Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

Modification of radiation damage of bacteria by folic acid antagonists

Pittillo, Robert F. (Southern Research Institute, Birmingham, Ala.), Mary Lucas, Robert T. Blackwell, and Carolyn Woolley. Modification of radiation damage of bacteria by folic acid antagonists. J. Bacteriol. 90:1548-1551. 1965.-The folic acid analogues, 2,4-diamino-6-methylpteridine, amethopterin, and aminopterin, have been found to sensitize certain bacteria, especially Escherichia coli, to the lethal action of ionizing irradiation. Data are presented which indicate that (i) the compounds must be present during the irradiation period for maximal sensitization to be observed, (ii) the sensitizing effect can be nullified by cysteine or cysteamine, (iii) the sensitizing effect occurs in a number of diverse bacterial genera, and (iv) folic acid neither sensitizes bacteria to irradiation nor prevents the sensitization caused by these antifolic agents.     

Deoxycytidylate hydroxymethylase: purification, properties, and the role of a thiol group in catalysis

Deoxycytidylate (dCMP) hydroxymethylase from Escherichia coli infected with a T-4 bacteriophage amber mutant has been purified to homogeneity. It is a dimer with a subunit molecular weight of 28,000. Chemical modification of the homogeneous enzyme with N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) leads to complete loss of enzyme activity. dCMP can protect the enzyme against NEM inactivation, but the dihydrofolate analogues methotrexate and aminopterin alone do not afford similar protection. Compared to dCMP alone, dCMP plus either methotrexate or aminopterin greatly enhances protection against NEM inactivation. DTNB inactivation is reversed by dithiothreitol. For both reagents, inactivation kinetics obey second-order kinetics. NEM inactivation is pH dependent with a pKa for a required thiol group of 9.15 +/- 0.11. Complete enzyme inactivation by both reagents involves the modification of one thiol group per mole of dimeric enzyme. There are two thiol groups in the totally denatured enzyme modified by either NEM or DTNB. Kinetic analysis of NEM inactivation cannot distinguish between these two groups; however, with DTNB kinetic analysis of 2-nitro-5-thiobenzoate release shows that enzyme inactivation is due to the modification of one fast-reacting thiol followed by the modification of a second group that reacts about 5-6-fold more slowly. In the presence of methotrexate, the stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group. It appears that the two equally sized subunits are arranged asymmetrically, resulting in one thiol-containing active site per mole of dimeric enzyme.     

The Structure of the Spermatozoon of the Japanese Quail,Coturnix coturnix L., var. japonica

The spermatozoa of the Japanese quail conform, in their general ‘sauropsid’ plan, to that of other non-passerine birds. They are notable, however, in that the flagellum is very elongated (208 μm) and carries approximately 2,500 mitochondria in an extensive midpiece. Ultrastructurally, the acrosome and acrosome-nucleus junction is exactly as reported for other galliform birds. The neck region contains two centrioles arranged almost in-line; this unusual layout apparently occurs also in guinea fowl sperm. At the tip of the axoneme, beyond the termination of the central pair microtubules, is a structure—the tip granule—previously recognized in sperm of the domestic fowl. Trypsin digestion splits the axonemal cylinder, and the doublets then spiralize. This response has been reported before, in the sperm tails of other avian species.     

Alternating torsions in a living '9 + 2' flagellum

Live spermatozoa of the Japanese quail were observed as they swam in highly viscous salines. Under these conditions, torsions of the flagellum were readily seen. The torsions had a characteristic magnitude (nominally 180 degrees) and pattern of incidence. As a cycle of bending propagated over it, each position on the flagellum experienced first a sinistral torsion and, later, a restoring dextral torsion. The two zones of torsion were each associated with bending; between them was a torsion-free zone that tended to be straight. The amount of interdoublet sliding needed to generate the torsions may be as little as 10 nm. These dynamic propagating torsions have been detected by following the angular displacements of individual (swollen) mitochondria lying adjacent to the axoneme. It is suggested that torque generation is a primary outcome when the unconstrained ''9 + 2'' axoneme is activated.     

Thermal melting of chromatin from the pancreas and liver of guinea pigs treated with 1-methyl-1-nitrosourea

The carcinogen 1-methyl-1-nitrosourea (MNU) can cause pancreatic cancer in guinea pigs. We have examined the relative damage produced by MNU treatment on the chromatin from pancreas and liver of these animals. Thermal denaturation of chromatin from guinea pig pancreas and liver was studied following parenteral administration of MNU in several doses. Estimates of single strand breakage were also obtained by examination of the fluorescence of intercalated ethidium bromide. Oligomeric chromatin melted with a main Tm at 78 degrees C, with additional components at 48 degrees C, 55 degrees C and 65 degrees C. Repetitive treatment with MNU at several doses between 20 mg/kg and 70 mg/kg produced destabilization of pancreatic chromatin as shown by a shift from 78 degrees C to lower melting components. The liver by contrast was relatively unaffected. In addition, pancreatic chromatin showed an increase in alkali-induced strand unwinding with MNU treatment, probably due to an increase in single strand breaks, while there was no change in this regard in the liver. The data indicate that the pancreas is more susceptible to damage by MNU than the liver.     

Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways

There are several pathways for the incorporation of sugars into glycosphingolipids (GSL). Sugars can be added to ceramide that contains sphinganine (dihydrosphingosine) synthesized de novo (pathway 1), to ceramide synthesized from sphingoid bases produced by hydrolysis of sphingolipids (pathway 2), and into GSL recycling from the endosomal pathway through the Golgi (pathway 3). We reported previously the surprising observation that SW13 cells, a human adrenal carcinoma cell line, synthesize most of their GSL in pathway 2. We now present data on the synthesis of GSL in four additional cell lines. Approximately 90% of sugar incorporation took place in pathway 2, and 10% or less in pathway 1, in human foreskin fibroblasts and NB41A3 neuroblastoma cells. In contrast, approximately 50-90% of sugar incorporation took place in pathway 1 in C2C12 myoblasts. The C2C12 cells divide more rapidly and synthesize 10-14 times as much GSL as the other three cell lines. In C6 glioma cells, approximately 30% of sugar incorporation occurred in pathway 1 and 60% in pathway 2. There was no relation between the utilization of pathways for GSL and sphingomyelin synthesis in foreskin fibroblasts and C2C12 cells. In both cells pathways 1 and 2 each accounted for 50% of incorporation of choline into sphingomyelin. In five of the six cell lines that we have studied, most GSL synthesis takes place in pathway 2. We suggest that when the need for synthesis is relatively low, as in slowly dividing cells, GSL are synthesized predominantly from sphingoid bases salvaged from the hydrolytic pathway. When cells are dividing more rapidly, the need for increased synthesis is met by upregulating the de novo pathway.      

Effect of cartilage proteoglycans on human collagenase activities.

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.