Description of the oöcysts of Eimeria arnyi n. sp. (Apicomplexa: Eimeriidae) from the eastern ringneck snake Diadophis punctatus arnyi (Serpentes: Colubridae)

Coccidian oöcysts recovered from the faeces of eastern ringneck snakes, Diadophis punctatus arnyi, from Kansas, USA were found to represent a previously unreported eimerian. Oöcysts of Eimeria arnyi n. sp. are subspherical, 16.9×15.1 (15–18.5×13.5–16) m, with a thin, single-layered wall and a shape-index (length/width) of 1.1 (1.1–1.3). A micropyle and öocyst residuum are absent but a large polar granule is present. The sporocysts are elongate, 13.2×6.9 (12–14.5×6.5–7) m, with Stieda and substieda bodies and a shape-index of 1.9 (1.7–2.3). Each sporozoite contains one to two anterior and a single posterior refractile bodies. Sporulation was exogenous and complete within four days at 23°C.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Novel DNA structures resulting from dTam3 excision in tobacco

A Tam3 two-element system has been designed by combining an immobilized Tam3 element with a non-autonomous dTam3 element inserted into the HPT gene. The phenotypic assay employed, restored hygromycin resistance, indicated thattrans-activation of the non-autonomous dTam3 element occurred. Molecular analyses of the excision sites revealed that the ends of the dTam3 element remain in the empty donor sites. The predominant consequence of this type of excision appears to be that excised fragments fail to re-integrate into the tobacco genome. Only one case of dTam3 re-integration could be detected. The ends of this element had been degraded upon integration into the tobacco genome. Either the altered structure of the Tam3 derivatives or tobacco host factors are influencing thetrans-activation of a dTam3 element, resulting in aberrant excision.     

The effects of endogenous or exogenous catecholamines on blood respiratory status during acute hypoxia in rainbow trout (Oncorhynchus mykiss)

Summary An extracorporeal circulation of rainbow trout (Oncorhynchus mykiss) was utilized to continuously monitor the rapid and progressive effects of endogenous or exogenous catecholamines on blood respiratory/acid-base status, and to provide in vivo evidence for adrenergic retention of carbon dioxide (CO₂) in fish blood (cf. Wood and Perry 1985). Exposure of fish to severe aquatic hypoxia (final P _wO₂=40–60 torr; reached within 10–20 min) elicited an initial respiratory alkalosis resulting from hypoxia-induced hyperventilation. However, at a critical arterial oxygen tension (P _aO₂) between 15 and 25 torr, fish became agitated for approximately 5 s and a marked (0.2–0.4 pH unit) but transient arterial blood acidosis ensued. This response is characteristic of abrupt catecholamine mobilization into the circulation and subsequent adrenergic activation of red blood cell (RBC) Na⁺/H⁺ exchange (Fievet et al. 1987). Within approximately 1–2 min after the activation of RBC Na⁺/H⁺ exchange by endogenous catecholamines, there was a significant rise in arterial PCO₂ (P _aCO₂) whereas arterial PO₂ was unaltered; the elevation of P _aCO₂ could not be explained by changes in gill ventilation. Pre-treatment of fish with the -adrenoceptor antagonist phentolamine did not prevent the apparent catecholamine-mediated increase of P _aCO₂. Conversely, pre-treatment with the -adrenoceptor antagonist sotalol abolished both the activation of the RBC Na⁺/H⁺ antiporter and the associated rise in P _aCO₂, suggesting a causal relationship between the stimulation of RBC Na⁺/H⁺ exchange and the elevation of P _aCO₂. To more clearly establish that elevation of plasma catecholamine levels during severe hypoxia was indeed responsible for causing the elevation of P _aCO₂, fish were exposed to moderate hypoxia (final P _wO₂=60–80 torr) and then injected intraarterially with a bolus of adrenaline to elicit an estimated circulating level of 400 nmol·l^-1 immediately after the injection. This protocol activated RBC Na⁺/H⁺ exchange as indicated by abrupt changes in arterial pH (pHa). In all fish examined, P _aCO₂ increased after injection of exogenous adrenaline. The effects on P _aO₂ were inconsistent, although a reduction in this variable was the most frequent response. Gill ventilation frequency and amplitude were unaffected by exogenous adrenaline. Therefore, it is unlikely that ventilatory changes contributed to the consistently observed rise in P _aCO₂. Pretreatment of fish with sotalol did not alter the ventilatory response to adrenaline injection but did prevent the stimulation of RBC Na⁺/H⁺ exchange and the accompanying increases and decreases in P _aCO₂ and P _aO₂, respectively. These results suggest that adrenergic elevation of P _aCO₂, in addition to the frequently observed reduction of P _aO₂ are linked to activation of RBC Na⁺/H⁺ exchange. The physiological significance and the potential mechanisms underlying the changes in blood respiratory status after addition of endogenous or exogenous catecholamines to the circulation of hypoxic rainbow trout are discussed.Abbreviations P _aCO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen tension - P _da dorsal aortic pressure - pHa arterial pH - P _wO₂ water oxygen tension - RBC red blood cell - V _f breathing frequency     

Fetal lung surfactant lipid synthesis from glycogen during organ culture

The role of fetal lung glycogen as a precursor for lipids during late gestational development was explored by a combination of in vivo labeling with [U-14C]glucose, administered directly to rat fetuses at 18.5 days, and in vitro assessment using an organ explant culture system. Our major objectives were to demonstrate that radioactivity was transferred specifically and preferentially to surfactant lipids, as glycogenolysis occurred, and to determine the molecular distribution of 14C labeling in newly synthesized phosphatidylcholine (PC). Surfactant and residual (non-surfactant) lipids were separated by sucrose density gradient centrifugation, and other subcellular fractions such as microsomes were isolated by subsequent centrifugations. After 72 h of culture, there was a 5.7-fold increase in the concentration of PC in the surfactant fraction, which contributed 8.8% of total PC at the beginning and 29.6% (P less than 0.001) at the end of the 72 h period. The labeling of PC in the surfactant fraction increased markedly during culture, but there was no significant change in the residual fraction or microsomal PC. Hydrolysis of surfactant PC indicated that the radioactivity was predominantly located in the fatty acyl portion of the molecule, both before and after culture; however, PC glycerol labeling also increased significantly during culture. The distribution of PC radioactivity was similar in the residual fraction and microsomes, with the majority of 14C in the fatty acids. Neutral lipid radioactivity also increased significantly in both the surfactant (240%) and residual (136%) fractions. Quantitation of the changes in radioactivity among subcellular components during lung explant culture indicated that the greatest decrease occurred in glycogen, whereas only lipids, particularly those of the surfactant fraction, were found to show significant increases. These results support the hypothesis that glycogen, which accumulates in fetal lung prior to augmented surfactant production, can supply precursors for synthesis of functionally essential pulmonary phospholipids.     

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [¹⁴C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12–14-day-old plants was calculated to be 330 μmol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22°C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

Circadian rhythms in Neurospora crassa: the effects of point mutations on the proteolipid portion of the mitochondrial ATP synthetase

Summary Five oligomycin-resistant (oli ^r) mutant strains of Neurospora crassa were analyzed for their growth rate and for the periodicity of their circadian rhythm. The most resistant strains had periods of 18–19 h while the least resistant strain had a normal period of 21.0 h. There was a rough correlation between the in vivo degree of oligomycinresistance and the amount of change in the period. Several of the oli ^r mutations have been previously described by Sebald et al. (1977) in terms of known amino acid changes in the primary structure of the proteolipid, or DCCD-binding protein, found in the F₀ membrane portion of the mitochondrial ATP synthetase. Amino acid changes in the structure of this protein are reported here for two other oli ^r mutations. The proteolipid isolation procedures were slightly modified to include a delipidation step, and an HPLC procedure was developed to separate the hydrophobic peptides of this protein. Analysis of heterocaryons carrying both the oli ^r and oli ^s markers indicated that the oli ^r and oli ^s mutations were codominant to each other in terms of period and growth rate. The changes in the primary structure of this DCCD-binding protein reported here are the first known examples of changes in the primary structure of a protein which alter the period of a circadian rhythm.     

The presence and photoregulation of protochlorophyllide reductase in green tissues

Summary The enzyme protochlorophyllide (pchlide) reductase has been identified amongst the peptides, resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), of chloroplast membranes from oat and barley plants. In support of this identification the enzymic activity associated with the enzyme has also been measured in the same preparations. A higher level of enzyme was found in plants which had been darkened prior to extraction. Based on this data, mechanisms for the light regulated diurnal variation of the reductase are discussed.     

Splenic natural killer-cell activity in mice infected with Leishmania donovani

Several strains of inbred mice were infected with the protozoan parasite Leishmania donovani, and, at several points during the infection, spleens of groups of these mice were tested for natural killer (NK)-cell activity vs lymphoma target cells in vitro and were evaluated for parasite burdens. Generally, elevated followed by normal (compared to uninfected control mice) or subnormal NK responses occurred as the result of infection. Elevated NK responses were not accompanied by high circulating levels of interferon, yet infected mice responded to an injection of an interferon inducer with interferon production as great as control mice. No consistent correlations among susceptibility phenotype to L. donovani infection, spontaneous NK activity phenotype, and infection-induced NK activation/depression patterns were detected among the various strains of mice.     

Tubulin-colchicine complexes differentially poison opposite microtubule ends

The kinetics of radiolabeled guanosine 5'-triphosphate-tubulin dimer addition to preformed microtubule copolymers, containing large numbers of tubulin-colchicine complexes (TCs), were examined at apparent equilibrium. The results indicated that radiolabeled dimer addition to copolymers occurs predominantly by a "treadmilling" reaction, analogous to that described for unpoisoned microtubules, and that some labeled dimer uptake also occurs by equilibrium exchange. The data further showed that TCs decrease the steady-state treadmilling reaction in a concentration-dependent manner. Since microtubule copolymers exhibited a treadmilling reaction, it was possible to differentially radiolabel opposite copolymer ends with [3H]- and [14C]guanine nucleotides and thus to measure the effects of TCs on dimer loss from opposite copolymer ends upon copolymer dilution. Dimer loss from both copolymer ends was inhibited in a concentration-dependent manner, but dimer loss from copolymer net assembly (A) ends (defined under steady-state conditions) was inhibited to a far greater extent than that from the opposite, net disassembly (D) copolymer ends. TCs therefore exhibited a graded, polar poisoning action, with copolymer A-end association and dissociation rate constants being far more susceptible to TC inhibition than those at the opposite copolymer D ends. The potential significance of this TC effect for regulating microtubule spatial orientation in vivo is discussed.