A brief history of vertebrate functional morphology

The discipline of functional morphology grew out of a comparativeanatomical tradition, its transformation into a modern experimentalscience facilitated largely by technological advances. Earlymorphologists, such as Cuvier, felt that function was predictablefrom organismal form, to the extent that animals and plantsrepresented perfect adaptations to their habits. However, anatomyalone could not reveal how organisms actually performed theiractivities. Recording techniques capable of capturing fast motionwere first required to begin to understand animal movement.Muybridge is most famous for his pioneering work in fast photographyin the late 19th century, enabling him to "freeze" images ofeven the fastest horse at a full gallop. In fact, contemporarykinematic analysis grew directly out of the techniques Muybridgedeveloped. Marey made perhaps an even greater contribution toexperimental science through his invention of automatic apparatifor recording events of animal motion. Over the first half ofthe 20th century, scientists developed practical methods torecord activity patterns from muscles of a living, behavinghuman or animal. The technique of electromyography, initiallyused in clinical applications, was co-opted as a tool of organismalbiologists in the late 1960s. Comparative anatomy, kinematicanalysis and electromyography have for many years been the mainstayof vertebrate functional morphology; however, those interestedin animal form and function have recently begun branching outto incorporate approaches from experimental biomechanics andother disciplines (see accompanying symposium papers), and functionalmorphology now stands at the threshold of becoming a truly integrative,central field in organismal biology.     

Patterns of sex ratio, virginity and developmental mortality in gregarious parasitoids

Theory considering sex ratio optima under ‘strict local mate competition with offspring groups produced by a single foundress’ makes a suite of predictions, one of which is a mean female bias. Treating individual offspring as discrete units, theory further predicts sex ratios to have low variance (precise sex ratio) and to equal the reciprocal of clutch size (one male per clutch). The maternal decision may be complicated by imperfect control of sex allocation, limited insemination capacity of sons and offspring developmental mortality: each can lead to virgin daughters (with zero fitness) and consequently select for less biased sex ratios. When clutches are small and/or developmental mortality is common, appreciable proportions of virgins are expected, even when control of sex allocation is perfect and the mating capacity of males is unlimited. This suite of predictions has been only partially tested. We provide further tests by examining sex ratios and developmental mortalities within and across species of locally mating parasitoids. We find a wide range of mean developmental mortalities (6–67%), but mortality distributions are consistendy overdispersed (have greater than binomial variance) and sexually differential mortality appears to be absent. Sex ratios are female biased and have low variance, but are not perfectly precise and variance is increased by mortality within species and (equivocally) across species. Sex ratios less biased than the reciprocal of clutch size are observed; probably due to a maternal response to developmental mortality in one species, and to limited insemination capacity in others. Cross species comparisons indicate that mean proportions of mortality and virginity are positively correlated. Virginity is more prevalent than predicted among species with higher mortalities but not among lower mortality species. Predicted relationships between virginity and clutch size are supported in species with lower mortalities but only partially supported when mortality rates are higher.     

Chlordiazepoxide inhibition of reflex vagal bradycardia in the cat

The effect of chlordiazepoxide (CDZ) on phenylephrine-induced reflex vagal bradycardia was studied in cats anesthetized with chloralose. The sympathetic component of the reflex was eliminated by either pretreating the animals with propranolol (1 mg/kg, i.v.) or sectioning the spinal cord. In animals pretreated with propranolol, CDZ (3, 10 and 20 mg/kg, i.v.) produced a dose-related inhibition of phenylephrine-induced bradycardia. These doses of CDZ had no significant effect on phenylephrine-induced pressor responses. Similar results were obtained with CDZ in animals with spinal cords transected. Chlordiazepoxide did not prevent bradycardia evoked by electrical stimulation of the peripheral cut-end of the right vagus nerve. These results indicate that CDZ can inhibit reflex vagal bradycardia and that the inhibition involves a central action of the drug.     

A highly Ca2+-sensitive pool of granules is regulated by glucose and protein kinases in insulin-secreting INS-1 cells

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca(2+) to directly measure the Ca(2+) sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca(2+) sensitivity of release in that a small proportion of granules makes up a highly Ca(2+)-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca(2+). A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca(2+) are not located close to Ca(2+) channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca(2+)-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca(2+), suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca(2+) sensor that is poised to respond to modest, global elevations of [Ca(2+)](i).     

Noonan syndrome type I with PTPN11 3 bp deletion: structure-function implications

Noonan syndrome was recently reported to be caused by mutations in the PTPN11 gene in 40% of the cases. This gene encodes the nonreceptor-type protein tyrosine phosphatase SHP-2 and has been shown to be self down-regulated with the concurrency of two SH2 domains. Insertion of a specific loop (D'EF) from N-terminal SH2 domain into the SHP-2 active-site is responsible for the reversible inhibition of the phosphatase activity. Here we report the first in frame trinucleotide deletion resulting in the removal of Aspartate 61 (D61del), a key residue of the N-terminal SH2 D'EF loop. Energetic-based structural analysis and electrostatic calculations carried out on the wild-type and mutant proteins predict lower stability of the D'EF loop for the D61del variant as compared to the wild type indicating better access to the active site and most likely an enzyme activated for longer extent. Similar computations were performed on the previously functionally characterized gain-of-function D61Y mutant and similar behaviors were observed. The simulation data for the D61del and D61Y mutants suggest that both variants could yield more catalytic cycles than the wild-type molecule in the same timespan because of the opening of the active site. It also supports the notion that D61 plays a major role for proper down-regulation of the protein tyrosine phosphatase activity of SHP-2.     

Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins

DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.     

Monoamine release by neurons of a primitive nervous system: an amperometric study

We measured monoamine release from dissociated neurons of the sea pansy Renilla koellikeri, a representative of the most evolutionarily ancient animals with nervous systems, by real-time monitoring of exocytosis using the amperometric method with carbon-fiber microelectrodes. Depolarization-induced, as well as spontaneously active, neurons exhibited calcium-dependent exocytotic events at both the soma and the terminal bulb of neuritic processes. All spontaneously active neurons exhibited a bursting activity pattern in which amplitudes of exocytotic events appeared to be distributed in a quantal-like fashion. Fast Fourier transform analysis of bursting activity in 20 such neurons revealed burst harmonics with a major frequency of 8 Hz and a dominant rate of 95 Hz for individual exocytotic events within bursts. The results suggest that exocytotic transmitter release is as ancient as neurons and that endogenously bursting neurons in the sea pansy are as complex as those of higher animals. In addition, the observation that both soma and neuritic terminals of the same neuron can release transmitter suggests that local release sites in these cnidarian neurons are not critical for nerve net function.