Three-Dimensional Culture of Rhipicephalus (Boophilus) microplus BmVIII-SCC Cells on Multiple Synthetic Scaffold Systems and in Rotating Bioreactors

Tick cell culture facilitates research on the biology of ticks and their role as vectors of pathogens that affect humans, domestic animals, and wildlife. Because two-dimensional cell culture doesn’t promote the development of multicellular tissue-like composites, we hypothesized that culturing tick cells in a three-dimensional (3-D) configuration would form spheroids or tissue-like organoids. In this study, the cell line BmVIII-SCC obtained from the cattle fever tick, Rhipicephalus (Boophilus) microplus (Canestrini, 1888), was cultured in different synthetic scaffold systems. Growth of the tick cells on macrogelatinous beads in rotating continuous culture system bioreactors enabled cellular attachment, organization, and development into spheroid-like aggregates, with evidence of tight cellular junctions between adjacent cells and secretion of an extracellular matrix. At least three cell morphologies were identified within the aggregates: fibroblast-like cells, small endothelial-like cells, and larger cells exhibiting multiple cytoplasmic endosomes and granular vesicles. These observations suggest that BmVIII-SCC cells adapted to 3-D culture retain pluripotency. Additional studies involving genomic analyses are needed to determine if BmVIII-SCC cells in 3-D culture mimic tick organs. Applications of 3-D culture to cattle fever tick research are discussed.

The Two Conserved Cysteine Residues of the Triple Gene Block Protein 2 Are Critical for Both Cell-to-Cell and Systemic Movement of Bamboo mosaic virus

The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which is known to be required for the cell-to-cell movement of potexviruses. This protein has two conserved Cys residues, Cys-109 and Cys-112, at its C-terminal tail, which is supposed to be exposed on the outer surface of the endoplasmic reticulum (ER) membrane and ER-derived granular vesicles. In this study, we investigated the importance of these two Cys residues on the cell-to-cell and systemic movement of BaMV. Our results indicate that the Cys-to-Ala substitutions in TGBp2 make the cell-to-cell movement of BaMV relatively inefficient and the systemic movement of BaMV severely inhibited. Moreover, the defect in systemic movement is attributed to the inefficient transport of viral RNA in the phloem of petiole. Clearly, TGBp2 is critical not only for the cell-to-cell but also for the systemic movement of BaMV. In addition, the conserved Cys residues are important for the functioning of TGBp2.

Mutations in the Central Domain of Potato Virus X TGBp2 Eliminate Granular Vesicles and Virus Cell-to-Cell Trafficking

ABSTRACT Most RNA viruses remodel the endomembrane network to promote virus replication, maturation, or egress. Rearrangement of cellular membranes is a crucial component of viral pathogenesis. The PVX TGBp2 protein induces vesicles of the granular type to bud from the endoplasmic reticulum network. Green fluorescent protein (GFP) was fused to the PVX TGBp2 coding sequence and inserted into the viral genome and into pRTL2 plasmids to study protein subcellular targeting in the presence and absence of virus infection. Mutations were introduced into the central domain of TGBp2, which contains a stretch of conserved amino acids. Deletion of a 10-amino-acid segment (m2 mutation) overlapping the segment of conserved residues eliminated the granular vesicle and inhibited virus movement. GFP-TGBp2m2 proteins accumulated in enlarged vesicles. Substitution of individual conserved residues in the same region similarly inhibited virus movement and caused the mutant GFP-TGBp2 fusion proteins to accumulate in enlarged vesicles. These results identify a novel element in the PVX TGBp2 protein which determines vesicle morphology. In addition, the data indicate that vesicles of the granular type induced by TGBp2 are necessary for PVX plasmodesmata transport.

Cleavage in vivo and in vitro in the Marsupial Macropus eugenii

In the tammar wallaby, transport down the oviduct takes less than 24 h after fertilization and a mucoid coat is deposited within a few hours of fertilization, with excess spermatozoa trapped in the mucoid layer. The mucin coat thickens as the zygote passes down the oviduct. A proteinaceous shell is laid down outside the mucin coat in the utero-tubal region of the tract. The fertilized zygote enters the uterus in the pronuclear stage with cleavage proceeding in the uterus. In vivo, the first cleavage takes place two days post coitum (p.c.) (approximately 24 h after ovulation) but the next three cleavage stages may be completed within 24 h (between 48 h and 72 h p.c.). Thus, cell-doubling time appears to be around 8 h for 2-8-cell stages. Cleavage in vitro can occur with, or without, the shell membrane. Cleavage in early embryos of the tammar in vitro is slower than that occurring in vivo, and in vitro there may be a '4-cell block' in early development, as in dasyurids. The pattern of cleavage differs markedly from that of dasyurid marsupials in that there is no extrusion of yolk material from the cells and no separation of the blastomeres during the first cleavage stages to the 8-cell stage. The blastomeres are characterized by numerous vesicular structures and lipid droplets, but no yolk bodies. Polarity is not marked in early cleavage, but by the 8-cell stage polarity has developed with surface microvilli and numerous granular vesicles and mitochondria in the cortical regions at one pole of the cells, but sparse microvilli on the inner surfaces and at the other pole. There are complex intervillous interdigitations of microvilli between cells. However, clear identification of cells as pluriblast or trophoblast cells is not possible up to the 8-cell stage examined. These results demonstrate that this macropodid marsupial has a distinctive pattern of early development which differs from that of Didelphis and of the dasyurid marsupials so far described.

Ultrastructural localization of nitric oxide synthase in canine small intestine and colon

The ultrastructural distribution and subcellular localization of nitric oxide synthase (NOS) immunoreactivity and its possible colocalization with vasoactive intestinal polypeptide (VIP) and substance P in the muscularis externa in canine ileum and colon were studied by using polyclonal antisera raised against VIP, substance P, and cerebellar NOS. Immunogold staining, with or without silver enhancement, was carried out directly on ultrathin sections using single and two-faced double immunogold methods. NOS immunoreactivity was observed in nerve profiles in myenteric plexus and circular muscle layer. Immunoreactivity was occasionally detected in smooth muscle cells and interstitial cells of Cajal. The double immunostaining revealed NOS and VIP in the same nerve varicosities but never in the same organelles. NOS was localized in electron-dense material of undetermined nature, whereas VIP was associated with large granular vesicles. Substance P and NOS were never found in the same nerves. These results indicate that NOS is present in the enteric nerves containing VIP but in different organelles and that nitric oxide release probably does not occur by an exocytotic mechanism.

Innervation of interstitial cells of Cajal by vasoactive intestinal polypeptide containing nerves in canine colon

The hypothesis was tested, through structural and functional studies, that interstitial cells of Cajal receive and can respond to direct innervation from nerves containing the vasoactive intestinal polypeptide neuromediator. The submucosal network of interstitial cells of Cajal has been postulated to provide pacemaking activity for the circular muscle and to be involved in neurotransmission from noradrenergic, noncholinergic nerves for which vasoactive intestinal polypeptide is a putative mediator. The distribution of vasoactive intestinal polypeptide and substance P immunoreactive material in nerve profiles of the enteric nervous system of the canine colon was examined. In addition, electrophysiological studies were done on the interstitial cells bordering the submucosal side of the circular muscle layer after they were electrically isolated using heptanol. The vasoactive intestinal polypeptide immunoreactivity, located exclusively in nerve large granular vesicles, was found throughout the enteric nervous system (myenteric plexus, submucous plexus, and circular muscle – submucosa interface). The highest proportion (38% compared with 22–24%) of profiles of large granular vesicles with vasoactive intestinal polypeptide immunoreactivity was found in nerve profiles of the circular muscle – submucosa interface. In contrast, substance P immunoreactivity was found in nerve profiles of myenteric plexus (33% of large granular vesicles were positive) but not associated with submucosal interstitial cell nerve network. The vasoactive intestinal polypeptide hyperpolarized interstitial cells by 9 mV when electrically isolated by 1 mM heptanol and markedly reduced (about 50%) their input membrane resistance. We conclude that the distribution of vasoactive intestinal polypeptide immunoreactivity and its action are consistent with a postulated role of the interstitial cells as a major site of neurally mediated inhibition of colonic pacemaker activity.Key words: enteric nervous system, interstitial cells of Cajal, inhibitory junction potential, nonadrenergic noncholinergic nerves.

The Fine Structure of Intraepithelial Free Nerve Endings in the Canine Vocal Cord Mucosa

Innervation of the epithelium on the undersurface of the canine vocal cords was investigated by transmission electron microscopy. In the subepithelial lamina propria, nerve bundles containing unmyelinated fibers were observed. The nerve bundles, encircled by basal lamina, were enclosed by a thin connective tissue layer and by flattened flbroblast-like cells. With nerve bundles approaching the epithelium, the axons divided repeatedly and entered the epithelial layer. In the epithelial cells, nerve axons formed knob-like swellings that contained a small number of large granular vesicles and a large number of small agranular vesicles. A sensory function responsive to irritant chemical stimuli and to mechanical stimuli is presumed for these vesicle-containing nerve processes.

Occurrence of FMRF amide-like immunoreactive nerve fibers in guinea pig small intestine.

We investigated the distribution of FMRF amide-like immunoreactivity in the small intestine of the guinea pig. Immunoreactive nerve fibers were found mainly in the myenteric and submucous plexuses and in the inner circular muscle layer. The labeled processes contained variable proportions of small clear vesicles 30-40 nm in diameter and large granular vesicles 80-120 nm in diameter. The large granular vesicles showed heavy immunoreactivity. The antisera against FMRF amide crossreact with peptides belonging to the pancreatic polypeptide family; it has therefore been suggested that the FMRF amide immunoreactivity demonstrated in the small intestine is caused by a peptide that is biosynthetically related to, but not necessarily a member of, the pancreatic polypeptide family.

Structural studies of innervation on nonpregnant rat uterus

Whole-mount preparations of the uterus and mesentery from nonpregnant rats were examined after staining with glyoxylic acid or acetylcholinesterase to demonstrate the innervation. Some uterine tissues were also evaluated by electron microscopy. Glyoxylic acid fluorescent nerves were present in the mesentery, mesometrium, and uterine wall exclusively around blood vessels. Acetylcholinesterase positive nerve fibers from Frankenhauser's plexus (cervical ganglia) were associated with blood vessels and muscle in the uterine wall but not in the mesentery. Electron microscopy revealed nerve varicosities with granular vesicles associated with blood vessels and varicosities with agranular vesicles located near blood vessels and muscle cells. Treatment of animals for 3 days with 5- and 6-hydroxydopamine, respectively, increased granular size and damaged the varicosities with granular vesicles but did not change nerves with agranular vesicles or induce the presence of gap junctions. The results of this study suggest that blood vessels in the uterus are highly innervated by both adrenergic and other types of nerves that probably control blood flow. Nonadrenergic but not adrenergic nerves may also directly control myometrial contractility.