In Vitro HIV-1 LTR Integration into T-Cell Activation Gene CD27 Segment and the Decoy Effect of Modified-Sequence DNA

Integration into the host genome is an essential step in the HIV-1 life cycle. However, the host genome sequence that is favored by HIV-1 during integration has never been documented. Here, we report that CD27, a T cell activation gene, includes a sequence that is a target for in vitro HIV-1 cDNA integration. This sequence has a high affinity for integrase, and the target nucleotides responsible for this higher affinity were identified using a crystal microbalance assay. In experiments involving a segment of the CD27 gene, integration converged in the target nucleotides and flanking sequence DNA, indicating that integration is probably dependent upon the secondary structure of the substrate DNA. Notably, decoy modified CD27 sequence DNAs in which the target nucleotides were replaced suppressed integration when accompanying the original CD27 sequence DNA. Our identified CD27 sequence DNA is useful for investigating the biochemistry of integrase and for in vitro assessment of integrase-binding inhibitors.     

Plant-growth regulators from common starfish (<Emphasis Type="Italic">Asterias amurensis</Emphasis> Lütken) waste

Starfish waste has been shown to be an effective compost material not only in the promotion of plant growth but also in terms of having insecticidal activity. In the present study, plant growth regulation by chemicals from starfish was examined. The aqueous fraction from a hot water extract of the starfish Asterias amurensis Lütken showed plant-growth activity, while the aqueous fraction from a methanol extract inhibited growth of Brassica campestris. The lipophilic fraction from the methanol extract also exhibited a plant growth-promoting effect. The active components from each extract were identified. Asterubine from the hot water extract promoted plant growth. A ceramide from the lipophilic fraction showed root growth promoting effect, and three glucocerebrosides had promotive effects on the entire plant. Asterosaponins were identified as the main growth inhibitors in the aqueous fraction of the methanol extract. These active compounds from starfish waste could be analyzed as potential plant growth regulators in agricultural applications in the future.     

Three types of vitronectin in human blood

Vitronectin is a cell-adhesive glycoprotein in serum and plasma, also termed serum spreading factor and complement S-protein. It consists of a mixture of a polypeptide of molecular weight 75 kilodalton (kDa) and its nicked product of 65 kDa plus 10 kDa. By a quantitative immunoblotting assay, human blood samples could be classified into three distinct vitronectin types; type I (58% of the population) was 75 kDa rich and 65 kDa poor, type II (35% of the population) contained approximately equal amounts of 75 kDa and 65 kDa, and type III (5% of the population) was 75 kDa poor and 65 kDa rich. The vitronectin type did not correlate with age, sex, or ABO blood type.     

Chromosomal mapping of 18S-28S rRNA genes and 10 cDNA clones of human chromosome 1 in the musk shrew (Suncus murinus).

The direct R-banding fluorescence in situ hybridization (FISH) method was used to map 18S-28S ribosomal RNA genes and 10 human cDNA clones on the chromosomes of the musk shrew (Suncus murinus). The chromosomal locations of 18S-28S ribosomal RNA genes were examined in the five laboratory lines and wild animals captured in the Philippines and Vietnam, and the genes were found on chromosomes 5, 6, 9, and 13 with geographic variation. The comparative mapping of 10 cDNA clones of human chromosome 1 demonstrated that human chromosome 1 consisted of at least three segments homologous to Suncus chromosomes (chromosomes 7, 10, and 14). This approach with the direct R-banding FISH method is useful for constructing comparative maps between human and insectivore species and for explicating the process of chromosomal rearrangements during the evolution of mammals.     

Structure and expression of human B cell stimulatory factor-2 (BSF-2/IL-6) gene.

The chromosomal DNA segment of human B cell stimulatory factor-2 (BSF-2/IL-6) was isolated and characterized by nucleotide sequence analysis. The human BSF-2/IL-6 gene consists of five exons and four introns and its organization shows a distinctive similarity to granulocyte colony-stimulating factor gene. The two genes have the same number of exons and introns and the size of each exon is strikingly similar. The BSF-2/IL-6 mRNA was found to be constitutively expressed in a human T cell leukemia virus-1 transformed T cell line, TCL-Na1, a bladder cell carcinoma line, T24, and an amnion derived cell line, FL. The BSF-2/IL-6 mRNA was also found to be inducible with interleukin-1 beta in an astrocytoma line, U373 and a glioblastoma line, SK-MG-4. S1 mapping and primer extension analyses showed the presence of multiple initiation sites and the preferential utilization of a different initiation site for each individual tissue tested.     

Postnatal development of the ovarian bursa of the golden hamster (Mesocricetus auratus): its complete closure and morphogenesis of lymphatic stomata

The golden hamster ovarian bursa was studied by light and electron microscopy to clarify the process of its complete closure and the development of lymphatics that leads to morphogenesis of stomata. The results were as follows. 1) The bursa completely closed at 9 days of age primarily due to development of the mesotubarium superius. 2) With the closure, the ovary and bursa became closely apposed, and most of the original bursal cavity disappeared. 3) Between 9 and 12 days of age U-shaped folds of the bursal mesothelium began to invade the connective tissue of the bursa. 4) Widening of the internal angle of the U-shaped folds contributed to reappearance of the bursal cavity, and thus separation of the bursa from the ovary. It also contributed to future geometrical proximity of lymphatics to the cavity of the bursa. 5) The separation of the bursa from the ovary began as early as 12 days of age in the cephalic half of the bursa. It occurred remarkably late in the caudal half. Juxtaposition of the window portion of the bursa to the ovary remained in some adult animals. 6) Development of lymphatics in the cephalic half of the bursa was divided into two stages, before and after days 21-24 of life. In the first stage, lymphatics grew in the submesothelial connective tissue, and the framework of lymphatics was formed. In the second stage, lymphatics extended small branches to form the submesothelial plexus or lymphatic lacuna. 7) Intercellular junctions between contiguous lymphatic endothelial cells were mostly tight and desmosomelike. Open junctions were, if they occurred at all, rare. (8) A smooth-surfaced area lined with the lymphatic endothelium was found in the bursa on day 27 of life, before the initiation of ovulation. Valvelike stomal orifices were absent before the initiation of ovulation and extremely rare even after the first ovulation. They were commonly present in the bursae after the fourth ovulation, however. The process of complete closure of the ovarian bursa is very complex and may be related to the later development of the bursal mesothelium and lymphatics. Some liplike stomal orifices are of purely developmental origin. However, all valvelike stomal orifices are assumed to be formed as a result of damage to the bursal mesothelium, as well as to the submesothelial connective tissue and lymphatics, by repetition of ovulation. It is possible that liplike stomal orifices may be formed in the process of repairing the damage.     

Phosphorylation of two isozymes of (Na+ + K+)-ATPase by inorganic phosphate

The phosphorylation of two isozymes (alpha(+) and alpha) of (Na+ + K+)-ATPase by 32Pi was studied under equilibrium conditions in various enzyme preparations from rat medulla oblongata, rat cerebral cortex, rat cerebellum, rat kidney, guinea pig kidney, and rabbit kidney. In ouabain-sensitive (Na+ + K+)-ATPases such as the brain, guinea pig kidney, and rabbit kidney enzymes, ouabain stimulated the Mg2+-dependent phosphorylation at lower concentrations, while a higher concentration was required for the stimulation of rat kidney (Na+ + K+)-ATPase, an ouabain-insensitive enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that two isozymes of the brain (Na+ + K+)-ATPase were also phosphorylated by 32Pi in the presence of ouabain. The properties of the phosphorylation were compared between the medullar oblongata (referred to as alpha(+] and the kidney (referred to as alpha) (Na+ + K+)-ATPases. The steady-state level of phosphorylation was achieved faster in the kidney enzymes than in the medulla oblongata enzyme. Phosphorylation without ouabain was greater in the kidney enzymes than in the brain enzymes. Furthermore, the former enzymes were inhibited by K+ much more than the latter. These findings suggest that the two isozymes of (Na+ + K+)-ATPase differ in their conformational changes during enzyme turnover.     

Heavy chain of human high molecular weight and low molecular weight kininogens binds calcium ion

An antibody subpopulation, anti high molecular weight (anti-HMW) kininogen-Ca2+ antibody able to bind specifically to the HMW kininogen-Ca2+ complex, was isolated from anti-HMW kininogen antiserum. Partially purified anti-HMW kininogen antibody was applied to a HMW kininogen-Sepharose column equilibrated with 40 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and anti-HMW kininogen-Ca2+ antibody was eluted with 5 mM ethylenediaminetetraacetic acid. As a result of characterization by enzyme-linked immunosorbent assay, this antibody specifically recognized the cyanogen bromide cleaved fragment 1 (CB-1) region (1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by metal ions such as Ca2+ and Mg2+ and that these changes were due to the conformational change of the CB-1 region of the heavy chain. The dissociation constant (Kd) for the heavy chain-Ca2+ measured by CD analysis at 214 nm was found to be 0.33 +/- 0.09 mM (mean +/- SD). The number of Ca2+-binding sites of heavy chain calculated from the Hill plot was 1.15 +/- 0.04 (mean +/- SD). Then, a possible Ca2+-binding site was found in the amino-terminal portion of the heavy chain of kininogen molecules.