Turnover of Extracellular DNA in Eutrophic and Oligotrophic Freshwater Environments of Southwest Florida

The turnover of extracellular DNA was investigated in oligotrophic springs of the Crystal River and the eutrophic Medard Reservoir of southwest Florida. The Medard Reservoir possessed large populations of bacterioplankton and phytoplankton (6.8 × 10⁹ cells per liter and 28.6 μg of chlorophyll a per liter, respectively), while the Crystal River springs only contained a fraction of the microbial biomass found in the Medard Reservoir. Although dissolved DNA values were greater in the Medard Reservoir, higher rates of DNA removal resulted in similar extracellular DNA turnover times in both environments (9.62 ± 3.6 h in the Crystal River and 10.5 ± 2.1 h in the Medard Reservoir). These results indicate that regardless of trophic status or microbial standing stock, extracellular DNA turns over rapidly in subtropical planktonic freshwater environments. Therefore, recombinant DNA sequences from released genetically engineered microorganisms might not be expected to survive for long periods of time in freshwater planktonic environments.     

Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure

The predicted conformation and position of the central transforming region (residues 55–67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1–166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an -helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.     

Cloning and expression of the toxin B gene ofClostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.     

Studies on new,centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimer's disease, and other memory disorders.     

Biotransformations of aromatic aldehydes by acetogenic bacteria

Vanillin was subject to O demethylation and supported growth of Clostridium formicoaceticum and Clostridium thermoaceticum. Vanillin was also stimulatory to the CO-dependent growth of Peptostreptococcus productus. The aldehyde substituent of vanillin was metabolized by routes which were dependent upon both the acetogen and a co-metabolizable substrate (e.g. carbon monoxide [CO]). C. formicoaceticum and C. thermoaceticum oxidized the aldehyde group of vanillin to the carboxyl level, while P. productus reduced the aldehyde group of vanillin to the alcohol level. In contrast, during CO-dependent growth, C. thermoaceticum reduced 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol while P. productus both reduced and oxidized 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol and 4-hydroxybenzoate, respectively. These metabolic potentials indicate aromatic aldehydes may affect the flow of reductant during acetogenesis.     

Delimitation ofPelargonium sect.Glaucophyllum (Geraniaceae)

Pelargonium otaviense Knuth andP. spinosum Willd. are excluded from sect.Glaucophyllum, whileP. grandiflorum (Andr.)Willd.,P. patulum Jacq. andP. tabulare (Burm. f.)L'Hérit. of sect.Eumorpha are included. Sect.Glaucophyllum is characterized by green to glaucous vegetative organs and zygomorphic white to pink corolla with five narrow petals. All the species have an identical pollen and chromosome morphology, the same basic chromosome number (x = 11) and similar flavonoid patterns. A close relationship between sect.Glaucophyllum and sect.Pelargonium is indicated by the occurrence of natural hybrids and concordant characters. Isorhamnetin and luteolin have been detected in the genus for the first time.     

Hensen's node,but not other biological signallers,can induce supernumerary digits in the developing chick limb bud

Summary The purpose of this study was to determine whether the organizer regions of early avian and amphibian embryos could induce supernumerary (SN) wing structures to develop when they were grafted to a slit in the anterior side of stage 19–23 chick wing buds. Supernumerary digits developed in 43% of the wings that received anterior grafts of Hensen's node from stage 4–6 quail or chick embryos; in addition, 16% of the wings had rods of SN cartilage, but not recognizable SN digits. The grafted quail tissue did not contribute to the SN structures. When tissue anterior or lateral to Hensen's node or lateral pieces of the area pellucida caudal to Hensen's node were grafted to anterior slits, the wings usually developed normally. No SN structures developed when Hensen's nodes were grafted to posterior slits in chick wing buds. Wings developed normally when pieces of the dorsal lip of the blastopore from stage 10–11.5 frog (Xenopus laevis and Rana pipiens) embryos were grafted to anterior slits. No SN digits developed when other tissues that have limb-inducing activity in adult urodele amphibians [chick otic vesicle, frog (Rana pipiens) lung and kidney] or that can act as heteroinductors in neural induction (rat kidney, lung, submaxillary gland and urinary bladder; mouse liver and submaxillary gland) were grafted to anterior slits in chick wing buds. SN digits also failed to develop following preaxial grafts of chick optic vesicles. These results suggest that although the anteroposterior polarity of the chick wing bud can be influenced by factors other than the ZPA (e.g., Hensen's node, retinoids), the wing is not so labile that it can respond to a wide variety of inductively-active tissues.     

Seed size variation: magnitude,distribution, and ecological correlates

Summary We examined seed-mass variation in 39 species (46 populations) of plants in eastern-central Illinois, USA. The coefficient of variation of seed mass commonly exceeded 20%. Significant variation in mean seed mass occurred among conspecific plants in most species sampled (by hierarchical ANOVA), averaging 38% of total variance. For most species, within-plant variation was the larger component of total variance, averaging 62% of total variance. Variation in seed mass among fruits within crops was significant in most species tested.We conclude that variation in seed mass among and within plants is widespread and common. There was little evidence of trade-offs between number of seeds and mean or variance of seed mass, and little correlational evidence of local competition for maternal resources. No consistent ecological (dispersal mode and growth form) correlates of variance of seed mass were evident.     

Homozygous osteogenesis imperfecta unlinked to collagen I genes

Summary In a consanguineous pedigree in which a severe type of osteogenesis imperfecta was segregating as an autosomal recessive trait, analysis of genetic markers for both collagen I structural loci COL1A1 and COL1A2 showed that the phenotype was unlinked to either locus.     

Spontaneous release of endogenous aspartate and glutamate from rat striatal slices is increased following destruction of local neurons by ibotenic acid

We sought to determine in rat striatum whether the release of neurotransmitter amino acids aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) were affected by local neurons. To do so, unilateral microinjections of ibotenic acid, an excitotoxin that destroys local neurons without affecting fibers of passage, were made into the striatum. Release of endogenous amino acids from lesioned and intact striatal slices were measured by HPLC one week later. The effectiveness and specificity of the lesion were confirmed by measuring the enzyme activity associated with extrinsic dopamine neurons (tyrosine hydroxylase; 111±14%), intrinsic GABA neurons (glutamic acid decarboxylase; 19±7%) and intrinsic acetylcholine neurons (choline acetyltransferase; 37±10%). Destruction of local striatal neurons markedly attenuated the release of GABA (41±12% of control) elicited by depolarization with K⁺ (35 mM), but did not significantly reduce the K⁺-evoked release of Asp (80±17%) and Glu (92±8%). However, spontaneous release of Asp and Glu was significantly greater than that observed in unlesioned tissue (159±18% and 209±27%, respectively), while the spontaneous release of GABA was not significantly reduced (75±43%). Although release of the neurotransmitter amino acids Asp, Glu and GABA were affected by the lesion, the release of the non-neurotransmitter amino acid tyrosine was unaffected. These data are consistent with the hypotheses that: 1) the predominant source of releasable stores of endogenous Asp and Glu in the striatum arises from extinsic neurons, and 2) that the spontaneous release of Asp and Glu from axon terminals in the striatum may be regulated, at least in part, by local inhibitory neurons.