Metabolic activities in germinated ancient lotus seeds

Seeds of Taizi lotus (Nelumbo nucifera Gaertn cv. Taizi) wereunearthed in a western suburb of Beijing in 1984, and determinedto be 58070-years-old by radiocarbon dating. Pretreatment withconcentrated sulphuric acid for 6 h promoted 75% of the seedstested to germinate. Over 3 weeks of incubation after the additionof water, storage starch, total soluble sugar and globulin inthe cotyledons decreased whereas albumin and soluble -aminonitrogen increased. These changes in cotyledonary componentswere similar to the changes seen in four present-day Indianlotus varieties, although the content of reducing sugar in Taizilotus was significantly lower. The polypeptide compositionsin extracts from the cotyledons and embryonic axis of Taiziseeds, as analysed by SDS-PAGE, were similar to those of present-dayJiangxi seeds. Taizi lotus also showed the ability to incorporateradioactive leucine into the trichloroacetic acid (TCA)-insolublefraction of a cotyledonary extract during the 24 h post-imbibitionperiod. The incorporation was inhibited when seeds of Taizilotus were allowed to rehydrate for 24 h in the presence of-amanitin or cycloheximide. Key words: Ancient lotus, Nelumbo nucifera, protein synthesis, seed germination, seed viability     

Identification and mapping of functional domains on human T-cell lymphotropic virus type 1 envelope proteins by using synthetic peptides.

To identify the regions that are important in human T-cell leukemia virus type 1 (HTLV-1) envelope function, we synthesized 23 kinds of peptides covering the envelope proteins and examined the inhibitory effect of each peptide on syncytium formation induced by HTLV-1-bearing cells. Of the 23 synthetic peptides, 2, corresponding to amino acids 197 to 216 on gp46 and 400 to 429 on gp21, inhibited syncytium formation induced by HTLV-1-bearing cells but did not affect syncytium formation induced by human immunodeficiency virus type 1-producing cells. The peptide concentrations giving 50% inhibition of syncytium formation for gp46 197 to 216 and gp21 400 to 429 were 14.9 and 6.0 microM, respectively. A syncytium formation assay with overlapping synthetic peptides containing amino acids 175 to 236 and 391 to 448 of the envelope proteins showed that syncytium formation was inhibited by peptides that contained the amino acid sequences 197 to 205 (Asp-His-Ile-Leu-Glu-Pro-Ser-Ile-Pro) and 397 to 406 (Gln-Glu-Gln-Cys-Arg-Phe- Pro-Asn-Ile-Thr). These observations suggest that the two regions corresponding to amino acids 197 to 216 and 400 to 429 are involved] in HTLV-1 envelope function.     

Dictyostelium mucoroides cells exhibit cell cycle-dependent sorting behavior as observed in D. discoideum development

Evidence has been obtained indicating that the cell's position in the cell cycle at the onset of starvation is a naturally occurring variable closely involved in the subsequent sorting and pattern formation during the development of Dictyostelium discoideum Ax2. It is of interest to know whether a similar phenomenon is also noticed in species other than D. discoideum and also without any treatment of cells for cell synchronization. For this, the sorting behavior of D. mucoroides-7 ( Dm7 ) cells and its relation to the cell-cycle phase at the onset of starvation were analyzed, using non-synchronized Dm7 cells pulse-labeled with 5'-bromo-2-deoxyuridine (BrdU). The results demonstrate that Dm7 cells starved at the early G2 phase aggregate most rapidly, but are eventually sorted out to the posterior prespore zone of migrating slugs. In contrast, cells starved at the mid late G2 phase exhibited slower aggregation, but were sorted out to the anterior zone (tip), this being basically similar to the sorting behavior of D. discoideum cells. Measurements of cell numbers and nuclearity provided evidence that approximately 80% of cells progressed their cell-cycle after the formation of multicellular structures (mounds), probably coupling with prespore differentiation as in the case of D. discoideum . Thus, cell cycle-dependent sorting during Dictyostelium development is most likely to be a common phenomenon in different species.     

Two genes controlling acute phase responses by the antitumor polysaccharide,lentinan

Lentinan, a -1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) andMus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence lenght polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 andltn1.2) responsible for DT-APR.ltn1.1 is closely linked toD3Mit11 on chromosome 3 andltn1.2 toD11Nds9 on chromosome 11 (P<0.001). The linkage analysis also suggested thatltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that theltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.     

Promotion of Cocklebur Seed Germination by Allyl, Sulfur and Cyanogenic Compounds

The effects of allyl, sulfur and cyanogenic compounds on thegermination of upper cocklebur (Xanthium pennsylvanicum Wallr.)seeds were examined. Mercaptoethanol and methylmercaptan aswell as KCN, substrates for rßcyanoalanine synthase(CAS), and H₂S and thiocyanate, the products of the CAS catalyzingreaction, were effective in promoting germination, suggestingthe involvement of CAS in germination. Most of allyl compounds, especially allylthiourea, as well asethylene which activated CAS [Hasegawa et al. (1994) Physiol.Plant. 91: 141], promoted the germination in an abnormal typewhich occurred by the predominant growth of cotyledons as didC₂H₄ [Katoh and Esashi (1975) Plant Cell Physiol. 16: 687].However, they failed to activate CAS unlike ethylene, and toliberate free ethylene during an incubation period. It was thuspossible that an C₂H₄-like double bond within allyl compoundscan act to promote seed germination. (Received June 10, 1996; Accepted August 21, 1996)     

Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.     

K-ras Gene Mutation Related to Histological Atypias in Human Colorectal Adenomas

To investigate the relationship of oncogene analysis to morphology, we analyzed K-ras gene mutations by dot-blot hybridization with and without consideration of histological atypias in individual colorectal adenomas. Each of 54 colon polyps were divided into two parts after fixation. One part was used as a mass to assess point mutations; the remaining portion of each polyp was paraffin-embedded, stained with hematoxylin and eosin, and examined for point mutations related to histological atypias. In the first part of our study, K-ras gene mutations at codon 12 were detected in 13 cases (24%). In the second part of our study, 12 cases had distinctly different histological atypias. From each of these 12 cases, two areas, one with higher or one with lower grade atypia in the same polyp were excised to analyze for K-ras gene mutation. Two of these 12 cases (17%) had the mutation in different areas of the same tumor. These two cases contained the mutation only in the areas with higher grade atypia, and only one case added information regarding ras mutation upon microdissection when compared to the entire biopsy. These results suggest that oligonucleotide hybridization can identify the majority of cases containing ras mutations despite regional morphologic variation. Individual cases, however, may contain clonal subpopulations within adenomas with different ras sequences from other regions within the same adenoma.     

Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli.

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.     

Penetration by bull spermatozoa into the zona pellucida of dead bovine oocytes recovered from frozen-thawed ovaries

The present study was carried out to determine if the zona pellucida of dead bovine oocytes obtained from ovaries stored at -196 degrees C could be used to assess penetrability of capacitated bull spermatozoa. Follicular oocytes were recovered from bovine ovaries which were frozen slowly in a box containing dry ice, plunged into liquid nitrogen, and thawed at 37 degrees C. The dead oocytes were inseminated with various concentrations of spermatozoa preincubated for 0 to 4 h. Sperm penetration rates of the dead oocytes were significantly altered by sperm concentration and preincubation time. Dead and living oocytes matured in vitro (control) gave similar patterns of penetrability based on sperm preincubation time. When sperm concentration was increased, the rate of multiple sperm penetration into the dead oocytes also increased significantly, but the rate of penetration into living oocytes did not alter significantly. All dead oocytes from ovaries stored at -196 degrees C for 1 d to 3 mo were penetrated at similar rates by spermatozoa preincubated for 1-h. Thus, we conclude that dead follicular oocytes recovered from frozen ovaries are useful for the assessment of sperm capacitation and/or the acrosome reaction in cattle.