A procedure to produce gnotobiotic calves by cesarean section.

An effective and economical method was developed for procuring and rearing calves in gnotobiotic conditions. To evaluate apparatuses and surgical techniques, three calves, 1 to 3, welf 1 was weak and pale at delivery and died within 5 hours after delivery. Calf 2 was delivered alive, but died from a human error at 3 days of age. It was free from demonstrable bacteria and fungi at that time. Calf 3 was also successfully delivered and raised. It was killed at 10 days of age, since fungi were isolated from the feces and waste materials from it.     

Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

Impact of phenolic compounds on hydrothermal oxidation of cellulose

The effect of phenolic compounds on hydrothermal oxidation of cellulose was studied using a batch reactor at 300 degrees C with H(2)O(2) as oxidant. Intermediate products, as well as the yields of acetic acid produced in the oxidation of cellulose, phenolic compounds, and cellulose-phenolic compound mixtures were examined. Phenolic compounds used were phenol, 1,4-benzenediol, 2-methoxy-4-methylphenol, and 2,6-di-tert-butyl-4-methylphenol. In the case of oxidation of cellulose-phenolic compound mixtures, (1) formic acid, a basic oxidation product from carbohydrates, decreased considerably, (2) 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde, acid-catalyzed dehydration products from carbohydrates, appeared, and (3) the yield of acetic acid increased compared to that in the oxidation of cellulose. From these results, phenolic compounds seem to inhibit the oxidation of cellulose under hydrothermal conditions. The inhibition of the oxidation of cellulose by phenolic compounds seems to be related closer to the stability of phenolic compounds under oxidation conditions rather than the ease to remove phenolic hydrogen on the OH group.     

Prevention of lymph node metastases by adoptive transfer of CD4+ T lymphocytes admixed with irradiated tumor cells

CD4⁺8^– T lymphocytes with potent antitumor activity in vivo were obtained in peritoneal exudate cells by immunizing mice with irradiated MM48 tumor cells admixed with OK-432. These immune CD4⁺ T cells were used in adoptive immunotherapy for prevention of lymph node metastases after removal of the primary tumor. Complete cure of metastases was obtained by adoptive transfer of CD4⁺ T cells admixed with irradiated MM48 tumor cells, but not by CD4⁺ T cells alone. To analyze the curative effect of admixing tumor cells on the prevention of metastases, a model of 1-day tumor inoculated with macrophages was used. Administration of immune CD4⁺ T cells alone resulted in the regression of local tumor in more than half of the mice, although all of them eventually died of lymph node metastases. On the other hand, adoptive transfer of immune CD4⁺ T cells plus irradiated tumor cells resulted in the complete regression of local tumors in all the mice, which survived without any sign of metastasis. The curative effect of the immune CD4⁺ T cells obtained by admixing irradiated tumor cells was tumor-specific. Macrophages induced by OK-432 (tumoricidal), implanted together with tumor, assisted tumor regression more than did macrophages elicited by proteose peptone (nontumoricidal) in the same adoptive transfer system. Administration of recombinant interleukin-2 instead of stimulant tumor cells did not enhance, but rather eliminated the constitutive antitumor activity of CD4⁺ T cells. On the other hand, exogenous recombinant interleukin-1 was more effective in the enhancement of antitumor activity of the CD4⁺ T cells as compared with stimulant tumor cell administration. In this case, the activating states of macrophages at the implanted tumor site had no influence on the therapeutic efficacy. A possible role of macrophages for induction of tumor-specific cytotoxic T cells that were mediated by tumor-specific CD4⁺ T cells is discussed.     

Effect of Theanine,r-Glutamylethylamide,on Brain Monoamines and Striatal Dopamine Release in Conscious Rats

Theanine, r-glutamylethylamide, is one of the major components of amino acids in Japanese green tea. Effect of theanine on brain amino acids and monoamines, and the striatal release of dopamine (DA) was investigated. Determination of amino acids in the brain after the intragastric administration of theanine showed that theanine was incorporated into brain through blood-brain barrier via leucine-preferring transport system. The concentrations of norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole acetic acid (5HIAA) in the brain regions were unaffected by the theanine administration except in striatum. Theanine administration caused significant increases in serotonin and/or DA concentrations in the brain, especially in striatum, hypothalamus and hippocampus. Direct administration of theanine into brain striatum by microinjection caused a significant increase of DA release in a dose-dependent manner. Microdialysis of brain with calcium-free Ringer buffer attenuated the theanine-induced DA release. Pretreatment with the Ringer buffer containing an antagonist of non-NMDA (N-methyl-D-aspartate) glutamate receptor, MK-801, for 1 hr did not change the significant increase of DA release induced by theanine. However, in the case of pretreatment with AP-5, (±)-2-amino-5-phosphonopentanoic acid; antagonist of NMDA glutamate receptor, the theanine-induced DA release from striatum was significantly inhibited. These results suggest that theanine might affect the metabolism and/or the release of some neurotransmitters in the brain, such as DA.     

Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus

Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80°C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3–4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP.