Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Structural relatedness of lysis proteins from colicinogenic plasmids and icosahedral coliphages

The host-lysis-inducing functions of phi X174 protein E and MS2 protein L were recently shown to reside on the N-terminal and C-terminal halves of the two respective lysis proteins. In the present study it is shown that the small lysis proteins encoded in various colicinogenic plasmids share local sequence similarities and certain structural characteristics with the essential peptides of their coliphage-coded counterparts. Despite their dissimilar sizes and origins, it is suggested that the colicinogenic lysis proteins are functionally analogous and evolutionarily related to those of icosahedral single- stranded DNA and RNA phages.      

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Use of antisense RNA to help identify a genomic clone for the 5' region of mouse beta-glucuronidase

It was possible to gauge the inhibition of mouse beta-glucuronidase expression by injecting RNA, made from both strands of subclones of a cosmid containing the complete gene, into mouse blastomeres at the four-cell stage. Although our initial screen did not identify the 5' region, we were able to isolate a subclone containing homology to 20 bp coding for N-terminal amino acids of rat and human beta-glucuronidase structural genes. Antisense RNA prepared from one strand of the 350 bp Pst I subclone inhibited beta-glucuronidase expression by 89% while RNA prepared from the other strand had little effect. The subclone appears to correspond to the 350 bp fragment identified by others as one including the ATG start site of mouse beta-glucuronidase.     

Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of newly synthetized oocyte protein and (2) cytochemical F-actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP-associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP-associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to α-chymotrypsin, the amount of newly synthesized actin displayed by cumulus-enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP-associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.     

Ferritin H and L mRNAs in human neoplastic tissues

The typical tissue isoferritin pattern varies during neoplastic transformation, usually shifting toward a more acidic profile. To investigate the molecular basis of this phenomenon, we have analyzed the steady-state levels of the H and L mRNAs in several neoplastic tissues. By using specific probes for the two ferritin subunits, we have found, in three different adenocarcinomas and in a case of Hodgkin lymphoma, a two- to four-fold increase of the H and L mRNA levels compared to those found in normal human liver.     

Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor

Regulation of the fibrinolytic system of cultured human umbilical vein endothelial cells (HUVECs) by recombinant interleukin 1 beta (rIL-1 beta) and tumor necrosis factor alpha (rTNF alpha) was investigated. Functional and immunologic assays indicated that both cytokines decreased HUVEC tissue-type plasminogen activator (tPA) and increased type 1 plasminogen activator inhibitor (PAI-1) in a dose- and time-dependent manner. Maximal effects (50% decrease in tPA antigen; 300-400% increase in PAI-1 activity) were achieved with 2.5 units/ml rIL-1 beta and 200 units/ml rTNF alpha. Combinations of rIL-1 beta and rTNF alpha were not additive at these maximal concentrations. After a 24-h pretreatment with rIL-1 beta, HUVECs secreted tPA at one-quarter of the rate of control cells and released PAI-1 at a rate that was 5-fold higher than controls. Neither the basal rate of PAI-1 release nor the increased rate of release of PAI-1 in response to rIL-1 beta was affected by subsequently treating the cells with secretagogues (e.g. phorbol myristate acetate) suggesting that PAI-1 is not contained within a rapidly releasable, intracellular storage pool. Northern blot analysis using a PAI-1 cDNA probe indicated that the cytokines increased the steady-state levels of the 3.2- and 2.3-kb PAI-1 mRNA species, but with a preferential increase in the larger mRNA form. The fact that both rIL-1 beta and rTNF alpha act in a similar manner strengthens the hypothesis that the local development of inflammatory/immune processes could reduce endothelial fibrinolytic activity.     

Molecular analysis of gene deletion in aniridia-Wilms tumor association

Summary Hybrid clones were produced from the fusion of Chinese hamster cells and human fibroblasts from a patient with the aniridia-Wilms tumor association (AWTA). The DNA from the parental cells and the hybrid clones was screened by Southern blot and DNA hybridization with probes for the human insulin and Ha-ras-1 genes. Two alleles for the Ha-ras-1 gene were shown to exist in the AWTA cells by restriction fragment length polymorphism. One hybrid clone, containing a single allele for Ha-ras-1 was shown to contain a single chromosome 11 with a cytogenetically visible deletion at 11p13. The DNA from this hybrid contained the human genes for insulin, ^A, ^G, Ha-ras-1, and calcitonin, but lacked any human sequences homologous to a human catalase cDNA. This clone was also shown to express human lactate dehydrogenase A (LDH A) activity. These data indicate that the deletion of the affected chromosome in this AWTA patient begins distal to LDH A and includes band 11p13, but does not extend to calcitonin or other genes thought to be located in the distal half of chromosome 11p.     

Collagen remodeling during decidualization in the mouse

Summary An ultrastructural study of the features and distribution of collagen fibrils was performed in the endometrium of virgin and pregnant (2nd to 11th day) mice. Collagen-containing structures were observed in the cytoplasm of fibroblasts on the 2nd day of pregnancy. Treatment of tissues with lanthanum nitrate established that these structures were intracytoplasmic. Their association with lysosome-like bodies suggested the occurrence of intracellular digestion of collagen, probably connected with remodeling of the endometrial stroma prior to decidualization. On the 4th day of pregnancy, very few collagen fibrils were present in the intercellular space. From the 6th day of pregnancy onwards, thick collagen fibrils were observed between decidual cells. The diameter of these fibrils measured up to 300 nm whereas the fibrils present in the endometrium of virgin mice measured 40–68 nm.