Partial purification of cell wall α-galactosidases and α-arabinosidases from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes

Two protein fractions with activity as α-galactosidase (EC 3.2.1.22) and α-arabinosidase (EC 3.2.1.55), respectively, were identified in the proteins of cell wall of Cicer arietinum L. cv. Castellana extracted with 3 M LiCl. These fractions were partially purified by gel filtration chromatography (Bio Gel P-150), increasing the specific arabinosidase activity 57-fold and the α-galactosidase activity 6-fold. Other protein fractions with glucosidase (EC 3.2.1.21) and glucanase (EC 3.2.1.6) activity also appeared. According to earlier authors, α-arabinosidases and α-galactosidases are related to alterations in linkages occurring in cell walls, since the enzymes are able to hydrolyze isolated wall polymers. However, our preparations hydrolyze intact cell walls only to a very limited extent, such that their participation in the autolytic processes of cell walls can be ruled out.     

Translational activation of uncapped mRNAs by the central part of human eIF4G is 5' end-dependent.

Translation initiation factor (eIF) 4G represents a critical link between mRNAs and 40S ribosomal subunits during translation initiation. It interacts directly with the cap-binding protein eIF4E through its N-terminal part, and binds eIF3 and eIF4A through the central and C-terminal region. We expressed and purified recombinant variants of human eIF4G lacking the N-terminal domain as GST-fusion proteins, and studied their function in cell-free translation reactions. Both eIF4G lacking its N-terminal part (aa 486-1404) and the central part alone (aa 486-935) exert a dominant negative effect on the translation of capped mRNAs. Furthermore, these polypeptides potently stimulate the translation of uncapped mRNAs. Although this stimulation is cap-independent, it is shown to be dependent on the accessibility of the mRNA 5' end. These results reveal two unexpected features of eIF4G-mediated translation. First, the C-terminal eIF4A binding site is dispensable for activation of uncapped mRNA translation. Second, translation of uncapped mRNA still requires 5' end-dependent ribosome binding. These new findings are incorporated into existing models of mammalian translation initiation.     

Oocytes Preserve the Ability of Mouse Cumulus Cells in Culture to Synthesize Hyaluronic Acid and Dermatan Sulfate

A soluble factor(s) produced by fully grown oocytes is essential, together with follicle stimulating hormone (FSH), to stimulate in vitro hyaluronic acid (HA) synthesis by mouse cumulus cells (CCs). The stability of the response to this stimulus by CCs in culture was investigated. The data showed that preculture for 8 hr in basal medium reduced to approximately 30% the ability of CCs to synthesize HA in response to FSH or dibutyryl cyclic AMP (Bt₂cAMP) and soluble oocyte factor(s). However, if CCs were precultured for the same period of time as intact cumulus cell-oocyte complexes, or in the presence of fully grown oocytes, or in medium conditioned by fully grown oocytes, their ability to synthesize HA was 75-95% preserved. In vitro stimulation of dermatan sulfate (DS) synthesis by CCs does not require oocyte factors and is induced by FSH or Bt₂cAMP treatment alone. However, the preservation of such activity, like that of HA synthesis, depended on the presence of a soluble oocyte factor(s) during preculture. The presence of isolated oocytes or of oocyte-conditioned medium also prevented the spreading of CCs in culture. However, inhibiting CC spreading by culture on agar-coated plates or in serum-free medium did not preserve their HA or DS synthetic activity, thus suggesting that the two oocyte actions on CCs are independent. Growing oocytes were unable both to induce HA synthesis in freshly isolated CCs stimulated with FSH and to preserve the ability to synthesize HA and DS in 8-hr precultured CCs. The results suggest that the stability of the differentiated state of mouse CCs in vitro depends upon continued exposure to a soluble factor(s) produced by fully grown oocytes.     

Intracellular progesterone receptors are differentially regulated by sex steroid hormones in the hypothalamus and the cerebral cortex of the rabbit

The aim of this study was to examine the role of sex steroid hormones in the regulation of intracellular progesterone receptors (PR) in the rabbit central nervous system. We determined PR concentration in cytosol preparations from the hypothalamus, the frontal, tempo-parietal and occipital cortex, by using the specific binding of the synthetic progestin [³H]ORG 2058. PR concentration was higher in the hypothalamus of intact adult females than in that of adult males and prepubertal females, whereas no significant differences were observed in the cerebral cortex of these animals. PR concentration was similar in the three cortical regions analyzed, indicating a homogeneous distribution of PR in the cerebral cortex. The administration of estradiol to ovariectomized animals increased PR concentration in the hypothalamus but not in the cortex. The administration of progesterone to ovariectomized rabbits did not modify PR concentration in any region, however when progesterone was administered after estradiol, it induced a significant diminution in hypothalamic PR concentration without effects in the cortex. These findings suggest that in the rabbit, PR are estrogen regulated in the hypothalamus but not in the cerebral cortex. In the latter, PR are not regulated by progesterone, whereas in the former the estrogen-induced PR are down-regulated by progesterone. Interestingly, hypothalamic PR constitutively expressed in ovariectomized animals are progesterone-insensitive.     

Author index for volume 93

The presence of Ca²⁺ is essential for survival in culture of fully grown oocytes isolated from mouse ovaries but not for survival of small, meiotically incompetent oocytes, metaphase II oocytes, and early embryos. Ninety percent of fully grown ovarian oocytes die within 2 hr when cultured in calcium-free medium (CFM). CFM death does not occur if other cations (1 mM La³⁺ or 10 mM Sr²⁺, but not 12 mM Mg²⁺ nor 1 mM D-600) replace Ca²⁺ in the medium. Sensitivity to CFM is progressively acquired by the oocyte during the growth phase, in parallel with the acquisition of meiotic competence, and is lost after 2 hr of culture in the presence of at least 0.5 mM Ca²⁺. The loss of sensitivity to CFM during in vitro culture is not related to the concomitant spontaneous resumption of meiosis, since the oocyte becomes resistant to CFM even if germinal vesicle breakdown is prevented by the addition of dibutyryl cAMP to the culture medium. Some hypotheses are put forward to explain the peculiar and transient high calcium requirements of fully grown oocytes.     

single cotyledon (sic) mutants of pea and their significance in understanding plant embryo development

Angiosperms are divided into two distinct classes—the dicotyledons (dicots) and monocotyledons (monocots)—based in part on the number of cotyledons in mature embryos. In this paper, we describe single‐cotyledon pea mutants, termed sic (single cotyledon), all of which show a degree of fusion between the cotyledons. The fusion in sic1 is along the margin of one cotyledon and is less complete than in sic2 embryos, but the effects of the mutations are additive in the double mutant. Occasionally sic2 mutants will show fusion of the two cotyledons into one cylindrical embryo in which the shoot apex becomes surrounded by the cotyledons. Both sic1 and sic2 mutants produce fertile plants. In the sic3 embryo, a single cotyledon is generated under the shoot apex that breaks the vascular connection between root and shoot, causing embryo lethality. The pattern of cotyledon development in all these mutants is identified by in situ mRNA hybridization and antibody labeling, using the storage protein vicilin as a cotyledon‐specific marker. These patterns indicate that the joining of the cotyledons was due to zonal growth. The results indicate that there are genes in pea that influence the positioning and the morphology of the cotyledon. A model for cotyledon development in pea is proposed that is based on the regulation of the positioning of cell clusters by the sic genes. Dev. Genet. 25:11–22, 1999. © 1999 Wiley‐Liss, Inc.     

MFASAT: a new alphoid DNA sequence isolated from Macaca fascicularis (Cercopithecidae, Primates).

A new highly repeated DNA fragment isolated from Macaca fascicularis (MFASAT) is described. Our findings obtained by sequencing, Southern blot analysis, and fluorescent in situ hybridization (FISH) on metaphasic chromosomes strongly suggest that MFASAT can be considered as a member of the alphoid DNA family characteristic of Old World monkeys. The chromosomal localization of MFASAT, obtained by FISH, showed that this alphoid DNA is present in the peri-centromeric area of all the chromosomes. MFASAT showed a high degree of conservation when compared, by sequence alignment, to other Macaca species and Papio papio as expected for species with considerable genome conservation. A low degree of homology has been found comparing M. fascicularis alphoid DNA with a more distantly related Cercopithecidae species such as Cercopithecus aethiops.     

Superior activity of the type C class of ISS in vitro and in vivo across multiple species

CpG-C are a novel class of CpG motif-containing immunostimulatory sequences (ISS) that includes both a 5'-TCG element and a CpG-containing palindrome. CpG-C drive all known ISS activities and, in particular, are potent enhancers of IFN-alpha from plasmacytoid dendritic cells (PDCs). In our examination of CpG-C sequence requirements, we determined that optimal IFN-alpha-inducing activity could be achieved with longer palindromes. Longer palindromes also correlated with maintenance of the double-stranded (ds) form despite concentration and pH changes, indicating a preference for ds oligodeoxynucleotides (ODNs) by the ISS-induced signaling mechanism for IFN-alpha synthesis. This correlation did not hold for all arms of the ISS-induced immune response, since we did not observe increased B cell activity with the longer palindrome CpG-C ODNs. We further demonstrated that CpG-C retained activity in an in vitro primate system and induced the expression of several cytokines and IFN-alpha-inducible genes when CpG-C were administered in vivo to mice and primates. In conclusion, we have shown CpG-C to exert several types of immune functions across multiple species, and this novel class is thus an attractive candidate for ISS-based therapeutic strategies.