5-bromodeoxyuridine induction of hematopoietic neoplasia and retrovirus activation in the soft-shell clam, Mya arenaria

Hematopoietic neoplasia and virus replication were induced in shoft-shell clams, Mya arenaria, by 5-bromodeoxyuridine (5-BrdUrd). Eighty clams were found to be nonneoplastic by the in vivo bleeding method. These clams were randomly distributed into four aquaria and maintained in seawater at 1°C. 5-BrdUrd was added to aquaria in the following concentrations: 0, 20, 100, and 200 μg/ml. After 4 days of treatment, the aquaria were drained, rinsed, and fresh sea water without 5-BrdUrd was added. Sea water was changed on a weekly basis thereafter. The clams were diagnosed for neoplasia by the in vivo method at days 4, 9, 16, and 23 of the experiment. Results showed that on day 23, neoplasia was not found in the aquarium without 5-BrdUrd, but in the aquaria containing 20, 100, and 200 μg/ml of 5-BrdUrd the incidences of neoplasia were 37, 79, and 35%, respectively. 5-BrdUrd-induced neoplastic tissue was homogenized and subjected to differential ultracentrifugation. Retrovirus-like particles resembling ones previously shown to induce neoplasia were isolated from neoplastic clams which were induced by 5-BrdUrd. When these particles were inoculated into healthy clams, the clams developed neoplasia. Neoplastic hemocytes, cultured in sea water containing 50 μg/ml of 5-BrdUrd, formed pseudopodia after 4 days. Pseudopod formation is a trait of normal hemocytes. This indicates that differentiation of neoplastic hemocytes may be induced by the drug.     

The course and mortality of a hematopoietic neoplasm in the soft-shell clam,Mya arenaria

Results from two experiments containing approximately 280 Mya arenaria indicated that significantly higher (P < 0.05) mortality occurred within the neoplastic clam population than in the nonneoplastic clam population. Using an in vivo blood cytological technique, five levels of severity were established. The levels were based on the number of neoplastic cells in the circulation with level 1 as the lowest severity and level 5 as the highest severity. Neoplastic clams that were diagnosed as the lowest level had higher survival rates (60%) than those clams diagnosed as the highest level (0%). The hematopoietic neoplasm in M. arenaria followed one of three courses: (1) in approximately 50% of the neoplastic clams the disease progressed to a higher severity and resulted in death; (2) in approximately 40% of the neoplastic clams the disease was chronic, i.e., remained at a stable level; and (3) in approximately 10% of the neoplastic clams the disease diminished in severity or disappeared entirely. In addition, 10% of the clams diagnosed as nonneoplastic at the beginning of the experiments were neoplastic by the termination of the experiment. The contraction of the disease may have been de novo or by stimulation of latent infections.The prevalence of the neoplasm in clams collected from an epizootic area followed a biphasic seasonal pattern. The highest prevalences occurred in October, November, and in May. The hematopoietic neoplasm in M. arenaria was also age and species specific.     

The biosynthesis of lubimin from [1-14C]isopentenyl pyrophosphate by cell-free extracts of potato tuber tissue inoculated with an elicitor preparation from Phytophthora infestans

A cell-free enzyme system, which catalyses the incorporation of radiolabel from [$\text{1}\text{2}$-¹⁴C]isopentenyl pyrophosphate into the sesquiterpenoid phytoalexin lubimin, has been prepared from tuber tissue of Solanum tuberosum inoculated with an elicitor preparation from Phytophthora infestans. Biosynthesis of lubimin is optimum at pH 7.$\text{3}\text{2}$-7.5 and is dependent upon Mg²⁺ and NADPH. Lubimin labelling by cell-free enzyme system prepared from tissue 48 hr after treatment with elicitor rises rapidly to a maximum over the first 30 min of incubation and does not decline for a further 150 min. The biosynthetic capacity for lubimin in cell free extracts can be observed as early as 3 hr after inoculation of tuber tissue, and rises to a maximum at about 48 hr after treatment, declining thereafter. Lubimin labelling is inhibited by iodoacetamide, the effect of which is reversed by 3,3-dimethylallylpyrophosphate. Preliminary observations on the cell-free system show that it will also catalyse the incorporation of [2-¹⁴C]mevalonic acid into lubimin in the presence ofan ATP-generating system.     

Presence of four toxins in red tide infested clams and cultured Gonyaulax tamarensis cells.

Four toxins have been isolated by Sephadex G-15 and high pressure ion exchange chromatography from the soft shell clams, $\text{Mya arenaria}$, which were collected during 1972 and 1974 red tide outbreaks on the New England coast. One of the toxins was saxitoxin and the rest seem to be new toxins. The same toxins were isolated from the extract of cultured $\text{Gonyaulax tamarensis}$.     

Effect of glycl-tRNA concentration on in vitro serine incorporation into the peptidoglycan of S. epidermidis

Seven species of seryl-tRNA have been isolated from $\text{S. epidermidis}$ strain Texas 26 by the use of Plaskon CTFE powder as the support for Adogen 464 in reversed-phase chromatography. All but one of these seven seryl-tRNA incorporated ¹⁴C-serine into $\text{in vitro}$ biosynthesized peptidoglycan. This suggested that at least one of the functional roles of six of the seven seryl-tRNA peaks isolated may be involved in $\text{in vivo}$ cell wall synthesis. It has also been shown that the ratio of glycine to serine in the pentapeptide bridge depended upon the concentration of glycyl-tRNA in the assay mixture. By increasing the concentration of glycyl-tRNA the amount of serine inserted into the bridge is increased. Thus the amount of serine incorporated into the pentapeptide bridge appears to be dependent upon the concentration of glycyl-tRNA.     

Hydrophobic ion probe studies of membrane dipole potentials

Hydrophobic anions of dipicrylamine and of sodium tetraphenylborate have been employed as probes of interfacial dipole potential variations in lipid bilayer membranes. Systematic variation of dipole potentials has been achieved by introduction of compounds incorporating N⁺ and B^? charge centers. Distribution of hydrophilic and and hydrophobic groups relative to these charge centers has been shown to control the orientation in the membrane/solution interface of the electric dipole moment formed by these centers. Thus triphenyl-[4-trimethylphenylammonium] borate orients with the B^? center, surrounded by phenyl groups, embedded in the membrane, while the smaller methylated N⁺ center is directed toward the aqueous phases. This orientation has been confirmed using dipicrylamine probe ions. Results obtained in this system have been interpreted quantitatively using a previously developed model incorporating discrete charge effects. A second class of compounds, $\text{tri}\text{-n-}\text{alkylamine}$ borane (T_nAB) complexes having the generic formula (C_nH_{$\text{2n+1}$})₃N⁺B^?H₃, have also been synthesized for this study, using even-carbon alkyls ranging from ethyl to decyl. Molecular orientation of the complex is with the N⁺ center and its associated alkyl groups directed into the membranes, while the protonated B^? center is directed toward the aqueous phases, as confirmed by use of tetraphenylborate ions as probes.     

Effect of E. coli endotoxin on the structure-function of fatty acid synthetase lipoprotein

The lipoprotein structure of the fatty acid synthetase complex from Ceratitis capitata has been used as a model to $\text{val}\text{i}$ date the claim that phospholipids from membranes assume a $\text{signif}\text{i}$ cant role in the cell-endotoxin interactions. The enzyme-complex was exposed to a ¹⁴C-lipopolysaccharide preparation and the $\text{inte}\text{r}$ action was followed by a) circular dichroism spectra, b) enzyme activity and c) gel filtration chromatography. It should be $\text{emph}\text{a}$ sized that the E. coli endotoxin modifies all these properties of the enzyme complex and that a model involving phospholipids and phase transitions has been proposed to account for these $\text{intera}\text{c}$ tions.     

Translation of avian sarcoma virus RNA in Xenopus laevis oocytes.

Evidence for the synthesis and processing of Pr76 (precursor to group-specific antigens p27, p19, and $\text{p12}\text{15}$, upon injection of avian sarcoma virus 70S or 35S RNA into $\text{Xenopus}$ oocytes has been presented. Further, we show that tRNA^trp primer, bound to 35S RNA, does not block translation of virion RNA under these conditions.     

Chlorophyll-protein complexes of a Codium species,including a light-harvesting siphonaxanthin-Chlorophylla ab-protein complex,an evolutionary relic of some Chlorophyta

Eight chlorophyll-protein complexes were isolated from thylakoid membranes of a Codium species, a marine green alga, by mild SDS-polyacrylamide gel electrophoresis. CP 1a¹, CP 1a², CP 1a³ and CP 1a⁴ were partially dissociated Photosystem (PS) I complexes, which in addition to the core reaction centre complex, CP 1, possessed PS I light-harvesting complexes containing chlorophyll (Chl) a, Chl b and siphonaxanthin. LHCP¹ and LHCP³ are orange-brown green chlorophyll $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 0.66) that contain siphonaxanthin and its esterified form, siphonein. CP a and CP 1, the core reaction centre complexes of PS II and PS I, respectively, had similar spectral properties to those isolated from other algae or higher plants. These P-680- or P-700-Chl a-proteins are universally distributed among algae and terrestrial plants; they appear to be highly conserved and have undergone little evolutionary adaptation. Siphonaxanthin and siphonein which are present in the Codium light-harvesting complexes of PS II and PS I are responsible for enhanced absorption in the green region (518 and 538 nm). Efficient energy transfer from both xanthophylls and Chl b to only Chl a in Codium light-harvesting complexes, which have identical fluorescence emission spectra at 77 K to those of the lutein-Chl $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 1.2) of most green algae and all higher plants, proved that the molecular arrangement of these light-harvesting pigments was maintained in the isolated Codium complexes. The siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ allow enhanced absorption of blue-green and green light, the predominant light available in deep ocean waters or shaded subtidal marine habitats. Since there is a variable distribution of lutein, siphonaxanthin and siphonein in marine green algae and siphonaxanthin is found in very ancient algae, these novel siphonein-siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ may be ancient light-harvesting complexes which were evolved in deep water algae.     

Filter-feeding bivalves can remove avian influenza viruses from water and reduce infectivity

Avian influenza (AI) viruses are believed to be transmitted within wild aquatic bird populations through an indirect faecal–oral route involving contaminated water. This study examined the influence of filter-feeding bivalves, Corbicula fluminea, on the infectivity of AI virus in water. Clams were placed into individual flasks with distilled water inoculated 1:100 with a low pathogenic (LP) AI virus (A/Mallard/MN/190/99 (H3N8)). Viral titres in water with clams were significantly lower at 24 and 48 h post-inoculation compared to LPAI-infected water without clams. To determine whether clams affected the infectivity of AI viruses, 18 wood ducks (Aix sponsa) were divided into test groups and inoculated with a variety of treatments of clam supernatants, whole clams and water exposed to a high pathogenic (HP) AI (A/whooper swan/Mongolia/244/05 (H5N1)). None of the wood ducks inoculated with HPAI-infected water that was filtered by clams or that was inoculated with or fed tissue from these clams exhibited morbidity or mortality. All wood ducks exposed to either HPAI-infected water without clams or the original viral inoculum died. These results indicate that filter-feeding bivalves can remove and reduce the infectivity of AI viruses in water and demonstrate the need to examine biotic environmental factors that can influence AI virus transmission.     

Control of the bacterium Serratia marcescens in an insect host-parasite rearing program

Two occluded viruses of the Entomopoxvirus (D)/¹: ¹/¹: $\text{X}\text{X}\text{:}\text{I}\text{O}$ (= Vagoiavirus) group have been found in larvae of Dermolepida albohirtum (Scarabaeidae: Melolonthinae) and Aphodius tasmaniae (Scarabaeidae: Aphodiinae) from northern Queensland and northern Tasmania, Australia, respectively. Electron microscopical studies have been made of thin sections of occluded (mature) and nonoccluded virus particles within the fat body tissue of living diseased D. albohirtum larvae and of occluded virus particles within a dead, field-collected A. tasmaniae larva. The morphology and development of the known Australian entomopoxviruses are compared with previously known entompox or spheroidosis viruses from various insects.     

Incorporation of cinnamic acid-2-[14C] into tylophorine

Tylophora indica plants have been shown to contain phenanthroindolizidine alkaloids of the tylophorine type. Cinnamic acid-[2-¹⁴C]was incorporated efficiently into these alkaloids supporting the hypothesis that ring A and C-10 and C-6$\text{\$}\text{?}$of tylophorine are derived from phenylalanine.     

The roles of cytochrome b5 in a reconstituted N-demethylase system containing cytochrome P-450.

Compound 102804 isolated from $\text{Bacillus cereus}$ has been found to be a potent inhibitor of the N⁵-methyltetrahydrofolate-homocysteine transmethylase isolated from $\text{Escherichia coli}$ B. This inhibition was noted when 102804 was added to the enzyme reaction mixture after the reaction started or concurrently with the preparation of the mixture. Chemically inactivated 102804 has no activity as an inhibitor of this enzyme system.     

Development of mutagenicity test using Escherichia coli K-12 as indicator organism

A derivative of Escherichia coli K-12 (strain $\text{343}\text{113}$) has been developed in which mutations in several genes can be detected simultaneously by plating parts of the bacterial population on different selective media. The mutation types include reversions from differently induced auxotrophies (nad^-, arg^-) aand (forward) mutations leading to resistance against 5-methytryptophan and to gal⁺ phenotype. It is assumed that many types of DNA alteration, including deletions and changes involving gross DNA regions, will lead to viable detectable mutants.The usefulness of strain E. coli$\text{343}\text{113}$ was tested in spot tests, in liquid tests, in tests with extracts of mammalian organs, and in mammalian-mediated tests. It is concluded that strain $\text{343}\text{113}$ is at least as useful in routine mutagenicity testing (especially in mammalian-mediated assays) as other present bacterial strains.     

Occurrence and distribution of deoxyribonucleic acid-hydrolyzing bacteria in sea water

A large proportion of the heterotrophic bacteria isolated from sea water sampled from the Pacific Ocean and the neritic Sea of Japan were able to hydrolyze deoxyribonucleic acid (DNA). Their numbers ranged from 10² to $\text{10}{}^4\text{100 ml}$ in the upper layers of the water profile and were less than $\text{10}{}^2\text{100 ml}$ in deeper water in the open sea. In neritic regions, such as Aburatsubo Inlet, their numbers were higher, ranging between 10⁵ and $\text{10}{}^7\text{100 ml}$. These organisms formed a constant proportion of the total heterotrophic bacterial populations and showed few seasonal fluctuations.In incubation experiments it was shown that the DNA occurring in sea water was extensively degraded in situ by the indigenous bacterial flora. More detailed examination of the degradation, using an isolated marine Vibrio sp., has demonstrated that purine and pyrimidine bases and inorganic orthophosphate are released into the medium. The bacterium totally assimilated the cytosine released and appeared to convert adenine to hypoxanthine. In contrast guanine and thymine attained constant concentrations in the medium.     

Extraction of pure ribosomal protein and removal of Coomassie blue from acrylamide gels

A method has been developed to recover pure ribosomal proteins from two-dimensional polyacrylamide electrophoresis slabs. Proteins and Coomassie blue were extracted from the gels and separated by passing them through a Sephadex G-25 column. All the steps were performed in the presence of 66% ($\text{v}\text{v}$) acetic acid. The technique has been applied to Escherichia coll ribosomal proteins labeled with ¹²⁵I.     

Anomalous side chain amidation in plasma membrane proteins of simian virus 40-transformed lymphocytes indicated by isoelectric focussing and laser Raman spectroscopy.

Plasma membranes isolated from normal hamster lymphocytes and lymphoid cells transformed by SV40¹ have been compared. Isoelectric focussing in 1% Triton $\text{X-100}\text{8M}$ urea reveals higher isoelectric points than normal for the non-glycosylated proteins in the membranes of transformed cells. This suggests greater amidation of membrane aspartates and/or glutamates. The focussing patterns also reveal a shift to lower pH for the isoelectric points of most glycosylated proteins suggesting increased silalylation. The Amide I – Amide II laser Raman spectra of the two membrane categories are consistent with greater side chain amidation in the membranes of neoplastic lymphocytes.     

Serotonin action on short-circuit current and ion transport across isolated rabbit ileal mucosa.

Serotonin has previously been implicated as the cause of the diarrhea associated with carcinoid syndrome and the amine has been shown by others to be an intestinal secretagogue in preparations of intestinal loops $\text{in}$$\text{vivo}$. In the present paper the action of serotonin on isolated segments of rabbit ileal mucosa stripped of muscle layers was studied $\text{in}$$\text{vitro}$. Serotonin (10^?4M) caused an abrupt significant rise in short-circuit current (Isc) across the mucosal epithelial cell layer but this effect was transient. No change was observed in tissue conductance. In this preparation, serotonin did not alter ²²Na, ³⁶Cl or residual ion fluxes across the mucosa. High blood serotonin levels for a period of several days also did not alter ion fluxes or Isc in isolated rabbit ileum. Therefore, it is concluded that serotonin must cause its secretory activity observed $\text{in}$$\text{vivo}$ by some mechanism other than a direct action on epithelial cell transport mechanisms.     

The isolation of acyl carrier protein from the pigeon liver fatty acid synthetase complex1 II

A low molecular weight protein of less than 10, 000 Daltons has been isolated from Subunit I (β-ketoacyl thioester reductase) of the pigeon liver fatty acid synthetase complex and purified to homogeneity. This protein contains all of the [¹⁴C]-labeled pantetheine incorporated into the fatty acid synthetase on injection of [¹⁴C]-labeled pantetheine into pigeons. It also has one β-alanine and one sulfhydryl group. This protein is an acceptor of an acetyl group from acetyl-CoA and a malonyl group from malonyl-CoA in the presence of Subunit II (transacylase). In these respects it is very similar to $\text{E. coli}$ acyl carrier protein.