Bioenergetics in two pulmonate snails, Helisoma and Physa

Energy budgets constructed for the pulmonate snails, Physa gyrina and Helisoma trivolvis, indicate:Abstract1. 1. Assimilation efficiency in wild type Physa (88[middle dot]9 per cent) and Helisoma (73[middle dot]2 per cent) are exceptionally high when compared to reported values for invertebrates while assimilation efficiency in an albino strain of Helisoma, although above average for most invertebrates, is comparatively low (59[middle dot]8 per cent).2. 2. With a single exception, there are no relationships between weight or weight gain and food (energy) ingested, wastes (energy) egested, assimilation efficiency or energy devoted to reproduction.3. 3. Daily food energy ingested by wild P. gyrina (27[middle dot]5 cal/day) and H. trivolvis (27[middle dot]6 cal/day) are similar and significantly higher than daily food energy ingested by albino H. trivolvis (15[middle dot]9 cal/day).4. 4. Albino H. trivolvis devoted more energy to egg production (2[middle dot]1 cal/day) than did the wild variety (1[middle dot]1 cal/day).5. 5. Secondary productivity of both varieties of H. trivolvis were exceptionally high (17[middle dot]8 and 32[middle dot]6 per cent).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22019/1/0000435.pd

In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix–turn–helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3′ ends and two nucleotides inside the 5′ ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3′ end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes

A Revised Design for Microarray Experiments to Account for Experimental Noise and Uncertainty of Probe Response

Background Although microarrays are analysis tools in biomedical research, they are known to yield noisy output that usually requires experimental confirmation. To tackle this problem, many studies have developed rules for optimizing probe design and devised complex statistical tools to analyze the output. However, less emphasis has been placed on systematically identifying the noise component as part of the experimental procedure. One source of noise is the variance in probe binding, which can be assessed by replicating array probes. The second source is poor probe performance, which can be assessed by calibrating the array based on a dilution series of target molecules. Using model experiments for copy number variation and gene expression measurements, we investigate here a revised design for microarray experiments that addresses both of these sources of variance. Results Two custom arrays were used to evaluate the revised design: one based on 25 mer probes from an Affymetrix design and the other based on 60 mer probes from an Agilent design. To assess experimental variance in probe binding, all probes were replicated ten times. To assess probe performance, the probes were calibrated using a dilution series of target molecules and the signal response was fitted to an adsorption model. We found that significant variance of the signal could be controlled by averaging across probes and removing probes that are nonresponsive or poorly responsive in the calibration experiment. Taking this into account, one can obtain a more reliable signal with the added option of obtaining absolute rather than relative measurements. Conclusion The assessment of technical variance within the experiments, combined with the calibration of probes allows to remove poorly responding probes and yields more reliable signals for the remaining ones. Once an array is properly calibrated, absolute quantification of signals becomes straight forward, alleviating the need for normalization and reference hybridizations

Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other α/β-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.

Combined extracellular matrix cross-linking activity of the peroxidase MLT-7 and the dual oxidase BLI-3 is critical for post-embryonic viability in <i>Caenorhabditis elegans</i>

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation

Carriages for skylines

Carriages may be classified as slackpulling or non-slackpulling, and the slackpulling carriages may be further classified by how they accomplish the slackpulling function. Slackpulling carriages provide the skyline with lateral yarding capability and therefore can be used in a variety of silvicultural prescriptions. Non-slackpulling carriages can include either chokers or grapples and are used in clearcuts. For a skyline system to operate efficiently, the carriage must be matched to the number of lines and the line diameters on the yarder

In polycistronic Qβ RNA, single-strandedness at one ribosome binding site directly affects translational initiations at a distal upstream cistron

In Qβ RNA, sequestering the coat gene ribosome binding site in a putatively strong hairpin stem structure eliminated synthesis of coat protein and activated protein synthesis from the much weaker maturation gene initiation site, located 1300 nucleotides upstream. As the stability of a hairpin stem comprising the coat gene Shine–Dalgarno site was incrementally increased, there was a corresponding increase in translation of maturation protein. The effect of the downstream coat gene ribosome binding sequence on maturation gene expression appeared to have occurred only in cis and did not require an AUG start codon or initiation of coat protein synthesis. In all cases, no structural reorganization was predicted to occur within Qβ RNA. Our results suggest that protein synthesis from a relatively weak translational initiation site is greatly influenced by the presence or absence of a stronger ribosome binding site located elsewhere on the same RNA molecule. The data are consistent with a mechanism in which multiple ribosome binding sites compete in cis for translational initiations as a means of regulating protein synthesis on a polycistronic messenger RNA

The Pheromone of the Cave Cricket, Hadenoecus cumberlandicus, Causes Cricket Aggregation but Does Not Attract the Co-Distributed Predatory Spider, Meta ovalis

Food input by the cave cricket, Hadenoecus cumberlandicus Hubble & Norton (Orthoptera: Rhaphidophoridae), is vital to the cave community, making this cricket a true keystone species. Bioassays conducted on cave walls and in the laboratory show that clustering in H. cumberlandicus is guided by a pheromone, presumably excreta. This aggregation pheromone was demonstrated by using filter paper discs that had previous adult H. cumberlandicus exposure, resulting in > 70% response by either nymphs or adults, prompting attraction (thus, active component is a volatile), followed by reduced mobility (arrestment) on treated surfaces. Adults were similarly responsive to pheromone from nymphs, agreeing with mixed stage composition of clusters in the cave. Effects of [0.001M – 0.1M] uric acid (insect excreta's principle component) on H. cumberlandicus behavior were inconsistent. This pheromone is not a host cue (kairomone) and is not used as a repellent (allomone) as noted through lack of responses to natural H. cumberlandicus pheromone and uric acid concentrations by a co-occurring predatory cave orb weaver spider, Meta ovalis Gertsch (Araneae: Tetragnathidae). This pheromone is not serving as a sex pheromone because nymphs were affected by it and because this population of H. cumberlandicus is parthenogenic. The conclusion of this study is that the biological value of the aggregation pheromone is to concentrate H. cumberlandicus in sheltered sites in the cave conducive for minimizing water stress. Rather than signaling H. cumberlandicus presence and quality, the reduced mobility expressed as a result of contacting this pheromone conceivably may act as a defense tactic (antipredator behavior) against M. ovalis, which shares this favored habitat site

Bioenergetics of the cave cricket, Hadenoecus subterraneus

1. 1. In late spring, cave crickets in Mammoth Cave National Park were investigated in order that their bioenergetics might be elucidated.2. 2. A predictive relation between crop-empty live weight and hind femur length independent of sex was found.3. 3. Long-term weight loss patterns were 1.55 and 1.19mg/hr for females and males, respectively, suggesting feeding intervals of 11.5 and 9.9 days.4. 4. Calorific assimilation efficiencies were found to be 80.4% for females and 82.5% for males.5. 5. The estimated maximal metabolic rates were half that expected for insects of similar mass.6. 6. The mixed waste calorific levels were found to be exceptionally low.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26417/1/0000504.pd

Water budgets of cave crickets, Hadenoecus subterraneus and camel crickets, Ceuthophilus stygius

1. 1. Studies of cave and camel crickets from cave entrances in Mammoth Cave National Park have produced estimates of total water budgets and component contributions to water balance.2. 2. Weight specific (mg/g crop-free live weight/hr) total water loss (3.200 vs 2.220) and water gained in food (2.393 vs 1.902) are greater in cave than in camel crickets, respectively.3. 3. Weight specific evaporative water loss in both humid, still air (2.269 vs 1.325) and dry, moving air (15.28 vs 9.85) is greater in cave than in camel crickets, respectively.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26939/1/0000505.pd