolved nocturnal behavior, presumably to exploit night-flowering plants or avoid predators. Like their day-active relatives, they have apposition compound eyes, a design usually found in diurnal insects. The insensitive optics of apposition eyes are not well suited for nocturnal vision. How well then do nocturnal bees and wasps see? What optical and neural adaptations have they evolved for nocturnal vision? RESULTS: We studied female tropical nocturnal sweat bees (Megalopta genalis) and discovered that they are able to learn landmarks around their nest entrance prior to nocturnal foraging trips and to use them to locate the nest upon return. The morphology and optics of the eye, and the physiological properties of the photoreceptors, have evolved to give Megalopta's eyes almost 30 times greater sensitivity to light than the eyes of diurnal worker honeybees, but this alone does not explain their nocturnal visual behavior. This implies that sensitivity is improved by a strategy of photon summation in time and in space, the latter of which requires the presence of specialized cells that laterally connect ommatidia into groups. First-order interneurons, with significantly wider lateral branching than those found in diurnal bees, have been identified in the first optic ganglion (the lamina ganglionaris) of Megalopta's optic lobe. We believe that these cells have the potential to mediate spatial summation. CONCLUSIONS: Despite the scarcity of photons, Megalopta is able to visually orient to landmarks at night in a dark forest understory, an ability permitted by unusually sensitive apposition eyes and neural photon summation.     

Immunohistochemical detection of secretoneurin, a novel neuropeptide endoproteolytically processed from secretogranin II, in normal human endocrine and neuronal tissues

Summary An antiserum raised against a synthetic peptide derived from the primary amino sequence of rat secretogranin II (chromogranin C) was used for immunological (quantitative radioimmunoassay analysis) and immunohistochemical studies of normal human endocrine and nervous tissues. This antibody recognized a novel and biologically active neuropeptide which was coined as secretoneurin. In endocrine tissues, secretoneurin was mainly co-localized with chromogranin A and B with some exceptions (e.g., parathyroid gland). Secretoneurin was demonstrated immunohistochemically in the adrenal medulla, thyroid C cells, TSH- and FSH/LH-produting cells of the anterior pituitary, A and B cells of pancreatic islets, in endocrine cells of the gastrointestinal tract and the bronchial mucosa, and the prostate. Immunoreactivity determined by radioimmunoassay analysis revealed high secretoneurin levels in the anterior and posterior pituitary and lower levels in pancreatic and thyroid tissue. A strong secretoneurin immunoreactivity was also found in ganglion cells of the submucdsal and myenteric plexus of the gastrointestinal tract, and in ganglionic cells of dorsal root ganglia, peripheral nerves, and ganglion cells of the adrenal medulla. Thus, secretoneurin may serve as a useful marker of gangliocytic/neuronal differentiation.     

Effects of Lactococcus lactis on Composition of Intestinal Microbiota: Role of Nisin

This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.