Thermogenic responses to body cooling during fever induced by Staphylococcus aureus cell walls in rabbits

Hypothalamic temperature (T _hypo) and metabolic heat production (M) were measured in seven conscious rabbits injected intravenously with either saline or with Staphylococcus aureus, (8 · 10⁷ cell walls · kg⁻¹) while being subjected to a 3-h period of ramp-like total body cooling using a chronically implanted intravascular heat exchanger. In pyrogen-injected animals cooling started (1) at the time of injection or (2) 70 min after injection. In (1) the fall in T _hypo induced by heat extraction was similar (1.0 °C) in afebrile and febrile animals. In (2) there was a transient increase in T _hypo of about 0.5 °C at a time corresponding to the start of fever resulting in a significantly smaller fall in T _hypo at the end of the 3-h cooling period (0.5 °C vs 0.9 °C, P < 0.05, n = 5). At this time in both (1) and (2) M was lower than theoretically expected from the increase in shivering threshold during fever. However, most of this effect can be explained when available data showing a decrease in thermosensitivity during S. aureus-induced fever are taken into account. After cessation of cooling in both groups of febrile animals T _hypo rose to about 1 °C higher than the precooling level, which is comparable to the fever level in a separate series of experiments with S. aureus injection without cooling (1.2 °C). Accepted: 23 September 1997     

Elevated intra-rumen pressure and secondary rumen contractions in sheep (Ovis aries)

1. Sheep rumens were insufflated with nitrogen to 5, 10, 15, and 20 cm water pressure and sustained at each pressure for 10 min. 2. Measurements included rumen motility, reticulorumen myoelectric activity, eructation frequency and volume, changes in tracheal pressure and rumen contraction amplitude. 3. As intra-rumen pressure increased, contractions designated as special secondary contractions appeared. 4. At a pressure of approximately 15 cm water, most of the special secondary contractions became regular secondaries; therefore, the special secondaries were called pro-secondary contractions. 5. Increased intra-rumen pressure was associated with respiratory distress. The recovery phase following, rumen insufflation was accompanied by hyperpnea.     

Cell cycle analysis of human peripheral blood T lymphocytes in long-term culture

We have studied the cell cycle of resting T lymphocytes from long-term (LT) cultures following stimulation with phytohemagglutinin (PHA) and recombinant Interleukin 2 (IL-2). We examined the kinetics of entry into S phase by autoradiography, the accumulation of cellular RNA by microfluorometric techniques, and ultrastructural morphology by electron microscopy. In addition, we examined the expression at the mRNA level of six cell cycle-dependent growth-regulated genes (c-fos, c-myc, KC-1, JE-3, vimentin, and histone H3). We show that T lymphocytes of LT cultures respond differently to mitogenic stimulation than the T lymphocytes of freshly isolated peripheral blood mononuclear cell cultures. At the ultrastructural, biochemical, and molecular levels, resting T lymphocytes of LT cultures can be distinguished from physiological (G0) lymphocytes of peripheral blood.     

Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)

The MNK (Menkes disease protein; ATP7A) is a major copper- transporting P-type ATPase involved in the delivery of copper to cuproenzymes in the secretory pathway and the efflux of excess copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in Menkes disease, a fatal neurodegenerative copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic detergent DDM (n-dodecyl beta-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 microM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6 microM; h=5.0). The purified MNK demonstrated active ATP-dependent vectorial 64Cu transport when reconstituted into soya-bean asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian copper-transporting P-type ATPase associated with a human disease.     

Loopholes in the regulation of invasive species: genetic identifications identify mislabeling of prohibited aquarium plants

Numerous invasive aquatic species introductions can be traced to the aquarium trade. Many potentially harmful aquarium species may be difficult to identify based on morphology alone. As such, some prohibited or invasive species may be available for purchase if they are mislabeled as species without restrictions. Here we compare molecular identifications to internet vendors’ identifications for accessions of a popular genus of aquarium plants that are difficult to distinguish morphologically (Myriophyllum; watermilfoils). Specifically, we identified the extensive mislabeling of M. heterophyllum—an invasive species in the northeastern and western US. Furthermore, genotypes of M. heterophyllum found in our aquarium survey have also been found in invasive populations, suggesting their potential introduction through escape from aquaria, water gardens, or nurseries. Two additional taxa were sold under incorrect names. Finally, our survey revealed that Myriophyllum taxa present in the aquarium trade generally have poorly known distributions and ecologies, and therefore their invasive potential is unknown. Our study confirms that molecular identification methods can provide a valuable tool to survey commercial pathways for potentially harmful species that are otherwise difficult to identify.     

Structure and regulation of the sheep metallothionein-Ia gene

Screening of a sheep genomic lambda library with a sheep metallothionein-I cDNA clone resulted in the isolation of a 13,200-base-pair fragment containing a metallothionein gene which DNA sequence analysis identified as the gene encoding the cloned cDNA. The two introns occur at identical positions to those in other mammalian metallothioneins but are considerably larger. The first intron contains a DNA element that is present in a related but not identical form in many places in the sheep genome. Comparison of the promoter sequences of this gene (sMT-Ia) with the promoters of metallothionein genes from other species identified a number of conserved regions which may be important in the regulation of this gene by heavy metals, glucocorticoids and alpha-interferon. In sheep fibroblasts, the levels of sMT-Ia mRNA was found to be maximally elevated (95-fold) in the presence of zinc or cadmium and elevated 30-fold in the presence of copper. Dexamethasone had no effect upon mRNA levels. Thus this gene shows a pattern of regulation similar to the human MT-If gene, but distinct from the other human and mouse metallothionein genes so far reported.     

Proline bis-amides as potent dual orexin receptor antagonists

A series of OX(2)R/OX(1)R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead. Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions were discovered. N-methylation of the benzimidazole moiety within the lead structure significantly reduced P-gp susceptibility while increasing potency, giving rise to good brain penetration. A compound from this series has demonstrated in vivo central activity when dosed peripherally in a pharmacodynamic model of orexin activity.