Chromosome elimination in three Baltic,south Pacific and north-east Pacific hagfish species

We have confirmed that chromosome elimination occurs in the cells ofMyxine glutinosa, collected from the Baltic Sea off Sweden,Eptatretus cirrhatus from the south Pacific Ocean off the east coast of New Zealand, andE. stoutii from the north-east Pacific Ocean off Canada, similar to cells of four Japanese hagfish species. InM. Glutinosa, E. cirrhatus type A,E. cirrhatus type B andE. stoutii, the differences in chromosome number between spermatogonia (44, 72, 80 and 48) and somatic cells (28, 34, 34 and 34) were 16, 38, 46 and 14 respectively. The amount of DNA eliminated from presumptive somatic cells averaged 43.5%, 48.7%, 54.6% and 52.8% respectively. Euchromatic chromosomes and/or parts of chromosomes in addition to heterochromatic chromosomes were clearly eliminated inE. cirrhatus andE. stoutii. Adding our previous observations of four Japanese hagfish species, chromosome elimination occurs in all seven of the hagfish species. These results suggest that this phenomenon, chromosome elimination, generally occurs in the order Myxinida. In addition, B-chromosomes were observed in the germ cells ofE. cirrhatus andE. stoutii, similar to the cells ofE. okinoseanus, E. burgeri andParamyxine atami (E. atami). This fact suggests that B-chromosomes might exist generally in the family Eptatretidae.     

Evolutionary development of lymphocyte heterogeneity: leucocyte subpopulations in the Pacific hagfish

Flow cytometric analysis of forward angle versus 90 degree scatter patterns of hagfish peripheral blood revealed two distinct leucocyte populations with size characteristics analogous to mammalian monocytes/granulocytes (hagfish large leucocytes) and small lymphocytes (hagfish small leucocytes). A cell population enhanced for the small leucocytes was obtained by density gradient centrifugation. Over 70% of the small leucocyte population consistently stained with a rabbit antiserum directed against polypeptide determinants on hagfish immunoglobulin, while staining of the large cell population was greatly reduced (less than 10%). A panel of monoclonal antibodies raised against a crude hagfish leucocyte preparation distinguished the two cell populations and revealed the existence of subpopulations of both small and large leucocytes.     

Production and specificity of antibodies to streptococci in the Pacific hagfish, Eptatretus stoutii.

Specific antibody activity at high levels to group A streptococcal carbohydrate was detected by radioimmunoassay in the sera of certain hagfish immunized with streptococcal whole cells. Antigen binding activity could be depleted by absorption of immune serum with streptococcal cells but not with sheep erythrocytes. Sugar inhibition studies indicated that rhanmose was the immunodominant sugar recognized in the A carbohydrate by hagfish antibody. This was in contrast to previous identification of N-acetylglucosamine as the immunodominant sugar recognized by antibodies from mammalian species.     

Histamine release from peripheral blood leucocytes in egg-allergic patients

Twelve egg-allergic patients were selected on the basis of a clinical history of egg allergy and a positive skin-prick test (SPT) to whole egg. A study was then carried out on the ability of the patients’ washed leucocytes to release histamine in the presence of whole egg, ovomucoid, ovalbumin and ovotransferrin. Histamine release (HR) from washed leucocytes was demonstrated in ten out of twelve patients, but only four out of ten released over 40% of their total histamine. Spontaneous HR ranged from 2·1–14·5% with a mean of 7·5%. There was good agreement between positive and negative HR, skin test and radioallergosorbent test (RAST) results. Concordance between the HR and skin test was found in 83%, HR and RAST in 71% and skin test and RAST in 78% of patients. However there was poor quantitative agreement between these three tests. When skin-prick tests and HR thresholds were compared, ovomucoid elicited the greatest skin test sensitivity in five out of six patients, whereas five of the same six were more sensitive to ovalbumin when judged by HR.     

Ventilation in Pacific hagfish (Eptatretus stoutii) during exposure to acute hypoxia or hypercapnia

A technique was developed to measure ventilation in unrestrained Pacific hagfish (Eptatretus stoutii) by inserting and fastening into the nostril a flexible tube fitted with an ultrasonic flow probe. This technique permitted the continuous measurement of ventilation (respiratory) frequency (fR), stroke volume and minute ventilation () in real time in fish exposed to acute hypoxia or hypercapnia. Exposing fish to acute hypoxia (final Pw_O2=21.0±3.4 mm Hg) caused hypoxaemia and a marked increase in of 350 ± 71 ml min⁻¹ kg⁻¹ (from 235 to 585 ml min⁻¹ kg⁻¹) owing exclusively to an increase in fR of 44 ± 7 min⁻¹ (from 19 to 63 min⁻¹). Because O₂ consumption (0.4 mmol kg⁻¹ h⁻¹) was unaltered during hypoxia, there was an associated marked increased in the ventilation convection requirement from 36.7 to 81.8 l mmol⁻¹. Injecting the O₂ chemoreceptor stimulant NaCN into inspired water (external CN⁻) or pre-branchial blood (internal CN⁻) evoked ventilatory responses that were similar to those observed during hypoxia although of a lesser magnitude. With external CN⁻, increased maximally by 146 ± 46 ml min⁻¹ kg⁻¹ and fR increased by 20 ± 2 min⁻¹. With internal CN⁻, the maximal increase in was 93 ± 30 ml min⁻¹ kg⁻¹ and fR increased maximally by 19 ± 6 min⁻¹. Exposure to acute hypercapnia (final PwC = 7.0 ± 0.2 mm Hg) caused an increase in of 169 ± 60 ml min⁻¹ kg⁻¹. These results provide compelling evidence for chemoreceptor-mediated control of breathing in hagfish and suggest that ventilatory responses to environmental hypoxia and hypercapnia in the vertebrates arose in the myxine lineage.     

An immunocytochemical investigation of glial morphology in the Pacific hagfish: radial and astrocyte-like glia have the same phylogenetic age

Summary This study attempts to reconstruct the early phylogenetic history of macroglial cells among craniates. Since glia does not fossilize, such a reconstruction must be based on a cladistic comparison of glial characters in the Recent craniate taxa (hagfishes, lampreys, and gnathostomes); however, there are only few data on glial morphology and none on glial immunocytochemistry in hagfishes. Therefore, we investigated the presence and localization of various macroglia-specific epitopes in the brain and spinal cord of the Pacific hagfish,Eptatretus stouti (Myxinoidea) by means of immunocytochemistry. Antibodies directed against S100-pfotein and vimentin showed no cross reactivity. Antibodies directed against glial fibrillary acidic protein and glutamine synthetase labelled various glial structures. Glial fibrillary acidic protein-like immunoreactivity was observed in ependymal cells with radially oriented processes in some regions. However, throughout the entire CNS, labelling of non-ependymal cells and their processes prevailed. The processes of these cells made occasional vascular contacts and they also made contacts with neuronal perikarya. Glutamine synthetase-like immunoreactivity was also found in some processes with radial orientation and in ependymal cells; but the antibody stained mainly non-ependymal cells which gave rise to a felt-like meshwork of interdigitating fine and very fine processes penetrating the neuropil of the entire brain. Additionally, there was labelling in the walls of blood vessels and in processes enwrapping individual neurons. The occurrence of glial fibrillary acidic protein- and glutamine synthetase-like immunoreactivity in non-ependymal glial elements in the brain of hagfishes and the relative scarcity of labelling in radial glial elements necessitates a re-interpretation of the evolutionary history of glial cells. Non-ependymal macroglia with immunocytochemical and morphological characters resembling typical (mammalian) astrocytes appears to be as primitive as the various forms of radial ependymal glia.     

Immunoglobulin receptors on human leucocytes. III. Comparative study of human bone marrow and blood B cells: role of IgM receptors

A comparative study of B cells present in human bone marrow and blood was performed. In both compartments the cells carrying the Ig receptors were found to be small mononuclear cells. Predominance of IgM receptors was found on bone marrow cells whereas Ig receptors present on peripheral blood cells were predominantly of the IgG class. Bone marrow lymphoid cells of non-sensitized donors were capable of binding a primary antigen, keyhole limpet haemocyanin (KLH) and could be retained on glass bead columns coated with either KLH or with goat anti-human IgM antiserum but not with anti-IgG. Whereas bone marrow cells of donors immunized with KLH 16–27 days earlier lacked KLH reactive cells, the latter cells could be demonstrated in the blood. It is concluded that human bone marrow B cells carrying IgM receptors are essential for the early antigen recognition step following which recruitment of these cells into the circulation takes place.     

Histamine receptors on human peripheral blood leucocytes.

Human blood leucocytes were tested for their capacity to bind histamine coupled as a rabbit serum albumin conjugate (H-RSA) to formalized ox red cells. The percentage of rosette-forming target cells was directly related to the concentration of erythrocyte-bound H-RSA. Under optimal experimental conditions the average number of rosettes was 52% for monocytes, 3 for lymphocytes, 25% for neutrophils and 59% for eosinophils. Free histamine partially inhibited rosette formation by blood monocytes in a dose-dependent fashion from 10-3 to 10-5 mol/l, and complete inhibition was achieved by the H-RSA conjugate. In contrast, three amines chemically related to histamine, L-histidine, imidazoleacetic acid and N-acetylhistamine, had no inhibitory effect. The histamine H1 antagonists, mepyramine and chlorpheniramine, and the H1 receptor agonist, 2-(2-aminoethyl)-thiazole, all inhibited rosette formation by blood monocytes in a dose-dependent fashion. However, the H2 receptor antagonists, burimamide and metiamide, and the H2 receptor agonists, Dimaprit and 4-methyl-histamine, were inactive. There was no statistical difference in the percentage of histamine rosettes for monocytes. lymphocytes, neutrophils or eosinophils between atopic and non-atopic individuals.     

Antibody formation by adoptively transferred mouse peripheral blood leucocytes

Peripheral blood leucocytes from C3H mice that had been injected intraperitoneally with bovine serum albumin in Freund's adjuvant were transferred to irradiated, syngeneic recipients. Determination of bovine serum albumin antibody titres in the recipients showed that as the time between immunization and transfer increased, fewer peripheral blood leucocytes were needed to produce appreciable amounts of antibody. Secondary stimulation of the donor mice before transfer with aqueous bovine serum albumin resulted in greater antibody synthesis in the recipient.

When the peripheral blood leucocytes from mice immunized with bovine serum albumin were mixed with 0.1 mg of the antigen in vitro and the mixture injected into recipients no detectable antibody was produced. An in vitro booster of 0.05 mg bovine serum albumin resulted in greatly increased amounts of antibody when compared to peripheral blood leucocytes transferred without added antigen.

     

Cartilage in the atlantic hagfish,Myxine glutinosa

Light and electron microscopic observations and biochemical analysis of the lingual cartilages from the Atlantic hagfish, Myxine glutinosa, reveal two different types of cartilage, designated types 1 and 2, respectively. The anterior and medial lingual are type 1, while the posterior lingual cartilage is type 2. Chondrocytes in type 1 cartilage are similar to those found in other vertebrate cartilages. The presence within the Golgi elements of material that resembles a component of the extracellular matrix suggests the involvement of active chondrocytes in the synthesis of the matrix. The matrix of the type 1 cartilage contains fibrils arranged to form concentric lamellae in the territorial matrix and irregularly arranged, branched fibrils in the interterritorial matrix. Biochemical analysis of the type 1 cartilage reveals that it is composed primarily of a cyanogen bromide (CNBr)-insoluble protein of unique composition that we have termed “myxinin.” Myxinin appears to be similar, but not identical, to lamprin. Type 2 cartilage bears no resemblance to any other known vertebrate cartilage. The principal cells are hypertrophied and are characterized by masses of cytoplasmic filaments. The appearance of the organelles in smaller nest cells suggests that nest cells are active in the production of some of the matrix, which consists primarily of collagen. Microfibrils and a basal lamina-like material are also present. Biochemical analysis of the type 2 cartilage reveals that the CNBr-insoluble material is different from myxinin. Comparisons of lamprey and hagfish cartilages prompt the concept that these two agnathans probably followed long-independent evolutionary histories.     

Effect of serotonin on the chemiluminescence response of rat peripheral blood leucocytes

Serotonin (5-HT) has been shown to exert various immunomodulatory effects. In this study, the effects of 5-HT, 5-hydroxy-DL-tryptophan (5-HTP) and dl-p-chlorophenylalanine (PCPA) on the chemiluminescence (CL) responses of rat peripheral blood leucocytes (PBL) activated by phorbol myristate acetate (PMA), opsonized zymosan or latex beads were assessed. The CL responses were measured following in vitro treatment with 0.01-100 μM 5-HT, and either 1 h after the last i.p. administration of 5-HT (0.05, 0.5, 1, 2.5, 5 or 10 mg/kg for 4 days), 5-HTP (25 or 100 mg/kg for 4 days) or PCPA (200 mg/kg for 4 days, n = 5), or 48 h after a single 200 mg/kg PCPA injection. A concentration-dependent decrease in CL responses was noted following in vitro 5-HT treatment. In vivo treatment of rats with 5-HT produced a reverse bell-curve inhibiting effects on the CL response with a maximal inhibition in rats receiving 1 mg/kg/day 5-HT and a weaker response of PMA-activated PBL. In vivo treatment with high-dose 5-HTP increased CL response of opsonized zymosan-activated PBL, while low-dose 5-HTP decreased CL response of opsonized zymosan and latex beads-activated PBL. No effect was observed in PMA-activated PBL from rats treated with 5-HTP. By contrast, in vivo treatment with PCPA increased CL responses induced by PMA or latex beads, whereas CL responses using opsonized zymosan were not significantly affected. These results suggest that 5-HT modulates the CL response of rat leucocytes to particulate stimuli.