Evidence for a KCl-Stimulated, Mg-ATPase on the Golgi of Corn Coleoptiles

Membranes of corn (Zea mays, cv Trojan 929) coleoptiles were fractionated by sucrose density gradient centrifugation and the locations of organelles were determined using marker enzymes and electron microscopy. Latent IDPase (or UDPase) was selected as the Golgi marker and UDPG-sterol glucosyl transferase was selected as the plasma membrane (PM) marker, because they were clearly separable from markers for the other organelles. Golgi-rich and PM-rich fractions were studied in relation to their ATPase activities. The pH optimum of the KCl, Mg²⁺-ATPase of the PM-rich fraction from a step gradient was 6.0 to 6.5, while the Golgi-rich fraction had peaks at pH 6.0 to 6.5 and pH 7.5. It is hypothesized that the peak at pH 6.0 to 6.5 for the Golgi-rich fraction is due to PM-contamination, while the peak at pH 7.5 represents the activity of a Golgi ATPase. To reduce PM contamination, Golgi-rich fractions obtained from step or rate-zonal gradients were recentrifuged isopycnically on linear sucrose gradients. The distribution of KCl, Mg²⁺-ATPase activity was measured at pH 6.5 and 7.5. The pH 6.5 ATPase was coincident with UDPG-sterol glucosyl transferase, a PM marker, while the pH 7.5 ATPase overlapped with latent UDPase, a Golgi marker. These results provide strong evidence for a KCl, Mg²⁺-ATPase, active at pH 7.5, associated with the Golgi membranes of corn coleoptiles.     

Identification of the Archaeoglobus fulgidus endonuclease III DNA interaction surface using heteronuclear NMR methods.

BACKGROUND: Endonuclease III is the prototype for a family of DNA-repair enzymes that recognize and remove damaged and mismatched bases from DNA via cleavage of the N-glycosidic bond. Crystal structures for endonuclease III, which removes damaged pyrimidines, and MutY, which removes mismatched adenines, show a highly conserved structure. Although there are several models for DNA binding by this family of enzymes, no experimental structures with bound DNA exist for any member of the family. RESULTS: Nuclear magnetic resonance (NMR) spectroscopy chemical-shift perturbation of backbone nuclei (1H, 15N, 13CO) has been used to map the DNA-binding site on Archaeoglobus fulgidus endonuclease III. The experimentally determined interaction surface includes five structural elements: the helix-hairpin-helix (HhH) motif, the iron-sulfur cluster loop (FCL) motif, the pseudo helix-hairpin-helix motif, the helix B-helix C loop, and helix H. The elements form a continuous surface that spans the active site of the enzyme. CONCLUSIONS: The enzyme-DNA interaction surface for endonuclease III contains five elements of the protein structure and suggests that DNA damage recognition may require several specific interactions between the enzyme and the DNA substrate. Because the target DNA used in this study contained a generic apurinic/apyrimidinic (AP) site, the binding interactions we observed for A. fulgidus endonuclease III should apply to all members of the endonuclease III family and several interactions could apply to the endonuclease III/AlkA (3-methyladenine DNA glycosylase) superfamily.     

Variation in basal heat shock protein 70 is correlated to core temperature in human subjects

Heat shock proteins are highly conserved proteins and play an important chaperone role in aiding the folding of nascent proteins within cells. The heat shock protein response to various stressors, both in vitro and in vivo, is well characterised. However, basal levels of heat shock protein 70 (Hsp70) have not previously been investigated. Monocyte-expressed Hsp70 was determined every 4 h, over a 24 h time period, in 17 healthy male subjects (177 ± 6.4 cm, 75.7 ± 10.9 kg, 19.8 ± 4.3 years) within a temperature and activity controlled environment. Core temperature was measured at 5-min intervals during the 24 h period. Hsp70 showed significant diurnal variation (F = 7.4; p < 0.001), demonstrating peaks at 0900 and 2100 hours, and a nadir at 05.00. Core temperature followed a similar temporal trend (range = 35.96–38.10°C) and was significantly correlated with Hsp70 expression (r _s = 0.44; p < 0.001). These findings suggest a high responsiveness of Hsp70 expression in monocytes to slight variations in core temperature.     

Restricted domain mobility in the Candida albicans Ess1 prolyl isomerase

Ess1 is a peptidyl prolyl cis/trans isomerase that is required for virulence of the pathogenic fungi Candida albicans and Cryptococcus neoformans. The enzyme isomerizes the phospho-Ser-Pro linkages in the C-terminal domain of RNA polymerase II. Its human homolog, Pin1, has been implicated in a wide range of human diseases, including cancer and Alzheimer's disease. Crystallographic and NMR studies have demonstrated that the sequence linking the catalytic isomerase domain and the substrate binding WW domain of Pin1 is unstructured and that the two domains are only loosely associated in the absence of the substrate. In contrast, the crystal structure of C. albicans Ess1 revealed a highly ordered linker that contains a three turn α-helix and extensive association between the two tightly juxtaposed domains. In part to address the concern that the marked differences in the domain interactions for the human and fungal structures might reflect crystal lattice effects, NMR chemical shift analysis and ¹⁵N relaxation measurements have been employed to confirm that the linker of the fungal protein is highly ordered in solution. With the exception of two loops within the active site of the isomerase domain, the local backbone geometry observed in the crystal structure appears to be well preserved throughout the protein chain. The marked differences in interdomain interactions and linker flexibility between the human and fungal enzymes provide a structural basis for therapeutic targeting of the fungal enzymes.     

Modulation of temperature-sensitive TRP channels

Animals sense temperature--either cold or hot--by the direct activation of temperature-sensitive members of the TRP family of ion channels, the thermo-TRPs. To date, six TRP channels--TRPV1-4, TRPM8 and TRPA1--have been reported to be directly activated by heat and to be involved in thermosensation. Temperature sensing can be modulated by phosphorylation of intracellular residues by protein kinases or by insertion of new channels into the cell membrane. In this review we provide a brief overview of the properties of thermo-TRPs, and we summarise signalling pathways involved in their regulation.     

Removal of blood components from cervical smears: implications for cancer diagnosis using FTIR spectroscopy

Red and white cell lysis buffers were investigated to determine their ability to remove blood components from cervical smears prior to IR microspectroscopy. A white cell lysis buffer was effective in increasing the spectral reproducibility and sample homogeneity and in reducing the presence of inflammatory exudate, particularly leukocytes, from cervical smears. The reduction of leukocytes appeared to cause abnormal samples to be grouped with normal samples, resulting in poor discrimination. Despite differences between the cytological and histological diagnoses of cervical abnormalities, the results indicate that the differences seen in the spectra of exfoliated malignant and nonmalignant cervical cells could be due to the presence of inflammation.