Fine structure of the Caenorhabditis elegans secretory-excretory system

The secretory-excretory system of C. elegans, reconstructed from serial-section electron micrographs of larvae, is composed of four cells, the nuclei of which are located on the ventral side of the pharynx and adjacent intestine. (1) The pore cell encloses the terminal one-third of the excretory duct which leads to an excretory pore at the ventral midline. (2) The duct cell surrounds the excretory duct with a lamellar membrane from the origin of the duct at the excretory sinus to the pore cell boundary. (3) A large H-shaped excretory cell extends bilateral canals anteriorly and posteriorly nearly the entire length of the worm. The excretory sinus within the cell body joins the lumena of the canals with the origin of the duct. (4) A binucleate, A-shaped gland cell extends bilateral processes anteriorly from cell bodies located just behind the pharynx. These processes are fused at the anterior tip of the cell, where the cell enters the circumpharyngeal nerve ring. The processes are also joined at the anterior edge of the excretory cell body, where the excretory cell and gland are joined to the duct cell at the origin of the duct. Secretory granules may be concentrated in the gland near this secretory-excretory junction. Although the gland cells of all growing developmental stages stain positively with paraldehyde-fuchsin, the gland of the dauer larva stage (a developmentally arrested third-stage larva) does not stain, nor do glands of starved worms of other stages. Dauer larvae uniquely lack secretory granules, and the gland cytoplasm is displaced by a labyrinth of large, transparent spaces. Exit from the dauer stage results in the return of active secretory morphology in fourth-stage larvae.     

Physiological compensation in unilateral eyestalk ablated crayfish, Cherax quadricarinatus

The eyestalks of crustaceans contain neurosecretory cells involved in the regulation of molting. In crayfish, bilateral ablation results in increased molting frequency and weight gain whereas unilateral ablation typically has no effect on molting frequency and weight gain. The effects of unilateral ablation were examined in juvenile Australian freshwater crayfish, Cherax quadricarinatus. As observed for other crayfish species, molting frequency and weight gain of unilateral ablated crayfish were not significantly different from control (intact) crayfish. Survival of unilateral ablated crayfish, however, was reduced compared to controls and was likely due to stress associated with the surgical procedure itself. Using radiolabeling techniques, protein synthesis was determined for neural tissues from the remaining eyestalk of ablated crayfish and compared to protein synthesis of neural tissues from eyestalks of control, non-ablated crayfish. Protein synthesis of ablated crayfish neural tissues was significantly higher (ca. 45%) than protein synthesis of control neural tissues. Electrophoretic analysis (SDS-PAGE and autoradiography) further demonstrated that protein synthesis increased linearly for all proteins in the remaining eyestalk of ablated crayfish. Together, these results suggest that a compensatory response occurred in unilateral ablated crayfish allowing normal physiological functions, particularly those involved in regulating growth cycles, to be maintained. J. Exp. Zool. 289:184-189, 2001.     

Lipocalin signaling controls unicellular tube development in the Caenorhabditis elegans excretory system

Unicellular tubes or capillaries composed of individual cells with a hollow lumen perform important physiological functions including fluid or gas transport and exchange. These tubes are thought to build intracellular lumina by polarized trafficking of apical membrane components, but the molecular signals that promote luminal growth and luminal connectivity between cells are poorly understood. Here we show that the lipocalin LPR-1 is required for luminal connectivity between two unicellular tubes in the Caenorhabditis elegans excretory (renal) system, the excretory duct cell and pore cell. Lipocalins are a large family of secreted proteins that transport lipophilic cargos and participate in intercellular signaling. lpr-1 is required at a time of rapid luminal growth, it is expressed by the duct, pore and surrounding cells, and it can function cell non-autonomously. These results reveal a novel signaling mechanism that controls unicellular tube formation, and provide a genetic model system for dissecting lipocalin signaling pathways.     

Post-infection Development and Morphology of Meloidogyne cruciani

The development and life stages of Meloidogyne cruciani on tomato was studied at 28 C. Roots of 2-wk-old ''Rutgers'' tomato seedlings were exposed to inoculum for 24 h, rinsed, and the seedlings repotted. No major changes in juvenile development were observed prior to 8 days after inoculation. At 11 days the second-stage juvenile had enlarged considerably. The genital primordium had not yet asumed the V-shape characteristic of developing females, but the presence of rectal glands identified the juveniles as females. At this time (11 days), two additional, previously undescribed esophageal lobes were first observed; they were adjacent to the dorsal and subventral glands. After molting from second to third stage, the stylet cone, shaft, and the lumen of the stylet knobs are shed and remain attached to the second-stage cuticle. The excretory duct of the third-stage juveniles was directed anteriorly from the excretory pore of the second-stage cuticle and appear attached to the body wall of the third-stage juveniles opposite the procorpus. At 19 days after inoculation, the last molt took place. The adult female possessed a new stylet, a large five-gland esophagus, a prominent excretory system ending in a unicellular gland and a fully developed reproductive system.     

Genes required for osmoregulation and apical secretion in Caenorhabditis elegans

Few studies have investigated whether or not there is an interdependence between osmoregulation and vesicular trafficking. We previously showed that in Caenorhabditis elegans che-14 mutations affect osmoregulation, cuticle secretion, and sensory organ development. We report the identification of seven lethal mutations displaying che-14-like phenotypes, which define four new genes, rdy-1-rdy-4 (rod-like larval lethality and dye-filling defective). rdy-1, rdy-2, and rdy-4 mutations affect excretory canal function and cuticle formation. Moreover, rdy-1 and rdy-2 mutations reduce the amount of matrix material normally secreted by sheath cells in the amphid channel. In contrast, rdy-3 mutants have short cystic excretory canals, suggesting that it acts in a different process. rdy-1 encodes the vacuolar H+-ATPase a-subunit VHA-5, whereas rdy-2 encodes a new tetraspan protein. We suggest that RDY-1/VHA-5 acts upstream of RDY-2 and CHE-14 in some tissues, since it is required for their delivery to the epidermal, but not the amphid sheath, apical plasma membrane. Hence, the RDY-1/VHA-5 trafficking function appears essential in some cells and its proton pump function essential in others. Finally, we show that RDY-1/VHA-5 distribution changes prior to molting in parallel with that of actin microfilaments and propose a model for molting whereby actin provides a spatial cue for secretion.     

Morphology of the excretory system of Hysterothylacium haze (Nematoda: Anisakidae: Raphidascaridinae)

The morphology of the excretory system of Hysterothylacium haze was examined by serial histological sections. The excretory system was H-shaped and glandular, consisting of lateral filaments and a commissure, with the exretory pore opening posterior to the nerve ring. A large excretory nucleus was present in the left filament. The cuticularized excretory duct was confined to the left side of the commissure. The glandular excretory system is rare among the Raphidascaridinae.     

Distinct immunohistochemical expression of osteopontin in the adult rat major salivary glands

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70–100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the compostion of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell–cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.Nicholas Obermüller and Nikolaus Gassler contributed equally to this work.     

Q开关Nd:YAG 1064nm和532nm激光对犬心肌切割作用的研究

目的 :研究 10 64nm和 53 2nm波长激光在激光能量为 14 0mJ/pulse(脉冲 )时对犬心肌切割效率。方法 :用Q开关Nd :YAG 10 64和 53 2nm波长脉冲激光分别照射犬离体和在体心肌组织 ,光学显微镜和偏振光学显微镜行组织学分析 ,观察不同条件下激光切割组织的深度和光热对组织的损伤。结果 :离体和在体实验 ,10 64nm波长激光的切割效率高于 53 2nm(p <0 .0 1)。在体和离体实验显示 10 64nm激光能量和重复率相同时 ,所致的切割效率无明显差异 (p >0 .0 5) ,血液对 10 64nm激光的切割效率影响较小。相反 ,在 53 2nm时血液对其影响较大 ,相同的激光能量和重复率 ,离体实验切割效率高于在体 (p <0 .0 1)。 10 64nm激光所致的光热和机械损伤均轻于 53 2nm激光。结论 :在切割效率方面 ,10 64nm激光比 53 2nm更适用于TMLR。 10 64nmQ开关Nd :YAG激光可通过光导纤维传输 ,是TMLR的一个有潜力的激光源     

A biochip platform for cell transfection assays

In this paper, we describe the development and characterization of a biochip platform for cell transfection assays. Silicon wafers were surface modified by plasma polymerization of allylamine plasma polymer (ALAPP) and grafting of a protein-resistant layer of poly(ethylene oxide) (PEO) on the plasma polymer surface. Excimer laser ablation was then used to pattern ALAPP-PEO coated samples for spatially controlled protein adsorption and subsequent cell attachment. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface modifications before and after excimer laser ablation. Experiments confirmed the creation of a two-dimensionally controlled surface chemistry on the biochip. Cell culture experiments using human embryonic kidney (HEK 293) cells showed that cells attached exclusively to laser ablated areas. In addition, cells confined to ablated areas were successfully transfected with plasmid DNA containing the gene for green fluorescent protein (GFP). The cell transfection efficiencies of cells growing in a culture flask and cells confined on the biochip were determined to be 21 and 13%, respectively.     

Evaluation of bone healing following Er:YAG laser ablation in rat calvaria compared with bur drilling

The Er:YAG laser is currently used for bone ablation. However, the effect of Er:YAG laser irradiation on bone healing remains unclear. The aim of this study was to investigate bone healing following ablation by laser irradiation as compared with bur drilling. Rat calvarial bone was ablated using Er:YAG laser or bur with water coolant. Er:YAG laser effectively ablated bone without major thermal changes. In vivo micro‐computed tomography analysis revealed that laser irradiation showed significantly higher bone repair ratios than bur drilling. Scanning electron microscope analysis showed more fibrin deposition on laser‐ablated bone surfaces. Microarray analysis followed by gene set enrichment analysis revealed that IL6/JAK/STAT3 signaling and inflammatory response gene sets were enriched in bur‐drilled bone at 6 hours, whereas the E2F targets gene set was enriched in laser‐irradiated bone. Additionally, Hspa1a and Dmp1 expressions were increased and Sost expression was decreased in laser‐irradiated bone compared with bur‐drilled bone. In granulation tissue formed after laser ablation, Alpl and Gblap expressions increased compared to bur‐drilled site. Immunohistochemistry showed that osteocalcin‐positive area was increased in the laser‐ablated site. These results suggest that Er:YAG laser might accelerate early new bone formation with advantageous surface changes and cellular responses for wound healing, compared with bur‐drilling.      

Ultrastructure of the excretory system of the marine nematode Monhystera disjuncta

The excretory system of Monhystera disjuncta is a single ventral gland in the pharyngo-intestinal region. Its ultrastructural morphology is described. The posterior part is swollen, contains the nucleus and many secretory granules. This part gradually narrows anteriorly to form the cell neck, in the apical part of which a valve structure is differentiated. This pear-shaped valve structure opens into a cuticular duct which is embedded in an accompanying cytoplasmic sheath. The cuticular duct opens to the exterior by a pore between the two subventral inner labial papillae.     

The effects of adrenalectomy on the harderian gland of the Wistar albino rats

Harderian glands of the Wistar albino rats normal and adrenalectomized were investigated by light microscopy. In normal, these glands have a tubuloalveolar structure. The gland is located in the medio posterior aspect of the orbit. It is lobulated and appears homogeneous in colour and texture. Harderian gland consist of tubules with wide lumina lined by a single layer of columnar epithelial cells surrounded by myoepithelial cells within their basal lamina. It contains porphyrin pigment which is stored as solid intraluminal deposits. The glandular epithelium possesses two cell types, termed A and B. Type A cells are more numerous. The single excretory duct of the gland is directly continuous with endpieces at the hilus and opens nasally and ventrally to the third eyelid. The excretory duct is accompanied by many acini of small serous glands around it. The tubuloalveoli of the gland is not divided into lobules. There is no branched duct system within the gland. The secretion seems to be associated with porphyrins, is essentially released by exocytosis, but holocrine secretion also occurs. The single excretory duct is lined by a stratified epithelium. The gland is surrounded by a collagenous capsule. The adrenalectomy, caused degenerative changes in the glands. Epithelial height was lower than in normal gland epithelium. Most of the acini were completely disorganised. The acinar lumina were filled with porphyrin debris. The results suggest that rat harderian glands are sensitive to adrenal androgen changes in both male and female rats.     

Label-retaining cells in the rat submandibular gland.

To identify stem cells in salivary glands, label-retaining cells (LRCs) were established in rat submandibular glands. Developing and regenerating glands were labeled with bromodeoxyuridine (BrdU). To cause gland regeneration, the glands were injured by duct obstruction. BrdU LRCs were observed in all the parenchymal structures except for the acinus of the glands labeled during regeneration. Among these LRCs, a few, but not many, expressed neither keratin18 (K18; an acinar/duct cell marker) nor alpha-smooth muscle actin (alphaSMA; a myoepithelial cell marker), and thus were putative stem cells. These (K18 and alphaSMA)(neg) LRCs were invariably observed in the intercalated duct and the excretory duct. In the intercalated duct, they were at the proximal end bordering the acinus (the neck of the intercalated duct). Next, to test the above identification, gland extirpation experiments were performed. LRCs were established by labeling developing glands with iododeoxyuridine (IdU) in place of BrdU. Removal of one submandibular gland forced the IdU-LRCs in the remaining gland to divide. They were labeled with chlorodeoxyuridine (CldU). The (K18 and alphaSMA)(neg) LRCs in the neck of the intercalated duct and in the excretory duct did not change in number or in IdU label. The CldU label appeared in these cells and then disappeared. These results indicate that the (K18 and alphaSMA)(neg) LRCs have divided asymmetrically and are thus considered salivary gland stem cells.     

Ectodermal interactions during neurogenesis in the glossiphoniid leech Helobdella triserialis

Morphogenetic cell interactions during development were studied by combining cell ablation and cell lineage tracing techniques in embryos of the leech Helobdella triserialis. Ablation of an identified ectodermal teloblast, or teloblast precursor blastomere, on one side of an early embryo was often found to result in the later abnormal migration of the progeny cells of the corresponding contralateral, nonablated teloblast to the ablated side of the embryo; such abnormal migration was termed “midline violation.” Two different kinds of midline violation were observed. Crossover: after ablation of an N teloblast individual stem cell progeny of the contralateral N teloblast sometimes cross the ventral midline of the germinal plate of the embryo. Switching: after ablation of an OPQ teloblast precursor bandlets of stem cells produced by the contralateral O, P, or Q teloblasts sometimes switch to the germinal band of the ablated side at the site of origin of the germinal bands. The occurrence of crossover and switching shows that the eventual site occupied by a progeny cell of a particular teloblast is not automatically determined by its lineage, but also depends on interactions with other cells. Midline violation in the leech embryo CNS does not constitute true regulation, however, since the restoration of neurons to the ablated side is accompanied by a neuron deficit on the nonablated side. The occurrence of the two distinct kinds of midline violation, crossover and switching, may be explained by the relative position of the stem cell bandlets within the germinal bands, and by the geometrical features of the formation of the germinal plate from the germinal bands.     

Cell migration in the formation of the pronephric duct in Xenopus laevis

To determine if cell migration is involved in the formation of the pronephric duct in Xenopus, we used morphometry, ablation, and videomicroscopy of vitally stained cells to study duct formation. In St 23-24 (Nieuwkoop and Faber, 1956) embryos, a ridge of cells forms caudal to the pronephric rudiment. The ridge lengthens at approximately the same rate as the embryonic trunk from St 23 to St 31. Ablation experiments demonstrated that the ridge constitutes the pronephric duct rudiment (PDR); when the ridge was ablated at St 23-24, little or no duct formation occurred, whereas a duct formed when the pronephric rudiment was ablated and the ridge left intact. Vital dye injections showed that the PDR forms from the intermediate mesoderm ventral to myotomes IV-VIII. From St 29/30 to St 33/34, the PDR actively elongates along the ventral edge of the myotomes as far as myotome XIV, where it joins the cloaca as the pronephric duct. Videomicroscopy of vitally stained cells showed that the PDR elongates throughout its length and does not incorporate additional cells from the mesoderm over which it elongates. The results strengthen the case for a common mode of pronephric duct formation among amphibian species.     

Histology and mucosubstance histochemistry of ferret lingual glands.

The histology and mucosubstance histochemistry of the ferret lingual glands were studied. Both serous and mucous minor salivary glands were present in the posterior part of the tongue. In serous glands, acinar cells and a very few cells of the excretory ducts contained granules which gave reactions for neutral mucopolysaccharides only. The mucous glands, including the duct system, contained mainly weakly sulphated acidic mucin, some neutral mucin but no carboxylated mucin. Occasional goblet cells were present in the excretory ducts of both serous and mucous glands. They contained weakly sulphated mucin.     

Chemical screening reveals Ronidazole is a superior prodrug to Metronidazole for nitroreductase-induced cell ablation system in zebrafish larvae

The Metronidazole (MTZ)/nitroreductase (NTR)-mediated cell ablation system is the most commonly used chemical-genetic cell ablation method in zebrafish. This system can specifically ablate target cells under spatial and temporal control. The MTZ/NTR system has become a widely used cell ablation system in biological, developmental, and functional studies. However, the inadequate cell-ablation ability of some cell types and the side effects of high concentration MTZ impede extensive applications of the MTZ/NTR system. In the present study, the US drug collection library was searched to extend the NTR system. Six MTZ analogs were found, and the cell-ablation ability of these analogs was tested in zebrafish larvae. The results revealed that two of the NTR substrates, Furazolidone and Ronidazole, ablated target cells more efficiently than MTZ at lower concentrations. Furthermore, the working concentration of Ronidazole, but not Furazolidone and MTZ, did not affect axonal bridge formation during spinal cord regeneration. Our results, taken together, indicate that Ronidazole is a superior prodrug to MTZ for the NTR system, especially for the study of neuron regeneration in zebrafish larvae.