Factor in urinary extracts from pregnant women that inhibit mouse oocyte maturation in vitro

Mouse oocyte maturation inhibitory factors, on the basis of inhibitory activity of spontaneous germinal vesicle breakdown (GVBD) of denuded mouse oocytes in culture, were extracted and partially purified by reversed-phase resin adsorption and Sephadex G-100 and G-50 column chromatographies from the urine of pregnant women. Denuded oocytes obtained from ovaries of ICR mice underwent spontaneous GVBD by cultivation for 3 h in modified Krebs–Ringer's buffered solution, while this spontaneous GVBD was found to be inhibited by adding the final preparation (U-D-4) of urine. The inhibition was dose dependent, ranging from 0.6 to 10 μg protein/ml medium. Oocytes treated with U-D-4 and resuspended in control medium resumed GVBD. The molecular mass of U-D-4 was estimated to be less than 2,000 Da with gel filtration. Ether treatment failed to extract inhibitory factor(s) from U-D-4 and pepsin treatment inactivated U-D-4, indicating that inhibitory factor(s) in U-D-4 are peptide-like substances. The inhibitory effect of U-D-4 on spontaneous GVBD was partially reversed in the presence of naloxone, a potent opioid antagonist. U-D-4s obtained from urine samples of pregnant women, nonpregnant women, and men showed the inhibitory effect on spontaneous GVBD; however, the activity of U-D-4 obtained from pregnancy urine was significantly more potent than those of the other urine samples. © 1993 Wiley-Liss, Inc.     

Impacts of iron on phosphate starvation-induced root hair growth in Arabidopsis

In Arabidopsis, phosphate starvation (-Pi)-induced responses of primary root and lateral root growth are documented to be correlated with ambient iron (Fe) status. However, whether and how Fe participates in -Pi-induced root hair growth (RHG) remains unclear. Here, responses of RHG to different Fe concentrations under Pi sufficiency/deficiency were verified. Generally, distinct dosage effects of Fe on RHG appeared at both Pi levels, due to the generation of reactive oxygen species. Following analyses using auxin mutants and the phr1 mutant revealed that auxin and the central regulator PHR1 are required for Fe-triggered RHG under −Pi. A further proteomic study indicated that processes of vesicle trafficking and auxin synthesis and transport were affected by Fe under −Pi, which were subsequently validated by using a vesicle trafficking inhibitor, brefeldin A, and an auxin reporter, R2D2. Moreover, vesicle trafficking-mediated recycling of PIN2, an auxin efflux transporter, was notably affected by Fe under -Pi. Correspondingly, root hairs of pin2 mutant displayed attenuated responses to Fe under -Pi. Together, we propose that Fe affects auxin signalling probably by modulating vesicle trafficking, chiefly the PIN2 recycling, which might work jointly with PHR1 on modulating -Pi-induced RHG.     

Inhibition of Kinesin Synthesis In Vivo Inhibits the Rapid Transport of Representative Proteins for Three Transport Vesicle Classes into the Axon

Abstract: We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin-like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign a specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide-containing synaptic vesicles, the amyloid precursor protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations.     

Persistent Enhancement of Sustained Calcium-Dependent Glutamate Release by Phorbol Esters: Role of Calmodulin-Independent Serine/Threonine Phosphorylation and Actin Disassembly

Abstract: The phorbol ester 4β-phorbol 12,13-dibutyrate increases the final extent of Ca²⁺-dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca²⁺. Previous work from our laboratory demonstrated that this 4β-phorbol 12,13-dibutyrate-dependent enhancement of synaptic vesicle recycling persists following the removal of 4β-phorbol 12,13-dibutyrate, requires localized Ca²⁺ entry through voltage-regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C- and multifunctional Ca²⁺/calmodulin-dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4β-phorbol 12,13-dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca²⁺/calmodulin-dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester-dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca²⁺-dependent glutamate release through a kinase-independent mechanism. Given our finding that the actin-depolymerizing agent cytochalasin D totally occluded the effect of 4β-phorbol 12,13-dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cytoskeletal protein kinase C binding proteins.     

Alterations in Phosphatidylcholine Metabolism of Stretch-Injured Cultured Rat Astrocytes

Abstract: The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic-bottomed tissue culture wells in medium that was usually supplemented with 10 µM unlabeled arachidonate. Cell injury was produced by stretching (5–10 mm) the Silastic membrane with a 50-ms pulse of compressed air. Stretch-induced cell injury increased the incorporation of [³H]choline into PC in an incubation time- and stretch magnitude-dependent manner. PC biosynthesis was increased three- to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch-induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A₂ (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch-induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretrating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n-acetylcysteine). Arachidonate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non-arachidonate-enriched cells. In summary, the data suggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-catalyzed Haber-Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch-induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch-induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.     

The protein content and morphogenesis of zymogen granules

When zymogen granules, the secretion granules of pancreatic acinar cells, fill, secretory product is accumulated in immature granules, condensing vacuoles. Mature granules are formed when this product (protein) condenses into an osmotically inactive aggregate and, bulk water is expelled. This hypothesis for granule morphogenesis has two elements. The first is that immature granules are precursors to mature granules. The second is that a particular maturational event, condensation, which involves the aggregation of protein, takes place. These hypotheses lead to two straightforward predictions. One, that condensing vacuoles on average, should contain less protein than filled or mature granules. And two, that, due to condensation, mature granules should contain protein at a common concentration. In the current work, both of these predictions were tested using measurements of the protein content of individual granules acquired by X-ray microscopy. Neither prediction was affirmed by the experimental results. First, there was no distinguishable difference in the distribution of protein between immature and mature granules. Second, the protein concentration of mature granules varied widely between preparations, although granules from the same preparation had similar concentrations. From the data we conclude that: 1) mature granules and condensing vacuoles are different, though not necessarily unrelated, types of secretory vesicle, and not two forms of the same object; 2) as such, condensing vacuoles are not precursors to mature granules; 3) all granules do not contain protein at one particular concentration when full, or mature; 4) granule maturation does not involve a condensation step; 5) concentration is not determined by such physical limits as the space available for protein packing or condensation; and 6) the amount of protein contained is physiologically regulated.     

Phosphatidylcholine could be the source of 1,2-DAG which activates protein kinase C in EGF-stimulated colon carcinoma cells (HT29)

In our previous study (A. Balogh et al, Cell. Signalling 5 (6), 795-802, 1993.), we have shown that epidermal growth factor (EGF) increased protein kinase C (PKC) activities in colon carcinoma cell line (HT29), possibly through the increased 1,2-diacylglycerol (1,2-DAG) production via phosphatidylcholine (PC). Here we investigate the effect of the well-known PKC activator 12-O-tetradecanoyl-2 phorbol-13-acetate (TPA), on the levels of ³²P incorporation into EGF induced phosphatidylinositols (PI, PI4P, PI4, 5P₂) and different phospholipids (PC, PA, PS) as well as on induced tyrosine kinase activity. TPA significantly decreased the effects of EGF and it had the biggest inhibitory effect on EGF induced PC level. These data support our contention that PC plays an important role in the activation of PKC via 1,2-DAG production in the EGF stimulated pathway.     

Determination of the labyrinth in different amphibian species and its correlation with determination of the other ectoderm derivatives

The process of labyrinth determination has been studied in three urodelean and seven anuran species by means of homoplastic transplantation of ear region epidermis, defined as the piece of epidermal layer containing the material of the prospective ear vesicle (labyrinth rudiment). The ear region epidermis was grafted onto the abdominal wall of embryos of the same developmental stage. The earliest stage of operation resulting in ectopic ear vesicle formation was determined, suggesting the appearance of organ-specific properties in the ear ectoderm. These properties were enhanced in the further course of development, as indicated by the frequency of ear vesicle formation and by the volume and degree of complexity that the vesicles reached. The data obtained allowed us to arrange the species studied in a sequence, ranging from most Ranidae and Bufo viridis, in which organ-specific properties appear earlier and are most strongly expressed, to Triturus vulgaris in which their expression is least pronounced. Comparison of properties of the material giving rise to the ear vesicle or to several other ectodermal derivatives led to the conclusion that species-specific differences in determination of different ectodermal rudiments are due to species specific properties of the whole ectoderm. These differences appear to be determined by an evolutionary shift of the beginning of gastrulation towards later cleavage cycles.Deceased in November 1993     

牛胰多肽与CL／PC脂质体作用后二级结构的变化

用傅立叶变换红外光谱研究了ＢＰＰ与膜作用后其二级结构的变化,通过对红外光谱中酰胺Ｉ谱带进行解卷积、微分及曲线拟合等处理,结果表明,ＢＰＰ与脂膜作用后,其二级结构中α－螺旋成分增多,无规卷曲成分减少。     

Characterisation of phosphatidylcholine/phosphatidylinositol sonicated vesicles. Effects of phospholipid composition on vesicle size

Phosphatidylinositol (PI), dipalmitoylphosphatidylcholine (DPPC) and mixed lipid (DPPC plus PI) sonicated vesicles have been prepared covering a range of composition. The vesicles were characterised by gel filtration, electron microscopy and photon correlation spectroscopy. The dimensions of the vesicles as measured by electron microscopy were in good accord with those obtained from photon correlation spectroscopy measurements. The number average diameters of the vesicles increase on increasing the PI content and range from approx. 30–80 nm as the weight % of PI is increased from 0 to 100. Gel filtration on Sepharose 4B columns gave anomalous results indicating that PI-containing vesicles were retarded on the gel possibly due to an interaction between the inositol headgroup and the gel matrix. Electrophoretic measurements on multilamellar vesicles show that the surface charge density increases with the PI content of the vesicles upto 50 weight % PI and remains constant thereafter. The radii of sonicated vesicles also increase with PI content which reflects a decreasing liposome curvature with increasing surface charge density.