NON-RANDOM ASSOCIATION OF MITOTIC CELLS FROM CULTURED HUMAN LYMPHOCYTE SUSPENSIONS

By using chromosome markers in two separate cell lines of a human dispermic chimera, it was shown that 4.9% of metaphases in suspensions of PHA-cultured lymphocytes were paired because of non-random factors. A similar amount of metaphase pairing occurred in cultured lymphocytes of normal donors, and evidence from the relative mitotic cycles of the paired cells indicated that some of this pairing was non-random. Such non-random pairing could be a source of bias in cell kinetic and other studies involving metaphase cells in lymphocyte cultures.     

A high incidence of mitotic chiasmata in endoreduplicated Bloom's syndrome cells

Summary The frequency of mitotic chiasmata is compared in endoreduplicated and non-endoreduplicated Bloom's syndrome fibroblasts and in endoreduplicated Fanconi's anemia lymphocytes. The incidence of mitotic chiasmata in BS diplochromosomes is greatly increased over that in diploid BS cells and is much higher than in FA or normal diplochromosomes. The distribution of chiasmata among the BS diplochromosomes is not significantly different from that expected if crossing-over occurs at random along the chromosomes. This is in contrast to the distribution of chiasmata in chromosomes of diploid BS cells which is highly non-random among chromosomes and chromosome regions (Kuhn 1976). Mitotic crossing-over is increased in endoreduplicated cells from all sources compared to diploid cells, but the incidence is highest in endoreduplicated BS cells. This provides evidence against the idea that the high rate of mitotic crossing-over in diploid BS lymphocytes is primarily due to an increase in chromosome pairing. BS chromosomes apparently have a greater tendency to undergo mitotic exchange than normal or FA cells, both in diplo-chromosomes and in accidentally paired homologous segments in diploid cells.     

Distribution of different cell types within the rat thymus in the neonatal period of life

Summary This study attempted to define reciprocal positions of cell types within the thymus. Random or non-random contacts between specific cell types were analyzed by means of graph theory. For analysis, thymus blocks were sectioned serially and, then, thymus cells were categorized into types, based on morphological criteria. The distribution of individual cell types within the cortex, cortico-medullary zone and medulla was presented in form of a map. In the analysis, three types of epithelial cell, characteristic of each thymus zone, macrophages, Langerhans-like cells and lymphocytes were found in non-random relations to one another. Moreover, characteristic groups of cells associated with one another were also demonstrated.This study was supported by the Polish Academy of Sciences     

Paired cytoskeletal patterns in an epithelium of siamese twin cells.

The epidermis of some insects is a sheet of siamese twin cells which are formed by conserving the midbody between siblings after cell division. We have found that for about 36 h after ecdysis to the 5th stage, the cells of Calpodes caterpillars contain one to five or more actin bundles. The variation in number of bundles occurs in an epithelium that is presumed to be otherwise genetically and developmentally homogeneous. The number of bundles is paired in adjacent cells (P less than 0.005, n = 617). Confocal microscopy shows midbodies between paired but not between unpaired cells. The pairing is reminiscent of the paired nucleolar patterns in these siamese twin cells (Locke, M., H. Leung, Tissue and Cell 17, 573-588 (1985)) or the mirrored patterns of stress fibers in newly divided 3T3 cells (Albrecht-Buehler, G., J. Cell Biol. 72, 595-603 (1977)). The pairing provides further evidence for the operation of transiently heritable factors as determinants for cell pattern.     

Comparative study of the proliferative activity of human lymphocytes from different pairs of donors in a mixed culture in vitro

The proliferative response of human lymphocytes was studied in one-way and two-way mixed lymphocyte cultures (MLC). The maximal proliferation was shown to be attained in a two-way MLC containing unequal quantities of the cells from two paired donors. It was also demonstrated in a one-way MLC that the cells of one of the two paired donors are more effective responders. The most powerful proliferation in the MLC of these donors was observed in the cultures containing the excess of more effective cells. Thymosine increased the response of human lymphocytes in the two-way MLC. In vitro cell preincubation reduced the response in the cultures with a high proliferative response in the control. It has been thus demonstrated that lymphocytes from paired donors possess different functional activity in the MLC.     

Hippocampal place cells acquire location-specific responses to the conditioned stimulus during auditory fear conditioning

We recorded neurons from the hippocampus of freely behaving rats during an auditory fear conditioning task. Rats received either paired or unpaired presentations of an auditory conditioned stimulus (CS) and an electric shock unconditioned stimulus (US). Hippocampal neurons (place and theta cells) acquired responses to the auditory CS in the paired but not in the unpaired group. After CS-US pairing, rhythmic firing of theta cells became synchronized to the onset of the CS. Conditioned responses of place cells were gated by their location-specific firing, so that after CS-US pairing, place cells responded to the CS only when the rat was within the cell's place field. These findings may help to elucidate how the hippocampus contributes to context-specific memory formation during associative learning.     

The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE

During the cell cycle, the Golgi, like other organelles, has to be duplicated in mass and number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. Here we show that in Drosophila S2 cells, the Golgi likely duplicates in mass to form a paired structure during G1/S phase and remains so until G2 when the two stacks separate, ready for entry into mitosis. We show that pairing requires an intact actin cytoskeleton which in turn depends on Abi/Scar but not WASP. This actin-dependent pairing is not limited to flies but also occurs in mammalian cells. We further show that preventing the Golgi stack separation at G2 blocks entry into mitosis, suggesting that this paired organization is part of the mitotic checkpoint, similar to what has been proposed in mammalian cells.     

Non-homologous chromosome pairing and crossover formation in haploid rice meiosis

While many studies have provided significant insight into homolog pairing during meiosis, information on non-homologous pairing is much less abundant. In the present study, fluorescence in situ hybridization (FISH) was used to investigate non-homologous pairing in haploid rice during meiosis. At pachytene, non-homologous chromosomes paired and formed synaptonemal complexes. FISH analysis data indicated that chromosome pairing could be grouped into three major types: (1) single chromosome paired fold-back as the univalent structure, (2) two non-homologous chromosomes paired as the bivalent structure, and (3) three or more non-homologous chromosomes paired as the multivalent structure. In the survey of 70 cells, 65 contained univalents, 45 contained bivalents, and 49 contained multivalent. Moreover, chromosomes 9 and 10 as well as chromosomes 11 and 12 formed non-homologous bivalents at a higher frequency than the other chromosomes. However, chiasma was always detected in the bivalent only between chromosomes 11 and 12 at diakinesis or metaphase I, indicating the pairing between these two chromosomes leads non-homologous recombination during meiosis. The synaptonemal complex formation between non-homologs was further proved by immunodetection of RCE8, PAIR2, and ZEP1. Especially, ZEP1 only loaded onto the paired chromosomes other than the un-paired chromosomes at pachytene in haploid.     

Meiosis in haploid barley — An interpretation of non-homologous chromosome associations

Detailed meiotic studies were conducted on ten haploid plants representing six different genotypes of barley (Hordeum vulgare, 2n=14). At pachytene stages the non-homologous chromosomes were observed to pair as intimately as homologous chromosomes in many cells. Foldback pairing, involving single chromosomes, and multivalent associations were common. At diplotene, up to 4 chiasmatalike structures were observed in paired chromosomes but it is not likely that they resulted from crossing over. At diakinesis the bivalent frequency mean was from 1 to 1.3 per cell whereas by metaphase I the paired associations were rare with a single rod bivalent being observed in 3 to 5% of the cells. The frequencies of various types of secondary associations at metaphase were also recorded. — The origin and significance of bivalents and secondary associations in haploids is reviewed and discussed. Caution is urged in the interpretation that low levels of chromosome pairing in haploids is evidence of homology. It is concluded that very little chromosome duplication is likely to be found within the haploid set of barley chromosomes and that the basic chromosome number is seven.     

Target cell contact suppresses neurite outgrowth from soma-soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma-soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non-target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth-promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12-24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non-target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma-soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre- and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma-soma paired cells was transient, and neuronal sprouting began after a delay of 48-72 h. In contrast, when paired with VD1, both RPeD1 and this non-target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma-soma paired cells was target cell contact/synapse specific and Ca(2+) dependent. Specifically, soma-soma pairing in CM containing either lower external Ca(2+) concentration (50% of its control level) or Cd(2+) resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre- and postsynaptic neurons in a time-dependent and cell-specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca(2+) concentration.     

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.     

Meiotic Analysis of the Regenerated Plants Derived from a Young Spike of Octoploid Triticale ×Secale cereale F1 Hybrid

The regenerated plants had high frequencies of changes in chromosome number and the pairing variation. The chromosome number was more or less variably decreased in different pollen mother cells. Most of regenerated plants were mixoploids. Some had higher frequency of homolgous chromosome pairing because double doses of rye genome had inhibited the effect of Ph gene. But, there was obvious different of chromosome pairing among regenerted plants. The difference was related to numerical chromosome change. Meiosis in a regenerant was analysed with Giemsa-C banding technigue. Partial rye chromosomes did not pair homologously or only very loosely paired. There was moderate level of wheat-wheat homologous partial pairing, however, wheat-rye chromosome pairing also occurred.     

美味猕猴桃原生质体再生植株细胞遗传学研究 Ⅲ .母株减数分裂前期Ⅰ染色体配对的光镜观察

首次报道在光镜下观察美味猕猴桃 (品种 :No.2 6原生质体植株的母株 )花粉母细胞( PMC)染色体在减数分裂前期的配对 ,发现其配对和凝缩有明显不同步性。不同细胞间染色体配对形式变化较大 ,一般以二价联会为主 ,其次由其它多种配对方式 (包括有复合配对、重复配对、着丝点或端粒处联合和多价联会 )形成多价体 ,还有少数未配对或发生内配对 (偶见 )的单价体和几条二价体之间的次级配对。粗线期观察到少数染色体有缺失 (或重复 )、倒位、易位和疏松配对等结构性改变。表明该植株是一个复杂的区段异源六位体 ,少数染色体在结构上累积有变异。还认为该植株是研究减数分裂染色体配对和联会机制的好材料。     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000     

Formation of new cortico-rubral synapses as a possible mechanism for classical conditioning mediated by the red nucleus in cat

1.) By extracellular and intracellular recordings of the red nucleus (RN) cell activity, we investigated enhancement of signaling effectiveness at the cortico-rubral synapses underlying the establishment of classical conditioning mediated by RN in the cat. The classical conditioning of forelimb flexion was produced by pairing the conditioned stimulus (CS) to the cerebral peduncle (CP) with the unconditioned stimulus (US) to the forelimb skin at an interval of 100 msec for about a week. 2.) The increased responsiveness of RN cells to the CS was correlated with acquisition of the conditioned forelimb flexion, i.e. RN cells responded to the CS with higher firing probability in the animals which received the paired conditioning than those in the animals which received the CS alone or pairing of the CS and the US at random intervals or those in the naive animals which did not receive any training. 3.) Monosynaptic excitation of RN cells in response to the single pulse to CP was most enhanced in the animals which received the paired conditioning. By contrast, response of RN cells, as well as the behavioral response, induced by stimulation of the cerebellar interpositus nucleus (IP) was not enhanced after the paired conditioning. The difference between the responses to the stimulation of CP and IP suggested that the primary site of neuronal change is the cortico-rubral synapses. 4.) In the animals that received the paired conditioning, the excitatory postsynaptic potentials (EPSPs) induced by stimulation of CP had fast-rising components superimposed on the normal slow-rising EPSPs. On the other hand, most of the CP-EPSPs recorded in the naive animals showed a slow time course. The slow time course of the CP-EPSPs has been attributed to the peripheral localization of the cortico-rubral synapses on the dendrites of RN cells. 5.) The electrotonic length of RN cells in the animals which received the paired conditioning was not shorter than that in the naive animals. Therefore, it was suggested that the appearance of the fast-rising component in the CP-EPSPs is cause by formation of the new cortico-rubral synapses on proximal portions of the soma-dendritic membrane of RN cells. 6.) Since it has been established that new synapses formed by collateral sprouting are retained for more than several months, the formation of new synaptic connections could underlie long-lasting behavioral modification.     

Regularities in the primary structure of proteins

In this paper the latest protein database consisting of more than a million amino acids is analyzed to characterize the short range regularities in the primary structure. The amino acid distributions along the polypeptide chain and among the proteins have been studied first. Their influence on the amino acid pair statistics was taken into account. We are primarily interested in the distances of the covalent structure, where the amino acid pair frequencies show non-random characters. The amino acid pairs separated by at least 20 residues in the covalent structure exhibit an exact Gaussian distribution. We found that there is a range of non-random pairing in the covalent structure. We conclude that the pair preference characters are different for each of the 20 x 20 amino acid pairs. The range of the non-random pairing varies from pair to pair, and in most cases it does not extend beyond the 9th neighbour. The preferences of a certain pair in a certain position can not be derived from the character of that pair in another position. The preference values of 400 amino acid pairs are listed for up to the pairs in 9th neighbour position. Some fields of potential application of these data have also been discussed.     

Models for antigen receptor gene rearrangement. III. Heavy and light chain allelic exclusion

The extent of allelic exclusion in Ig genes is very high, although not absolute. Thus far, it has not been clearly established whether rapid selection of the developing B cell as soon as it has achieved the first productively rearranged, functional heavy chain is the only mechanism responsible for allelic exclusion. Our computational models of Ag receptor gene rearrangement in B lymphocytes are hereby extended to calculate the expected fractions of heavy chain allelically included newly generated B cells as a function of the probability of heavy chain pairing with the surrogate light chain, and the probability that the cell would test this pairing immediately after the first rearrangement. The expected fractions for most values of these probabilities significantly exceed the levels of allelic inclusion in peripheral B cells, implying that in most cases productive rearrangement and subsequent cell surface expression of one allele of the heavy chain gene probably leads to prevention of rearrangement completion on the other allele, and that additional mechanisms, such as peripheral selection disfavoring cells with two productively rearranged heavy chain genes, may also play a role. Furthermore, we revisit light chain allelic exclusion by utilizing the first (to our knowledge) computational model which addresses and enumerates B cells maturing with two productively rearranged kappa light chain genes. We show that, assuming that there are no selection mechanisms responsible for abolishing cells expressing two light chains, the repertoire of newly generated B lymphocytes exiting the bone marrow must contain a significant fraction of such kappa double-productive B cells.