Correlation between [U-14C]glucose metabolism and function in perfused cat brain

In brain perfusion experiments conducted with blood containing [U-14C]glucose the relative specific activity (RSA) of blood glucose carbon incorporated in brain intermediate metabolites was measured. It was demonstrated that the so-called metabolic pattern of Geiger is not constant, but it bears a close relation to the function of the brain. The results of the study may be summarized briefly as follows. (1) In a group (A) of cats with a high level of brain function, the RSA of lactic acid was 75 per cent; that of glutamic acid 80 per cent; aspartic acid 75 per cent; glutamine 61 per cent; GABA 43 per cent; and respiratory CO2 55 per cent. It was observed that the major part of the carbon of amino acids, such as glutamic acid and aspartic acid, which are directly associated with the tricarboxylic acid cycle are derived from blood glucose. (2) In a group (B) showing a low level of brain function, the RSA of each amino acid was considerably lowered. The RSA of glutamic acid and aspartic acid was about 50 per cent and that of respiratory CO2 was 27 per cent. (3) In a group (C) with a still lower level of brain function, each amino acid as well as the respiratory CO2 had still lower RSA values. (4) The metabolic pattern of Geiger corresponds to values obtained during low functional activity of the brain in our experiment.     

Enzymological characteristics of avian influenza A virus neuraminidase

Neuraminidases of 18 strains of avian influenza A virus were examined by both colorimetric and fluorometric assays using fetuin and 4-methylumbelliferyl-N-Ac-alpha-D-neuraminide as substrates, respectively, to compare them with those of human influenza A and B viruses. The ratios of the neuraminidase activity of avian influenza virus measured by the colorimetric assay method to that measured by the fluorometric assay were distributed in the range of 2.4-20.3. The enzyme of avian influenza virus showed calcium-ion dependence in both assay methods. These results suggest that neuraminidase of avian influenza A virus is varies greatly from one strain to another in substrate specificity as compared with those of human influenza A and B viruses, and that some strains of avian influenza A virus have a neuraminidase with unique enzymological characteristics different from that of human influenza A virus as well as that of influenza B virus.     

Lymphocyte subpopulations in high endothelial venules and lymphatic capillaries of bronchus-associated lymphoid tissue (BALT) in the rat

The subpopulations of lymphocytes and non-lymphoid cells in high endothelial venules (HEV) and in lymphatic capillaries surrounding lymphoid follicles in bronchus-associated lymphoid tissue (BALT) were examined by electron microscopy after preembedding the tissue and staining with an immunoperoxidase technique. The results were compared with those obtained in gut-associated lymphoid tissue (GALT) reported previously. Monoclonal mouse-anti-rat T cell, IgG, IgM, IgA, and Ia antisera were used. Plasma cells that were reactive to anti-IgG, anti-IgM, and anti-IgA were detected as cells in which the 3',3'-diaminobenzidine tetrahydroxychloride reaction product was localized in rough endoplasmic reticulum and perinuclear spaces but not on plasma membranes. These plasma cells did not occur in either lymphatic capillaries or HEV in BALT as they did in GALT. Cells with surface Ig (sIg cells), T-cell antigen (T cells), and Ia antigen (Ia cells) were present in BALT. T cells were located predominantly in the follicular area opposite the bronchial epithelium; IgM- and IgG-reactive cells were found in the follicular area adjacent to the bronchial epithelium; and IgA-positive cells were found in the lateral part of the area where the T cells were localized (T-cell area). Ia cells were abundant throughout BALT and in moderate numbers in the epithelium. A striking observation was the presence of "nurse-cell"-like structures in the periphery of BALT. The percentages of T, sIgG, sIgM, and sIgA cells in the HEV were 54.7%, 2.4%, 28.9%, and 27.3%, respectively, and in the lymphatic capillaries, 41.2%, 3.8%, 38.2%, and 21.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoreactive corticotropin-releasing hormone, adrenocorticotropin and cortisol in human plasma during pregnancy and delivery and postpartum

The activity of the hypothalamo-pituitary adrenal axis was examined, by measuring the levels of immunoreactive (IR) corticotropin-releasing hormone (CRH), adrenocorticotropin (ACTH) and cortisol (F) in human plasma during normal pregnancy and after delivery with or without complications and during normal postpartum using a specific RIA. The level of IR-CRH in maternal plasma increased progressively during pregnancy, increased further at delivery and declined rapidly to the non-pregnant level on the 1st day postpartum. The level of IR-F in maternal plasma also increased progressively during pregnancy, increased further at delivery, but decreased slowly postpartum, not returning to the non-pregnant level within 5 days. Significant correlations were found between the level of IR-CRH and IR-ACTH, IR-CRH and IR-F, and IR-ACTH and IRF in maternal plasma both during pregnancy and after delivery. It is noteworthy that the concentration of IR-CRH in the maternal plasma at delivery was higher in multiple pregnancy than in normal pregnancy, and that the level of IR-CRH in the umbilical cord in uncomplicated cases was much lower than that in the maternal plasma, and was significantly lower than those in the umbilical cord plasma in cases of asphyxia, IUGR or premature delivery. The level of IR-F, not IR-CRH and IR-ACTH, at normal vaginal delivery was significantly higher than that at elective cesarean section. On these results, we investigated the feto-maternal-hypothalamo-pituitary adrenal axis during pregnancy and delivery, in which CRH plays an important role.     

U-14C]glucose metabolism of the perfused cat brain with blood flow disturbances

—In order to study changes of the glycolytic-respiratory system and amino acid metabolism associated with blood flow disturbance, the cat brain perfusion was conducted with artificial blood containing [U-¹⁴C]glucose and the results were compared with those of standard perfusion keeping the cerebral blood flow at constant rate. The findings of the present study are briefly summarized: (1) In blood flow disturbance there was observed an accumulation of lactate just as seen in the low functional state observable in the standard perfusion. However the increase in relative specific activity of lactate was not so marked as the rise in cerebral lactate content, and this indicates that there is an increase of lactate production from substrates other than glucose as well as an increase of net flow of glucose carbon to lactate. (2) In blood flow disturbance relative specific activities of glutamate, aspartate, glutamine and respiratory CO₂ were decreased as compared with those in the brain of high functional state. The relative specific activity of GABA in the reduced blood flow brain was at the same level as that of the brain at high functional state and it was higher than the relative specific activity of glutamate. (3) The relative specific activity and content of alanine were increased in the low function brain with standard perfusion.     

Role of the fly in the transport of Yersinia enterocolitica.

Yersinia enterocolitica were isolated from flies collected from a piggery and a kitchen of farm and from ham hung in a piggery. The cultures were identified as Y. enterocolitica biovar 4 and serovar 3 by biochemical and serological characteristics. From these results it is suggested that flies may play an important role in food contamination by Y. enterocolitica. In this study, the probable donors of Y. enterocolitica to the flies were swine.     

Detection and Quantification of Male-Specific Fetal DNA in the Serum of Pregnant Cynomolgus Monkeys (Macaca fascicularis)

Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research. We have developed a specific, highly sensitive PCR system for detecting and quantifying male-specific fetal DNA in pregnant cynomolgus monkeys. We used multiplex quantitative real-time PCR to analyze cell-free DNA in maternal blood serum obtained from 46 pregnant monkeys at gestational weeks 5, 12, and 22. The presence of SRY gene and DYS14 Y chromosomal sequences was determined in 28 monkeys with male-bearing pregnancies. According to confirmation of fetal sex at birth, the probe and primers for detecting the Y chromosomal regions at each time point revealed 100% specificity of the PCR test and no false-positive or false-negative results. Increased levels of the SRY-specific sequences (mean, 4706 copies/mL serum DNA; range, 1731 to 12,625) and DYS14-specific sequences (mean, 54,814 copies/mL serum DNA; range, 4175–131,250 copies) were detected at week 22. The SRY- and DYS14-specific probes appear to be an effective combination of markers in a multiplex PCR system. To our knowledge, this report is the first to describe the detection of cell-free DNA in cynomolgus monkeys.Abbreviations: C_t, threshold cycleAnalysis of cell-free circulating nucleic acids in human maternal plasma or serum has led to the development of risk-free methods for prenatal genetic diagnosis and the assessment of several fetal and maternal conditions, for example, sex determination for paternally inherited diseases, pregnancy-associated complications, sex-linked disorders for ambiguous genitalia, and embryo tracking.^{1,4,12,14,18,19} Technical challenges associated with detecting fetal DNA arise due to the low concentration of fetal DNA in maternal plasma during pregnancy and the difficulty of differentiating the genetic material of the fetus from that of the mother.^5,13,20 Fetal sex determination using sequences derived from the Y chromosome only is relatively simple and has a reported accuracy rate in humans of approximately 99.0% at 7 wk of gestation and 100% after 20 wk, depending on the protocol and methods used.^3,5,17,20 In other species, researchers have used real-time PCR assays during pregnancy to predict fetal sex from cell-free DNA at an accuracy of 100%.^9,10,11 Cell-free fetal DNA in the maternal circulation represents only 3% to 6% of the total free DNA obtained from plasma throughout pregnancy; however, this percentage is variable between pregnancies.^5,13,20In clinical biomedical research, it is essential to develop animal models for human diseases to reveal their mechanisms.^16,22 Continued progress in surgical intervention and molecular medicine suggests that it may soon be possible to develop potential treatments or even cures for several fetal genetic diseases at an early stage of pregnancy.¹⁵ Fetal developmental research during early pregnancy might be facilitated by using cell-free fetal DNA in the maternal blood rather than other methods, such as serum screening and ultrasonography. Nonhuman primates, especially macaques, are useful model animals for studying fetal development because of the similarity of the reproductive characteristics, placental structure, and developmental events between these animals and humans.^9,10 These developmental similarities highlight the importance of the study of cell-free fetal DNA in nonhuman primates and its usefulness as a marker to obtain genetic information about the fetus.In the current study, we investigated the presence of cell-free fetal DNA in the maternal plasma of cynomolgus monkeys by developing and using a standardized PCR system. To this end, we selected the SRY (sex-determining region Y) gene and DYS14 sequences of the cynomolgus monkey to use as sex-associated markers. The Y chromosome-specific sequences in the single-copy sex determination region of SRY and the multicopy (thus yielding increased sensitivity) sequences of DYS14 in the TSPY (testis-specific protein, Y-linked) gene have had wide clinical use in humans as molecular markers for detecting and quantifying cell-free fetal DNA.^3,7 In addition, TSPY has been used in bovines for detecting cell-free fetal DNA² and in rhesus macaques for long-term evaluation of microchimerism.⁸ Given the reports of fetal sex determination in rhesus macaques^9,10 and sheep¹¹ by analyzing Y chromosome-specific sequences from cell-free DNA, we hypothesized that we could predict the fetal sex of cynomolgus monkeys at different stages of gestation. This information has been extremely useful in optimizing the design of experimental studies in biomedical research and in managing a nonhuman primate breeding colony.¹⁰ Because cynomolgus and rhesus macaques are closely related members of the same genus, the current experiments are similar to a previous study.⁹We developed an efficient 2-color multiplex PCR system to detect and quantify fetal DNA in the maternal serum of cynomolgus monkeys during pregnancy. We used 2 loci on the Y chromosome in a single PCR test to minimize the likelihood of false-positive signals. Here we report the results of detection and analysis of fetal DNA at various weeks of gestation and evaluate our PCR system for its ability to determine fetal sex from pregnant monkeys’ cell-free DNA.