Leishmania mexicana pifanoi: in vivo and in vitro interactions between amastigotes and macrophages

Macrophages of the cell line J774 were used in a comparative study of virulence involving amastigote stages of Leishmania mexicana pifanoi isolated from macrophages (AMA-M) of the aforementioned cell line, amastigote forms grown in the UM-54-cell-free medium (AMA-C), and promastigote stages. The macrophage cultures were inoculated with AMA-M and AMA-C at the culture cell to parasite ratios of 1:3, 1:5, and 1:10. The macrophages were exposed to either kind of amastigotes for 24, 48, and 72 h. At the end of each of these periods, and for each dilution, the percentages of macrophages harboring the parasites within their cytoplasm and the mean numbers of intracellular parasite/macrophage were estimated on the basis of examination of 200 phagocytes. When either AMA-M or AMA-C were employed, after 24 h, the percentages of infected macrophages were, respectively, 84.5%, 89.0%, and 94.5% for the three aforementioned dilutions, the majority of the phagocytes containing 1-5 parasites. After 48- and 72-h exposures, the macrophages harbored 6-11 and 11-20 amastigotes/cell, respectively. Evidently intracellular multiplication of the amastigotes has taken place. In contrast to the results obtained with amastigote forms, after inoculations of the macrophages cultures with promastigotes at the dilutions previously used for amastigotes, only 48-78 phagocytes were found to contain intracellular stages within their cytoplasm. Many macrophages were parasite-free, especially when exposed to fewer promastigotes. Experiments in which 5 X10(6) promastigotes, AMA-M, or AMA-C were inoculated into the footpads of hamsters yielded the following results with regard to terminal footpad volumes: 1.57, 3.31, and 3.32 cm3, respectively. Evidently both kinds of amastigotes had equal virulence for hamsters; however, the promastigote stages were much les virulent for these experimental hosts.     

HLA-JY328: Mapping studies and expression of a polymorphic HLA class I gene

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk^– cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from,L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed —a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.     

Regulation of rabbit fetal glycogen: effect of in utero fetal decapitation on the metabolism of glycogen in fetal heart, lung, and liver

To understand the control mechanisms involved in the regulation of fetal glycogen, we have studied the effect of in utero fetal decapitations on glycogen metabolism in rabbit fetal heart, lung, and liver. In utero fetal decapitations were performed between days 18 and 21 of gestation. Two to four fetuses on one side of the horn were decapitated. Fetuses were delivered between days 23 and 26 or between days 28 and 30 of gestation. Fetal heart, lungs, and liver were analyzed for DNA, protein, glycogen, glycogen synthase (I and D forms), glycogen phosphorylase (a and b forms), phosphofructokinase, pyruvate kinase, and lactic dehydrogenase. In fetal heart and lung, no difference was observed in any of the above measurements in the intact and decapitated fetuses. In contrast, fetal liver does not appear to develop the glycogen system as indicated by the very low levels of glycogen (0.02 mg/mg DNA) in decapitated fetuses as compared with intact fetuses (0.4 mg/mg DNA). Similarly the levels of glycogen synthase and phosphorylase were two to three times lower in livers from decapitated fetuses as compared with the livers from intact fetuses. The three enzymes phosphofructokinase, pyruvate kinase, and lactic dehydrogenase were not affected by fetal decapitation in all three tissues. These results indicate that the fetal hypothalamic-pituitary-adrenal (thyroid) axis is not required at least after day 18 of gestation for the normal accumulation and subsequent utilization of glycogen in fetal heart and lungs, while it is an absolute requirement for the development of the fetal liver glycogen system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age differences in responsiveness of brainstem chemosensitive neurons to extracellular pH changes

The sensitivity of neurons in the caudal chemosensitive area on the ventrolateral surface of the medulla oblongata (VMS) to extracellular pH changes was examined in newborn and young developing kittens and compared to that of adult cats. The pH was varied by superfusion of the VMS with mock cerebrospinal fluid (CSF) of pH 7.4 (control), 7.0 (acid) and 7.8 (alkaline). A total of 97 neuronal units in the three age groups changed their firing rates inversely in response to extracellular fluid (ECF) pH changes. The greatest sensitivity was found in the adult group where acid superfusion caused an increase in neuronal activity. The least sensitivity was observed in the newborn group (1-6 days old), whereas the young kitten group (4-6 weeks old) exhibited an intermediate sensitivity. Neurons of kittens older than 7 weeks of age demonstrated a response pattern characteristic of the adult group. Neurons of neonates older than seven days, exhibited a response pattern characteristic of the young kitten group.     

Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication

Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.     

Kinetics of the sodium-dependent glutamine transporter in human intestinal cell confluent monolayers.

The intestinal epithelium metabolism of glutamine plays a critical role in inter-organ nitrogen flow. Although it is known that glutamine is the primary oxidative energy source and nucleotide precursor in intestinal cells, the luminal uptake of glutamine by the apical surface of enterocytes is poorly understood. In this study we have uncovered the sodium-dependent transporter system responsible for L-glutamine uptake by the apical membrane of a human intestinal epithelial cell line. The sodium-dependent Michaelis constant (Km) = 247 +/- 45 microM glutamine, and Jmax = 4.44 +/- 0.65 x 10(-9) mole min-1(mg protein)-1 (37 degrees C). Glutamine shares the transporter with alanine, as demonstrated by unlabeled glutamine inhibition of [3H]alanine uptake kinetics with a purely competitive-type inhibition pattern, and glutamine inhibition Ki = 205 +/- 18 microM by Dixon analysis. The inhibition pattern for a series of amino acid analogs indicated that this intestinal apical membrane sodium-dependent transporter for glutamine is distinct from any other transport system found in membranes of non-intestinal cells.     

Preliminary study of the distribution of the toxic elements As,Cd, and Hg in human hair and tissues by RNAA

In order to study the relationships between trace element concentrations of hair and internal body burdens, a radiochemical NAA technique has been used for determination of the elements As, Cd, and Hg in autopsy samples of liver, kidney-cortex, lung, and hair from 24 male persons who died by accident. High significant positive correlations were observed between the As concentration in hair and in kidney-cortex, and between Cd and Zn concentrations in kidney-cortex. The contents of Cd, both for lung and kidney-cortex, were related to the smoking habits of the subjects.

     

皮层第二体感区下行活动对尾核神经元伤害性反应的影响

本实验用清醒麻痹的家猫观察到,皮层第二体感区(SⅡ区)的下行活动对尾核痛相关单位的自发放电和伤害性反应均有影响。在35个痛相关单位中,刺激SⅡ区后,62.7％的自发放电受到影响(p<0.05)。在72个痛相关单位中,刺激SⅡ区后,77.8％的伤害性反应受到抑制(p<0.01),其中抑制痛兴奋单位的伤害性反应最为明显。而用利多卡因局部阻滞SⅡ区后,33个痛相关单位中,51.5％的伤害性反应增强(p<0.01)。本结果提示:(1)SⅡ区的下行活动不仅对尾核痛相关单位的自发活动有影响,而且对痛相关单位的伤害性反应有以抑制为主的下行性调制作用。(2)在正常机能状态下,SⅡ区对伤害性信息向尾核的传递有一定的紧张性的下行性抑制作用。