The Asteroidea (Echinodermata) of the Muschelkalk (Middle Triassic of Germany)

Trichasteropsis Eck, from the Muschelkalk of Germany is the only Triassic asteroid known from more than fragmentary material. Most spécimens representT. weissmanni (Münster) whereasT. senfti ECK,T. bielertorum n. sp., andBerckhemeraster charistikos n. gen. et n. sp., are each known from few individuals.Parsimony analysis hère treats the füll Ordovician to Récent history of the Asteroidea using a somasteroid (a pre-asteroid stelleroid) outgroup. Ambulacral évolution is critical in echinoderm history; the ambulacral arrangement of crown-group asteroids first appears in Paleozoic sister groups, and the subclass Ambuloasteroidea n. subcl. is proposed for Paleozoic and younger taxa with critical ambulacral apomorphies. Muschelkalk asteroids are assigned to the family Trichasteropsiidae n. fam., superorder Forcipulatacea. The recently described Triassic genusNoriaster belongs to the extant family Poraniidae, superorder Valvatacea.Trichasteropsis andNoriaster represent separate major phylogenetic branches of the post-Paleozoic infraclass Neoasteroidea, and together they indicate that diversification of modern-type asteroids was under-way during the Triassic, although the Mesozoic marine révolution largely was a Jurassic and later event. Post-Paleozoic asteroids appear to hâve returned to Paleozoic life modes in spite of new morphological expressions. Trichasteropsis is skeletally robust, suggesting protection from wave impact or predators. It is found in sédiments associated with shell banks but not from within the banks.Trichasteropsis senfti commonly occurs with brachiopods whereasT. weissmanni does not, although brachiopods are found in associated strata. Aspects of morphology of both species are similar to those of récent predatory Asteriidae suggesting similar behavior, but feeding habits ofTrichasteropsis are unverified.     

Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.     

Physical map of 1p36, placement of breakpoints in monosomy 1p36, and clinical characterization of the syndrome

Monosomy 1p36 is the most common terminal deletion syndrome. This contiguous gene deletion syndrome is presumably caused by haploinsufficiency of a number of genes. We have constructed a contig of overlapping large-insert clones for the most distal 10.5 Mb of 1p36, evaluated the deletion sizes in 61 subjects with monosomy 1p36 from 60 families, and created a natural deletion panel. We found pure terminal deletions, interstitial deletions, derivative chromosomes, and more complex rearrangements. Breakpoints were "binned" into 0.5-Mb regions. Analyses revealed some clustering of breakpoints but no single common breakpoint. Determination of the parental origin showed that 60% of de novo 1p36 terminal deletions arose from the maternally inherited chromosome. Of the 61 subjects, 30 were examined systematically through a protocol at the Texas Children's Hospital General Clinical Research Center. Specifically, we report hearing evaluations, palatal and ophthalmological examinations, echocardiograms, neurological assessments, and thyroid function tests. To our knowledge, this systematic molecular and clinical characterization of monosomy 1p36 is the largest and most comprehensive study of this deletion syndrome to date. Many cytogenetically visible, apparent terminal deletions are more complex than anticipated by cytogenetics, as revealed at the molecular level by our study. Our clinical findings allow for the more accurate recognition of the syndrome and for proper medical evaluation.     

Comparative biological responses to human Sonic, Indian, and Desert hedgehog

In this report, we describe the involvement of the quail neuroretina 1 (QN1) protein in retinal development. The Qn1 cDNA was isolated as a gene specifically expressed at the onset of neuronal cell cycle withdrawal (Bidou et al., Mech. Dev. 43 (1993) 159). Qn1 is located in the cytoplasm in proliferating cells during the early stages of the development. Its distribution changes, becoming predominantly nuclear, in neurons during establishment of the quiescent state upon the differentiation. We decreased the amount of QN1 protein by an antisense strategy in vitro or in vivo. This decrease of the amount of QN1 protein results in additional mitosis and in severe abnormalities such as retinal dysplasia. Our results suggest that QN1 plays a key role at the onset of neuronal cell cycle withdrawal.     

Mechanically unfolding proteins: the effect of unfolding history and the supramolecular scaffold

The mechanical resistance of a folded domain in a polyprotein of five mutant I27 domains (C47S, C63S I27)(5)is shown to depend on the unfolding history of the protein. This observation can be understood on the basis of competition between two effects, that of the changing number of domains attempting to unfold, and the progressive increase in the compliance of the polyprotein as domains unfold. We present Monte Carlo simulations that show the effect and experimental data that verify these observations. The results are confirmed using an analytical model based on transition state theory. The model and simulations also predict that the mechanical resistance of a domain depends on the stiffness of the surrounding scaffold that holds the domain in vivo, and on the length of the unfolded domain. Together, these additional factors that influence the mechanical resistance of proteins have important consequences for our understanding of natural proteins that have evolved to withstand force.     

Caulobacter crescentus synthesizes an S-layer-editing metalloprotease possessing a domain sharing sequence similarity with its paracrystalline S-layer protein

Strains of Caulobacter crescentus elaborate an S-layer, a two-dimensional protein latticework which covers the cell surface. The S-layer protein (RsaA) is secreted by a type I mechanism (relying on a C-terminal signal) and is unusual among type I secreted proteins because high levels of protein are produced continuously. In efforts to adapt the S-layer for display of foreign peptides and proteins, we noted a proteolytic activity that affected S-layer monomers with foreign inserts. The cleavage was precise, resulting in fragments with an unambiguous N-terminal sequence. We developed an assay to screen for loss of this activity (i.e., presentation of foreign peptides without degradation), using transposon and traditional mutagenesis. A metalloprotease gene designated sap (S-layer-associated protease) was identified which could complement the protease-negative mutants. The N-terminal half of Sap possessed significant similarity to other type I secreted proteases (e.g., alkaline protease of Pseudomonas aeruginosa), including the characteristic RTX repeat sequences, but the C-terminal half which normally includes the type I secretion signal exhibited no such similarity. Instead, there was a region of significant similarity to the N-terminal region of RsaA. We hypothesize that Sap evolved by combining the catalytic portion of a type I secreted protease with an S-layer-like protein, perhaps to associate with nascent S-layer monomers to "scan" for modifications.     

Walking and running at resonance

Humans and other animals can temporarily store mechanical energy in elastic oscillations, f(el), of body parts and in pendulum oscillations, f(p) = const sq.rt (g/L), of legs, length L, or other appendages, and thereby reduce the energy consumption of locomotion. However, energy saving only occurs if these oscillations are tuned to the leg propagation frequency f. It has long been known that f is tuned to the pendulum frequency of the free-swinging leg of walkers. During running the leg frequency increases to some new value f = f(r). We propose that in order to maintain resonance the animal, mass M, actively increases its leg pendulum frequency to the new value f(p,r) =const sq.rt (a(y)/L)=f(r), by giving its hips a vertical acceleration a(y)= F(y)/M. The pendulum frequency is increased if the impact force F(y) of the stance foot is larger than Mg, explaining the observation by Alexander and Bennet-Clark (1976) that F(v) becomes larger than Mg when animals start to run. Our model predictions of the running velocity U(r) as function of L, F(v), are in agreement with measurements of these quantities (Farley et al. 1993). The leg's longitudinal elastic oscillation frequency scales as f(el) = const sq.rt (k/M). Experiments by Ferris et al., (1998) show that runners adjust their leg's stiffness, k, when running on surfaces of different elasticity so that the total stiffness k remains constant. Our analysis of their data suggests that the longitudinal oscillations of the stance leg are indeed kept in tune with the running frequency. Therefore we conclude that humans, and by extension all animals, maintain resonance during running. Our model also predicts the Froude number of walking-running transitions, Fr = U(2)/gL approximately 0.5 in good agreement with measurements.     

Visual competition

Binocular rivalry--the alternations in perception that occur when different images are presented to the two eyes--has been the subject of intensive investigation for more than 160 years. The psychophysical properties of binocular rivalry have been well described, but newer imaging and electrophysiological techniques have not resolved the issue of where in the brain rivalry occurs. The most recent evidence supports a view of rivalry as a series of processes, each of which is implemented by neural mechanisms at different levels of the visual hierarchy. Although unanswered questions remain, this view of rivalry might allow us to resolve some of the controversies and apparent contradictions that have emerged from its study.     

A co-operative interaction between Neisseria gonorrhoeae and complement receptor 3 mediates infection of primary cervical epithelial cells

Little is known about the pathogenesis of gonococcal infection within the lower female genital tract. We recently described the distribution of complement receptor 3 (CR3) on epithelia of the female genital tract. Our studies further indicate that CR3-mediated endocytosis serves as a primary mechanism by which N. gonorrhoeae elicits membrane ruffling and cellular invasion of primary, human, cervical epithelial cells. We have extended these studies to describe the nature of the gonococcus-CR3 interaction. Western Blot analysis demonstrated production of alternative pathway complement components by ecto- and endocervical cells which allows C3b deposition on gonococci and its rapid conversion to iC3b. Anti-iC3b and -factor I antibodies significantly inhibited adherence and invasion of primary cervical cells, suggesting that iC3b covalently bound to the gonococcus serves as a primary ligand for CR3 adherence. However, gonococcal porin and pili also bound to the I-domain of CR3 in a non-opsonic manner. Binding of porin and pili to CR3 were required for adherence to and invasion of cervical epithelia. Collectively, these data suggest that gonococcal adherence to CR3 occurs in a co-operative manner, which requires gonococcal iC3b-opsonization, porin and pilus. In conjunction, these molecules facilitate targeting to and successful infection of the cervical epithelium.