Dynamics of the ‘echo’ effect in a phytoplankton system with nitrogen fixation

Consideration of nitrogen fixation adds a positive nonlinear feedback to plankton ecosystem models. We investigate the consequences of this feedback for secondary phytoplankton blooms and the response of phytoplankton dynamics to physical forcing. The dynamics of phytoplankton, Trichodesmium (the nitrogen fixer), and nutrients is modeled with a system of three differential equations. The model includes two types of nonlinear interactions: the competition of phytoplankton and Trichodesmium for light, and the positive feedback resulting from Trichodesmium recycling. A typical simulation of the model in time, with forcing by a varying mixed-layer depth, reveals a clear successional sequence including a secondary or ‘echo’ bloom of the phytoplankton. We explain this sequence of events through the stability analysis of three different steady states of the model. Our analysis shows the existence of a critical biological parameter, the ratio of normalized growth rates, determining the occurrence of ‘echo’ blooms and the specific sequence of events following a physical perturbation. The interplay of positive and negative feedbacks appears essential to the timing and the type of events following such a perturbation.     

NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times (T₂) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P₀=0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P₀=1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo (T₂=3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T₂=8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P/P₀=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T₂ of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study

The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.     

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, β-sheets, and 3₁₀-helices by strategically using ²H-labeled amino acids and SDSL. In this study, we have explored the use of ¹³C-labeled residues as the NMR active nuclei for this approach for the first time. ¹³C-labeled d₅-valine (Val) or ¹³C-labeled d₆-leucine (Leu) were substituted at a specific Val or Leu residue (i), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i-2 and i-3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The ¹³C ESEEM peaks in the FT frequency domain data were observed for the i-3 samples, and no ¹³C peaks were observed in the i-2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures.     

Nanoscale protein domain motion and long-range allostery in signaling proteins—a view from neutron spin echo spectroscopy

Many cellular proteins are multi-domain proteins. Coupled domain–domain interactions in these multidomain proteins are important for the allosteric relay of signals in the cellular signaling networks. We have initiated the application of neutron spin echo spectroscopy to the study of nanoscale protein domain motions on submicrosecond time scales and on nanometer length scale. Our NSE experiments reveal the activation of protein domain motions over a long distance of over more than 100 Å in a multidomain scaffolding protein NHERF1 upon binding to another protein, Ezrin. Such activation of nanoscale protein domain motions is correlated with the allosteric assembly of multi-protein complexes by NHERF1 and Ezrin. Here, we summarize the theoretical framework that we have developed, which uses simple concepts from nonequilibrium statistical mechanics to interpret the NSE data, and employs a mobility tensor to describe nanoscale protein domain motion. Extracting nanoscale protein domain motion from the NSE does not require elaborate molecular dynamics simulations, nor complex fits to rotational motion, nor elastic network models. The approach is thus more robust than multiparameter techniques that require untestable assumptions. We also demonstrate that an experimental scheme of selective deuteration of a protein subunit in a complex can highlight and amplify specific domain dynamics from the abundant global translational and rotational motions in a protein. We expect NSE to provide a unique tool to determine nanoscale protein dynamics for the understanding of protein functions, such as how signals are propagated in a protein over a long distance to a distal domain.     

Diastolic time – frequency relation in the stress echo lab: filling timing and flow at different heart rates

 

A cutaneous force-frequency relation recording system based on first heart sound amplitude vibrations has been recently validated. Second heart sound can be simultaneously recorded in order to quantify both systole and diastole duration.

Aims

1- To assess the feasibility and extra-value of operator-independent, force sensor-based, diastolic time recording during stress.

Methods

We enrolled 161 patients referred for stress echocardiography (exercise 115, dipyridamole 40, pacing 6 patients). The sensor was fastened in the precordial region by a standard ECG electrode. The acceleration signal was converted into digital and recorded together with ECG signal. Both systolic and diastolic times were acquired continuously during stress and were displayed by plotting times vs. heart rate. Diastolic filling rate was calculated as echo-measured mitral filling volume/sensor-monitored diastolic time.

Results

Diastolic time decreased during stress more markedly than systolic time. At peak stress 62 of the 161 pts showed reversal of the systolic/diastolic ratio with the duration of systole longer than diastole. In the exercise group, at 100 bpm HR, systolic/diastolic time ratio was lower in the 17 controls (0.74 ± 0.12) than in patients (0.86 ± 0.10, p < 0.05 vs. controls). Diastolic filling rate increased from 101 ± 36 (rest) to 219 ± 92 ml/m²* s^-1 at peak stress (p < 0.5 vs. rest).

Conclusion

Cardiological systolic and diastolic duration can be monitored during stress by using an acceleration force sensor. Simultaneous calculation of stroke volume allows monitoring diastolic filling rate. Stress-induced "systolic-diastolic mismatch" can be easily quantified and is associated to several cardiac diseases, possibly expanding the spectrum of information obtainable during stress.     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Selection of optimal variants of Gō-like models of proteins through studies of stretching

The Gō-like models of proteins are constructed based on the knowledge of the native conformation. However, there are many possible choices of a Hamiltonian for which the ground state coincides with the native state. Here, we propose to use experimental data on protein stretching to determine what choices are most adequate physically. This criterion is motivated by the fact that stretching processes usually start with the native structure, in the vicinity of which the Gō-like models should work the best. Our selection procedure is applied to 62 different versions of the Gō model and is based on 28 proteins. We consider different potentials, contact maps, local stiffness energies, and energy scales—uniform and nonuniform. In the latter case, the strength of the nonuniformity was governed either by specificity or by properties related to positioning of the side groups. Among them is the simplest variant: uniform couplings with no i, i + 2 contacts. This choice also leads to good folding properties in most cases. We elucidate relationship between the local stiffness described by a potential which involves local chirality and the one which involves dihedral and bond angles. The latter stiffness improves folding but there is little difference between them when it comes to stretching.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.     

Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

The kinetics of renaturation of the β₂-subunit of Escherichia coli tryptophan-synthetase (l-serine hydrolyase (adding indole) E.C. 4.2.1.20) and those of its two proteolytic fragments F₁ and F₂ are studied and compared. Steps corresponding to the refolding of F₁, to the association of the folded F₁ and F₂ fragments, and to an isomerization of the associated protein are identified. These steps are ordered on the pathway of renaturation and some of their kinetic parameters are determined. This leads to a tentative kinetic model for the renaturation of nicked-β₂ starting from the denatured F₁ and F₂ fragments.The step corresponding to the refolding of the F₁ domain, as well as that corresponding to the last rate-limiting isomerization leading to the native protein, is shown to be the same in the refolding of the entire, uncleaved β₂-protein. It is concluded that the refolded F₁ fragment corresponds to a folding intermediate on the pathway of renaturation of the β₂-subunit.     

Determination of the number of degrees of freedom of the trapeziometacarpal joint–An in vitro study

ObjectiveMost of the studies about trapeziometacarpal joint assume that it exhibits only two independent degrees of freedom, but the experimental or theoretical support for considering a two-degrees of freedom model is not always clear.Materials and methodsTherefore, an in vitro kinematic study has been designed to demonstrate, from experimental data, that only two of the trapeziometacarpal degrees of freedom (i.e., flexion/extension and adduction/abduction) are non-null and independent. Several movements of maximal amplitude in flexion, abduction and circumduction have been realized and the relative position and orientation of the segment coordinate system embedded on the first metacarpal with respect to that embedded on the trapezium have been collected using electromagnetic sensors. The trapeziometacarpal rotations have been described using a joint coordinate system and the joint displacements have been evaluated on the axes of this coordinate system.ResultsThe root mean square (RMS) values of the joint displacement components have been found small enough to assume that the trapeziometacarpal joint has no translation degrees of freedom. A paraboloid coupling equation has been found between the internal/external rotation angle and the two other, flexion/extension and adduction/abduction, angles.ConclusionThus, this study demonstrates that the trapeziometacarpal joint has only two independent rotational degrees of freedom, and further, the described methodology could also be used to determine the coupling laws between degrees of freedom of various joints.     

Some aspects of mechanism of inhibition of cholesterol absorption by β-sitosterol

(1) Mixed bile salt micelle solubilized either cholesterol or β-sitosterol to a comparable extent. When added simultaneously, β-sitosterol restricted the micellar solubility of cholesterol. (2) β-Sitosterol also reduced the cholesterol content in the aqueous (micellar) phase of the intestinal contents of rats, the extent of reduction being comparable with that observed in vitro. The intestinal uptake of cholesterol in vivo was equivalent to the micellar incorporation of cholesterol both in vitro and in vivo. (3) β-Sitosterol had no inhibitory effect on cholesterol absorption from the micellar solution in jejunal loops in situ, whereas the rate of β-sitosterol uptake was only about one-fifth that of cholesterol. (4) The intestinal uptake of β-sitosterol intubated into the stomach of rats was about one-fifth that of cholesterol. The intestinal brush-border membrane discriminated these sterols. These results suggest that the restriction of the micellar solubility of cholesterol, rather than the inhibition of uptake from brush-border membrane, is the major determinant for the interference of β-sitosterol with cholesterol absorption.     

Diagnostic Utility of the Impact of Event Scale–Revised in Two Samples of Survivors of War

The study aimed at examining the diagnostic utility of the Impact of Event Scale-Revised (IES-R) as a screening tool for post-traumatic stress disorder (PTSD) in survivors of war. The IES-R was completed by two independent samples that had survived the war in the Balkans: a sample of randomly selected people who had stayed in the area of former conflict (n = 3,313) and a sample of refugees to Western European countries (n = 854). PTSD was diagnosed using the MINI International Neuropsychiatric Interview. Prevalence of PTSD was 20.1% in the Balkan sample and 33.1% in the refugee sample. Results revealed that when considering a minimum value of specificity of 0.80, the optimally sensitive cut-off score for screening for PTSD in the Balkan sample was 34. In both the Balkan sample and the refugee sample, this cut-off score provided good values on sensitivity (0.86 and 0.89, respectively) and overall efficiency (0.81 and 0.79, respectively). Further, the kappa coefficients for sensitivity for the cut-off of 34 were 0.80 in both samples. Findings of this study support the clinical utility of the IES-R as a screening tool for PTSD in large-scale research studies and intervention studies if structured diagnostic interviews are regarded as too labor-intensive and too costly.