Early interactions of Flavobacterium psychrophilum with macrophages of rainbow trout Oncorhynchus mykiss

The early interactions of a low and a highly virulent Flavobacterium psychrophilum strain with head kidney and spleen macrophages of rainbow trout Oncorhynchus mykiss were characterized. The highly virulent strain was killed 5.8 to 11 times less frequently than the low virulent strain. The head kidney macrophages showed a microbicidal activity approximately twice as high as that of the spleen macrophages. A 2- to 3-fold higher production of reactive oxygen species (ROS) was induced by the highly virulent strain than by the low virulent one. The head kidney macrophages produced approximately twice as much ROS as the spleen macrophages. The low virulent strain was killed approximately 10 times more frequently by H2O2 than was the highly virulent strain. In spleen macrophages, the highly virulent strain caused twice as much cytotoxic effects compared to the low virulent strain. In conclusion, virulence in F. psychrophilum appears to be correlated with higher O. mykiss macrophage cytotoxicity and resistance to ROS and, therefore, with enhanced resistance to bacterial killing. Moreover, due to lower ROS production, spleen macrophages have a lower antimicrobial action against F. psychrophilum, compared to head kidney macrophages and, thus, might form a 'safe site' in which bacteria can reside.     

通用引物PCR检测临床常见致病菌的实验研究

通用引物可一次性扩增18种临床常见致病菌和耐药菌株的DNA,扩增片段长度在220bp左右,18种特异性探针分别与18种标准菌株的PCR扩增产物杂交结果显示探针都具有高度特异性;5种37例经法国梅里埃API细菌鉴定系统确定的临床分离菌株进行杂交鉴定,鉴定结果与分离株一致,表明设计的探针具有高度特异性及准确性。80例临床标本分别用法国梅里埃API细菌鉴定系统及PCR杂交法进行检测,阳性率分别为(52.5%)和(67.5%),表明PCR结合寡核苷酸杂交法比传统的生物学培养法更为灵敏,值得推广。     

Formation of Fine Oil-in-Water Emulsions by the Gel Emulsification Method with Saccharide and Protein

An emulsification method using a gel-like phase of a saccharide and protein mixture has been developed. In the method, which is called a gel emulsification method, an oil is added to the highly concentrated saccharide solution containing protein to form a clear gel-like phase, which followed by dilution with water to form a fine oil-in-water emulsion. This emulsion was investigated as to its emulsifying activity and emulsion stability as compared with that obtained by high-shear equipment, which was called a homomixer method. The emulsifying activity of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The emulsions prepared by both methods were highly stable in terms of the stability against coalescence. On the other hand, the stability against creaming of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The surface hydrophobicity of the protein and the unfreezable water content in the highly concentrated saccharide solution containing protein were not correlated to the emulsifying properties of the emulsions prepared by the gel emulsification method, which appeared to be dependent on the viscosity of the highly concentrated saccharide solution containing protein.     

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

Sulphated glycosaminoglycans expression in the basement membranes of colorectal adenocarcinomas. Preliminary study: correlation with histological grading

Summary The expression of sulphated glycosaminoglycans was studied at the ultrastructural level by the high iron diamine technique in the basement membranes of 26 colorectal adenocarcinomas (10 well-differentiated, 7 moderately-differentiated, 9 poorlydifferentiated). Sulphated glycosaminoglycan expression was highly variable. It was scored as regular (5 cases), slightly irregular (6 cases), highly irregular (15 cases). In general, poor histological differentiation could be correlated with absent or highly irregular expression. However, in a limited number of cases, severe alterations of basement membranes were also present in well-differentiated (2 cases) and moderately-differentiated (4 cases) tumours. Such a variability shows up a heterogeneity which is not revealed by histological grading.     

Effect of progesterone (norethindrone) only oral contraceptive on mouse uterus--a SEM study

Impact of progesterone (norethindrone) only oral contraceptive (MICRONOR) on mouse uterus was investigated by SEM. The three dimensional SEM photographs revealed that, flat wave like mucous folds of the uterus (control) become more thick and highly convoluted after 50 days of MICRONOR feeding. The uteri became highly secretory as indicated by increased thickness of nonciliated secretory cell population with their increased microvilli. Low doses of norethindrone in combination with circulating estrogen, instead of causing regression, maintained a steady and highly secretory state which may interfere implantation/gamete transport.     

Patterns of leg muscle recruitment vary between novice and highly trained cyclists

This study compared patterns of leg muscle recruitment and coactivation, and the relationship between muscle recruitment, coactivation and cadence, in novice and highly trained cyclists. Electromyographic (EMG) activity of tibialis anterior (TA), tibialis posterior (TP), peroneus longus (PL), gastrocnemius lateralis (GL) and soleus (SOL) was recorded using intramuscular fine-wire electrodes. Four experimental conditions of varying cadence were investigated. Differences were evident between novice and highly trained cyclists in the recruitment of all muscles. Novice cyclists were characterized by greater individual variance, greater population variance, more extensive and more variable muscle coactivation, and greater EMG amplitude in periods between primary EMG bursts. Peak EMG amplitude increased linearly with cadence and was not different at individual preferred cadence in either novice or highly trained cyclists. However, EMG amplitude in periods between primary EMG bursts, as well as the duration of primary EMG bursts, increased with increasing cadence in novice cyclists but were not influenced by cadence in highly trained cyclists. Our findings suggest that muscle recruitment is highly skilled in highly trained cyclists and less refined in novice cyclists. More skilled muscle recruitment in highly trained cyclists is likely a result of neuromuscular adaptations due to repeated performance of the cycling movement in training and competition.     

Detection of benzylic adducts in DNA and nucleotides from 7-sulfooxymethyl-12-methylbenz[a]anthracene and related compounds by 32P-postlabeling using new TLC systems

7,12-Dimethylbenz[a]anthracene (DMBA) is a highly potent experimental carcinogen, that must be transformed to its ultimate carcinogenic form in vivo. The meso-region theory of aromatic hydrocarbon carcinogenesis predicts that 7-hydroxymethyl sulfate (7-HMBA) ester plays a major role in the metabolic activation, benzylic DNA adduct formation and complete carcinogenicity of HMBA and DMBA. This study was undertaken to detect highly lipophilic benzylic DNA adducts resulting from the reaction between 7-hydroxymethy sulfate ester of HMBA (7-SMBA) and DNA as well as determine their DNA base selectivity. Synthetic 7-SMBA was incubated with DNA (800 microg/ml) and individual deoxynucleoside 3'-monophosphates (600 microg/ml) and benzylic adducts were analyzed by 32P-postlabeling/TLC following their enrichment with butanol extraction. Dilute ammonium hydroxide-based solvents were developed to detect the highly lipophilic aralkyl adducts. The reaction with DNA, dGp and dAp gave rise to multiple adducts; dCp and dTp showed no significant adducts. Chromatographic comparison revealed that the major DNA adduct was derived from dG. The methodology developed was also found applicable for highly lipophilic adducts resulting from sulfate esters of structurally-related metabolites of DMBA.     

Rapid screening of libraries with radiolabeled DNA sequences generated by PCR using highly degenerate oligonucleotide mixtures.

The PCR technique can use protein-derived oligonucleotide sequences as primers to develop probes for screening recombinant libraries. Here we report a method with highly degenerate mixtures of oligonucleotides as primers for the PCR that eliminates the need to identify or isolate the DNA sequences derived by PCR. The method uses the pool of PCR-generated DNA sequences radiolabeled during the extension reaction as a probe, combined with highly stringent hybridization and wash conditions that permit only homologous sequences to hybridize and therefore target desired clones. This technique was used successfully to clone the receptor for tumor necrosis factor.     

Investigation of the salivary 18-hydroxycorticosterone:aldosterone ratio in man using a direct assay

A method for the direct determination of 18-hydroxycorticosterone (18OHB) in human saliva has been developed and validated. Saliva was collected at 30 min and 1 h intervals between 0600 and 2200 h from healthy men and women for the determination of 18OHB (SHB), aldosterone (SA) and glucocorticoids (SGC = cortisol + cortisone). SHB was highly correlated with SA (r = 0.75; P less than 0.001) but even more highly with SGC (r = 0.89; P greater than 0.001). Multiple regression analysis confirmed that SGC was a more important determinant of SHB than was SA. Though the concentrations of 18OHB and aldosterone were highly correlated there was considerable variation in the 18OHB:aldosterone ratio during the period of saliva collection. This ratio tended to be highest in the morning and lowest in the evening and was weakly correlated with SGC level (r = 0.62; P less than 0.01). The 18OHB:aldosterone ratio in saliva approximates to, and is highly correlated with, that in plasma. We suggest that the fluctuations in SHB:SA ratio correspond to the relative rates of secretion of 18OHB and aldosterone and that this ratio is modulated either by ACTH or by cortisol. Whether this indicates that 18OHB is a by-product of glucocorticoid as well as aldosterone metabolism, or whether this implies a separate physiological role for the steroid remains to be clarified.     

A sensitive high-performance liquid chromatographic procedure with fluorometric detection for the analysis of decarboxylated S-adenosylmethionine and analogs in urine samples

A highly sensitive HPLC method for the determination of decarboxylated S-adenosylmethionine (dc-SAM) by fluorometric detection was developed. The reaction of dc-SAM and its analogs with chloroacetaldehyde leads to the corresponding 1,N6-etheno derivatives. These highly fluorescent derivatives were fully characterized through their proton nuclear magnetic resonance spectra and/or mass spectra. This derivatization procedure has been applied to the analysis of dc-SAM in rat and human urine. After a simple cation exchange column prepurification, the urine extracts were derivatized with chloroacetaldehyde and analyzed by reversed-phase HPLC with fluorometric detection. The method allowed the determination of subpicomole amounts of dc-SAM and was shown to be highly reproducible with the use of decarboxylated S-adenosylethionine as internal standard. The application of the method to the analysis of urine of rats treated with MDL 72175, a potent ornithine decarboxylase inhibitor, showed that the dc-SAM levels increased in a dose-related fashion.     

Acetylcholinesterase and its association with lipid.

Human erythrocyte acetylcholinesterase preparations which vary in lipid content, from lipid-rich to lipid-poor, have been successfully prepared using deoxycholate. It was found that the lipid content of the enzyme decreased gradually as the deoxycholate concentration used in its preparation was increased. The binding of lipid to lipid-poor preparations of the enzyme has been investigated. It was found that the activity of such preparations was highly dependent on their phospholipid contents. Maximum specific activity was associated with a fixed phospholipid content. The lipid-poor enzyme was highly activated by addition of either endogenous(membrane) or exogenous lipid. Based on the data presented, it was concluded that acetylcholinesterase is a phospholipoprotein, and its activity is highly dependent on its phospholipid component.     

Shifts in configuration of the 5'-noncoding region of a mouse messenger RNA under translational control

Several major mRNA species of mouse and other mammalian cells occur both as small untranslated ribonucleoprotein particles and as functional molecules associated with ribosomes in polysomes. One of these, that codes for a 21-kDa polypeptide, was analyzed with respect to distribution of sites accessible to RNase T1 in the 5'-noncoding region. This region, which is about 100 nucleotides long, contains several sites that are highly sensitive to the enzyme, as well as many G residues not susceptible to cleavage. The distribution of highly sensitive sites was compared in the active and inactive states of the P21 mRNA present in cytoplasmic extracts by subjecting the extract to limited nuclease digestion followed by separation of partially fragmented polysomes from free messenger ribonucleoprotein particles. The mRNA in polysomes contained two highly sensitive sites, one near the 5' terminus and the other in the middle of the region, next to a sequence potentially capable of Shine-Dalgarno interaction. The untranslated molecules lacked the 5'-proximal site but had several highly accessible sites not present in the active molecules. The initiation AUG showed little accessibility both in polysomes and in messenger ribonucleoproteins. Both forms were quite different from the deproteinized mRNA with respect to distribution of nuclease-sensitive sites. Our results indicate that interaction of the mRNA with cytoplasmic factors strongly affects its conformation in the 5'-noncoding region and that a particular conformation may be important for effective interaction with ribosomal particles during polypeptide chain initiation.     

Hypersensitive response, cell death and histochemical localisation of hydrogen peroxide in host and non-host seedlings infected with the downy mildew pathogen Sclerospora graminicola

Hypersensitive response, cell death and release of hydrogen peroxide as measures of host and non‐host defense mechanisms upon inoculation with the downy mildew pathogen Sclerospora graminicola were studied histochemically at the light microscopy level. The materials consisted of coleoptile tissues of the highly susceptible (cv. HB3), highly resistant (cv. IP18293) and induced resistant pearl millet host seedlings and non‐host sorghum (cv. SGMN10/8) and cotyledon of french bean (cv. S9). Resistance up to 80% protection against the downy mildew pathogen was induced in the highly susceptible HB3 cultivar of pearl millet by treating the seeds with 2% aqueous leaf extract of Datura metel for 3 h. Time course study with the pathogen inoculated highly resistant pearl millet cultivar revealed the appearance of hypersensitive response in 20% of seedlings as necrotic spots as early as 2 h after inoculation. In contrast, a similar reaction was observed in the highly susceptible pearl millet cultivar only 8 h after inoculation with the pathogen. In induced resistant seedlings, appearance of hypersensitive response was recorded 4 h after inoculation. Delayed hypersensitive response was observed in both the non‐host species at 10 h after inoculation. Hypersensitive response in the seedlings of the highly resistant pearl millet cultivar 24 h after inoculation showed 100% hypersensitive response, which was not observed in susceptible and non‐host species, although the induced resistant seedlings showed 90% hypersensitive response after that period of time. Cell death in the tissues of the test seedlings was also observed to change with time. Statistical analysis revealed that the tissues of highly resistant pearl millet seedlings required 2.9 h to attain 50% cell death. Tissues of induced resistant and highly susceptible pearl millet seedlings required 4.65 and 6.50 h respectively. In non‐hosts, 50% cell death was not recorded. Quantification of hydrogen peroxide in the tissue periplasmic spaces of the test seedlings revealed 2.94 h as the time required for 50% hydrogen peroxide accumulation in the tissues of highly resistant pearl millet seedlings. Tissues of induced resistant and highly susceptible pearl millet seedlings needed 3.76 and 5.5 h respectively. Fifty percent hydrogen peroxide localisation in non‐hosts could not be recorded. These results suggested the involvement of hydrogen peroxide, cell death and hypersensitive response in pearl millet host defense against S. graminicola.     

Curved helix segments can uniquely orient the topology of supertwisted DNA

To show that DNA containing sharp sequence-directed curvature can preferentially establish ends of supertwisted domains, a highly curved DNA from Crithidia fasciculata was cloned into two sites separated by 28% in pBR325. When this construct (pJGC2) was examined by electron microscopy, 63% of the supercoiled molecules were branched with three or more arms, and the remaining molecules appeared as linear interwound rods. The distance between the tips of two of the arms for the branched molecules measured within 2% of 28% of the DNA contour for 32% of the pJGC2 molecules, as contrasted with only 6.6% for the poorly branched pBR325 DNA. When one of the curved segments in pJGC2 was replaced by a highly curved fragment from SV40, similar results were obtained.     

The protein concentration of crude cell and tissue extracts as estimated by the method of dye binding: comparison with the Lowry method.

This paper describes a new, inexpensive, and highly sensitive voltammetric assay for aromatic l-amino acid decarboxylase (AADC) activity in rat and human brains by highperformance liquid chromatography (hplc). l-Dopa was used as substrate and d-dopa for the blank. After isolating dopamine formed enzymatically from l-dopa on a small Amberlite CG-50 column, the dopamine in the column eluate was assayed by hplc with a voltammetric detector. Dihydroxybenzylamine was added to each incubation mixture as an internal standard and therefore this assay was highly reproducible. The peak height in hplc was linear from 100 fmol to 100 pmol of dopamine. The values obtained by this method agreed with those by radioassay using [1-¹⁴C]dopa. The enzyme activity in rat cerebral cortex and in Parkinsonian caudate nucleus, in which the activity was too low to be measured even with the radioassay, could be measured accurately.     

Cloning and characterization of the SIL promoter

The SIL gene undergoes a site-specific rearrangement with the SCL gene in 25% of patients with T cell acute lymphoblastic leukemia (ALL). The functional result of this rearrangement is that the SIL regulatory elements aberrantly drive expression of the SCL gene. We have cloned and sequenced the human SIL promoter, cloned a murine homolog, found the sequence to be highly conserved, and defined a minimal promoter region. Both the cloned murine and human sequences were found to be highly active in either human or murine cells. SCL mRNA, driven by a cloned SIL promoter, could be downregulated by DMSO in stably transfected F4-6 murine erythroleukemia cells. The SIL promoter was found to be partially unmethylated in proliferating tissues, in which it is highly expressed, and more highly methylated in post-mitotic tissues, in which SIL is not expressed. The isolation of the SIL promoter provides an important tool for the study of both the SIL gene expression as well as the role of the SIL promoter in leukemogenesis.     

Purification by cibacron blue F3GA dye affinity chromatography and comparison of NAD(P)H:quinone reductase (E.C.1.6.99.2) from rat liver cytosol and microsomes

The dicoumarol-sensitive NAD(P)H:quinone reductase (E.C.1.6.99.2), often referred to as DT-diaphorase, has been purified from both the cytosolic and microsomal fractions from rat liver using a novel, highly efficient, two-step purification procedure utilizing immobilized Cibacron Blue F3GA dye affinity chromatography as the principal step. Under the conditions reported here, this dye affinity resin, generally recognized as preferentially binding nucleotide-dependent proteins, was highly selective in the recovery of up to 95% of the NAD(P)H:quinone reductase directly from the cytosol as a preparation which was often greater than 90% pure. Further purification by gel exclusion chromatography resulted in pure protein preparations with final recoveries approaching 80%. Similar results were obtained during the purification of this quinone reductase activity from microsomal extracts. Evidence is presented which suggests that the enzyme isolated from each cellular fraction are highly homologous, if not identical; data are consistent with genetic evidence.     

Effects of High Levels of DNA Adenine Methylation on Methyl-Directed Mismatch Repair in ESCHERICHIA COLI

Two methods were used in an attempt to increase the efficiency and strand selectivity of methyl-directed mismatch repair of bacteriophage lambda heteroduplexes in E. coli. Previous studies of such repair used lambda DNA that was only partially methylated as the source of methylated chains. Also, transfection was carried out in methylating strains. Either of these factors might have been responsible for the incompleteness of the strand selectivity observed previously. In the first approach to increasing strand selectivity, heteroduplexes were transfected into a host deficient in methylation, but no changes in repair frequencies were observed. In the second approach, heteroduplexes were prepared using DNA that had been highly methylated in vitro with purified DNA adenine methylase as the source of methylated chains. In heteroduplexes having a repairable cI/+ mismatch, strand selectivity was indeed enhanced. In heteroduplexes with one chain highly methylated and the complementary chain unmethylated, the frequency of repair on the unmethylated chain increased to nearly 100%. Heteroduplexes with both chains highly methylated were not repaired at a detectable frequency. Thus, chains highly methylated by DNA adenine methylase were refractory to mismatch repair by this system, regardless of the methylation of the complementary chain. These results support the hypothesis that methyl-directed mismatch repair acts to correct errors of replication, thus lowering the mutation rate.     

Cross-Category Adaptation: Objects Produce Gender Adaptation in the Perception of Faces

Adaptation aftereffects have been found for low-level visual features such as colour, motion and shape perception, as well as higher-level features such as gender, race and identity in domains such as faces and biological motion. It is not yet clear if adaptation effects in humans extend beyond this set of higher order features. The aim of this study was to investigate whether objects highly associated with one gender, e.g. high heels for females or electric shavers for males can modulate gender perception of a face. In two separate experiments, we adapted subjects to a series of objects highly associated with one gender and subsequently asked participants to judge the gender of an ambiguous face. Results showed that participants are more likely to perceive an ambiguous face as male after being exposed to objects highly associated to females and vice versa. A gender adaptation aftereffect was obtained despite the adaptor and test stimuli being from different global categories (objects and faces respectively). These findings show that our perception of gender from faces is highly affected by our environment and recent experience. This suggests two possible mechanisms: (a) that perception of the gender associated with an object shares at least some brain areas with those responsible for gender perception of faces and (b) adaptation to gender, which is a high-level concept, can modulate brain areas that are involved in facial gender perception through top-down processes.