Connecting the protein structure universe by using sparse recurring fragments

The quest to order and classify protein structures has lead to various classification schemes, focusing mostly on hierarchical relationships between structural domains. At the coarsest classification level, such schemes typically identify hundreds of types of fundamental units called folds. As a result, we picture protein structure space as a collection of isolated fold islands. It is obvious, however, that many protein folds share structural and functional commonalities. Locating those commonalities is important for our understanding of protein structure, function, and evolution. Here, we present an alternative view of the protein fold space, based on an interfold similarity measure that is related to the frequency of fragments shared between folds. In this view, protein structures form a complicated, crossconnected network with very interesting topology. We show that interfold similarity based on sequence/structure fragments correlates well with similarities of functions between protein populations in different folds.     

Synaptic current between neuromuscular junction folds

Measurements of membrane infoldings of vertebrate subsynaptic membranes were taken to evaluate the possible electrophysiological implications. The shapes of standard interfolds of different neuromuscular junctions were established from micrographs available in the literature. Electrical properties were estimated using published fibre membrane and myoplasm electrical values. Models of synaptic current pathways were designed taking into account the small size of the postsynaptic patch activated by a transmitter quantum. This analysis reveals a resistance "in series" between the ACh-sensitive interfold crest and the remainder of the muscle fibre. The calculated cytoplasmic resistance of an interfold is between 0.2 and 3 Mohms which is in the same range as the fibre DC input resistance. The calculated interfold resistance appears to be dependent on the fibre type, the age and the pathology. Functional roles of junctional folds and dendritic spines are discussed.     

Spatial organization of intestinal microbiota in the mouse ascending colon

Complex microbial populations are organized in relation to their environment. In the intestine, the inner lining (mucosa) is a potential focal point for such organization. The proximal murine colon contains mucosal folds that are known to be associated with morphologically distinct microbes. To identify these microbes, we used the technique of laser capture microdissection (LCM) to sample microbes associated with these folds (interfold region) and within the central lumen (digesta region). Using 16S rRNA gene tag pyrosequencing, we found that microbes in the interfold region were highly enriched for the phylum Firmicutes and, more specifically, for the families Lachnospiraceae and Ruminococcaceae. Other families such as Bacteroidaceae, Enterococcaceae and Lactobacillaceae were all enriched in the digesta region. This high-resolution system to capture and examine spatial organization of intestinal microbes should facilitate microbial analysis in other mouse models, furthering our understanding of host–microbial interactions.     

Epithelial cell proliferation in the intestine of the winter flounder,Pseudopleuronectes americanus

Summary To study epithelial cell proliferation in the North American flounder (Pseudopleuronectes americanus), fed and fasted fish received intravenous injections of ³H-thymidine and were killed 11/2 to 2 h later. Radioautographs of proximal, middle, and distal intestinal segments revealed proliferating epithelial cells at all levels of intestinal folds including the crest although labelled nuclei were most abundant in the epithelial cells on the lower half of folds and between folds. Mature appearing goblet cells with labelled nuclei were observed at all levels of the folds. The mean labelling index was greater in the epithelium of fed than fasted flounder. In fed flounder the mean labelling index was greatest in the proximal segment and least in the distal segment; no substantive differences in mean labelling indices were observed in the various segments of intestine from fasted fish. Electron microscopy revealed no major structural differences among epithelial cells along the base of folds compared to cells near the crest of folds. These findings indicate that 1) epithelial cell proliferation occurs at all levels of the folds of flounder intestine and is not compartmentalized to the base of the folds and interfold epithelium as reported in other teleosts, and 2) epithelial cell proliferation in the flounder intestine varies with feeding status.Supported be research grants AM 17537 and RR 05764 from the National Institutes of Health, Bethesda, Maryland and grant DEB7826821 AO1 from the National Science Foundation, Washington, D.C.The authors are grateful to Dr. Michael Field for stimulating discussions and suggestions and for providing facilities for collecting material from fish     

Elasticity and stress relaxation of a very small vocal fold

Across mammals many vocal sounds are produced by airflow induced vocal fold oscillation. We tested the hypothesis that stress-strain and stress-relaxation behavior of rat vocal folds can be used to predict the fundamental frequency range of the species' vocal repertoire. In a first approximation vocal fold oscillation has been modeled by the string model but it is not known whether this concept equally applies to large and small species. The shorter the vocal fold, the more the ideal string law may underestimate normal mode frequencies. To accommodate the very small size of the tissue specimen, a custom-built miniaturized tensile test apparatus was developed. Tissue properties of 6 male rat vocal folds were measured. Rat vocal folds demonstrated the typical linear stress-strain behavior in the low strain region and an exponential stress response at strains larger than about 40%. Approximating the rat's vocal fold oscillation with the string model suggests that fundamental frequencies up to about 6 kHz can be produced, which agrees with frequencies reported for audible rat vocalization. Individual differences and time-dependent changes in the tissue properties parallel findings in other species, and are interpreted as universal features of the laryngeal sound source.     

INFLUENCE OF INSTANTANEOUS AND INTEGRATED LIGHT-FLUX DENSITY ON LEAF ANATOMY AND PHOTOSYNTHESIS

Experiments were conducted to examine whether leaf adaptation to light in Fragaria virginiana (Rosaceae) was determined by peak photon-flux density or by total quanta received during the day. Leaf structure and apparent photosynthesis rates were similar under environments where total energy received was the same even though peak photon-flux density was different. When peak photon-flux density was held constant and total quanta varied, significant differences were noted in apparent photosynthesis, leaf thickness, specific leaf weight, mesophyll cell volume, and A^mes/A ratio. High total quanta produced high-light or sun-type leaves even at relatively low peak intensities. Thus, total light energy received during the day has a greater influence on leaf adaptation to light than does peak photon-flux density.     

Rate of quantal transmitter release at the mammalian rod synapse.

Under scotopic conditions, the mammalian rod encodes either one photon or none within its integration time. Consequently the signal presented to its synaptic terminal is binary. The synapse has a single active zone that releases neurotransmitter quanta tonically in darkness and pauses briefly in response to a rhodopsin isomerization by a photon. We asked: what minimum tonic rate would allow the postsynaptic bipolar cell to distinguish this pause from an extra-long interval between quanta due to the stochastic timing of release? The answer required a model of the circuit that included the rod convergence onto the bipolar cell and the bipolar cell''s signal-to-noise ratio. Calculations from the model suggest that tonic release must be at least 40 quanta/s. This tonic rate is much higher than at conventional synapses where reliability is achieved by employing multiple active zones. The rod''s synaptic mechanism makes efficient use of space, which in the retina is at a premium.     

Effect of the ventricular folds in a synthetic larynx model

Within the human larynx, the ventricular folds serve primarily as a protecting valve during swallowing. They are located directly above the sound-generating vocal folds. During normal phonation, the ventricular folds are passive structures that are not excited to periodical oscillations. However, the impact of the ventricular folds on the phonation process has not yet been finally clarified.An experimental synthetic human larynx model was used to investigate the effect of the ventricular folds on the phonation process. The model includes self-oscillating vocal fold models and allows the comparison of the pressure distribution at multiple locations in the larynx for configurations with and without ventricular folds.The results indicate that the ventricular folds increase the efficiency of the phonation process by reducing the phonation threshold level of the pressure below the vocal folds. Two effects caused by the ventricular folds could be identified as reasons: (1) a decrease in the mean pressure level in the region between vocal and ventricular folds (ventricles) and (2) an increase in the glottal flow resistance.The reason for the first effect is a reduction of the pressure level in the ventricles due to the jet entrainment and the low static pressure in the glottal jet. The second effect results from an increase in the glottal flow resistance that enhances the aerodynamic energy transfer into the vocal folds. This effect reduces the onset threshold of the pressure difference across the glottis.     

On the pH-dependence of the reaction of hemoglobin with carbon monoxide.

Flash-photolysis experiments were performed on solutions of carbonmonoxy hemoglobin (human Hb A) as function as pH. The fraction of fast reaction and the amount of photodissociation as produced by a given amount of light quanta has been analyzed in terms of the allosteric model of ligand binding by Monod, Wyman and Changeux. It is shown how the switch-over point of the allosteric transition from the T or R state is controlled by the protons, which act as allosteric effectors.     

Microvillar components of light adaptation in blowflies

The process of light adaptation in blowfly photoreceptors was analyzed using intracellular recording techniques and double and triple flash stimuli. Adapting flashes of increasing intensity caused a progressive reduction in the excitability of the photoreceptors, which became temporarily suppressed when 3 x 10(6) quanta were absorbed by the cell. This suppression was confirmed by subsequently applying an intense test flash that photoactivated a considerable fraction of the 10(8) visual pigment molecules in the cell. The period of temporary desensitization is referred to as the refractory period. The stimulus intensity to render the receptor cell refractory was found to be independent of the extracellular calcium concentration over a range of 10(-4) and 10(-2) M. During the refractory period (30-40 ms after the adapting flash) the cell appears to be "protected" against further light adaptation since light absorption during this period did not affect the recovery of the cell's excitability. Calculations showed that the number of quantum absorptions necessary to induce receptor refractoriness is just sufficient to photoactivate every microvillus of the rhabdomere. This coincidence led to the hypothesis that the refractoriness of the receptor cells is due to the refractoriness of the individual microvilli. The sensitivity of the receptor cells after relatively weak adapting flashes was reduced considerably more than could be accounted for by the microvilli becoming refractory. A quantitative analysis of these results suggests that a photoactivated microvillus induces a local adaptation over a relatively small area of the rhabdomere around it, which includes several tens of microvilli. After light adaptation with an intense flash, photoactivation of every microvillus by the absorption of a few quanta produced only a small receptor response whereas photoactivation of every rhodopsin molecule in every microvillus produced the maximum response. The excitatory efficiency of the microvilli therefore increases with the number of quanta that are absorbed simultaneously.     

Role of temperature in quanta mechanisms of facilitation in the frog neuromuscular junction

The results of computer simulations on the Double Barrier Synapse (DBS) model are presented which quantify the relationship between the synapse parameters and the quanta transfer process. The DBS model is applicable to a variety of states of synaptic activity, and by changing the synapse parameters it is possible to simulate various conditions of quanta transmission. The influence of the bathing solution temperature change on the synaptic parameters under different conditions of transmitter release in the frog neuromuscular junction is investigated. Simulations demonstrate that several synaptic parameters, including the parameters of the presynaptic membrane, are not affected by the temperature change. It is shown that a stimulation frequency exists at which the steady-state level of facilitation during a long train of stimuli is the same for a wide range of temperatures. Received: 2 August 1996 / Accepted in revised form: 19 February 1998     

A unifold, mesofold, and superfold model of protein fold use.

As more and more protein structures are determined, there is increasing interest in the question of how many different folds have been used in biology. The history of the rate of discovery of new folds and the distribution of sequence families among known folds provide a means of estimating the underlying distribution of fold use. Previous models exploiting these data have led to rather different conclusions on the total number of folds. We present a new model, based on the notion that the folds used in biology fall naturally into three classes: unifolds, that is, folds found only in a single narrow sequence family; mesofolds, found in an intermediate number of families; and the previously noted superfolds, found in many protein families. We show that this model fits the available data well and has predicted the development of SCOP over the past 2 years. The principle implications of the model are as follows: (1) The vast majority of folds will be found in only a single sequence family; (2) the total number of folds is at least 10,000; and (3) 80% of sequence families have one of about 400 folds, most of which are already known.     

Role of bronchial basement membrane in airway collapse

Bronchial basement membrane is an elastic structure that has the potential to be load bearing and thus to contribute to the mechanical stiffness of the bronchus. To investigate this possible role, the membrane was modeled as a thin-walled linearly elastic tube surrounded by a uniform liquid on the outside and by air on the inside. When the external pressure on such a tube exceeds the internal pressure by a critical amount, the tube buckles reversibly into two or more folds. The critical buckling pressure varies as the square of the number of folds. The analysis was used to investigate the collapse behavior of the model tube into patterns ranging from 2 to 24 folds. This showed that the resistance to collapse increases rapidly as the number of folds increases. Data in the literature lead to the conclusion that the pressures involved in collapsing the tubes are probably in the physiological range. It is suggested, on the basis of the model results reported here, that bronchial hyperresponsiveness could be related to the number of folds into which the basement membrane buckles when the bronchial muscle contracts. A reduced number of folds would yield an increased response.     

Using scores derived from statistical coupling analysis to distinguish correct and incorrect folds in de-novo protein structure prediction

Distinguishing native from non-native folds remains a challenging problem for protein structure prediction. We describe a method, SCA-distance scoring, based on results from statistical coupling analysis which discriminates between native and non-native folds produced by a de novo protein structure prediction method for four out of five test proteins. The method is particularly good at discriminating non-native folds which are close in RMSD to the true fold but contain a change in an internal structural element. SCA-distance scoring is a useful addition to the tools available for distinguishing native from non-native folds in protein structure prediction.     

Neural fold formation at newly created boundaries between neural plate and epidermis in the axolotl

According to a recent model, the cortical tractor model, neural fold and neural crest formation occurs at the boundary between neural plate and epidermis because random cell movements become organized at this site. If this is correct, then a fold should form at any boundary between epidermis and neural plate. To test that proposition, we created new boundaries in axolotl embryos by juxtaposing pieces of neural plate and epidermis that would not normally participate in fold formation. These boundaries were examined superficially and histologically for the presence of folds, permitting the following observations. Folds form at each newly created boundary, and as many folds form as there are boundaries. When two folds meet they fuse into a hollow "tube" of neural tissue covered by epidermis. Sections reveal that these ectopic folds and "tubes" are morphologically similar to their natural counterparts. Transplanting neural plate into epidermis produces nodules of neural tissue with central lumens and peripheral nerve fibers, and transplanting epidermis into neural plate causes the neural tube and the dorsal fin to bifurcate in the region of the graft. Tissue transplanted homotypically as a control integrates into the host tissue without forming folds. When tissue from a pigmented embryo is transplanted into an albino host, the presence of pigment allows the donor cells to be distinguished from those of the host. Mesenchymal cells and melanocytes originating from neural plate transplants indicate that neural crest cells form at these new boundaries. Thus, any boundary between neural plate and epidermis denotes the site of a neural fold, and the behavior of cells at this boundary appears to help fold the epithelium. Since folds can form in ectopic locations on an embryo, local interactions rather than classical neural induction appear to be responsible for the formation of neural folds and neural crest.     

Quantum relations in photoreactivation of Colpidium

1. The amount of visible or long ultraviolet light (UV) required to photoreactivate Colpidium colpoda injured with known dosages of short UV (2654 A) was determined. 2. The effect of the short UV was tested by the delay in division of exposed animals compared to controls. Photoreactivation was tested by the effect of postillumination on the delay of division of treated colpidia compared to controls. 3. Colpidia were used in two physiological states: well fed and starved in balanced medium for 48 hours. The latter are much more sensitive to short UV although less susceptible of photoreactivation. 4. Photoreactivation occurred over the entire span from 3350 A to 4350 A for the well fed colpidia, from 3130 A to 5490 (green) for starved colpidia. 5. The photoreactivating effect of a single quantum of blue (4350 A) or long UV (3660 A) delivered per quantum of 2654 A used to injure colpidia was too slight to be considered significant. The effect of 10 quanta was usually more pronounced, but only after 100 quanta had been delivered was the photoreactivation nearly maximal for well fed colpidia. 6. The quantum requirement for maximal photoreactivation of the starved animals was greater at all wave lengths tried: 3660, 4050, 4350, and 5460 A being of the order of 800 incident quanta per incident quantum of 2654 A. 7. The transmission of UV(2654 A), blue, yellow, and red light by a suspension of colpidia was determined. 8. Large dosages of blue, violet, or long UV were slightly injurious to starved colpidia. In a few cases large dosages of 3660 A killed starved colpidia, especially after a non-lethal dose of short UV(2654 A). 9. Photoreactivation seems to be a balance between the slight injurious effect produced by the visible light or UV of long wave lengths and the injury produced by short wave length UV. 10. Possible reasons for the large number of quanta of photoreactivating light required per quantum of short UV are discussed.     

QUANTITATIVE ASPECTS OF TRANSMITTER RELEASE

The opener-stretcher motor neuron in crayfish makes 50 endings upon each of 1200 muscle fibers. We have calculated the quantal content of junctional potentials produced by individual terminals and by the whole cell at various physiological frequencies. The results show that when the motor neuron is active at 20 impulses/second, it releases 50 quanta/impulse per muscle fiber, or a total of 4.5 x 10⁹ quanta/hr. These figures are similar to those for vertebrate muscles per fiber, but larger for the entire neuron because the opener motor unit is so large. On the basis that the quanta correspond to synaptic vesicles each containing 10³–10⁴ molecules of transmitter, the release rate must be around 10^-11 mole/hr. This value is within an order of magnitude of the release figures obtained for mammalian neurons by collecting transmitter in perfusates, but it is far lower than the value reported for a crustacean inhibitory neuron. If the membrane materials surrounding each vesicle were lost in the release process, the replacement synthesis would involve 24 mm² of membrane/hr. We conclude that the metabolic load in terms of transmitter synthesis is probably sustainable, but that the release mechanism must operate in such a way that vesicle membrane materials are neither lost nor incorporated into the terminal membrane.     

Relationships among cell chlorophyll content,photosystem II light-harvesting and the quantum yield for oxygen production in Chlorella

Cells of the green alga Chlorella vulgaris were grown under conditions where total Chl/cell varied by a factor of almost 80; from 0.02 fmol/cell to nearly 1.6 fmol/cell. The change in Chl/cell was accomplished by an approximately 11-fold increase in RCII/cell along with a 7-fold increase in Chl/RCII. The effective absorption cross section per RCII at 596 nm varied by a factor of 6, increasing with Chl/cell from a minimum of 20 A² to a maximum of 116 A². In contrast, over the same range of Chl/cell, the quantum requirement for O₂ production remained relatively constant at 10.4±1.8 quanta absorbed/O₂ evolved. The results are well described by a simple model in which changes in Chl/cell are produced by coordinated changes in reaction center and light-harvesting complexes. The model predicts that between 20 and 40% of the light-harvesting chlorophyll-protein complexes commonly assigned to PSII, do not function as antenna for PSII.This paper is dedicated to the memory of Warren L. Butler, a pioneer in the field of photobiology. His creativity and innovativeness as a scientist were matched only by his patience as a mentor.