Infrared video thermography: a technique for assessing cold adaptation in insects

Insects can survive subzero temperatures by two main strategies: freeze tolerance and freeze avoidance. An array of techniques have been used to investigate the physiological limits of insects to low temperatures, such as differential scanning calorimetry, temperature-controlled cooling apparatus, thermocouples, and computer-controlled chart recording equipment. However, these techniques require animals to be stationary, precluding behavioral data. We used infrared video thermography to investigate cold adaptation in an alpine insect, expanding such investigations to include behavioral response as an indicator of physiological stress. This technique is noninvasive and provides a large amount of physiological information, such as supercooling points, lower lethal temperatures, and hemolymph melting points. Insect supercooling points in response to a constant cooling rate were variable; however, temperatures at the initiation of behavioral stress response were less variable. Assessments of supercooling points and lower lethal temperatures obtained in this way are more biologically meaningful because allowing unhindered movement of insects more closely resembles natural environments.     

Apolipophorin-III-like protein expressed in the antenna of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae)

Antennal proteins of the male fire ant (Solenopsis invicta) were analyzed by two-dimensional gel electrophoresis, with the objective of identifying pheromone-binding proteins, which have not previously been found in ant antennae. The major low-molecular weight protein found in the male fire ant antenna was subjected to Edman degradation to determine the N-terminal amino acid sequence. Degenerate PCR primers based on this sequence were used to obtain a cDNA sequence corresponding to the full-length protein sequence. In-gel trypsin digestion followed by MALDI-TOF mass spectrometry and HPLC-ESI/MS/MS demonstrated that the protein gel spot contained only the protein corresponding to the cDNA sequence obtained by PCR. The sequence is similar to apolipophorin-III, an exchangeable lipid-binding protein. Fire ant apolipophorin-III is expressed in the antenna as well as the head, thorax and abdomen.     

Remote homolog detection using local sequence-structure correlations

Remote homology detection refers to the detection of structural homology in proteins when there is little or no sequence similarity. In this article, we present a remote homolog detection method called SVM-HMMSTR that overcomes the reliance on detectable sequence similarity by transforming the sequences into strings of hidden Markov states that represent local folding motif patterns. These state strings are transformed into fixed-dimension feature vectors for input to a support vector machine. Two sets of features are defined: an order-independent feature set that captures the amino acid and local structure composition; and an order-dependent feature set that captures the sequential ordering of the local structures. Tests using the Structural Classification of Proteins (SCOP) 1.53 data set show that the SVM-HMMSTR gives a significant improvement over several current methods.     

GAA repeat instability in Friedreich ataxia YAC transgenic mice

Friedreich ataxia (FRDA) is primarily caused by an unstable GAA repeat-expansion mutation within intron 1 of the FRDA gene. However, the exact mechanisms leading to this expansion and its consequences are not fully understood. To study the dynamics of this mutation, we have generated two lines of human FRDA YAC transgenic mice that contain GAA repeat expansions within the appropriate genomic context. We have detected intergenerational instability and age-related somatic instability in both lines, with pronounced expansions found in the cerebellum. The dynamic nature of our transgenic GAA repeats is comparable with previous FRDA patient somatic tissue data. However, there is a difference between our FRDA YAC transgenic mice and other trinucleotide-repeat mouse models, which do not show pronounced repeat instability in the cerebellum. This represents the first mouse model of FRDA GAA repeat instability that will help to dissect the mechanism of this repeat.     

Glutamate-cysteine ligase attenuates TNF-induced mitochondrial injury and apoptosis

Glutathione (GSH) is important in free radical scavenging, maintaining cellular redox status, and regulating cell survival in response to a wide variety of toxicants. The rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which is composed of catalytic (GCLC) and modifier (GCLM) subunits. To determine whether increased GSH biosynthetic capacity enhances cellular resistance to tumor necrosis factor-alpha- (TNF-alpha-) induced apoptotic cell death, we have established several mouse liver hepatoma (Hepa-1) cell lines overexpressing GCLC and/or GCLM. Cells overexpressing GCLC alone exhibit modest increases in GCL activity, while cells overexpressing both subunits have large increases in GCL activity. Importantly, cells overexpressing both GCL subunits exhibit increased resistance to TNF-induced apoptosis as judged by a loss of redox potential; mitochondrial membrane potential; translocation of cytochrome c to the cytoplasm; and activation of caspase-3, caspase-8, and caspase-9. Analysis of the effects of TNF on these parameters indicates that maintaining mitochondrial integrity mediates this protective effect in GCL-overexpressing cells.     

The carbonate radical anion-induced covalent aggregation of human copper, zinc superoxide dismutase, and alpha-synuclein: intermediacy of tryptophan- and tyrosine-derived oxidation products

In this review, we describe the free radical mechanism of covalent aggregation of human copper, zinc superoxide dismutase (hSOD1). Bicarbonate anion (HCO3-) enhances the covalent aggregation of hSOD1 mediated by the SOD1 peroxidase-dependent formation of carbonate radical anion (CO3*-), a potent and selective oxidant. This species presumably diffuses out the active site of hSOD1 and reacts with tryptophan residue located on the surface of hSOD1. The oxidative degradation of tryptophan to kynurenine and N-formyl kynurenine results in the covalent crosslinking and aggregation of hSOD1. Implications of oxidant-mediated aggregation of hSOD1 in the increased cytotoxicity of motor neurons in amyotrophic lateral sclerosis are discussed.     

A postmitotic role for Isl-class LIM homeodomain proteins in the assignment of visceral spinal motor neuron identity

LIM homeobox genes have a prominent role in the regulation of neuronal subtype identity and distinguish motor neuron subclasses in the embryonic spinal cord. We have investigated the role of Isl-class LIM homeodomain proteins in motor neuron diversification using mouse genetic methods. All spinal motor neuron subtypes initially express both Isl1 and Isl2, but Isl2 is rapidly downregulated by visceral motor neurons. Mouse embryos lacking Isl2 function exhibit defects in the migration and axonal projections of thoracic level motor neurons that appear to reflect a cell-autonomous switch from visceral to somatic motor neuron character. Additional genetic mutations that reduce or eliminate both Isl1 and Isl2 activity result in more pronounced defects in visceral motor neuron generation and erode somatic motor neuron character. Thus, an early phase of high Isl expression and activity in newly generated motor neurons permits the diversification of visceral and somatic motor neuron subtypes in the developing spinal cord.     

Effective use of preparative chiral HPLC in a preclinical drug synthesis

Access to a key 3-aryl-delta-lactone intermediate in enantiopure form using preparative chiral chromatography allowed expedited preparation of an important drug discovery target. A preclinical drug discovery strategy that combines rapid route discovery with effective use of preparative chiral chromatography can result in significant savings of both time and labor.