Occurrence of ubiquitin during spermiogenesis in Chara vulgaris

Experiments with an anti-ubiquitin antibody proved the presence of ubiquitin in spermatids at all spermiogenesis stages in Charta vulgaris. Its level increased before marked ultrastructural changes of spermatids correlated with disappearance of somatic proteins (histones) and appearance of protamine-type generative proteins. The obtained results seem to confirm our earlier hypotheses concerning a significant role of ubiquitin-proteasome system in Chara spermatozoid differentiation.     

Cytochemical studies on histone-type and protamine-type proteins during spermiogenesis in Chara vulgaris and Chara tomentosa

Comparative studies concerning detection of histone-type and protamine-type proteins were carried out on Chara species (C. vulgaris, C. tomentosa). Analysis of antheridia during spermiogenesis (stages I-X) of both Charta species showed very similar staining patterns obtained after reactions revealing the examined proteins. Cytochemical studies showed a replacement of lysine-rich histone proteins by more basic arginine-rich ones during medium spermiogenesis (st. VI-VIII) in two Charta species, while late spermiogenesis (st. IX) and mature spermatozoids (st. X) were characterised by the presence of protamine-like proteins only.     

Immunocytochemical and ultrastructural analyses of the function of the ubiquitin-proteasome system during spermiogenesis with the use of the inhibitors of proteasome proteolytic activity in the alga, Chara vulgaris

Spermiogenesis in Chara vulgaris and in animals share many common features, including exchange of nucleohistones into nucleoprotamines, remodeling and extreme condensation of chromatin, formation of flagellae and of microtubule manchette, and decrease in cytoplasm volume. In C. vulgaris, spermiogenesis is not preceded by meiosis since this alga is a haplobiont. In the present work we showed that in early spermiogenesis characterized by a significant metabolic activity of spermatids, the inhibitors of proteasomes did not visibly change their ultrastructure but significantly prolonged this process. At late stages of spermiogenesis, MG-132 and epoxomicin dramatically changed the structure of nuclei: regular fibrillar and lamellar structure of chromatin was disturbed and clusters of grains corresponding to aggresomes appeared, but the nucleus shape and cytoplasm structure were the same as in the controls. Immunocytochemical studies revealed that these inhibitors blocked disappearance of histones from nuclei while the structures corresponding to aggresomes were clusters of undegraded ubiquitinated histones, since they gave positive immunosignals indicating the presence of ubiquitin and histones.     

Occurrence of calreticulin during the exchange of nucleohistones into protamine-type proteins in Chara vulgaris spermiogenesis

During spermiogenesis of an alga Chara vulgaris, which resembles that of animals, nucleohistones are replaced by protamine-type proteins. This exchange takes place in a spermatid nucleus during the key V spermiogenesis stage, in which rough endoplasmic reticulum is the site of protamine-type protein synthesis and is also the pathway guiding the proteins to their destination, nucleus. In the present work, it was shown that a chaperon protein, calreticulin (CRT), abundantly present at this significant V stage of spermiogenesis in a few cellular compartments, i.e., a nucleus, lumen of cisternae, and vesicles of significantly swollen ER as well as outside these structures, e.g., in Golgi apparatus, could have taken part in the process of exchange of nuclear proteins. Colocalization of two proteins, protamine-type proteins, crucial for reproduction, and CRT, was especially visible in a nucleus, mainly on its peripheries where condensed chromatin was present. Localization of protamine-type proteins and CRT in nucleus is in agreement with our previous results showing that protamine-type proteins were twofold more labelled in the peripheral area in comparison to the nucleus center occupied by noncondensed chromatin. The role of CRT in the reproduction of both plants and animals is also discussed.     

Electron microscope studies on surface activity in cells of Chara vulgaris

Summary Small fleck-like structures have been observed in the cell walls of developing lateral branches of Chara vulgaris. These were orientated with their flat surface parallel to the plasmalemma, and appeared to lie in definite layers at different depths in the wall. It was considered that these structures were membranous material periodically incorporated into the wall by deposition of other material. In the same cells membranous material was observed on the outside of the plasmalemma. It is thought that these residues may subsequently become buried in the wall to form the flecks. Charasomes, consisting of an invagination of the plasmalemma containing distinct tubules, were frequently observed alongside longitudinal walls of cells of more mature laterals. The possible function of these organelles is discussed.With 8 Figures in the Text     

Further ultrastructural research of Chara vulgaris spermiogenesis: endoplasmic reticulum,structure of chromatin, 3H-lysine and 3H-arginine incorporation

On the basis of morphological features, 10 consecutive structural phases of spermatids were identified in Chara vulgaris spermiogenesis. They were schematically presented. In early and middle spermiogenesis, i.e. during the period preceding formation of fibrillar structure of mature spermatozoid nucleus, a slight remodelling of chromatin, accompanied by proplastid transformation into an amyloplast as well as by development of 2 flagella and a microtubular manchette, is observed. First, condensed chromatin concentrates around the nuclear envelope (phases III-V) and then it transforms into a network-like structure (phase VI). This change in chromatin structure is preceded by nucleolar extrusion to the cytoplasm where nucleoli become degraded (phase IV) and by a dynamic development of rough endoplasmic reticulum (RER) (phase V) which is continuous with the nuclear envelope and with RER of the adjacent spermatids via plasmodesmata. The inner membrane of the nuclear envelope invaginates into the nucleoplasm in which "nuclear reticulum" appears. It all happens during increased 3H-arginine and 3H-lysine incorporation into proteins which are rapidly translocated into the nucleus. In medium-late spermiogenesis (phases VI-VIII), network-like condensed chromatin disappears. Next, the structure of the nucleus changes dramatically. Short, randomly positioned fibrils (phase VII) appear and gradually become longer (phase VIII), thicker (phase IX) and more distinct, lying parallel to the surface of elongating and curling nucleus. Membranes of the nuclear envelope become closer to each other and a distinct dark layer--probably lamin--appears adhering to the inner membrane of the nuclear envelope. Towards the end of spermiogenesis (phase X), very densely packed parallel helices, ca 2 nm in diameter, are visible. The surfaces of flagella and the spermatozoid are covered with diamond-shaped larger and smaller scales, respectively. Helically coiled spermatozoids are liberated from antheridial filament cells through earlier created (phase VIII) "liberation pores" with pads of unknown nature.     

GA3 content in antheridia of Chara vulgaris at the proliferative stage and in spermiogenesis estimated by capillary electrophoresis

In male sex organs of Chara vulgaris L., the gibberellic acid (GA3), was identified by capillary zone electrophoresis. The antheridia at cell division stage of antheridial filaments leading to formation of spermatids contain 0.09 microg GA3 per antheridium, i.e. 5.3 times more than antheridia at differentiation stage of spermatozoids (spermiogenesis). Spermiogenesis is not regulated by gibberellins.     

Salinity response of a freshwater charophyte, Chara vulgaris

Abstract. Chara vulgaris L. growing in an oligohaline lake was adapted to laboratory conditions and subjected to long-term salinity treatments ranging from 0 to 350 mol m ³ NaCl added to the lake water (40–680 mosmol kg ¹). Osmotic potential and concentration of the main osmotically active solutes (K⁺, Na⁺, Mg²⁺, Cl and sucrose) in the vacuolar sap of the central internodal cells were estimated. C. vulgaris did regulate turgor but incompletely. Turgor decreased from 335 mosmol kg ¹ under control conditions to 52–111 mosmol kg ¹ at 350 mol m ³ NaCl. The enhancement of πⁱ was achieved by increase in both ions and sucrose. Sterile and fertile plants differed in their response to osmotic stress. In sterile plants, the ions accounted for about 87% of the vacuolar osmotic potential. The increase of πⁱ under osmotic stress was exclusively due to an accumulation of Na⁺ and Cl^-. In fertile plants, sucrose accounted for about 35% of πⁱ and ions for about 51% Under osmotic stress, sucrose content increased together with the ionic content of Na⁺ and Cl^-.     

The influence of testosterone on the alkaline proteolytic activity in rat skeletal muscle

The effects of testectomy and subsequent administration of testosterone propionate on the activity of the alkaline proteinases in rat skeletal muscle were investigated. Castration of the mature rat was followed by a short-term delay in protein accretion in skeletal muscle tissue as measured by the protein/DNA ratio and was paralleled by a 2–3 fold increase in specific activity of the alkaline proteinase(s). This increase of proteolytic activity was equally significant when expressed relative to μg DNA. Although the gain in body weight was significantly lower in the castrated rats, nevertheless the protein/DNA ratio in muscle after 6 weeks approximated the values of sham-operated control rats without normalization of the proteolytic activity.Treatment of the castrated rats with testosterone propoinate resulted in restoring normal levels of previously elevated levels of alkaline proteolytic activity in muscle tissue. The normalization of enzyme activity as well as protein accretion in muscle was dose-dependent. Treatment of the rats with a low dose (0.1 mg/day) of testosterone propionate failed to restore the proteolytic activity, but led to a small increase of the protein/DNA ratio as well as to a progressive increase in body weight. These data indicate a regulatory role of testosterone in the adaptive behaviour of the alkaline proteolytic system in rat skeletal muscle.     

Study on the phenology of Chara vulgaris in Xin’an Spring, north China

The phenology of Chara vulgaris was studied.The specimens were collected four times from April 2004 to January 2005 (one sampling per season).The water temperature,pH,dissolved oxygen,specific conductance,maximum width and maximum depth were monitored at every sampling time.Eighteen main morphological characteristics of the Chara vulgaris were also observed and measured under the microscope.The results showed that all the environmental factors had different seasonal patterns and some of the morphological characteristics had significant fluctuations,indicating differences in their seasonality.At the same time,some morphological characteristics were affected by the environmental parameters to some extent and the effect was primarily exhibited in its vegetative proportions; there was little effect on the sexual reproductive characteristics.Therefore,the relatively stable sexual reproductive characteristics can be used to identify the species.     

Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulfate, facilitating proteolytic fingerprinting

Normally trypsin has negligible activity after being dissolved in sodium dodecyl sulfate (SDS), and so it has had little utility for proteolytic fingerprinting during gel electrophoresis. Here it is demonstrated that trypsin retained activity in SDS if it was first complexed to either of two soybean-derived protease inhibitors: trypsin inhibitor (Kunitz) or trypsin-chymotrypsin inhibitor (Bowman-Birk). The inhibitors alone did not cause proteolysis. Heating or acidification in SDS inactivated the inhibitor-dependent tryptic activity, as did prior treatment with tosyl lysine chloromethyl ketone, a covalent affinity reagent for trypsin. Quenching of samples with acid at intervals prior to gel electrophoresis revealed that proteolysis did not occur in sample buffer (pH 6.8), but only at higher pH and during gel electrophoresis. Exposure of trypsin to SDS prior to addition of trypsin inhibitor resulted in an irreversible loss of activity with a half-life of about 10 s. It is proposed that the trypsin inhibitors stabilize trypsin by retarding its denaturation in SDS. The substrate for these experiments was the alpha subunit of the Na,K-ATPase. The same pattern of Na,K-ATPase fragments was obtained with bovine and porcine trypsin and with rat and porcine Na,K-ATPases. Different fragments resulted when chymotrypsin or elastase were substituted for trypsin; these proteases were active in the absence of an inhibitor, and were not markedly stabilized by interaction with soybean trypsin-chymotrypsin inhibitor (Bowman-Birk).     

Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis

Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.     

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth,biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml^?1) inhibited Trichosporon growth. RIT (100 μg ml^?1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml^?1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.     

Activation of bean (Phaseolus vulgaris) alpha-amylase inhibitor requires proteolytic processing of the proprotein.

Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the alpha-amylases of mammals and insects. This alpha-amylase inhibitor (alpha AI) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M(r)) 15,000 to 18,000. We report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, we found that antibodies to alpha AI recognize large (M(r) 30,000-35,000) polypeptides as well as typical alpha AI processing products (M(r) 15,000-18,000). Alpha AI activity was found in all extracts that had the typical alpha AI processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant alpha AI in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-alpha AI when the gene is expressed in tobacco. When pro-alpha AI was separated from mature alpha AI by gel filtration, pro-alpha AI was found not to have alpha-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule.     

The interactive effects of irradiance and photoperiod on Chara vulgaris L.: concerted responses in morphology, physiology, and reproduction

Both the single and interactive effects of irradiance and photoperiod on a worldwide charophyte species, Chara vulgaris L., were investigated in a greenhouse experiment. Under high light intensity, plants exhibited shortened shoot, compact canopy and low Chl a/Caro. In contrast, elongated shoot, expanded canopy and low Chl a/Chl b corresponded to low light intensity. In addition, both ash mass ratio and relative growth rate (RGR) were positively related to light intensity. The effect of photoperiod on plants was relatively complex. Although photoperiod did not affect morphological characters, an increased photoperiod significantly decreased Chl a/Chl b and Chl a/Caro ratios. Two-way analysis of variance (ANOVA) analysis indicated that significant interaction of irradiance and photoperiod was present on ash mass ratio, RGR, Chl a/Chl b, Chl a/Caro and Chl (a + b)/Caro ratios. The high RGR found for 50% sunlight, L:D 16:8 (50/16) conditions along with the significant interaction of irradiance and photoperiod on RGR indicated that the effect of irradiance was more important than photoperiod for plant growth. And finally, both irradiance and photoperiod had positive effects on the emergence of sex organs (♂♀) and the maturation of oospores, except that increased photoperiod did not accelerate the maturation of oospores. In summary, for both plant growth and reproduction, C. vulgaris was able to acclimate morphologically and physiologically to different irradiance levels and photoperiods. This study can partly explain the broad geographic distribution of C. vulgaris. Handling editor: S. M. Thomaz