Hyperplastic cellular components of a hemangiopericytoma. An ultrastructural study

Based on ultrastructural features of cellular components of a hemangiopericytoma, hyperplastic cells are classifiable into fibroblast-like (group I), endotheloid (group II) and pericyte-like (group III) cells. The transformation of the group I cells to the group II, or to the group III cells, is pronounced in our electron micrographs and this may imply that the group I cell is the principal cell of origin in this neoplasm. The smooth muscle-like (group IV) cells comprising the media of the arteries and veins in this neoplasm may represent modified, possibly de-differentiated smooth muscle cells reacted to the neoplastic proliferation of the surrounding adventitial (group I) cells.     

Role of multipotent fibroblasts in the healing colonic mucosa of rabbits. Ultrastructural and immunocytochemical study.

Light- and electron microscopy and immunocytochemistry were used to study the healing colonic mucosa of rabbits after experimental excision. Between 3 and 5 days, abundant young fibroblasts which retained many features of mesenchymal cells invaded the growing capillaries into the loose connective tissue of the healing colonic mucosa. Our electron microscopy revealed the transformation of these young fibroblasts into smooth muscle cells, into histiocyte-like cells involved in phagocytotic activity, and into vasoformative cells incorporated into the growing capillaries. The mitotic proliferation of pre-existing smooth muscle cells at the ulcer margin did not seem to be the major reason for re-establishment of the muscular tissue. The present immunocytochemistry revealed an active production of fibronectin in rough endoplasmic reticulum in the young fibroblasts. This may mean that this glycoprotein is involved in the re-establishment of both connective and muscular tissues by enhancement of adhesion and chemoattractant activities of such cells. In addition, the immunoreaction of endothelial cells of the growing capillaries suggests a role of this glycoprotein in the acceleration of the neocapillarization.     

Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells.

An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin II or III. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP753) but not by CGP42112A. In parallel, a transient and dose-dependent induction of c-fos was demonstrated not only with angiotensins II and III but also with angiotensin I. Both angiotensins II and III exerted their maximal effect at 1 microM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-fos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin II, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins II and III act directly on smooth muscle cells.     

Characterization of three different flavodoxins from Azotobacter vinelandii

The flavodoxins from Azotobacter vinelandii cells grown N2-fixing and from cells grown on NH4OAc have been purified and characterized. The purified flavodoxins from these cells are a mixture of three different flavodoxins (Fld I, II, III) with different primary structures. The three proteins were separated by fast protein liquid chromatography; Fld I eluted at 0.38 M KCl, Fld II at 0.43 M KCl and Fld III at 0.45 M KCl. The most striking difference between the three flavodoxins was the midpoint potential (pH 7.0, 25 degrees C) of the semiquinone/hydroquinone couple, which was -320 mV for Fld I and -500 mV for the other two flavodoxins (Fld II and Fld III). All three flavodoxins were present in cells grown on NH4OAc. In cells grown on N2 as N source only Fld I and Fld II were found. The concentration of Fld II was 10-fold higher in N2-fixing cells than in cells grown on NH4OAc. Evidence has been obtained that Fld II is involved in electron transport to nitrogenase. As will be discussed, our observation that preparations of Azotobacter flavodoxin are heterogeneous, has consequences for the published data.     

A method of quantitating Paneth cell metaplasia of the stomach by image analysis

Paneth cells are one of the histologic components of intestinal metaplasia of the stomach, as are mucin-producing goblet cells. With the aid of an image quantifier, the distribution of Paneth cells histochemically labeled with acid fuchsin was analyzed for a gastrectomy specimen containing an adenocarcinoma of the intestinal type; the topographic distribution of goblet cells histochemically labeled with Alcian blue (pH 2.5) was also analyzed. The specimen was cut into 63 blocks (0.5 X 4.0 cm) in four zones; antrum (zone I), intermediate region (zone II) and fundus (zones III and IV). Paneth cells were found only in sections containing mucin-producing goblet cells. Paneth cells were found in 12.5% of the 16 sections from the antral zone I containing Alcian blue-positive goblet cells. The rates were 44.4% for the intermediate zone II and 55.5% for the distal fundic zone III. The total area occupied by Paneth cells was significantly lower in the gastric mucosa as compared to the duodenal mucosa. The "Paneth cell index" (total Paneth cell area/total goblet cell area) was highest in the duodenum, followed by the distal fundic zone III. This method of quantitating Paneth cell metaplasia of the stomach will be used to investigate the topographic distribution of those cells in populations with low and high incidences of intestinal metaplasia.     

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5β1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III(1), III(13), and III(14)), with III(13) and III(14) assumed to be the major sites. The interaction between III(2-3) and III(12-14) was inhibited by DP, whereas the interaction between I(1-5) and III(12-14) was specifically and strongly enhanced by DP. Because the interaction between III(2-3) and III(12-14) is involved in forming a globular conformation of fibronectin, and that between I(1-5) and III(12-14) is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.     

Immunochemical characterization and cellular localization of pepsinogens in cat and dog.

The antigenic relationships and cellular localization of cat and dog pepsinogens were investigated by electrophoretic analysis, immunodiffusion, immunoelectrophoresis, immunoabsorption, and by immunofluorescence, respectively. Rabbit antiserum to human and hog group I (Pg I) and group II pepsinogens (Pg II) had been previously prepared. Electrophoretic analysis revealed at least eight distinct proteases in extracts of gastric and proximal duodenal mucosa, resistant to alkalinization but destroyed by sequential accidification and neutralization. Rabbit antiserum to Pg I (anti-Pg I) and Pg II (anti-Pg II) produced a single precipitin arc against each extract forming a line of nonidentity. Immunoelectrophoresis of extracts produced a single precipitin arc against anti-Pg I or anti-Pg II. The specificity of the antibodies for the group I or group II pepsinogens was confirmed by immunoabsorption. By immunofluorescnece, both Pg I and Pg II were present in mucous neck and chief cells in fundic mucosa, in the pyloric gland cells in antral mucosa, and Brunner's glands in the proximal duodenum. The results indicate that canine and feline pepsinogens are electrophoretically heterogenous, that canine and feline Pg I share antigenic determinants with each other but not with Pg II, that a similar positive relationship exists for Pg II, and that both Pg I and Pg II are localized to the peptic cell mass, consisting of four types of cells.     

The effect of adjuvant and specific or non-specific vaccination on development of protective immunity of rabbits against Trichostrongylus colubriformis infection.

Adult New Zealand rabbits were vaccinated subcutaneously with one dose of 100 micrograms adult nematode phosphate buffered saline-soluble proteins (PBS-ASP, groups I and II), a detergent-soluble fraction of adult somatic proteins (DS-ASP, group III) or three doses of 1 mg normal rabbit serum proteins (group IV). Injections of the immunogens in groups II, III and IV were accompanied with beryllium hydroxide, Be(OH)2 as an adjuvant. Vaccinated rabbits and also those of group V (naive) were challenged orally with 10,000 infective larvae of T. colubriformis 14 days after antigen injection and necropsied 2 weeks later. A single dose of PBS-ASP induced 33.5% protection when the antigen was given alone (group I) and 69.4% when injected with Be(OH)2 (group II). A detergent-soluble fraction of ASP given with the adjuvant provided 87.2% protection (group III), whilst non-specific vaccination with serum proteins plus Be(OH)2 elicited 99% protection (group IV). Mesenteric lymph node leukocyte responses were measured using a leukocyte migration inhibition assay. A significant response was observed only in group IV. In ELISA tests IgA antibodies specific to PBS-ASP reached the highest level in the intestinal mucosa of groups I and II and in the bile of groups I and III. Antibody levels of IgG isotype were similar in the intestinal mucosa of all the immunized groups. Nematode antigen was detected using a 'sandwich' ELISA method in faecal protein extracts of rabbits of groups II and III on days 2-6 after challenge.     

Vascular role of vasopressin in the presence and absence of influence from angiotensin II or alpha-adrenergic system

The effects of a vasopressin (AVP) pressor antagonist, d(CH2)5Tyr(Me)AVP, on mean arterial pressure (MAP), total peripheral resistance (TPR), cardiac output (CO), and the distribution of CO were investigated by the microsphere technique in three groups of pentobarbital anesthetized rats: intact (I), saralasin pretreated (II), and phentolamine pretreated (III). Saralasin and phentolamine were infused intravenously to inactivate the renin-angiotensin and alpha-adrenergic systems, respectively. The AVP antagonist decreased MAP and TPR in all groups and it caused a greater depressor effect in groups II and III than in group I. In group I, AVP antagonist increased blood flow (BF) to the stomach and skin. In group II, AVP antagonist increased BF to the muscle and skin. In group III, AVP antagonist markedly increased BF to the muscle. Therefore, the degree of vasoconstrictor influence exerted by AVP in different vascular beds varies depending on endogenous vasomotor tone from the renin-angiotensin and (or) sympathetic nervous systems.     

Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: II. characterization of cells cultured ex vivo and their contribution to the in vivo immunomodulation

Summary Burkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines (LCL). We studied three groups of BL lines for their capacity to interact with allogeneic lymphocytes. The results showed that as long as the lines have the group I phenotype, they do not stimulate allogeneic T lymphocytes irrespective whether they carry the EBV genome. The group II and III cells are stimulatory. Generally there was no correlation between sensitivity to lymphocyte-mediated lysis and the phenotype of the lines. In one set of lines, the group I cells had higher sensitivity to both natural killer and lymphokine-activated killer effectors compared to the group II or III lines. However, such correlation could not be seen with the other two sets of lines. Among the phenotypic features investigated, expression of the adhesion molecules LFA-1 and LFA-3 correlated with the tendency for cell aggregation.     

An ultrastructural study on gastric endocrine cells in the stomach gland patch of the koala Phascolarctos cinereus

The endocrine cells in the stomach gland patch of the koala (Phascolarctos cinereus) were studied ultrastructurally. They were classified into 3 types based on the ultrastructural profiles of their endocrine granules and tentatively categorized as type I, II, and III endocrine cells. Type I cells contained round granules that were for the most part larger than those observed in the other 2 cell types. The granules ranged from moderate to relatively high in electron density. Type II cells were angular in shape and characterized by the presence of granules that were polymorphous in profile. Contents of the endocrine granules in type II cells also showed a range of high to moderate electron density. Type III cells were oval or pyramidal in shape. They contained highly polymorphous granules that were round, oval, dumbbell-like or comma in shape and characterized by the presence of a clear space or halo separating the high to low electron-dense core from the limiting membrane of granules. Type III cells were observed most often whereas type I and II cells were a less frequent observation.     

Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping

In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response.     

Effect of oxidative stress on the spermatogenic process and hsp70 expressions in mice testes

The effect of oxidative stress on the process of spermatogenesis in terms of hsp70 expression was studied. For creating different oxidative stressed mice, three selenium (Se) levels viz., deficient (group I), adequate (group II) and excess (group III) were fed for 8 weeks in a yeast-based diet. After completion of diet feeding, Se level was significantly decreased in group I and significantly increased in group III, as compared to group II. Glutathione peroxidase (GSH-Px) activity was significantly decreased in both liver and testis in group I animals; however, the activity was comparable in groups II and III. Significant increase in the testis glutathione-S-transferase (GST) activity was observed in group I. No change was seen in group III, when compared to group II. Histological analysis of testis revealed a significant decrease in the germ cell population in group I, as compared to group II, with a predominant effect on spermatid and mature sperm numbers. In group III, displacement of germ cell population was observed. ELISA assays for hsp70 level showed increase in group I as compared to group II, whereas no significant change was observed in group III, as compared to group II. Immunohistochemical analysis revealed intense localization of hsp70 only in spermatid and sperm cells. The expression in groups II and III was homogeneous with slightly increased expression around lumen in group III. The data indicate that excessive oxidative stress in Se deficient group, affects the spermatogenesis process, especially affecting the mature sperm number which in turn leads to infertility.     

Comparative genomic analysis reveals novel facts about Leptospirillum spp. cytochromes

Chemolithoautotrophic acidophilic bacteria, which belong to the genus Leptospirillum, can only grow with Fe(II) as electron donor and oxygen as an electron acceptor. Members of this genus play an important role in bioleaching sulfide ores. We used nearly complete genome sequences of Leptospirillum ferrooxidans (group I), Leptospirillum rubarum, Leptospirillum '5-way CG' (group II) and Leptospirillum ferrodiazotrophum (group III) to identify cytochromes that are likely involved in electron transfer chain(s). The results show the presence of genes encoding a number of c-type cytochromes (18-20 genes were identified in each species), as well as bd and cbb? oxidases. Genes encoding cbb? oxidase are clustered, with predicted genes involved in cbb? maturation proteins. Duplication of cbb? encoding genes (ccoNO) was detected in all four genomes. Interestingly, these micro-organisms also contain genes that potentially encode bc? and b?f-like complexes organized into two putative operon structures. To date, the Leptospirillum genus includes the only organisms reported to have genes coding for two different bc complexes. This study provides detailed insights into the components of electron transfer chains of Leptospirillum spp., revealing their conservation among leptospirilla groups and suggesting that there may be a single common pathway for electron transport between Fe(II) and oxygen.     

Fine structure of the elasmobranch renal tubule: neck and proximal segments of the little skate

This is the first in a series of studies that examines the renal tubular ultrastructure of elasmobranch fish. Each subdivision of the neck segment and proximal segment of the renal tubule of the little skate (Raja erinacea) has been investigated using electron microscopy of thin sections and freeze-fracture replicas. Flagellar cells, characterized by long, wavy, flagellar ribbons, were observed in both nephron segments. They were found predominantly in the first subdivision of the neck segment, which suggests that propulsion of the glomerular filtrate is a primary function of this part of the renal tubule. In the non-flagellar cells of the neck segment (subdivisions I and II), there were bundles of microfilaments, a few apical cell projections, and, in subdivision II, numerous autophagosomes. In the proximal segment, the non-flagellar cells varied in size, being low in subdivision I, cuboidal in II, tall columnar in III, and again low in IV. Apical cell projections were low and scattered in subdivisions I and IV and were highest in III where the basolateral plasma membrane was extremely amplified by cytoplasmic projections. Furthermore, in these cells the mitochondria were numerous with an extensive matrix and short cristae. A network of tubules of the endoplasmic reticulum characterized the apical region of the non-flagellar cells in subdivisions I, II, and IV. In the late part of subdivision II and the early part of III, the cells were characterized by numerous coated pits and vesicles, large subluminal vacuoles, and basally located dense bodies, all of which are structures involved in receptor-mediated endocytosis. Freeze-fracture replicas revealed gap junctions restricted to the cells of the first three subdivisions of the proximal segment. The zonulae occludentes were not different in the neck and proximal segments, being composed of several strands, suggesting a moderately leaky paracellular pathway.     

Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis

In contrast to healthy conditions, intestinal epithelial cells (IECs) stimulate proinflammatory CD4+ and CD8+ T cells during Crohn's disease (CD). The underlying regulatory mechanisms remain unknown. Here we investigated the epithelial expression of major histocompatibility complex (MHC) I and MHC II and its interference with endocytic pathways, in vivo. During ileoscopy, ovalbumin (OVA) was sprayed onto ileal mucosa of CD patients (ileitis and remission) and controls. The epithelial traffic of OVA and MHC I/II pathways were studied in biopsies using fluorescence and electron microscopy. We found MHC I and MHC II to accumulate within multivesicular late endosomes (MVLE) of IECs. Faint labeling for these molecules was seen in early endosomes and lysosomes. MVLE were entered by OVA 10 min after exposure. Exosomes carrying MHC I, MHC II, and OVA were detected in intercellular spaces of the epithelium. OVA trafficking and labeling patterns for MHC I and MHC II in IECs showed no differences between CD patients and controls. Independent of inflammatory stimuli, MHC I and MHC II pathways intersect MVLE in IECs, which were efficiently targeted by luminal antigens. Similar to MHC II-enriched compartments in professional antigen presenting cells, these MVLE might be critically involved in MHC I- and MHC II-related antigen processing in IECs and the source of epithelial-released exosomes. The access of luminal antigens to MHC I in MVLE might indicate that the presentation of exogenous antigens by IECs must not be restricted to MHC II but might also occur as "cross-presentation" via MHC I to CD8+ T cells.     

A steady-state study on the formation of Compounds II and III of myeloperoxidase

The reaction between native myeloperoxidase and hydrogen peroxide, yielding Compound II, was investigated using the stopped-flow technique. The pH dependence of the apparent second-order rate constant showed the existence of a protonatable group on the enzyme with a pKa of 4.9. This group is ascribed to the distal histidine imidazole, which must be deprotonated to enable the reaction of Compound I with hydrogen peroxidase to take place. The rate constant for the formation of Compound II by hydrogen peroxide was 3.5.10(4) M-1.s-1. During the reaction of myeloperoxidase with H2O2, rapid reduction of added cytochrome c was observed. This reduction was inhibitable by superoxide dismutase, and this demonstrates that superoxide anion radicals are generated. When potassium ferrocyanide was used as an electron donor to generate Compound II from Compound I, the pH dependence of the apparent second-order rate constant indicated involvement of a group with a pKa of 4.5. However, with ferrocyanide as an electron donor, protonation of the group was necessary to enable the reaction to take place. The rate constant for the generation of Compound II by ferrocyanide was 1.6.10(7) M-1.s-1. We also investigated the reaction of Compound II with hydrogen peroxide, yielding Compound III. Formation of Compound III (k = 50 M-1.s-1) proceeded via two different pathways, one of which was inhibitable by tetranitromethane. We further investigated the stability of Compound II and Compound III as a function of pH, ionic strength and enzyme concentration. The half-life values of both Compound II and Compound III were independent of the enzyme concentration and ionic strength. The half-life value of Compound III was pH-dependent, showing a decreasing stability with increasing pH, whereas the stability of Compound II was independent of pH over the range 3-11.     

Transition between moult and migration in a long-distance migratory passerine: organ flexibility in the African wintering area

Phenotypic flexibility of organs in migratory birds has been documented for a variety of species of different genera during the migratory period. However, very little is known about phenotypic mass changes of organs with respect to other events within the annual cycle. This seems particularly interesting when birds face different physiological challenges in quick succession. We investigated mass changes of 13 organs from garden warblers (Sylvia borin) during the transition from moult to migration. These long-distance migratory birds perform a complete moult within their wintering area just shortly before the onset of spring migration. Birds were sampled in three successive stages according to their moult status: group I consisted of birds with growing primary or secondary wing feathers, group II consisted of birds with completed wing moult but with still moulting body feathers, and group III consisted of birds that had completed wing moult and body moult. Size-corrected flight muscle, kidney mass, and pancreas mass differed significantly among the three groups. Flight muscle was heaviest in birds that were about to leave their wintering area (group III) compared with birds still in body moult (group II). Kidney and pancreas showed a pattern similar to each other, with the heaviest mass occurring in birds with moulting wing feathers (group I) and significantly reduced mass in birds that had completed wing moult (group II) or both wing and body moult (group III). Mass reductions of kidney and pancreas during the transition from moult to migration are considered to be related to the demands of moult, while increased flight muscle may be due to moult, migration, or both. Phenotypic mass changes of organs in birds occur during their migration, but they also occur during the transition between other phases of the annual cycle such as moult and migration and are not restricted to the flight muscle.     

Mitochondrial Complex II Can Generate Reactive Oxygen Species at High Rates in Both the Forward and Reverse Reactions

Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.     

Reconstruction of the muscle system in Antygomonas sp. (Kinorhyncha,Cyclorhagida) by means of phalloidin labeling and cLSM

In the present investigation the entire muscle system of the cyclorhagid kinorhynch Antygomonas sp. was three-dimensionally reconstructed from whole mounts by means of FITC-phalloidin labeling and confocal scanning microscopy. With this technique, which proved to be especially useful for microscopically small species, we wanted to reinvestigate and supplement the findings obtained by histological and electron microscopical methods. The organization of the major muscle systems can be summarized as follows: 1) All muscle fibers, apart from the intestinal ones, the spine, and the mouth cone muscles, show a cross-striated pattern; 2) Dorsal longitudinal muscle fibers as well as segmentally arranged dorsoventral fibers occur from segment III to XIII; 3) Diagonal muscle fibers are located laterally in segments III to X; 4) Two rings of circular fibers are present in segment II, forming the closing apparatus in Cyclorhagida. Further circular muscles are present in segment I, forming the mouth cone and the eversible introvert, and in the pharyngeal bulb.