Measurement of pulsatile flows and variable volumes by bolus injection of indicator in a model of the pulmonary circulation

The reliability and accuracy of the bolus injection-dye dilution technique were assessed for a physiological range of frequencies (13-49 min-1) and stroke volumes (60-160 ml) on a glass model where flows and volumes varied as a preset function of time (n = 320). We found that the technique overestimates flow by about 8% with a 95% confidence interval of +/- 10% for one measurement. Mean transit times are accurate within a +/- 7% confidence interval for one measurement. In a time-dependent flow and volume system this technique measures the mean volume as related to time with fluctuations up to +/- 30% around the mean. Results are independent of time and site of injection. The double injection-single sampling technique gives results that are equivalent to those obtained by single injection and sampling of dye.     

Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laevis

Maturation-promoting factor (MPF) was purified 20- to 30-fold from unfertilized eggs of Xenopus laevis, by ammonium sulfate precipitation and chromatography on pentyl-agarose and arginine-agarose. The final material induces maturation in 50% of the recipient oocytes when 5 ng of protein is injected in a volume of 20 ml. The maturation response includes precocious germinal vesical breakdown, elevated protein phosphorylation, amplification of cytoplasmic MPF, and formation of an activatable egg blocked at second meiotic metaphase. These eggs are capable of cleavage and, in some cases, of gastrulation. A quantitative in vivo assay of MPF is described and a unit of MPF activity is defined as that amount causing a 50% maturation frequency when oocytes are injected each with a 20-nl test volume. Maturation frequency has a very high-order dependence on MPF concentration. The purification procedure selects simultaneously for endogenous protein phosphorylation systems containing kinases, protein substrates, and phosphatases. This fact, as well as the finding that ATP enhances MPF activity at least twofold when included in the dilution medium for assay, is discussed in terms of the possible involvement of protein phosphorylation in MPF activation and inactivation.     

Reversed-phase high-performance liquid chromatography procedure for the simultaneous determination of S-adenosyl-l-methionine and S-adenosyl-l-homocysteine in mouse liver and the effect of methionine on their concentrations

An improved reversed-phase high-performance liquid chromatography (HPLC) procedure with ultraviolet detection is described for the simultaneous determination of S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH) in mouse tissue. The method provides rapid resolution of both compounds in a 25-μl perchloric acid extract of the tissue. The limits of detection in 25-μl injection volumes were 22 and 20 pmol for SAM and SAH, respectively. The limits of quantitation in 25-μl injection volumes were 55 and 50 pmol for SAM and SAH, respectively, with recovery consistently >98%. The assay was validated over linear ranges of 55–11 000 pmol for SAM and 50–10 000 pmol for SAH. The intra-day precision and accuracy were ≤6.4% relative standard deviation (RSD) and 99.9–100.0% for SAH and ≤6.7% RSD and 100.0–100.1% for SAM. The inter-day precision and accuracy were ≤5.9% RSD and 99.9–100.6% for SAH and ≤7.0% RSD and 99.5–100.1% for SAM. Compared to earlier procedures, the HPLC method demonstrated significantly better separation, detection limit and linear range for SAM and SAH determination. The assay demonstrated applicability to monitoring in mice the time-course of the effect of methionine on SAM and SAH levels in the liver. Administering methionine to mice increased by 10-fold the liver concentration of SAM and SAH within 2 h, which then rapidly decreased to the control levels by 8 h. This indicated that methionine was promptly converted to SAM and then rapidly catabolized into SAH. Thus, the metabolism of methionine to SAM should be considered in the supplementation of methionine to maintain SAM levels in the body.     

Sperm penetration in vitro into ovarian and tubal oocytes from mice of the inbred KE and C57 strains

The method of in vitro fertilization was applied to test a previous suggestion that the lowered fertilizability of the tubal oocytes of female KE strain mice and the high resistance of their zona pellucida to proteolytic enzymes, are due to the premature cortical reaction taking place near the time of ovulation. Therefore higher fertilizability of ovarian oocytes is expected. The effectiveness of F₁ hybrid sperm penetration into ovarian and tubal KE oocytes confirmed these assumptions. The ovarian KE oocytes recovered 9–10 hours after administration of human chorionic gonadotropin (HCG) showed significantly higher penetrability (70–83%) than did the tubal oocytes recovered 12 hours after HCG (about 50%) and 14–16 hours after HCG (20%). Similar results were obtained with C57 oocytes. Sperm penetration into ovarian oocytes (10 hours after HCG) was much more effective (67%) than into tubal oocytes (18%); this finding correlated with more rapid zona dissolution by chymotrypsin. On the basis of these results one might speculate that premature cortical reaction takes place also in the C57 strain.     

Ionic Osmoregulation during Salt Adaptation of the Cyanobacterium Synechococcus 6311

The mechanisms of salt adaptation were studied in the cyanobacterium Synechococcus 6311. Intracellular volumes and ion concentrations were measured before and after abrupt increases of external NaCl concentrations up to 0.6 molar NaCl. Equilibrium volumes, measured with a rapid and accurate electron spin resonance spin probe method, showed that at low NaCl concentrations the cells did not shrink as expected for an impermeable solute. However, when the NaCl concentration exceeded a critical value, volume losses occurred. These losses were not fully reversed by hypoosmotic treatment, suggesting membrane damage. The critical value of irreversible volume loss paralleled the increase in salinity during cell growth. Rapid mixing experiments showed that exposure of Synechococcus 6311 to non-damaging NaCl concentrations caused water extrusion from the cells; the volume decreases were time resolved to about 200 milliseconds. Subsequently, volumes increased rapidly as NaCl moved into the cells. Controls recovered their volumes within 15 seconds, while salt-adapted cells grown at 0.6 molar NaCl required 1 minute for volume equilibration. This decrease in the rate of cell volume recovery indicates that salt adaptation is accompanied by changes in cell membrane properties. Subsequent to these initial rapid volume changes, a more gradual sequence of ion movement and sugar accumulation was observed. Under conditions for photoautotrophic growth, significant Na⁺ extrusion was observed 30 min after salt shock. Sucrose accumulation reached a maximum value after 16 hours and K⁺ accumulation reached equilibrium after 40 hours. The final concentrations of K⁺ and Na⁺ and sucrose and glucose inside the 0.6 molar NaCl-grown cells indicate that the inorganic ions and organic `compatible' solutes are the major osmotic species which account for the adaptation of Synechococcus 6311 to salt.     

Loss of habitats,naturalness and species diversity in Eurasian forest landscapes

Man has exploited land and forests in Western and Central Europe longer and more intensively than in Northern Europe and further east in Eurasia. We estimated forest naturalness and modelled expected biodiversity loss in seven different landscapes (2500 km² each) in the Netherlands, Sweden, Finland, Poland, St. Petersburg (Western European Russia), Perm (Eastern European Russia), and Irkutsk (Central Siberia) across the distribution of Pinus sylvestris L. in Eurasia. Field inventories showed that the mean living tree volumes were relatively similar in the studied sites, but the volumes of dead wood differed greatly. In Irkutsk and Perm the volume of dead trees per ha was about 5–10 times larger than in Central and Western European regions. The studied forests were generally young in all regions except for Irkutsk, where about half of the study plots had trees older than 120 years. Signs of recent forest fires were found almost exclusively on Russian sites. According to Landsat satellite image-based land-cover classifications the amount of remaining forest habitat in the studied landscapes varied from 25% in the Netherlands to 93% in Irkutsk. Estimated by forest patch size and density of cut stumps, forests were also more fragmented and heavily managed in the western study landscapes compared to eastern ones. Based on species–area relationship functions, we calculated that the proportion of forest-dwelling species already extinct or expected to become extinct due to habitat loss ranges from 1–2% in Irkutsk to 13–24% in the Netherlands study landscape. For saproxylic species, which depend on dead wood, the extinction estimates were calculated based on remaining dead wood volume in the landscape. The modelled expected loss of saproxylic species ranged from 7–14% in Irkutsk to 35–58% in the Netherlands.     

Ovulated oocytes collected from PMSG/hCG-treated and cycling Djungarian or Siberian hamsters (Phodopus sungorus) are structurally similar with no evidence of polar body formation,indicating arrest in meiosis I

This study was undertaken (1) to devise a method of inducing multiple follicular development and subsequent ovulation in the Djungarian or Siberian hamster (Phodopus sungorus) and (2) to assess the quality of ovulated oocytes collected from PMSG/hCG treated animals in comparison to naturally ovulating animals. Hamsters (4–5 weeks; n = 70) received 5 IU PMSG followed 50–52 hr later by 10 IU hCG. Ovulated oocytes were collected 14–20 hr after hCG injection. Ovulated oocytes were flushed from oviducts of cycling animals (7–12 weeks; n = 30) exhibiting two consecutive estrous cycles. Oocytes were fixed and subjected to triple fluorescence immunostaining using anti-tubulin antibodies, fluorescein phalloidin, and Hoechst 33258. The mean number of ovulated oocytes collected from cycling animals was 4.8 ± 0.4 (range 1–7). Ovulation occurred in 73% of the PMSG/hCG-stimulated animals. The mean number of oocytes ovulated from stimulated animals was 9.2 ± 0.8 (range 0–22). The ovaries of animals that did not ovulate or that ovulated few oocytes did respond to PMSG, as indicated by the presence of multiple follicular development and pre-ovulatory stigmata. There was no evidence of a polar body in ovulated oocytes collected from PMSG/hCG-treated or cycling animals, indicating that oocytes were arrested in meiosis I. In the majority (80%) of ovulated oocytes from PMSG/hCG-treated and cycling animals, cortically placed chromosomes were aligned on a metaphase plate equidistant from a bipolar spindle. Sparse f-actin staining was observed in the region of the ooplasm surrounding the chromosomes. As the interval between hCG injection and the time of collection increased, chromosomes lost their proper alignment and migrated away from the cortex of the oocyte concomitant with a disruption of spindle integrity. This collapse of proper chromosome alignment and disruption of spindle architecture also characterized aging oocytes collected from cycling animals. These data show that in the Djungarian or Siberian hamster (Phodopus sungorus), (1) there is individual animal variation in responsiveness to hCG following PMSG treatment, (2) there are no cytological differences in the quality of oocytes collected from hormonally treated animals when compared to cycling animals, and (3) oocytes are ovulated in meiosis I. © 1995 Wiley-Liss, Inc.     

The volumes of anatomical components in leaves oftypha angustifolia L. andtypha latifolia L.

The volumes of tissues in leaves of two species ofTypha with different habitus were measured. It was found that air cavities occupy more than 50%, parenchyma about 20% and photosynthetic tissue about 10% of the total volume of the leaf. If the cavities are omitted, parenehyma amounts to approximately 50% and photosynthetic tissue to little more than 20% of the total volume of tissues. The relative volumes of tissues vary continuously from the base to the tip of the leaf. The differences between the two species studied were not large. Besides the relative volumes, the real ones of leaves and tissues were calculated.     

Studies on the appearance and nature of a maturation-inducing factor in the cytoplasm of amphibian oocytes exposed to progesterone

Full-grown oocytes of amphibians respond in vitro to exogenous progesterone by undergoing physiological maturation (breakdown of the germinal vesicle (GVBD), meiosis, and acquisition of the capacity for activation). Both cytoplasm and “cytosol” from maturing oocytes have been shown to produce similar events when injected into unstimulated oocytes. This activity appeared within 4 hr after hormone treatment in Rana pipiens and Xenopus laevis and represents the earliest detectable, specific response of the oocyte yet observed, i.e., 6–8 hr before GVBD in Rana. Maturing oocytes retained activity as long as 100 hr after exposure to progesterone, and activity was also obtained from ovulated eggs and cleaving embryos. In addition, cytoplasm from Rana pipiens, Xenopus laevis, or Ambystoma mexicanum was effective in inducing maturation in oocytes of each other, indicating a lack of specificity.Recipient oocytes of Xenopus laevis consistently began to mature within 1.5–3 hr after injection of maturing cytoplasm, well before progesterone-treated controls. The timing of the response was closely related to the quantity of cytoplasm transferred, suggesting the presence of both a minimum and threshold level of cytoplasmic factor. Serial cytoplasmic transfer in Xenopus oocytes showed no significant loss of activity through 10 injections.     

酶电极流动注射分析法测定葡萄糖

 一种酶电极流动注射分析系统(EFIA)用于血糖和发酵葡萄糖的快速测定。研究了酶电极及其工作系统的性能和各种影响参数,,奠定了实用化基础。     

Stem cells do not play a significant role in repopulation of adult human cardiomyocytes

Currently, there are two points of view on the ability of adult human heart to regenerate. One of them holds that the myocardium has a poor ability to regenerate. According to the other, the myocardium can rapidly regenerate due to the presence of resident stem cells in it. The purpose of this study was to test these hypotheses by investigating the distribution of cardiomyocytes by size and ploidy in human beings of different age. Using cytofluorometry and interferometry, we determined the dry weight, volume, and ploidy of myocytes isolated from the left ventricle of a normal heart of 12 men at the age of 20–30 (n = 7) and 40–50 (n = 5) years. The mean dry weight of cardiomyocytes was 6906 ± 182 pg (10^–12 g) in the 20- to 30-yearold men and 9126 ± 263 pg in 40- to 50-year-old men; the myocyte volume was 55250 ± 1457 and 73005 ± 2106 µm³, respectively. Cells with volumes intermediate between the cells at the stage of “dividing myocytes” and mature myocytes were absent. The number of cardiomyocytes in the left ventricle was (3.18 ± 0.05) × 10⁹ in the 20–30-year-old age group and (2.06 ± 0.6) × 10⁹ in the 40–50-year-old group. The largest subset (41.3%) of the myocyte population was represented by mononuclear cells with tetraploid nuclei. The proportion of myocytes of different ploidy classes and their mean ploidy did not change in the range of 20–50 years. On the basis on these data, we concluded that stem cells do not play a significant role in restoring the number of lost myocytes. Hypertrophy of myocytes caused by the increase in their cytoplasm is the main mechanism of compensation of the function of the left ventricle of the heart in aging human beings.     

Genetic diversity analysis and population structure in apricot (Prunus armeniaca L.) grown under north-western himalayas using ISSR markers

Investigation of genetic variability and population relationship of 50 accessions of the apricot (Prunus armeniaca L.) was carried out using ISSR markers. The results revealed that the number of alleles per locus varied from 4 to 8 with a mean value of 6.75, and the mean effective number of alleles (Ne) per locus was 1.54. Similarly, the polymorphic information content (PIC) values ranged from 0.464 to 0.424, with a mean value of 0.424. The mean heterozygosity, marker index, resolving power, and effective multiplex ratio (EMR) ranged from 0.001 to 0.002, 0.01–0.06, 1.76–3.84, and 1–4.12. The dendrogram clustered genotypes into two main clades based on their origins. The population structure revealed two sub-populations with some admixtures. The average expected heterozygosity and population differentiation within two sub-populations was 0.1428 and 0.216, respectively. The results outcome reveals that the four ISSR markers comprehensively separated the indigenous germplasm from the exotic germplasm. The genetic divergence within indigenous genotypes and exotic genotypes could allow for future insights into apricot breeding programs.     

Plasma membrane water permeabilities of human oocytes: the temperature dependence of water movement in individual cells.

Membrane water permeability values were measured in individual fresh human pre-ovulatory oocytes using real time microscopy in a microscope diffusion chamber. The cells were exposed to anisosmotic conditions, their volume responses measured, and from these data the Lp values were computed employing the Kedem-Katchalsky analyses of irreversible thermodynamics. Lp values were measured at four temperatures for each oocyte between 37 degrees C and 10 degrees C, and the temperature-related Arrhenius activation energy (Ea) calculated. It was apparent that individual oocytes exhibited a wide range of Lp values; at 37 degrees C Lp values ranged between 0.33 and 1.80 microns/atm/min. However, each oocyte exhibited the expected inverse linear correlation between Lp and temperature, with high linear correlations (R2 values between 0.73 and 0.96). A mean value for Ea of 8.61 +/- 5.11 Kcal/mol was computed. It is apparent that pre-ovulatory human oocytes express a range of biological diversity in terms of membrane water transport, and this fact needs to be considered when attempting to formulate cryopreservation protocols for storage of these oocytes.     

Relations of Bromine,Iron, Rubidium,Strontium, and Zinc Content to Morphometric Parameters in Pediatric and Nonhyperplastic Young Adult Prostate Glands

The variation with age of the Br, Fe, Rb, Sr, and Zn mass fractions and some histological characteristics of intact prostate glands of 50 subjects aged 0–30 years was investigated by an energy-dispersive X-ray fluorescence and a quantitative morphometric analysis. Mean values?±?standard error of the mean (M?±?SΕΜ) for the mass fractions (in milligrams per kilogram wet-mass basis) of these trace elements in pre-puberty were: Br—10.5?±?1.3, Fe—28.6?±?4.1, Rb—3.05?±?0.27, Sr—0.42?±?0.08, and Zn—32.9?±?3.2. During puberty and postpuberty, when there is a significant increase in circulating androgens, the mean values were: Br—5.60?±?0.57, Fe—19.3?±?1.6, Rb—3.50?±?0.28, Sr—0.24?±?0.03, and Zn—113?±?10. Mean values (M?±?SΕΜ) of percent volumes (%) of the stroma, epithelium, and lumen in the prostate before puberty were 73.4?±?2.6, 20.4?±?1.7, and 4.45?±?0.94, respectively, versus 46.5?±?2.5, 38.5?±?1.9, and 14.9?±?1.2 during puberty and postpuberty. A significant positive correlation between the prostatic Zn and percent volume of both glandular epithelium (r?=?0.573, p?≤?0.001) and glandular lumen (r?=?0.725, p?≤?0.001) was found. For the first time, it has been demonstrated that the glandular lumen is a main pool of Zn accumulation, and that the stroma is a main pool of Br and Fe accumulation in the normal human prostate, for the age range 14 to 30 years. It was concluded that the Zn binds tightly within the prostatic fluid because the volume of glandular lumen reflects the volume of prostatic fluid.     

Trout coelomic fluid suitability as Goldfish oocyte extender can be determined by a simple turbidity test

Regeneration technologies such as androgenesis, intracytoplasmic sperm injection, and nuclear transfer require that handling conditions do not alter oocyte ability to sustain embryo development. One important parameter in the maintenance of oocyte quality in fish is the possibility to prevent oocytes activation during manipulation. In Cyprinid, such activation is known to be delayed when Salmonid coelomic fluid is used as incubation medium. Coelomic fluid however is a biological fluid whose ability to sustain oocyte quality during in vitro incubation may be variable. The purpose of the present work was to explore this variability using Rainbow Trout (Oncorhynchus mykiss) coelomic fluid (TCF) and Goldfish (Carassius auratus) oocytes, and to set up a test which would reflect TCF suitability for Goldfish oocyte incubation. We showed that different TCF induced very different development rates after oocyte incubation for 30 min at 20 °C: at 24h post fertilization (pf) and at hatching, rates ranged between 35% and 110% of the non-incubated controls. When TCF (1 volume) was mixed with tap water (9 volumes), a precipitate developed whose extent was measured by spectrophotometry. This turbidity test proved to be highly correlated to development rates after Goldfish oocyte incubation in TCF (r² = 0.83 at hatching, n = 150): TCF with the highest turbidity (> 1.5 absorbance unit at 400 nm) were the ones which altered the most the development rates after incubation (less than 50 % at hatching). This easy and rapid turbidity test can therefore be used as a reliable estimator of TCF suitability for Goldfish oocyte incubation and manipulation.     

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.     

Stereological estimation of ovarian oocyte volume, surface area and number: application on mice treated with nandrolone decanoate

Changes in the number and size of oocytes can lead to fertilization problems. The present study aimed to evaluate the number, volume, and surface area of oocytes in healthy as well as nandrolone decanoate-treated (ND) mice using stereological methods. Five control mice received vehicle, and five ND-treated mice received ND. Using the 'isotropic Cavalieri' design', the ovary was sectioned. The volume of the ovary (cortex and medulla) was estimated. The oocytes' volume and surface area were estimated using the invariator. The number of the oocytes was estimated using an optical disector. The volumes of the ovary, cortex, and medulla decreased ~50% in the ND-treated mice. The mean number (coefficient of variation) of preantral, antral, and atretic oocytes in the control ovary were 1,690 (0.29), 2,100 (0.52), and 3,900 (0.2), respectively, which decreased ~54%, ~87%, and ~91%, respectively in the ND-treated animals. The mean volume (coefficient of variation) of the preantral, antral, and atretic oocytes were 86,000 (0.27), 110,000 (0.48), and 27,000 (0.33) μm3, respectively. The mean surface area (coefficient of variation) of the three types of oocytes were 9,000 (0.24), 9,900 (0.28), and 4,700 (0.21) μm2, respectively. These parameters remained unchanged in the ND-treated mice. ND induces reduction in the number of oocytes, but not in the volume or the surface area.     

Fertilization and development in vitro of bovine oocytes following intracytoplasmic injection of heat-dried sperm heads

This study investigated the development of bovine oocytes following intracytoplasmic injection of sperm heads from spermatozoa dried by heating. When sperm suspension was heated in a dry oven at 50, 56, 90, and 120 degrees C, the mean amounts of residual water were about 0.3 g water/g dry weight within 8 h, 6 h, 1.5 h, and 20 min of heating, respectively. Oocyte activation, cleavage of oocytes, and development of cleaved embryos to the morula stage were better in oocytes injected with spermatozoa stored at 25 degrees C for 7-10 days following drying at 50 and 56 degrees C than at 90 and 120 degrees C; however, only a small proportion of oocytes developed to the blastocyst stage. When spermatozoa were dried at 50 degrees C for 16 h, activation, male pronucleus (MPN) formation, cleavage, and development to the morula stage were less good than when spermatozoa were dried for 8 and 10 h and no blastocysts were obtained. The development of oocytes was significantly better when spermatozoa were stored for 7-10 days at 4 degrees C than 25 degrees C after drying at 50 degrees C for 8 h. Longer storage (7 days-12 mo) of heat-dried spermatozoa at 4 degrees C did not affect MPN formation in activated oocytes, but blastocyst development was significantly lower when spermatozoa were stored for 3 mo or more. These results demonstrate that bovine oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop at least to the blastocyst stage.     

The volume of cavities in proteins and virus capsids

An improved algorithm for the calculation of the volume of internal cavities within protein structures and virus capsids as well as the volumes occupied by single amino acid residues were presented. The geometrical approach was based on atomic van der Waals radii. The results obtained with two sets of the radii, those proposed by Pauling and those determined by Tsai et al were compared. The main improvement compared with our previous approach is a more elaborate treatment of the regions at the very boundary of the cavities, which yields a more accurate volume estimate. The cavity volume of a number of Plant Pathogenesis‐Related proteins of class 10 (PR‐10) were reevaluated and the volumes and other geometrical parameters for about 400 capsids of icosahedral viruses were reported. Using the same approach the volumes of amino acid residues in polypeptides as mean values averaged over multiple conformations of the side chain were also estimated. Proteins 2016; 84:1275–1286. © 2016 Wiley Periodicals, Inc.