Purification of Masked Temperature data from Humans: Some Preliminary Observations on a Comparison of the use of an Activity Diary, Wrist Actimetry, and Heart Rate Monitoring

Fourteen ambulatory subjects, varying in their amount of habitual physical activity, were studied for 24h during a total of 25 “typical” days. Rectal temperature was recorded every 6 minutes, an activity diary was filled in every half hour, and wrist activity and heart rate were monitored every minute. Actimetry and heart rate data generally showed close parallelism with each other and with the masking effects on body temperature. Psychological stressors such as public speaking produced a greater effect on heart rate and body temperature than on wrist movement, while typing produced high values for wrist movement, but affected heart rate and temperature much less. When data for the circadian rhythm of body temperature were purified, the diary, actimetry, and measurement of heart rate were all useful in reducing masking effects, but the present evidence indicates that heart rate can be more successful than actimetry-as judged by the closeness of the purified data to a sinusoid. This superiority of heart rate monitoring over wrist activity as a method of purification might be because core temperature can be increased by stressor-induced thermogenesis, as well as by physical activity, and because wrist movement can, with some activities, give an inaccurate estimate of the factors that contribute to whole-body thermogenesis.     

Synthetic peptides induce antibodies in sheep against Taenia ovis

Summary Two peptides corresponding to putative protective regions located at the N- and C-termini of the host-protectiveT. ovis recombinant antigen, 45W, were synthesized. Antibodies raised against 45W and 45WB/X, a truncated from of 45W, were found to react strongly with the N-terminal peptide. When sheep were immunised with each peptide alone, the N-terminal peptide was found to be highly immunogenic, whereas the C-terminal peptide required conjugation to a carrier protein to be immunogenic. Both these immunogens elicited antibodies that cross-reacted with the parent protein; however, only antibodies directed toward the N-terminal peptide were able to bind antigens from theT. ovis oncosphere. Significant protection against challenge infection was not provided by any of the peptide immunogens used.     

UVB-induced apoptosis in normal human keratinocytes: role of the erbB receptor family

Exposure of human keratinocytes to ultraviolet B (UVB) light leads to the activation of a variety of cell-surface receptors; however, the biologic consequences of these activated receptors are still unclear. It was previously reported that inhibition of cellular tyrosine kinase activity suppressed UVB-dependent effects in human skin. We confirmed that the same suppression of UVB-induced apoptosis occurs in normal human keratinocytes grown in culture. Furthermore, we sought to determine the role of erbB receptor tyrosine kinases in human keratinocytes following UVB irradiation. Using a specific inhibitor of the erbB family of tyrosine kinase receptors, DAPH, we investigated the effects of UVB-dependent activation of these receptors on keratinocyte biology. The addition of DAPH to keratinocytes resulted in the concentration-dependent protection of UVB-induced apoptosis. The protection from apoptosis was not due to the induction of keratinocyte differentiation, the loss of keratinocyte viability, or inhibition of the proliferative potential of keratinocytes by DAPH. The effect of DAPH on apoptosis was specific for UVB as it had no effect on bleomycin-induced apoptosis. Furthermore, the inhibition of UVB-induced apoptosis could also be observed using neutralizing antibodies to either erbB1 or erbB2. Finally, we demonstrated that DAPH could also inhibit UVB-induced apoptosis in an epidermal organotypic model system. These studies suggest an important role for the erbB receptors in UVB-induced apoptosis of human keratinocytes.     

Pro-apoptotic proteins released from the mitochondria regulate the protein composition and caspase-processing activity of the native Apaf-1/caspase-9 apoptosome complex

The apoptosome is a large caspase-activating ( approximately 700-1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is approximately 135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9(1-130)) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9(1-130), demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6-8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.     

Developmental effects of embryonic exposure to toxaphene in the American alligator (Alligator mississippiensis)

A variety of organochlorine pesticides have been shown to adversely affect embryonic development. A number of abnormalities have been documented in alligators (Alligator mississippiensis) from highly-contaminated Lake Apopka, FL, USA that are similar to the results of experimental studies exposing embryos to pesticides. In the current study, we exposed developing alligator embryos to varying concentrations of toxaphene, a broad-spectrum pesticide found in relatively high concentration in Lake Apopka alligator egg yolk. The toxaphene, dissolved in 50 microl of ethanol, was applied topically to the eggshell just prior to the sex-determining period of development. Shortly after hatching, we examined a number of morphological and physiological endpoints to determine the consequences of sub-lethal embryonic exposure to toxaphene. Our results indicate that toxaphene had little or no effect on the morphological endpoints examined including body mass (BM) and size, liver, thyroid, and gonad development. In addition, toxaphene failed to affect sexual differentiation, or in vitro thyroxin, testosterone (T), and estradiol production. However, male plasma T concentration was higher in animals treated with 10 and 0.01 microg toxaphene/kg (based on mean egg mass) than control males. Because in vitro T production was not different among control groups, we suggest the difference in plasma T could be due to differences in hypothalamic-pituitary stimulation of the gonad or hepatic steroid degradation. This study indicates that technical grade toxaphene, at the applied doses, does not induce the same developmental abnormalities associated with alligators living in Lake Apopka. Future studies should consider the effects of embryonic exposure to a mixture of chemicals, including toxaphene metabolites, on development in alligators to better evaluate the consequences of environmental contamination.