[Percutaneous endoscopic gastrostomy: a 7 years experience long-term tube feeding. Follow-up]. / Gastrostomía Endoscópica Percutánea: 7 años de experiencia en nutrición enteral a largo plazo. Seguimiento clínico.

Percutaneous endoscopic gastrostomy (PEG) is the first choice method for long-term enteral feeding when the digestive tube is undamaged and the patients' survival is longer than 2 months. There are increasing series and indications reported and although it is a safe technique we should take into account an appropriate patient selection. The aim of this work was to analyze the follow-up of the patients undergoing percutaneous endoscopic gastrostomy (PEG) at a hospital with high assistance level and one of the referents for organ transplantation in our community, with the peculiarities that this may have on our series. We analyze a cohort of 73 patients submitted to PEG during the years 2000-2007 at the Reina Sofía Hospital of Córdoba. Neurological and ENT neoplasms and upper GI tract neoplasms are among the most frequent causes, with similar results to those reported in other series. We highlight the number of young patients with cystic fibrosis (CF) in our series with nutritional support through PEG complementing oral and nocturnal feeding, which has lead to improved nutritional parameters before lung transplant. In consequence, we therefore analyze patients' selection and their characteristics, the ethical and moral implications in some of these patients, such in neurological patients, their complications, mortality, and we mention as being of interest the transient and well tolerated indication in a subgroup of CF patients that deserves special mention.

Activation of rye 5RL neocentromere by an organophosphate pesticide.

An interstitial constriction located on the long arm of rye chromosome 5R (5RL) shows neocentromeric activity at meiosis. In some meiocytes this region is strongly stretched orienting with the true centromere to opposite poles at metaphase I, and keeping sister chromatid cohesion at anaphase I. We found previously that the frequency of neocentric activity varied dramatically in different generations suggesting the effect of environmental factors. Here we studied the behavior of the 5RL neocentromere in mono- and ditelosomic 5RL, and mono-, and disomic 5R wheat-rye addition lines, untreated and treated with an organophosphate pesticide. The treated plants form neocentromeres with an about 4.5-fold increased frequency compared to untreated ones, demonstrating that the pesticide promotes neocentric activity. The neocentromere was activated irrespectively of the pairing configuration or the presence of a complete or truncated 5R centromere. Fluorescence in situ hybridization (FISH) with 2 repetitive sequences (UCM600 and pSc119.2) present at the constriction showed kinetic activity at several locations within this region. Immunostaining with anti-α-tubulin showed that treated plants have abnormal spindles in 46% of the metaphase I cells, indicating that disturbances in spindle formation might promote neocentromere activation.

Neocentrics and holokinetics (holocentrics): chromosomes out of the centromeric rules.

The centromere appears as a single constriction at mitotic metaphase in most eukaryotic chromosomes. Holokinetic chromosomes are the exception to this rule because they do not show any centromeric constrictions. Holokinetic chromosomes are usually forgotten in most reviews about centromeres, despite their presence in a number of animal and plant species. They are generally linked to very intriguing and unusual mechanisms of mitosis and meiosis. Holokinetic chromosomes differ from monocentric chromosomes not only in the extension of the kinetochore plate, but also in many other peculiar karyological features, which could be understood as the 'holokinetic syndrome' that is reviewed in detail. Together with holokinetic chromosomes we review neocentromeric activity, a similarly intriguing case of regions able to pull chromosomes towards the poles without showing the main components reported to be essential to centromeric function. A neocentromere is a chromosomal region different from the true centromere in structure, DNA sequence and location, but is able to lead chromosomes to the cell poles in special circumstances. Neocentromeres have been reported in plants and animals showing different features. Both in humans and Drosophila, neocentric activity appears in somatic cells with defective chromosomes lacking a functional centromere. In most cases in plants, neocentromeres appear in chromosomes which have normal centromeres, but are active only during meiosis. Because of examples such as spontaneous or induced neocentromeres and holokinetic chromosomes, it is becoming less surprising that different structures and DNA sequences of centromeres appear in evolution.

Chromosomes with a life of their own.

B chromosomes (Bs) can be described as 'passengers in the genome', a term that has been used for the repetitive DNA which comprises the bulk of the genome in large genome species, except that Bs have a life of their own as independent chromosomes. As with retrotransposons they can accumulate in number, but in this case by various processes of mitotic or meiotic drive, based on their own autonomous ways of using spindles, especially in the gametophyte phase of the life cycle of flowering plants. This selfish property of drive ensures their survival and spread in natural populations, even against a gradient of harmful effects on the host plant phenotype. Bs are inhabitants of the nucleus and they are subject to control by 'genes' in the A chromosome (As) complement. This interaction with the As, together with the balance between drive and harmful effects makes a dynamic system in the life of a B chromosome, notwithstanding the fact that we are only now beginning to unravel the story in a few favoured species. In this review we concentrate mainly on recent developments in the Bs of rye and maize, two of the species currently receiving most attention. We focus on their population dynamics and on the molecular basis of their structural organisation and mechanisms of drive, as well as on their mode of origin and potential applications in plant biotechnology.

Meiotic loss of the B chromosomes of maize is influenced by the B univalent co-orientation and the TR-1 knob constitution of the A chromosomes.

The suppression of meiotic loss when the maize B chromosomes are unpaired is genetically determined. Two genotypes were selected in 1B x 0B crosses: the H line where the B transmission rate is Mendelian (50%) and the L line where the B is present in only about 40% of the progeny. Using the ZmBs probe located at the centromere and at the distal portion of the B chromosome in FISH, we found that the centromeric and telomeric ends of the B univalent co-orient at metaphase I. This feature seems to promote proper centromere orientation causing the lack of meiotic loss of the unpaired B. The co-orientation was observed in both lines, however in the L line the B univalents were not always properly oriented, showing amphitelic orientation in about 25% of the metaphase I cells. We also studied plants of the H and L lines with FISH to test the possible relation between the knob constitution and B loss. It has been found that the plants of both lines are similarly variable for the 180-bp knob repeat, but they differ in the TR-1 350-bp repeat, the L line having more TR-1 knobs. The use of a 45S rDNA probe which labels chromosome 6, allowed us to determine that this chromosome shows the main variability between the two lines: the L line has TR-1 in both arms, showing a large TR-1 knob on the long arm. The H line has only one, generally located on the short arm besides the NOR.

The high variability of subtelomeric heterochromatin and connections between nonhomologous chromosomes, suggest frequent ectopic recombination in rye meiocytes.

The position of telomeres, centromeres and subtelomeric heterochromatin (SH) has been studied by FISH in rye meiocytes. We compare the morphology of the signals from zygotene to telophase II mainly to determine differences in SH and telomere positions between plants with and without neocentromeres. Plants from two varieties were used: Paldang showing neocentromeres, and Puyo without neocentromeres but with two B chromosomes. In both varieties, at zygotene and pachytene the SH is observed forming clumps often including two or more bivalent ends. At diplotene the SH is stretched suggesting that it is close to the nuclear envelope. In these cases, the telomere signals are not stretched and lay behind the SH. Frequently, two or more bivalents are joined by conspicuous SH connections at diplotene strongly suggesting ectopic recombination. Probably as a result, differential distribution of the SH between recombinant homologues or the whole meiotic products is observed. From diplotene onwards, the large heterochromatic blocks cover the telomeres, the SH being the morphological end of the bivalents, both in plants with or without neocentromeres. The Bs are tightly associated only at the telomeric end of the long arm from diplotene to metaphase I. The high variability between homologous chromosomes and the frequent nonhomologous bindings of SH, strongly suggest that rye SH is in dynamic state and frequently changes in chromosome position during meiosis.

Mitotic microtubule development and histone H3 phosphorylation in the holocentric chromosomes of Rhynchospora tenuis (Cyperaceae).

In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.

Mitotic instability in wheat x Thinopyrum ponticum derivatives revealed by chromosome counting, nuclear DNA content and histone H3 phosphorylation pattern.

To evaluate the mitotic stability of Triticum aestivum x Thinopyrum ponticum derivatives (BC(2)F(7) and BC(2)F(5) doubled haploids), chromosome counting by both conventional and immunostaining techniques, and measurement of DNA content were performed. The wheat progenitor line, PF 839197, the wheat recurrent parent CEP 19 and the control Chinese Spring were also investigated. In the hybrid derivatives, chromosome number ranged from 2n=36 to 60, with a predominance of chromosome numbers higher than 2n=42, that was confirmed by determination of nuclear DNA content. Chinese Spring' and PF 839197 were stable, but CEP 19 showed chromosome number variation (20%). Analyses of non-pretreated cells revealed the presence of anaphase bridges, lagging chromatids, chromosome fragments and micronuclei. Immunostaining with an antibody recognizing histone H3 phosphorylated showed dicentric chromatids forming anaphase bridges and pericentromeric phosphorylation at centric chromosome fragments but not at lagging chromatids. The possible causes of the observed mitotic instability are discussed.

Movement ability of rye terminal neocentromeres.

Rye terminal neocentromeres were analyzed in various aspects. Plants with and without neocentromeres were crossed to determine the possible genetic control on their formation. The segregation obtained in our work is consistent with the hypothesis of two trans-acting genes determining neocentric activity in such a way that individuals with no neocentromeres at all would carry all non-activating alleles, whereas one activating allele might permit the activation of a few neocentromeres. Individuals with four activating alleles would show the maximum frequency of neocentromeres per cell. Anti-tubulin immunolabelling was used to visualize the interaction between the neocentromeres and the microtubules. In most cases an end-on interaction between neocentromeres and microtubules was observed, but a few neocentromeres were observed free of them. Spikes were irradiated at early meiosis to determine whether acentric fragments carrying subtelomeric heterochromatin were able to behave as neocentromeres. In no case were acentric fragments observed to form an extension polewards as they did in whole chromosomes. Broken chromosomes joined by a thin thread of chromatin to the centromeric region

The parasitic effects of rye B chromosomes might be beneficial in the long term.

Rye B chromosomes (Bs) have strong parasitic effects on fertility. B carrying plants are less fertile than 0B ones, whereas the Bs have no significant effects on plant vigour. On the other hand, it has been reported that B transmission is under genetic control in such a way that H line plants transmit the Bs at high frequency, whereas the Bs in the low B transmission rate line (L) fail to pair at metaphase I and are frequently lost. In the present work we analyse variables affecting vigour and fertility considering not only the number of Bs of each plant, but also its H or L status and the B number of its maternal parent. Our results show that the Bs not only decrease female fertility of the B carrier, but the fertility of its progeny, with the exception of 0B plants coming from a 4B mother, which are the most fertile. In this way B chromosomes can be considered as a selective factor. Pollen abortion was higher in B carriers, in the progeny of B carriers and in H plants, but 4B plants coming from B carrying mothers produce less aborted pollen, indicating that a high B number is more deleterious if it is transmitted in the pollen grains. A similar result was obtained for endosperm quality estimated as grain weight, because it is negatively influenced by the Bs in 4B plants coming from a 0B mother. H plants were always less fertile than L ones, indicating that alleles increasing the loss of Bs in the L line will be probably selected as a defence of the A genome against the invasive Bs of the H line. Flower number is not affected by the Bs.

One gene determines maize B chromosome accumulation by preferential fertilisation; another gene(s) determines their meiotic loss.

Genotypes of high (H(m)) and low (L(m)) male B transmission rate (B-TR) were obtained. B-TR segregation in the F2 is reported, showing that the H(m) and L(m) lines differ in a single locus we call mBt (male B transmission), controlling B preferential fertilisation in maize. The egg cells control which one of the sperm nuclei is going to fertilise them, mBt(h) egg cells being preferentially fertilised by the sperm nucleus carrying the supernumerary B chromosomes (Bs). It is hypothesised that the mBt gene is involved in the normal fertilisation of maize but the parasitic Bs take advantage of the mBt(h) allele to increase their own transmission. Selection was also carried out when the Bs were transmitted on the female side (H(f) and L(f) lines). The F1 hybrids show that the gene(s) that we call fBt (female B transmission), controlling female B-TR, is located on the A chromosomes acting at diploid level, the fBt(l) allele(s) for low transmission being dominant. This allele causes the loss of Bs at meiosis, which is shown using a specific B molecular probe to determine B presence/absence in microspores of both lines and hybrids. Maize Bs are a nice example of intragenome conflict, because the mBt and fBt loci are a polymorphic system of attack and defence between A and B chromosomes.

Nature and evolution of B chromosomes in plants: A non-coding but information-rich part of plant genomes.

This work reviews recent advances providing insights on the origin and evolution of B chromosomes (Bs) in representative plant species. Brachyome dichromosomatica has large and micro Bs. Both carry an inactive ribosomal gene cluster. The large Bs contain the B-specific Bd49 family, mainly located at the centromere. Multiple copies are present in the A chromosomes (As) of related species, whereas only a few copies exist in B. dichromosomatica As. The micro Bs share sequences with the As, the large Bs and have the B-specific repeats Bdm29 and Bdm54. It seems that the large and micro Bs are related in origin. It is very unlikely that the Bs originated by simple excision from the As. Rye Bs are composed of sequences predominantly shared with the As. B-specific sequences are located at the heterochromatic end of the long arm. Probably, they originated from the As after many rearrangements, with a tendency for duplication. The E3900 family derives from a Ty3 gypsy retrotransposon, but the D1100 family shows no evidence of genic origin. The overall composition of maize As and Bs is similar suggesting a common origin. Several B-specific sequences have been found, the most studied being pZmBs, which is located at the B centromere. It shows partial homology to the centromere of chromosome 4 and to the knobs. It is not known whether the B centromere derives from centromere 4, or whether both have a more distant common origin. The dynamics of Bs in populations depends on their non-Mendelian mechanisms of transmission, their effects on carrier fitness and on A genes modulating their parasitic properties. Three representative examples are reviewed. The Bs of Allium schoenoprassum are transmitted at a mean lower than Mendelian and adversely affect vigour and fertility. However, there is a differential selection operating in favour of B-containing seedlings. Rye Bs undergo strong drive, which is counteracted by harmful effects on fertility and instabilities at meiosis. Both nondisjunction and meiotic behaviour, and consequently the establishment of B polymorphisms, mainly depend on the Bs themselves. B nondisjunction in maize is controlled by the B, but the As control preferential fertilisation. Considering the non-equilibrium model, the Bs of Allium seem to have been neutralised by the A genome, the As of maize provide defence against B attack, whereas the Bs of rye are only slightly neutralized.

Is maize B chromosome preferential fertilization controlled by a single gene?

In previous work, genotypes for high and low B chromosome transmission rate were selected from a native race of maize. It was demonstrated that the B transmission is genetically controlled. The present work reports the fourth and fifth generations of selection and the F1 hybrids between the lines. The native B is characterized by a constant behaviour, with normal meiosis and nondisjunction in 100% of postmeiotic mitosis. It is concluded that genetic variation for B transmission between the selected lines is due to the preferential fertilization process. The F1 hybrids show intermediate B transmission rate between the lines. They are uniform, the variance of the selected character being one order of magnitude lower than that of the native population. In addition, 0B x 2B and 2B x 2B crosses were made to study the effect of the presence of B chromosomes in the female parent, resulting in non-significant differences. Several crosses were made both in Buenos Aires and in Madrid to compare the possible environmental effect, but significant differences were not found. Our results are consistent with the hypothesis of a single major gene controlling B transmission rate in maize, which acts in the egg cell at the haploid level during fertilization. It is also hypothesized that maize Bs use the normal maize fertilization process to promote their own transmission.

Neocentric activity of rye 5RL chromosome in wheat.

The neocentric activity of a constriction located on the long arm of rye 5R chromosome (5RL) was analysed. It is not observed in normal rye but it is unusually stretched in bivalents involving 5RL telosomes in wheat-ditelosomic 5RL addition lines. In 20% of metaphase I cells, the 5RL bivalent presents the centromeres oriented to one pole and the constrictions oriented towards the opposite pole with a strong tension. In 5% of the cells, the constriction was able to orient the bivalent to the poles without tension in the centromeres. Sister chromatid cohesion, which is one of the distinct features of centromeric function, is persistent at the constriction in delayed 5RL chromosomes at anaphase I. Neither the elongation of the constriction nor the neocentric activity was observed at second meiotic division or mitosis. FISH studies showed that the 5RL constriction lacked detectable quantities of two repetitive DNA sequences, CCS1 and the 180-bp knob repeat, present at cereal centromeres and neocentromeres, respectively. We propose that, under special conditions, such as the wheat background, the normally non-centromeric DNA present at this region of 5RL acquires a specific chromatin structure, differentiated as an elongated constriction, which is able to function as a centromere.

The chromosomal distribution of phosphorylated histone H3 differs between plants and animals at meiosis.

Plant (Secale cereale, Triticum aestivum) and animal (Eyprepocnemis plorans) meiocytes were analyzed by indirect immunostaining with an antibody recognizing histone H3 phosphorylated at serine 10, to study the relationship between H3 phosphorylation and chromosome condensation at meiosis. To investigate whether the dynamics of histone H3 phosphorylation differs between chromosomes with a different mode of segregation, we included in this study mitotic cells and also meiotic cells of individuals forming bivalents plus three different types of univalents (A chromosomes, B chromosomes and X chromosome). During the first meiotic division, the H3 phosphorylation of the entire chromosomes initiates at the transition from leptotene to zygotene in rye and wheat, whereas in E. plorans it does so at diplotene. In all species analyzed H3 phosphorylation terminates toward interkinesis. The immunosignals at first meiotic division are identical in bivalents and univalents of A and B chromosomes, irrespective of their equational or reductional segregation at anaphase I. The grasshopper X chromosome, which always segregates reductionally, also shows the same pattern. Remarkable differences were found at second meiotic division between plant and animal material. In E. plorans H3 phosphorylation occurred all along the chromosomes, whereas in plants only the pericentromeric regions showed strong immunosignals from prophase II until telophase II. In addition, no immunolabeling was detectable on single chromatids resulting from equational segregation of plant A or B chromosome univalents during the preceding anaphase I. Simultaneous immunostaining with anti-tubulin and anti-phosphorylated H3 antibodies demonstrated that the kinetochores of all chromosomes interact with microtubules, even in the absence of detectable phosphorylated H3 immunosignals. The different pattern of H3 phosphorylation in plant and animal meiocytes suggests that this evolutionarily conserved post-translational chromatin modification might be involved in different roles in both types of organisms. The possibility that in plants H3 phosphorylation is related to sister chromatid cohesion is discussed.

Chromosome nondisjunction and instabilities in tapetal cells are affected by B chromosomes in maize.

Abnormal mitosis occurs in maize tapetum, producing binucleate cells that later disintegrate, following a pattern of programmed cell death. FISH allowed us to observe chromosome nondisjunction and micronucleus formation in binucleate cells, using DNA probes specific to B chromosomes (B's), knobbed chromosomes, and the chromosome 6 (NOR) of maize. All chromosome types seem to be involved in micronucleus formation, but the B's form more micronuclei than do knobbed chromosomes and knobbed chromosomes form more than do chromosomes without knobs. Micronuclei were more frequent in 1B plants and in a genotype selected for low B transmission rate. Nondisjunction was observed in all types of FISH-labeled chromosomes. In addition, unlabeled bridges and delayed chromatids were observed in the last telophase before binucleate cell formation, suggesting that nondisjunction might occur in all chromosomes of the maize complement. B nondisjunction is known to occur in the second pollen mitosis and in the endosperm, but it was not previously reported in other tissues. This is also a new report of nondisjunction of chromosomes of the normal set (A's) in tapetal cells. Our results support the conclusion that nondisjunction and micronucleus formation are regular events in the process of the tapetal cell death program, but B's strongly increase A chromosome instability.

Relationship between pachytene synapsis, metaphase I associations, and transmission of 2B and 4B chromosomes in rye.

2B rye plants selected for high (H) or low (L) B transmission rate were studied at pachytene and metaphase I of meiosis to determine the relationship between synapsis, bivalents at metaphase I, and B transmission rate. The results show that the 2 B chromosomes (Bs) form bivalents at pachytene in both the H and L lines, whereas the frequency of bivalents at metaphase I is much higher in the H than in the L line. This demonstrates that B transmission is mainly related to the proper association of Bs at metaphase I, as well as that synapsis of the 2 Bs in the L line is normal, but the bivalent is not consolidated by a chiasma in most cases. Crosses were made between 2B plants of the H and L lines in all combinations (H x H, H x L, L x H, and L x L) to obtain 4B plants. Similarly, bivalent formation at pachytene and metaphase I was studied. The results show that 4B plants of the H x H and L x L classes differ significantly at pachytene and metaphase I since the former forms more bivalents. The heterozygous 4 Bs of the H x L and L x H classes show intermediate values. The relation H x H > H x L > L x H > L x L was consistently found for the variables transmission rate, bivalents at pachytene, bivalents at metaphase I, and B mean chiasma frequency. A maternal effect was also found. Our data suggest that there are two separate mechanisms acting upon synapsis and chiasma formation in H and L B chromosomes: (i) there is variable efficiency of the control of synapsis at early stages of meiosis; and (ii) there is variable efficiency of the control of the number of chiasmata.

[Evaluation of alpha 1-antitrypsin phenotypes in Castilla-León: study of a population of newborn infants]. / Valoración de fenotipos de alfa 1-antitripsina en Castilla-León: estudio en una población de recién nacidos.

We have screened a population of newborn babies without any previous apparent pathology in order to discover the distribution of alpha 1-anti-trypsin phenotypes (AAT) in our community. The research has been done on 457 babies born in the province of Valladolid, determining the plasma levels of AAT and the corresponding phenotype. AAT quantification was done by nephelometry, and the phenotypic study was done by the technique described by Jeppsson. It has been possible to identify 8 different phenotypes, PIM being the one that had the highest incidence rate (72.42%), followed by PIMS (19.69%). In relation to the serum levels of AAT, the highest values were found for PIM (220 +/- 60 mg/dl) and the lowest for PIZ (43 +/- 2 mg/dl). Finally, the results obtained by our laboratory are compared with those of other authors.