Light and Electron Microscopic Observations of Gametogenesis in Hastigerina pelagica (Foraminifera)*

SYNOPSIS. During gametogenesis mother individuals of Hastigerina pelagica (d'Orbigny) undergo significant morphological changes. Thirty h before gamete release, the cytoplasm changes from pale orange to bright red, possibly due to transport of stored lipids from the inner region to more peripheral parts of the cytoplasm. During the next 10 to 15 h the bubble capsule which surounds the calcareous shell is discarded. After all bubbles have disappeared, the individual sheds its spines by resorbing the spine bases close to the shell surface. A single mother nucleus divides into some hundreds of thousands of gamete nuclei within a span of ～ 20 h. A bulge of cytoplasm is extruded from the aperture and increases in size during the next 5 to 10 h. This bulge consists of cytoplasmic strands in which gametes and spherical bodies are embedded. The gametes and spherical bodies mature and are released during the afternoon and early evening. The gametes have 2 unequal acronematic flagella. A previously undescribed structure in foraminiferal reproduction is the spherical body which consists of a large vacuole surrounded by a thin cytoplasmic layer in which several nuclei, various typical cell organelles and multiple flagella are present. The spherical bodies are believed to play a role as receptacles of waste material, possibly including residual digestive enzymes, thereby protecting the gametes from lysis during the reproductive process. Fusion of gametes and further development into the next generation have not been observed.     

Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [¹²⁵I]mRNA, [³H]Met-tRNA_f, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNA_f to the 40 S ribosomal subunit. The initial Met-tRNA_f binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented.     

Concentration of iron and manganese in rice plants as influenced by hexachlorocyclohexane applied to soil

Summary Application of a granular formulation of hexachlorocyclohexane (HCH) to the potted soil at flooding decreased the concentration of iron and. to some extent, manganese in rice plants, especially at concentrations above 3 ppm active ingredient (a.i.) Likewise, HCH, applied to rice fields at transplanting (several days after submergence) caused a significant decrease in the concentration of iron, and not manganese, in the rice plant but only at concentrations above 12.5 kg a.i./ha despite high levels of reduced iron in the soil. Inhibition of iron reduction by HCH was more pronounced when applied at flooding than at several days after flooding.     

The nature of formate metabolism in greening barley leaves

The metabolism of [³H]formate has been examined in etiolated and greening leaves of barley (Hordeum vulgare), dwarf bean (Phaseolus vulgarls), broad bean (Vicia faba) and corn (Zea mays). Tritium was extensively incorporated by primary leaves incubated for 20-min periods in light or dark. The organic acids and free amino acids were the principal products of formate metabolism but these and other products were more heavily labelled in green tissues. Time course experiments with barley leaves revealed a rapid labelling of serine, accompanied by increasing amounts of ³H in glycine and aspartate as the feeding period was extended. These amino acid products were formed throughout a 4-day greening period with an approximate doubling in total incorporation being due to large accumulations of tritiated glycine and aspartate. The involvement of tetrahydrofolate-dependent reactions in formate metabolism was indicated by inhibition of [¹⁴C] and [³H]formate incorporation by the folate antagonist, aminopterin. Labelling of glycine and serine was also strongly inhibited (up to 90%) when the leaves were incubated with increasing concentrations of isonicotinylhydrazide.     

A simple and sensitive colorimetric method for the determination of long-chain free fatty acids in subcellular organelles

A colorimetric assay for the determination of long-chain free fatty acids (FFA) is described. The FFA were extracted from subcellular organelles with chloroform:heptane:methanol. The copper soaps of FFA were determined colorimetrically with diphenylcarbazide. There are three advantages to employing the present modified procedure. (a) The sensitivity has been increased approximately twofold over that of the previous procedure of K. Falholt, B. Lund, and W. Falholt (1973, Clin. Chim. Acta46, 105–111); (b) it takes less time to complete the assay compared to the tedious procedures currently available; and (c) the presence of bovine serum albumin, a known FAA-binding protein, does not interfere with the assay procedure. The assay shows a linear response over the range of 10 to 130 nmol of FFA. The recovery of free fatty acids from mitochondria is 99%.     

Purification of lipoamide dehydrogenase from Ascaris muscle mitochondria and its relationship to NADH:NAD+ transhydrogenase activity

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3) has been isolated from Ascaris suum muscle mitochondria. This activity has been purified to apparent homogeneity from both the pyruvate dehydrogenase complex and from 150,000g mitochondrial supernatants which were devoid of pyruvate dehydrogenase complex activity. The enzymes from both sources exhibited similar kinetic, catalytic, and regulatory properties and appear to be identical as judged by polyacrylamide gel electrophoresis. The native enzyme acts as a dimer, containing 2 mol of FAD, and has a subunit molecular weight of 54,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel chromatography. The enzyme also possesses substantial NADH:NAD⁺ transhydrogenase activity. Heat denaturation and differential solubilization experiments imply that the transhydrogenase activity previously reported is, in fact, associated with the lipoamide dehydrogenase moiety of the Ascaris pyruvate dehydrogenase complex. Whether or not this activity functions physiologically in hydride ion translocation, as previously suggested, remains to be demonstrated.     

Fluence response relationship of carotenogenesis inNeurospora crassa

The fluence response of the blue light induced carotenoid synthesis inNeurospora is biphasic. Using fluence rates between 0.3 and 40 Wm^-2, increasing illumination times beyond 16 min (at 20°C) result in a second rise of the amount of carotenoids synthesized in the subsequent dark period. On altering the temperature, the transition point to the second phase of the response is shifted to shorter/longer illumination periods with increasing/decreasing temperature, respectively. The transition point can also be shifted by administering high fluence rates of near UV light: The start of the second phase is already triggered after an irradiation time of 2 min. The findings suggest that elements of the transduction sequence become depleted and senstivity recovers in a temperature-dependent process. The biphasic response and the effects of UV light are discussed in relation to the transduction mechanism and to the ecological significance.Presented in part at the meeting of the Deutsche Botanische Gesellschaft, September 1978, Marburg, Federal Republic of Germany     

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide (E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta253, 489–497) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. (A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J.173, 597–605) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem.253, 8702–8707).     

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.