The measurement of gamma-glutamyl hydrolase (conjugase) activity in rat brain

An assay using the artificial substrate, 2,4-diamino-10-methyl-pteroylglutamyl-gamma-glutamate (MTX-G₁), was developed to measure gamma-glutamyl hydrolase (conjugase), which hydrolyzes folylpolyglutamates. This assay allows us to: 1) measure conjugase for the first time in rat brain and 2) measure conjugase in a reliable, sensitive and inexpensive manner. The MTX-binding assay results were compared to samples analyzed by HPLC and found to vary by only 13%. The artificial substrate, MTX-G₁, had a lower rate of hydrolysis than pteroylglutamyl-gamma-glutamate (Pte-G₂), 70.7±0.64 and 92.6±0.22 nmoles/hr/mg protein respectively. Conjugase was semi-purified 24 fold in H₂O and found to have a pH optimum of 5.0.     

The Effect of Familiarity on Behavior of Kenneled Dogs During Interactions With Conspecifics

Kenneled environments often prevent direct physical contact between dogs, potentially causing stress, and so it has been recommended that such contact should be provided. This study examined the effect of familiarity on the behavior of dogs during off-lead interaction. Kenneled dogs (3 breeds) were given 15-min off-lead interactions with a familiar dog and an unfamiliar dog; the behavior of the focal dog and the distance between the dogs were recorded. More time in contact and interaction behaviors were recorded at 0 to 3 min with unfamiliar dogs than with familiar dogs. At 9 to 12 min, familiar pairs spent more time within 5 body lengths and more time being followed than unfamiliar pairs, who spent more time at more than 5 body lengths apart. This suggests that the initial interaction is more important when dogs are unfamiliar, but once this “greeting” has occurred, unfamiliar pairs are more likely to investigate their surroundings independently rather than together. Breed differences were observed only at 0 to 3 min. The study suggests that familiarity should be taken into account when assessing the effectiveness of conspecific contact as a potential enrichment for kennel-housed dogs.     

Histidine residue modification inhibits binding of murine β nerve growth factor to its receptor

The reaction of the β subunit of murine nerve growth factor (NGF) with diethylpyrocarbonate (DEP) results in the quantitative modification of histidine residues and the loss of binding to rabbit superior cervical ganglia microsomes. No conformational changes accompanied the conversion as judged by fluorescence spectra. Hydroxylamine converted the carbethoxy derivatives back to the unmodified imidazoles and simultaneously restored the capacity of NGF to bind to its receptor. Modification of des (1–9) NGF, from which His-4 and His-8 have been quantitatively removed, results in the same loss in binding activity, suggesting that His-75 and/or His-84 may play an important role in hormone-receptor interactions.     

Conjugative Transfer of theEscherichia coli–Clostridium perfringensShuttle Vector pJIR1457 toClostridium botulinumType A Strains

An RP4-oriT shuttle vector pJIR1457 originally developed forClostridium perfringenswas successfully transferred by conjugation fromEscherichia colitoClostridium botulinumtype A strains and to a nontoxigenicC. botulinumtype A–transposon Tn916mutant strain lacking the entire toxin gene cluster. The light chain (LC) of botulinum toxin was highly expressed in the toxin deletion mutant strain from a pJIR1457 construct containing the recombinant botulinal gene for LC. This shuttle vector system will be valuable for genetic analysis ofC. botulinumand will enable genetic manipulation and recombinant expression studies of botulinum neurotoxins as pharmaceutical agents.