Aspects of the life history of Cercopithifilaria johnstoni (Nematoda:Filarioidea)

and 1988. Aspects of the life history of Cercopithifilaria johnstoni (Nematoda:Filarioidea). International Journal for Parasitology 18: 1087–1092. Cercopithifilaria johnstoni (Nematoda:Filarioidea) occurs in the subcutaneous connective tissues of a spectrum of native murid and marsupial hosts in Eastern Australia. Life cycle studies revealed that: (i) microfilaria occur in lymphatic capillaries and extravascular connective tissue of the dermis (= ‘skin-inhabiting’), (ii) ixodid ticks, particularly Ixodes trichosuri, are intermediate hosts in nature, (iii) development from microfilariae to infective third-stage larva occurs only while the tick is off the host, that is, during ecdysis from larva to nymph or from nymph to adult. Transmission of C. johnstoni in a wild population of bush rats (Rattus fuscipes) occurred in summer and winter, and was associated with peaks in the number of larval and/or nymphal stages of ticks on rats. C. johnstoni was transmitted experimentally to bandicoots (Isoodon macrourus, Perameles nasuta), bush rats and laboratory rats (R. norvegicus), indirectly by subcutaneous inoculation of third-stage larvae and directly by tick feeding. The prepatent period was approximately 3 months and the longest duration of microfilariae in the ‘ skin’ was more than 25 months. Dermal and ocular lesions were observed in R. fuscipes. The host-parasite relationship has the potential for development as an inexpensive and practical model for human onchocerciasis.     

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.     

Cytochalasin D and cationized ferritin as probes for the morphological investigation of blebbing in two human cell lines

Summary The potent fungal metabolite cytochalasin D (CD) and cationized ferritin (CF) are used in combination to test for negative charge distribution on blebs (knobs). Two established human epithelial cell lines, WISH and HeLa, that display blebs in various phases of the cell cycle or under certain culture conditions (37,46) are investigated. CD alone, applied at a low concentration (1.0 μg/ml) and for a short time period (3 min), causes blebs to appear as the prevalent surface feature. These are filled mainly with free ribosomes. Additionally, feltlike mats, presumed to be disorganized, compacted microfilaments, are formed directly beneath the cell membrane. These are especially evident in the cortical cytoplasm below the blebs or bleb clusters. CF (0.345 mg/ml), applied for a 5-min period after CD administration (1.0 μg/ml) for 3 min, appears along the surface of microvilli, at the base of blebs, and in vesicles beneath the bleb clusters. In some cases, microfilaments (6 nm in diameter) are closely related to the vesicles. CF does not preferentially bind to the apical cell membrane of blebs. Above areas of the subplasmalemmal microfilaments, CF membrane binding is apparent, even under circumstances where the filaments are disorganized by cytochalasin treatment. These results seem to show the following: (a) bleb membranes are different from the remainder of the cell and do exhibit a loss of negative charge and (b) surface charge may be dependent on the presence or structural integrity of membrane-related 6-nm microfilaments. The support of this research by a grant from the Baylor College of Dentistry and The Oklahoma College of Osteopathic Medicine and Surgery is gratefully acknowledged. The assistance of Dr. J. H. Martin, Department of Pathology, Baylor University Medical Center, is also greatly appreciated.     

Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s)

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.     

Sialic acid moiety is responsible for the charge heterogeneity and the biological potency of rat lutropin

To elucidate a possible role of sialic acid moiety in the electrical heterogeneity of rat pituitary lutropin, seven components separated were individually treated with neuraminidase. Some intermediates with isoelectric points corresponding to the native components were concomitantly seen at the serial stages of the enzyme treatment. All the treated components showed an isoelectric point of about 10.0 which was the same to the isoelectric point of one of the seven components. Desialylation of the components with less biological activity caused enhancement of the in vitro cyclic AMP producing- and testosterone producing-activities as well as the binding activity to the receptor. It is concluded that the number of sialic acid moiety in lutropin is responsible for the charge heterogeneity and the biological potency of the hormone.     

Melittin lysis of red cells

Summary This paper describes experiments designed to explore interactions between human red blood cell membranes and melittin, the main component of bee venom. We found that melittin binds to human red cell membranes suspended in isotonic NaCl at room temperature, with an apparent dissociation constant of 3×10^–8 m and maximum binding capacity of 1.8×10⁷ molecules/cell. When about 1% of the melittin binding sites are occupied, cell lysis can be observed, and progressive, further increases in the fraction of the total sites occupied lead to progressively greater lysis in a graded manner. 50% lysis occurs when there are about 2×10⁶ molecules bound to the cell membrane. For any particular extent of melittin binding, lysis proceeds rapidly during the first few minutes but then slows and stops so that no further lysis occurs after one hour of exposure of cells to melittin. The graded lysis of erythrocytes by melittin is due to complete lysis of some of the cells, since both the density and the hemoglobin content of surviving, intact cells in a suspension that has undergone graded melittin lysis are similar to the values observed in the same cells prior to the addition of melittin. The cells surviving graded melittin lysis have an increased Na and reduced K, proportional to the extent of occupation of the melittin binding sites. Like lysis, Na accumulation and K loss proceed rapidly during the first few minutes of exposure to melittin but then stops so that Na, K and hemoglobin content of the cells remain constant after the first hour. These kinetic characteristics of both lysis and cation movements suggest that melittin modifies the permeability of the red cell membrane only for the first few minutes after the start of the interaction. Direct observation of cells by Nomarsky optics revealed that they crenate, become swollen and lyse within 10 to 30 sec after these changes in morphology are first seen. Taken together, these results are consistent with the idea that melittin produces lysis of human red cells at room temperature by a colloid osmotic mechanism.     

Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.     

Further studies of human whole-body radiofrequency absorption rates

Further studies of human whole-body radiofrequency (RF) absorption rates were carried out using a TEM-cell exposure system. Experiments were done at one frequency near the grounded resonance frequency (approximately 40 MHz), and at several below-resonance frequencies. Absorption rates are small for the K and H orientations of the body, even when grounded. For the body trunk in an E orientation, the absorption rate of a sitting person is about half of the rate for the same person standing with arms at the sides; the latter in turn is about half the rate for the same subject standing with arms over the head. Two-body interactions cause no increase in absorption rates for grounded people. They do, however, increase the absorption rates for subjects in an E orientation in free space; the largest interaction occurs when one subject is lambda/2 behind the other (as seen by the incident wave). When these results are applied to practical occupational exposure situations, the whole-body specific absorption rate does not exceed the ANSI limit of 0.4 W/kg for exposures permitted by the ANSI standard (C95.1-1982) at frequencies from 7 to 40 MHz.     

In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.     

The development of the human spleen

Summary Splenic tissue of human fetuses from the 14^th to the 24^th week of gestation (menstrual age) were investigated by light- and electron microscopy to describe the development of the red and white pulp in close relationship to the differentiation of the vascular tree. Special interest is focussed on the differentiation of the T-cell- and the B-cell regions and their specific stationary cells.The preliminary stage, here called the primary vascular reticulum, lasts up to the 14^th gestational week (gw). Numerous erythrocytes, normoblasts and macrophages are seen among a network of mesenchymal cells and argyrophilic fibers. Hematopoiesis, especially erythropoiesis, can be recognized.The characteristic organ structure becomes established during the subsequent transformation stage of the fetal spleen, beginning with the 15^th gw. Splenic lobules begin to form during the 15^th to 17^th gw. They consist of a central artery, surrounded by a sheath of lightly stained stationary cells which resemble myofibroblasts. At the periphery of these lobules the red pulp forms. Initially mobile cells are distributed throughout the reticulum. Soon they begin to accumulate in the venous sinuses, which develop from lacunae among the reticular network and come into contact with the venous system. The endothelial wall of these sinuses remains discontinuous, confirming the theory of the open vascularization of the spleen. The development of the larger veins is correlated with the differentiation of the splenic trabeculae.The development of the white pulp is correlated with the stage of lymphoid colonization within the spleen, beginning around the 18^th gw. An accumulation of lymphocytes around the central arteries can be recognized during the 19^th and 20^th gw. These lymphoid cells show morphological and immunohistochemical characteristics of T-precursor cells. Within the now assembling periarterial lymphoid sheath (PALS) a few precursors of interdigitating cells (IDC) are recognizable, giving evidence for the differentiation of the T-cell region.Around the 23^rd gw the assemblage of primary follicles is discernible at the periphery of the PALS. Precursors of the follicular dendritic reticulum cell (FDRC), the specific stationary cell of the B-cell region, have been recognized. This observation leads to the conclusion that the small primary follicles represent the beginning formation of the B-cell region.The significance of the vascular system for the differentiation of the specific splenic organization is discussed.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 111)The authors appreciate the contribution of human fetal material from Dr. von Hollweg and Dr. Körner from the Hospital Heidberg, Hamburg, and the excellent technical assistance of Mrs. H. Hansen, Mrs. I. Knauer, Mrs. M.v. Kolszynski, Mrs. J. Quitzau, Mrs. H. Siebke and Mrs. H. Waluk