Biomedical subjects

Daniel H Shain

Publications and source records attributed to Daniel H Shain.

11 recordsLinked to original sources

PubMed2006

Structural model of an antistasin/notch-like fusion protein from the cocoon wall of the aquatic leech, Theromyzon tessulatum.

The aquatic leech, Theromyzon tessulatum, secretes a proteinaceous cocoon with extraordinary physical properties (e.g., proteolytic, thermal resiliency). The deduced amino acid sequence of a major protein (Tcp-Theromyzon cocoon protein) from the T. tessulatum cocoon wall has been used to model the endogenous structure of the Tcp protein. The Tcp protein sequence comprises six internal repeats, each containing 12 ordered Cys residues. Amino acid alignments suggest that the region Cys1-->6 is homologous to antistasin, a leech anticoagulant, and Cys7-->12 is homologous to an epidermal growth factor-like domain found in notch-class proteins, which play critical roles in development, signaling, and adhesion throughout the Animalia. Modeling of individual domains (i.e., antistasin and notch) positions multiple hydrophobic and charged residues on the surface. When the antistasin and notch domains were fused, hydrophobic pockets appeared that may facilitate a polymerization mechanism. Collectively, the predicted features of our Tcp model are consistent with the physical properties of the leech cocoon wall.

Animals↗

PubMed2005

Two-dimensional ultrastructural elements lead to three-dimensional reconstruction of protuberances on the cocoon membrane of the leech, Theromyzon tessulatum.

Protuberances on the cocoon surface of the leech, Theromyzon tessulatum, are roughly parallel rows of triangular prisms arranged equidistantly to each other on the outer surface of the cocoon membrane. The distance between neighboring protuberances is approximately 1.6 microm, the height approximately 0.5 microm and the semi-width approximately 0.3 microm. The fibrillar arrangement within the protuberance maintains some elements of the helicoids found within the cocoon membrane but a high proportion of large holes disrupt the symmetry of the protuberance ultrastructure. A procedure for 3D reconstruction of the protuberance using the complementarity between the paratangential and normal sections through the cocoon is presented. Our results demonstrate that the ultrastructure of protuberances show elements of a twisted fibrillar arrangement, but the demands of filling a narrow space ruled by acute angles appears to cause a high degree of ultrastructural disorganization.

Animals↗

PubMed2005

Quantitating adenylate nucleotides in diverse organisms.

Quantitation of cellular adenylate levels (i.e., ATP, ADP, AMP) has widespread applications in physiological, metabolic and energetic studies. We have compared classical adenylate extraction procedures (i.e., perchloric acid, boiling) with a previously unreported proteinase K-based extraction technique. Our results suggest that all three techniques are comparable in soft animal tissue, but proteinase K-based extractions consistently generated higher adenylate yields from a broad range of organisms, particularly those containing a cell wall (e.g., alga, bacteria, fungi, plant).

Adenine Nucleotides↗

PubMed2004

Distinctions in adenylate metabolism among organisms inhabiting temperature extremes.

Microbiota from multiple kingdoms (e.g., Eubacteria, Fungi, Protista) thrive at temperature optima ranging from 0-20 degrees C (psychrophiles) to 40-85 degrees C (thermophiles). In this study, we have monitored changes in adenylate levels and growth rate as a function of temperature in disparate thermally adapted organisms. Our data indicate that growth rate and adenylate levels increase with temperature in mesophilic and thermophilic species, but rapid losses of adenosine 5'-triphosphate (ATP) occur upon cold or heat shock. By contrast, psychrophilic species decrease adenylate levels but increase growth rate as temperatures rise within their viable range. Moreover, psychrophilic ATP levels fell rapidly upon heat shock, but dramatic gains in ATP (approximately 20-50%) were observed upon cold shock, even at sub-zero temperatures. These results suggest that energy metabolism in thermophiles resembles that in mesophiles, but that elevated adenylate nucleotides in psychrophiles may constitute a compensatory strategy for maintaining biochemical processes at low temperature.

Acclimatization↗

PubMed2004

Four kingdoms on glacier ice: convergent energetic processes boost energy levels as temperatures fall.

A diverse group of glacially obligate organisms coexist on temperate glaciers between Washington State and Alaska. A fundamental challenge for these and other cold-adapted species is the necessity to maintain an energy flux capable of sustaining life at low physiological temperatures. We show here that ice-adapted psychrophiles from four kingdoms (Animalia, Eubacteria, Fungi, Protista) respond to temperature fluctuations in a similar manner; namely, ATP levels and the total adenylate pool increase as temperatures fall (within their viable temperature limits, respectively), yet growth rate increases with temperature. By contrast, mesophilic representatives of each kingdom respond in an opposite manner (i.e. adenylates increase with temperature). These observations suggest that elevated adenylate levels in psychrophiles may offset inherent reductions in molecular diffusion at low physiological temperatures.

Adaptation, Physiological↗

PubMed2004

Cell interactions that affect axonogenesis in the leech Theromyzon rude.

The leech nervous system comprises a relatively simple network of longitudinal (connective) and transverse (segmental) nerves. We have followed the normal pattern of axon development in the glossiphoniid leech Theromyzon rude by immunostaining embryonic preparations with antibody to acetylated alpha-tubulin. The dependence of the normal pattern of axon growth on cells in the mesodermal (M) and ectodermal (N, O, P and Q) lineages was examined by selectively ablating subsets of these lineages in developing embryos. We found that ablating mesoderm severely disrupted overall axonogenesis, while various ectodermal ablations induced a range of more specific phenotypes. In particular, formation of the posterior segmental nerve (PP) was abnormal in embryos deficient in primary neuroectoderm (N lineage). More specific ablations demonstrated that a subset of N-derived cells were required for establishing the PP nerve root. Previous studies have shown that the PP nerve root is normally pioneered by an O lineage-derived neuron (PD). Our results suggest that the role of the N lineage-derived cells is to induce the migration of neuron P(D) to its normal position in the posterior compartment of the hemiganglion.

Animals↗

PubMed2004

A cysteine-rich protein in the Theromyzon (Annelida: Hirudinea) cocoon membrane.

The aquatic leech, Theromyzon rude, secretes a flexible, proteinaceous cocoon that is resistant to a broad range of denaturing conditions (e.g. heat, denaturing chemicals). We have partially solubilized the Theromyzon cocoon membrane in 10% acetic acid and identified two major protein fragments. Microsequencing of both Theromyzon cocoon protein (Tcp) fragments generated an identical stretch of the amino-terminal sequence that was used to clone the corresponding gene. The predicted linear amino acid sequence of the resulting cDNA contained an unusually high cysteine content (17.8%). Sequence analysis identified six internal repeats, each comprising 12 ordered Cys residues in a approximately 62 amino acid repeating unit. Sequence comparisons identified homology with undescribed, Cys-rich repeats across animal phyla (i.e. Arthropod, Nematoda).

Amino Acid Sequence↗

PubMed2004

Molecular adaptation in the ice worm, Mesenchytraeus solifugus: divergence of energetic-associated genes.

The ice worm, Mesenchytraeus solifugus, is the largest glacially obligate metazoan and among only a few metazoan species that complete their life cycle at 0 degrees C. We conducted a large-scale sequencing analysis of cDNAs isolated from ice worm anterior segments. Sequence comparisons among an available group of ice worm, arthropod, chordate, and nematode homologues suggest that ice worms encode proteins that are less bulky, are less polar, and contain fewer charged residues. Also, subunits of the catalytic F1 ATP synthase complex appear to have diverged more rapidly than other ice worm genes examined, suggesting a role in cold-temperature adaptation. Modeling of F1 ATP synthase beta and gamma subunits identified nonconservative, ice worm-specific amino acid substitutions at subunit contact sites and at sites proximal to the catalytic site.

Amino Acid Sequence↗

PubMed2004

The ice worm, Mesenchytraeus solifugus, elevates adenylate levels at low physiological temperature.

The ice worm, Mesenchytraeus solifugus, is among a few metazoan species that survive exclusively in glacier ice/snow. In this study, we demonstrate that ice worm adenylate levels [i.e. adenosine 5'-triphosphate (ATP), ADP and AMP] are maintained at levels well above their mesophilic counterparts, and that their response to temperature change is distinctly opposite, namely, ice worms increase energy levels as temperatures fall. Initially, this response is characterized by a sharp spike in [ATP] and the adenylate energy charge (even at sub-zero temperatures), which is followed by corresponding increases in [ADP] and [AMP] within a few days. These results suggest that ice worms have evolved a compensatory mechanism by which gains in adenylate nucleotides off-set, at least in part, the inherent lethargy and death usually associated with cold temperature.

Acclimatization↗

PubMed2004

Ultrastructural properties of the Theromyzon (Annelida: Hirudinae) cocoon membrane.

The cocoon of the leech Theromyzon trizonare consists of fibrils packed into an arrangement that produces both C- and S-like patterns of bow-shaped lines in sections oblique through the membrane. Sections normal to the cocoon membrane show layers containing cross-sections of fibrils (approximately 16 nm dia.) that are separated by a center-to-center distance of approximately 23 nm. In cross-section, each fibril presents a central hole approximately 5 nm in diameter. A structureless layer covers most of the exterior surface of the cocoon membrane, and short protuberances are apparent in some zones.

Animals↗

PubMed2004

Changes in gene expression at the precursor --> stem cell transition in leech.

The glossiphoniid leech, Theromyzon trizonare, displays particularly large and accessible embryonic precursor/stem cells during its early embryonic cleavages. We dissected populations of both cell types from staged embryos and examined gene expression profiles by differential display polymerase chain reaction methodology. Among the approximately 10,000 displayed cDNA fragments, 56 (approximately 0.5%) were differentially expressed at the precursor --> stem cell transition; 29 were turned off (degraded, precursor-specific); and 27 were turned on (transcribed, stem cell-specific). Several putative differentially expressed cDNAs from each category were confirmed by Northern blot analysis on staged embryos. DNA sequencing revealed that 19 of the cDNAs were related to a spectrum of genes including the CCR4 antiproliferation gene, Rad family members, and several transcriptional regulators, while the remainder encoded hypothetical (10) or novel (27) sequences. Collectively, these results identify dynamic changes in gene expression during stem cell formation in leech and provide a platform for examining the molecular aspects of stem cell genesis in a simple invertebrate organism.

Animals↗