An antibody probe to determine the native species of glycinamide ribonucleotide transformylase in chicken liver

Antibody probes of Western blots [Renart, J., Reiser, J., & Stark, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3116] of chicken liver homogenates under various conditions revealed that glycinamide ribonucleotide transformylase can be rapidly proteolyzed in such homogenates. These findings, along with molecular weight measurements by ultracentrifugation, identify the true form of glycinamide ribonucleotide transformylase as a monomeric protein of 117000 daltons. This protein has been purified 400-fold in 44% yield from chicken liver in one step on an affinity column of 10-formyl-5,8-dideazafolate-Sepharose. Native glycinamide ribonucleotide transformylase retains full activity after proteolytic cleavage to a form (Mr 55000) similar to fragments seen in the Western blot of the homogenates. This phenomenon may be responsible for the previous identification of glycinamide ribonucleotide (GAR) transformylase as a dimer of 55000-dalton subunits. Similar analyses using antibodies to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase [Mueller, W. T., & Benkovic, S. J. (1981) Biochemistry 20, 337] and trifunctional enzyme [Smith, G. K., Mueller, W. T., Wasserman, G. F., Taylor, W. D., & Benkovic, S. J. (1980) Biochemistry 19, 4313] confirm that these two proteins were isolated in their native forms.     

Autumn stomatal closure in six conifer species of the Central Rocky Mountains

Summary Environmental and water relations parameters during fall were monitored for six conifer tree species common to the central Rocky Mountains growing naturally at the same location (Pinus contorta, Pinus ponderosa, Pinus flexilus, Pseudotsuga menziesii, Abies lasiocarpa, Picea engelmannii). Subsequent to what appeared to be the beginning of seasonal stomatal closure, leaf conductance to water vapor declined sharply following the onset of freezing air temperatures at night. A coincident rapid decline in morning xylem pressure potentials (_p) also occurred which resulted in values that were considerably below afternoon _p. Continuing decreases in maximum leaf conductance during the day were highly correlated with corresponding decreases in minimum nocturnal air temperatures of the preceding night. By mid-December, morning _p returned to values very near afternoon _p and were only slightly lower than before the onset of subfreezing nights. A preliminary model is proposed which interprets the qualitative interaction between air and soil temperatures, soil and plant water potentials, and leaf conductance during seasonal stomatal closure in fall.     

Discriminative stimulus properties of the serotonin agonist 1-(3-trifluoromethylphenyl)piperazine (TFMPP)

Using a standard two-lever drug discrimination procedure, twelve rats were trained to discriminate 1.0 mg/kg of the serotonin (5-HT) agonist TFMPP from saline. Once trained, the animals displayed a dose-related decrease in discriminative performance upon administration of lower doses of TFMPP. Tests of stimulus generalization were performed using the purported 5-HT agonist RU-24, 969 and 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). While TFMPP produced stimulus effects similar to those of RU-24,969, these effects seem to be dissimilar to those of DOM. The results of the present study suggest that the discriminative stimulus effects of TFMPP may involve a 5-HT1-related mechanism.     

Gene amplification in Bacillus subtilis

A strain of Bacillus subtilis that carries in its genome a staphylococcal chloramphenicol acetyltransferase gene (from pC194) responds to growth at different concentrations of chloramphenicol by an alteration in the number of copies per genome of the sequences encoding the gene. Growth at 20 micrograms chloramphenicol ml-1 results in a 15-fold amplification of the sequences, whereas growth in the absence of chloramphenicol results in their loss. The mechanism of in situ amplification probably has much in common with that involved in 'R factor transitioning'. The hybridization procedures that have been used for accurately determining the number of copies of the amplified DNA sequences are potentially useful for plasmid copy number determination. The findings reported here also provide a potentially useful alternative to more conventional cloning strategies that are based on autonomous plasmids in B. subtilis. The particular advantages that can be envisaged include enhanced stability of the cloned sequences and control of the number of copies that are present.     

Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.     

Human Brain Phenol Sulfotransferase: Biochemical Properties and Regional Localization

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catecholamines and phenol and catechol drugs. The human blood platelet contains a thermolabile (TL) form of PST that catalyzes the sulfate conjugation of dopamine and other monoamines and a thermostable (TS) form that catalyzes the sulfate conjugation of micromolar concentrations of phenol and p-nitrophenol. Experiments were performed to determine whether the brain contains forms of PST analogous to the TL and TS forms found in the human platelet, and to determine whether there are regional variations in human brain PST activity. We found that the human brain contains at least two forms of PST, forms that are similar to the platelet TS and TL forms of the enzyme with respect to substrate specificity, apparent Km constants, thermal stability, and sensitivity to inhibitors. Optimal conditions were determined for the measurement of these two activities in brain homogenates. The stability of PST activities in the human brain after death was determined in five samples of cerebral cortex that were obtained during clinically indicated neurosurgical procedures. An average of 76 +/- 8% and 80 +/- 9% (mean +/- SEM) of the basal TL and TS PST activities, respectively, remained in these five samples of cerebral cortex after 8 h of storage under simulated post-mortem conditions. Six human brains were then obtained less that 8 h after death from patients who had no neurological disease prior to death. The mean activities of the TL and TS forms of PST were measured in 17 different regions of the six brains. If the pituitary was excluded from consideration, TL and TS PST activities both varied approximately fivefold among these regions, and both activities were highest in cerebral cortex. However, the average TS activity in the anterior pituitary, a tissue of non-neural origin embryologically, was 6.5-fold greater than the highest average TS PST activity found in cerebral cortex.     

The molecular basis for the difference in immune hemolysis activity of the Chido and Rodgers isotypes of human complement component C4

Human C4 displays a structural polymorphism which is consistent with there being two closely linked genetic loci coding for this protein. These give rise to two C4 isotypes, designated C4A and C4B, which can be distinguished by charge and apparent m.w. differences in their respective alpha-chains and by the presence or absence of the Chido/Rodgers blood group antigens. Previous qualitative studies of C4 immune hemolysis activity in whole plasma had suggested that the C4B isotype was functionally more active. By using purified C4A and C4B isolated from individual donors known serologically to possess only one of the C4 isotypes, we examined the molecular basis for the differences in their respective hemolytic activities. It was found that the C4B:C4A hemolytic activity ratio was approximately 4:1. This fourfold difference could not be accounted for by a commensurate difference in the cleavage rate of the two isotypes by C1s by differences in the kinetics of assembly or intrinsic decay of the respective C3 convertase enzymes, or by differences in the rate of isotypic C4b cleavage by factor I in the presence of C4bp . However, the fourfold greater deposition efficiency of nascent C4b of the C4B isotype onto the surface of C1-bearing sheep erythrocytes quantitatively accounted for the observed difference in immune hemolysis function. It was further found that the thioester bond of nascent C4b of the C4A isotype preferentially transacylates onto amino group nucleophiles, whereas in the C4B isotype, acylation of hydroxyl groups is strongly preferred. Thus, the difference in immune hemolysis activity between the two C4 isotypes does not necessarily indicate an impairment of function in C4A; it may merely be a reflection of the relative abundance at the surface of a C1-bearing target of hydroxyl and amino groups capable of being acyl acceptors for nascent C4b. Finally, we also present evidence showing that the apparent m.w. difference between the alpha-chains of the C4A and C4B isotypes is not due to differences in protein glycosylation.     

Evolution of the albumin: alpha-fetoprotein ancestral gene from the amplification of a 27 nucleotide sequence

The genes for alpha-fetoprotein and albumin arose by duplication of an ancestral gene that contained three genetic domains. These domains were generated by the triplication of a primordial genetic domain composed of five exons or subdomains. That the primordial domain itself arose by amplification of a simpler sequence is suggested by nucleotide sequence homologies among the subdomains of the mouse alpha-fetoprotein gene. A detailed analysis of these homologies reveals that each of the five subdomain families contains remnants of a 27-base-long repeat from which the entire alpha-fetoprotein coding sequence has been assembled. A consensus sequence for the 27 nucleotide repeat is derived, and the positions of the repeats within each subdomain are described. A model is proposed for the evolution of the primordial domain by the amplification and divergence of the 27 base-pair sequence, along with the condensation of the repeats into subdomains separated by intervening sequences. It is postulated that the role of intervening sequences may be to limit sequence amplification in genes such as alpha-fetoprotein and albumin whose protein products cannot tolerate size variation.     

Replication and recombination in adenovirus-infected cells are temporally and functionally related.

We have studied the temporal and functional relationships between DNA replication and recombination in adenovirus-infected cells by using Southern blot hybridization to detect recombinant products among intracellular viral genomes. The data show that recombination can be detected soon after DNA replication has commenced and that the proportion of recombinant products increases thereafter. To determine the functional relationship between DNA replication and recombination, replication was blocked with the protein synthesis inhibitor anisomycin, the replication inhibitor cytosine arabinoside, and conditionally lethal mutations in either the virus-specified DNA-binding protein or the DNA polymerase. All treatments that directly or indirectly blocked DNA replication caused a delay in the appearance of recombinant products and a marked decline in their abundance relative to products of parental genotype. These data strongly suggest that DNA replication and recombination are interrelated, either because both processes share functions or because DNA structures produced by replication are suitable substrates for recombination. In addition, we have shown that some recombination function(s) is intrinsically thermolabile at 40.9 degrees C, even in wild-type crosses, since the appearance of recombinant products is delayed and their extent is reduced compared with that from crosses performed at 39.9 degrees C.     

Changes in binding of a 27-kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm

Motility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein-glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27-kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27-kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed the loss of a 27-kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the ¹²⁵I-distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.