Quantitative determination of Ca2+-dependent Mg2+-ATPase from sarcoplasmic reticulum in muscle biopsies.

The possibility of quantifying the total concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum was investigated by measurement of the Ca2+-dependent steady-state phosphorylation from [gamma-32P]ATP and the Ca2+-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) activity in crude muscle homogenates. The Ca2+-dependent phosphorylation at 0 degree C (mean +/- S.E.) was 40.0 +/- 2.5 (n = 6) and 6.2 +/- 0.7 (n = 4) nmol/g wet wt. in rat extensor digitorum longus (EDL) and soleus muscle, respectively (P less than 0.001). The Ca2+-dependent 3-O-MFPase activity at 37 degrees C was 1424 +/- 238 (n = 6) and 335 +/- 56 (n = 4) nmol/min per g wet wt. in rat EDL and soleus muscle, respectively (P less than 0.01). The molecular activity calculated from these measurements amounted to 35 +/- 5 min-1 (n = 6) and 55 +/- 10 min-1 (n = 4) for EDL and soleus muscle respectively. These values were not different from the molecular activity calculated for purified Ca2+-ATPase (36 min-1). The Ca2+-dependent 32P incorporation in soleus muscle decreased in the order mice greater than rats greater than guinea pigs. In EDL muscles from hypothyroid rats at a 30% reduction of the Ca2+-dependent phosphorylation was observed. The Ca2+-dependent phosphorylation in vastus lateralis muscle from three human subjects amounted to 4.5 +/- 0.8 nmol/g wet wt. It is concluded that measurement of the Ca2+-dependent phosphorylation allows rapid and reproducible quantification of the concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum. Since only 20-60 mg of tissue is required for the measurements, the method can also be used for biopsies obtained in clinical studies.     

Interactions of amiloride and small monovalent cations with the epithelial sodium channel. Inferences about the nature of the channel pore.

The voltage dependence of amiloride-induced inhibition of current flow through apical membrane sodium channels in toad urinary bladder was studied at different ionic conditions. The "inert" salt N-methyl-D-glucamine HCl (NMDG HCl) affected neither the apparent inhibition constant (Kl) for the amiloride-induced current inhibition nor the apparent fraction of the transmembrane voltage that falls between the mucosal solution and the amiloride-binding site (delta). When NMDG+ was replaced with Na+, Kl increased, reflecting amiloride-Na+ competition, whereas delta was unchanged. Similar results were obtained with another permeant cation, Li+. When NMDG+ was replaced by K+, an impermeant but channel-blocking cation, Kl increased whereas delta decreased. Similar results were obtained using another impermeant, channel-blocking cation guanidinium. The results are interpreted on the premise that Na+ and K+ compete with amiloride by binding to cation binding sites within the channel lumen such that ion occupancy of these sites vary with voltage. Occupancy by K+, which cannot traverse the channel, will increase as the mucosal solution becomes positive, relative to the serosal solution. Occupancy by Na+, which can traverse the channel, is comparatively voltage independent. Ion movement through the channels was simulated using discrete-state kinetic models. Two types of models could describe the shape of the current-voltage relationship and the voltage dependence of the amiloride-induced channel block. One model had a single ion-binding site with a broad energy barrier at the inner (cytoplasmic) side of the site. The other model had two binding sites separated from each other and from the aqueous solutions by sharp energy barriers.     

Reduction of hexavalent chromium in a reconstituted system of cytochrome P-450 and cytochrome b5

The reduction of hexavalent chromium (Cr(VI] by the monooxygenase components was studied. Both a reconstituted system of cytochrome P-450 (P-450) and cytochrome b5 (b5) with NADPH was capable of reducing Na2CrO4 (30 microM) provided anaerobic atmosphere. The rates were 1.29 nmol Cr.min-1 nmol P-450(-1) and 0.73 nmol Cr.min-1 nmol b5(-1). Using NADH instead of NADPH gave very low reducing activities, confirming the enzymic nature of the P-450 dependent Cr(VI) reductase reaction. Oxygen, 22% (air) and 0.1% gave 89% and 69% inhibition of Cr(VI) reducing activity, respectively. Carbon monoxide (100%) caused an inhibition of about 37% and 44% for P-450 and b5, respectively. Externally added flavin mononucleotide (FMN) (3 microM) or Fe-ADP (10 microM) to the complete system stimulated the enzymatic reaction about 2-fold and 3-fold, respectively.     

Electron microscopical studies of membrane injuries in blue fox spermatozoa subjected to the process of freezing and thawing

Disintegration of blue fox sperm membranes is studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In unfrozen spermatozoa studied by SEM, the plasmalemma and the acrosome appeared to be intact, except for a few cases of disruption of the former structure at the anterior part of the head. In semen frozen in 0.5-ml plastic straws by use of N2 vapor after dilution with Tris-fructose-citric acid with 8 vol % glycerol and 20 vol % egg yolk and thawed at 70 degrees C for 8 sec, the spermatozoa displayed different degrees of membrane damage. These alterations could be classified into three main categories of which the first included only minor changes in the plasmalemma, but vesiculation and disintegration of the outer part of the acrosomal membrane. In the second category (also the most frequent one) the outer part of the acrosomal membrane was extensively vesiculated, and the plasmalemma was discharged proximal to the equatorial segment. Extensive loss of plasmalemma and complete absence of the outer part of the acrosomal membrane characterized the last category of membrane damage. The functional implications of the three categories of membrane alterations are discussed.     

Determination of the enantiomeric composition of (R*,S*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate, the male-produced aggregation pheromone of Sitophilus granarius

The enantiomeric composition of sitophilate, the granary weevil [Sitophilus granarius (L.)] male-produced aggregation pheromone [(R^*,S^*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate)], was determined by three methods: (1) bioassaying the synthetic (2S,3R) and (2R,3S) enantiomers of the active (R^*,S^*) diastereomer; (2) ¹H NMR spectroscopy of Mosher ester derivatives of the natural pheromone and synthetic (2S,3R)-and (2R,3S)-sitophilate; and (3) capillary GLC comparisons of the retention times of derivatized natural pheromone and the two synthetic enantiomers. The combined methods confirmed the (2S,3R) enantiomer as the active form of sitophilate. Male granary weevils were shown to produce >96% (2S,3R)-sitophilate. No significant attraction of S. granarius by the (2R,3S) enantiomer was observed. Rice and maize weevils [S. oryzae (L.) and S. zeamais Motschulsky] were not attracted by (2S,3R)-sitophilate. S. granarius L. est un déprédateur important des grains stockés. Le (R^*,S^*)-1-éthylpropyl 2-méthyl-3-hydroxypentanoate a été identifié en 1987 comme le principal composé du sitophilate, la phéromone mâle d'agrégation de S. granarius. La composition énantiométrique du sitophilate a été déterminée par 3 méthodes:

Metal-dependent binding of a nuclear factor to the rat metallothionein-I promoter.

Genomic footprinting studies in vivo and experiments using synthetic metal regulatory elements (MREs) in vitro suggest protein binding to the MREs of the mouse and rat metallothionein I (MT-I) genes. Using gel-retardation assays of promoter fragments, we observe a cadmium-dependent binding factor for the rat MT-I promoter in rat hepatoma cells. This factor is present in extracts from both uninduced and cadmium-induced cells, but requires the presence of cadmium to bind to the promoter. The formation of a cadmium-dependent complex is competed by an oligonucleotide containing two MREs. This competition is lost when when one of the MREs is mutated, indicating a requirement for at least two MREs for binding of this factor. The cadmium-dependent factor dissociates more rapidly from the MT-I promoter than does a factor that binds to a consensus Sp1 site present on the same DNA fragment. UV crosslinking analysis using nuclear extracts from cadmium induced cells, in the presence of an oligonucleotide probe containing both 5-bromodeoxyuridine and 32P-deoxycytidine, identifies a 39 kDalton protein associated with the metal inducible complex.     

Intergeneric rosettes: sequestered surface recognition among human periodontal bacteria

The human oral bacteria, Streptococcus sanguis and Bacteroides loescheii, when mixed in equal numbers in vitro, formed large settling coaggregates. As the relative number of each cell type was changed, coaggregates became smaller until at cell-type ratios of 10 to 1, rosettes formed. Rosettes consisting of a streptococcal cell in the center surrounded by bacteroides cells exhibited surface recognition properties of only the bacteroides, which coaggregated with many other cell types such as Actinomyces naeslundii, and formed large settling multigeneric aggregates. The ecological significance of these results derives from the following: (i) the direct demonstration that intergeneric coaggregates can protect the central cell from or prevent its access to other cells in the environment, and (ii) the potential for these effects to occur during bacterial succession of various cell types observed in progressively more severe stages of human periodontal disease.     

Measurement of exchange of trace metals between the body and its surroundings with the use of trace methodology

Application of general tracer theory to the problem of estimating fluxes of tracee between the gastrointestinal tract and the body proper, from observations of the movement of tracer, shows that a number of assumptions must be fulfilled. Two specific sets of assumptions are discussed and, in both cases, measurement of tracer fluxes yields information on the integrated absorption of the tracee.     

Organotin compounds induce aneuploidy in human peripheral lymphocytes in vitro

In vitro exposure of PHA-stimulated human lymphocytes to organotin compounds resulted in statistically significant increases in the frequencies of hyperdiploid cells. When taken together with our previous study demonstrating spindle inhibiting effects of the same organotin compounds by an indirect method (Jensen et al., 1989), the present study strongly indicates that organotin compounds are able to induce aneuploidy, probably by affecting spindle function.