Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes

Summary A hybrid phage (Sda1), containing an 8.1 kb EcoRI DNA fragment from the Escherichia coli chromosome, was selected on the basis of its ability to suppress bacterial thermosensitivity caused by the dnaA46 mutation. We have shown that this suppression is due to a recA ⁺-dependent amplification of the 8.1 kb fragment; consistent with this observation, cloning of the 8.1 kb fragment into a high copy number plasmid (pBR325) leads also to suppression of dnaA46. In the suppressed strains growing at high temperature, bidirectional replication starts in or near the oriC region and requires the presence of the DnaA polypeptide. These findings suggest that the overproduction of a gene product(s), encoded by the cloned 8.1 kb fragment, can restore dnaA-dependent initiation of replication at high temperature in the oriC region. Genetic mapping shows that the groES (mopB) and groEL (mopA) genes are located on the 8.1 kb suppressor fragment. Further analysis, including in vitro mutagenesis and subcloning, demonstrates that the amplification of the groES and groEL genes is both necessary and sufficient to suppress the temperature sensitive phenotype of the dnaA46 mutation.     

Role of mevalonate-5-pyrophosphate decarboxylase in the regulation of chick intestinal cholesterogenesis

The response to different dietary conditions of the enzymes responsible for the transformation of mevalonic acid to isopentenyl pyrophosphate has been studied for the first time in the small bowel of the chick to elucidate the role of these enzymes in the regulation of intestinal cholesterogenesis. Feeding a 2% cholesterol diet from hatching resulted in a small but significant inhibition of mevalonate-5-pyrophosphate decarboxylase, while mevalonate kinase and mevalonate-5-phosphate kinase remained unaltered. Similar results were obtained for the three enzymes when 13-day-old chicks fed a standard fat-free diet were switched to a 5% cholesterol diet. Starved chicks exhibited lower intestinal decarboxylase activity than chicks fed a standard diet, while refeeding resulted in levels of activity similar or slightly greater than controls. None of the enzymes effecting the conversion of mevalonate to isopentenyl pyrophosphate in the small intestine presented diurnal variations. Results obtained suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in the regulation of cholesterol synthesis in the small intestine.     

Evidence for titratable gating charges controlling the voltage dependence of the outer mitochondrial membrane channel,VDAC

Summary A voltage-dependent anion-selective channel, VDAC, is found in outer mitochondrial membranes. VDAC's conductance is known to decrease as the transmembrane voltage is increased in either the positive or negative direction. Charged groups on the channel may be responsible for this voltage dependence by allowing the channel to respond to an applied electric field. If so, then neutralization of these charges would eliminate the voltage dependence. Channels in planar lipid bilayers which behaved normally at pH 6 lost much of their voltage dependence at high pH. Raising the pH reduced the steepness of the voltage dependence and raised the voltage needed to close half the channels. In contrast, the energy difference between the open and closed state in the absence of a field was changed very little by the elevated pH. The groups being titrated had an apparent pK of 10.6. From the pK and chemical modification, lysine epsilon amino groups are the most likely candidates responsible for VDAC's ability to respond to an applied electric field.     

Channel formation in phospholipid bilayer membranes by the toxin ofHeminthosporium maydis,race T

Summary Southern Corn Leaf Blight is caused by a toxin produced by a virulent form ofHelminthosporium maydis (Race T). The toxin has been shown to uncouple oxidative phosphorylation and dissipate Ca²⁺ gradients in mitochondria isolated from susceptible, but not resistant, corn. The possibility that the toxin acted by increasing the permeability of membranes to ions was tested using a planar bilayer membrane system. Addition of the toxin to the bilayer system, under voltage-clamp conditions, resulted in stepwise increases in current across the phospholipid bilayer, a response characteristic for channel formers. Single-channel conductance in 1m KCl is 27 pS which corresponds to 1.7×10⁷ ions sec^–1 channel^–1 at 100 mV applied potential. The toxin channels are: (i) fairly uniform in conductance, (ii) ideally selective for K⁺ over Cl^–, and (iii) most conductive to H⁺. The channel showed the following selectivity for alkali metal cations: Rb⁺>K⁺>Cs⁺>Na⁺>Li⁺ (169731) based on the most frequently observed conductance in 1m chloride salts. The toxin showed no voltage dependence over the range of –100 to +100 mV. Channel formation was also a property of a synthetic analog (Cmpd IV) of the toxin. The ability of the native toxin to form channels may be a mode of toxin action on mitochondrial membranes from susceptible corn.     

Histochemical changes of substance P, FRAP, serotonin and succinic dehydrogenase in the spinal cord of rats with adjuvant arthritis

Various histochemical changes were found in spinal segments L4-L5 of rats with adjuvant arthritis, predominantly 30 days after inoculation. A slight to marked increase of substance P immunoreactivity occurred in laminae I, II and X. FRAP activity was enhanced in lamina II. Serotonin immunoreactivity was heavier in laminae I, VIII and IX in a few animals. The intensity of the histoenzymological reaction for succinic dehydrogenase increased in certain laminae VIII and X neurons. At day 15 of the disease the increase of substance P and FRAP activities was chiefly restricted to the medial portion of the superficial dorsal horn. There was a significant positive correlation between the scratching behaviour of arthritic rats and the substance P immunoreactivity in laminae X and I. If one accepts that scratching is pain-related, the data are consistent with a possible role of substance P in the chronic pain associated with adjuvant arthritis. They leave undetermined the significance of the other histochemical changes.     

Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase

The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.     

Further characterization of [3H]fulunitrazepam binding sites on cultured mouse astroglia

Astroglial cells in primary cultures bind [³H]flunitrazepam with a high affinity on a single type of site and on a number of binding sites which increased during astroglial growth and differentiation. These binding sites show a particular pharmacological spectrum characterized by an inhibition of high affinity by RO-5-4864 (4-chlorodiazepam), an anticonvulsant of the benzodiazepine family and by an inhibition of binding of lower affinities by diazepam clonazepam and clobazam. RO-5-4864 and clonazepam compete for the same binding site in astroglia. The heat stability and the hormonal modulation by thyroxine are similar for astroglia and neuronal-cells. Benxodiazepines modulate the astroglial 5-HT receptor. Such an effect could be a possible physiological response to benzodiazepines for astroglial cells in primary cultures.     

New enzymatic changes of L-cystathionine catalyzed by bovine tissue extracts

L-Cystathionine was used as substrate for enzyme systems prepared by heating bovine tissue extracts in the presence of pyruvate at 60 degrees C for 10 min. Analysis of the products indicated that the systems converted L-cystathionine into the cyclic ketimine form which was detected by its spectral properties and by chromatography on the amino acid analyzer. Alanine, alpha-aminobutyrate and cystine were also produced. Pyruvate and alpha-ketobutyrate enhance the production of the ketimine by liver, kidney and heart extracts, and are necessary for the brain extracts: alpha-Ketoglutarate is much less effective and its presence favors the production of homocystine by all the extracts. Homocystine was found in the brain incubates when any of the ketoacids assayed were added. The overall reaction is explained by the action of heat stable cystathionine gamma-lyase and beta-synthase which produce alpha-ketobutyrate and pyruvate used for the transamination of the remaining cystathionine to the monoketoacid. This last compound cyclizes spontaneously into the ketimine form thus avoiding the removal of the second amino group. This represents a new nontransulfurative path leading to the production of a seven membered etherocyclic product whose biochemical implications are yet unexplored.