Magnetic resonance studies on the mitochondrial divalent cation carrier.

Measurements of water proton spin relaxation enhancements (epsilon) can be used to discriminate high-affinity binding of Mn-2+ or Gd-3+ to biological membranes, from low-affinity binding. In rat liver mitochondria, epsilon b values of approx. 11 are observed upon binding of Mn-2+ to the inner membrane, while internal or low-affinity binding remains invisible to this technique. Energy-driven Mn-2+ uptake by liver mitochondria results in the subsequent decay of epsilon. Comparison of epsilon with the initial velocity of Mn-2+ uptake in rat liver mitochondria reveals a linear correlation, which holds at all temperatures between 0 degrees C and 40 degrees C, regardless of the mitochondrial protein concentration. Consequently, enhancement appears to reflect the binding of Mn-2+ to the divalent cation pump. Binding of Mn-2+ to blowfly flight muscle also results in substantial epsilon, which is associated with the glycerol-1-phosphate dehydrogenase instead of divalent cation transport. Consequently, no decay in epsilon due to uptake occurs after Mn-2+ is bound. Lanthanide ions are also bound and transported by mitochondria. Addition of Gd-3+ to pigeon heart or rat liver mitochondria results in epsilon b approximately equal to 5-6, which decays with similar kinetics in both systems. The uptake velocity of Gd-3+ in rat liver mitochondria is about 1/6 the rate with which Mn-2+ is transported. Lanthanides also diminish epsilon due to the addition of Mn-2+, and greatly retard the Mn-2+ uptake kinetics. The presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone depresses epsilon upon addition of Mn-2+ or Gd-3+ and also uncouples energy-driven uptake. On the other hand, prolonged anaerobic incubation in the presence of antimycin and rotenone exhausts the mitochondria of their energy stores, blocks the uptake of Mn-2+, but does not affect epsilon significantly. Evidently, the uncoupler-induced disappearance of divalent cation binding sites is not the result of "de-energization". Measurements of epsilon at several NMR frequencies indicate a correlation time (tau b) for carrier-bound Mn-2+ in rat liver mitochondria between 20 ns and 4 ns as one varies the temperature between 10 degrees C and 30 degrees C. The 13 Kcal/mole activation energy for tau b suggests that the 11 ns time constant at room temperature represents the movement of the Mn-11-carrier comples. On the other hand, tau b is probably approx. 100 times too short to represent the rotational motion of a carrier protein. Apparently, Mn-2+ binds to a small arm of the carrier which moves independent     

Investigation into the use of Aspergillus oryzae S1 nuclease in the presence of solvents which destabilize or prevent DNA secondary structure: Formaldehyde,formamide, and glyoxal

The activity of Aspergillus oryzae S₁ nuclease in solutions which destabilize DNA secondary structure was investigated. S₁ nuclease is able to degrade single-stranded DNA in the presence of various concentrations of formaldehyde, formamide, and glyoxal. It is further shown that S₁ nuclease can be used: (1) in the presence of formaldehyde to generate cleavage products from partially denatured duplex DNA; (2) to obtain thermal-melting profiles in the presence of formamide.     

ULTRASTRUCTURAL OBSERVATIONS OF VITELLOGENESIS IN THE SPIDER CRAB, LIBINIA EMARGINATA L

Ovaries from the spider crab, Libinia emarginata L. were studied to learn more of vitellogenesis in crustaceans. Oogonia and previtellogenic oocytes were found in the core of the ovaries. Vitellogenic oocytes are located more peripherally. Profiles of the endoplasmic reticulum are abundant in the vitellogenic oocytes. The granular and agranular reticulum as well as the Golgi complex are active in yolk synthesis. As vitellogenesis proceeds, yolk precursors are incorporated into the egg by micropinocytosis at the egg surface. Thus, in Libinia, yolk materials appear to be derived from both intra- and extraoocytic sources.     

Sources of mortality for a cynipid gall-wasp (Dryocosmus dubiosus (Hymenoptera: Cynipidae)): The importance of the Tannin/Fungus interaction

Summary Emergence success was determined for 1300 galls of the cynipid waspDryocosmus dubiosus. Galls were collected throughout a single host tree (a California coast live oakQuercus agrifolia). Each gall was reared individually in small gelatin capsules. For each gall data was recorded on 17 parameters characterizing hyperparasitism, fungal infestation, leaf tannin levels, inter- and intraspecific competition, and spatial position within the tree. Using contingency table analysis and logistic regression, we determined that the most significant factors influencing the success ofD. dubiosus galls are 1) fungal infestation and 2) chalcid hyperparasitism, both having negative effects. Of the factors investigated we found that leaf tannin level had the strongest influence on the degree of fungal infestation. Fungal infestation, in turn, is lowest in regions of high leaf tannins.     

Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii

A new nitrogenase from Azotobacter vinelandii has been isolated and characterized. It consists of two proteins, one of which is almost identical with the Fe protein (component 2) of the conventional enzyme. The second protein (Av1'), however, has now been isolated and shown to differ completely from conventional component 1, i.e., the MoFe protein. This new protein consists of two polypeptides with a total molecular weight of around 200,000. In place of Mo and Fe it contains V and Fe with a V:Fe ratio of 1:13 +/- 3. The ESR spectrum of Av1' also differs from conventional component 1 in that lacks the g = 3.6 resonance that arises from the FeMo cofactor but contains an axial signal with gav less than 2 as well as inflections in the g = 4-6 region possibly arising from an S = 3/2 state. This new enzyme can reduce dinitrogen, protons, and acetylene but is only able to utilize 10-15% of its electrons for the reduction of acetylene.     

Individual variations in the content of giant secretory polypeptides in salivary glands of Chironomus

Salivary glands in aquatic larvae of Chironomus are responsible for formation of a fiber that larvae use to construct feeding tubes. Major constituents of this fiber include a family (the sp-I family) of high M _r (1 × 10⁶) secretory polypeptides. Because of our interest in the polypeptide composition and polymerization of the salivary fiber we conducted a survey of the electrophoretic pattern of sp-I components found in salivary glands obtained from individual larvae. The survey encompassed ten strains of Chironomus tentans, three strains of Chironomus pallidivittatus and four strains of Chironomus thummi. Salivary glands from C. tentans and C. pallidivittatus contained at least four sp-I components (sp-Ia, sp-Ib, sp-Ic and sp-Id) that behave identically with regard to their electrophoretic mobility and detectability when larvae were exposed to galactose or glycerol. Sp-I components in C. thummi were generally fewer and not directly comparable by electrophoretic mobility to sp-I components in the other two species. During this survey two important alterations were observed in the electrophoretic pattern of sp-I components obtained from C. tentans and C. pallidivittatus. First, all four sp-I components exhibited, with a low frequency, double bands that appeared as slow-versus-fast electrophoretic variants of a particular component. Secondly, the relative steady-state level of each sp-I component fluctuated in comparison to other sp-I components in the same extract. This fluctuation varied such that any one sp-I component might appear as a single prominent component. Sp-I components are encoded by a multigene family located in Balbiani rings (BRs). Results presented here, in conjunction with known nucleotide sequence data from BR genes, led us to the following conclusions. The slow and fast electrophoretic variants observed for each sp-I component suggest that each corresponding BR gene may be able to expand and/or contract in size. The observed degree of independent fluctuation in the steady-state level of each sp-I component suggests that each BR gene may be able to regulate its expression independently from the others. Finally, the observation that salivary glands sometimes contained only one prominent sp-I component led us to hypothesize that, whereas salivary fibers might typically be heteropolymers comprised of two or more types of sp-I components, homopolymers comprised of only one sp-I component may also exist.     

Contrasting mechanisms of envZ control of mal and pho regulon genes in Escherichia coli.

The envZ11 missense mutation in the regulatory gene envZ pleiotropically repressed synthesis of OmpF, alkaline phosphatase, and several proteins of the maltose regulon. Procaine treatment of wild-type cells resulted in the same phenotype through an envZ+-mediated mechanism. Here we show that envZ11-procaine act differently on the mal and pho regulons. In the mal system, the expression of the positive regulator gene malT, measured as beta-galactosidase activity of a malT-lac+ operon fusion, was drastically reduced by procaine treatment or by the envZ11 mutation. In contrast, expression of the positive regulator of the pho regulon phoB was not reduced by procaine treatment. The products of the regulatory genes phoM, phoR, and phoU were also not required for procaine action. Procaine and envZ11 inhibited expression of only two products of the pho regulon, alkaline phosphatase and the PhoE porin. The conclusion that envZ11-procaine act differently on the mal and the pho regulons is supported by our ability to isolate second-site mutations with a Mal+ PhoA- phenotype in an envZ11 strain.     

Plasma lactate and ventilation thresholds in trained and untrained cyclists

Six trained male cyclists and six untrained sedentary men were studied to determine whether the plasma lactate threshold (PLT) and ventilation threshold (VT) occur at the same work rate in both fit and unfit populations. The PLT was determined from a marked increase in plasma lactate concentration ([La]) and VT from a nonlinear increase in expired minute ventilation (VE) during incremental leg-cycling tests; work rate was increased 30 W every 2 min until volitional exhaustion. The trained subjects' mean VO2 max (63.8 ml O2 X kg-1 X min-1) and VT (65.8% VO2 max) were significantly higher (P less than 0.05) than the untrained subjects' mean VO2max (35.5 ml O2 X kg-1 X min-1) and VT (51.4% VO2 max). The trained subjects' mean PLT (68.8% VO2 max) and VT did not differ significantly, but the untrained subjects' mean PLT (61.6% VO2 max) was significantly higher than their VT. The trained subjects' mean peak [La] (10.5 mmol X l-1) did not differ significantly from the untrained subjects' mean peak [La] (11.5 mmol X l-1). However, the time of appearance of the peak [La] during passive recovery was inversely related to VO2 max. These results suggest that variance in lactate diffusion and/or removal processes between the trained and untrained subjects may account in part for the different relationships between the VT and PLT in each population.