Nephridial and gonoduct distribution patterns in Nerillidae (Annelida: Polychaeta) examined by tubulin staining and cLSM

The distribution and configuration of nephridia and gonoducts are described for seven species from seven genera of the interstitial polychaete family Nerillidae. The ciliated nephridia and gonoducts were identified by tubulin staining and examined with a confocal laser scanning microscope. The following species of the seven to nine-segmented nerillids were examined: Leptonerilla prospera, Nerilla antennata (nine segments); Nerillidium mediterraneum, Trochonerilla mobilis, Gen. sp. A (eight segments); and Aristonerilla brevis, Paranerilla limicola (seven segments). Two of the examined species are hermaphroditic (N. mediterraneum and Gen. sp. A). Segmented nephridia can be found from the first to the last segment, with a total of two to five pairs. One to three pairs of segmented spermioducts are present in all species. One pair of gonoducts is found in all species, except for P. limicola, where they are absent. Nephridia vary in length from half to almost twice the length of a segment and may be curled up in loops. In A. brevis and P. limicola the nephridia are discontinuously ciliated. The distribution and configuration of spermioducts and gonoducts are also variable, although to a lesser extent. The spermioduct distribution is generally consistent within genera and therefore of systematic significance. Nephridia and gonoducts are never found together in the same segments, and the results indicate that gonoducts and nephridia have developed from the same anlagen. The distribution patterns of nephridia and gonoducts are discussed with respect to segmentation, systematics, and development.     

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.     

Further analogues of plant peptide hormone phytosulfokine-alpha (PSK-alpha) and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor of structure H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In studies on the structure/activity relationship of PSK-alpha the synthesis was performed of a series of a further 23 analogues modified in position 1, 3 or 4 as well as simultaneously in positions 1 and 3 of the peptide chain. Peptides were synthesized by the solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK-alpha according to the test of Matsubayashi et al. Among these peptide analogues, [H-Phe(4-Cl)1]-PSK-alpha (IV), [H-Phe(4-I)1]-PSK-alpha (VII), and [Phe(4-Cl)3]-PSK-alpha (XI) retained 30% PSK-alpha activity. Analogue [Tyr(PO3H2)3]-PSK-alpha (IX) showed 10% of PSK-alpha activity.     

C(alpha)-hydroxymethyl methionine: synthesis, optical resolution and crystal structure of its (+)-N(alpha)-benzoyl derivative.

(R, S)-Methionine was transformed into C(alpha)-hydroxymethyl methionine by a route involving C(alpha)-hydroxymethylation of 2-phenyl-4-methylthioethyl-5-oxo-4,5-dihydro-1,3-oxazole. The absolute configuration of (-)-C(alpha)-hydroxymethyl methionine was elucidated to be (S) by chemical correlation with (S) (-)-C(alpha)-ethyl serine. Absolute structure determination (by single crystal X-ray diffraction) on N(alpha)-benzoyl-C(alpha)-hydroxymethyl methionine confirmed the (R)-configuration for the (+)-enantiomer. In addition, the X-ray diffraction analysis showed that the C(alpha,alpha)-disubstituted glycyl residue adopts the fully extended (C5) conformation.     

Chirality transferring [3,3] sigmatropic rearrangement of (1-Acyloxy-2-alkenyl)trianlkylsilane synthesis of optically active vinylsilane-containing alpha-amino acid

A vinylsilane-containing alpha-amino acid and alpha,alpha-disubstituted alpha-amino acid 2 having two contiguous asymmetric carbon centers at their alpha and beta positions were synthesized in an optically active form by ester-enolate Claisen rearrangement of the alpha-acyloxysilane 1 as the key step, where the chirality of an alpha-acyloxy-TBDMS group was completely transferred to the rearranged product.     

Effects of sodium fluoride (NaF) on the cilia and microtubular system of Tetrahymena

The effect of the nucleophilic reagent NaF on the microtubular system of Tetrahymena was studied by using scanning electron microscopy (SEM), confocal microscopy, and flow cytometry. Treatments with 40 mM NaF significantly reduced the amount of alpha-tubulin while 80 mM treatment did not alter its quantity. One possible explanation for this alpha-tubulin overexpression is that the higher amount of alpha-tubulin enables this organism to carry out the appropriate function of the cytoskeleton under this undesirable influence of higher amounts of 80 nM NaF. However, the amount of acetylated tubulin increased in a dose-dependent manner. The cilia became fragile under the effect of 80 mM NaF. Confocal microscopy revealed that after 40 mM NaF treatment transversal microtubule bands (TMs) and longitudinal microtubule bands (LMs) as well as basal bodies (BBs) were extremely strong decorated with anti-acetylated tubulin antibody and TM-localization abnormalities were visible. In the 80 mM NaF-treated cells, the deep fiber of oral apparatus was very strongly labeled, while the TMs and LMs were less decorated with anti-acetylated tubulin antibody, and LM deformities were visible. It is supposed that post-translational tubulin modifications (e.g., acetylation) defend the microtubules against the NaF-induced injury. NaF is able to influence the activity of several enzymes and G-proteins, therefore is capable to alter the structure, metabolism, and the dynamics of microtubular system. The possible connection of signaling and cytoskeletal system in Tetrahymena is discussed.     

BH(4) (tetrahydrobiopterin)-dependent activation, but not the expression, of inducible NOS (nitric oxide synthase)-2 in proinflammatory cytokine-stimulated, cultured normal human astrocytes is mediated by MEK-ERK kinases

Nitric oxide (NO) from astrocytes is one of the signalers used by the brain's extensive glial-neuronal-vascular network, but its excessive production by pro-inflammatory cytokine-stimulated glial cells can be cytodestructive. Here, we show how three pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) together stimulated the activation, but not the prior expression, of NOS-2 protein via a mechanism involving MEK-ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS-2 mRNA which were followed by bursts of MEK-ERK activities, synthesis of the NOS-2 co-factor tetrahydrobiopterin (BH(4)), a build-up of NOS-2 protein and from it active NOS-2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS-2 expression nor NOS-2 protein accumulation but stopped BH(4) synthesis and the assembly of the NOS-2 protein into active NOS-2 enzyme. The complete blockage of active NOS-2 production by the brief exposure to U0126 was bypassed by simply adding BH(4) to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS-2 gene expression and accumulation of NOS-2 protein and the second being the synthesis of the BH(4) factor needed to dimerize the NOS-2 protein into active, NO-making NOS-2 enzyme.     

Maternal gamma (gamma)-tubulin is involved in microtubule reorganization during bovine fertilization and parthenogenesis

In this study, gamma-tubulin distribution was determined chronologically in conjunction with microtubule dynamics during bovine fertilization and parthenogenesis. In unfertilized bovine oocytes, gamma-tubulin was identified in the cytoplasm, mainly in the cortex and concentrated in the meiotic spindle. Following sperm penetration, gamma-tubulin in the cytoplasm was recruited by a sperm component. During pronuclear apposition, gamma-tubulin was localized as spots at the spindle poles. gamma-tubulin spots were observed in blastomeres of embryos cleaved in vitro. Following electrical stimulation, gamma-tubulin and microtubule matrix were noted in oocyte cortex. In the late pronuclear stage, considerably less gamma-tubulin and microtubules were detected in the cytoplasm. At the mitotic metaphase of parthenotes, gamma-tubulin was recruited to the condensed chromatin and concentrated in the spindle. The gamma-tubulin spots were not detected until the 8-cell stage of parthenotes. This suggests that maternal gamma-tubulin is recruited by a sperm component to reconstitute the zygotic centrosome. In the absence of sperm components, the cell cycle-related assembly of gamma-tubulin organizes microtubule nucleation for positioning the pronucleus and spindle protein of mitotic metaphase during the first cell cycle of bovine parthenotes.     

Structure of the nervous system in Tubiluchus troglodytes (Priapulida)

Abstract. The nervous system of the meiobenthic priapulid species Tubiluchus troglodytes is described by immunohistochemistry and confocal laser scanning microscopy. The brain is circumpharyngeal, consisting of a central ring of neuropil and both anterior and posterior somata. From the brain emerges a ventral nerve cord, which shows ganglion-like swellings in the neck and caudal region. The introvert includes longitudinal neurite bundles running below and between the rows of scalids, with a small cluster of sensory cells under each scalid. In the body wall of the neck and trunk region, longitudinal and circular neurite bundles are present in an orthogonal pattern. The tail is innervated from the caudal swelling of the ventral nerve cord; it also includes longitudinal and circular bundles in an orthogonal pattern. The pharynx has a reticulated system of neurite bundles running between the pharyngeal teeth and fimbrillae. Below each tooth and fimbrilus is a ganglion-like cluster of somata. The intestine is surrounded by a nerve net. The data on the nervous system are compared within other priapulids and with other species of Scalidophora (Kinorhyncha and Loricifera).     

Chemical constituents of rice (Oryza sativa) hulls and their herbicidal activity against duckweed (Lemna paucicostata Hegelm 381)

Four new compounds, stigmastanol-3beta-p-glyceroxydihydrocoumaroate (1), stigmastanol-3beta-p-butanoxydihydrocoumaroate (2), lanast-7,9(11)-dien-3alpha,15alpha-diol-3alpha-D-glucofuranoside (3) and 1-phenyl-2-hydroxy-3,7-dimethyl-11-aldehydic-tetradecane-2-beta-D-glucopyranoside (4), along with several known compounds were isolated from the methanol extract of hulls of Oryza sativa. The new structures were established by one- and two-dimensional NMR and in combination with IR, EI/MS, FAB/MS and HR-FAB/ MS. Compound (3) strongly inhibited the growth of duckweed (Lemna paucicostata Hegelm 381), whilst compounds (2) and (4) exhibited weak inhibition.     

Osteoclast responses to lipopolysaccharide, parathyroid hormone and bisphosphonates in neonatal murine calvaria analyzed by laser scanning confocal microscopy.

Because the development and activity of osteoclasts in bone remodeling is critically dependent on cell-cell and cell-matrix interactions, we used laser confocal microscopy to study the response of osteoclasts to lipopolysaccharide (LPS; 10 microg/ml), parathyroid hormone (PTH; 10(-8) M), and bisphosphonates (BPs; 1-25 microM clodronate or 0.1-2.5 microM risedronate) in cultured neonatal calvaria. Following treatment with LPS or PTH (<48 hr), osteopontin (OPN) and the alphavbeta3 integrin were found colocalized with the actin ring in the sealing zone of actively resorbing osteoclasts. In contrast, non-resorbing osteoclasts in BP-treated cultures showed morphological abnormalities, including retraction of pseudopods and vacuolization of cytoplasm. In the combined presence of LPS and BP, bone-resorbing osteoclasts were smaller and the sealing zone diffuse, reflecting reduced actin, OPN, and beta3 integrin staining. Depth analyses of calvaria showed that the area of resorbed bone was filled with proliferating osteoblastic cells that stained for alkaline phosphatase, collagen type I, and bone sialoprotein, regardless of the presence of BPs. These studies show that confocal microscopy of neonatal calvaria in culture can be used to assess the cytological relationships between osteoclasts and osteoblastic cells in response to agents that regulate bone remodeling in situ, avoiding systemic effects that can compromise in vivo studies and artifacts associated with studies of isolated osteoclasts.     

Quantitative Analysis of Immunofluorescent Signals for Dystrophin, β-Dystroglycan and Myosin in Skeletal Muscle by Epifluorescence Microscopy

Quantitative analysis of signal intensities in immunostained sections has been performed in only a few studies owing to difficulties with quantifying amounts of antigen present. We determined correlations between fluorescent signal intensities and amounts of antigen in muscle cryosections by altering section thickness from 4 to 10 μm. Fluorescent signals of dystrophin. β-dystroglycan and myosin were detected with monoclonal and/or poly-clonal primary antibodies using routine procedures. Confocal laser microscopy demonstrated that these signals were distributed uniformly along the z-axis suggesting that the antibodies permeated well through the sections. Epifluorescence microscopy with microfluor-ometry demonstrated a positive correlation between the optical density of signals and section thickness. These findings suggest that immunofluorescent signals can be quantitated by epifluorescence microscopy.     

Anti-initiating activity of 3beta-methoxy-13alpha,14alpha-epoxyserratan-21beta-ol (PJJ-34) from the stem bark of Picea jezoensis Carr. var. jezoensis

Ultraviolet light is the major cause of skin cancers in human, and several effects of ultraviolet light B (UVB) are thought to contribute to skin photocarcinogenesis. 3Beta-methoxy-13alpha,14alpha-epoxyserratan-21beta-ol (PJJ-34; 1) isolated from Picea jezoensis Carr. var. jezoensis showed the strongest antitumor-promoting activity among naturally occurring triterpenoids in the in vivo two-stage mouse skin-carcinogenesis test. To investigate the anti-initiating activity, we further studied mouse models initiated with ultraviolet-B (UVB) and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). Oral administration of the PJJ-34 (1) during a period before and after the three times of UVB irradiation led to a remarkable effect: oral administration of a 0.0025% solution of 1 only to the test group, which started one week before and ended one week after the irradiation, showed less than half papillomas, and inhibition of tumor incidence and tumor multiplicity in comparison to the control group. Therefore, it was recognized that PJJ-34 (1) showed strong anti-initiating activity as well as anti-promoting activity. After all, 1 seems to be useful as cancer-chemopreventive agent.     

Synthesis and biological activity profile of new analogues of the cyclic opioid peptide H-Tyr-c[D-Cys-Gly-Phe(pNO2)-D-Cys]-NH2 containing (S)-alpha-hydroxymethylcysteine in place of cysteine.

To examine the effect on biological activity of replacing D-Cys in the opioid peptide H-Tyr-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]-NH(2) in position 2 or/and 5 with alpha-hydroxymethylcysteine (alpha-Hmc), three analogues were synthesized. These compounds exhibit agonist activity at both mu and delta receptors. However, the most active analogue, with (S)-alpha-Hmc residue in position 5, was 3360- and 2190-fold less active than the parent peptide in the GPI and MVD assays, respectively.     

The structure of the muscular and nervous systems of the male Intoshia linei (Orthonectida)

Orthonectida is a small group of parasites, which, according to recent studies, may be phylogenetically close to Annelida. Here, we describe the musculature and serotonin-like immunoreactive (SLIR) nervous system of male adults of Intoshia linei (Orthonectida) using immunohistochemistry and confocal laser scanning microscopy. The whole muscular system consists of four outer longitudinal and eight pairs of inner semicircular muscle fibres. Immunohistochemistry revealed six serotonin-like cells at the anterior part of the body, and two backward lateral longitudinal nerves, merging at the posterior end. Compared to females, the organization of the nervous system is modified and its progenetic origin seems unlikely. The general neuromuscular organization corresponds to the pattern of small-sized annelids, suggesting their possible phylogenetic affinity. Free-living males and females of the orthonectid I. linei may present a good example of a highly simplified Bilaterian with fully functioning nervous and muscular systems. This simplicity is secondary and is caused by two factors—the parasitic life style and miniaturization of free-living sexual stages.     

Structure and hydration of the amylopectin trisaccharide building blocks--Synthesis, NMR, and molecular dynamics

To gain insight into the molecular details and hydration of amylopectin, the five constituting trisaccharides have been chemically synthesized as their methyl alpha-glycosides. All five trisaccharides were subjected to 950 MHz NMR spectroscopy for complete assignment and nanosecond molecular dynamics trajectories were calculated to study the structure and dynamics of the trisaccharides in aqueous solution. Systematic analysis of the simulation data revealed several examples of bridging water molecules playing an important role in the stabilization of specific amylopectin conformations, which was also supported by the experimental NMR data such as interresidue NOE's and heteronuclear scalar couplings between nuclei from neighboring residues. Although alpha-maltotriose, alpha-iso-maltotriose, alpha-panose and alpha-isopanose are relatively well characterized structures, the study also includes one less characterized trisaccharide with the structure alphaGlcp(1-->4)alphaGlcp(1-->6)alphaGlcp. This trisaccharide, tentatively labelled alpha-forkose, is located at the branch point of amylopectin, forking the amylopectin into two strands that align into double-helical segments. The results show that the conformation of alpha-forkose takes a natural bend form which fits well into the structure of the double-helical segment of amylopectin. As the only trisaccharide in this study the structure of alpha-forkose is not significantly influenced by the hydration. In contrast, alpha-isopanose takes a restricted, but rather extended form due to an exceptionally strong localized water density. The two homo-linkage oligomers, alpha-maltotriose and alpha-iso-maltotriose, showed to be the most extended and the most flexible trimers, respectively, providing regular structure for crystalline domains and maximum linker flexibility for amorphous domains.     

Soluble tubulin complexes in oocytes of the common leopard frog, Rana pipiens, contain gamma-tubulin

Oocytes of the leopard frog, Rana pipiens, contain soluble tubulin which was previously shown to exist predominantly in megadalton (MDa) fractions and that fails to readily assemble in vitro. In order to further characterize these tubulin complexes, DEAE Sepharose chromatography, Sephacryl S-300 size exclusion columns and specific immunoprecipitation were used. The results revealed the presence of alpha-, beta-, and gamma-tubulin associated with several other proteins in the soluble fraction of Rana pipiens ovarian oocytes. These Rana oocyte tubulin complexes appear to be analogous to those recently reported in Xenopus ovulated eggs as gamma-tubulin ring complexes. This seems true since both size (estimates, i.e. approximately 2MDa) and protein components are similar. Furthermore, both alpha- and gamma-tubulin antibodies immunoprecipitated identical protein bands from Rana oocyte soluble fraction. These putative Rana gamma-tubulin ring proteins include 107, 97, 95, 90 and 75 kDa components which are similar in size to those found in Xenopus and other species. Rana appears to belong to a select group in which gamma-tubulin complexes contain significant alpha- and beta-tubulin (i.e., Xenopus and sheep), while other species such as Drosophila, Aspergillus, Saccharomyces, human cells and many other mammalian cells tested lack the other tubulin components. The heterogeneity in both size and protein components of Rana oocyte gamma-tubulin ring complexes may reflect different states of tubulin complex assembly. The lower vertebrate oocyte is hypothesized to act as a repository and prestaging point for the assembly of gamma-tubulin ring complexes which will become the maternal contribution to the centrosomes of the embryo. While the gamma-tubulin ring complexes of vertebrate eggs have been described previously, this is the first report biochemically characterizing soluble gamma-tubulin complexes in vertebrate ovarian oocytes.     

Disruption of the beta-sheet structure of a protected pentapeptide, related to the beta-amyloid sequence 17-21, induced by a single, helicogenic C(alpha)-tetrasubstituted alpha-amino acid.

Fifteen years ago it was shown that an alpha-aminoisobutyric acid (Aib) residue is significantly more effective than an L-Pro or a D-amino acid residue in inducing beta-sheet disruption in short model peptides. As this secondary structure element is known to play a crucial role in the neuropathology of Alzheimer's disease, it was decided to check the effect of Aib (and other selected, helix inducer, C(alpha)-tetrasubstituted alpha-amino acids) on the beta-sheet conformation adopted by a protected pentapeptide related to the sequence 17-21 of the beta-amyloid peptide. By use of FT-IR absorption and 1H NMR techniques it was found that the strong self-association characterizing the pentapeptide molecules in weakly polar organic solvents is completely abolished by replacing a single residue with Aib or one of its congeners.