The naevoid basal-cell carcinoma syndrome (Gorlin syndrome) is a chromosomal instability syndrome

We report the establishment and preliminary characterization of a stable steroidogenic granulosa cell line, JC-410. This cell line was obtained by spontaneous immortalization of a primary culture of porcine granulosa cells. Cultured JC-410 cells produced less progesterone than granulosa cells in primary culture. Progesterone synthesis by JC-410 cells was approximately 10% and 1% of the amount produced by granulosa cells from small and medium sized follicles, respectively. Although FSH and LH did not change progesterone levels in cultured JC-410 cells, forskolin and cholera toxin induced a 2.6- and 2.75-fold increase, respectively, versus control. The JC-410 cells responded to 0.1, 1 and 5 mM cAMP with an increase in progesterone synthesis of 2.5-, 28- and 49-fold versus control, respectively, after a 24 h incubation. No detectable levels of estradiol-17beta were found in JC-410 cells after 48 h in culture. However, addition of 0.01, 0.1 and 1 microM androstenedione elevated the levels of estradiol-17beta to 0.028, 0.3 and 1.21 pg/microg protein, respectively. The level of expression of 3betaHSD, aromatase and P450scc genes in JC-410 cells is of similar magnitude to the level of expression in granulosa cells in primary culture. The JC410 cells have been maintained in culture for more than one year during which their population doubled over 100 times. We conclude that JC-410 is a stable cell line that lost responsiveness to the gonadotropins during the process of immortalization, but retained its steroid biosynthetic capability and the expression of key steroidogenic genes. These characteristics may reflect features of cells arrested in an early stage of granulosa cell differentiation.     

Genetic instability in radiation-induced leukaemias: mouse models

Ivermectin is not lethal to the adult worms of Onchocerca volvulus or to those of O. ochengi, a cattle parasite closely related to O. volvulus. Although ivermectin penetrates the nodules in which the adults of these nematodes live, it is not known what levels of the drug enter the worms. Adult male O. ochengi were incubated in [3H]ivermectin in a saturated solution of unlabelled ivermectin (11.44 microM), to measure uptake by the oral and transcuticular routes, and in [3H]inulin, to ascertain if oral ingestion occurs in vitro. Uptake of [3H]ivermectin was high [1040 disintegrations/min (d.p.m.) at 3 h, representing a mean total of 86 pmoles ivermectin/worm] and occurred predominantly by the transcuticular route. Viability of worms was not reduced by this exposure, and uptake continued for up to 12 h. Only low levels of [3H]inulin (four d.p.m.) were detected in worms, indicating that the gut is probably not functional in vitro. Scanning and transmission electron microscopy revealed that the epicuticle of both sexes had an irregular surface which was pitted with a honeycomb structure in males, and rough and abundantly folded in females. These structures greatly increased the absorptive surface of the worms. In conclusion, ivermectin is able to enter adult O. ochengi males at concentrations sufficient to kill non-filarial nematodes.     

Radiation induced chromosomal instability in human T-lymphocytes

Rats transgenic for HLA-B27 and human beta 2-microglobulin develop a spontaneous, multisystem, inflammatory disease that resembles human B27-associated disease and that involves the gut mucosa. This model predominantly affects the colon and is characterized by an extensive infiltration of the mucosa by inflammatory cells, largely composed of mononuclear cells. In addition, an increased plasma level of nitric oxide (NO)-derived metabolites was described in this model. Deficiency in the anti-inflammatory cytokine, interleukin-10 (IL-10), leads to the development of colitis in IL-10 knockout mice, suggesting that IL-10 plays a major role in the control of gut inflammation. The objectives of this work were to study the mechanisms of the inflammatory bowel disease (IBD) in HLA-B27 rats and to determine the effects of treatment with IL-10. The 33-3 line of HLA-B27 recombinant rats with established disease was treated in two consecutive experiments with murine recombinant IL-10 for five weeks. Assessment of the effect of this treatment was performed, based on clinical, histological and biological (myeloperoxidase and inducible NO synthase activities; tumor necrosis factor-alpha, interferon-delta, CD3, iNOS and beta-actin mRNA expression. In 33-3 rats with established disease, mesenteric lymph nodes were hyperplastic, and colonic cellularity and MPO and iNOS activities in the colonic mucosa were increased without any detectable effects of IL-10 administration. IFN-gamma and iNOS mRNA were only detected in the colon of transgenic rats. Despite a lack of effect on disease expression, IL-10 strikingly reduced the level of IFN-gamma mRNA in gut mucosa. Up-regulation of IFN-gamma mRNA suggests that the IBD of HLA-B27 rats is mediated by T-helper 1 lymphocytes. Sustained administration of IL-10, in HLA-B27 rats with established disease, efficiently inhibited IFN-gamma mRNA expression but did not influence disease expression: these results indicate that IFN-gamma may exert a critical role at an earlier stage of the disease rather in the maintenance of the lesions.     

The target is straw or the arrow is crooked.

Comments on the article by H. J. M. Hermans and H. J. G. Kempen (see record 1998-12442-002) which argued that cross-cultural psychology conceptualizes cultural difference in terms of strict, stable dichotomies defining homogenous groups while making final recommendations on the subject, namely that researchers should examine cultural contact zones and complex cultural identities. Although the present authors agree with these suggestions, they argue that Hermans and Kempen's "challenge" to cross-cultural psychology seems either (1) aimed at a "straw man" or (2) overstated and misguided. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The subthalamic nucleus: a possible target for stereotaxic surgery in Parkinson''s disease

Agenesis of the sacrum is a rare anomaly that is associated with numerous visceral abnormalities, spinal cord malformation, and lower limb defects. A fatal case of sacral and lower lumbar agenesis in a 3-day-old female infant born at 38 weeks of gestation is reported. The extraneural malformations comprised an imperforate anus, a rectovaginal fistula, and musculoskeletal abnormalities, including several thoracocervical hemivertebrae and aplasia of the sacrum and the fourth and fifth lumbar vertebrae. The cervical and high thoracic spinal cord segments were normal. Disruption of secondary neurulation, possibly due to notochord dysfunction, was suggested by malformation of the ventral half of the lower thoracic spinal cord with relative preservation of the dorsal horns and, more caudally, by loss of all normal histological landmarks, including the central canal. Neither skeletal muscle nor myoblasts were found in muscle compartments that would normally have received motor innervation from the levels of the spinal cord from which anterior horn cells were absent, indicating parallel, segmental failure of myotomal differentiation in the caudal eminence.     

Impact of genomic imprinting on genomic instability and radiation-induced mutation

A cross-sectional survey conducted among evening students was used to determine the prevalence of Wuchereria bancrofti infection in Maceió, capital of the State of Alagoas, northeast Brazil. A single thick-blood smear was used, being collected between 10 p.m. and 12 a.m. From a total of 29,551 students enrolled at evening elementary schools in the 33 city sectors, 16,569 (56.4%) were random selected for inclusion in the study. From those, 10,857 (65.5%) were interviewed and examined and 73 (0.7%) were found to have microfilaraemia. Autochthonous W. bancrofti carriers live in 10 of the 33 city sectors, suggesting a focal distribution. Moreover, 84% of infections were diagnosed among 29% of all students examined, inhabiting three contiguous sectors at the city central area, presenting infection rates up to 5.3%. Students living in city sectors with prevalence of microfilariae carriers greater than 1% were found to have a higher risk for infection when compared to students from the rest of the town [Relative Odds (RO) 12.8, 95% CI 6.7-25.1]. Eleven positive individuals from non endemic areas were living in Maceió for more than 10 years; time of residence in the area was a major risk factor for infection among students not born in the region (p < 0.01). Regarding sex, male students presented a higher proportion of positive (RO 1.7, 95% CI 1.1-2.9).     

The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus

Neuropeptide Y (NPY) immunoreactive (-ir) nerve fibers densely innervate hypophysiotropic TRH perikarya and dendrites in the hypothalamic paraventricular nucleus (PVN). To evaluate the contribution of the arcuate nucleus (Arc) to this innervation, the effect of Arc ablation by neonatal monosodium glutamate (MSG) treatment on the density of NPY-fibers contacting TRH neurons in the PVN was investigated. After the lesioned animals and vehicle-treated controls reached adulthood, the number of contacts between NPY-ir boutons and TRH-ir perikarya in the PVN was determined in double-immunostained sections. In controls, numerous contacts between NPY-ir terminals and TRH perikarya and dendrites were observed, confirming earlier findings. MSG treatment resulted in a marked reduction of the size of the Arc and also the number of NPY-perikarya with a concomitant reduction of 82.4 +/-2.1% in the relative number of NPY terminals contacting TRH perikarya and first order dendrites in the medial parvocellular and periventricular subdivisions of the PVN. In contrast, lesioning of the ascending adrenergic bundle in the brain stem caused no statistically significant change in the number of NPY-terminals in close apposition to hypophysiotropic TRH neurons in the PVN. These data confirm earlier findings that NPY-containing axon terminals innervate TRH neurons in the PVN and further demonstrate a potentially important anatomical relationship between NPY-producing neurons in the Arc and hypophysiotropic TRH neurons.     

A new retroviral vector for detecting mutations and chromosomal instability in mammalian cells

A retroviral vector carrying both forward (neo) and backward (herpes simplex virus thymidine kinase or HSV-TK gene) selection markers was constructed as a substrate for mutational assay in mammalian cells. The cells infected with this virus are first selected with G418, mutagenized and then selected with the anti-herpes drug acyclovir (ACV). Since HSV-TK, but not the host TK, is capable of converting ACV to a toxic metabolite, cells retaining the intact HSV-TK gene fail to survive, while the cells carrying a mutated HSV-TK gene or which have lost the gene can form colonies in the presence of ACV, making it possible to detect the genetic defects in a positive manner. It is also possible to discriminate between small mutations and large deletions by checking the presence of the linked marker, neo. As a model experiment, we prepared an uncloned pool of rat fibroblast cells (CREF) infected with this virus and irradiated them with increasing doses of ultraviolet light. Dose-dependent increases in the number of ACV-resistant colonies were observed. Structural analysis of the HSV-TK gene in these clones revealed point mutations or small deletions in the majority of the cases. Since it requires no pre-existing genetic markers in the host cells, this system may be used for a wide variety of mammalian cells and provides a useful tool to assess both their susceptibility to various mutagens and their genomic instability.     

Categorical thinking is the target.

Replies to the commentaries by R. G. Tweed, L. G. Conway, III, and Andrew G. Ryder (see record 1999-11644-006), T. L. Holdstock (see record 1999-11644-007), and R. J. Smith (see record 1999-11644-008) on Hermans and Kempen's article (see record 1998-12442-002) on the issues of globalization and localization in cross cultural psychology. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Protein phosphatase 2A is required for the initiation of chromosomal DNA replication

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract resulted in strong inhibition of DNA replication. PP2A was required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A.     

Molecular cloning and chromosomal localization of Chinese hamster telomeric protein chTRF1. Its potential role in chromosomal instability

Chinese hamster cells frequently have altered karyotypes. To investigate the basis of recent observations that karyotypic alterations are related to telomeric fusions, we asked whether these alterations are due to lack of telomere repeat binding factor/s. Further, Chinese hamster chromosomes contain large blocks of interstitial telomeric repeats, which are preferentially involved in chromosome breakage and exchange, rendering it an interesting model for such studies. Here, we report on the cloning and the chromosomal localization of the Chinese hamster telomere repeat binding factor, chTRF1. The sequence analysis revealed, similar to human TRF1 (hTRF1), an N-terminal acidic domain, a TRF1 specific DNA binding motif and a C-terminal Myb type domain. Unlike mouse TRF1 (mTRF1), chTRF1 shows 97.5% identity to hTRF1. chTRF1 gene was localized on the long arm of chromosome 5. In vitro translation of chTRF1 resulted in protein product similar in molecular weight to hTRF1. Immunostaining of Chinese hamster ovary cells (CHO) with anti-TRF1 antibody revealed punctate nuclear staining. At metaphase, antibodies failed to detect TRF1 on most of the chromosome ends and the interstitial telomeric repeat bands. These studies suggest that chTRF1 does not bind the interstitial telomeric repeats, and its presence at the metaphase chromosome ends is limited. The later could be a factor contributing to frequent karyotypic alterations observed in Chinese hamster cells.     

Long-term in vivo transmission of alpha-particle-induced chromosomal instability in murine haemopoietic cells

A study was made on the dentin bonding of MMA/PMMA resin using sulfinic acid/BPO/amine polymerization initiator, to examine: 1) the effect of ferric and copper chloride contained in phosphoric acid or citric acid dentin conditioners, and 2) the effect of the addition of acidic monomers to the resin. Tensile bond strength was significantly improved by conditioning dentin with 10% phosphoric acid containing 3% ferric chloride. The citric acid-based conditioners and the addition of the copper salt to the acidic conditioners were less effective in improving bond strength. The mechanism of the improved bond strength is discussed in terms of polymerization promoting action of ferric ion at the dentin-resin interface. For improving bond strength, the presence of a suitable acidic monomer was essential. Its effectiveness depended on the types of acids: Two phosphoric acid ester monomers were effective but methacrylic acid had little effect.     

Inhibitory effect of folinic acid on radiation-induced micronuclei and chromosomal aberrations in V79 cells

Folinic acid (FA), clinically called leucovorin, has been widely used as a nutrient supplement in dietary intake and is capable of inhibiting cytotoxicity and chromosomal damage induced by chemicals. However, data on its antigenotoxic effect on radiation-induced chromosomal damage are limited. The present study was, therefore, performed to investigate the effect of FA on radiation-induced (X-rays and UV radiation) micronuclei (MN) and structural chromosomal aberrations (SCA) concurrently in V79 Chinese hamster lung cells. Exponentially growing cells were exposed to five doses of X-rays (1-12 Gy) and UV radiation (50-800 microJ x 10(2)/cm2) and post-treated with 5 or 50 micrograms FA/ml of culture medium for 16 h. The slides were analyzed for the presence of MN and SCA using standard procedures. The results showed that X-ray treatment alone produced dose-related cytotoxicity as measured by nuclear division index (NDI) and mitotic index (MI). X-rays produced a clear dose-related clastogenicity as measured by percent of micronucleated binucleated cells (MNBN) (5-79%) and percent of aberrant cells (11-92%). FA at 5 micrograms/ml slightly decreased X-ray induced chromosomal damage in both assays; however, the inhibition was significant (12-46% of MNBN, 14-48% in aberrant cells) only when X-ray-treated cultures were post-treated with 50 micrograms FA/ml. Post-treatment of FA had no effect on X-ray induced cytotoxicity as measured by NDI and MI. A similar a dose-related increase in % MNBN (0.5-10.3%) and percent aberrant cells (6-35%) was produced by UV radiation treatment alone. There were significant percentages of MNBN and aberrant cell inhibitions at both 5 and 50 micrograms/ml in both assays. As in the case of X-ray-treated cells, there was a clear dose-related cytotoxicity in UV-treated cells alone. No reduction in NDI or MI was found when UV-exposed cells were post-treated with 5 or 50 micrograms of FA. These data demonstrate the beneficial effect of FA in decreasing radiation-induced chromosomal damage.     

An intestinal mucin is the target substrate for a baculovirus enhancin

An invertebrate intestinal mucin (IIM) was identified from a lepidopterous insect, Trichoplusia ni. The IIM is a major protein constituent of the peritrophic membrane that facilitates the digestive process, as well as protecting invertebrate digestive tracts from microbial infections. The IIM demonstrated biochemical characteristics similar to vertebrate mucins, but exhibited strong association with the chitin-containing peritrophic membrane matrix. We have demonstrated that a baculovirus enhancin, which is encoded and carried by specific baculoviruses, has mucin-degrading activity both in vitro and in vivo. The in vivo degradation of IIM by enhancin was correlated with the enhancement of baculovirus infections in insects. These findings have shown that viruses have evolved a novel strategy to overcome intestinal mucinous barriers against microorganisms by utilizing a mucin-degrading enzyme.     

The aged suprachiasmatic nucleus is phase-shifted by cAMP in vitro

The cyclic adenosine monophosphate (cAMP) analog, 8-bromo-cAMP, phase advanced circadian neuronal rhythms in both aged and adult rat suprachiasmatic nuclei (SCN) by approximately 2 h in vitro. Rhythm amplitude was 20% lower in aged compared to adult SCN. The diminished efficacy of serotonergic agonists to phase shift behavioral rhythms of aged animals may be due to decrements in signal transduction mechanisms proximal to cAMP.     

The cytoplasmic membrane is a primary target for the staphylocidal action of thrombin-induced platelet microbicidal protein

Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner.     

The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase

The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues. We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.     

Calpain activation is upstream of caspases in radiation-induced apoptosis

The molecular events involved in apoptosis induced by ionizing radiation remain unresolved. In this paper we show that the cleavage of fodrin to a 150 kDa fragment is an early proteolytic event in radiation-induced apoptosis in the Burkitts' Lymphoma cell line BL30A and requires 100 microM zVAD-fmk for inhibition. Caspases-1, -3, -6 and -7 were shown to cleave fodrin to the 150 kDa fragment in vitro and all were inhibited by 10 microM zVAD-fmk. We also show that the in vitro cleavage of fodrin by calpain is inhibited by 100 microM zVAD-fmk as was the calpain-mediated hydrolysis of casein. We demonstrate that calpain is activated within 15 min after radiation exposure, concomitant with the cleavage of fodrin to the 150 kDa fragment whereas caspase-3 is activated at 2 h correlating with the cleavage of fodrin to the 120 kDa fragment. These results support a role for calpain in the early phases of the radiation-induced apoptosis pathway, upstream of the caspases.