Modulation of the crayfish escape reflex--physiology and neuroethology

We review here factors that control the excitability of thegiant neuron-mediated tail-flip escape behavior in crayfish,focusing especially on recent findings concerning serotonergicmodulation. Serotonin can either facilitate or inhibit escapedepending on concentration and pattern of application. Low concentrationsfacilitate while high ones inhibit; however, if high concentrationsarise gradually they facilitate instead of inhibiting. The effectsof serotonin can also be altered by social experience, withapplication regimens that cause facilitation in social isolatescoming to produce inhibition after an extended period of livingas a subordinate. Attempts to understand both the possible physiologicalbasis of some of these complexities and their possible functionare discussed. Neuroethological investigations indicate thatgiant neuron-mediated escape is inhibited during the initialfights that establish social relationships and is facilitatedin their immediate aftermath. Once the relationship of a pairis well-established, the presence of the dominant tends to suppressgiant neuron-mediated escape (but not tail-flip escape mediatedby non-giant circuitry) in the subordinate, but the presenceof the subordinate has relatively little effect on the dominant.These patterns of modulation can be seen as consistent withthe known variations in serotonin's effect as a function ofconcentration and social experience and may provide a biologicalreason for these variations.     

Toxoplasma gondii cyclic GMP-dependent kinase: chemotherapeutic targeting of an essential parasite protein kinase

The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) has in vivo activity against the apicomplexan parasites Toxoplasma gondii and Eimeria tenella in animal models. The presumptive molecular target of this compound in E. tenella is cyclic GMP-dependent protein kinase (PKG). Native PKG purified from T. gondii has kinetic and pharmacologic properties similar to those of the E. tenella homologue, and both have been functionally expressed as recombinant proteins in T. gondii. Computer modeling of parasite PKG was used to predict catalytic site amino acid residues that interact with compound 1. The recombinant laboratory-generated mutants T. gondii PKG T761Q or T761M and the analogous E. tenella T770 alleles have reduced binding affinity for, and are not inhibited by, compound 1. By all other criteria, PKG with this class of catalytic site substitution is indistinguishable from wild-type enzyme. A genetic disruption of T. gondii PKG can only be achieved if a complementing copy of PKG is provided in trans, arguing that PKG is an essential protein. Strains of T. gondii, disrupted at the genomic PKG locus and dependent upon the T. gondii T761-substituted PKGs, are as virulent as wild type in mice. However, unlike mice infected with wild-type T. gondii that are cured by compound 1, mice infected with the laboratory-generated strains of T. gondii do not respond to treatment. We conclude that PKG represents the primary molecular target responsible for the antiparasitic efficacy of compound 1.     

Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors

Heart septation and valve malformations constitute the most common anatomical birth defects. These structures arise from the endocardial cushions within the atrioventricular canal (AVC) through dynamic interactions between cushion cells and the extracellular matrix (termed cardiac jelly). Transformation of endothelial cells to mesenchymal cells is essential for the proper development of the AVC and subsequent septation and valve formation. Atrioventricular septal defects can result from incomplete endocardial cushion morphogenesis. We show that hyaluronan-deficient AVC explants from Has2(-/-) embryos, which normally lack mesenchyme formation, are rescued by heregulin treatment, which restores phosphorylation of ErbB2 and ErbB3. These events were blocked using a soluble ErbB3 molecule, as well as with an inhibitor of ErbB2, herstatin. We show further that ErbB3 is activated during hyaluronan treatment of Has2(-/-) explants. These data provide a link between extracellular matrix-hyaluronan and ErbB receptor activation during development of early heart-valve and septal mesenchyme.     

Running energetics of the North American river otter: do short legs necessarily reduce efficiency on land?

Semi-aquatic mammals move between two very different media (air and water), and are subject to a greater range of physical forces (gravity, buoyancy, drag) than obligate swimmers or runners. This versatility is associated with morphological compromises that often lead to elevated locomotor energetic costs when compared to fully aquatic or terrestrial species. To understand the basis of these differences in energy expenditure, this study examined the interrelationships between limb morphology, cost of transport and biomechanics of running in a semi-aquatic mammal, the North American river otter. Oxygen consumption, preferred locomotor speeds, and stride characteristics were measured for river otters (body mass=11.1 kg, appendicular/axial length=29%) trained to run on a treadmill. To assess the effects of limb length on performance parameters, kinematic measurements were also made for a terrestrial specialist of comparable stature, the Welsh corgi dog (body mass=12.0 kg, appendicular/axial length=37%). The results were compared to predicted values for long legged terrestrial specialists. As found for other semi-aquatic mammals, the net cost of transport of running river otters (6.63 J kg(-1)min(-1) at 1.43 ms(-1)) was greater than predicted for primarily terrestrial mammals. The otters also showed a marked reduction in gait transition speed and in the range of preferred running speeds in comparison to short dogs and semi-aquatic mammals. As evident from the corgi dogs, short legs did not necessarily compromise running performance. Rather, the ability to incorporate a period of suspension during high speed running was an important compensatory mechanism for short limbs in the dogs. Such an aerial period was not observed in river otters with the result that energetic costs during running were higher and gait transition speeds slower for this versatile mammal compared to locomotor specialists.     

Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae

Protein kinases are coded by more than 2,000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues. At least 30% of all proteins are thought to contain covalently bound phosphate. Despite the importance and widespread occurrence of this modification, identification of sites of protein phosphorylation is still a challenge, even when performed on highly purified protein. Reported here is methodology that should make it possible to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment. Proteins are digested with trypsin and the resulting peptides are then converted to methyl esters, enriched for phosphopeptides by immobilized metal-affinity chromatography (IMAC), and analyzed by nanoflow HPLC/electrospray ionization mass spectrometry. More than 1,000 phosphopeptides were detected when the methodology was applied to the analysis of a whole-cell lysate from Saccharomyces cerevisiae. A total of 216 peptide sequences defining 383 sites of phosphorylation were determined. Of these, 60 were singly phosphorylated, 145 doubly phosphorylated, and 11 triply phosphorylated. Comparison with the literature revealed that 18 of these sites were previously identified, including the doubly phosphorylated motif pTXpY derived from the activation loop of two mitogen-activated protein (MAP) kinases. We note that the methodology can easily be extended to display and quantify differential expression of phosphoproteins in two different cell systems, and therefore demonstrates an approach for "phosphoprofiling" as a measure of cellular states.     

Distribution of mating type alleles in the wheat pathogen Mycosphaerella graminicola over spatial scales from lesions to continents

A total of 2035 Mycosphaerella graminicola strains collected from 16 geographic locations on four continents were assayed for the mating type locus. RFLP fingerprints were used to identify clones in each population. At the smallest spatial scale analyzed, both mating types were found among fungal strains sampled from different lesions of the same leaf as well as from different pycnidia in the same lesion. At larger spatial scales, the two mating types were found at equal frequencies across spatial scales ranging from several square meters to several thousand square kilometers. Though the absolute frequencies of the two mating types sometimes varied for different sampling units within the same spatial scale in the hierarchy (plots within a field, fields within a country, or different continents of the world), none of the differences were statistically significant from the null hypothesis of equal frequencies for the two mating types. The evolutionary forces likely to maintain the even distribution of the two mating types in this pathogen were discussed.     

The circle of the soul: the role of spirituality in health care

This paper examines the critical attitude of behavioral professionals toward spiritual phenomena, and the current growing openness toward a scientific study of spirituality and its effects on health. Health care professionals work amidst sickness and suffering, and become immersed in the struggles of suffering persons for meaning and spiritual direction. Biofeedback and neurofeedback training can facilitate relaxation, mental stillness, and the emergence of spiritual experiences. A growing body of empirical studies documents largely positive effects of religious involvement on health. The effects of religion and spirituality on health are diverse, ranging from such tangible and easily understood phenomena as a reduction of health-risk behaviors in church-goers, to more elusive phenomena such as the distant effects of prayer on health and physiology. Psychophysiological methods may prove useful in identifying specific physiological mechanisms mediating such effects. Spirituality is also a dimension in much of complementary and alternative medicine (CAM), and the CAM arena may offer a window of opportunity for biofeedback practice.     

Synthesis of the first diethylenetriaminepentahydroxamic acid (DTPH) bifunctional chelating agent

Synthesis of a new pentahydroxamic acid bifunctional chelating agent (BCA), constructed on the aminoazaalkyl core of diethylenetriaminepentaacetic acid (DTPA), is reported. Rational modifications in the structure of DTPA, which could result in an enhancement of its chelation properties, add to the collection of diagnostic and therapeutic metals bound by this chelator, and might implement significant improvements in the in vivo behavior of this compound, are described. Further improvements in the stability of the ligand-metal complexes of DTPA may improve both diagnostic and therapeutic outcomes such as tumor-to-normal tissue ratios and target-delivered radioactivity. A combination of hydroxamate functions with the azaalkyl backbone of DTPA might be a suitable approach to generate such higher stabilities. This rationale may be justified by the well-known affinity of hydroxamates against different transition metals and favorable properties of DTPA as a versatile chelator. Thus, the N(4),N(alpha),N(alpha),N(epsilon),N(epsilon)-pentakis[[((N-hydroxy-N-methyl]carbonyl)methyl]-2, 6-diamino-4-azahexanoic hydrazide (5, DTPH) was designed and synthesized through a convergent synthesis and in 40.7% overall yield. Conjugation of this compound to the monoclonal antibody (MAb) Delta Ch2HuCC49, used as a model protein, was carried out to evaluate the efficiency of this molecule as a BCA. Radiolabeling of the DTPH-Delta CH2HuCC49 conjugate with lutetium-177 ((177)Lu) and biodistribution of the labeled conjugate in athymic nude mice, bearing LS174T human colon carcinoma xenografts, are reported.     

Proteomic characterization of the small subunit of Chlamydomonas reinhardtii chloroplast ribosome: identification of a novel S1 domain-containing protein and unusually large orthologs of bacterial S2, S3, and S5

To understand how chloroplast mRNAs are translated into functional proteins, a detailed understanding of all of the components of chloroplast translation is needed. To this end, we performed a proteomic analysis of the plastid ribosomal proteins in the small subunit of the chloroplast ribosome from the green alga Chlamydomonas reinhardtii. Twenty proteins were identified, including orthologs of Escherichia coli S1, S2, S3, S4, S5, S6, S7, S9, S10, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 and a homolog of spinach plastid-specific ribosomal protein-3 (PSRP-3). In addition, a novel S1 domain-containing protein, PSRP-7, was identified. Among the identified proteins, S2 (57 kD), S3 (76 kD), and S5 (84 kD) are prominently larger than their E. coli or spinach counterparts, containing N-terminal extensions (S2 and S5) or insertion sequence (S3). Structural predictions based on the crystal structure of the bacterial 30S subunit suggest that the additional domains of S2, S3, and S5 are located adjacent to each other on the solvent side near the binding site of the S1 protein. These additional domains may interact with the S1 protein and PSRP-7 to function in aspects of mRNA recognition and translation initiation that are unique to the Chlamydomonas chloroplast.     

Yeast genes controlling responses to topogenic signals in a model transmembrane protein

Yeast protein insertion orientation (PIO) mutants were isolated by selecting for growth on sucrose in cells in which the only source of invertase is a C-terminal fusion to a transmembrane protein. Only the fraction with an exocellular C terminus can be processed to secreted invertase and this fraction is constrained to 2-3% by a strong charge difference signal. Identified pio mutants increased this to 9-12%. PIO1 is SPF1, encoding a P-type ATPase located in the endoplasmic reticulum (ER) or Golgi. spf1-null mutants are modestly sensitive to EGTA. Sensitivity is considerably greater in an spf1 pmr1 double mutant, although PIO is not further disturbed. Pmr1p is the Golgi Ca(2+) ATPase and Spf1p may be the equivalent ER pump. PIO2 is STE24, a metalloprotease anchored in the ER membrane. Like Spf1p, Ste24p is expressed in all yeast cell types and belongs to a highly conserved protein family. The effects of ste24- and spf1-null mutations on invertase secretion are additive, cell generation time is increased 60%, and cells become sensitive to cold and to heat shock. Ste24p and Rce1p cleave the C-AAX bond of farnesylated CAAX box proteins. The closest paralog of SPF1 is YOR291w. Neither rce1-null nor yor291w-null mutations affected PIO or the phenotype of spf1- or ste24-null mutants. Mutations in PIO3 (unidentified) cause a weaker Pio phenotype, enhanced by a null mutation in BMH1, one of two yeast 14-3-3 proteins.