Intracellular transport of vitellogenin in Xenopus oocytes: An autoradiographic study

The role of primordial yolk platelets (PYPs) in the transport of the yolk precursor vitellogenin to the yolk platelets in Xenopus laevis oocytes has been demonstrated by electron microscopic autoradiography. Within 20 min after exposure of the oocyte to ³H-labeled-vitellogenin, silver grains are associated with small PYPs which are formed by the fusion of endosomes. At 40 min after incorporation of ³H-labeled vitellogenin, autoradiographic silver grains are associated with larger PYPs and with the superficial layer of yolk platelets. Thus, the results demonstrate that PYPs are an intermediate in the transport of vitellogenin from endosomes to yolk platelets. These observations are consonant with the general hypothesis that vitellogenin first associates (binds?) with the plasma membrane, then is incorporated by endocytosis into endosomes which fuse to form PYPs, and finally the contents of the PYPs are eventually deposited into yolk platelets.     

Oogenesis in Fundulus heteroclitus: I. Preliminary observations on oocyte maturation in vivo and in vitro

During the breeding season, the ovary of Fundulus heteroclitus contains follicles of all sizes up to around 1.8 mm in diameter and continuously ovulates eggs (1.7 mm) into the ovarian lumen. Vitellogenic follicles appear to extend up to 1.3 mm, while those larger than 1.4 mm are undergoing hydration concomitant with maturation. If feeding ceases, vitellogenesis appears to stop and maturational stages flush out of the ovary. Refeeding causes reappearance of maturational stages. A culture procedure was developed for dissected F. heteroclitus follicles and a size range (1.3–1.4 mm) was defined which responded to added hormones by undergoing the same maturational changes (germinal vesicle breakdown, fusion, and peripheral detachment of oil droplets, appearance of activability) as occurs in vivo, although the extent of hydration was not quite as extensive. Ovulation, however, did not consistently occur in vitro. Both 11-deoxycorticosterone and progesterone are potent inducers of maturation in vitro, with median effective doses being 10–20 ng/ml. Hydrocortisone is about one-tenth as active. Human chorionic gonadotropin promotes maturational changes in about 25% of the follicles over a broad concentration range (1–100 IU/ml). The environmental and hormonal mechanisms which influence oocyte maturation in teleosts are discussed in relation to our findings for F. heteroclitus.     

Expression of embryonic hemoglobin genes in mice heterozygous for α-thalassemia or β-duplication traits and in mice heterozygous for both traits

Hemoglobins of mouse embryos at 11.5 through 16.5 days of gestation were separated by electrophoresis on cellulose acetate and quantitated by a scanning densitometer to study the effects of two radiation-induced mutations on the expression of embryonic hemoglobin genes in mice. Normal mice produce three kinds of embryonic hemoglobins. In heterozygous α-thalassemic embryos, expression of EI (x₂y₂) and EII (α₂y₂) is deficient because the x- and α-globin genes of one of the allelic pairs of Hba on chromosome 11 was deleted or otherwise inactivated by X irradiation. Simultaneous inactivation of the x- and α-globin genes indicates that these genes must be closely linked. Reduced x- and α-chain synthesis results in an excess of y chains that associate as homotetramers. This unique y₄ hemoglobin also appears in β-duplication embryos where excess y chains are produced by the presence of three rather than two functional alleles of y- and β-globin genes. In double heterozygotes, which have a single functional allele of x- and α-globin genes and three functional alleles of y- and β-globin genes, synthesis of α and non-α chains is severely imbalanced and half of the total hemoglobin is y₄. Mouse y₄ has a high affinity for oxygen, P₅₀ of less than 10 mm Hg, but it lacks cooperativity so is inefficient for oxygen transport. The death of double heterozygotes in late fetal or neonatal life may be due in large part to oxygen deprivation to the tissues.     

Nickel complexes of sepiapterin and 6-acetyldihydrohomopterin, a pyrimidodiazepine from Drosophila

The nonfluorescent pyrimidodiazepine in Drosophila melanogaster 6-acetyldihydrohomopterin (H₂Ahp) was studied using ultraviolet and infrared spectroscopy. The H₂Ahp was unstable in 3% NH₄Cl whereas a related pteridine sepiapterin was stable. Since Ni²⁺ stabilized H₂Ahp completely, the structure of the H₂Ahp · Ni complex was examined. Among 15 pterins, including sepiapterin, the spectral properties in the presence of Ni²⁺ reflect the pK_a's and the reactive group on the side chain but for H₂Ahp the spectral properties are rather different from the pteridines and they indicate that the seven-membered ring seemed to have the predominant influence. The Ni²⁺ comples of H₂Ahp resulted in a shift in the absorption maximum from 383 to 436 nm. The corresponding spectral shift of the pteridines due to Ni²⁺ was much less. From the infrared spectra of H₂Ahp and sepiapterin in the presence and absence of Ni²⁺, the sites of interaction of Ni²⁺ with H₂Ahp were shown to be the phenolic oxygen and N5 in the ring. In the absence of Ni²⁺ an internal hydrogen bond in sepiapterin was indicated that may involve the carbonyl oxygen and the secondary alcoholic oxygen on the side chain. Other metal ions were tested (Cd²⁺, Zn²⁺) but were not as effective as Ni²⁺ in stabilizing H₂Ahp.     

Properties of microsomal acyl coenzyme A reductase in mouse preputial glands

Synthesis of long-chain fatty alcohols in preputial glands of mice is catalyzed by an NADPH-dependent acyl coenzyme A (CoA) reductase located in microsomal membranes; sensitivity to trypsin digestion indicates that the reductase is on the cytoplasmic side of the membrane. Results with pyrazole and phenobarbital demonstrate the reaction is not catalyzed by a nonspecific alcohol dehydrogenase or an aldehyde reductase. Acyl-CoA reductase activity is sensitive to sulfhydryl and serine reagent modification, is stimulated by bovine serum albumin, and produces an aldehyde intermediate. The activity is extremely detergent sensitive and cannot be restored even after removal of the detergents. Phospholipase C or asolectin treatment does not release the acyl-CoA reductase from microsomal membranes, but causes a significant decrease in the activity recovered in the membrane pellet. Glycerol does not solubilize the reductase activity, nor does 3.0 m NaCl; however, the combination of glycerol and 3.0 m NaCl did release about 50% of the acyl-CoA reductase from the microsomal pellet. Substrate concentration curves obtained in the presence or absence of bovine serum albumin show significant differences in enzyme activities. The reductase is sensitive to the concentration of palmitoyl-CoA and is progressively inhibited at levels beyond the critical micellar concentration of the substrate. The apparent K_m for acyl-CoA reductase is 14 μm; however, the maximum velocity varies with the concentration of albumin used. Expression of enzyme activity in delipidated microsomes requires specific phospholipids, which suggests that in vivo regulation of acyl-CoA reductase activity could be achieved through modifications in membrane lipid composition.     

Regulation of acyl coenzyme A reductase by a heat-stable cytosolic protein during preputial gland development

A low-molecular-weight protein located in the cytosol of mouse preputial glands has been shown to stimulate the activity of a microsomal acyl coenzyme A (CoA) reductase in the gland. This cytoplasmic protein was stable to heating and lyophilization, but was destroyed by trypsin digestion. It was able to bind palmitoyl-CoA and gel elution behavior indicated it had a molecular weight of 10,000–12,000. The level of this stimulatory cytosolic protein and the activity of acyl-CoA reductase were shown to correlate with differentiation of the preputial gland during development of puberty in male mice; the acyl-CoA reductase activity first appeared at 4 weeks of age and increased dramatically up to 6 weeks of age. By 8 weeks, when sexual maturity was attained, the reductase activity decreased to that level found in mature male mice. The cytosol from the preputial glands of the youngest mice (3 weeks) contained sufficient heat-stable acyl-CoA binding protein to stimulate acyl-CoA reduction; however, the 3-week-old preputial gland microsomes had little or no acyl-CoA reductase activity. As the animal matured, the stimulatory capacity in the heat-treated cytosol increased, reaching a maximum at 6 weeks; by 8 weeks, the stimulatory capacity of the soluble fraction had decreased to that found in mature male mouse. Results of this study suggest that the concentration of acyl-CoA, cytoplasmic acyl-CoA binding protein, and acyl-CoA reductase activity regulate the level of fatty alcohols in vivo and that the reductase activity and binding protein have similar patterns of development during puberty.     

Novel features of hamster pinealocyte ultrastructure

Pineal glands from young adult hamsters (Mesocricetus auratus Water-house) kept on a 12 : 12 light : dark photoperiod were examined by electron microscopy. Ultrastructural features of nuclei, mitochondria, and centrioles that have not been reported previously are presented in this paper. Of particular interest is the presence of modified centrioles in normal hamster pinealocytes.     

The diversion of dimethylailylpyrophosphate from polyisoprenoid to cyclopiazonic acid biosynthesis in Penicillium cyclopium westling

During the production of α-cyclopiazonic acid (αCA) by Penicillium cyclopium, dimethylallyltransferase (EC. 2.5.1.1.) T, isopentenyl pyrophosphate isomerase (EC. 5.3.3.2) I, and a prenyl-aryltransferase, S, which produces β-cyclopiazonic acid (βCA) are all induced at the same time. This last enzyme appears maximally before the highest rate of α- or βCA production. Both transferases are not utilized to their maximum capacity, and the production of their end products seems to bear no relationship to their concentrations. Other controls therefore must play an important role in the utilization of their common substrate dimethylallylpyrophosphate (DMAPP). There are two possible control systems: (a), a direct competition by S and T for DMAPP; and (b), control by compartmentation. The first possiblility is the more likely, in view of some of the controls that could apply to the deflection. The three enzymes were separated so that possible controls on the deflection of DMAPP from polyisoprenoids could be studied. They all possessed a subunit structure and exhibited maximum molecular weithts (in the absence of divalent cations and presence of a thiol reductant) of 96 000 (S) and 64 000 (I and T) daltons. Mg²⁺ caused a diminution in size to 75 000 (S) and 50 000 (I and T) daltons. Mg²⁺ had the same effect on I and T but caused major disruptive changes to S. These effects were reversible by addition of EDTA. S was quite specific for DMAPP and cycloacetoacetyl-l-tryptophan (cAATrp) and exhibited Michaelis constants as follows; K_m^cAATrp, 6.0μM and K_m^DMAPP 2.0 μM. It had no obvious requirement for a divalent cation and had an isoelectric point of 5.3. I had a K_m of 6.7 μM and an isoelectric point of 4.5, and either Mg²⁺ or Mn²⁺ was essential. The Michaelis constants for T could not be given but its isoelectric point was 5.1. The enzyme carried out the two reactions normally associated with it (i.e., two additions of IPP to produce farnesyl pyrophosphate) and required Mg²⁺ to do so. The pH optima for S, I, and T were 6.5–7.5, 6.0, and 8.0 respectively. The early and major controlling factor was the appearance of the cosubstrate of S, cAATrp. Other factors were: (a), the appearance of αCA which inhibited T more effectively than S; (b), the removal of free Mn²⁺ and Mg²⁺, both essential for I and T but not for S, possibly brought about by chelation with cAATrp, α- and βCA; (c), the observed low pH of 6.0 when the activity of S was unaltered, I was at its highest, and T exhibited 50% of its maximum; and (d), an activation of I by low physiological levels of βCA and cAATrp which would enhance the rate of appearance of DMAPP to react with an existing pool of cAATrp.     

A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the K_a for chorismate and the K_is for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.     

Differential effect of UV irradiation on induction of intragenic and intergenic recombination during commitment to meiosis in Saccharomyces cerevisiae

A comparison was made between the induction of intragenic and intergenic recombinations during meiosis in a wild-type diploid of Saccharomyces cerevisiae. Under non-irradiated normal conditions, production of both intragenic and intergenic recombinants greatly increased in the cells with commitment to meiosis. The susceptibility of cells to the induction of both the spontaneous intra- and intergenic recombinations in meiotic cells was similar. However, under condition of UV irradiation, there were striking differences between intra- and intergenic recombinations. Susceptibility to induction of intragenic recombination by UV irradiation was not enhanced at meiosis compared with mitosis, and was not altered through commitment to meiotic processes. In contrast, however, susceptibility to the induction of intergenic recombination by UV irradiation was enhanced markedly during commitment to meiosis compared with mitosis. Genetic analysis suggested that the enhanced susceptibility to recombination during meiosis is specifically concerned with reciprocal-type recombination (crossing-over) but not non-reciprocal-type recombination (gene conversion). Hence it is concluded that the meiotic process appears to be intimately concerned with the mechanism(s) of induction of recombination, especially reciprocal-type recombination.