Nucleation‐based prediction of the protein folding rate and its correlation with the folding nucleus size

The problem of protein self‐organization is in the focus of current molecular biology studies. Although the general principles are understood, many details remain unclear. Specifically, protein folding rates are of interest because they dictate the rate of protein aggregation which underlies many human diseases. Here we offer predictions of protein folding rates and their correlation with folding nucleus sizes. We calculated free energies of the transition state and sizes of folding nuclei for 84 proteins and peptides whose other parameters were measured at the point of thermodynamic equilibrium between their unfolded and native states. We used the dynamic programming method where each residue was considered to be either as folded as in its native state or completely disordered. The calculated and measured folding rates showed a good correlation at the temperature mid‐transition point (the correlation coefficient was 0.75). Also, we pioneered in demonstrating a moderate (‐0.57) correlation coefficient between the calculated sizes of folding nuclei and the folding rates. Predictions made by different methods were compared. The established good correlation between the estimated free energy barrier and the experimentally found folding rate of each studied protein/peptide indicates that our model gives reliable results for the considered data set. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Quantitative protein changes in the hypothalamic neurons of pubertal male rats, castrated neonatally.

The effect of neonatal castration of male rats on the sexual differentiation of the hypothalamus at puberty was studied. Male rats were castrated on days 1, 5 and 7 after birth. Their brains were processed for study on days 83-85. The neurons and cell nuclei of the preoptic area, mediobasal and ventromedial nuclei were assessed for changes in cell and nuclear sizes and dry weight (calculated using interferometric methods). Neonatal castration resulted in size as well as dry weight increase in the neurons of the anterior and mediobasal hypothalamus. The dry weight increased by 34% (P less than 0.001) in the medial preoptic area, by 25% (P less than 0.001) in the arcuate neurons and by 22% (P less than 0.001) in the ventromedial nucleus. The cell nuclei exhibited perceptible weight increase too--in the medial preoptic area 68% (P less than 0.001); 55% in the arcuate neurons (P less than 0.001), and 39% in the ventromedial region. The weight and size increases in neonatally castrated males were equal to those of females of the same age. In rats castrated on day 7, the cell sizes and dry weights of the ventromedial nucleus increased but the cell nuclei exhibited only little change. It is assumed that the changes in the dry weight may be the result of increased synthetic processes in these groups of neurons which are connected with the tonic and cyclic release of gonadotropins. These changes also point to the hypothalamic differentiation shifting to the female type in the absence of the inducing effect of androgens.     

The hypothalamic neurosecretory system of the marine teleost fish, Mugil auratus risso.

The hypothalamic neurosecretory system of the marinefish, Mugil auratus, consists of two nuclei, viz., the nucleus praeopticus and the nucleus laterlis tuberis. Both are paired, and while those of the nucleus praeopticus are vertically arranged as L-shaped bodies, their strand-like counterparts in the nucleus lateralis tuberis extend in an antero-posterior direction. The two constituent bodies of the nucleus praeopticus lie on both sides of the third cerebral ventricle. Each is differentiated into a dorsal pars magnocellularis and a ventral pars parvocellularis. A nervous tract, the hypothalamo-hypophysial tract, extends posteriorly from each body, but it is not until after they penetrate the pituitary gland that they fuse into one structure, the neurohypophysis. Many neurosecretory granules accumulate in the neurohypophysis adjacent to the meta-adenohypophysial region, and fewer scattered granules of varying sizes are also present along the hypothalamo-hypophysial tracts. Inner to these hypothalamo-hypophysial tracts extend the two bodies of the nucleus lateralis tuberis along the infundibular region. Axons from this nucleus extend sideways, and as they merge with those adjacently disposed of the hypothalamo-hypophysial tracts, they enter the pituitary gland as a unified structure.     

The Monte Carlo simulation of the biological effect of the 10B(n, alpha)7Li reaction in cells and tissue and its implication for boron neutron capture therapy

The energy deposition in the nucleus of cells exposed to the 10B(n, alpha)7Li neutron capture reaction has been calculated and compared to the measured biological effect of this reaction. It was found that a considerable distribution of hit sizes to the nucleus occurs. The comparison of hit size frequency with the observed survival indicates that not every hit, independent of its size, can lead to cell death. This implies the existence of a hit size effectiveness function. The analysis shows that the location of boron relative to the radiation-sensitive volume of the cell is of great importance and that average dose values alone are of limited use for predicting the biological effect of this reaction. Boron accumulating in the cell nucleus is much more efficient in cell killing than the same amount of boron uniformly distributed; its presence in one cell, however, has little effect on its neighboring cells in a tissue. When boron is present on the cell surface of a tissue (as presumably delivered by antibodies), its cell-killing effect is greatly reduced compared to that in uniform distribution. However, in this case much of the dose to one cell comes from neutron capture reactions occurring on the surface of its neighbor cells. These data have implications for the choice of boron carries in neutron capture therapy. The mathematical analysis carried out here is similar to that proposed recently for low-level exposure effects of radiation, taking mutation and/or carcinogenesis as biological effects. The results here show that high-level exposure to high-LET particles (resulting in cell killing) should be treated in an analogous manner.     

Changes in volume,surface area,and frequency of nuclear pores on the vegetative nucleus of tobacco pollen in fresh,hydrated, and activated conditions

The vegetative nucleus (VN) of Nicotiana tabacum L. has been qualitatively and quantitatively studied in fresh, hydrated, and activated pollen. Techniques included the use of optical sectioning by confocal scanning laser microscopy to obtain volume and surface area measurements, and stereoscopic pairs; and freeze-etch electron microscopy to estimate the frequency of nuclear pores per m² in the vegetative nucleus. Several morphological changes were observed to occur in pollen grain nuclei during the early processes of tube growth. In freshly dehisced pollen grain, the vegetative and generative nuclei were side by side, but following hydration and activation of the grain, the elongated generative nucleus became partially surrounded by the vegetative nucleus. It was found that during hydration, the surface area of the vegetative nucleus increased and there was a decrease in the frequency of nuclear pores. The calculated total number of pores remained similar. After activation and pollen-tube growth, the vegetative nucleus retained the same surface area as in the hydrated state but the frequency of nuclear pores decreased; therefore, the calculated total number of pores was significantly lowered. When considered alongside complementary biochemical data, these morphological results indicate that RNA production in the vegetative nucleus decreases following germination.Abbreviations VN vegetative nucleus (nuclei) - GN generativenucleus - GC generative cell - CSLM confocal scanning laser microscope We acknowledge research support by the Biotechnology Action Programm of the Commission of European Communities, and CNR for the fellowship awarded to Dr. Wagner. We would also like to thank Mrs. C. Faleri for the expert technical help.     

The structural characteristics of the phrenic nucleus in the cat and their functional significance]

The structural elements of phrenic nucleus have been compared under the horseradish peroxidase introduction into the phrenic muscle and using a silver staining method. All the neurons of nucleus have been proposed to be motoneurons. The characteristic features of phrenic nucleus organisation are created by the groups of motoneurons and their dendritic bundles. Each nucleus contains about 800 motoneurons. Their sizes have been determined in three planes. Some functional characteristics of phrenic nucleus on the basis of its structural architecture are under discussion.     

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.     

Follicular growth and kinetics during the estrous cycle, pregnancy and postpartum in the Indian mole rat (Bandicota bengalensis)

Follicular growth and kinetics were studied in detail in the ovaries of the Indian mole rat (Bandicota bengalensis) during various stages of the estrous cycle; days 7, 12, 15, 19, and 21 of pregnancy; and day 2 postpartum. The sizes of follicles, oocytes, nuclei, and nucleoli were measured. In all rats, regression coefficients, a, and intercepts, b, were calculated in oocyte/follicle, oocyte nucleus/follicle and oocyte nucleus/oocyte regressions. The oocyte reached its maximum size when the average follicle diameter was 117 microns in nonpregnant rats and 131 microns in pregnant rats. The oocyte nucleus attained maximum size when the follicle diameter was 110 microns during the estrous cycle and 111 microns during pregnancy and postpartum. Maximum values of the diameter of the largest antral follicle and average diameter of the four largest antral follicles were observed during proestrus (473 and 442 microns, respectively) and on day 21 of pregnancy (611 and 538 microns, respectively). Chi 2 analysis showed that distribution of various types of follicles was not independent of the stage of the estrous cycle and pregnancy. In estrus and metestrus most of the follicles were between stages I and V. However, by diestrus and proestrus, follicles of all size groups developed. The numbers of stage I and II follicles did not differ as pregnancy advanced. More stage V follicles were present on day 12 than on day 7 of pregnancy; however, their numbers decreased by day 15. Afterwards, progressive increase of stage V and (VI + VII) follicles was observed until day 21. This was accompanied by the shift of follicles from stage (III + IV) on days 19 and 21 of pregnancy and even of stage II on day 2 postpartum. Wherever possible, the results have been compared with previous observations in various rodent species.     

Determination of size of folding nuclei of fibrils formed from recombinant Aβ(1-40) peptide

We have developed a highly efficient method for purification of the recombinant product Aβ(1-40) peptide. The concentration dependence of amyloid formation by recombinant Aβ(1-40) peptide was studied using fluorescence spectroscopy and electron microscopy. We found that the process of amyloid formation is preceded by lag time, which indicates that the process is nucleation-dependent. Further exponential growth of amyloid fibrils is followed by branching scenarios. Based on the experimental data on the concentration dependence, the sizes of the folding nuclei of fibrils were calculated. It turned out that the size of the primary nucleus is one “monomer” and the size of the secondary nucleus is zero. This means that the nucleus for new aggregates can be a surface of the fibrils themselves. Using electron microscopy, we have demonstrated that fibrils of these peptides are formed by the association of rounded ring structures.     

Topographic organization of the karyometric response to neonatal castration of the male mouse in the paraventricular and ventromedial hypothalamic nuclei

We have analysed the karyometric development of four topographic subdivisions of the paraventricular nucleus (rostral, medial, lateral and caudal) and three topographic subdivisions of the ventromedial nucleus (ventral, central and dorsal) of the hypothalamus in neonatally castrated male mice. Castration at birth produces a decrease of the nuclear sizes from the 10th to the 35th postnatal day, but after this age, no differences were detected in comparison with the sizes of the control male mice. Neonatal gonadal hormone deprivation also produces changes in the nuclear shape of the nuclei, that tend to be more spherical in both centres, as we could establish after the study of the form factor parameter (perimeter/area ratio). We have found differences in the response of both hypothalamic nuclei to neonatal castration and differences in the response of the topographic subdivisions of both of them.     

A direct comparison of the masculinizing effects of testosterone,androstenedione, estrogen,and progesterone on the development of the zebra finch song system

To assess which hormones are capable of masculinizing the neural song system of zebra finch hatchlings, we implanted female hatchlings with estrogen (estradiol [E2], 75 μg, n = 9), testosterone (T, 75–88 μg, n = 13), androstenedione (AE, 75 μg, n = 7), progesterone (P, 117 μg, n = 10), or nothing (Blanks, n = 10) and compared these to unimplanted males (n = 7). Implants, consisting of a hormone and Silastic mixture encased in polyethylene tubing, were placed under the skin of the breast on the day of hatching. Birds were killed when they were subadult (58 to 68 days old). We measured volumes of area X, the higher vocal center (HVC), and the robust nucleus of the archistriatum (RA); measured soma sizes in the lateral magnocellular nucleus of the neostriatum (IMAN), HVC, and RA: and counted RA neurons. E2 masculinized all measures in the song system and nearly sex-reversed the size of RA neurons. T masculinized volumes of nuclei and soma sizes but not the number or spacing of RA neurons. E2 was always at least as effective as T in masculinizing measures of the song system and was usually more effective. AE and P did not significantly masculinize any measure. These data suggest that E2 is more potent than aromatizable androgens or P in masculinizing the female song system in development and that the action of E2 alone may be sufficient to masculinize the volume of song control nuclei and the size and number of neurons. © 1995 John Wiley & Sons, Inc.     

Light and Electron Microscopic Observations on Encystment of Acanthamoeba palestinensis, Reich

SYNOPSIS. The ultrastructural changes occurring during encystment of Acanthamoeba palestinensis have been investigated. The cyst wall consists of endocyst and exocyst, both having the same fine structure. At irregular intervals in the cyst wall ostioles occupied by opercula are present. The nuclear membrane forms bulb-shaped projections and releases vesicles bounded by double membranes into the cytoplasm. Dense nucleolus-like bodies of different sizes and variable numbers are found in the nucleus of every cyst. The importance of the cyst structure as a taxonomic criterion is discussed.     

Unusual nuclear structure of the spermatozoon in a marsupial,Sminthopsis crassicaudata

The organization of sperm chromatin in the dasyurid marsupial, Sminthopsis crassicaudata, was investigated using various morphological techniques. Transmission electron microscopy indicates two quite distinct chromatin regions became evident late in spermiogenesis with an outer globular region containing blocks of very electron-dense chromatin. Fluorescent light microscopical studies after staining with DNA dyes and 7-amino actinomycin D of testicular, caput, and cauda epididymal spermatozoa showed that this region fluoresced less brightly than the rest of the nucleus, indicating the presence of fewer DNA binding sites. Freeze fracture showed that the chromatin in most of the nucleus had randomly arranged particles of various sizes, but that of the outer region was composed entirely of small particles. This outer region was more resistant to low concentrations of the ionic detergent, SDS, whereas both guanidine hydrochloride and urea together with sodium chloride generally dispersed all the chromatin except that in the outer globular region and in a localized area of the nucleus beneath the acrosome. This study has thus revealed that the outer globular chromatin of these spermatozoa responds differently to ionic detergents and protein denaturing agents and has a different chromatin organization than most of the rest of the nucleus. The significance of these differences remains, however, to be determined. © 1994 Wiley-Liss, Inc.     

10种鸣禽控制鸣啭神经核团大小与鸣唱复杂性的相关性

为进一步揭示鸣禽鸣唱行为的神经生物学机制 ,本实验先对 8个科 10种鸣禽的鸣唱行为进行了观察和录音 ,并借助声谱软件分析了每种鸣禽的鸣唱复杂性。鸣唱语句复杂性的评价指标包括 :短语总数、每个短语中所含的平均音节数及音节种类数、所有短语的总音节数及音节种类数、最长短语的音节数及音节种类数。然后 ,测定了前脑三个鸣啭学习控制核团和一个与发声无关的视觉参考核团体积 ,分析了鸣唱语句复杂性和这些核团大小间的相关关系。结果表明 :1)HVC和HVC/Rt与 7种鸣唱语句复杂性指标无关 ;RA和RA/Rt与总音节种类数相关 ;AreaX与总音节数及音节种类数相关 ;2 )HVC/RA和HVC/X比值与多个鸣唱语句复杂性指标相关。结果提示 :鸣禽鸣唱复杂性不同特征可能受不同神经控制     

Structural changes in the nuclei of human lymphocytes under the effects of ionizing radiation within the range of doses that cause the adaptive response

Low-level radiation either from external, or from incorporated sources is shown to cause a non-monotonous change in some intranuclear parameters of human peripheral blood lymphocytes. For instance, radiation induces changes in the parameters that characterize the location of perecentromeric regions within the interphase nucleus and a non-monotonous increase in nuclear sizes. The exposure to doses exceeding 5 cGy impairs the relationship between the nuclear sizes and location therein of the perecentromeric regions of interphase chromosomes which exist in the control and after exposure to 2.5 cGy. Differences between the dose ranges of 1.5-3.5 and 17-25 cGy are manifested by the kinetics of restoration of the pattern of distribution of lymphocyte nuclear sizes.     

Alignment of Sfi I sites with the Not I restriction map of Schizosaccharomyces pombe genome.

A Sfi I restriction map of the fission yeast Schizosaccharomyces pombe genome was aligned with the Not I restriction map. There are 16 Sfi I sites in the S. pombe genome. Three Sfi I sites are on chromosome III which is devoid of Not I sites. The sizes of the entire genome and individual chromosomes, calculated from the Sfi I fragment sizes, are consistent with that calculated from the Not I fragment sizes. The Sfi I map provides greater physical characterization of the S. pombe genome and further validates the use of S. pombe chromosomal DNA as size standard. These maps have allowed detection of polymorphism on all three chromosomes.     

Projections from neurons of the medial terminal nucleus of the accessory optic tract to the caudate nucleus in the cat revealed by combining techniques of retrograde axonal transport and experimentally induced degeneration

Neurons of the medial terminal nucleus of the accessory optic tract receiving direct retinal inputs were shown to project to the heat and body of the caudate nucleus in the cat using techniques of retrograde horseradish peroxidase axonal transport and experimentally induced degeneration. These primarily ipsilateral projections are evenly distributed throughout the aforementioned areas of the nucleus. Neurons of the medial terminal nucleus forming synaptic connections with caudate nucleus cells are distinguished by their varied shapes and sizes, ranging from 20 × 10 to 37.5 × 18 µm and are located in both the ventral and dorsal subdivisions of the nucleus. The supposed functional significance of these projections for the regulation of muscle tonus tension is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 214–219, March–April, 1986.     

Life histories, neighbourhood sizes, and variance structure in some North American conifers

Neighourhood sizes and variance structure were determined for ten North American coniferous forest tree species, using information on seed dispersal, patterns of life history, and demography. A synthetic cohort of reproductive individuals belonging to a critical age group was constructed using normal yield tables. Data on reproductive individuals in a synthetic cohort were used to determine the neighbourhood sizes for each species. The neighbourhood sizes varied from 1244 to 50 118 individuals. Effective population sizes were calculated from data on neighbourhood sizes and replacement patterns of individuals in various species, the interdeme differentiation, F _st ranged from 0.0002 to 0.007. The results suggest that large neighbourhood and effective population sizes, and hence panmixia, may be virtually common among the species examined. These conclusions are consistent with recent findings on population differentiation and estimates of gene flow in confers using allozyme markers.     

Method of determining the concentration of glycogen, DNA, 3H-thymidine label and dry weight in one and the same cell]

A method is proposed for determination of glycogen, DNA, 3H-thymidine incorporation and dry weight in the same cell, the technique being based on successive discovery and measuring of each of these indices. Cells are obtained from animals, previously injected with 3H-thymidine, to be charted on preparation, made pictures and measured in square units. Then on preparations embedded into glycerine or vaseline oil, the optical path difference of rays for the nucleus and cytoplasm of selected cells is measured with the interferencial microscope. This is followed by the fluorescent PAS reaction and the content of glycogen is registered microfluorimetrically in the same cells. Preparations after that are treated with a freshly prepared water solution of 0.025% borohydride sodium, stained with the routine or fluorescent Feulgen reaction, and DNA content is determined in the same cells in which glycogen and delta delta were previously measured. The stained nuclei are photographed, their areas are measured and the dry weight of the nucleus and cytoplasm of marked cells is calculated from the values of the nuclear areas and of delta delta. Eventually the preparations are covered by emulsion and exposed, and 3H-thymidine-containing nuclei are determined, the index of marked nuclei and the marking intensity over the nucleus are calculated. As a result, a precise and reliable determination of glycogen, DNA, dry weight and 3H- or 14C-thymidine incorporation is made in either of the marked cell.