Single Transition-to-single Transition Polarization Transfer (ST2-PT) in [15N,1H]-TROSY

This paper describes the use of single transition-to-single transition polarization transfer (ST2-PT) in transverse relaxation-optimized spectroscopy (TROSY), where it affords a sensitivity enhancement for kinetically stable amide 15N-1H groups in proteins. Additional, conventional improvements of [15N,1H]-TROSY include that signal loss for kinetically labile 15N-1H groups due to saturation transfer from the solvent water is suppressed with the water flip back technique and that the number of phase steps is reduced to two, which is attractive for the use of [15N,1H]-TROSY as an element in more complex NMR schemes. Finally, we show that the impact of the inclusion of the 15N steady-state magnetization (Pervushin et al., 1998) on the signal-to-noise ratio achieved with [15N,1H]-TROSY exceeds by up to two-fold the gain expected from the gyromagnetic ratios of 1H and 15N.     

Suppression of anti-TROSY lines in a sensitivity enhanced gradient selection TROSY scheme

A simple modification of the TROSY pulse transfer scheme, suggested by Yang and Kay [J. Biomol. NMR 13 (1999) 3–10], is proposed which results in the suppression of unwanted anti-TROSY lines without any extra loss in sensitivity. The higher sensitivity of this TROSY transfer scheme therefore becomes available for 2D [¹⁵N, ¹H] TROSY correlation and 3D/4D ¹⁵N separated NOESY type experiments where complete suppression of the broad anti-TROSY lines is essential.     

Backbone resonance assignment in large protonated proteins using a combination of new 3D TROSY-HN(CA)HA, 4D TROSY-HACANH and 13C-detected HACACO experiments

A new method for backbone resonance assignment suitable for large proteins with the natural ¹H isotope content is proposed based on a combination of the most sensitive TROSY-type triple-resonance experiments. These techniques include TROSY-HNCO, ¹³C-detected 3D multiple-quantum HACACO and the newly developed 3D TROSY multiple-quantum-HN(CA)HA and 4D TROSY multiple-quantum-HACANH experiments. The favorable relaxation properties of the multiple-quantum coherences, signal detection using the ¹³C antiphase coherences, and the use of TROSY optimize the performance of the proposed set of experiments for application to large protonated proteins. The method is demonstrated with the 44 kDa uniformly ¹⁵N,¹³C-labeled and fractionally (35%) deuterated trimeric B. Subtilis Chorismate Mutase and is suitable for proteins with large correlation times but a relatively small number of residues, such as membrane proteins embedded in micelles or oligomeric proteins.     

An enhanced-sensitivity pure absorption gradient 4D 15N, 13C-edited NOESY experiment

Summary An enhanced-sensitivity gradient 4D ¹⁵N, ¹³C-edited NOESY experiment is presented. Gradients are employed to suppress artifacts, eliminate the intense H₂O signal and select for the coherence transfer pathway involving ¹⁵N magnetization. The latter use of the gradients results in a decrease in the number of phase cycle steps by a factor of two relative to the previously published 4D sequence (Kay et al., 1990) allowing for the recording of spectra with increased resolution per unit measuring time. Theoretical sensitivity enhancements of as much as a factor of can be expected over the previously published sequence, neglecting the effects of relaxation and pulse imperfections.     

Sugar-to-base correlation in nucleic acids with a 5D APSY-HCNCH or two 3D APSY-HCN experiments

A five-dimensional (5D) APSY (automated projection spectroscopy) HCNCH experiment is presented, which allows unambiguous correlation of sugar to base nuclei in nucleic acids. The pulse sequence uses multiple quantum (MQ) evolution which enables long constant-time evolution periods in all dimensions, an improvement that can also benefit non-APSY applications. Applied with an RNA with 23 nucleotides the 5D APSY-HCNCH experiment produced a complete and highly precise 5D chemical shift list within 1.5 h. Alternatively, and for molecules where the out-and-stay 5D experiment sensitivity is not sufficient, a set of out-and-back 3D APSY-HCN experiments is proposed: an intra-base (3D APSY-b-HCN) experiment in an MQ or in a TROSY version, and an MQ sugar-to-base (3D APSY-s-HCN) experiment. The two 3D peak lists require subsequent matching via the N1/9 chemical shift values to one 5D peak list. Optimization of the 3D APSY experiments for maximal precision in the N1/9 dimension allowed matching of all ¹⁵N chemical shift values contained in both 3D peak lists. The precise 5D chemical shift correlation lists resulting from the 5D experiment or a pair of 3D experiments also provide a valuable basis for subsequent connection to chemical shifts derived with other experiments.     

Sequential resonance assignment of medium-sized15 N/13C-labeled proteins with projected 4D triple resonance NMR experiments

We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K. (1996) J. Am. Chem. Soc., 118, 8146–8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments for the efficient sequential resonance assignment of ¹⁵N/¹³C-labeled proteins. A 3D ^{/ /}(CO)NHN experiment is recorded either in conjunction with 3D HNN< > or with the newly presented 3D HNN scheme. The first combination yields sequential assignments based on the measurement of¹³ C chemical shifts and provides a complete ¹H, ¹³C and ¹⁵N resonance assignment of polypeptide backbone and CH_n moieties. When employing the second combination, ¹³C=O chemical shifts are not measured, but the sequential assignment relies on both ¹³C and¹ H chemical shifts. The assignment is performed in a semi-automatic fashion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile mutual interconversion of single-quantum and zero/double-quantum frequencies detected in conventional and reduced-dimensionality spectra, respectively. In particular, SPSCAN comprises a peak picking routine tailored to cope with the distinct peak patterns of projected NMR experiments performed with simultaneous acquisition of central peaks. Data were acquired at 13 °C for the N-terminal 63-residue polypeptide fragment of the 434 repressor. Analysis of these spectra, which are representative for proteins of about 15 kDa when working at commonly used temperatures around 30 °C , demonstrates the efficiency of our approach for the assignment of medium-sized¹⁵ N/¹³C doubly labeled proteins.     

Linkage relationships and allelic associations of the cystic fibrosis locus and four marker loci

Summary The linkage relationships between the cystic fibrosis (CF) locus and four marker loci (MET-H, MET-D, D7S8 and D7S16), allelic associations between these loci and the extent of informativity at these marker loci were investigated in a sample of 206 families with at least one child affected by CF. The data were contributed by 11 laboratories from Europe and Israel. The maximum lod scores and recombination frequency estimates ( ) (and confidence limits of ) were: 18.3 at =0.007(0.001–0.038) for CF vs. MET, 11.0 at (0.001–0.068) for CF vs. D7S8, and 5.7 at =0.0(0.0–0.064) for CF vs. D7S16. A gene order of CF-MET-D7S8 was best supported by the data, but its preference to the order D7S8-CF-MET is mainly based on one single family. There are significant allelic associations between CF, MET, D7S8 and D7S16; these allelic associations affect the risk of random individuals to be carriers of CF.     

Aboveground biomass and root/shoot ratio regulated drought susceptibility of ecosystem carbon exchange in a meadow steppe

Background and aims

Nitrogen deposition and altered precipitation regime are likely to change plant growth, biomass allocation and community structure, which may influence susceptibility of ecosystem functions (i.e. ecosystem carbon exchange) to extreme climatic events, such as drought.

Methods

In a meadow steppe, we deployed a drought treatment on a long-term water and nitrogen addition experiment to investigate resource abundance changes induced variation in the sensitivity of ecosystem carbon exchange to extreme drought.

Results

Compared to the control plots, long-term water and nitrogen addition caused a strong increase in biomass, and a reduction in diversity and root/shoot ratio. Net ecosystem CO₂ exchange (NEE) in water and nitrogen addition plots were more sensitive to drought stress than the control plots. The enhanced NEE drought sensitivity (S_NEE) in nitrogen fertilization habitat is associated with changes in aboveground biomass and root/shoot ratio, rather than variation in species diversity, while S_NEE in the unfertilized plots was controlled by root/shoot ratio. Compared to the water and nitrogen addition plots, the control plots had the highest percentage recovery of ecosystem carbon exchange (R_NEE) during the rehydration period. R_NEE is likely determined by aboveground biomass and level of damage in the photosynthetic organ.

Conclusion

These findings suggest that long-term changes in precipitation regimes and nitrogen deposition may significant alter the susceptibility of key ecosystem processes to drought stress.

     

Cell Counting and Viability Assessment of 2D and 3D Cell Cultures: Expected Reliability of the Trypan Blue Assay

Background

Whatever the target of an experiment in cell biology, cell counting and viability assessment are always computed. The Trypan Blue (TB) assay was proposed about a century ago and is still the most widely used method to perform cell viability analysis. Furthermore, the combined use of TB with a haemocytometer is also considered the standard approach to estimate cell population density. There are numerous research articles reporting the use of TB assays to compute cell number and viability of 2D and 3D cultures. However, the literature still lacks studies regarding the reliability of the TB assay in terms of assessment of its repeatability and reproducibility.

Methods

We compared the TB assay's measurements obtained by two biologists who analysed 105 different samples in double-blind for a total of 210 counts performed. We measured: (a) the repeatability of the count performed by the same operator; (b) the reproducibility of counts performed by the two operators.

Results

There were no significant differences in the results obtained with 2D and 3D cell cultures: we estimated an approximate variability of 5% when the TB assay was used to assess the viability of the culture, and a variability of around 20% when it was used to determine the cell population density.

Conclusions

The main aim of this study was to make researchers aware of potential measurement errors when TB is used with a haemocytometer for counting and viability measurements in 2D and 3D cultures. We believe that these results can help researchers to determine whether the expected reliability of the TB assay is compliant with their applications.

     

A new technique for continuous measurement of the heat of fermentation

Summary A special temperature control system has been developed and applied to continuous measuring of the heat evolved during a fermentation process. In this system, the fermentation broth was overcooled by a given constant cooling water flow. The excess heat removed from the fermentor was then made up by an immersion electrical heater. The action of the temperature controller was precisely monitored as it varied in response to the amount of heat produced by the microbial activities.The technique was used for determining the heat evolution byEscherichia coli grown on glucose. The ratio between quantities of total heat release and total oxygen consumption has been determined to be 0.556 MJ/mol O₂.The newly developed technique can be employed as an online sensor to monitor the microbial activities of either aerobic or anaerobic fermentation systems.Symbols C_c Heat capacity of cooling water (MJ/kg · °C) - C_p Heat capacity (MJ/kg · °C) - I Current of immersion heater (A) - K Constant in Equation (2) (h) - K Constant in Equation (13) (m³ · h · °C/MJ) - Q_c Flow rate of cooling water (m³/h) - Heat of agitation (MJ/m³ · h) - Heat dissipated by the bubbling gas (MJ/m³ · h) - Heat removal by the action of controller (MJ/m³ · h) - Heat of fermentation (MJ/m³ · h) - Heat loss to the surroundings (MJ/m³ · h) - Q_pass Constant average power dissipated by the immersion heater (MJ/m³ · h) - Fluctuating power dissipated by the immersion heater (MJ/m³ · h) - Power dissipated by the immersion heater (MJ/m³ · h) - T Temperature of fermentation broth (°C) - Constant average temperature of fermentation broth (°C) - Fluctuating temperature of fermentation broth (°C) - T_a Temperature of the ambient air (°C) - T_c Inlet temperature of cooling water (°C) - U₁A₁ Specific heat transfer coefficient for determination of heat loss to the surroundings (MJ/m³ · h · °C) - U₂A₂ Specific heat transfer coefficient for cooling surfaces (MJ/m³ · h · °C) - U₃A₃ Constant in Equation (16) (MJ/m³ · h · °C) - V Voltage of immersion heater (V) - V_L Liquid volume (m³) - OUR Oxygen uptake rate (mol O₂/m³ · h) Greek Letters H_fo The ratio between the total heat release and the total oxygen uptake (MJ/mol O₂) - _c Density of cooling water (kg/m³) - Time constant defined in Equation (6) (h) - _iM_iC_pi Heat capacity of system components (fermentation broth + fermentor jar + stainless steel) (MJ/m³ · °C)     

The role of dopamine D2 receptors in the amygdala in metabolic and behavioral responses to stress in male Swiss-Webster mice

Objective

The D2 dopamine receptor is found in different parts of the amygdala. However, its contribution to stress is unknown. Thus, in the present study, we examined the effects of excitation and inhibition of D2 dopamine receptors in the amygdala on the metabolic and hormonal changes in response to stress.

Methods

Bilateral amygdala cannulation was carried out in Swiss-Webster mice (n = 7). On recovery, different doses of the dopamine D2 receptor antagonist, sulpiride (1, 5 and 10 μg/mouse) or the dopamine D2 receptor agonist, bromocriptine (1, 5 and 10 μg/mouse) were injected into the amygdala. The animals were then placed in stress apparatus (communication box) where they received an electric shock (10 mV voltage, 10 Hz frequency and 60 s duration) after 30 min. The animal's activities were recorded for 10 min before and 10 min after the stress induction. Locomotion, rearing and freezing were investigated. Metabolic changes, such as food and water intake and anorexia, were studied.

Results

The results show that stress increased the concentration of plasma corticosterone, which was followed by a decrease in locomotion and rearing and an increase in freezing behavior. Furthermore, both weight and water and food intake were reduced. Administration of bromocriptine led to a reduction of corticosterone at doses of 1 and 5 μg/mouse and an increase of corticosterone at 10 μg/mouse. Additionally, lower doses of bromocriptine (1 and 5 μg/mouse) caused an increase in locomotion and rearing and a decrease in freezing behavior. Similar results were observed with sulpiride injection.

Conclusion

D2 dopamine receptors can play a major role in the amygdala in stress. Both an agonist and an antagonist of the D2 receptor attenuate the metabolic and hormonal changes observed in response to stress

     

Improved sensitivity and coherence selection for [15N,1H]-TROSY elements in triple resonance experiments

In experiments with proteins of molecular weights around 100 kDa the implementation of [¹⁵N,¹H]-TROSY-elements in [¹⁵N]-constant-time triple resonance experiments yields sensitivity enhancements of one to two orders of magnitude. An additional gain of 10 to 20% may be obtained with the use of sensitivity enhancement elements. This paper describes a novel sensitivity enhancement scheme which is based on concatenation of the ¹³ C ¹⁵N magnetization transfer with the ST2-PT element, and which enables proper TROSY selection of the ¹⁵N multiplet components.     

Inter- and intra-population differences in the effects of temperature on postdiapause development of Delia radicum

Canadian populations of D. radicum differ in their response to temperature during postdiapause development. Populations that are primarily of the early-emerging type ( ) (St-Jean, Quebec; London, Ontario) have high values for the parameters describing this response: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaamOuamaaBa% aaleaacaWGTbaabeaakiabg2da9aaa!38F2!$$R_m = $$ 12.7–13.3; 28.0–31.8 °C ; T = 10.3–14.2 ( , the maximum developmental rate at the temperature, [ °C ] where the developmental rate is highest, and T , the parameter which gives the shape of the truncated normal curve fitted to the data), a low degree-day requirement for emergence (160–232 ), and may lack a developmental delay at temperatures above ca. 21 °C . Populations of the late-emerging type (Kildare, Prince Edward Island) have low parameter values ( , °C ; T = 6.4), high degree-day requirements (530 ), and a developmental delay at high temperatures. The parameters for the early-emergers in the population from Winnipeg, Manitoba (74% early) were intermediate ( , °C , T = 10.7, ), but resembled the early rather than the late type. This population varied from 31 to 90% early type over a 10-year period and the rate of postdiapause development at 20 °C was directly related to the percentage early. In the year with the most rapid development (90% early), development was significantly slower than in the populations from other locations with predominantly early populations, and the year with the slowest development (31% early) showed significantly faster development than that from Kildare, Prince Edward Island (100% late). Therefore the parameters for early and late types of development will not be accurate for use in mixed populations, and the parameters in mixed populations will change among years. Populations of D. radicum in North America and Europe (67 locations by years) varied from 0–100% early. At Winnipeg, the percentage early was directly related to the annual temperature accumulation ( ) during the growing season. The calculation of developmental parameters for the early-emergers of mixed populations provides a more accurate basis for estimating the times of first emergence and the first peak of emergence than parameters based on the whole population. Since postdiapause developmental rates vary both among and annually within locations, developmental models should be designed to include such variations.     

Circadian variation of insensible perspiration in man

Body weight loss was measured continuously for 24 hours in lightly clothed subjects at a room temperature of 26°C and relative humidity of 60% using the bed scale (model 33B, James Addison Potter). One male, aged 38 years and four females, aged 22 years served as subjects. Skin temperatures of the chest, thigh, leg and arm were measured every ten min. and mean skin temperature was calculated. Water loss through the skin was estimated by deducting body weight loss due to respiratory gas exchange and respiratory water loss from total weight loss. Respiratory water loss was estimated by using the formula (44-P_a) where _e is the rate of evaporative water loss in the expired air (g/min), the oxygen uptake (L/min STPD) and P_a is the ambient water vapor pressure. Insensible water loss through skin was found to be significantly lower between 2:00–5:00 h than in the daytime. These findings contradict the results that the value of insensible water loss was higher at night than during the day.     

Allozyme variation among and within eleven Orchis species (fam. Orchidaceae), with special reference to hybridizing aptitude

The electrophoretic variability of nine enzymatic loci was studied in 31 populations of eleven species of the genus Orchis (fam. Orchidaceae). This orchid group shows a higher degree of genetic variability ( ) than reported by Gottlieb (Progr. Phytochem. 7:1–46, 1981) for other outbreeding plant species ( ).Furthermore, the Nei coefficients of genetic identity show a higher biochemical-genetic similarity between species pairs of which we found the hybrids ( ) than between other species pairs ( ). These results seem to indicate a correlation between hybridizing aptitude and degree of genetic similarity. High values could be partially attributed to the presence of a gene flow between two species which, by hybridizing, would join their genetic pools. But they could also indicate that hybridizing species are not well differentiated: the isozyme similarity would reflect a more general similarity of the gene pool that could lead to hybrid formation.     

Gas exchange in the incubation mounds of megapode birds

Summary The brush turkey (Alectura lathami) and mallee fowl (Leipoa ocellata) are megapode birds that incubate their eggs by burying them in mounds. Respiratory gas exchange between the buried eggs and the atmosphere occurs mainly by diffusion through about 60 cm of decomposing forest litter (brush turkey) or sand (mallee fowl).Gas fluxes in the brush turkey mound are greatly influenced by the respiration of thermophilic microorganisms which consume O₂ at rates over eight times that of all of the eggs. The respiratory exchange ratio ( ) of the microorganisms is 0.75 and theQ ₁₀ for metabolism is 2.56. Fermentation and nitrogen fixation do not occur in the mounds.If the mound becomes too wet, gas tensions near the eggs can become critical because water increases rates of microbial respiration and impedes gas diffusion. However, field mounds are relatively dry, possibly because the adult bird modifies the shape of the mound and affects the entry of rain water. At egg level in field mounds, and are about 132 and 21 Torr, respectively, in both species. Embryonic respiration decreases and increases about 5 Torr in the immediate environment of individual eggs in late development. Due to a high eggshell gas conductance, which increases during incubation, the gas tensions within the shell of late embryos ( ca. 108 Torr, ca. 47 Torr) are not far from the mean values found in species that nest above ground.     

Activity and oxygen consumption during hypoxic exposure in high altitude and lowland sceloporine lizards

Summary Resting rates of O₂ consumption against , exercise endurance times and during recovery from vigorous exercise were measured inSceloporus occidentalis captured near sea level and inS. graciosus captured above 2850 m. Oxygen consumption against was also measured inS. occidentalis captured above 2850 m. When was recorded continuously, as ambient was slowly reduced from 155 Torr, it became directly dependent upon ambient between 110 and 120 Torr. The critical for the high altitude lizards was lower than that for the lowland lizards, which enabled the former to maintain relatively higher 's when ambient was reduced below 120 Torr. The high altitude lizards also had significantly greater endurance when stimulated to exercise at 1600 m ( 130 Torr). Both the higher under hypoxia and the greater endurance roughly parallel a significantly greater maximum in the high altitude lizards. At a simulated altitude of 3600 m ( 100 Torr), maximum and rate of recovery of the O₂ debt calculated from post active were significantly reduced in the lowland but not the high altitude lizards. The effects of simulated altitude conditions on the lowland but not the mountaine animals indicate adaptations to altitude in these sceloporine lizards. We did not find any consistent relationship between organ/body weight ratios or hematocrit and our measures of endurance or the altitude at which the lizards were captured.     

Optimal vitamin D spurs serotonin: 1,25-dihydroxyvitamin D represses serotonin reuptake transport (<Emphasis Type="Italic">SERT</Emphasis>) and degradation (<Emphasis Type="Italic">MAO-A</Emphasis>) gene expression in cultured rat serotonergic neuronal cell lines

Background

Diminished brain levels of two neurohormones, 5-hydroxytryptamine (5-HT; serotonin) and 1,25-dihydroxyvitamin D₃ (1,25D; active vitamin D metabolite), are proposed to play a role in the atypical social behaviors associated with psychological conditions including autism spectrum disorders and depression. We reported previously that 1,25D induces expression of tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway to 5-HT, in cultured rat serotonergic neuronal cells. However, other enzymes and transporters in the pathway of tryptophan metabolism had yet to be examined with respect to the actions of vitamin D. Herein, we probed the response of neuronal cells to 1,25D by quantifying mRNA expression of serotonin synthesis isozymes, TPH1 and TPH2, as well as expression of the serotonin reuptake transporter (SERT), and the enzyme responsible for serotonin catabolism, monoamine oxidase-A (MAO-A). We also assessed the direct production of serotonin in cell culture in response to 1,25D.

Results

Employing quantitative real-time PCR, we demonstrate that TPH-1/-2 mRNAs are 28- to 33-fold induced by 10 nM 1,25D treatment of cultured rat serotonergic neuronal cells (RN46A-B14), and the enhancement of TPH2 mRNA by 1,25D is dependent on the degree of neuron-like character of the cells. In contrast, examination of SERT, the gene product of which is a target for the SSRI-class of antidepressants, and MAO-A, which encodes the predominant catabolic enzyme in the serotonin pathway, reveals that their mRNAs are 51–59% repressed by 10 nM 1,25D treatment of RN46A-B14 cells. Finally, serotonin concentrations are significantly enhanced (2.9-fold) by 10 nM 1,25D in this system.

Conclusions

These results are consistent with the concept that vitamin D maintains extracellular fluid serotonin concentrations in the brain, thereby offering an explanation for how vitamin D could influence the trajectory and development of neuropsychiatric disorders. Given the profile of gene regulation in cultured RN46A-B14 serotonergic neurons, we conclude that 1,25D acts not only to induce serotonin synthesis, but also functions at an indirect, molecular-genomic stage to mimic SSRIs and MAO inhibitors, likely elevating serotonin in the CNS. These data suggest that optimal vitamin D status may contribute to improving behavioral pathophysiologies resulting from dysregulation of serotonergic neurotransmission.

     

A physiologically-based flow network model for hepatic drug elimination III: 2D/3D DLA lobule models

Background

One of the major issues in current pharmaceutical development is potential hepatotoxicity and drug-induced liver damage. This is due to the unique metabolic processes performed in the liver to prevent accumulation of a wide range of chemicals in the blood. Recently, we developed a physiologically-based lattice model to address the transport and metabolism of drugs in the liver lobule (liver functional unit).

Method

In this paper, we extend our idealized model to consider structural and spatial variability in two and three dimensions. We introduce a hexagonal-based model with one input (portal vein) and six outputs (hepatic veins) to represent a typical liver lobule. To capture even more realistic structures, we implement a novel sequential diffusion-limited aggregation (DLA) method to construct a morphological sinusoid network in the lobule. A 3D model constructed with stacks of multiple 2D sinusoid realizations is explored to study the effects of 3D structural variations. The role of liver zonation on drug metabolism in the lobule is also addressed, based on flow-based predicted steady-state O₂ profiles used as a zonation indicator.

Results

With this model, we analyze predicted drug concentration levels observed exiting the lobule with their detailed distribution inside the lobule, and compare with our earlier idealized models. In 2D, due to randomness of the sinusoidal structure, individual hepatic veins respond differently (i.e. at different times) to injected drug. In 3D, however, the variation of response to the injected drug is observed to be less extreme. Also, the production curves show more diffusive behavior in 3D than in 2D.

Conclusion

Although, the individual producing ports respond differently, the average lobule production summed over all hepatic veins is more diffuse. Thus the net effect of all these variations makes the overall response smoother. We also show that, in 3D, the effect of zonation on drug production characteristics appears quite small. Our new biophysical structural analysis of a physiologically-based 3D lobule can therefore form the basis for a quantitative assessment of liver function and performance both in health and disease

     

High-resolution 3D HNCOCA experiment applied to a 28 kDa paramagnetic protein

Summary A new triple-resonance 3D HNCOCA pulse scheme is presented, designed to identify the backbone nuclei (H^N, N, CO, C) of doubly labelled proteins. The two carbon frequencies are labelled along the same indirect dimension and the corresponding dwell times can be independently scaled in order to account for the relaxation properties and chemical shift ranges of the CO and C. If one takes advantage of the symmetry properties of the spectra in the course of the peak picking, this 3D scheme has the same sensitivity as the 4D experiment, but with an improved resolution. The sequence is illustrated on a 0.5 mM sample of Rhodobacter capsulatus cytochrome c a homodimeric paramagnetic protein of 2×14 kDa. A resonance assignment strategy, based on a low-concentration ¹³C/¹⁵N-labelled sample and a more concentrated ¹⁵N-labelled sample, is proposed for proteins where the expression system shows a limited efficiency.