Cytoskeletal control of calcium absorption across the rat small intestine

1. The effect of colchicine, cytochalasin-B and procaine on calcium transport across the rat small intestine was investigated. The results obtained show the following: 2. Colchicine and cytochalasin-B at different concentrations inhibited significantly (P less than 0.001) calcium accumulation in rat intestinal cells, whereas procaine at different concentrations increased significantly (P less than 0.001) calcium accumulation in the rat small intestine. 3. Unidirectional influx of calcium across the rat small intestine was significantly inhibited (P less than 0.01) in the presence of colchicine and cytochalasin-B in the preincubation medium. Procaine, on the other hand, caused a significant increase (P less than 0.01) in the unidirectional influx of calcium across the rat intestinal cells. 4. The cell water content was not altered in the presence of the different drugs indicating that the changes in calcium transport across the rat intestinal cells are not due to alterations in the structure of the cell membrane.     

Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of cholesterol and its analogues

Electron paramagnetic resonance (EPR) spectra of nitric oxide (NO) complexes of ferrous cytochrome P-450scc were measured at 77 K for the first time without using the rapid-mixing and freeze-quenching technique. Without substrate the EPR spectra were very similar to those of cytochrome P-450cam (from Pseudomonas putida) and cytochrome P-450LM (from rat liver microsomes) with rhombic symmetry; gx = 2.071, gz = 2.001, gy = 1.962, and Az = 2.2 mT for 14NO complexes. Upon addition of substrates [such as cholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and 22-ketocholesterol], the EPR spectra exhibited many variations having rhombic symmetry in the major component and an additional minor component with less rhombic symmetry. Furthermore, addition of 20(S)-hydroxycholesterol caused a striking change in the EPR spectrum. The component with rhombic symmetry disappeared completely, and the component with less rhombic symmetry dominated (gx = 2.027, gz = 2.007, gy = 1.984, and Az = 1.76 mT for 14NO complexes). These observations suggest the existence of the following physiologically important natures: (1) the conformational flexibility of the active site of the enzyme due to the steric interaction between the substrate and the heme-bound ligand molecule and (2) the importance of the hydroxylation of the cholesterol side chain at the 20S position to proceed the side-chain cleavage reaction in cytochrome P-450scc.     

Serum retinol and the inverse relationship between serum cholesterol and cancer.

Several human studies have shown an inverse relation between vitamin A intake (and serum concentrations of retinol and carotene) and cancer. Serum cholesterol concentrations have also been reported in inverse relation to cancer. In a study of 3102 people in Evans County, Georgia, who were followed for over 12-14 years to assess the incidence of cancer there was an inverse association between the risk of cancer and both serum retinol and serum cholesterol concentrations. The data also showed an unexpectedly strong correlation between serum retinol and total cholesterol concentrations. The inverse relationship with cancer was stronger with serum retinol than with cholesterol, which suggested that the association with cholesterol might be secondary. This suggestion may also explain the cholesterol-cancer association reported in several other cohort studies. Further studies of the relation between serum concentrations of cholesterol, retinol, and carotene and the incidence of cancer are needed.     

Protein folding pathways of adenylate kinase from E. coli: hydrostatic pressure and stopped-flow studies.

Adenylate kinase (AKe) from E. coli is a small, single-chain, monomeric enzyme with no tryptophan and a single cysteine residue. We have constructed six single-Trp mutants of AKe to facilitate optical studies of these proteins and to specifically examine the interrelationship between their structure, function, dynamics, and folding reactions. In this study, the effects of hydrostatic pressure on the folding reactions of AKe were studied. The native structure of AKe was transformed to a non-native, yet pressure stable, conformation by hydrostatic pressure of about 300 MPa. This pressure lability of AKe is rather low for a monomeric protein and presumably may be attributed to substantial conformational flexibility and a correspondingly large volume change. The refolding of AKe after pressure-induced denaturation was reversible under ambient conditions. At low temperature (near 0 degrees C), the refolding process of pressure-exposed AKe mutants displayed a significant hysteresis. The observation of a slow refolding rate in the 193 region and a faster folding rate around the active site (86, 41, 73 regions) leads us to suggest that in the folding process, priority is afforded to functional regions. The slow structural return of the 193 region apparently does not hinder the more rapid return of enzymatic activity of AKe. Circular dichroism studies on the pressure-denatured Y193W mutant show that the secondary structure (calculated from far-UV spectra) returned at a rapid rate, but the tertiary structure alignment (calculated from near-UV spectra) around the 193 region occurred more slowly at rates comparable to those detected by fluorescence intensity. Denaturation of AKe mutants by guanidine hydrochloride and subsequent refolding experiments were also consistent with a much slower refolding process around the 193 region than near the active site. Fast refolding kinetic traces were observed in F86W, S41W, and A73W mutants using a fluorescence detection stopped-flow rapid mixing device, while only a slow kinetic trace was observed for Y193W. The results suggest that the differences in regional folding rates of AKe are not derived from the specific denaturation methods, but rather are inherent in the structural organization of the protein.     

Histochemical detection of horseradish peroxidase activity in the rat by the vibratome-paraplast method

A method is described for the histochemical detection of horseradish peroxidase in Paraplast Plus embedded brain sections. The procedure uses 150-micron-thick Vibratome-cut slices of glutaraldehyde-paraformaldehyde-fixed brain tissue. Tetramethylbenzidine stabilized by diaminobenzidine/cobalt/H2O2 is used as chromogen. The Vibratome-cut slices are dehydrated through a graded series of acetone, cleared in toluol and flat-embedded in Paraplast Plus embedding medium. Serial sections can be cut as thin as 5-7 micron. The method is universal in its application and permits optimal visualization of labeled neurons with great morphological detail at the light-microscopic level.     

Chromosome counts in some Alchemilla species from north-east Anatolia

This paper presents the results of karyological analysis of seven Alchemilla species collected from north-east Anatolia, Turkey, belonging to Alchemilla sect. Alchemilla subsect. Heliodrosium ser. Vulgares and subsect. Calycanthum ser. Elatae and ser. Calycinae. The following chromosome numbers were determined: A. haraldi 2n = 85–105, A. heterophylla 2n = 85–97, A. hirtipedicellata 2n = 86–100, A. oriturcica 2n = 86–102, A. persica 2n = 78–99, A. procerrima 2n = 69–78 and A. trabzonica 2n = 78–88. The chromosome numbers of three of these seven species are presented for the first time.     

Identification of respiratory vagal feedback in awake normal subjects using pseudorandom unloading.

Evidence of the Hering-Breuer reflex has been found in humans during anesthesia and sleep but not during wakefulness. Cortical influences, present during wakefulness, may mask the effects of this reflex in awake humans. We hypothesized that, if lung volume were increased in awake subjects unaware of the stimulus, vagal feedback would modulate breathing on a breath-to-breath basis. To test this hypothesis, we employed proportional assist ventilation in a pseudorandom sequence to unload the respiratory system above and below the perceptual threshold in 17 normal subjects. Tidal volume, integrated respiratory muscle pressure per breath, and inspiratory time were recorded. Both sub- and suprathreshold stimulation evoked a significant increase in tidal volume and inspiratory flow rate, but a significant decrease in inspiratory time was present only during the application of a subthreshold stimulus. We conclude that vagal feedback modulates respiratory timing on a breath-by-breath basis in awake humans, as long as there is no awareness of the stimulus.     

Diet changes in the carbon and nitrogen content of the copepod Metridia lucens

Diel changes in the near-surface (0–50 m) abundance, prosomelength, and carbon and nitrogen content of the copepod Metridialucens were measured in Deep Cove, Doubtful Sound, New Zealand(45°27'S, 167°9'E) between 3 and 6 September 1996. Metridialucens showed maximal abundance a night, suggesting a patternof normal diel vertical migration (DVM). The change in abundancesuggested that the descent of the population occurred –1h prior to dawn and the ascent –1 h after dusk. However,a proportion of the population remained near the surface duringthe day. Although there was no diel pattern in the prosome lengthof M.lucens collected near the surface, there was a marked dielcycle in the measured carbon and nitrogen contents, with maximalvalues being measured towards the end of the night prior tothe downward migration. We suggest that this diet cycle in themeasured elemental content was caused by DVM occurring morestrongly in those animals which had a better body condition,i.e. a higher elemental content per unit length.