Measurements of macromolecule-bound and ultra-filtrable polyamines in rat liver homogenized without buffer

Ultra-filtrable and macromolecule-bound polyamines in rat liver homogenates, made without buffer, were determined, using Potter-Elvehjem homogenizer and commercially available, pressure-aided ultrafiltration device with a membrane pore size that allows passage of particles of molecular weight no larger than 5000. About 90% of polyamines in the liver were shown to be equilibrated with externally added 15N-labeled polyamines, based on the difference in the ratio of the natural to 15N-labeled polyamine in the liver homogenate and the ultrafiltrate. The entire amount of ultrafiltrate in the homogenized liver, required for calculation of the amounts of ultra-filtrable and macromolecule-bound polyamines, was estimated to be about 0.25 g in one gram of the homogenate, using a limited dilution curve of spermine in the ultrafiltrate with phosphate buffered saline and distilled water. With this value, ultra-filtrable polyamines in normal rat liver homogenate were calculated as about 25%, 8%, and 2% of the total amount of putrescine, spermidine, and spermine, respectively. The method was then used to measure ultra-filtrable and macromolecule-bound polyamines in regenerating rat liver homogenates, to examine possible changes of polyamines during cell growth. The method was also applied to measure other ultra-filtrable compounds such as amino acids and inorganic ions in rat liver homogenate.     

Cellular prion protein promotes Brucella infection into macrophages

The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.     

A case report of a scalp dermoid cyst containing watery-clear fluid

We report a case of a 7-month-old female with a dermoid cyst on the anterior fontanelle. She was born with a vacuum extractor. Two weeks later, her scalp on the anterior fontanelle bulged. A doctor was consulted when she was 3 months old, because the tumor was enlarging. Magnetic resonance image (MRI) showed a cystic mass on the anterior fontanelle. She was brought to our institute. At the first medical examination, she was alert and had no neurological deficit. The tumor was suspected to be a sinus pericranii or a pseudo meningocele. She was observed as an out patient, but the tumoral size become more enlarged. When she was seven months old, we punctured the cystic tumor and the tumor collapsed. But, two weeks later, it enlarged again. The cystic fluid was watery clear and the composition differed from cerebrospinal fluid (CSF). The tumor was resected totally. The histological examination revealed a dermoid cyst which involved ducts of the eccrine glands and folliculi pili. The cystic fluid was thought to be secreted from the eccrine glands.     

Increased serum cholesteryl ester transfer protein in obese children

OBJECTIVE: To determine whether serum cholesteryl ester transfer protein (CETP), which is one of the physiologically active gene products secreted from adipose tissue, is increased and associated with atherogenic lipoprotein profile in obese children. RESEARCH METHODS AND PROCEDURES: Subjects were 42 consecutive outpatient Japanese obese children, 29 boys and 13 girls, ranging in age from 5 to 14 years, and 25 age-matched non-obese children, 13 boys and 12 girls, as the control group for measuring CETP mass. Blood was drawn after an overnight fast and, at the same time, and anthropometric measurements including height, body weight, waist girth, hip girth, and triceps and subscapular skinfold thicknesses were taken. Paired samples were obtained from 15 obese children who underwent psychoeducational therapy. Serum CETP mass was assayed by an enzyme-linked immunosorbent assay. RESULTS: The serum levels of triglyceride, total cholesterol (TC), low-density lipoprotein cholesterol, TC/high-density lipoprotein cholesterol (HDLC), apolipoproteins (apo) B, apo B/apo A(1), and insulin in obese children were significantly higher than the respective reference values. Serum CETP level was approximately 2-fold higher (98.7 +/- 3.6 vs. 50.9 +/- 4.0 nM, means +/- SEM, p < 0.001) in the obese children than in the controls. In 15 obese children, whose percentage of overweight declined during therapy, CETP levels decreased significantly. CETP level was correlated with HDLC, TC/HDLC, and insulin, and with percentage of overweight when the data of the obese and non-obese children were combined. DISCUSSION: CETP is increased and associated with the atherogenic lipoprotein profile in obese children.     

Increase in hepatic content of oleic acid induced by dehydroepiandrosterone in the rat.

The effects of dehydroepiandrosterone (DHEA) on the acyl composition of lipids in rat liver were studied. The content of oleic acid (18:1) in hepatic lipids was increased markedly by feeding rats a diet containing 0.5% (w/w) DHEA for 14 days. Treatment of rats with DHEA caused an increase in the activity of the terminal desaturase of the stearoyl-CoA desaturation system, without changing either the activity of NADH-cytochrome b5 reductase or the microsomal content of cytochrome b5. Among the changes observed in hepatic lipids, the increase in 18:1 content in phosphatidylcholine (PtdCho) was the most prominent; an approximately 2.5-fold increase in the proportion of 18:1 was induced at position 2, but not at position 1, by DHEA. This selective elevation of 18:1 at position 2 of PtdCho seems to be produced by the concerted actions of the induced 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase and the induced stearoyl-CoA desaturase. The content of 18:1 in serum lipids was unchanged by DHEA treatment, suggesting that secretion of lipids containing 18:1 into the circulation was not affected by DHEA. These results suggest that the elevation of hepatic content of 18:1 caused by DHEA treatment is mainly due to the induction of stearoyl-CoA desaturase.     

Effects of ketamine on renal nerve activity, arterial pressure and heart rate in rats

We recorded renal nerve activity (RNA) together with arterial pressure (AP) and heart rate (HR) in 24 Wister rats anesthetized with nitrous oxide to investigate the effects of ketamine on the sympathetic nerve activity and the cardiovascular dynamics. The magnitude and time course of the responses to four graded doses of ketamine (1, 5, 10, 25 mg/kg) were studied in 19 rats. RNA responded biphasically, initially decreasing dose-dependently to minimal values of 89 +/- 4.4, 77 +/- 8.2, 54 +/- 5.2, and 17 +/- 3.7% of control for 1, 5, 10, and 25 mg/kg, respectively, and then increasing above control dose-independently. AP showed a biphasic response. HR first decreased dose-dependently but then increased slightly. In the remaining five rats, we compared the effects of ketamine 5 mg/kg on RNA, AP, and HR before surgical baroceptor denervation with those after the denervation. The denervation abruptly increased RNA, AP, and HR. Ketamine decreased RNA, AP, and HR in the denervated state and returned them to pre-ketamine values without overshoot. The finding that in the nerve intact state ketamine produced the characteristically biphasic response of RNA could be explained by the following mechanisms: (1) ketamine depresses the vasomotor center causing the initial decrease in RNA; (2) ketamine depresses the inhibitory effects of baroreflex causing the successive increase in RNA. The biphasic change in AP could be partly attributed to biphasic responses of RNA to ketamine.     

Carotid sinus nerve terminals which are tyrosine hydroxylase immunoreactive are found in the commissural nucleus of the tractus solitarius

Summary Tyrosine hydroxylase immunoreactive sensory neurons in the petrosal ganglion selectively innervate the carotid body via the carotid sinus nerve. Central projections of the carotid sinus nerve were traced with horseradish peroxidase. The commissural nucleus of the tractus solitarius was examined by dual labelling light and electron microscopy. Dense bilateral labelling with horseradish peroxidase was found in the tractus solitarius and commissural nucleus of the tractus solitarius. Horseradish peroxidase was found in unmyelinated axons, myelinated axons, and nerve terminals. About 88% of horseradish peroxidaselabelled carotid sinus nerve axons were unmyelinated. Tyrosine hydroxylase immunoreactivity was identified in unmyelinated axons, myelinated axons, dendrites, perikarya, and nerve terminals. Most tyrosine hydroxylase immunoreactive axons (93%) in the commissural nucleus of the tractus solitarius were unmyelinated. Tyrosine hydroxylase immunoreactivity was simultaneously identified in carotid sinus nerve unmyelinated axons, myelinated axons, and nerve terminals. These double-labelled terminals comprised 28% of the number of tyrosine hydroxylase immunoreactive terminals in the commissural nucleus of the tractus solitarius, and 55% of transganglionically-labelled terminals. Therefore, there are both central and peripheral sources of tyrosine hydroxylase immunoreactive nerve terminals in the commissural nucleus of the tractus solitarius. These data support the hypothesis that peripheral tyrosine hydroxylase immunoreactive neurons are involved in the origination of the chemoreceptor reflex. Axo-axonic synapses between peripheral carotid sinus nerve afferent terminals and central terminals containing tyrosine hydroxylase immunoreactivity were observed in 22% of the axo-axonic synapses observed. Thus, central tyrosine hydroxylase immunoreactivity neurons are involved in the modulation of the chemo- and/or baroreceptor reflexes. Synaptic contacts were not observed between carotid sinus nerve afferents and tyrosine hydroxylase immunoreactive perikarya of dendrites. Catecholaminergic neurons are thus unlikely to be the second order neurons of either the chemo- or baroreceptor reflex in the commissural nucleus of the tractus solitarius.     

Establishment of an antitoxoplasma state by stable expression of mouse indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-degrading enzyme, is inducible by various interferons (IFNs). IDO-mediated tryptophan degradation, but not the formation of IDO-catalyzed tryptophan metabolites, has been suggested as a mechanism for the antiparasitic action of IFN-gamma. To determine whether the IFN-gamma-induced IDO alone is sufficient for establishing the antiparasitic state, we constructed a mouse IDO expression plasmid containing a heavy metal-responsive metallothionein promoter and obtained a stable transformant (C6) by transfection of this plasmid into mouse rectal cancer (CMT-93) cells. In the presence of 100 microM ZnSO4, C6 cells yielded a high level of IDO; and after a 2-day culture period, the enzyme induction resulted in complete depletion of tryptophan from the culture medium. Under these conditions, the growth of Toxoplasma gondii in C6 cells infected with the organisms on day 3 after enzyme induction was completely blocked. In the absence of ZnSO4, however, IDO induction was negligible in C6 cells, and T. gondii continued to grow. Furthermore, in a transformant (CC10) carrying an antisense mouse IDO plasmid or in parental CMT-93 cells, IDO was not induced at all even in the presence of 100 microM ZnSO4, and T. gondii continued to grow in these cells as well. These results taken together indicate that complete depletion of tryptophan from the culture by IDO alone is sufficient to establish the antitoxoplasma state in mouse cells.     

Enzyme-linked immunosorbent assay to differentiate the antibody responses of animals infected with Brucella species from those of animals infected with Yersinia enterocolitica O9

Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.