A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

DNA fragmentation during exposure of rat cerebella to ethanol under hypoxia imposed in vitro

To gain a better understanding into the mechanisms of damage incurred by neurons in periods following heavy alcohol exposure during development, we used an in vitro system to monitor the effects of alcohol and hypoxia on cell survival and DNA integrity. Samples representing the first few hours of exposure to alcohol and hypoxia were compared to those resulting from hypoxia alone. Measurements were taken from cell counts using Trypan blue exclusion and TUNEL assays as well as digital scans of the ethidium bromide fluorescence of genomic DNA isolated from the treated tissue. We found that DNA degradation from hypoxia was accelerated by several hours in the presence of 100 mM ethanol. This result depended on age, with adult animals (>8 months) having a similar response to 4-day postnatal animals, while the effect on 10-day postnatal animals and those of intermediate age (45 days postnatal) was increasingly delayed. Different methods of inducing the processive degradation of DNA produced laddering typical of apoptosis, a biphasic degradative process, or patterns usually associated with necrosis.     

Elemental composition of human milk from mothers of premature and full-term infants during the first 3 months of lactation

To examine longitudinal and gestational effects of mineral content in human milk, we analyzed human milk from lactating mothers of premature (PRT,n = 24, < 2000g birth weight, < 37 wk gestation) and fullterm (FT,n = 19, > 2500g, 39–41 wk gestation), living in Newfoundland, Canada. Samples were collected once a week for 8 wk with one final sample collected at 3 mo. Milk samples collected in acid-washed containers were wet ashed with concentrated HNO₃, and barium, cadmium, calcium, cesium, cobalt, copper, cerium, lanthanum, magnesium, manganese, molybdenum, nickel, lead, rubidium, tin, strontium, and zinc were measured using inductively coupled plasma-mass spectrometry. Data were analyzed using standard multiple-regression procedures with correlated data analyses to take account of the relationship between successive weeks. Results indicated lower Ca and Pb in PRT milk. Calcium was the only nutritionally significant element to differ between groups. Molybdenum in both PRT and FT milk showed a definite decrease with time, suggesting that the Mo content in milk is homeostatically regulated. However, Ce, La, Ba, and Sn did not display any pattern indicative of biological regulation and potential human requirement.     

Feeling by sight or seeing by touch?

We have addressed the role of occipital and somatosensory cortex in a tactile discrimination task. Sight-ed and congenitally blind subjects rated the roughness and distance spacing for a series of raised dot patterns. When judging roughness, intermediate dot spacings were perceived as being the most rough, while distance judgments generated a linear relation. Low-frequency rTMS applied to somatosensory cortex disrupted roughness without affecting distance judgments, while rTMS to occipital cortex disrupted distance but not roughness judgments. We also tested an early blind patient with bilateral occipital cortex damage. Her performance on the roughness determination task was normal; however, she was greatly impaired with distance judgments. The findings suggest a double-dissociation effect in which roughness and distance are primarily processed in somatosensory and occipital cortex, respectively. The differential effect of rTMS on task performance and corroborative clinical evidence suggest that occipital cortex is engaged in tactile tasks requiring fine spatial discrimination.     

Sampling accuracy of reef resource inventory technique

A new technique called the reef resource inventory (RRI) was developed to map the distribution and abundance of benthos and substratum on reefs. The rapid field sampling technique uses divers to visually estimate the percentage cover of categories of benthos and substratum along 2×20 m plotless strip-transects positioned randomly over the tops, and systematically along the edge of reefs. The purpose of this study was to compare the relative sampling accuracy of the RRI against the line intercept transect technique (LIT), an international standard for sampling reef benthos and substratum. Analysis of paired sampling with LIT and RRI at 51 sites indicated sampling accuracy was not different (P>0.05) for 8 of the 12 benthos and substratum categories used in the study. Significant differences were attributed to small-scale patchiness and cryptic coloration of some benthos; effects associated with sampling a sparsely distributed animal along a line versus an area; difficulties in discriminating some of the benthos and substratum categories; and differences due to visual acuity since LIT measurements were taken by divers close to the seabed whereas RRI measurements were taken by divers higher in the water column. The relative cost efficiency of the RRI technique was at least three times that of LIT for all benthos and substratum categories and as much as 10 times higher for two categories. These results suggest that the RRI can be used to obtain reliable and accurate estimates of relative abundance of broad categories of reef benthos and substratum.

Biotelemetry: a mechanistic approach to ecology

Remote measurement of the physiology, behaviour and energetic status of free-living animals is made possible by a variety of techniques that we refer to collectively as 'biotelemetry'. This set of tools ranges from transmitters that send their signals to receivers up to a few kilometers away to those that send data to orbiting satellites and, more frequently, to devices that log data. They enable researchers to document, for long uninterrupted periods, how undisturbed organisms interact with each other and their environment in real time. In spite of advances enabling the monitoring of many physiological and behavioural variables across a range of taxa of various sizes, these devices have yet to be embraced widely by the ecological community. Our review suggests that this technology has immense potential for research in basic and applied animal ecology. Efforts to incorporate biotelemetry into broader ecological research programs should yield novel information that has been challenging to collect historically from free-ranging animals in their natural environments. Examples of research that would benefit from biotelemetry include the assessment of animal responses to different anthropogenic perturbations and the development of life-time energy budgets.     

Psychological impact of genetic testing for breast cancer susceptibility in women of Ashkenazi Jewish background: a prospective study

The recognition that the prevalence of three founder mutations in the BRCA1 and BRCA2 genes is over 2% in Ashkenazi Jews has resulted in numerous epidemiological research studies of this ethno-religious group. To determine the effects of incorporating research into clinical practice, a psychological impact study of women participating in an epidemiological study was conducted. Sixty women of Ashkenazi Jewish background who underwent genetic testing for founder mutations were assessed using mailed, self-administered questionnaires with validated measures of psychological outcome. Forty-three women elected to learn their results and 17 women declined to do so. Women who elected to learn their results were also assessed 7-10 days, 4 months, and 12 months after results disclosure. Women who chose to learn their results had significantly higher baseline breast cancer anxiety, compared to those who elected not to learn their results (z = -2.27; p = 0.023). Unaffected women who elected to learn their results showed a significant decrease in breast cancer anxiety 4 months (z = -2.37, p = 0.018) and 12 months (z = -3.06, p = 0.002) post-notification compared to baseline. Genetic testing for mutations common in Ashkenazi Jewish women with result disclosure does not lead to adverse psychological outcomes.     

During apoptosis bcl-2 changes membrane topology at both the endoplasmic reticulum and mitochondria

In healthy cells the antiapoptotic protein Bcl-2 adopts a topology typical of tail-anchored proteins with only the hydrophobic carboxyl terminus inserted into the membrane, as shown by labeling cell lysates with a membrane-impermeant sulfhydryl-specific reagent. Induction of apoptosis in cells triggered a change in the conformation of Bcl-2 such that cysteine 158 near the base of helix 5 inserted into the lipid bilayer of both endoplasmic reticulum and mitochondria where it was protected from labeling. Addition of a peptide corresponding to the BH3 domain of the proapoptotic protein Bim to cell lysates triggered a similar conformational change in Bcl-2, demonstrating that preexisting, membrane-bound Bcl-2 proteins change topology.