Morphological and Molecular Identification of Spirometra Tapeworms (Cestoda: Diphyllobothriidae) from Carnivorous Mammals in the Serengeti and Selous Ecosystems of Tanzania

Spirometra tapeworms (Cestoda: Diphyllobothriidae) collected from carnivorous mammals in Tanzania were identified by the DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and internal transcribed spacer 1 (ITS1), and by morphological characteristics. A total of 15 adult worms were collected from stool samples and carcasses of Panthera leo, Panthera pardus, and Crocuta crocuta in the Serengeti and Selous ecosystems of Tanzania. Three Spirometra species: S. theileri, S. ranarum and S. erinaceieuropaei were identified based on morphological features. Partial cox1 sequences (400 bp) of 10 specimens were revealed. Eight specimens showed 99.5% similarity with Spirometra theileri ({"type":"entrez-nucleotide","attrs":{"text":"MK955901","term_id":"1796114604","term_text":"MK955901"}}MK955901), 1 specimen showed 99.5% similarity with the Korean S. erinaceieuropaei and 1 specimen had 99.5% similarity with Myanmar S. ranarum. Sequence homology estimates for the ITS1 region of S. theileri were 89.8% with S. erinaceieuropaei, 82.5% with S. decipiens, and 78.3% with S. ranarum; and 94.4% homology was observed between S. decipiens and S. ranarum. Phylogenetic analyses were performed with 4 species of Spirometra and 2 species of Dibothriocephalus (=Diphyllobothrium). By both ML and BI methods, cox1 and ITS1 gave well supported, congruent trees topology of S. erinaceieuropaei and S. theileri with S. decipiens and S. ranarum forming a clade. The Dibothriocephalus species were sisters of each other and collectively forming successive outgroups. Our findings confirmed that 3 Spirometra species (S. theileri, S. ranarum, and S. erinaceieuropaei) are distributed in the Serengeti and Selous ecosystems of Tanzania.     

Selective allele loss and interference between cauliflower mosaic virus DNAs

Summary Some cauliflower mosaic virus (CaMV) alleles are selectively lost during growth of the virus in mixedly infected turnip plants. Viral DNA from plants co-inoculated with DNA of the cabbage S isolate and infectious cabbage S DNA with an extra EcoRI restriciion site lacked the extra site. The EcoRI allele was also lost in most plants co-inoculated with a non-infectious mutant of cabbage S DNA while little selective allele loss was observed with two other non-infectious mutant DNAs. Plants co-inoculated with DNAs of closely-related isolates (CM4-184 and W) contained both parental viral DNAs and some DNAs with characteristics of both parents. Interference, scored as a reduced frequency of infection or a delay in symptom appearance relative to plants inoculated with wild-type DNA, occurred when plants were inoculated with wild-type and mutant DNAs covalently attached to one another in partial dimer plasmid DNAs. Similarities in the conditions leading to selective allele loss and those leading to interference suggest that both may have been due to active gene conversion between CaMV DNA molecules.     

Disruption of phospholipid asymmetry in erythrocyte vesicles deficient in spectrin

The transbilayer distribution of phospholipids in right-side-out and inside-out vesicles derived from human erythrocytes was studied by phospholipase A2 digestion assays and by staining with the fluorescent dye merocyanine 540. In both types of vesicles, the normal asymmetric distribution of phospholipids characteristic of intact cells was disrupted. Because both types of vesicles are deficient in spectrin, the major protein of the cytoskeletal network which normally underlies the membrane, these results support the contention that spectrin is involved in the maintenance of phospholipid asymmetry.     

The Metabolism of Oat Leaves during Senescence: III. The Senescence of Isolated Chloroplasts

The changes in chlorophyll and protein in senescing chloroplasts isolated from the first leaves of 7-day-old oat (Avena sativa) seedlings have been investigated. In darkness the chlorophyll in these plastids is highly stable, losing only 5 to 10% of its content after 7 days at 26 C. This result contrasts with the behavior of chlorophyll in intact leaves, in which about 80% of the pigment would have disappeared in that time. The protein is less stable than the chlorophyll, though more stable than in the leaf; probably a small amount of protease is present in the plastids. Some protein is also being synthesized in the chloroplasts along with its breakdown; gains of up to 38% in protein and 13% in chlorophyll were observed under different conditions. l-Serine, which actively promotes senescence in the leaf, has only a very slight effect on the chloroplasts, and kinetin antagonizes it. Kinetin also has a small but significant effect in preserving the protein from breakdown. Acid pH somewhat promotes the breakdown, both of chlorophyll and protein. A loss of chlorophyll and protein comparable to that occurring in the senescence of the leaf could not be induced in the chloroplasts by suspending them in malate, in cytoplasmic extract, or in any of a number of enzymes tested alone. Incubation with a mixture of four enzymes was the only treatment which approximated the senescent process in the leaf, causing 34% loss of chlorophyll at pH 5 and 40% loss of protein at pH 7.4, both in 72 hours.In white light, the chlorophyll and the carotenoids, but not the protein, disappear rapidly. This disappearance was shown to be prevented in an atmosphere of nitrogen or in air by a number of reducing agents, of which ascorbic acid was the most effective. It is, therefore, ascribed to photooxidation rather than to normal senescence.     

MA 160 and EB 33 cell lines: HeLa cell contaminants,hybrids or prostatic epithelial cells?

Summary Studies of acid phosphates produced by cell lines MA 160 and EB 33 demonstrated immunochemically their prostatic origin. MA 160 and EB 33, rather than being HeLa contaminants, may be hybrids of prostatic epithelial and HeLa cells or true prostatic cell lines with chromosomal changes common to all long-term cultivated cell lines. This research was supported by NIH (Cancer) Research Grants Nos. 18748 and 16426; and Detroit General Hospital Research Corporation.     

Nuclear magnetic resonance and molecular modeling studies on O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-se rine, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans

The solution conformation of O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-ser ine (GXS), a carbohydrate-protein linkage region fragment from connective tissue proteoglycans, was investigated by two-dimensional NMR spectroscopy and molecular modeling calculations. Specifically, the 1H and 13C resonances were assigned by 2D-COSY and by 1H-13C heteronuclear correlation spectroscopy methods. 2D-NOESY was used to generate distance constraints between the galactose and xylose and between the xylose and serine residues. The 1H vicinal coupling constants for the sugars and the serine were also determined. A general molecular modeling methodology suitable for complex carbohydrates was developed. This methodology employed molecular dynamics and energy minimization procedures together with the application of inter-residue spatial constraints across the linkages derived from 2D-NOESY. The first step in this methodology is the generation of a wide variety of starting conformations that span the (phi, psi) space for each linkage. In the present study, nine such conformations were constructed for each linkage using the torsion angles phi and psi corresponding to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined molecular dynamics/energy minimization refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. Characterization of GXS using this methodology identified a single family of conformations that are consistent with the solution phase NMR data on this molecule.     

Adaptation of human immunodeficiency virus type 1 to cells expressing a binding-deficient CD4 mutant (lysine 46 to aspartic acid).

Human immunodeficiency virus (HIV-1) was adapted to replicate efficiently in cells expressing an altered form of the CD4 viral receptor. The mutant CD4 (46 K/D) contained a single amino acid change (lysine 46 to aspartic acid) in the CDR2 loop of domain 1, which results in a 15-fold reduction in affinity for the viral gp120 glycoprotein. The ability of the adapted virus to replicate in CD4 46 K/D-expressing cells was independently enhanced by single amino acid changes in the V2 variable loop, the V3 variable loop, and the fourth conserved (C4) region of the gp120 glycoprotein. Combinations of these amino acids in the same envelope glycoprotein resulted in additive enhancement of virus replication in cells expressing the CD4 46 K/D molecule. In cells expressing the wild-type CD4 glycoproteins, the same V2 and V3 residue changes also increased the efficiency of replication of a virus exhibiting decreased receptor-binding ability due to an amino acid change (aspartic acid 368 to glutamic acid) in the gp120 glycoprotein. In neither instance did the adaptive changes restore the binding ability of the monomeric gp120 glycoprotein or the oligomeric envelope glycoprotein complex for the mutant or wild-type CD4 glycoproteins, respectively. Thus, particular conformations of the gp120 V2 and V3 variable loops and of the C4 region allow postreceptor binding events in the membrane fusion process to occur in the context of less than optimal receptor binding. These results suggest that the fusion-related functions of the V2, V3, and C4 regions of gp120 are modulated by CD4 binding.     

Photoprotective effect of kinetin on pigment content and photochemical activities of wheat chloroplasts agingin vitro

The effects of kinetin (Kn) on pigment content and electron transport activities (ETA) in wheat leavesin vivo and chloroplastsin vitro aging in light was investigated. Excised wheat leaves were infiltrated with Kn for 3 h under irradiation. The treatment increased zeaxanthin (Zx) content by 40% and also increased chlorophyll (Chia, Chib) and major carotenoid (Car) contents in the leaves (per fresh mass unit). Chloroplasts isolated from Kn treated leaves, when incubated in light for 4 h showed relatively lower pigment loss and slower loss of ETA compared to the chloroplasts of untreated leaves. These observations suggest photoprotective action of Kn. The photoprotection was more prominent when Kn was applied directly to the irradiated chloroplastsin vitro. Moreover, chloroplasts agingin vitro under irradiation without Kn treatment lost pigments and ETA. Within 3 h of irradiation, both whole chain (H₂O to methylviologen) electron transport as well as photosystem (PS) 2 activity were completely lost. However, in the chloroplasts treated with Kn, the loss of pigments was slow and even after 4 h of irradiation the chloroplasts retained 15 % of PS 2 and 9 % of whole chain ETA. In the untreated chloroplasts, the loss of Zx after 4 h of irradiation was 49 % whereas in Kn treated samples its level was 1.3 times higher than that of control. Since a higher level of Zx was maintained in Kn treated chloroplasts, photoprotective action of Kn is possibly mediated through Zx. One of us (NKC) thanks Sambalpur University for study leave and Department of Biological Sciences, Mankato State University, Mankato for labortory facilities.