snRNP: Rich Nuclear Bodies in Hyacinthus orientalis L. Microspores and Developing Pollen Cells.

The aim of the present work was the characterization of nuclear bodies in the microspore and developing pollen cells of Hyacinthus orientalis L.. The combination of Ag-NOR, immunofluorescence and immunogold techniques was used in this study. The obtained results showed the presence of highly agyrophylic extranucleolar bodies in microspore and developing pollen cells, which were finally identified as Cajal bodies. In all cases, a strong accumulation of snRNP-indicating molecules including TMG cap, Sm proteins and U2 snRNA, was observed in the examined nuclear bodies. In contrast to their number the size of the identified structures did not change significantly during pollen development. In the microspore and the vegetative cell of pollen grains CBs were more numerous than in the generative cell. At later stages of pollen development, a drastic decrease in CB number was observed and, just before anthesis, a complete lack of these structures was indicated in both pollen nuclei. On the basis of these results, as well as our previous studies, we postulate a strong relationship between Cajal body numbers and the levels of RNA synthesis and splicing machinery elements in microspore and developing pollen cells.

Transcriptional activity and distribution of splicing machinery elements during Hyacinthus orientalis pollen tube growth.

The localization of newly formed transcripts and molecules participating in pre-mRNA splicing, i.e., small nuclear ribonucleoproteins (snRNPs) and SC35 protein, in growing pollen tubes of Hyacinthus orientalis L. were analyzed in vitro and in vivo. The results indicated that the restart of RNA synthesis occurred first in the vegetative and then in the generative nucleus of both in vitro and in vivo growing pollen tubes. Changes in RNA synthesis were accompanied by the redistribution of splicing machinery elements in both vegetative and generative nuclei of the growing pollen tube. At stages of pollen tube growth when the vegetative and generative nuclei were transcriptionally active, clear differences in the distribution pattern of the splicing system components were observed in both pollen nuclei. While both small nuclear RNA with a trimethylguanosine cap on the 5' end and SC35 protein were diffusely distributed in the nucleoplasm in the vegetative nucleus, the studied antigens were only present in the areas between condensed chromatin in the generative nucleus. When the transcriptional activity of both pollen nuclei could no longer be observed at later stages of pollen tube growth, snRNPs and SC35 protein were still present in the vegetative nuclei but not in the generative nuclei. We, therefore, investigated potential differences in the spatial organization of splicing system elements during pollen tube growth. They clearly reflect differences in gene expression patterns in the vegetative and the generative cells, which may be determined by the different biological roles of angiosperm male gametophyte cells.

Distribution of poly(A) RNA and splicing machinery elements in mature Hyacinthus orientalis L. pollen grains and pollen tubes growing in vitro.

The localization of poly(A) mRNA and molecules participating in pre-mRNA splicing, i.e., small nuclear ribonucleoproteins (snRNPs) and the SC35 protein, in mature Hyacinthus orientalis L. pollen grains before anthesis and pollen tubes germinating in vitro were analyzed. The observations indicated a pattern of poly(A) mRNA distribution in mature pollen grains before anthesis which differed from that in germinating pollen grains. Directly before anthesis, poly(A) mRNA was homogeneously distributed throughout the whole cytoplasm, whereas after rehydration, it accumulated at one of the pollen poles. In the pollen tube, poly(A) mRNA was present in the cytoplasm, mainly in the areas beneath the cell membrane and the apical zone. Both before anthesis and during growth of the pollen tube, splicing snRNPs and SC35 protein were localized mainly in the area of the pollen nuclei. During anthesis and just after rehydration of the pollen grains, the pattern of labeling and the levels of the investigated antigens in the areas of the vegetative and generative nuclei were similar. During growth of the pollen tube, a change was observed in the distribution and an increase in the levels of trimethylguanosine snRNA and SC35 protein in the vegetative nucleus. Such a pattern of localization of the splicing machinery suggests resumption of transcription and/or maturation of pre-mRNA in the growing pollen tube.

Immunocytochemical evidence of calreticulin-like protein in pollen tubes and styles of Petunia hybrida Hort.

With a polyclonal antibody raised against calreticulin (CRT) the locations where the protein occurs in unpollinated and pollinated styles of Petunia hybrida were localized. The epitopes binding the CRT antibody were immunolocalized preferentially in pollen tubes. In transmitting tract cells, both before and after pollination, the level of CRT was low. The protein was mainly localized in the cytosol and around dictyosomes of transmitting-tract cells. In pollen tubes, a high level of CRT was found at their tips rich in endoplasmatic reticulum, cisternae piles of reticular and/or dictyosomal origin, and vesicles. Binding sites of the CRT antibody were also found in the internal callosic cell wall of the pollen tube. These results indicate a role of CRT in cells directly participating in pollen-pistil interaction.

Immunocytochemical localization of esterified and unesterified pectins in unpollinated and pollinated styles of Petunia hybrida Hort.

Localization of pectins in the style of Petunia hybrida before and after pollination was investigated by immunocytochemistry using two primary monoclonal antibodies specific to highly (JIM7) and weakly (JIM5) methylesterified pectins. In the unpollinated style, esterified pectins occurred mainly in the cell walls of cortex tissue, while unesterified pectins were present mainly in the extracellular matrix (ECM) of the transmitting tract. After pollination no remarkable differences were found in pectin distribution in the ground tissue of the style. On the other hand, in the transmitting tract a reduction in the quantity of unesterified pectins was observed. Unesterified pectins in the extracellular regions of the transmitting tissue decreased before the penetration of the pollen tubes, indicating that pollination induces a reduction in the amount of unesterified pectins in the transmitting-tract ECM. The correlation between the degradation of strongly Ca2+-binding pectins and the growing level of those ions in the extracellular regions of the transmitting tract in the pollinated pistil of P. hybrida (M. Lenartowska et al. 1997) suggests that this process may constitute a mechanism for creating an optimum calcium medium for in vivo-growing pollen tubes. Both pectin categories were localized in pollen tubes. Esterified pectin epitopes were localized mainly in the vesicles of the tip cytoplasm. Unesterified pectin epitopes were found in the external fibrillar wall of pollen tubes.

Localization of the calreticulin gene mRNA in unpollinated and pollinated styles of Petunia hybrida Hort.

The objective of our research on Petunia hybrida is to understand the role of calreticulin in the growth of pollen tubes in the pistil. The aim of this study was the first step: finding out whether CRT gene expression takes place in unpollinated and pollinated styles. It was revealed by in situ hybridization that the transcription of the calreticulin gene takes place in the transmitting cells of unpollinated and pollinated styles and in pollen tubes growing in vivo. The mRNA transcripts of the CRT gene were localized mainly on the surface of endoplasmic reticulum (ER) membranes, both in transmitting cells and in the tip cytoplasm of pollen tubes. The results of this study show that calreticulin can be involved in pollen - pistil interaction in vivo.

Immunocytochemical localization of pectins in the maturing anther of Allium cepa L.

Distribution of pectins in cell walls of maturing anther of Allium cepa L. was investigated. The monoclonal antibodies against defined epitopes of pectin were used: JIM5 recognizing unesterified pectin and JIM7 recognizing esterified pectin. It has been found that the cell walls of all anther tissues mainly contain esterified pectins. In the somatic tissues only small amounts of unesterified pectins are present in the cell wall junctions and adjacent middle lamellae and in the cell walls of the connective tissue. Thickening of the epiderm cell walls and growth of trabeculae in endothecium are completed through deposition of esterified pectins. In the cell walls of the middle layer and tapetum, unesterified pectins have been found only prior to their disintegration. The primary wall of microsporocytes is made up mainly of esterified pectins. Unesterified pectins occur outside microsporocytes only prior to the callose isolation stage. The presence of esterified pectins has also been detected on the surface of the callose wall surrounding dividing microsporocytes. Lysis of those pectins takes place after microsporogenesis, simultaneously with the lysis of the callosic walls. Before these processes pectins are unesterified. In the sporoderm of pollen grains mainly esterified pectins occur. They have been localized in the intine and aperture. The level of unesterified pectins in the intine is markedly lower.

Calcium in pollen-pistil interaction in Petunia hybrida Hort. III. Localization of Ca2+ ions and Ca(2+)-ATPase in pollinated pistil.

Studies were carried out of CA2+ and CA(2+)-ATPase localization in pollinated (6 and 48 h after pollination) pistils of Petunia hybrida. The results were confronted with CA2+ localization in mature pollen grain and in unpollinated pistil. It has been found that after pollination the number of CA2+ sequestered in the stigmal exudate and in the sporoderm of the pollen grain gets lower. That phenomenon was associated with the appearance of a large number of Sb/Ca precipitates in the submembrane cytoplasm of the germinating pollen. In the vacuolized pollen grain, i.e. grown into a pollen tube, there were only a few precipitates. In the pollen tube, CA2+ were found in the organelles of the tip cytoplasm and in the external pectin cell wall. Studies with the use of 45Ca2+ have revealed that the source of calcium ions incorporated into the pollen tube tip and its pectin wall is the transmitting tract of the style. In the transmitting tract overgrown with pollen tubes, Ca2+ were located in the intercellular matrix and in the transmitting cells. Sb/Ca precipitates occurred in the nuclei, around the secretory vesicles and on the plasmalemma in the transverse walls region. Elevated Ca2+ level was found in degenerating cells (inhibited pollen tubes, transmitting cells, nucellar cells). The progressing degeneration process of the cells of the transmitting tract of the pollinated pistil was associated with a decrease in the activity of plasmalemmal Ca(2+)-ATPase.

Calcium in pollen-pistil interaction in Petunia hybrida Hort. II. Localization of Ca2+ ions and Ca(2+)-ATPase in unpollinated pistil.

The localization of Ca2+ and Ca(2+)-ATPase activity in the unpollinated pistil of Petunia hybrida has been studied. Free or loosely sequestered Ca2+ ions have been found: (1) in the stigma exudate, (2) in the pistil transmitting tract, (3) on the surface of ovule, (4) in the cell wall of the embryo sac. The sites of Ca2+ sequestration in the transmitting cells were the plastids and vacuoles. In some cells, the Ca2+ ions were associated with the tonoplast and plasmalemma underlying the transverse walls which connect the transmitting cells. The sites of particular accumulation of Ca2+ were the surfaces of the placenta and of the ovule. In the ovule, numerous precipitates occurred in the extended walls connecting the embryo sac with the cells of the nucellus. Ca(2+)-ATPase was commonly localized on the plasmalemma (1) of the cells of the transmitting tract of the stigma and style, (2) of the placenta and (3) of the somatic cells of the ovule. The plasmalemma of the embryo sac cells was nearly completely devoid of the reaction product.

Calcium in pollen-pistil interaction in Petunia hybrida Hort. I. Localization of Ca2+ ions in mature pollen grain using pyroantimonate and autoradiographic methods.

The localization of Ca2+ in the mature pollen grain and the flow of these ions from the somatic tissues of the anther to the pollen grains has been studied using pyroantimonate and autoradiographic methods. In the pollen grain, Ca2+ ions have been localized in the sporoderm and in the cytoplasmic vesicles of probably dictyosomal origin. Calcium ions were transported into the sporoderm together with the compounds of degenerating tapetum. The material of degenerating tapetum forms pollen coat surrounding the mature pollen grain.

Localization of calcium-dependent ATPase in germinating pollen grain and pollen tube in Vicia faba.

It was demonstrated by a cytochemical method that the calcium-dependent ATPase was present in the germinating pollen grain. Immediately after hydration the enzyme was observed in the pollen plasmalemma. During germination, the chief sites of the enzyme activity were the plasmalemma around the aperture site and the cisternae of endoplasmic reticulum. In the pollen grain with a grown tube, the enzyme was localized in the plasmalemma of the tube tip, in the endoplasmic reticulum below the tip, and in the tonoplast of the vacuoles formed in the pollen grain after tube germination.

Localization of calcium on the stigma surface of Ruscus aculeatus L. : Studies using chlorotetracycline and X-ray microanalysis.

By use of chlorotetracycline and X-ray microanalysis it is demonstrated that the receptive surface of the stigma of Ruscus aculeatus is rich in calcium. The high level of calcium is found in the epidermal cells and in the exudate covering the stigma. These results indicate that in vivo, as in vitro, calcium takes part in the regulation of pollen grain germination.

Results after resection of postinfarction left ventricular aneurysms.

Although left ventricular aneurysmectomy (LVA) is a common surgical procedure, the late functional and hemodynamic results have not been well defined. This presentation describes our results with LVA in 135 patients operated between 1969 and 1979. Associated procedures were performed in 57 (42%) including coronary bypass grafting in 50, valve replacement in 5, closure of ventricular septal defect in 2, or combinations of these in 3 patients. One hundred four of the 122 hospital survivors were followed from 2 to 107 months (mean = 37 months). There were 13 hospital deaths (9.6%), 12 late deaths (9.8%) and an actuarial 5-year survival rate of 77%. Clinical improvement of preoperative heart failure occurred in 82%, and of angina in 70%. Only 33 patients (30%) returned to normal work. Bicycle exercise testing in 70 patients showed normal working capacity in 41 (59%). Recatheterization in 49 patients showed no significant changes in left ventricular end-diastolic pressure or cardiac index, and a borderline reduction of the total ejection fraction. Ventricular arrhythmias were detected by long-term ECG in 70% of all patients after surgery. Of those with preoperative life-threatening arrhythmias, rhythm improvement was noted in 50%, but only 2 of 13 patients were free of arrhythmias after operation. This study demonstrates a greater frequency of postoperative symptomatic and functional improvement as compared to hemodynamic and ECG improvement. Ventricular tachyarrhythmias originating from post-infarct scars increased intra- and postoperative risk and aneurysmectomy alone is considered insufficient for treatment of these disturbances. Further electrophysiologic investigations are needed and additional surgical measures may be necessary to improve the subset of patients with life-threatening arrhythmias.