L-Threonine dehydrogenase from goat liver. Feedback inhibition by methylglyoxal

L-Threonine dehydrogenase, which forms aminoacetone from L-threonine and NAD, has been extensively purified from goat liver. A feedback inhibition of this enzyme has been observed with methylglyoxal. Kinetic data and other experiments indicate that methylglyoxal acts at a site other than the active site of the enzyme. The enzyme contains a single subunit of Mr 89,000. The apparent Km values of the enzyme for L-threonine and NAD were found to be 5.5 and 1 mM, respectively.     

Basomedial amygdaloid lesions and p-CPA induced muricidal aggression

Bilateral lesions of basomedial amygdaloid nuclei are capable of significantly inhibiting muricidal aggression induced by oral p-chlorophenylalanine (p-CPA) in male rats. Rats lesioned in extra-amygdaloid structures or sham-lesioned show the usual p-CPA-induced muricidal activity, which ranges from 70 to 80% of treated animals. The results obtained indicate that basomedial amygdaloid nuclei play an important role in regulating p-CPA-induced muricidal aggression, even though the effect lasts for a relatively limited period of time. This fact is probably due to the intervention of still unidentified compensatory mechanisms.     

On the origin of the lactate dehydrogenase induced rate effect

To evaluate the ability of lactate dehydrogenase to facilitate the bond making/breaking steps for both the addition of pyruvate enol to NAD (pyruvate adduct reaction) and the normal redox reaction, the ability of the enzyme to facilitate the tautomerization of bound pyruvate is assessed. In addition, the equilibrium constants for the adduct reaction are obtained for both bound and free reactants from the ratio of the rate constants in the forward and reverse reactions (at pH 7). The latter comparison indicates that the enzyme facilitates bond making/breaking in the (forward) pyruvate adduct reaction by a factor of about 10(11) M. Similar comparisons suggest that reactant immobilization accounts for about 1000 M of this 10(11) M rate effect. Since the (pH-independent) rate constant for the ketonization of bound pyruvate enol assisted by the external buffer, imidazolium ion, is 2 X 10(7) M-1 s-1 and the corresponding rate constant for free pyruvate enol, again assisted by imidazolium ion, is 35 M-1 s-1 [Burger, J. W., II, & Ray, W. J., Jr. (1978) Biochemistry 17, 1664], the enzyme facilitates the bond making/breaking steps associated with the conversion of bound HO-C less than to bound O = C less than by a factor of about 10(6)-fold. The product of the above two rate enhancement factors and the rate factor suggested previously for the environmental effect on NAD produced by its binding to lactate dehydrogenase, 100-fold, is 10(11) M, and it accounts for the bond making/breaking effects exerted by the enzyme in the pyruvate adduct reaction. The rate constant for oxidation of ethanol (a model for lactate) by 1-methylnicotinamide (a model for NAD) is about 5 X 10(-12) M-1 s-1 at 25 degrees C in pure ethanol (delta H for this reaction is about 30 kcal/mol). The ratio of the rate constants for E X NAD X Lac----E X NADH X Pyr and the above model reaction is estimated as about 10(14) M in water; i.e., the LDH-induced rate effect is about 10(14) M. The product of the values for the above rate factors for the normal redox reaction is about 10(12) M. Although the value of this product is less certain than that for the adduct reaction, these rate factors do account for much of the LDH-induced rate effect.     

Sister-chromatid exchange induction by sodium selenite: reduced glutathione converts Na2SeO3 to its SCE-inducing form

Sodium selenite (Na2SeO3) is an anticarcinogenic/antimutagenic agent that exhibits carcinogenic/mutagenic properties in some short-term test systems used for the detection of DNA-damaging agents. One such test system is sister-chromatid exchange (SCE) induction. Na2SeO3 induces SCEs only if red blood cells (RBCs) are present to 'activate' it to its SCE-inducing form. Here, the ability of reduced glutathione, a major component of RBCs, to serve as an RBC substitute in the activation of Na2SeO3 was determined. Reduced glutathione (10(-4) and 10(-3) M) was shown to be as capable as RBCs in activating Na2SeO3 (7.95 X 10(-6) M) to its SCE-inducing form. These data suggest strongly that the pathway normally utilized by RBCs in the metabolism of Na2SeO3 is the same as that in which Na2SeO3 is converted to its SCE-inducing form.     

Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin

The origin of replication for the viral strand of bacteriophage M13 DNA is contained within a 507 base-pair intergenic region of the phage chromosome. The viral strand origin is defined as the specific site at which the M13 gene II protein nicks the duplex replicative form of M13 DNA to initiate rolling-circle synthesis of progeny viral DNA. Using in vitro techniques we have constructed deletion mutations in M13 DNA at the unique AvaI site which is located 45 nucleotides away on the 3' side of the gene II protein nicking site. This deletion analysis has identified a sequence near the viral strand origin that is required for efficient replication of the M13 genome. We refer to this part of the intergenic region as a "replication enhancer" sequence. We have also studied the function of this sequence in chimeric pBR322-M13 plasmids and found that plasmids carrying both the viral strand origin and the replication enhancer sequence interfere with M13 phage replication. Based upon these findings we propose a model for the mechanism of action of the replication enhancer sequence involving binding of the M13 gene II protein.     

Differential expression of murine leukemia virus loci in chemically induced hybrid cells.

The time course of murine leukemia virus production after chemical induction was determined in hamster-mouse somatic cell hybrids containing the xenotropic murine leukemia virus induction locus Bxv-1 or the ecotropic locus Akv-2. By using these hybrids, induction could be studied in the absence of secondary virus spread because xenotropic viruses cannot infect hybrid cells and ecotropic viruses cannot infect hybrids which have lost mouse chromosome 5. After induction, hybrids with Bxv-1 produced only a transient burst of virus, whereas those with Akv-2 continued to produce virus for periods in excess of 3 months. The presence or absence of other mouse chromosomes in the hybrid lines did not alter these induction patterns. Thus, endogenous murine leukemia virus loci differ in their response to induction, and both inducibility and the kinetics of virus expression are controlled at or near these proviral loci.     

Two genetically transmitted BALB/c mouse mammary tumor virus genomes located on chromosomes 12 and 16.

We have examined EcoRI-restricted cellular DNA from BALB/c mouse-hamster somatic cell hybrids by blot hybridization for the presence of mouse mammary tumor virus-related sequences. Results of this analysis show that mouse mammary tumor virus-related proviral copies are located on chromosomes 16 (16-kilobase-pair fragment) and 12 (10.5- and 7.7-kilobase-pair fragments).     

Lymphatic endothelial celline (CH3) from a recurrent retroperitoneal lymphangioma

Summary An endothelial cell line derived from a massive recurrent chyle-containing retroperitoneal lymphangioma was isolated in monolayer culture. Scanning and transmission electron microscopy and immunohistochemistry confirmed a close resemblance to blood vascular endothelium with typical cobblestone morphology, positive immunofluorescence staining for endothelial marker Factor VIII-associated antigen and fibronectin, and prominent Weibel-Palade bodies. The endothelial cells also exhibited other ultrastructural features characteristic of lymphatic endothelium, including sparse microvillous surface projections, overlapping intercellular junctions, and abundant intermediate filaments. This endothelial cell line represents a new source of proliferating lymphatic endothelium for future study, including structural and functional comparison to blood vascular endothelium. Supported in part by Arizona Disease Control Research Commission contracts 8277-000000-1-1-AT-6625 and ZB-7492. Presented in part at the 10th International Congress of Lymphology in Adelaide, Australia, August 1985.     

Pentylenetetrazol: Effect on carbaryl-induced changes of serotonin metabolism in pons-medulla of rat brain

Oral administration of carbaryl to adult male albino rats produced a dose dependent increase in the steady state level of 5-hydroxytryptamine (5-HT) at 1.00 h in pons-medulla (PM). 5-Hydroxyindole acetic acid (5-HIAA) concentration was significantly elevated only in response to a higher dose of this pesticide under similar conditions. A time course study with carbaryl and pentylenetetrazol (PTZ) showed a characteristic elevation of the steady state level of 5-HT in PM, but the 5-HIAA level was significantly elevated at 0.5 h only after carbaryl treatment. No significant change of the 5-HIAA level was evident after administration of PTZ alone or in combination with carbaryl. Tryptophan concentration was significantly elevated in PM at 0.5 h after carbaryl treatment and at 1.0 h after carbaryl + PTZ treatment. No significant change of tryptophan concentration was evident after the administration of PTZ alone under similar conditions. Measurement of (1) pargyline induced (a) accumulation of 5-HT and (b) depletion of 5-HIAA levels, and (2) probenecid-induced accumulation of 5-HIAA level in presence and absence of carbaryl and revealed that carbaryl accelerated the synthesis as well as the breakdown of 5-HT, whereas PTZ alone or in combination with carbaryl accelerated the synthesis of 5-HT without affecting its catabolism. The potency of this pesticide in elevating the pargyline-induced accumulation of 5-HT is in the order of carbaryl + PTZ greater than PTZ congruent to carbaryl. These results suggest that the carbaryl-induced increase in the synthesis of 5-HT is potentiated, and the turnover is reduced, in PM when PTZ is administered to the carbaryl-intoxicated rats.