Recently, three distinct isoforms of phospholipase D (PLD) were identified in Arabidopsis thaliana. PLD α represents the well-known form found in plants, while PLD β and γ have been only recently discovered (Pappan et al., 1997b; Qin et al., 1997). These isoforms differ in substrate selectivity and cofactors required for activity. Here, I report that PLD β and γ isoforms were active toward N-acylphosphatidylethanolamine (NAPE), but PLD α was not. The ability of PLD β and γ to hydrolyze NAPE marks a key difference from PLD α. N-acylethanolamines (NAE), the hydrolytic products of NAPE by PLD β and γ, inhibited PLD α from castor bean and cabbage. Inhibition of PLD α by NAE was dose-dependent and inversely proportional to acyl chain length and degree of unsaturation. Enzyme kinetic analysis suggested non-competitive inhibition of PLD α by NAE 14:0. In addition, a 1.2-kb tobacco (Nicotiana tabacum L.) cDNA fragment was isolated that possessed a 74% amino acid identity to Arabidopsis PLD β indicating that this isoform is expressed in tobacco cells. Collectively, these results provide evidence for NAE producing PLD activities and suggest a possible regulatory role for NAE with respect to PLD α.

The flagellum of Trypanosoma brucei contains a family of antigenically related EF-hand calcium-binding proteins which are called the calflagins. Genomic Southern blots indicated that multiple copies of calflagin genes occur in T brucei. All of the copies were contained in a single 23 kb Xhol-Xhol fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. Two new members of the calflagin family were found from genomic clone sequences. The deduced amino acid sequences of the genomic clones showed the calflagin genes were arranged tandemly along the genomic fragments and were similar to previously described calflagins. The calflagin genes were related by two unrelated 3' flanking sequences. An open reading frame that was unrelated to any calflagin was found at the 5' end of the 2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. In general, variability among the T. brucei calflagins is greater than related proteins in T. lewisii and T. cruzi. This variability results from amino acid substitutions at the amino and carboxy termini, and duplication of internal segments.

A behavioral model for acute responses in bivalves, was developed using time series analysis for use in a real-time biomonitoring unit. Stressed bivalves closed their shell and waited for the stressful conditions to pass. Baseline data showed that group behavior of fifteen bivalves was periodic, however, individuals behaved independently. Group behavior did not change over a period of 20 minutes more than 30 percent, however, following toxic exposures the group behavior changed by more than 30 percent within 20 minutes. Behavior was mathematically modeled using autoregression to compare current and past behavior. A logical alarm applied to the behavior model determined when organisms were stressed. The ability to disseminate data collected in real time via the Internet was demonstrated.

Utilization of cyanide as a nutritional nitrogen source in P. fluorescens NCIMB 11764 was shown to involve a novel metabolic mechanism involving nonenzymatic neutralization outside of cells prior to further enzymatic oxidation within. Several cyanide degrading enzymes were produced by NCIMB 11764 in response to growth or exposure to cyanide, but only one of these cyanide, oxygenase (CNO), was shown to be physiologically required for assimilation of cyanide as a growth substrate.

This research utilized population genetic analyses (protein starch-gel electrophoresis and DNA sequencing of the cytochrome b mtDNA gene), host-parasite specificity (lice coevolution), remote sensing of satellite data, and geographic information systems (GIS) to characterize newly discovered populations of pocket gophers (genus: Geomys) in Arkansas. These populations are isolated and occur in seemingly unsuitable habitat in the Ozark Mountains of Arkansas. Analyses of electrophoretic and ectoparasite data suggested the populations in the Ozark Mountains represented isolates allied to Geomys bursarius, a species not known to occur in Arkansas. Comparison of mitochondrial DNA sequence data of the cytochrome b gene with that of other taxa and morphometric analyses confirmed that these populations are most closely allied to G. bursarius occurring to the north in Missouri. Moreover, these mtDNA sequence analyses indicated a degree of differentiation typical of that between other subspecies of pocket gophers. Therefore, these populations represent a distinct genetic entity in an intermediate stage of speciation and should be designated as a new subspecies, Geomys bursarius ozarkensis. Molecular clock analysis revealed a time of lineage divergence for this new subspecies as approximately 511,000 YBP. Due to the isolated nature and limited distribution of this subspecies, an evaluation of critical habitat …

Bacterial aspartate transcarbamoylases (ATCase's) are divided into three classes that correspond to taxonomic relationships within the bacteria. The opportunistic pathogen Moraxeila catarrhalis has undergone several reclassifications based on traditional microbiological criteria. The previously uncharacterized ATCase from M. catarrhalis was purified to homogeneity and its chemical properties characterized. The ATCase from M. catarrhalis is a class C ATCase with an apparent molecular mass of 480-520 kDa. The M. catarrhalis ATCase is a dodecomer composed of six 35 kDa polypeptides and six 45 kDa polypeptides. The enzyme has an unusually high pH optimum of greater than pH 10. The enzyme exhibited hyperbolic kinetic with a Km for aspartate of 2 mM. A single, separate 78 kDa dihydroorotase from M. catarrhalis was identified and it was not associated with ATCase. These data support the reclassification of M. catarrhalis out of the Neisseriaceae family.

Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the interconversion of carbon dioxide and carbonate and has been found to play a wide range of roles in animals, plants and bacteria. Cotton genomic and cDNA libraries were screened for the plastidial isoform of carbonic anhydrase. The nucleotide sequences of two 1.2 Kb partial cDNA clones were determined. These clones exhibit high homology to carbonic anhydrases from other dicot plants and possess all the expected peptide motifs. For example, serine and threonine rich chloroplastic targeting peptide and conserved zinc binding residues are both present. These clones were utilized to isolate two carbonic anhydrase genes that were shown to encode different isoforms by PCR and RFLP analysis.

A study was undertaken to explore relationships between wetland characteristics which make them efficient water purifiers versus their ability to serve as wildlife habitat. The effects of designing constructed wetlands for improved habitat on water treatment efficiencies were quantified. Results indicate that some sacrifice in treatment efficiency is required and that the degree of efficiency reduction is dependant upon pollutant loading rates. However, sacrifice in efficiency is much smaller than increase in habitat quality, and can be offset by increasing wetland area. A practical, theoretical application was then attempted.

The proposed biology curriculum for non-majors has one main objective, namely to improve scientific literacy among college students. The National Science Education Standards defines scientific literacy as "the knowledge and understanding of scientific concepts and processes required for personal decision making, participation in civic and cultural affairs, and economic productivity". The suggested strategies to accomplish this goal are to limit the number of topics covered, introduce relevant scientific terminology, emphasize general biological concepts and themes, and hone critical thinking and problem-solving skills. Activities such as group projects, written and oral assignments, and class discussions are effective tools to assess student ability to communicate scientifically. It is also important for students to make connections between the course subject matter and how it affects real life events.

N-Acylphosphatidylethanoiamine (NAPE) is synthesized in the microsomes of cotton seedlings by a mechanism that is possibly unique to plants, the ATP-, Ca2+-, and CoA-independent acylation ofphosphatidylethanolamine (PE) with unesterified free fatty acids (FFAs), catalyzed by NAPE synthase. A photoreactive free fatty acid analogue, 12-[(4- azidosalicyl)amino]dodecanoic acid (ASD), and its 125I-labeled derivative acted as substrates for the NAPE synthase enzyme.

A publication by the National Academy of Sciences, USA (1979) outlined some of the research need for a great variety of economically important woody species whose remaining genetic resources need urgently to be collected and conserved. A viable regeneration system was established via tissue and cell suspension culture for Albizia falcataria and A. lebbeck, two important wood yielding leguminous tree species. The culture medium was standardized after several trials to obtain callus from the leaflet explants of these two tree species. The optimum use of casein hydrolysate (w/v) and coconut milk (v/v) in addition to 6-Benzylaminopurine and Indole-3-butyric acid could induce morphogenesis and somatic embryogenesis in the cultured tissue. This reports the first observation on somatic embryogenesis ofA. lebbeck using leaflets as the explants. Scanning Electron Microscopy and histological studies were done on the different stages plant development following standard techniques. Embryogenesis in suspension culture followed regeneration of plantlets in A. lebbeck. In A.falcaaria the regenerative process followed via organogenesis from the shoot buds developed on the leaf explants. After hardening the regenerated plants were transferred to the greenhouse. Some of the trees grew more than 25 feet tall within a few months outside the greenhouse. Karyotype of the three …

In the United States, biodiversity impact assessment has historically received little attention. Responding in 1993, the Council on Environmental Quality (CEQ) released guidelines on incorporating biodiversity into environmental impact assessment under the National Environmental Policy Act of 1969. The objectives of the study here were to identify the level of documentation of biodiversity impact assessment in sample Environmental Impact Statements (EISs); identify whether in the years following the release of 1993 CEQ guidelines any significant changes have taken place in assessment of biodiversity; identify deficiencies, and if the need exists, formulate appropriate recommendations and approaches for addressing biodiversity in EISs. The study involved a systematic review of 30 EISs published since the release of CEQ guidelines, and five EISs published prior to it. The review involved answering a series of standard questions, which attempted to ascertain the level of biodiversity impacts included in each impact statement. Trends in approaches to biodiversity impact assessment were investigated and deficiencies summarized. The analysis resulted in a series of recommendations for improving the manner in which biodiversity impact assessment can be approached.

The lumbar motor activity in isolated spinal cords of 72 postnatal Balb/C mice aged 2, 5, 10 and 21 days (PN2-21) was electroneurographically recorded (ENG) via bilateral ventral roots following treatment with three different concentrations (25, 100 and 200 pM) of the neurotransmitter, 5-hydroxytryptamine (5-HT), i.e., serotonin, to determine its effects on spinal pattern generation.

This research evaluated the potential of activation and attachment, as sequential companion biomarkers of phagocytosis by earthworm, Lumbricus terrestris, immunoactive coelomocytes for use in immunotoxicology. The potential was assessed by exposing earthworms to sublethal concentrations of CuSO4 and Arochlor 1254®, chemicals used as reference or standard immunotoxicants.

There is a transition nationwide on the analysis of forensic evidentiary stains containing biological material from traditional serology to Polymerase Chain Reaction (PCR) methodologies. The increased sensitivity of PCR, the limited number of alleles at each locus, and the necessity of producing unambiguous data for entry into the FBI's Combined DNA Index System make this study of extraction procedures of utmost importance. A "single tube" extraction procedure for blood stains collected onto FTA™ paper and a modified differential nonorganic extraction method from spermatozoa containing mixed stains were analyzed and compared. The extraction success was evaluated by amplification and typing of the amplified fragment length polymorphism, D1S80. These modifications of the nonorganic method utilized gave an improved separation of the spermatozoa-containing mixed stains.

To further understand the ATCase/DHOase bifunctional complex formed in Streptomyces, the genes encoding these and other pyrimidine enzymes were identified and characterized. Polymerase chain reaction (PCR) was utilized in this effort. Primers were constructed by selecting conserved regions of pyrimidine genes from known gene and protein sequences of a wide variety of organisms. These sequences were then optimized to Streptomyces codon usage. PCR products were obtained from internal sites within pyrimidine genes and also from primer combinations of different genes. The size, orientation, and partial sequence of the resulting products shows that Streptomyces has a gene organization of pyrR followed by pyrB, pyrC, carA, carB, and pyrF in an operon similar to that found in other Gram-positive bacteria.

Spontaneously active primary cultures obtained from dissociated embryonic medial medulla tissue were grown on microelectrode arrays for investigating burst patterns and pharmacological responses of respiratory-related neurons. Multichannel burst rates and spike production were used as primary variables for analysis. Pacemaker-like neurons were identified by continued spiking under low Ca++/high Mg++conditions. The number of pacemakers increased with time under synaptic blocking medium. Sensitivity to CO2 levels was found in some neurons. Acetylcholine changed activity in a complex fashion. Curare, atropine and gallamine modified ACh effects. Eserine alone was ineffective, but potentiated ACh-induced responses. Norepinephrine caused channel-specific increases or decreases, whereas dopamine and serotonin had little effect at 30 μM. GABA and glycine stopped most spiking at 70 μM. Developmental changes in glycine sensitivity (increasing with age) were also observed. It is concluded that pacemaker and chemosensitive neurons develop in medial medulla cultures, and that these cultures are pharmacologically histiotypic.

A 2 x 2 factorial design investigated effects of sediment nitrogen and water potassium levels on autofragment production. Reduced nitrogen levels significantly increased autofragment production whereas potassium levels did not significantly alter production. Up to 50% of autofragment production abscised from parent plants grown under low nitrogen conditions compared to 12% or less under high nitrogen conditions.

Recent studies regarding research on oral microorganisms and the oral diseases are presented. The normal flora of the mouth and the oral environment are first described. Dental plaque and dental caries are primary causes of oral disease. Streptococcus mutans is the major contributor in the initiation and progression of dental caries. Lactobacillus, Actinomyces, and Veillonella are other genera of bacteria linked to dental caries. Periodontitis and gingivitis are periodontal diseases that are caused by oral microorganisms. New research has indicated that various antimicrobial agents and techniques to eliminate or lessen the severity of periodontal diseases. Premature delivery of low birth weight babies in pregnant women has been strongly linked to periodontal disease. Present and future microbiological tests are available to easily determine the causative organisms for most oral diseases that help in diagnosis and treatment of a particular disease.

The nucleotide sequence for the pDKl TOL plasmid region encoding toluate-1,2-dioxygenase (Xy1XYZ, TO) was determined. TO is the first enzyme in the meta-cleavage operon, responsible for the conversion of toluates and benzoates to their carboxy-substituted diols. DNA sequence analysis revealed the presence of three open reading frames (ORF). The three ORFs correspond to xylX (1353 bp), xylY (486 bp) and xylZ (1008 bp), encoding predicted protein products of 51370 Da, 19368 Da and 36256 Da, respectively.

Aspartate transcarbamoylase (ATCase) of Streptomyces was characterized and its interaction with other pyrimidine enzymes explored.

Purified S6/H4 kinase (Mr 60,000) requires autophosphorylation for activation. A rabbit anti-S6/H4 kinase peptide (SVIDPVPAPVGDSHVDGAAK) antibody recognized both the S6/H4 kinase holoenzyme and catalytic domain. Immunoreactivity with p60 kinase protein, and S6/H4 kinase activity were precisely correlated in fractions obtained from ion exchange chromatography of P1798 lymphosarcoma extracts. An enzyme which catalyzed the MgATP-dependent phosphorylation and activation of S6/H4 kinase coeluted with immunoreactivity from Mono 5, but not Mono Q chromatography. Since S6/H4 kinase is homologous with rac-activated PAK65, the observation that phosphorylation is also required for activation suggests a complex mechanism for in vivo activation of the S6/H4 kinase.

The effects of cannabinoid agonists on spontaneous neuronal network activity were characterized in murine spinal cord and auditory cortical cultures with multichannel extracellular recording using photoetched electrode arrays. Different cultures responded reproducibly with global decreases of spiking and bursting to anandamide and methanandamide, but each agonist showed unique minor effects on network activity. The two tissues responded in a tissue-specific manner. Spontaneous activity in spinal tissue was terminated by 1 μM anandamide and 6.1 μM methanandamide. Cortical activity ceased at 3.5 μM and 2.8 μM respectively. Irreversible cessation of activity was observed beyond 8 μM for both tissues and test substances. Palmitoylethanolamide, demonstrated that CB2 receptors were not present or not responsive. However, the data strongly suggested the presence of CB1 receptors.

Cyanide was rapidly removed when added to culture supernatants of seven different Pseudomonas. The ability to remove cyanide was correlated with the accumulation of α-keto acids (pyruvate and α-ketoglutarate). These compounds react with cyanide forming less toxic cyanohydrins, thus conferring a mechanism for bacterial cyanide tolerance. When added to growth media the α-keto acids were shown also to serve as effective cyanide antagonists. While all bacteria tested accumulated α-keto acids, only those capable of utilizing cyanide as a nutritional nitrogen source were able to metabolize cyanohydrins. In P. fluorescens NCIMB 11764, the same enzyme (cyanide oxygenase) shown previously to be involved in cyanide metabolism appears responsible for cyanohydrin transformation. Keto acid excretion is believed to represent a new mechanism of bacterial cyanide detoxification with further enzymatic metabolism of the cyanohydrins helping to explain how cyanide can satisfy the nitrogen requirement in cyanide-utilizing bacteria.