Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site

Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP, is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of binding of NO to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used (15)NO and (14)NO and anaerobic sequential mixing-freeze-quench electron paramagnetic resonance to unambiguously confirm that the heme Fe is the target of the second NO. The linear dependence on NO concentration up to 600 s(-1) for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e., the proximal site of the heme, but also supports a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports the selective release of NO from the distal side of the transient bis-NO-sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO.     

How to grab a microtubule on the move

A recent paper in Cell highlights a second mechanism of regulation of separase in addition to the bound inhibitor securin. This second pathway involves separase phosphorylation and is dependent on CDC2.     

Two elongation responses to auxin respond differently to protein synthesis inhibition

The first and second responses to auxin react differently to the inhibition of protein synthesis by cycloheximide. It was determined that the protein with the shortest half-life, among the several necessary for the first response, is different from its counterpart among the several necessary for the second response. Specifically, the protein half-lives are 28 minutes and 11 minutes for the first and second responses, respectively.     

Leech responses to tissue transplantation

The aim of the present work is to describe histologically, histochemically and immunocytochemically, the sequence of events that lead to first and second set rejection of allo- or xenograft in leeches. Graft responses of leeches are comparable and are described following specific steps: inflammatory phase, rejection phase and granulation tissue formation (including re-epithelialisation, angiogenesis and fibroplasia).The responses to first and second graft in first set graft rejection as well as to the first transplant in second set graft experiments are identical and in the time span of a week all grafts are destroyed and disappear. In the second set graft rejection experiments the responses against the second transplant are markedly accelerated. The second graft shows massive structural alterations and it is rapidly rejected, within 3-4 days.Our results permit to highlight that in leeches there is a specific responsiveness of immune system similar to those described in highly divergent phyla.     

A kinetic theory approach to capturing interneuronal correlation: the feed-forward case

We present an approach for using kinetic theory to capture first and second order statistics of neuronal activity. We coarse grain neuronal networks into populations of neurons and calculate the population average firing rate and output cross-correlation in response to time varying correlated input. We derive coupling equations for the populations based on first and second order statistics of the network connectivity. This coupling scheme is based on the hypothesis that second order statistics of the network connectivity are sufficient to determine second order statistics of neuronal activity. We implement a kinetic theory representation of a simple feed-forward network and demonstrate that the kinetic theory model captures key aspects of the emergence and propagation of correlations in the network, as long as the correlations do not become too strong. By analyzing the correlated activity of feed-forward networks with a variety of connectivity patterns, we provide evidence supporting our hypothesis of the sufficiency of second order connectivity statistics. Action Editor: Carson C. Chow     

Evo-devo graduates to new levels

The second symposium on 'The Developmental Basis of Evolutionary Change' was held at the University of Chicago, IL, USA, from 25 to 28 October 2001.     

Improvements of Western blotting to detect monoclonal antibodies

A comparison of the effects of different factors on the sensitivity of Western blotting technique to detect monoclonal antibodies is described. The major improvements were obtained by: A) renaturating the antigen in the gel before transferring it in carbonate buffer at pH 10 onto nitrocellulose and B) using alkaline-phosphatase-conjugated second antibody instead of peroxidase-conjugated second antibody.     

The relation of nerves to multiple regeneration in a single newt limb.

Three amputation surfaces were formed on the lower arm of a single newt. Growth occurred on all combinations of these surfaces. The proximal surface (the only single surface to form regenerates) produced more regenerates (76% of the cases) than the two more distal surfaces. Blocking the proximal surface with whole skin greatly stimulates the production of accessory structures on the first and/or second more distal surfaces. The mean number of nerve fibers on the proximal surface is considerably higher than the nerve counts of the first more and second most distal surfaces. Limbs possessing a notch or digit(s) on the proximal surface and the absence of growth on the first more and second distal surfaces also show a decrease in nerve number on the first more and second most distal surfaces. An analysis of the mean number of nerve fibers on the blocked (proximal) surface shows a noticeable decrease in comparison with nerve fibers in an equivalent level on normal limbs. Nerve fiber counts on the first more and second most distal surfaces are markedly increased on those limbs where the proximal surface was blocked with whole skin. Threshold experiments suggest that the irregular occurrence of accessory structures on the first more distal and second most distal surfaces may be related to an insufficient number of nerve fibers on these surfaces. Similarly, a possible explanation for the regular occurrence of accessory structures on the proximal surface is that nerve number on this surface is always above threshold.     

Sensitivity of second harmonic generation from styryl dyes to transmembrane potential

In this article we present results from the simultaneous nonlinear (second harmonic generation and two-photon excitation fluorescence) imaging and voltage clamping of living cells. Specifically, we determine the sensitivity to transmembrane potential of second harmonic generation by ANEP-chromophore styryl dyes as a function of excitation wavelength and dye structure. We have measured second harmonic sensitivities of up to 43% per 100 mV, more than a factor of four better than the nominal voltage sensitivity of the dyes under "one-photon" fluorescence. We find a dependence of voltage sensitivity on excitation wavelength that is consistent with a two-photon resonance, and there is a significant dependence of voltage sensitivity on the structure of the nonchromophore portion of the dyes.     

Treatment with cyclophosphamide reverses acquired resistance to collagen arthritis subsequent to recovery from passive arthritis

Passive arthritis induced by anticollagen antibody was a mild, transient disease from which the animals normally recovered and the rats that had recovered from passive arthritis were resistant to develop a second phase of arthritis following a second administration of anticollagen antibody or the subsequent challenge with type II collagen. However, treatment of the recovered rats with cyclophosphamide (CY) shortly before a second administration of a serum concentrate induced a second episode of acute polyarthritis in 82% of the rats. In addition, CY treatment without further administration of a serum concentrate induced a recurrence of arthritis in 25% of the recovered rats. Similarly, treatment of the recovered rats with CY shortly before the challenge with type II collagen induced a second episode of arthritis in 83% of the rats. However, treatment with CY shortly before the challenge was unable to restore the suppressed humoral response to type II collagen. These results provide evidence that the resistant state is mediated, at least in part, by CY-sensitive events.     

Relation of the mental foramen to teeth on the mandibles belonging to the Byzantium period

Fifty mandibles belonging to the Byzantium period were studied in order to determine to racial differences about of the localization of the mental foramen according to the mandibular teeth. Mandibles of adult males without missing teeth, proximal decay and proximal attrition were used. On the right side, the most common position of the mental foramen was between the first and second lower teeth (50%), whereas on the left side, the most common position was in line with the longitudinal axis of the lower second premolar tooth (46%). Because of materials including mostly Alpine's and a few of Mediterranean subraces, two different positions of the mental foramen were determined in high similar ratios.     

The Response of Amino Acid Frequencies to Directional Mutation Pressure in Mitochondrial Genome Sequences Is Related to the Physical Properties of the Amino Acids and to the Structure of the Genetic Code

The frequencies of A, C, G, and T in mitochondrial DNA vary among species due to unequal rates of mutation between the bases. The frequencies of bases at fourfold degenerate sites respond directly to mutation pressure. At first and second positions, selection reduces the degree of frequency variation. Using a simple evolutionary model, we show that first position sites are less constrained by selection than second position sites and, therefore, that the frequencies of bases at first position are more responsive to mutation pressure than those at second position. We define a measure of distance between amino acids that is dependent on eight measured physical properties and a similarity measure that is the inverse of this distance. Columns 1, 2, 3, and 4 of the genetic code correspond to codons with U, C, A, and G in their second position, respectively. The similarity of amino acids in the four columns decreases systematically from column 1 to column 2 to column 3 to column 4. We then show that the responsiveness of first position bases to mutation pressure is dependent on the second position base and follows the same decreasing trend through the four columns. Again, this shows the correlation between physical properties and responsiveness. We determine a proximity measure for each amino acid, which is the average similarity between an amino acid and all others that are accessible via single point mutations in the mitochondrial genetic code structure. We also define a responsiveness for each amino acid, which measures how rapidly an amino acid frequency changes as a result of mutation pressure acting on the base frequencies. We show that there is a strong correlation between responsiveness and proximity, and that both these quantities are also correlated with the mutability of amino acids estimated from the mtREV substitution rate matrix. We also consider the variation of base frequencies between strands and between genes on a strand. These trends are consistent with the patterns expected from analysis of the variation among genomes. [Reviewing Editor: Dr. David Pollock]     

Effect of the passage of time on the contribution of initial response-outcome associations to instrumental performance

The effect of the passage of time on the contribution of initial response-outcome associations to subsequent instrumental performance was explored in three experiments with rats using outcome devaluation. Experiments 1 and 2 showed that a response that had been trained first with one outcome and then given identical training with a second outcome was more sensitive to devaluation of the second outcome than the first if the two training episodes were separated in time. Experiment 3 showed that inserting a delay between training with the second outcome and testing after outcome devaluation appeared to mitigate this effect of temporally separating first and second outcome training. Inserting this delay also made a response slightly more sensitive to devaluation of the first outcome than the second when there was no delay between the two training episodes. These results suggest that the passage of time can shift the balance between the contributions of first and second trained outcomes to instrumental performance.     

Arabidopsis ROOT PHOTOTROPISM2 Contributes to the Adaptation to High-Intensity Light in Phototropic Responses

Living organisms adapt to changing light environments via mechanisms that enhance photosensitivity under darkness and attenuate photosensitivity under bright light conditions. In hypocotyl phototropism, phototropin1 (phot1) blue light photoreceptors mediate both the pulse light-induced, first positive phototropism and the continuous light-induced, second positive phototropism, suggesting the existence of a mechanism that alters their photosensitivity. Here, we show that light induction of ROOT PHOTOTROPISM2 (RPT2) underlies photosensory adaptation in hypocotyl phototropism of Arabidopsis thaliana. rpt2 loss-of-function mutants exhibited increased photosensitivity to very low fluence blue light but were insensitive to low fluence blue light. Expression of RPT2 prior to phototropic stimulation in etiolated seedlings reduced photosensitivity during first positive phototropism and accelerated second positive phototropism. Our microscopy and biochemical analyses indicated that blue light irradiation causes dephosphorylation of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) proteins and mediates their release from the plasma membrane. These phenomena correlate closely with the desensitization of phot1 signaling during the transition period from first positive phototropism to second positive phototropism. RPT2 modulated the phosphorylation of NPH3 and promoted reconstruction of the phot1-NPH3 complex on the plasma membrane. We conclude that photosensitivity is increased in the absence of RPT2 and that this results in the desensitization of phot1. Light-mediated induction of RPT2 then reduces the photosensitivity of phot1, which is required for second positive phototropism under bright light conditions.     

Phagocytic response to fully controlled plural stimulation of antigens on macrophage using on-chip microcultivation system

To understand the control mechanism of innate immune response in macrophages, a series of phagocytic responses to plural stimulation of antigens on identical cells was observed. Two zymosan particles, which were used as antigens, were put on different surfaces of a macrophage using optical tweezers in an on-chip single-cell cultivation system, which maintains isolated conditions of each macrophage during their cultivation. When the two zymosan particles were attached to the macrophage simultaneously, the macrophage responded and phagocytosed both of the antigens simultaneously. In contrast, when the second antigen was attached to the surface after the first phagocytosis had started, the macrophage did not respond to the second stimulation during the first phagocytosis; the second phagocytosis started only after the first process had finished. These results indicate that (i) phagocytosis in a macrophage is not an independent process when there are plural stimulations; (ii) the response of the macrophage to the second stimulation is related to the time" delay from the first stimulation. Stimulations that occur at short time intervals resulted in simultaneous phagocytosis, while a second stimulation that is delayed long enough might be neglected until the completion of the first phagocytic process.     

Cell adhesion molecules and the transition from short- to long-term memory

Training chicks on a one-trial passive avoidance task results in a cascade of molecular and cellular processes in two forebrain regions, culminating within 60–90 min in post-translational glycosylation of synaptic membrane proteins and expression of immediate early genes c-fos and c-jun. We have now found a second window of vulnerability of memory to the protein synthesis inhibitor anisomycin, 4 h downstream of training. By 5.5 h post-training this window closes, to be replaced by a window of sensitivity to blockade of glycoprotein synthesis, presumably representing post-translational modification of the newly synthesised proteins. Amongst the pre- and post-synaptic membrane glycoproteins involved at both first and second time windows are the cell adhesion molecules, L1 (at both times) and NCAM (at the later). Molecular dissection of the external membrane domains of L1 distinguishes between a requirement for the IgG domain at the early time, the fibronectin-like domain at the later. The second time window only occurs if the animal is trained on a stimulus strong enough to be remembered for a long period. Weak memories do not persist beyond 6–8 h and the second wave of glycoprotein synthesis does not occur. Thus the second wave may represent the molecular processes required for the alterations in synaptic configuration, by way of the adhesion molecules amongst others, required for the morphological changes in neuronal connectivity hypothesised to encode memory.     

Studies on prenatal and postnatal development in rats exposed to 60-Hz electric fields

A series of three experiments was performed to determine the effects of 30-day exposures to uniform 60-Hz electric fields (100 kV/m) on reproduction and on growth and development in the fetuses and offspring of rats. In the first experiment, exposure of females for 6 days prior to and during the mating period did not affect their reproductive performance, and continued exposure through 20 days of gestation (dg) did not affect the viability, size, or morphology of their fetuses. In the second experiment, exposure of the pregnant rat was begun on 0 dg and continued until the resulting offspring reached 8 days of age. In the third experiment, exposure began at 17 dg and continued through 25 days of postnatal life. In the second and third experiments, no statistically significant differences suggesting impairment of the growth or survival of exposed offspring were detected. In the second experiment, a significantly greater percentage of the exposed offspring showed movement, standing, and grooming at 14 days of age than among-sham-exposed offspring. There was a significant decrease at 14 days in the percentage of exposed offspring displaying the righting reflex in the second experiment and negative geotropism in the third experiment. These differences were all transient and were not found when the animals were tested again at 21 days of age. Evaluation of the reproductive integrity of the offspring of the second experiment did not disclose any deficits.     

Ligand binding to ferrocytochrome c at high pH.

Ferrocytochrome c has been shown to bind two molecules of CO at pH 14. The second CO is thought to be bound only when the cytochrome c molecule is denatured, and once bound appears to be spectrally silent. Insolubilization of native cytochrome c prevents the binding of the second CO molecule. A scheme is proposed to explain these observations based on evidence from static titrations and flash-photolysis experiments, use of carboxymethyl cytochrome c and insoluble cytochrome c, and use of cyanide instead of CO as a ligand.     

Heritability of liability to cystic ovary development in Holstein cattle

Data from 249 and 152 records of first and second lactations, respectively, were collected from the files of Dairy Herd Improvement Association and veterinary clinical records of Holstein cows from the J. C. Penny farm of the University of Missouri-Columbia from 1977 through 1987. Incidences of cystic ovaries were .345 and .421 in the first and second lactations, respectively. Heritabilities of liability to cystic ovary development based on correlation of incidences among paternal half-sib relatives were 0.15 +/- 0.44 and 0.11 +/- 0.65 for first and second lactation records, respectively. Corresponding estimates by paternal half-sib analysis of variance, using either original or angle-transformed proportions, were assumed to be zero because sire components of variance were negative. The results suggest that individual selection based on the presence or absence of cystic ovary development would be, at best, very slow in bringing about a reduction in the incidence of this disease in dairy cattle herds.     

Tinea capitis due to Microsporum canis in infants

Three cases of scalp involvement by M. canis in infants are briefly reported. The first was a 2-month-old female with a few erythematous spots on the scalp, the second an 8-month-old male with lesions resembling seborrhoeic dermatitis and the third an 11-month-old male having areas of folliculitis of a kerion type. The father of the second case was found to have a tinea barbae infection due to the same fungus. Stray kittens were the most probable source of infection in all these cases.