Deregulation of cancer-related pathways in primary hepatocytes derived from DNA repair-deficient Xpa-/-p53+/- mice upon exposure to benzo[a]pyrene.

The current method to predict carcinogenicity of chemicals or drugs is the chronic 2-year rodent bioassay, which has disadvantages in duration, animal use, and specificity. An attractive alternative is the DNA repair-deficient Xpa(-/-)p53(+/-) mouse model that is sensitive to both genotoxic and nongenotoxic carcinogens. A next step in alternative carcinogenicity testing is the development of reliable in vitro systems. We investigated the use of primary hepatocytes, isolated from wild-type (WT) and Xpa(-/-)p53(+/-) mice, in combination with transcriptome analyses for their usefulness to predict carcinogenic features of compounds. As a proof of principle, we studied the response of hepatocytes to the genotoxic carcinogen benzo[a]pyrene (B[a]P). Upon treatment, both WT and Xpa(-/-)p53(+/-) hepatocytes appeared to be metabolically active. However, Xpa(-/-)p53(+/-) hepatocytes were more sensitive than WT hepatocytes to B[a]P treatment in terms of cell survival. In B[a]P-treated WT hepatocytes, DNA repair and cell cycle control genes were transcriptionally activated. Xpa(-/-)p53(+/-) hepatocytes were more responsive to B[a]P exposure, resulting in the downregulation of cancer-related pathways. Deregulation of mitogen-activated protein kinase signaling seems to play an essential role in this and might be the underlying reason for the increased susceptibility of Xpa(-/-)p53(+/-) mice toward carcinogens. Our conclusion is that primary hepatocytes combined with transcriptomics are promising to identify the carcinogenic features of chemicals. Furthermore, these cells seem suitable to gain further insight into the molecular mechanisms of the increased sensitivity of Xpa(-/-)p53(+/-) mice toward both genotoxic and nongenotoxic carcinogens.

The role of brain-derived neurotrophic factor receptors in the mature hippocampus: modulation of long-term potentiation through a presynaptic mechanism involving TrkB.

The neurotrophin BDNF has been shown to modulate long-term potentiation (LTP) at Schaffer collateral-CA1 hippocampal synapses. Mutants in the BDNF receptor gene trkB and antibodies to its second receptor p75NTR have been used to determine the receptors and cells involved in this response. Inhibition of p75NTR does not detectably reduce LTP or affect presynaptic function, but analyses of newly generated trkB mutants implicate TrkB. One mutant has reduced expression in a normal pattern of TrkB throughout the brain. The second mutant was created by cre-loxP-mediated removal of TrkB in CA1 pyramidal neurons of this mouse. Neither mutant detectably impacts survival or morphology of hippocampal neurons. TrkB reduction, however, affects presynaptic function and reduces the ability of tetanic stimulation to induce LTP. Postsynaptic glutamate receptors are not affected by TrkB reduction, indicating that BDNF does not modulate plasticity through postsynaptic TrkB. Consistent with this, elimination of TrkB in postsynaptic neurons does not affect LTP. Moreover, normal LTP is generated in the mutant with reduced TrkB by a depolarization-low-frequency stimulation pairing protocol that puts minimal demands on presynaptic terminal function. Thus, BDNF appears to act through TrkB presynaptically, but not postsynaptically, to modulate LTP.

High-voltage-activated calcium channel messenger RNA expression in the 140-3 neuroblastoma-glioma cell line.

Expression of calcium channel alpha1 subunits in oocytes or cell lines has proven to be a powerful method in the analysis of structure-function relations, but these experimental systems are of limited value in the examination of neuron-specific functions such as transmitter release. Cell lines derived from neurons are often capable of these functions, but their intrinsic calcium channel alpha1 subunits are complicating factors in experimental design. We have examined the biophysical and molecular properties of calcium channels in a little studied neuroblastoma-glioma hybrid cell line, 140-3, a close relative of the NG108-15 cell line, to test whether this cell line might serve a role as an expression system for neural mechanisms. This cell was selected as it contains an intact transmitter release mechanism yet secretes little in response to depolarization. Patch-clamp recording revealed only a prominent low-threshold, rapidly inactivating calcium current with a single-channel conductance of approximately 7 pS that was identified as T type. A search for calcium channel alpha1 subunit messenger RNA in the 140-3 cells with three different tests only revealed alpha1C, whereas alpha1A-alpha1C were present in the parent NG108-15 line. We made a particular effort to search for alpha1E, since this subunit has been associated with a low-voltage-activated current. Our findings suggest that, since the principal nerve terminal-associated calcium channels (alpha1A, alpha1B, alpha1E) are absent in the 140-3 cell, this cell line may prove a particularly useful model for the analysis of the role of high-voltage-activated calcium channels in complex functions of neuronal cells.

UDP-N-acetylglucosamine transferase and glutamine: fructose 6-phosphate amidotransferase activities in insulin-sensitive tissues.

Glutamine:fructose 6-phosphate amidotransferase (GFA) is rate-limiting for hexosamine biosynthesis, while a UDP-GlcNAc beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) catalyses final O-linked attachment of GlcNAc to serine and threonine residues on intracellular proteins. Increased activity of the hexosamine pathway is a putative mediator of glucose-induced insulin resistance but the mechanisms are unclear. We determined whether O-GlcNAc transferase is found in insulin-sensitive tissues and compared its activity to that of GFA in rat tissues. We also determined whether non-insulin-dependent diabetes mellitus (NIDDM) or acute hyperinsulinaemia alters O-GlcNAc transferase activity in human skeletal muscle. O-GlcNAc transferase was measured using 3H-UDP-GlcNAc and a synthetic cationic peptide substrate containing serine and threonine residues, and GFA was determined by measuring a fluorescent derivative of GlcN6P by HPLC. O-GlcNAc transferase activities were 2-4 fold higher in skeletal muscles and the heart than in the liver, which had the lowest activity, while GFA activity was 14-36-fold higher in submandibular gland and 5-18 fold higher in the liver than in skeletal muscles or the heart. In patients with NIDDM (n = 11), basal O-GlcNAc transferase in skeletal muscle averaged 3.8 +/- 0.3 nmol/mg.min, which was not different from that in normal subjects (3.3 +/- 0.4 nmol/mg.min). A 180-min intravenous insulin infusion (40 mU/m2.min) did not change muscle O-GlcNAc transferase activity in either group. We conclude that O-GlcNAc transferase is widely distributed in insulin-sensitive tissues in the rat and is also found in human skeletal muscle. These findings suggest the possibility that O-linked glycosylation of intracellular proteins is involved in mediating glucose toxicity. O-GlcNAc transferase does not, however, appear to be regulated by either NIDDM or acute hyperinsulinaemia, suggesting that mass action effects determine the extent of O-linked glycosylation under hyperglycaemic conditions.

[Atypical symptoms in patients with germinal testicular tumors]. / Atypische Symptomatik bei Patienten mit germinalen Hodentumoren.

The cardinal syndrome of a testicular germ cell tumour is typically scrotal enlargement. The present paper compares the group of patients with typical scrotal presentation and those who present with atypical symptoms caused by metastases. Among 284 retrospectively studied patients, 34 (12%) presented with extrascrotal symptoms. The most important were abdominal pain (n = 16) and pulmonary symptoms (n = 10). The group of patients with extrascrotal symptoms was characterized by the following parameters: percentage of pure seminoma in 35% (versus 56% in the patients with typical presentation), elevation of alpha-feto-protein in 47% (versus 27%), and elevation of beta-HCG in 61% (versus 29%). The outcome was lethal in 35% of the patients with atypical presentation, as opposed to 6% of those with typical presentation. In 22 patients with extrascrotal presenting signs a palpable testicular mass was found on clinical examination. Occult testicular tumour proved to be present in 9 patients, and burned-out tumours in 3. Unawareness of testicular cancer is a significant factor in diagnostic delay. Scrotal palpation should be part of every clinical examination in younger male patients with cancer from an unknown primary.

Cancer pain relief using chronic morphine infusion. Early experience with a programmable implanted drug pump.

Fourteen patients were implanted with drug pumps to provide chronic epidural or intrathecal morphine to relieve pain due to cancer. A new programmable pump was used in seven of the patients and a constant infusion device was used in the other seven patients. Results, judged by subjective pain reports (on a 0 to 10 scale), decrease in oral narcotics, and change in activity level, were excellent in eight patients, good in five patients, and poor in one patient. The programmable device has the obvious advantage of being able to vary dose according to patient need and requires less frequent refilling. Four programmable pump failures occurred, two requiring replacement.

[The pollen tube growth of Pisum mutants under consideration of the free amino acids]. / Das Pollenschlauch-Wachstum von Pisum-Mutanten unter Berücksichtigung der freien Aminosäuren.

The germination of the pollen grains and the growth rate of the pollen tubes of eight mutants of Pisum sativum were compared with the parent line by in vitro investigations. All of the mutants studied showed a retardation of pollen tube growth as compared to the parent line, resulting in competitive elimination of some of the gametes in plants heterozygous for the respective mutant genes. The deficit of recessive plants in the progenies of heterozygous mutant strains of peas can be attributed to this retardation. Marked differences between the various mutants with regard to the levels of various free amino acids in their pollen grains were found. Certain amino acids may be present in greatly reduced concentration or may be present in excess. In some genotypes pollen tube growth can be stimulated by adding the deficient amino acid. This is especially true for proline, valine and threonine. As far as the other amino acids are concerned, the mutants studied showed varying reactions. The germination rate of the pollen grains is reduced by proline and threonine; germination is completely inhibited by glutamine. The amino acids isoleucine, histidine and cysteine retard pollen tube growth in all the mutants investigated. This is also true for leucine which, however, has a stimulating effect in one of the chlorophyll mutants.