CGS8216 noncompetitively antagonizes the discriminative effects of diazepam in rats

The benzodiazepine antagonist properties of CGS8216 were evaluated in rats trained to discriminate between saline and 1.0 mg/kg of diazepam in a two-choice, stimulus-shock termination procedure. CGS8216 (0.3 to 100 mg/kg) administered alone, either s.c., p.o. or i.p., occasioned only saline-appropriate responding. When administered concomitantly with a constant 1.0 mg/kg dose of diazepam, CGS8216 produced dose-related decreases in drug-appropriate responding. CGS8216 was most potent by the i.p. route, and approximately tenfold less potent by the oral route. CGS8216 was dermatotoxic after s.c. administration. CGS8216 i.p. had a long duration of action. A dose of 30 mg/kg completely antagonized the discriminative effects of the 1.0 mg/kg training dose of diazepam when the antagonist was administered 8 hr before the start of the test session. In order to determine the type of antagonism by CGS8216, the dose-effect curve for diazepam was redetermined in the presence of varying doses of CGS8216 (0.3 to 3.0 mg/kg, i.p.). CGS8216 produced a dose-related rightward shift in the diazepam dose-effect curve, but also decreased the slope and appeared to decrease the maximal effect. These results are consistent with the interpretation that CGS8216 antagonizes diazepam in a noncompetitive manner. It may do so because either it interacts with a subpopulation of benzodiazepine receptors, it functions as a pseudo-irreversible antagonist due to its high affinity, or because it is an antagonist with agonist properties.     

Cloning of nisin resistance determinant and replication origin on 7.6-kilobase EcoRI fragment of pNP40 from Streptococcus lactis subsp. diacetylactis DRC3

The nisin resistance determinant and an origin of replication on pNP40, a plasmid of about 60 kilobases that is present in Streptococcus lactis subsp. diacetylactis DRC3, was cloned on a 7.6-kilobase EcoRI fragment. When self-ligated, this fragment existed as an independent replicon (pFM011) and contained a 2.6-kilobase EcoRI-XbaI fragment encoding nisin resistance.     

Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases

The proteolytic activity of four hemorrhagic metalloproteinases (Ht-a, c, d, and e) isolated from the venom of the Western diamondback rattlesnake (Crotalus atrox) was investigated using isolated extracellular matrix (ECM) proteins. We determined that all of the proteinases are capable of cleaving fibronectin, laminin, type IV collagen, nidogen (entactin), and gelatins. However, none of the proteinases were proteolytic against the interstitial collagen types I and III or type V collagen. With all of the substrates listed above Ht-c and Ht-d produced identical digestion patterns, as would be expected for these isoenzymes. With fibronectin, Ht-a produces a different ratio of products from Ht-c and Ht-d, while Ht-e produces a unique pattern of digestion. Ht-e and Ht-a produced nonidentical patterns with the laminin/nidogen preparation although some similarity was shared between them as well as with the Ht-c/d digestion pattern. Similar results were also observed for these proteinases with nidogen 150 as the substrate. The type IV collagen digestion patterns by Ht-e and Ht-a were similar to the pattern observed with Ht-c/d but differed by two bands. The digestion patterns of the three gelatins produced by the proteinases show differences between Ht-c and Ht-d when compared to Ht-e and Ht-a. This investigation clearly shows that several of the ECM proteins are efficiently digested by these toxins. The proteinases have some digestion sites in common but show differing specificities. In addition, the range of ECM proteins digested by these hemorrhagic proteinases is nearly identical to that demonstrated by the ECM proteinase stromelysin (MMP-3). From these data, and the knowledge of the roles these ECM proteins have in maintaining basement membrane structural/functional integrity, one can envision that the degradation of these ECM proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites.     

Extracellular ß-galactosidase production during growth of filamentous fungi on polygalacturonic acid

The ß-galactosidase enzymes of Aspergillus oryzae and Scopulariopsis sp. are secreted during growth of the fungi on polygalacturonic acid, but not during growth on a wide range of other substrates, including lactose. The enzymes are thermotolerant with temperature optima in the range 55–65°C. The results indicate that fungal extracellular ß-galactosidases are involved in fungal growth on complex polysaccharides but not on lactose.     

Rainbow trout protamines. Amino acid sequences of six distinct proteins from a single testis

All the protamines present in detectable amounts in a single mature testis from rainbow trout have been purified to homogeneity using acid extraction, gel filtration chromatography on Bio-Gel P-10, ion-exchange chromatography on carboxymethylcellulose and reverse-phase high-pressure liquid chromatography. Each of the six purified protamines was completely sequenced using automated gas-phase Edman degradation. Each protamine is two-thirds arginine and also contains proline, serine, valine and glycine. Three protamines also contain alanine while two contain isoleucine. Four of the protamines have 32 amino acids while the remaining two have 30. The six protamines have been classified into three families on the basis of their amino acid sequences.     

Characterization of lactose-fermenting revertants from lactose-negative Streptococcus lactis C2 mutants

Partial lactose-fermenting revertants from lactose-negative (lac(-)) mutants of Streptococcus lactis C2 appeared on a lawn of lac(-) cells after 3 to 5 days of incubation at 25 C. The revertants grew slowly on lactose with a growth response similar to that for cryptic cells. In contrast to lac(+)S. lactis C2, the revertants were defective in the accumulation of [(14)C]thiomethyl-beta-d-galactoside, indicating that they were devoid of a transport system. Hydrolysis of o-nitrophenyl-beta-d-galactoside-6-phosphate by toluene-treated cells confirmed the presence of phospho-beta-d-galactosidase (P-beta-gal) in the revertant. However, this enzyme was induced only when the cells were grown in the presence of lactose; galactose was not an inducer. In lac(+)S. lactis C2, enzyme induction occurred in lactose- or galactose-grown cells. The revertants were defective in EII-lactose and FIII-lactose of the phosphoenolpyruvate-dependent phosphotransferase system. Galactokinase activity was detected in cell extracts of lac(+)S. lactis C2, but the activity was 9 to 13 times higher in extracts from the revertant and lac(-), respectively. This suggested that the lac(-) and the revertants use the Leloir pathway for galactose metabolism and that galactose-1-phosphate rather than galactose-6-phosphate was being formed. This may explain why lactose, but not galactose, induced P-beta-gal in the revertants. Because the revertant was unable to form galactose-6-phosphate, induction could not occur. This compound would be formed on hydrolysis of lactose phosphate. The data also indicate that galactose-6-phosphate may serve not only as an inducer of the lactose genes in S. lactis C2, but also as a repressor of the Leloir pathway for galactose metabolism.