CULTURE AND HYBRIDIZATION STUDIES ON GIGARTINA PAPILLATA (RHODOPHYTA)1

The foliose red alga Gigartina papillata (C. Ag.) J. Ag. was studied in culture to determine its life history and possible relationship to the life history of Petrocelis middendorffii (Ruprecht) Kjellman. Carpospores cultured from individual female plants gave rise to either crustose Petrocelis-like plants that reproduced by tetraspores, or to another generation of foliose female (cystocarpic) plants that reproduced by carpospores. Apices cultured from blades of individual field-collected female plants produced either papillae with many procarps that developed cystocarps only when crossed with male plants, or papillae with few procarps that produced cystocarps in the absence of male plants. The results are interpreted to demonstrate that two types of life history occur in G. papillata: one, a sexual life history involving a crustose tetrasporophyte; the other, a possibly apomictic life history involving only cystocarpic plants. Hybridization experiments demonstrated, that G. papillata is interfertile with Gigartina-phase gametophytes cultured from tetraspores of P. middendorffii. Sexual plants of G. papillata are postulated to represent the naturally-occurring gametophyte of P. middendorffii in California. The possible relationships of the sexual and apomictic plants of G. papillata are discussed.     

Involvement of the neurointermediate lobe of the pituitary gland in the secretion of prolactin and luteinizing hormone in the rat

The relationship between prolactin (PRL) secretion and the neurointermediate lobe (NIL) of the pituitary gland was investigated. Plasma PRL concentrations in rats bearing anterior pituitaries autografted with or without the NIL to the renal capsule were elevated to equal extents at 1 through 6 weeks after surgery (p > 0.10). PRL levels in ovariectomized rats in which the NIL had been removed surgically (NIL-X) or only visualized (NIL-C) were 3–7 ng/ml 4, 7, and 28 days after surgery (p > 0.10); however, they were slightly higher in NIL-X $\text{vs.}$ NIL-C rats 14 days after surgery (p < 0.05). Plasma luteinizing hormone (LH) concentrations in NIL-C rats increased by 36% from 2 to 4 weeks after surgery (p < 0.05); this increase was not detected in NIL-X rats. PRL and LH surges were induced by estradiol implants in ovariectomized NIL-X and NIL-C rats; the profiles of the PRL surges were superimposable, although the magnitude of the LH surge was only 50% that in NIL-C rats (p < 0.05). These results cast doubt on the importance of the NIL in the regulation of PRL secretion either $\text{via}$ secreting hypophysiotropic hormones or $\text{via}$ conducting anterior pituitary hormones directly to the median eminence. However, the NIL may have a physiologically important role in the regulation of LH secretion.     

Some characteristics of a fast movement of auxin in intact tomato seedlings (Lycopersicon esculentum)

Using exogenous ¹⁴C-IAA, a fast movement of auxin was demonstrated in intact tomato plants ( Lycopersicon esculentum Mill, cv. Craigella) cultured in vitro. This movement is insensitive to or weakly enhanced by cold temperature (0°C) and not influenced by gravity. In short time experiments (10 min) the main part of the radioactivity which travels through the plant is IAA; in long time ones (6 h) the metabolism is much higher and leads mainly to the formation of IAAsp. The integrity of the plant is necessary for the possibility to observe such a fast movement. Isolated hypocotyl segments show only the classical polar and slow movement of auxin. Microautoradiographic techniques show that the fast movement takes place inside the plant. In the epicotyl, the vascular bundles, mainly phloem cells, are labelled; in contrast, the radioactivity detected in the hypocotyl is low, and the tracers can be visualized only inside the epidermal cells.     

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.     

Spore and crystal formation inBacillus thuringiensis var.thuringiensis during growth in cystine and cysteine

The effect of the addition of different concentratons of cystine and cysteine on sporulation and parasporal crystal formation inBacillus thuringiensis var.thuringiensis was studied. The effect was well pronounced when the cystine/cysteine additions were made after the stationary phase. Heat stable spores and crystals were formed when the culture was provided with a low concentration of cystine/cysteine (0.05 per cent w/v). At a moderate concentration of cystine or cysteine (0.15%), only heat labile spores were formed without the production of the crystal. When the cystine/cysteine concentration was high (0.25%), spore and crystal formation were completely inhibited. Partial reversal of inhibition of sporulation was brought about by sodium sulphate or Zinc sulphate and lead, copper, cadmium or cobalt acetate at 0.2 mM or at 0.2% of sodium or potassium pyruvate, citrate, cisaconitate, oxalosuccinate, ∞ -keto-glutarate, succinate, fumarate, malate, or oxalacetate. Glutamate (0.2%) overcame the inhibitory effect of cystine/cysteine completely. The structural changes observed using phase contrast microscopy were dependent upon the concentration of cystine/cysteine.     

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.     

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).     

A requirement for three cell types for in vitro generation of specific secondary cell-mediated cytotoxic response against SV40-induced tumor-associated antigens in mice

The present study demonstrates that a collaborative interaction among three cell types, namely, two distinct subsets of T cells and macrophages is needed for in vitro generation of specific secondary cell-mediated cytotoxicity against syngeneic SV40-transformed cells in mice. These data suggest a central role of three cell types in the generation of efficient antitumor immune response in syngeneic tumor systems.