Preferential expression of the neuropeptide Y Y1 over the Y2 receptor subtype in cultured hippocampal neurones and cloning of the rat Y2 receptor

1. Neuropeptide Y (NPY) and NPY receptors are most abundant in the hippocampal formation where they modulate cognitive functions. Expression of NPY receptors in rat cultured primary hippocampal cells was investigated in the present study by use of combined molecular, pharmacological and immunohistochemical approaches, including the cloning of the rat Y₂ receptor described here for the first time.
2. More than 70% of the hippocampal neurones were endowed with [¹²⁵I]-[Leu³¹,Pro³⁴]PYY Y₁-like receptor silver grain accumulations and Y₁ receptor immunostaining. These radio- and immuno-labelling signals were distributed over cell bodies and processes of bipolar, stellate and pyramidal-like neuronal cells, as confirmed by neurone-specific enolase and MAP-2 staining.
3. Competition binding profiles revealed that specific [¹²⁵I]-[Leu³¹,Pro³⁴]PYY binding was competitively displaced according to a ligand selectivity pattern prototypical of the Y₁ receptor sub-type with [Leu³¹,Pro³⁴]substituted NPY/PYY analogues>>C-terminal fragments=pancreatic polypeptides, with the non-peptide antagonist BIBP3226 being most potent. This profile excludes the possible labelling by [¹²⁵I]-[Leu³¹,Pro³⁴]PYY of the newly cloned Y₄, Y₅ and Y₆ receptors.
4. The expression of the genuine Y₁ receptor was confirmed by RT–PCR in hippocampal cultures. In contrast, negligible levels of Y₂-like/[¹²⁵I]-PYY_3–36 binding were detected in these cultures in spite of the presence of its mRNA, as characterized by RT–PCR. The expression of both the Y₁ and the Y₂ receptor mRNAs was also noted in normal embryonic hippocampal tissues showing that signals expressed in cultured neurones were also present in utero.
5. Taken together, these results suggest that the Y₁ receptor subtype may be of critical importance in the normal functioning of the rat hippocampus, especially during brain development and maturation.

     

Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture

Tamoxifen increases the risk of human endometrial cancer and is a potent carcinogen in rat liver, in which it produces DNA adducts and cytogenetic damage. Nevertheless its prophylactic use against breast cancer in healthy women is under investigation in several large trials. To investigate whether rat hepatocarcinogenicity predicts human hepatocarcinogenicity we used genetically engineered bacterial and mammalian target cells to investigate how alpha-hydroxy-tamoxifen, a major phase I metabolite of tamoxifen, is further metabolised by rat and human phase II enzymes, sulfotransferases, to mutagenic and DNA- adduct-forming species. We expressed rat hydroxysteroid sulfotransferase a, a liver-specific enzyme, and corresponding human sulfotransferase in bacteria (Salmonella typhimurium) and in a mammalian cell line (Chinese hamster V79 cells) and tested alpha- hydroxytamoxifen for DNA adduct formation and mutagenicity in these systems, using unmodified cells as controls. In cells that expressed rat hydroxysteroid sulfotransferase, alpha-hydroxytamoxifen was mutagenic and formed the same pattern of DNA adducts as that found in the liver of tamoxifen-treated rats. Alpha-hydroxytamoxifen was not activated, or was at least 20 times less active in cells expressing human hydroxysteroid sulfotransferase. All the other six known human xenobiotic-metabolising sulfotransferases were also expressed in S. typhimurium. None activated alpha-hydroxytamoxifen to a mutagen. These results suggest that the risk of DNA adduct formation, and cancer, in the human liver is low and explain why tamoxifen is a powerful carcinogen to the rat liver, and why standard short-term tests fail to detect its mutagenicity.      

Smoking-associated bulky DNA adducts in bronchial tissue related to CYP1A1 MspI and GSTM1 genotypes in lung patients

Relationships between smoking status and levels of bulky DNA adducts were investigated in bronchial tissue of lung patients in relation to their GSTM1 and CYP1A1 MspI genotypes. A total of 150 Hungarian patients undergoing pulmonary surgery were included in the study, 124 with lung malignancies and 26 with non-malignant lung conditions. There were significant relationships between smoking status and bulky DNA adduct levels, as determined by 32P-post-labelling analysis, in macroscopically normal bronchial tissues. There was a highly significant difference in the adduct levels of a combined group consisting of current smokers and short-term ex-smokers (< or = 1 year abstinence) compared with life-time non-smokers and long-term ex- smokers (> 1 year abstinence) (P = 0.0001). The apparent half-life was estimated to be 1.7 years for bulky DNA adducts in the bronchial tissue from ex-smokers. There were no statistically significant correlations between (i) daily cigarette dose and DNA adduct levels in current smokers, (ii) DNA adduct level and histological type of lung cancer, or (iii) GSTM1 and CYP1A1 MspI genotypes and DNA adduct levels after adjustment for either smoking status or malignancy. By multiple logistic regression analysis, smoking and GSTM1 null genotype were found to be risk factors for squamous cell carcinoma. However, bulky DNA adduct levels in bronchial tissue did not appear to be a statistically-significant risk factor for the major histological types of lung cancer.      

Metaphyseal growth arrest lines in psychosocial short stature

Metaphyseal growth arrest lines are seen in children who experience significant physical stress such as infection or malnutrition over a sufficient period of time. These lines have not been reported previously in children with psychosocial short stature (PSS). Two boys and a girl with PSS with metaphyseal growth arrest lines on skeletal radiographs at the time of maximal stress in their homes are described. All three had reversible growth hormone insufficiency during admission, which is pathognomic for PSS. Multiple growth arrest lines in the distal end of the radius or vertebrae should alert clinicians to an alternative diagnosis in a child with growth hormone insufficiency. This may provide a clue to the diagnosis of occult PSS.     

Radial maze proficiency of adult Wistar rats given prenatal complex magnetic field treatments

Exposure to sinusoidal (power-frequency) magnetic fields during prenatal development is implicated in adulthood behavioral impairments. However, the effects of prenatal exposure to weak-intensity, nonsinusoidal complex magnetic fields (CMFs), an increasingly common feature of the modern environment, have not been rigorously examined. In the present study, male and female Wistar-strain rats were exposed continually during prenatal development to one of three extremely low-frequency CMFs or a sham condition. As adults, rats were trained in an acquisition/reversal radial maze task. All rats exposed to the prenatal CMFs increased their commission of reference memory errors, but differences in working memory and motivation to complete the maze task were specific to the type of prenatal CMF. These results provide the first evidence that prenatal exposures to specific shapes of CMFs impair complex learning behaviors into adulthood.     

Radiographic damage in rheumatoid arthritis correlates with functional disability but not direct medical costs.

OBJECTIVE: Few longitudinal data exist on the relationship between radiographic damage and self-reported functional disability and direct medical costs in rheumatoid arthritis (RA). We assessed these relationships. METHODS: One hundred thirty patients with RA (at time of the first available radiograph, mean age 56.6 yrs, 16.9% male, mean disease duration 16.8 yrs) were followed for up to 13.4 years. Semiannually, they reported on functional disability (0 = no difficulty, 3 = unable to do), global severity (0 = very well, 100 = very poor), pain (0 = no pain, 3 = severe pain), and health services utilization through completion of the Stanford Health Assessment Questionnaire (HAQ). Concurrent hand radiographs were scored for erosions and joint space narrowing using the Genant method and a single score summing both erosions and joint space narrowing for both hands was calculated (0 = no damage, 200 = maximum damage). The univariate association of functional disability, global severity, pain, or direct medical costs with concurrent radiographic damage was assessed through Spearman correlations and hierarchical regression models. The hierarchical models permit exploitation of the between-patient and within-patient variation present in our longitudinal data. RESULTS: At the time of the first available radiograph, mean (SD) levels of functional disability, global severity, and pain were 1.3 (0.7), 39.4 (21.0), and 1.1 (0.7), respectively. At entry into the study, the average radiograph score was 49.7 and upon leaving the study it was 66.9. Patients were followed an average of 6.7 years, with radiograph scores increasing at an average rate of 2.5 units/yr. The Spearman correlation [95% confidence interval (CI)] between average per-patient radiograph score and average per-patient HAQ disability index, average per-patient global severity, average per-patient pain score, and average per-patient direct medical costs was, respectively, 0.42 (0.26, 0.55), 0.23 (0.06, 0.39), 0.20 (0.03, 0.36), and 0.06 (-0.11, 0.23). The mean slope (95% CI) for disability on radiograph score was 0.0186 (0.0132, 0.0226), for severity on radiographs 0.1889 (0.1295, 0.2498), and for pain on radiographs 0.0057 (0.0027, 0.0084). As an example, over 10 years, a 25 unit (i.e., 50%) increase in radiograph scores would, on average, be associated with a 0.46 unit (i.e., 35%) increase in disability, a 4.72 unit (12%) increase in global severity score, and a 0.14 unit (13%) increase in pain, all expressed on the HAQ scales. There was little association between radiograph score and direct medical costs. CONCLUSION: A clinically meaningful association exists between radiographic damage and self-reported functional disability, suggesting that interventions that slow radiographic progression may improve the patient's health status. Such a relationship was not observed between radiographic damage and direct medical costs.     

Laboratory integration and utilization of tandem mass spectrometry in neonatal screening: a model for clinical mass spectrometry in the next millennium

Clinical and neonatal screening methods using a tandem mass spectrometer are clearly a model for modern laboratory testing in the new Millennium. By the year 2000, more than 1 million blood and plasma samples will have been tested in laboratories throughout the world for a battery of metabolic disorders using a tandem mass spectrometer as the primary analytical device. A tandem mass spectrometer is considered the "ultimate" analytical detector in a variety of biochemical and clinical methods because of its very high accuracy, selectivity, precision, versatility and robust nature. The ability to achieve very high and reproducible sample throughput (∼600 samples/ instrument/24 h) has made this technology cost-effective for newborn screening. In order to reliably measure markers of inborn errors of metabolism while maintaining low costs and high efficiency, accuracy and quality, much attention needs to be placed on monitoring and maintenance of all components of the entire testing system. These components include specimen collection and sample preparation methods, analysis by LC tandem mass spectrometry, conversion of raw mass spectra (data) into clinically meaningful results (concentration), expert interpretation of these results so that the clinician can be provided with information to facilitate a diagnose, and follow-up and education so that the maximum benefits of newborn screening translate into prevention of disease symptoms or more effective treatments. Addressing each part of the whole system will produce a quality screening program that will detect a battery of disorders using tandem mass spectrometry with a disease frequency of nearly 1 in 4000 infants.     

Gelatinase B (MMP-9), but not its inhibitor (TIMP-1), dictates the growth rate of experimental thymic lymphoma.

Dysregulation of metalloproteinase production at tumor sites contributes to the modification of local stromal tissue necessary for tumor development. Gelatinase B (matrix metalloproteinase-9, MMP-9) is one of the key enzymes that have been associated with the progression of several tumors. Paradoxically, MMP-9 expression by tumor cells, most notably by lymphoma cells, is concomitant with the expression of its physiological inhibitor, TIMP-1. Not only are both genes often co-expressed in the most aggressive forms of lymphomas but also both are up-regulated upon contact with stromal cells. Since TIMP-1 is known to regulate growth in several cell types and some aggressive lymphoma cells express TIMP-1 constitutively without MMP-9, it is unclear whether the over-expression of MMP-9 is counterbalanced by TIMP-1 and whether TIMP-1 expression alone could favor the development of lymphoma. To gain further insight into the respective roles of MMP-9 and TIMP-1 in lymphoma, we generated lymphoma cell lines expressing constitutively high levels of MMP-9 or TIMP-1 and compared these cells for the ability to form thymic lymphoma in vivo. Moreover, we generated lymphoma cell lines expressing constitutively high levels of both MMP-9 and TIMP-1 to reproduce the net physiological balance resulting from the expression of both genes simultaneously and to determine which gene overrides the other. Our results show that mice injected with lymphoma cells expressing MMP-9 constitutively developed thymic lymphoma more rapidly than those injected with control lymphoma cells. Over-expression of TIMP-1 alone did not significantly influence tumor progression of lymphoma nor did it delay the capacity of MMP-9 to accelerate the development of thymic lymphoma.     

Characterization of the siRNAs associated with peach latent mosaic viroid infection

The peach latent mosaic viroid (PLMVd) is a small, circular RNA species that has been shown to induce RNA silencing in plants. With the goal of better understanding the biological mechanism underlying this process, the siRNAs found in PLMVd infected peach leaves were isolated and sequenced. Analysis of the resulting data prompted several conclusions, including: i. PLMVd strands of both polarities are substrates for the Dicer-like enzymes found in peach leaves; ii. the more highly structured regions of PLMVd trigger the activity of the Dicer-like enzymes; and, iii. the circular PLMVd conformers appear to be favored for transport into the cytoplasm.