CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : II. Photosynthetic Electron Transport

Photosynthetic electron transport is markedly affected in mixotrophic cells of ac-20 because they lack the capacity to form the wild-type level of cytochrome 559, as well as Q, the quencher of fluorescence of photochemical system II. The other components of the electron-transport chain, as well as reactions dependent upon photochemical system I, are unaffected in the mutant strain. These observations are discussed in terms of the previously reported effects of the ac-20 mutation on CO₂ fixation and ribulose-1,5-diphosphate carboxylase activity.     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : I. CO2 Fixation and Ribulose-1,5-Diphosphate Carboxylase Synthesis

A mutant strain of the green alga Chlamydomonas reinhardi, ac-20, is described in which both the rate of CO₂ fixation by whole cells and the rate of carboxylation of ribulose-1,5-diphosphate in cell-free extracts are reduced, particularly when sodium acetate is present in the growth medium. Of the enzymes of the reductive pentose phosphate cycle tested, only ribulose-1,5-diphosphate carboxylase activity is reduced in the mutant strain, and it appears that the low carboxylase activity limits the strain's rate of photosynthetic carbon metabolism. Evidence is presented to show that the fluctuation in the level of the enzyme activity in the presence or absence of acetate results from the fluctuation in the level of some factor(s) limiting the rate of synthesis of the protein.     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : III. Chloroplast Ribosomes and Membrane Organization

The fine structure of the ac-20 strain of Chlamydomonas reinhardi is described. Cells grown mixotrophically in the presence of acetate have a highly disordered chloroplast membrane organization and usually lack pyrenoids. Chloroplast ribosome levels are only 5–10% of wild-type levels. Cells grown phototrophically without acetate possess more chloroplast ribosomes and have more normal membrane and pyrenoid organization. Chloroplast ribosome levels rise rapidly when cells are transferred from acetate to minimal medium, whereas membrane reorganization occurs only after a lag. These results, combined with earlier studies of the photosynthetic properties of the mutant strain, suggest that proper membrane organization, Photosystem II activity, and ribulose-1,5-diphosphate carboxylase formation are dependent on the presence of chloroplast ribosomes. Other chloroplast components tested are unaffected by a 10-fold reduction in levels of chloroplast ribosomes.     

TRANSFER OF PROTEINS FROM THE CHLOROPLAST TO VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): A PATHWAY FOR DEGRADATION

Several chloroplast proteins were detected by immunoelectron microscopy within dense granules in cytoplasmic vacuoles in the alga Chlamydomonas reinhardtii Dangeard. Transfer from chloroplast to vacuoles of two major, pulse-labeled polypeptides, the large subunit of rubisco and the α subunit of ATPase, which are synthesized on chloroplast ribosomes, was demonstrated by the recovery of these polypeptides in vacuolar granules over a several-hour time period. The ultrastructure of cryofixed algal cells was examined to search for structures that would provide insight into the transfer of chloroplast proteins to vacuoles. Micrographs showed that the two membranes of the envelope were appressed, with no detectable intermembrane space, over most of the chloroplast surface. Protrusions of the outer membrane of the envelope were occasionally found that enclosed stroma, with particles similar in size to chloroplast ribosomes, but generally not thylakoid membranes. These observations suggest that chloroplast material, especially the stromal phase, was extruded from the chloroplast in membrane-bound structures, which then interacted with Golgi-derived vesicles for degradation of the contents by typical lysosomal activities. A protein normally targeted to vacuoles through the endomembrane system for incorporation into the cell wall was detected in Golgi structures and vacuolar granules but not the chloroplast.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : V. A Radioautographic Study of Membrane Growth in a Mutant of Chlamydomonas reinhardi y-1

The development of photosynthetic lamellae during greening of dark-grown Chlamydomonas y-1 cells was investigated by radioautography. Acetate-³H was used as a marker for membrane lipids. In short pulse-labeling experiments, about 50–60% of the radioactivity incorporated was found in the lipid fraction and about 25–50% in starch granules present in the chloroplast of these algae. The relative specificity of acetate-³H used as a marker for membranes was artificially increased through quantitative removal of the starch granules from fixed cells by amylase treatment. Analysis of turnover coefficients of different membrane constituents and of the contribution of turnover and net synthesis to the total label incorporated in pulse experiments indicated that the incorporation of acetate into specific lipids was mainly due to net synthesis. The distribution of radioactivity in the different lipid constituents at the end of a short pulse and after 30- and 60-min chases indicated that transacylation is minimal and may be disregarded as a possible cause of randomization of the label. Statistical analysis of radioautographic grain distribution and measurements of different structural parameters indicate that (a) the chloroplast volume and surface remain constant during the process, whereas the growth of the photosynthetic lamellae parallels the increase in chlorophyll; (b) the lamellae do not develop from the chloroplast envelope or from the tubular system of the pyrenoid; (c) all the lamellae grow by incorporation of new material within preexisting structures; (d) different types of lamellae grow at different rates. The pyrenoid tubular system develops faster than the thylakoids, and single thylakoids develop about twice as fast as those which are paired or fused to grana. It is concluded that growth of the membranes occurs by a mechanism of random intussusception of molecular complexes within different types of preexisting membranes.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : IV. Lipid and Pigment Changes during Synthesis of Chloroplast Membranes in a Mutant of Chlamydomonas reinhardi y-1

The glycolipid, phospholipid, pigment, and fatty acid content in whole y-1 cells during the greening process have been investigated. The time course of their changes indicates that phosphatidyl glycerol and glycolipids are the main lipids synthesized specifically during illumination of dark-grown cells, concomitant with an increase in the polyunsaturated C18:2 and C18:3 fatty acids. The pigment complex of light-grown cells consists mainly of chlorophylls a and b, lutein, β-carotene, violaxanthin, and neoxanthin. During the greening process, chlorophylls a and b are synthesized in constant proportions (ratio a/b equals 2.6), β-carotene and violaxanthin do not change significantly, and lutein and neoxanthin increase. The molar ratios of the different lipids and pigment to total chlorophyll during greening has been calculated. It was found that during the initial phase of greening when chlorophyll is synthesized at increasing rates, the molar ratios of various lipids and pigments to chlorophyll decrease and tend to become constant when chlorophyll and membrane synthesis proceed at constant rates. The implication of these findings with respect to the concept of membrane assembly through a spontaneous single step process is discussed     

CHLOROPLAST STRUCTURE OF THE CRYPTOPHYCEAE : Evidence for Phycobiliproteins within Intrathylakoidal Spaces

Selective extraction and morphological evidence indicate that the phycobiliproteins in three Cryptophyceaen algae (Chroomonas, Rhodomonas, and Cryptomonas) are contained within intrathylakoidal spaces and are not on the stromal side of the lamellae as in the red and blue-green algae. Furthermore, no discrete phycobilisome-type aggregates have thus far been observed in the Cryptophyceae. Structurally, although not necessarily functionally, this is a radical difference. The width of the intrathylakoidal spaces can vary but is generally about 200–300 A. While the thylakoid membranes are usually closely aligned, grana-type fusions do not occur. In Chroomonas these membranes evidence an extensive periodic display with a spacing on the order of 140–160 A. This periodicity is restricted to the membranes and has not been observed in the electron-opaque intrathylakoidal matrix.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : II. Plastid Differentiation during Greening of a Dark-Grown Algal Mutant (Chlamydomonas reinhardi)

Dark-grown cells of the y-1 mutant of Chlamydomonas reinhardi contain a partially differentiated plastid lacking the photosynthetic lamellar system. When exposed to the light, a rapid synthesis of photosynthetic membranes occurs accompanied by synthesis of chlorophyll, lipids, and protein and extensive degradation of the starch reserve. The process is continuously dependent on illumination and is completed within 6–8 hr in the absence of cell division. Photosynthetic activity (O₂ evolution, Hill reaction, NADP photo-reduction, and cytochrome f photooxidation) parallels the synthesis of pigment and membrane formation. During the greening process, only slight changes occur in the levels of soluble enzymes associated with the photosynthetic process (RuDP-carboxylase, NADP-linked G-3-P dehydrogenase, alkaline FDPase (pH 8)) as compared with the dark control. Also cytochrome f concentration remains almost constant during the greening process. The kinetics of the synthesis of chlorophyll, formation of photosynthetic membranes, and the restoration of photosynthetic activity suggest that the membranes are assembled from their constituents in a single-step process.     

SUBSTRUCTURE OF AMPHIBIAN MOTOR END PLATE : Evidence for a Granular Component Projecting from the Outer Surface of the Receptive Membrane

End-plate membrane has been examined at amphibian myoneural junctions by means of transmission electron microscopy of thin tissue sections. The postjunctional membrane exhibits morphologically specialized dense, convex patches which are located superficially facing the axon terminal but do not extend into the depths of the junctional folds. In the specialized regions the plasma membrane is ~ 120 Å thick and trilaminar. The outer dense lamina is thickened by the presence in it of granular elements ~60–120 Å in diameter which are spaced semiregularly at ~100–150-Å intervals and which border the junctional cleft directly. In these regions the concentration of the granules is of the order of ~ 10⁴/µm², which is in the same range as the estimated concentration of receptor sites at other vertebrate cholinergic junctions. Filamentous projections can sometimes be seen extending from the granules to the overlying basement membrane, and in oblique views a reticular pattern may appear both in these patches and in the basement membrane. The cytoplasmic surface of the specialized membrane is covered with an amorphous and filamentous dense material whose distribution coincides with that of the granules visible in the outer layer and which may be connected to them across the membrane. In unosmicated specimens stained with permanganate and uranyl acetate the specialized regions exhibit the same morphological features but stand out sharply in contrast to adjacent areas of unspecialized membrane which appear only faintly. Such preparations are particularly useful in assessing the extent of the specialized membrane. It is proposed that the granules visible at the outer surface of the end-plate membrane represent acetylcholine receptors and that in amphibians, as in annelids, the receptors at myoneural junctions are concentrated into patches which occupy less than the total postjunctional membrane surface area.     

Regulation of Penicillinase Synthesis: Evidence for a Unified Model

The kinetics of penicillinase induction in Bacillus cereus 569 was investigated. An increase in the rate of penicillinase synthesis was demonstrated within 30 sec of the addition of inducer (benzylpenicillin); however, the maximum induced rate of penicillinase synthesis was not attained until at least 30 min after the addition of inducer. In contrast to earlier claims, a quantitative estimate showed that the penicillinase messenger ribonucleic acid (mRNA) half-life is approximately 2 min. These findings strongly suggest that the rate of synthesis of penicillinase mRNA increases continuously during most of the 30-min latent period. A model for the regulation of penicillinase synthesis in three gram-positive organisms is presented which is consistent with a nondiffusible inducer, a short-lived mRNA, a relatively long latent period (i.e., an apparently slow inactivation of penicillinase repressor), and the existence of at least two regulatory genes.     

BIOGENESIS OF ENDOPLASMIC RETICULUM MEMBRANES : II. Synthesis of Constitutive Microsomal Enzymes in Developing Rat Hepatocyte

The constitutive enzymes of microsomal membranes were investigated during a period of rapid ER development (from 3 days before to 8 days after birth) in rat hepatocytes. The activities studied (electron transport enzymes and phosphatases) appear at different times and increase at different rates. The increase in the enzyme activities tested was inhibited by Actinomycin D and puromycin. G-6-Pase and NADPH-cytochrome c reductase activities appeared first in the rough microsomes, and subsequently in smooth microsomes, eventually reaching a uniform concentration as in adult liver. The evidence suggests that the enzymes are synthesized in the rough part, then transferred to the smooth part, of the ER. Changes in the fat supplement of the maternal diet brought about changes in the fatty acid composition of microsomal phospholipids but did not influence the enzymic pattern of the suckling. Microsomes from 8-day-old and adult rats lose 95% of PLP and 80% of NADH-cytochrome c reductase activity after acetone-H₂O (10:1) extraction. However, one-half the original activity could be regained by adding back phospholipid micelles prepared from purified phospholipid, or from lipid extracts of heart mitochondria, or of liver microsomes of 8-day or adult rats, thus demonstrating an activation of the enzyme by nonspecific phospholipid. The results suggest that during development the enzymic pattern is not influenced by the fatty acid or phospholipid composition of ER membranes.     

Regulation of Nitrogen Fixation in Klebsiella pneumoniae: Evidence for a Role of Glutamine Synthetase as a Regulator of Nitrogenase Synthesis

Mutations causing constitutive synthesis of glutamine synthetase (GlnC(-) phenotype) were transferred from Klebsiella aerogenes into Klebsiella pneumoniae by P1-mediated transduction. Such GlnC(-) strains of K. pneumoniae have constitutive levels of glutamine synthetase. Two of three GlnC(-) strains of K. pneumoniae studied, each containing independently isolated mutations that confer the GlnC(-) phenotype, continue to synthesize nitrogenase in the presence of NH(4) (+). One strain, KP5069, produces 30% as much nitrogenase when grown in the presence of 15 mM NH(4) (+) as in its absence. The GlnC(-) phenotype allows the synthesis of nitrogenase to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain. Glutamine auxotrophs of K. pneumoniae, that do not produce catalytically active glutamine synthetase, are unable to synthesize nitrogenase during nitrogen limited growth. Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F'133) simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase. These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae.     

A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization.     

Natural Selection as a Driver for the Genetic Component of Preeclampsia

Molecular Biology - Preeclampsia (PE) is a severe hypertensive pathology and affects 2–8% of pregnancies worldwide. Its etiopathogenesis is poorly understood, and prognostic biomarkers and...     

Direct Evidence for the Presence of Germ Cell Determinant in Vegetal Pole Cytoplasm of Xenopus laevis and in a Subcellular Fraction of It

To test for the presence of germ cell determinant in Xenopus embryos, vegetal pole cytoplasm containing the "germ plasm", or a subcellular fraction of it, was microinjected into single somatic blastomeres isolated from 32-cell embryos. Injected or non-injected (control) blastomeres were cultured in ³H-thymidine until normal control embryos reached the neurula stage. The labeled explants were then implanted into unlabeled host neurulae, which were allowed to develop to the tadpole stage. Labeled PGCs of explant origin in the genital ridges of the experimental tadpoles were examined by autoradiography.
Isolated blastomeres were injected with vegetal pole cytoplasm of 32-cell embryos or with a 20,000 g pellet made from vegetal pole cytoplasm of 2-cell embryos. Labeled PGCs were found in 7.6% and 2.3% of the experimental tadpoles, respectively. No labeled PGCs were found in the control tadpoles, except for one tadpole in the first experiment. These results strongly suggest that the vegetal pole cytoplasm and its subcellular fractions act as germ cell determinant.     

Mechanism of Folate Transport in Lactobacillus casei: Evidence for a Component Shared with the Thiamine and Biotin Transport Systems

Lactobacillus casei cells have been shown previously to utilize two separate binding proteins for the transport of folate and thiamine. Folate transport, however, was found to be strongly inhibited by thiamine in spite of the fact that the folate-binding protein has no measurable affinity for thiamine. This inhibition, which did not fluctuate with intracellular adenosine triphosphate levels, occurred only in cells containing functional transport systems for both vitamins and was noncompetitive with folate but competitive with respect to the level of folate-binding protein. Folate uptake in cells containing optimally induced transport systems for both vitamins was inhibited by thiamine (1 to 10 muM) to a maximum of 45%; the latter value increased to 77% in cells that contained a progressively diminished folate transport system and a normal thiamine system. Cells preloaded with thiamine could transport folate at a normal rate, indicating that the inhibition resulted from the entry of thiamine rather than from its presence in the cell. In a similar fashion, folate (1 to 10 muM) did not interfere with the binding of thiamine to its transport protein, but inhibited thiamine transport (to a maximum of 25%). Competition also extended to biotin, whose transport was strongly inhibited (58% and 73%, respectively) by the simultaneous uptake of either folate or thiamine; biotin, however, had only a minimal effect on either folate or thiamine transport. The nicotinate transport system was unaffected by co-transport with folate, thiamine, or biotin. These results are consistent with the hypothesis that the folate, thiamine, and biotin transport systems of L. casei each function via a specific binding protein, and that they require, in addition, a common component present in limiting amounts per cell. The latter may be a protein required for the coupling of energy to these transport processes.     

A GREEN DINOFLAGELLATE WITH CHLOROPHYLLS a and b: MORPHOLOGY,FINE STRUCTURE OF THE CHLOROPLAST AND CHLOROPHYLL COMPOSITION1

A green-colored marine unicell has been grown in unialgal culture and its morphology, chloroplast fine structure, and chlorophyll composition investigated. The organism is typical of dinoflagellates in its shape, flagellation, nucleus, mitochondria, and trichocysts. It is similar to Gymnodinium but possesses fine body scales. Chloroplasts and two kinds of vesicles bounded by double membranes, but no organelles obviously identifiable as nuclei or mitochondria, are associated in ribosome-dense cytoplasm separated by a double membrane from the dinophycean cytoplasm. The chloroplasts are unlike any previously reported for dinoflagellates. Each is enclosed by an envelope consisting of a double membrane. Chloroplast lamellae consist of three appressed thylakoids. Interlamellar pyrenoids are present. Pigment analysis reveals chlorophylls a and b but not chlorophyll c. It seems likely that the organism is an undescribed dinoflagellate containing an endosymbiont with chlorophylls a and b and that the reduction of the endosymbiont nucleus and mitochondria has permitted a more initmate symbiosis.