Sperm surface galactosyltransferase activities during in vitro capacitation

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as “decapacitation factors” when added back to in vitro fertilization assays. These glycoside “decapacitation factors” inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces “decapacitation factio” competition. On the other hand “conventional” low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of “decapacitation factos” is a necessary prerequisite for sperm binding to the zona pellucida.     

Spectral properties of a long-wavelength absorbing form of protochlorophyll in seeds of Cyclanthera explodens

The in vivo absorbance spectrum of the inner seed coat of Cyclanthera explodens Naud. showed a main peak in the red region at 671 nm and a weak shoulder at about 640 nm. The pigments were extracted with acetone. separated by paper chromatography and analysed spectrophotometrically. The only detectable pigment was protochlorophyll. The in vivo fluorsecence emission spectra had two main peaks, one at 632 and one at 691 nm. The relation between the two peaks was changed when the exvcitation wavelength was altered from 440 to 460 nm. Excitation at 420 nm gave an additional fluorescence emission peak at 595 nm. These data indicate the presence of at least three forms of protochlorophyll in the Cyclantera seed coat. The spectrum of circular dichroism had a very intence and characteristic signal in the red region with a negative asymmetrical Cotton effect (664 (+), 669 (0) and 687 (?) nm). This indicates that at least one of the protochlorophyll forms is present in a more or less crystalline form.     

Expression of XET-related genes and its relation to elongation in leaves of barley (Hordeum vulgare L.)

Five cDNA clones were isolated from barley (Hordeum vulgare L.) that encoded mRNAs related to xyloglucan endotransglycosylase (XET). One of the clones encoded a protein with XET activity in vitro. Sequence comparisons revealed five families of XET-related sequences, one of which (containing two of the barley genes) was novel. Hybridization studies using clone-specific probes indicated that the corresponding genes were represented once, or possibly twice, in the barley genome. Treatment of dwarf mutants with gibberellic acid (GA₃), or homozygosity at the ‘slender’ (sln1) locus, resulted in a 2.5-fold (approximately) stimulation of blade elongation rate. Three of the five clones detected mRNAs that were maximally expressed towards the base of the blade, and present in greater quantities in GA₃-treated or slender seedlings. The remaining two clones detected mRNAs that were maximally expressed in the middle of the blade. Relative elemental growth rate (REGR) profiles of leaves growing with or without GA₃ treatment revealed similar maximal REGR values despite a 2.5-fold difference in leaf elongation rate. Segments of GA₃-treated leaves attained their maximal REGR values more rapidly, this being associated with enhanced expression of the three ‘basal’ XET-related mRNAs. Highest XET activities were detected in the base of the elongation zone, and in GA₃-treated seedlings a second activity peak was observed near the distal end of the elongation zone. We conclude that there are likely to be several XET isoenzymes with different expression patterns, and identify those XET-related proteins potentially involved in leaf elongation.     

Physiological Reorganization and Post-Traumatic Regeneration in Stylonychia mytilus: Reflections on Developmental Constraints and the Evolutionary Origin of Alternative Modes of Asexual Morphogenesis

We investigated development of cortical ciliature in Stylonychia mytilus during starvation-induced physiological reorganization, and during regeneration following amputation of the anterior part of the cell. Cortical reorganization in the two processes is generally similar. The posterior part of the adoral zone of membranelles is resorbed and replaced with newly assembled membranelles. The pre-existing set of ventral cirri and dorsal bristles is entirely resorbed and replaced with new ones. Regenerants exhibit posterior displacement of the frontal-ventral-transverse cirri primordium and the undulating membrane primordium, and recruit basal bodies from ectopic locations for the development of these ciliature. This illustrates flexibility in the initiation site of ciliary primordia, and opportunism in utilizing building blocks. Such morphogenetic versatility of hypotrichs provides the basis for the operation of a global control of pattern formation, which governs cortical reorganization in dividers, and additionally, in the absence of the prerequisites for binary fission, alternative modes of cortical development such as physiological reorganization or regeneration. These considerations suggest that the three processes are homologous and that physiological reorganization and regeneration have evolved from binary fission. In physiological reorganization and regeneration, the micro- and macronuclei reorganize to resemble that in binary fission; these nuclear events are considered evolutionary relics of the nuclear development of binary fission. Tetrahymena also exhibits such morphogenetic flexibility; stomatogenesis is under global control, so that asexual cells can replace its oral apparatus without undergoing binary fission. Paramecium , on the other hand, adopts a more rigid strategy in relying heavily on pre-existing structures for morphogenetic cues; this could have imposed constraints in the exploration of alternative modes of asexual development.     

怀集石燕燕窝促细胞分裂活性的研究

1.借助Bio—Gel P—10柱层析法,由怀集石燕燕窝水提物部分纯化得一具有EGP活性的成分EGF-2。 2.在放射标记受体活性测定中,EGF-2与受体的竞争性结合曲线与小鼠EGF标准曲线相平行。EGF-2能显著刺激氚标记的胸腺嘧啶脱氧核苷对小鼠3T3成纤维细胞的掺入作用。这种活性不受抗小鼠EGF抗体的抑制。 3.借助Sephacry1-200 Superfine柱层析法,由上述水提物分离得一蛋白质组分(S-200Ⅰ+Ⅱ),对培养的人脐带淋巴细胞有促细胞分裂作用。并对培养的、经Con A转化的淋巴细胞有辅促细胞分裂作用。     

Influence of localized auxiliary heating on hand comfort during cold exposure

There is a needfor a hand-heating system that will keep the hands warm during coldexposure without hampering finger dexterity. The purpose of this studywas to examine the effects of torso heating on the vasodilativeresponses and comfort levels of cooled extremities during a 3-hexposure to 15°C air. Subjects were insulated, but theirupper extremities were left exposed to the cold ambient air. The effectof heating the torso [torso-heating test (THT)] on handcomfort was compared with a control condition in which no torso heatingwas applied, but Arctic mitts were worn [control test(CT)]. The results indicate that mean finger temperature, meanfinger blood flow, mean toe temperature, mean body skin temperature, body thermal comfort, mean finger thermal comfort, and rate of bodyheat storage were all significantly (P < 0.05) higher on average (n = 6)during THT. Mean body heat flow was significantly (P < 0.05) lower during THT. Therewere no significant differences (P  0.05) in rectal temperature between CT and THT. Mean unheated body skintemperature and mean unheated body heat flow (both of which did notinclude the torso area in the calculation of mean body skin temperatureand mean body heat flow) were also calculated. There were nosignificant differences (P  0.05) inmean unheated body skin temperature and mean unheated body heat flowbetween CT and THT. It is concluded that the application of heat to the torso can maintain finger and toe comfort for an extended period oftime during cold exposure.

     

Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA) function using a collection of mutants defective in all TCA cycle genes

The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomyces cerevisiae. We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins. The 15 major TCA cycle genes can be sorted into five phenotypic categories on the basis of their growth on nonfermentable carbon sources. We have previously reported a novel phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH). Null and nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the TCA cycle citrate synthase and in other genes of oxidative metabolism. The TCA cycle mutant collection was utilized to search for other genes that can suppress idh2 mutants and to identify TCA cycle genes that display a similar suppressible growth phenotype on glycerol. Mutations in 7 TCA cycle genes were capable of functioning as suppressors for growth of idh2 mutants on glycerol. The only other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype was IDH1, which encodes the companion Idh1p subunit of NAD-IDH. These results provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function.     

Occurrence of Peronospora sparsa (P. rubi) on cultivated and wild Rubus species in Finland and Sweden

An outbreak of a dryberry disease caused by Peronospora sparsa (syn. P. rubi) occurred in plantations of arctic bramble (Rubus arcticus subsp. arcticus) in Finland in the middle of 1990s. The disease persists and is most severe in cool and rainy summers. The disease has not been encountered in northern Sweden where cultivars (R. arcticus nothosubsp. stellarcticus) different from those in Finland are used. The occurrence of P. sparsa in wild Rubus spp. is virtually unknown in both areas and it is not known whether they constitute a potential infection source. Therefore, the aim of this study was to investigate the occurrence of P. sparsa on wild Rubus spp. growing in the vicinity of cultivations of arctic bramble. Symptomatic plants were sampled in 1997–1999. P. sparsa was detected using a light microscope, preceded by incubation of the sample in vitro if necessary, and by a polymerase chain reaction (PCR) based method. Plants of cultivated R. arcticus subsp. arcticus were commonly infected by P. sparsa in Finland. P. sparsa was also found on the cultivated R. arcticus nothosubsp. stellarcticus in Finland and Sweden. However, the infected plants of the cultivars of nothosubsp. stellarcticus seemed to be much less damaged than the cultivars of subsp. arcticus. Plants infected with P. sparsa were found in the populations of wild R. arcticus subsp. arcticus in both countries, and in cloudberry (R. chamaemorus) in natural habitats in Finland. In addition, P. sparsa was detected on specimens of R. arcticus subsp. arcticus (collected in 1966–1985) and R. chamaemorus (collected in 1899–1981) in Finnish herbaria. The samples of R. idaeus and R. saxatilis collected from the field in this study or investigated in the herbaria were not infected with P. sparsa. These data show that P. sparsa has not recently invaded Finland but has become an economically significant pathogen during the rapid expansion of cultivation of the apparently sensitive clones of arctic bramble.     

Rapid and simple method to determine chloroquine and its desethylated metabolites in human microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive and selective method was developed for the simultaneous determination of chloroquine (CQ) and its desethylated metabolites monodesethylchloroquine (DCQ) and bisdesethylchloroquine (BDCQ) in human liver microsomes. Analytes were separated on a C₁ column using methanol-water (70:30, v/v) and triethylamine (0.1% v/v) as the mobile phase. The fluorescence detector was set at 250 (excitation) and 380 nm (emission). Following protein precipitation with ice-cold acetonitrile, microsomal incubation supernatants were directly injected into the HPLC system. Typically, 200 μl of incubate were diluted with 200 μl of acetonitrile and 15 μl were injected. The limit of quantitation was 78 nM of CQ or metabolite. Intra-day variability averaged 2.9% for CQ, 1.5% for DCQ and 2.5% for BDCQ. Inter-day variability was 3.1% for CQ, 3.5% for DCQ and 3.7% for BDCQ. Mean accuracies were 100% for CQ and BDCQ and 102% for DCQ.     

Isolation of 15 polymorphic microsatellite loci in Acacia hybrid (Acacia mangium×Acacia auriculiformis)

This study reports the isolation of 15 microsatellites in Acacia hybrid (Acacia mangium×Acacia auriculiformis) based on the 5′ anchored polymerase chain reaction technique. Polymorphism of these loci was evaluated in a sample of 24 hybrid individuals. The level of polymorphism ranged from two to eight alleles with an observed heterozygosity ranging from 0.083 to 0.875. These loci were also characterized in both the parental species. The number of alleles ranged from two to six for both A. mangium and A. auriculiformis with the observed heterozygosity values ranging from 0.167 to 0.625 and 0.042 to 0.458 respectively. Five of these loci demonstrated Mendelian inheritance in a segregating F₁ population; four presented a distorted ratio and the remaining six did not segregate in progenies as they were homozygous in both parents.