Secondary structure prediction of 52 membrane-bound cytochromes P450 shows a strong structural similarity to P450cam

The secondary structure of 52 aligned cytochrome P450 sequences, all of which are membrane bound, is predicted and collectively compared with the crystal structure of the soluble cytochrome P450cam. Ten of 13 helical regions, 6 of 7 beta-pair regions, and beta-structure corresponding to a known beta-bulge near the active site of P450cam are predicted to exist in the membrane-bound P450s. Three turns associated with beta-structure in the soluble enzyme are also predicted for the membrane-bound forms. A strong structural similarity is evident between membrane P450s and the soluble P450cam. Consequently, a multitransmembrane structure involving much of P450 seems highly unlikely. A structure with two N-terminal transmembrane segments is compatible with these observations.     

SR Function in malignant hyperthermia

Malignant hyperthermia (MH) is a genetic disease in man and other animal species that predisposes to a catastrophic hypermetabolic syndrome that is triggered by certain anesthetic agents. A working hypothesis is that a defect in regulation of muscle cell calcium is the primary mechanism that initiates the MH syndrome. This paper reviews the evidence for a defect in muscle cell calcium as regulated by the sarcoplasmic reticulum membrane system. Skeletal muscle biopsied from MH man, pigs and dogs has abnormal in vitro contracture response to halothane and caffeine and these responses can be altered by lowering calcium content of the bathing solution and/or the muscle. Measurements of MH muscle cell Ca2+ by Ca2+-specific microelectrodes in vivo and fura-2 in vitro have demonstrated abnormal Ca2+ levels in resting and in caffeine-stimulated states. The SR membrane system is the primary calcium regulating organelle in skeletal muscle and a likely site for the defect in MH muscle. Two Ca2+ regulating functions of the SR have been explored in SR isolated from MH muscle. An abnormality of the 100K Ca2+-ATPase protein that functions to transport Ca2+ from myoplasm to inside the SR does not appear to be responsible for MH. The most probable defective site in the SR appears to be Ca2+ release channels and a Ca2+-induced Ca2+ release pathway has been shown to be abnormal in SR from MH human and pig muscle.     

Structure, Function, and Evolution of Proton-ATPases

Proton-ATPases are among the most important primary ion pumps in nature. There are three classes of these enzymes which are distinguished by their structure, function, mechanism of action, and evolution. They function in ATP formation at the expense of a protonmotive force generated by oxidative and photosynthetic electron transports, maintaining a constant pH in the cytoplasm, and forming acidic spaces in special compartments inside and outside the cell. The three classes of proton-ATPases evolved in a way that prevents functional assembly in the wrong compartment. This was achieved by a triple genetic system located in the nucleus, mitochondria and chloroplast, as well as delicate control of the proton pumping activity of the enzymes.     

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.     

Reduction of tumor growth following treatment with a glutamine antimetabolite

Assessment of arterial-venous differences across transplanted methylcholanthrene-induced sarcomas in rats revealed significant decreases in plasma concentrations of glutamine, serine and glucose. Treatment with the glutamine antimetabolite, acivicin, significantly reduced tumor weights by 65% at the conclusion of the experiment 34 days after tumor induction. These results suggest that glutamine is an essential metabolic substrate for tumor growth and that blockade of glutamine utilization can inhibit the growth of these transplantable sarcomas.     

Ferritin-iron increases killing of Chinese hamster ovary cells by X-irradiation

Abstract. Stationary-phase Chinese hamster ovary cells were cultured in medium containing ferritin (-19% iron by weight) added at concentrations ranging from 0 to 128 μ g/ml. One set of cultures was unirradiated, and another set was exposed to 4.0 Gy of X-ray. Clonogenic cell survival was assessed in each set of cultures. In the absence of added ferritin, 4.0 Gy killed approximately 50% of the cells. In the absence of radiation, ferritin was not toxic at less than 48 μ g/ml; above 48 μ g/ml, toxicity increased with concentration. Apoferritin was not toxic at any concentration tested (up to 1000 μ g/ml). Although 32 μg/ ml ferritin, reflecting only a 3–6 fold increase in iron concentration over normal serum, was not toxic, it reduced the survival of X-irradiated cells by an additional 75%. These results indicate that a sublethal concentration of ferritin can be a potent radiosensitizer. This suggests the possibility that high body iron stores may increase susceptibility to radiation injury in humans.     

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.     

Cationic probes: Specificity of distribution of metal-binding sites in bovine sperm

Salts of transition elements that alter the rate of sperm cell movement act at or near calcium-binding sites. After living bull sperm cells had been preincubated in VO₄^3?, Ni²⁺, Zn²⁺, Mn²⁺, and also La³⁺, they were then fixed. Crisply defined organelles and the absence of particulate deposits in the morphological controls contrasted sharply with the treated specimens; the latter contained regions of increased electron density, the nature and distribution of which depended on the test substance, reflecting the differential affinities of the specific ions. La³⁺ formed fine dense areas, mainly at the exocytic surface of the plasma membrane. VO₄^3? marks the cell surface but also left particulate densities within the cell. Ni²⁺ caused a nearly uniformly dense deposit at the surface and on the satellite fibers and axonemal microtubules. Zn²⁺ formed less uniform but coarser deposits, while in Mn²⁺ the distribution was similar to that in Zn²⁺ but much denser in the axonemal matrix and on the satellite fibers. Verapamil restricted the size and number of the opacities, while procaine permitted a similar distribution of slightly larger size reaction product. The differences in size and distribution of the enhanced densities were consistent and replicable for the individual assay substances. Vanadate, which specifically inhibits Na, K-ATPase, bound to ouabain-sensitive enzyme loci, however, completely disrupting the axonemal complex. This suggests that an important role of dynein in flagellar motion may relate to intracellular transport of Ca²⁺.     

Microhabitat utilization by two leptodactylid frogs in the Andes of central Chile

Summary Some basic parameters of the life history of Alsodes montanus and Alsodes tumultuosus (Anura-Leptodactylidae), were studied from 1977 to 1980 by periodic field observations at Farellones and La Parva (33–34° south lat.; 2,700–3,000 m above sea level). Special attention was paid to strategies of resource partitioning in relation to gross features of the environment. The latter was unstable with a relative short period favorable for activity of the animals. Physical environmental differences between the first and second season of this study, resulted in a decrease in total number of active adults, a reduction in the duration of larval activity and a shift in microhabitat preferences of larvae.During the favorable season, October to May, adults of both species showed spatial and temporal segregation, related to different physical features of the environment; larvae did not show temporal segregation. Larvae of both species were found in seven different microhabitats; only in one of these did they show significant difference in microhabitat preference, A. tumultuosus was found more often in crevices. Microhabitat dimensions were more important than time and food resources in the separation of the niches of the two species. The segregation of niche dimensions, microhabitat, diel and annual activity and food were not complementary.Coexistence was therefore observed with the species tending to use different resources. When the same resource was used, it was not limiting.     

Thermography as a predictive tool for laser treatment of port-wine stains

The argon laser, which has been proven both useful and safe for port-wine stain therapy, interacts with the hemoglobin of the vessels. In a percentage of cases, this treatment is still inefficient, and there is a lack of correlation between these bad results and clinical or histologic criteria. Thermography, which explores the vascularization of the port-wine stain, leads us to consider port-wine stains from a physical point of view. This very simple test shows no correlation with the clinical parameters of port-wine stain but is closely related to the results obtained with laser therapy. It seems to be a good criterion to estimate the argon laser treatment prognosis.