Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.

We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine. On the other hand, E. coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media. The high-activity plasmids also have an enhanced [3H]dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Desiccation as a mitigation tool to manage biofouling risks: trials on temperate taxa to elucidate factors influencing mortality rates

The desiccation tolerance of biofouling taxa (adults and early life-stages) was determined under both controlled and ‘realistic’ field conditions. Adults of the ascidian Ciona spp. died within 24 h. Mortality in the adult blue mussel Mytilus galloprovincialis occurred within 11 d under controlled conditions, compared with 7 d when held outside. The Pacific oyster Crassostrea gigas was the most desiccation-tolerant taxon tested (up to 34 d under controlled conditions). Biofouling orientated to direct sunlight showed faster mortality rates for all the taxa tested. Mortality in Mytilus juveniles took up to 24 h, compared with 8 h for Ciona, with greater survival at the higher temperature (18.5°C) and humidity (~95% RH) treatment combination. This study demonstrated that desiccation can be an effective mitigation method for a broad range of fouling taxa, especially their early life-stages. Further work is necessary to assess risks from other high-risk species such as algae and cyst forming species.     

Changes in Respiration of Seedling Roots Inoculated with Phytophthora cinnamomi

An increase in rate of respiration was recorded for intact roots of seven native Australian species 16 h after inoculation with Phytophthora cinnamomi. By 24 h the magnitude of the increase ranged from 2—159% above that of the uninoculated controls and was evidently not related to host susceptibility. A time sequence study of lesion extension and the associated increased respiration rates for both susceptible and tolerant eucalypts demonstrated a difference in response. The rate of respiration in the tolerant species increased 2 % and only at the site of inoculation, whereas in the susceptible species the respiration rate increased in a wave which began at the inoculation site and continued along the root with the advancing fungal invasion. Respiration rate only increased in regions of the root actually inhabited by the pathogen. The fungal contribution to the total respiration of infected roots was less than 1 % and was determined by measuring respiration of inoculated killed roots. Respiration rates were measured in the presence of potassium cyanide (KCN) and salicylhydroxamic acid (SHAM). Both KCN-sensitive and SHAM-sensitive respiration occurred in normal uninfected E. marginate seedlings. A large proportion of the increase in total respiration rate of infected seedlings compared with uninoculated controls was due to the alternate, SHAM-sensitive pathway. The physiological implications of these results are discussed.     

Met-enkephalin, age and anatomy: a microiontophoretic study in the hippocampus

Met-enkephalin, administered microiontophoretically, produced a greater increase in firing in cells in area CA 3-4 in the hippocampus of both young and aged Fisher 344 rats than it did in the CA 1 area. Furthermore, the effect of met-enkephalin on neuronal firing rates was not as great in old rats as it was in young rats. Finally, 20-40 nA of met-enkephalin produced an increase in firing in old rats that was equivalent to the difference (2.5 spikes/sec) in baseline firing between old (2.6 spikes/sec) and young rats (5.1 spikes/sec).     

Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.     

Concentration and relative molecular mass of hyaluronate in lymph and blood.

Human lymph was collected from patients with leaking lymph vessels after thoracic surgery. Ovine lymph was obtained from the mesenteric, lumbar, popliteal and prescapular lymph ducts by cannulation. The concentration of hyaluronate varied considerably (between 0.2 and 50 mg/l) and the highest concentrations were found in mesenteric lymph. The Mr of the polysaccharide showed a great polydispersity and variation between individuals and in different regions of the lymphatic system. High-Mr hyaluronate (greater than 10(6) was present in lymph both from man and sheep. Hyaluronate was also isolated by affinity chromatography in 70-80% yield from human serum and plasma obtained from healthy individuals and patients with rheumatoid arthritis and primary biliary cirrhosis. The weight (Mw)- and number (Mn)-average relative molecular masses were roughly the same in the three groups [(1.4-2.7) X 10(5) and (2.1-5.7) X 10(4) respectively]. The low Mr of hyaluronate in blood compared with that in lymph is explained by a preferential uptake of the large molecules by the liver endothelial cells.     

137Cs as a tracer of recent sedimentary processes in Lake Michigan

To determine recent sediment movement, we measured the levels of ¹³⁷Cs (an artificial radionuclide produced during nuclear weapons testing) of 118 southern Lake Michigan samples and 27 in Green Bay. These samples, taken from 286 grab samples of the upper 3 cm of sediment, were collected in 1975 as part of a systematic study of Lake Michigan sediment. ¹³⁷Cs levels correlated well with concentrations of organic carbon, lead, and other anthropogenic trace metals in the sediment. ¹³⁷Cs had a higher correlation with silt-sized than with clay-sized sediment (0.55 and 0.46, respectively). Atmospherically derived ¹³⁷Cs and trace metals are being redistributed by sedimentary processes in Lake Michigan after being incorporated in suspended sediment. We determined a distribution pattern of ¹³⁷Cs that represents areas of southern Lake Michigan where sediment deposition is occurring.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.