The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction

After force-feeding a protein-free diet to male rats for 5-7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.     

Spectral responses of sustaining fibers in the optic tracts of crayfish (Procambarus)

Summary Spectral response curves were recorded for 60–70 individual sustaining fibers in the optic nerve of the crayfish Procambarus. These cells belong to at least 8 of the 14 classes of sustaining fibers described by Wiersma and Yamaguchi (1966) on the basis of receptive fields. About 90 percent of the cells receive predominant input from yellow-green receptors and are maximally sensitive at 560 to 570 nm; a much smaller number receive principal input from blue receptors and are maximally sensitive near 460 nm.The wavelength sensitivity of optic fibers receiving their major input from yellow-green receptors depends on the state of dark adaptation of the animal and the intensity of illumination. Early in dark adaptation and at high intensities of stimulation the spectral response curve is distorted by light which has been filtered through the sleeves of red-brown shielding pigment. During dark adaptation a shift in maximum spectral response to shorter wavelengths parallels the retraction of the migratory pigment to the dark position and the development of retinal glow. The effects are reversed by injecting into a dark-adapted animal an extract of eyestalks containing the hormone controlling pigment migration: the pigment sleeves lengthen, retinal glow disappears, and shoulders or peaks of sensitivity appear in the red region of the spectrum.This work was supported by USPHS research grant EY 00222 to Yale University. A. E. R. W. was aided by a Fulbright-Hays travel grant. We are grateful to Prof. C. A. G. Wiersma and Dr. R. M. Glantz for a helpful demonstration of the recording technique.     

EVIDENCE FOR VARIATION IN THE QUANTITY OF DNA AMONG PLASTIDS OF ACETABULARIA

The DNA content of individual plastids of the giant unicellular algae Acetabularia mediterranea, and Polyphysa cliftoni was studied. Four methods were used for localizing DNA: acridine orange staining, radioautography following actinomycin D-³H treatment, electron microscopy of thin tissue sections, and electron microscopy of osomotically disrupted plastids. With each method, DNA was readily detected in 20–35% of plastids, but no DNA was observed in the remaining 65–80%. The results further showed that in those plastids with detectable DNA the amount of DNA present was variable. The sensitivity and reliability of the localization methods are discussed, and the possible implications of these findings are considered.     

Differential wavelength sensitivity in the receptive fields of sustaining fibers in the optic tract of the crayfishProcambarus

Summary Spike discharges were measured at 473 nm and at 573 nm in 40–50 individual sustaining fibers (slowly-adapting units signaling intensity levels over large receptive fields). The units belonged to five of the 14 classes of sustaining fibers recognized by Wiersma and Yamaguchi (1966) on the basis of the positions of their receptive fields. The test wavelengths were selected because they lie near the peaks of sensitivity of the two spectral types of receptor known to be present in the ommatida. Relative sensitivity was measured at 5 ° intervals as the test lights were moved around the eye on various arcs, and the receptive fields were described in terms of contours of equal sensitivity for each wavelength.No large differences in relative spectral sensitivity were observed as a function of position in the receptive field, but there was a consistent tendency for sensitivity to blue light to be relatively greater in the dorsal region of the eye. The difference was modest, generally being 0.5 log units or less. This effect could be caused either by regional variation in the population density of the blue and yellow-green receptors, or by weighting of inputs in the optic neuropile.This work was supported by USPHS research grant EY00222 to Yale University. A.E.R.W. was aided by a Fulbright-Hays travel grant.     

Ultrastructural features of the life cycle ofAcetabularia mediterranea

Summary The portion of the life cycle ofAcetabularia mediterranea from cyst formation to gamete release is described. During maturation, the cyst nucleus undergoes a series of mitoses in which the nuclear membrane remains intact, and the spindle microtubules are confined to the intranuclear space. There is a dark requirement for the completion of cyst maturation at the end of which the nuclei are in groups and centrioles are present. The final migration of the nuclei, cleavage of the cytoplasm into gametes, and the development of flagella is cxtremely rapid, taking six to twelve hours. Cysts which fail to germinate are blocked at a late stage in the maturation process, either before or after the final nuclear division.The observations are related to mitosis in other green algae (Chlorophyceae) and to gametogenesis inBryopsis hypnoides. Changes in the amount and distribution of heterochromatin in the nuclei are discussed in relation to the life cycle ofA. mediterranea.The substance of this paper was first presented orally at the Symposium onAcetabularia held at the Max-Planck-Institut für Zellbiologie, Wilhelmshaven, Federal Republic of Germany, July 12–15th, 1972.Supported in part by Grant GM 06637 from the U.S. Public Health Service.     

RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.

In vitro methylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies in rglB+ host strains C600 and DS410 by almost 2 orders of magnitude. By contrast, the rglB- derivative of DS410 showed no reduction in transformation efficiency with methylation while the rglB- derivative of C600 was partially tolerant to methylation. Further, we show that the 1.8 kilobase (kb) and 1.2 kb KpnI fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mC in vivo. Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with the rglB- host strains than with the rglB+ hosts. In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (from Petunia hybrida) when plated on rglB- hosts gave titres up to 222 times higher than on the rglB+ strains.