Hepatic DNA adduct dosimetry in rats fed tamoxifen: a comparison of methods

Liver homogenates from rats fed tamoxifen (TAM) in the diet were shared among four different laboratories. TAM-DNA adducts were assayed by high pressure liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES-MS/MS), TAM-DNA chemiluminescence immunoassay (TAM-DNA CIA), and (32)P-postlabeling with either thin layer ((32)P-P-TLC) or liquid chromatography ((32)P-P-HPLC) separation. In the first study, rats were fed a diet containing 500 p.p.m. TAM for 2 months, and the values for measurements of the (E)-alpha-(deoxyguanosin-N(2)-yl)-tamoxifen (dG-N(2)-TAM) adduct in replicate rat livers varied by 3.5-fold when quantified using 'in house' TAM-DNA standards, or other approaches where appropriate. In the second study, rats were fed 0, 50, 250 or 500 p.p.m. TAM for 2 months, and TAM-DNA values were quantified using both 'in house' approaches as well as a newly synthesized [N-methyl-(3)H]TAM-DNA standard that was shared among all the participating groups. In the second study, the total TAM-DNA adduct values varied by 2-fold, while values for the dG-N(2)-TAM varied by 2.5-fold. Ratios of dG-N(2)-TAM:(E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen (dG-N(2)-N-desmethyl-TAM) in the second study were approximately 1:1 over the range of doses examined. The study demonstrated a remarkably good agreement for TAM-DNA adduct measurements among the diverse methods employed.     

How do topical nasal corticosteroids improve sleep and daytime somnolence in allergic rhinitis?

Therapy for rhinitis improves sleep quality and symptoms of daytime sleepiness. This improvement with therapy may be secondary to anti-inflammatory effects, leading to a reduction of inflammatory mediators, or to a mechanical reduction of congestion directly leading to improvement in sleep disturbance. We combined our data from 3 placebo-controlled studies of intranasal corticosteroids in patients with perennial rhinitis to determine whether a correlation between the reduction of congestion and improved sleep and daytime somnolence existed. The pooled data of budesonide, flunisolide, and fluticasone demonstrated significantly decreased nasal congestion, sleep problems, and sleepiness in treated patients. The data demonstrated a correlation between a reduction in nasal congestion and an improvement of sleep (P < .01) and daytime somnolence (P = .01). Thus, topical intranasal corticosteroids should be used to decrease nasal congestion and to improve sleep and daytime somnolence in patients manifesting these symptoms.     

Malignant progression of B16 melanoma cells induced in vitro by growth factors produced by highly malignant cells

Four mouse B16 melanoma subclones (G3.15, G3.5, G3.12 and G3.26) exhibit progressively greater growth capacity in vitro and in vivo. Previously, non-metastatic G3.15 cells were sequentially converted, in monolayer cultures, to the moderately-metastatic G3.5 cells, and then to a highly-metastatic G3.5^* phenotype. Both conversions were induced by hypoxia followed by confluence, and also occurred in tumors. G3.5^* cells were comparable with, yet distinguishable from, G3.12 cells in being growth-autonomous in culture. In this study, the presumption that rapidly-growing G3.26 cells represented the ultimate progression step in this clonal system was examined. Both G3.12 and G3.5^* cells converted in vitro to the G3.26 phenotype during growth in serum-free medium conditioned by G3.26 cell growth. By selective filtration of conditioned medium and characterization of the stability of growth- and conversion-promoting activities, three distinct activities were found to promote a two-step G3.12 to G3.26 phenotype conversion: (1) a < 10 kDa filtrate stimulated slight attachment and proliferation of G3.12 cells, effects that were reversible, partly attributable to accumulated lactate, and fully mimicked by medium acidification to pH 6.5; (2) medium acidification, together with a heat- and acid-stable but partially trypsin-sensitive > 10 kDa activity, induced G3.12 G3.5^* conversion that resulted in acquisition of growth autonomy; and (3) a heat-, acid- and trypsin-sensitive > l0 kDa activity induced G3.5^* G3.26 conversion, characterized by anchorage-independent growth in soft agar, and potent lung colonization following intravenous injection. Phenotype analysis of G3.12 tumors and lung metastases revealed that G3.5^*-like cells were regularly present in tumors and metastases, whereas G3.26-like cells occurred almost exclusively in large lung metastases. While G3.12 cells might convert to G3.5^* cells in order to disseminate, G3.26 cells are apparently not involved in metastatic spread but probably account for the rapid growth of established metastases.     

Frontal lobe activation during object alternation acquisition

Object alternation (OA) tasks are increasingly used as probes of ventral prefrontal functioning in humans. In the most common variant of the OA task, subjects must deduce the task rule through trial-and-error learning. To examine the neural correlates of OA acquisition, the authors measured regional cerebral blood flow with positron emission tomography while subjects acquired an OA task, performed a sensorimotor control condition, or performed already learned and practiced OA. As expected, activations emerged in the ventral prefrontal cortex. However, activation of the presupplemental motor area was more closely associated with successful task performance. The authors suggest that areas beyond the ventral prefrontal cortex are critically involved in OA acquisition.     

Attention during looking and reaching as assessed by heart rate in 7(1/2)-month-old infants

Attention mediates the acquisition and encoding of information about the world and is central to motor action. Heart rate deceleration and behavioral inhibition are sensitive indices of the attentional process, but it is unknown whether these indices are valid in the context of overt action. The current study investigated the relationship between visual attention, action, and heart rate during reaching in 7(1/2)-month-old infants. We found that infants showed prolonged looking and large heart rate decelerations on reaching and looking trials. We conclude that overt action itself does not prevent the autonomic and behavioral changes that are also seen in attention to simple visual displays and that attention is maintained throughout the act of reaching.     

Canal-otolith interactions in the squirrel monkey vestibulo-ocular reflex and the influence of fixation distance

Natural head movements include angular and linear components of motion. Two classes of vestibulo-ocular reflex (VOR), mediated by the semicircular canals and otoliths (the angular and linear VOR, or AVOR and LVOR, respectively), compensate for head movements and help maintain binocular fixation on targets in space. In this study, AVOR/LVOR interactions were quantified during complex head motion over a broad range of fixation distances at a fixed stimulus frequency of 4.0 Hz. Binocular eye movements were recorded (search-coil technique) in squirrel monkeys while fixation distance (assessed by vergence) was varied using brief presentations of earth-fixed targets at various distances. Stimuli consisted of rotations around an earth-vertical axis and therefore always activated the AVOR. Horizontal and vertical AVORs were assessed when the head was centered over the axis of rotation and oriented upright (UP) and right-side-down (RD), respectively. AVOR gains increased slightly with increasing vergence in darkness, as expected given the small anterior position of the eyes in the head. Combined AVOR/LVOR responses were recorded when subjects were displaced eccentrically from the rotation axis. Eccentric rotations activated the AVOR just as when the head was centered, but added a translational stimulus which generated an LVOR component in response to interaural (IA) or dorsoventral (DV) tangential accelerations, depending on whether the head was UP or RD, respectively. When the head was eccentric and facing nose-out, the AVOR and LVOR produced ocular responses in the same plane and direction (coplanar and synergistic), and response magnitudes increased with increasing vergence. With the head facing nose-in, AVOR and LVOR response components were oppositely directed (coplanar and antagonistic). The AVOR dominated the response when fixation distance was far, and phase was compensatory for head rotation. As fixation distance decreased toward the rotation axis, responses declined to near zero, and when fixation distance approached even closer, the LVOR component dominated and response phase inverted. The same pattern was observed for both horizontal (head UP) and vertical (head RD) responses. The LVOR was recorded directly by rotating subjects eccentrically but in the nose-up (NU) orientation. The AVOR then generated torsional responses to head roll, coexistent with either horizontal or vertical LVOR responses to tangential acceleration when the subject was oriented head-out or right-side-out, respectively. Only the LVOR response components were modulated by vergence. A vectorial analysis of AVOR, LVOR, and combined responses supports the conclusion that AVOR and LVOR response components combine linearly during complex head motion. Received: 27 February 1997 / Accepted: 18 June 1997     

Spontaneous waves in the dentate gyrus of slices from the ventral hippocampus

Spontaneous negative-going potentials occurring at an average frequency of 0.7 Hz were recorded from the dentate gyrus of slices prepared from the temporal hippocampus of young adult rats. These events (here termed "dentate waves") in several respects resembled the dentate spikes described for freely moving rats during immobile behaviors and slow-wave sleep. Action potentials were observed on the descending portion of the in vitro waves and, as expected from this, whole cell recordings established that the waves were composed of depolarizing currents. Dentate waves appeared to be locally generated within the granule cell layer and were greatly reduced by antagonists of AMPA-type glutamate receptors or by lesions to the entorhinal cortex. Simultaneous recordings indicated that the waves were often synchronized in the inner and outer blades of the dentate gyrus. Knife cuts through the perforant path and the commissural/associational system did not eliminate synchronization, leaving electrotonic propagation via gap junctions as its probable cause. In accord with this, cuts that separated the two blades of the dentate eliminated synchronization between them, and a compound that inhibits gap junctions reduced wave activity. Dentate waves were regularly accompanied by sharp waves in field CA3 and were reduced in size by the acetylcholinesterase inhibitor, physostigmine. It is hypothesized that dentate waves occur when spontaneous glutamate release from dentate afferents produces action potentials in neighboring granule cells that then summate electrotonically into a population event; once initiated, the waves propagate, again electrotonically, and thereby engage a significant portion of the granule cell population.     

Transmission of HIV-1 infection between trophoblast placental cells and T-cells take place via an LFA-1-mediated cell to cell contact

HIV-1 vertical transmission is thought to mainly take place by virus crossing the placental barrier. However, the mechanism by which HIV-1-infects placental cells remains to be elucidated. We have found that purified cytotrophoblasts as well as trophoblastic cell lines are susceptible to infection by different HIV-1 isolates as detected by DNA-PCR and release of infectious virus, although with very low efficiency. Purified trophoblast or trophoblastic cell lines express low levels of chemokine receptors CCR-5 and CXCR-4 but not CD4 on the cell surface. To test if those molecules were used as receptors for HIV-1 infection, placental cells were pretreated with antibodies to CD4, CC-chemokines, C-X-C chemokines. None of those treatments inhibited HIV-1 infection. In contrast, we have found that HIV-1 infection of placental cells was increased in cocultures of infected T-cell blasts and placental cells. More interestingly, antibodies to beta(2) integrins and to LFA-1 were able to significantly block infection of placental cells. Cell surface expression of ICAM-1, an adhesion molecule involved in attachment of leukocytes to placenta, was upregulated in HIV-1-infected placental cells. Placental cells were able to transfer HIV-1 infection to T-cell blasts. This transmission required cell to cell contact and was also inhibited by anti-LFA-1 antibodies. In summary our results suggest that placental trophoblast could be infected by HIV-1 by a mechanism involving T cell to placental contact. Moreover, placental infection enhanced ICAM-1 expression and leukocyte adherence, an event which was required to transfer HIV-1 infection to T cells. This provides an explanation of the virus passing through the placental barrier during in utero HIV-1 vertical transmission.     

Somatic von hippel-lindau mutation in clear cell papillary cystadenoma of the epididymis

Papillary cystadenoma of the epididymis is an uncommon benign lesion that may occur sporadically or as a manifestation of von Hippel—Lindau (VHL) disease. Neither immunohistochemical studies nor molecular genetic analyses of the VHL gene have been reported previously for this lesion. The authors describe two cases of clear cell papillary cystadenoma of the epididymis, both of which were initially confused with metastatic renal cell carcinoma. Both lesions showed positive immunohistochemical staining for low and intermediate molecular weight keratins (Cam 5.2 and AE1/AE3), EMA, vimentin, α₁-antitrypsin, and α₁-antichymotrypsin. Each was negative for CEA. Because clear cell papillary cystadenoma is similar to renal cell carcinoma histologically, and because both occur as components of the von Hippel—Lindau disease complex, the authors analyzed both cases for the presence of mutations in the VHL gene. A somatic VHL gene mutation was detected in one of the two tumors by polymerase chain reaction followed by single-strand conformation polymorphism analysis. Direct sequencing revealed a cytosine to thymine transition at nucleotide 694, resulting in the replacement of an arginine with a stop codon after the sixth amino acid of exon 3. As the VHL gene is believed to function as a tumor suppressor gene, VHL gene mutations may play a role in the initiation of tumorigenesis in sporadic cystadenomas of the epididymis.