The corpora lutea proangiogenic state of VEGF system components is turned to antiangiogenic at the later phase of the oestrous cycle in cows

Blood vessel expansion and reduction in the corpus luteum (CL) is regulated by the vascular endothelial growth factor (VEGF) system and linked to the maintenance of the CL. The VEGF system has both angiogenic and antiangiogenic ligands and receptors. Our objective was to evaluate the relationship between the mRNA expression of angiogenic and antiangiogenic members of the VEGF system in the CL, throughout the luteal phase of the oestrous cycle in cows. The CL of 18 cows were collected by transvaginal surgery on days 4, 6, 9, 12, 15 and 18 of the oestrous cycle and the mRNA expression of VEGF system components was evaluated by quantitative real-time PCR. The mRNA expression of VEGF ligands and receptors increased (P<0.05) from the early- and mid-luteal phase (days 4 to 12) reaching its maximum expression on day 15 of the cycle. We found no expression of VEGF164b throughout the cycle. Expression of sVEGFR1 did not change during the oestrous cycle and exceeded that of the VEGFR1 by 100 times. Nonetheless, as VEGFR1 increased, the relationship between the soluble and membrane receptor decreased (P<0.01). In contrast, the expression of VEGFR2 was higher than that of its soluble isoform for all days studied, however, the ratio between the membrane-bound and its soluble counterpart decreased continuously throughout the cycle (P<0.01). Our results show that the expression levels for VEGF ligands, receptors and their antagonistic counterparts are adjusted during CL development and regression, to upregulate angiogenesis early in the oestrous cycle and restrict it at the time of luteolysis.     

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

A mannose-specific lectin from Vicia villosa seeds

A lectin specific to mannose has been purified from Vicia villosa seed by (NH₄)₂SO₄ fractionation, GalNAc-Sepharose and Man-Sepharose affinity chromatography. It was defined as VVL_M, which showed a single band on an acidic-PAGE stained with Coosmassie brilliant blue. The molecular weight of VVL_M was 50 kDa as determined by gel filtration on Biogel P-100 column. The VVL_M molecule consists of 2 distinct subunits with apparent molecular weight of 30 kDa and 22kDa determined by SDS-PAGE. VVL_M has at least four isolectins with similar haemagglutinating activity. Its extinction coefficient is calculated as A_1cm¹ = 16.4 at 280 nm. Sugars could not be detected phenol-sulfuric acid method. The circular dichroism analysis at far UV indicated that VVL_M was a β-sheet-rich protein, and gave no α-helix, 69% β-sheet, 14% β-turn by Provencher and Glockner method. The lectin was inhibited by α-methyl-d-mannose at 12.5 mM and glucose or GlcNAc at 50 mM. The carbohydrate binding specificity of VVL_M was investigated by using affinity chromatography on a VVL_M-Sepharose column. Among various Asn-linked oligosaccharides, core structure Manα1→3(Manα1→6)Manβ1→4GlcNAcβ1→4GlcNAc_OT were found to have high affinity for VVL_M-Sepharose. The antisera of VVL_M did not produce precipitin line with VVL_G in agar double diffusion plate indicating so serological relationship between VVL_M and VVL_G. However VVL_M showed similar immunodeterminants of some other lectins of mannose specificity such as Con A, PSL, LCA and VFL.     

Direct characterization of bovine microsatellites from cosmids: polymorphism and synteny mapping

A (TG)₈ oligonucleotide probe was used to screen 186 cosmids from a commercial bovine cosmid library. Of the 56 positive discovered, 7 were sequenced in the region of the microsatellite and analysed for polymorphism. These microsatellites, IDVGA-2, -3, -7, -8, -9, -10, -11 showed the following number of alleles and polymorphism information content (PIC) values (7/0.616, 8/0.693, 6/0.641, 5/0.643, 2/0.239, 10/0.844, 6/0.720). The microsatellites were also assigned to synteny groups as follows: IDVGA-2/U17, IDVGA-3/U16, IDVGA-7/U7, IDVGA-8/U29, IDVGA-9/U3, IDVGA-10/U19, IDVGA-11/A (probably U18).     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Reproductive exocrine and endocrine profiles and their seasonality in male langur monkeys (Presbytis entellus entellus)

The reproductive exocrine and endocrine profiles in male langurs are reported with an emphasis on seasonality. The animals showed positive response to electroejaculation throughout the year. The sperm concentration varied between 10–383 × 10⁶/ejaculation with wide fluctuations all through the year. No appreciable changes in the motility and percent live sperm were observed throughout the year. The levels of seminal fructose and magnesium remained unchanged throughout the year, while acid phosphatase showed wide fluctuations. Citric acid showed elevation during February and March and LDH showed elevated levels during April and May. The annual range of serum testosterone was 6–34 nMol/l with a peak during July. Cortisol ranged between 575–1587 nMol/l and prolactin ranged between 107–900 mU/l. Wide fluctuations were observed in hormonal levels. No seasonality was exhibited in the seminiferous tubule diameter, nuclear diameter of Sertoli cells and Leydig cells, and the cholesterol, glycogen, and sialic acid contents of testis. None of the parameters studied have shown any correlation with season. The results suggest that the male langurs lack seasonality in their reproductive exocrine and endocrine profiles and thus could be used as model for research in human reproduction.     

Activation of caspase-9 and its influencing factors in beef during conditioning

To study the activation of caspase-9 and its potential influence in conditioning, longissimus thoracis (LT), semitendinosus (STN) and psoas minor (PMi) muscles were used to analyze the ratio of pro-apoptotic bax to anti-apoptotic bcl-2 in fresh tissues and observe the changes in ATP, cytosolic cytochrome c and caspase-9 activity levels during storage at 4°C. Caspase-9 activity at 5 h is higher than the activity at 0 and 24 h in the muscles (P<0.001). The ATP content decreased between 0 and 3 h, between 8 and 14 h in the PMi and LT muscles (P<0.0001), whereas between 0 and 5 h, between 8 and 14 h in the STN muscle (P<0.0001). There is 60.2%, 55.3% and 43.1% available ATP in the STN, LT and PMi muscles at 5 h, respectively. The cytosolic cytochrome c level increased during 5 and 24 h storage in the LT and PMi muscles (P<0.0001), during 5 and 96 h in the STN muscle (P<0.0001). The cytosolic cytochrome c at 24 h (P<0.001) and ratio of bax to bcl-2 (P<0.05) was higher in the PMi than in other muscles. We concluded that the increase in cytosolic cytochrome c and available intracellular ATP should be responsible for the increase in caspase-9 activity; the activation of caspase-9 could be limited by the subsequent depletion of ATP; the postmortem release level of cytochrome c could be determined by the ratio of bax to bcl-2 in fresh tissues.     

Chemiluminescent Enzyme Immunoassay for Measuring Leptin

Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1–1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.