Centrally acting endogenous hypotensive substances in rats subjected to endotoxic shock.

Cerebroventricular perfusate (CVP) from rats subjected to endotoxic shock was infused into the lateral ventricle of the naive rat. This procedure produced a hypotensive response in the recipient rat which could be reversed by the intravenous injection of the opioid antagonist naltrexone. The degree of endotoxemic hypotension in the donor rat was attenuated by perfusing the cerebroventricular system with mock CSF. The results suggest the existence of endogenous hypotensive substances in rat central nervous system, possibly opioid in nature, which may play a critical role in the pathogenesis of endotoxic shock.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

Increase of plasma IL-6 concentration with age in healthy subjects.

The present study demonstrated that plasma IL-6 concentration was higher in older subjects than in younger ones and significantly in the male group (P = 0.02); Spearman rank correlation showed that plasma IL-6 concentration was positively correlated with age (r = 0.28, N = 55, P < 0.05); there was a highly significant correlation between the concentrations in plasma IL-6 and IL-1 alpha (r = 0.51, N = 52, P < 0.001). These findings suggest the possibility that age-related changes of plasma IL-6 and IL-1 alpha may provide a pathological basis for the susceptibility to such illness as commonly occurs in elderly people, especially Alzheimer's disease as the two interleukins can induce the production of alpha 1-antichymotrypsin and beta-amyloid protein precursor.     

Genotoxicity of heated cooking oil vapors.

Epidemiological studies of lung cancer in Chinese women indicated that factors other than cigarette smoking are related to lung cancer risk. A case-control study suggested that indoor air pollution, particularly from cooking oil emissions, may be involved. Condensates of volatile emissions from rapeseed and soybean cooking oils were prepared and found to be genotoxic in short-term tests including the Salmonella mutation assay, SV50 forward-mutation assay, and sister-chromatid exchange assay, as well as the micronucleus assay in mouse bone marrow. In contrast, condensates from rapeseed oil with butylated hydroxyanisole or hydrogenated rapeseed oil were not mutagenic, implicating oxidation products as the cause for mutagenicity. Peanut oil and lard condensates were not mutagenic in any assay. The association of exposure to Chinese rapeseed cooking-oil emissions and lung-cancer risk may be related to the mutagenic component of these condensates.     

Mitochondrial DNA alterations as ageing-associated molecular events.

Mitochondrial DNA (mtDNA) is a naked double-stranded circular extrachromosomal genetic element continuously exposed to the matrix that contains great amounts of reactive oxygen species and free radicals. The age-dependent decline in the capability and capacity of mitochondria to dispose these oxy-radicals will render mtDNA more vulnerable to mutations during the ageing process. During the past 3 years, more than 10 different types of deletions have been identified in the mtDNA of various tissues of old humans. Some of them were found only in a certain tissue but some others appeared in more than one organ or tissue. The 4977-bp deletion is the most prevalent and abundant one among these deletions. Skeletal muscle is the target tissue of most ageing-associated mtDNA deletions and has often been found to carry multiple deletions. The onset age of the various deletions in mtDNA varies greatly with individual and type of the deletion. The 4977-bp deletion has been independently demonstrated to occur in the mtDNA of various tissues of the human in the early third decade of life. However, the 7436-bp deletion was only detected in the heart mtDNA of human subjects in their late thirties. The others appeared only in older humans over 40 years old. No apparent sex difference was found in the onset age of these ageing-associated mtDNA deletions. The various ageing-associated deletions could be classified into two groups. Most of the deletions belong to the first group, in which the 5'- and 3'-end breakpoints of the deletion are flanked by 4-bp or longer direct repeats. The deletion in the second group occurs less frequently and shows no distinct repeat sequences flanking the deletion sites. These two groups of mtDNA deletions may occur by different mechanisms. The first group is most probably caused by internal recombination or slippage mispairing during replication of mtDNA by the D-loop mechanism. The deleted mtDNA and the deleted DNA fragment may be further degraded or escape from the mitochondria and get translocated into the nucleus. The latter route has been substantiated by many observations of inserted mtDNA sequences in the nuclear DNA. Thus, the fragments of migrating mtDNA may change the information content and expression level of certain nuclear genes and thereby promote the ageing process or cause cancer. Similar ageing-associated alterations of mtDNA have also been observed in aged animals and plants. I suggest that mtDNA deletions and other mutations to be discovered are molecular events generally associated with the ageing process.     

Transfer of a dominant gene for powdery mildew resistance and DNA from Hordeum bulbosum into cultivated barley (H. vulgare)

Summary In an attempt to transfer traits of agronomic importance from H. bulbosum into H. vulgare we carried out crosses between four diploid barley cultivars and a tetraploid H. bulbosum. Eleven viable triploid F₁ plants were produced by means of embryo rescue techniques. Meiotic pairing between H. vulgare and H. bulbosum chromosomes was evidenced by the formation of trivalents at a mean frequency of 1.3 with a maximum of five per cell. The resulting triploid hybrids were backcrossed to diploid barley, and nine DC₁ plants were obtained. Three of the BC₁ plants exhibited H. bulbosum DNA or disease resistance. A species specific 611-bp DNA probe, pSc119.2, located in telomeres of the H. bulbosum genome, clearly detected five H. bulbosum DNA fragments of about 2.1, 2.4, 3.4, 4.0 and 4.8 kb in size present in one of the BC₁ plants (BC₁-5) in BamHI-digésted genomic Southern blots. Plant BC₁-5 also contained a heterozygous chromosomal interchange involving chromosomes 3 and 4 as identified by N-banding. One of the two translocated chromosomes had the H. bulbosum sequence in the telomeric region as detected using in situ hybridization with pSc119.2. Two other BC₁ plants (BC₁-1 and BC₁-2) were resistant to the powdery mildew isolates to which the barley cultivars were susceptible. Seventy-nine BC₂ plants from plant BC₁-2 segregated 32 mildew resistant to 47 susceptible, which fits a ratio of 11, indicating that the transferred resistance was conditioned by a single dominant gene. Reciprocal crosses showed a tendency towards gametoselection that was relative to the resistance. Mildew resistant plant BC₁-2 also had a 1-kb H. bulbosum DNA fragment identified with a ten-base random primer using polymerase chain reaction (PCR). Forty-three BC₁ plants, randomly sampled from the 79 BC₁ plants, also segregated 2320 for the presence versus absence of this 1-kb H. bulbosum DNA fragment, thereby fitting a 11 ratio and indicating that the PCR product originated from a single locus. The 1-kb DNA fragment and disease resistance were independently inherited as detected by PCR analysis of bulked DNA from 17 resistant and 17 susceptible plants as well as by trait segregation in the 43 individual plants. The progenies produced could serve as an important resistant source in plant breeding. This is the first conclusive report of the stable transfer of disease resistance and DNA from H. bulbosum to H. vulgare.     

Maturation and fertilization of porcine oocytes in vitro

Four experiments were conducted to study 1) factors affecting porcine oocyte maturation in culture medium and 2) a new method for oocyte maturation outside the porcine body. In Experiment 1, five groups of oocytes were cultured in m-TCM199 or m-KRB medium for 24 to 28, 32 to 36 or 40 to 42 hours and then were fertilized in vitro. The cleavage rate (two to four-cell stage) of oocytes cultured for 32 to 36 hours was significantly higher than those of the other oocytes. The results indicate that a suitable culture period for the in vitro maturation of porcine oocytes is 32 to 36 hours. In Experiment 2, four groups of oocytes were cultured in m-KRB or m-KRB supplemented with PFF, PMSG or FSH for in vitro maturation, and the cleavage rates of oocytes were 7.94, 22.56, 30.23 and 23.26%, respectively, after in vitro fertilization. The results show that porcine follicular fluid (PFF) and gonadotrophins added to the culture medium promote porcine oocyte maturation in vitro. In Experiment 3, oocytes were cultured in m-KRB or m-TCM199, supplemented with both gonadotrophin and pocine folliclar fluid for maturation in vitro. After fertilization in vitro, the cleavage rates of oocytes were 26.32 and 27.93% for the two media. The results indicate that the difference between m-KRB and m-TCM199 was insignificant when the media were used to culture porcine oocytes. But there was a significant difference when PFF and gonadotrophins were added to the basic media. In Experiment 4, porcine oocytes were transferred into the reproductive tracts of other animals for maturation. After 34 to 36 hours, the oocytes were collected and fertilized in vitro. The cleavage rates of oocytes were 10.42, 28.45, 3.33 and 36.36%, respectively, for the oocytes matured in mouse uterine horns, rat uterine horns, rat oviducts or rabbit oviducts. The results show that porcine oocytes can be matured in the reproductive tracts of other animals.     

Growth and morphological changes in the stomach of newborn pigs during the first three days after birth.

Growth and morphological changes in the stomach of newborn pigs were examined during the first 3 days after birth. The stomach grew disproportionately faster than the body as a whole during this period. The growth was due to hyperplasia and hypertrophy during the first day and mainly to hyperplasia thereafter as gastric DNA content increased progressively after birth, and the protein:DNA and RNA:DNA ratios increased only on the first day. Histological and morphometric analyses revealed that the growth was more pronounced in the gastric body region than in the cardiac and pyloric regions, and more pronounced in the mucosal layer than in other layers. The percentage of mucosal volume occupied by parietal cells (volume density) and the number of parietal cells per unit volume of gastric mucosa (numerical density) increased significantly 3 days after birth in the cardiac and body regions, but not in the pyloric region, of the stomach. The observed morphological changes coincide with the known pattern of functional maturation during the immediate postnatal period. It is suggested that a high level of circulating gastrin and oral ingestion of milk-derived growth factors in the newborn pig contribute to these changes.     

链霉菌AC-57及其产生的阿克拉霉素A的研究

During the course of screening for new antitumor antibiotics, a new anthracycline antibiotic--aclacinomycin A was separated from the broth and mycelium of Streptomyces AC-57. The strain AC-57 was isolated from the soil collected in the Shanghai suburbs. According to its culture and physiological characteristics the producer was identified as Str. galilaeus AC-57. The broth and mycelium were extracted and treated with solvents as usual way. The aclacinomycin A was separated by silica-gel column chromatography eluted with chrolo-form-methanol. Aclacinomycin A, its aglycone and sugar components were identified by comparison of their physico-chemical and spectral data (MS, UV, IR, 1H-NMR, and 13C-NMR) with authentic compound, purified from the market sample.