Ultrastrukturen Und Cytochemie Der Pellikula Und Des Apikalkomplexes Der Kineten Von Babesia Bigemina Und Babesia Ovis In Hämolymphe Und Ovar Von Zecken*,†

SYNOPSIS The term kinete is used in this paper for the cigar-shaped, motile development stages (“vermicule”) of Babesia occurring intra- and extracellularly in hemolymph and ovary (including oocytes) of vectors, hard ticks (Ixodoidea). The structure of, and cyto-chemical activities of hydrolases (acid phosphatase, nonspecific esterase) in the pellicle and the apical complex were studied at the fine-structural level in kinetes of Babesia bigemina Smith & Kilborne, in hemolymph of female Boophilus microplus Canestrini. The cytochemistry of acid hydrolases was studied also in kinetes of Babesia ovis (Babes) Starcovici, in hemolymph and ovary of Rhipi-cephultis bursa Canestrini & Fanzago. The pellicle of the B. bigemina kinetes is composed of 3 membranes (pellicular complex): an outer membrane, ?8 nm thick (the plasmalemma) and 2 inner ones, each ?6 nm thick, lying closely together. The outer membrane appears to be covered by a structureless coat, 3 nm thick. The space between the inner double membrane and the plasmalemma is 7.5 nm. The whole pellicular complex is 30 nm in diameter. The 2 inner pellicular membranes appear to be derived from the endoplasmic reticulum (ER) for the following reasons: (a) a layer of hydrolase-active material is enclosed by these membranes; (b) in the spheroid parasite stages which transform from kinetes inside hemocytes, the inner double membrane is apparently replaced by an ER cisterna; (c) the thickness of each of the inner pellicular membranes is approximately the same as that of the ER membrane. There are circular openings in the pellicular double membrane with average diameters of 100 nm; despite some similarity to micropores, they have a specific structure. The term Intrapellikularfenster (IPF) (intrapellicular windows) or pseudomicropores is proposed for these pellicular differentiations. The margin of an FPF is formed by the 2 inner membranes folding into each other; cytoplasmic, electron-dense material is accumulated alongside this edge. Unlike that of micropores, the plasmalemma of the IPF is not invaginated. The IPF appears as a single, dark ring in tangential sections. At times, rhoptry-like bodies are associated with the openings. The function of the IPF is not known. An intrapellicular opening similar to the IPF, although wider, is present at the apex of the parasite. Its margin coincides with the inner edge of the apical ring. Typical subpellicular microtubuli were not observed in the Babesia kinetes. The apical complex of the B. bigemina kinetes consists of an Apikalschirm (apical umbrella), a crown of microtubuli beneath it, and rhoptries: micronemes are also present in large numbers. The Apikalschirm is located beneath the pellicle of the apical pole of the parasite. It is a wheel-like structure composed of spokes radiating from a wide, hub-like central ring (apical ring). It should be stressed that the apical ring is not identical with the polar ring described as an integral part of the pellicular complex in other Apicomplexa. Beneath each “rib” of the Apikalschirm there is one microtubule (subcostal microtubule). In kinetes of B. ovis the “ribs” are less well developed. In addition, the Apikalschirm is more pointed in kinetes of this species in tick oocytes and ova. The rhoptries of the kinetes are spindle-shaped and largely located directly beneath the Apikalschirm. They are arranged radially, each row being associated with a “rib”. A conoid was not observed. Occasionally, low hydrolytic activity could be detected in micronemes. The rhoptries and the Apikalschirm were always negative for phosphatase and esterase activity. With regard to the number and arrangement of its membranes and to its hydrolase activity, the pellicle of the kinetes of Babesia closely resembles the pellicular complex of the Coccidia. It differs from the latter by the presence of the IFF and by the lack of micropores and of true subpellicular microtubules. In the complexity of their pellicle and in some details of the organization of their apical complex (lack of a conoid; umbrella-like structure), the kinetes of Babesia resemble the ookinetes of the Haemosporidia.     

An Aphid-Secreted Salivary Protease Activates Plant Defense in Phloem

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Ant eating behavior of mountain gorillas

Eleven cases of feeding on driver ants (Dorylus sp.) by mountain gorillas (Gorilla gorilla beringei) are described. Ant eating provides the gorillas with more animal protein and other nutrients per unit feeding time than do other forms of insectivory that contribute to their diet, but it is so rare that it is unlikely to be of real nutritional significance. Gorillas obtain ants with their hands and do not use tools. Immature individuals (except infants) ate more ants than did adult females, and silverbacks were not seen to eat ants. These differences are more likely to reflect differences in individual taste and interest in novelty than differences in nutritional strategy. Not all gorillas in the Virungas population eat ants. Intra-population variability may be ecologically contingent, but ant eating appears to be a socially acquired and transmitted taste.     

Expression of human salivary protein genes

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamulaet al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to study the molecular and cellular biology of PRP production. Cell-free translation and mRNA characterization with EO27 indicate that the numerous PRPs seen in saliva are produced from relatively few, large precursors, probably by posttranslational cleavage. This supports an hypothesis originally proposed by Friedman and Karn in 1977 (Am. J. Hum. Genet. 29:44A;Biochem. Genet. 15:549) and later supported by biochemical studies (Karnet al., Biochem Genet. 17:1061, 1979) and molecular studies (Mamulaet al., Fed. Proc. 43:1522, 1984; Maedaet al., J. Biol. Chem. 260:1123, 1985). EO27 was also used in this study to localize PRP mRNA production to the acinar cells of the parotid gland byin situ hybridization.     

Transepithelial transport of nonelectrolytes in the rabbit mandibular salivary gland

Summary The characteristics of nonelectrolyte secretion by the rabbit mandibular salivary gland have been investigated in anin vitro perfused preparation. The concentrations of¹⁴C-labeled nonelectrolytes were measured in saliva samples collected over a range of flow rates during the secretory response of the gland to continuous acetylcholine infusion. Of the nine nonelectrolytes studied, the two particularly lipid-soluble molecules, ethanol and antipyrine, appeared in the saliva at approximately the same concentration as in the perfusate, regardless of the secretory flow rate. The more polar molecules (urea, ethanediol, thiourea, glycerol, erythritol, mannitol and sucrose) appeared at saliva/perfusate concentration ratios () which showed a strong dependence on flow. With the exception of thiourea, this could be attributed to the combined contributions of diffusion and solvent drag.For the polar nonelectrolytes, estimates have been obtained of both the permeability coefficients of the gland (P) and the solvent-drag filtration coefficients (1–). The relation between 1– and molecular radius suggests that small polar nonelectrolytes and the bulk of the secreted water cross the epithelium via aqueous channels that are approximately 0.8 nm in width. The location of the channels remains uncertain because tissue space measurements indicate that the nonelectrolytes most affected by solvent drag have access to both transcellular and paracellular pathways.     

True grit: A microwear experiment

Recently we noted the effects of experimental diets on microscopic dental wear in the American opossum and concluded that it might prove difficult to distinguish the microwear produced by an insectivorous diet from that produced by some kinds of herbivorous ones. We also noted that wear caused by gritty diets and those containing plant opal, although they might be confused with one another, are easily distinguished from other sorts of dietary wear. Our conclusions have been challenged on the basis that possibly we did not allow sufficient time in the experiments for diagnostic wear patterns to emerge. Additional data reported here show that this is not so. Even in our n “control” animals, fed a relatively soft unabrasive diet, enough time elapsed to produce significant dental wear. A new technique is described which for the first time allows the study of changing patterns of microscopic wear in a living animal over a period of time, thus allowing each animal to serve as its own control. A solution containing a broad-spectrum proteolytic enzyme when applied to the teeth of an anesthetized animal removes the proteinaceous coat (pellicle) which will otherwise obscure wear scratches. Precision dental impressions can then be made which reveal the details of the pattern of microwear on the teeth.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Nef genes of SIV

Molecular clones of SIVmac were constructed that differed only in sequences within the nef gene. DEAE-transfection of viral DNA containing an open from of nef yielded virus that replicated with similar kinetics and to a similar extent in macaque peripheral blood lymphocyte (PBL) cultures as virus with a deletion or stop codon within nef. Rhesus monkeys that received each kind of molecularly cloned virus became infected. Our results additionally suggest that mutant forms of virus are selected in vitro while open, functional forms are selected in vivo. In animals infected with virus containing a stop codon within nef, reversion of the stop codon to a coding codon was demonstrated in five of five clones analyzed. These results indicate that nef is playing some role crucial to the virus life cycle in vivo.     

Evaluation of hydroxyl radical-scavenging property of garlic

While antibiotics are broadly used in dental and medical therapy, little attention has been directed towards the potential toxic side effects of antibiotics on tissue regeneration. Here we examined the effect of a quinolone antibiotic, pefloxacin (Rhone Poulenc) on rat parotid gland responses to chronic isoproterenol treatment. Groups of rats received injections of isoproterenol to induce glandular growth, saline (controls), pefloxacin, or isoproterenol and pefloxacin in combination. Parotid gland weight decreased significantly after pefloxacin treatment for 7 days as well as inhibiting glandular enlargement provoked by isoproterenol. The same trend was observed for the rates of DNA synthesis, with the incorporation of [³H]-thymidine in isoproterenol/pefloxacin-treated rats reduced to 49% of isoproterenol treatment alone levels. Saline-treated animals were 42% of the rate of [³H]-thymidine incorporation into DNA observed in isoproterenol treated rats. While isoproterenol treatment increased steady-state mRNA levels for fos, jun, myc, src, c-erbB-2, ras and topo II, inclusion of pefloxacin with the isoproterenol regimen blocked these increases. Pefloxacin treatment by itself did not alter proto-oncogene mRNA levels in the parotid gland. Glandular amylase activity was decreased in the pefloxacin treated group, while the combination of isoproterenol with pefloxacin did not decrease glandular amylase levels to the extent of that observed with -agonist treatment alone. In acute experiments, pefloxacin significantly decreased the volume of saliva secreted by the parotid gland. These results suggest that quinolone-based antibiotics disturb the secretory function of the parotid gland and can inhibit cell proliferation and regeneration. (Mol Cell Biochem 165: 55–63, 1996)     

Establishment and characterization of a novel human myoepithelial cell line and matrix-producing xenograft from a parotid basal cell adenocarcinoma

Summary Myoepithelial cells exert important paracrine effects on epithelial morphogenesis and mitogenesis through direct cell-cell interactions and through synthesis of a basement membrane extracellular matrix. To study these effects further, this study established the first immortalized human myoepithelial cell line, HMS-1, and transplantable xenograft, HMS-X, from the rare parotid basal cell adenocarcinoma. The cell line exhibited a fully differentiated myoepithelial phenotype and the xenograft exhibited the rare property of accumulating an abundant extracellular matrix composed of both basement membrane and nonbasement membrane components with the latter predominating. With HMS-1 as a feeder layer, dramatic and specific induction of epithelial morphogenesis (sheroid formation) occurred with selected normal epithelial and primary carcinoma target cells. HMS-1 and HMS-X provide distinct advantages over the conventional murine matrices in existence. They will be invaluable in future studies of human tumor-myoepithelial and matrix interactions important for tumor cell growth, invasion, and metastasis.