Elemental composition of seston and nutrient dynamics in the Sea of Marmara

The Sea of Marmara, an intercontinental basin with shallow and narrowstraits, connects the Black and Mediterranean Seas. Data obtained during1991–1996 have permitted the determination of the elementalcomposition of seston in the euphotic zone and the N:P ratio of thesubhalocline waters of the Marmara Sea. Since primary production is alwayslimited to the less saline upper layer (15–20 m), of the Marmara Sea,the subhalocline waters of Mediteranean origin are always rich in nutrients(NO₃ + NO₂ = 8–10 μm, PO₄ = 0.8–1.2 μm) but depleted in dissolvedoxygen (30–50 μm) throughout the basin, yielding an -O_{_2} : N : P ratio of 178 : 9 : 1. Pollution of the surfacewaters since the 60s has modified the subhalocline nutrient chemistryslightly. In the euphotic zone, the N : P ratio of the seston changes from5.9 to 9.5 between the less and more productive periods. Though the biologyof the Marmara has changed significantly during the previous two decades,the close relationship observed between the elemental composition of thesurface seston and the NO₃ : PO₄ ratio of thesubhalocline waters strongly suggests that during the whole year primaryproduction throughout the basin and POM export to the lower layer remainnitrogen-limited. This suggestion needs to be confirmed by bio-assays,biological studies and sediment trap data from the upper subhaloclinedepths. Nonetheless, the counterflows in the Marmara basin possessrelatively low N : P ratios in both dissolved and particulate nutrients andextend as far as the adjacent seas. This revised version was published online in July 2006 with corrections to the Cover Date.     

The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface.

The VirB4 ATPase of Agrobacterium tumefaciens, a putative component of the T-complex transport apparatus, associates with the cytoplasmic membrane independently of other products of the Ti plasmid. VirB4 was resistant to extraction from membranes of wild-type strain A348 or a Ti-plasmidless strain expressing virB4 from an IncP replicon. To evaluate the membrane topology of VirB4, a nested deletion method was used to generate a high frequency of random fusions between virB4 and 'phoA, which encodes a periplasmically active alkaline phosphatase (AP) deleted of its signal sequence. VirB4::PhoA hybrid proteins exhibiting AP activity in Escherichia coli and A. tumefaciens had junction sites that mapped to two regions, between residues 58 and 84 (region 1) and between residues 450 and 514 (region 2). Conversely, VirB4::beta-galactosidase hybrid proteins with junction sites mapping to regions 1 and 2 exhibited low beta-galactosidase activities and hybrid proteins with junction sites elsewhere exhibited high beta-galactosidase activities. Enzymatically active VirB5::PhoA hybrid proteins had junction sites that were distributed throughout the length of the protein. Proteinase K treatment of A. tumefaciens spheroplasts resulted in the disappearance of the 87-kDa VirB4 protein and the concomitant appearance of two immunoreactive species of approximately 35 and approximately 45 kDa. Taken together, our data support a model in which VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains.     

Characterization of Calpain-Mediated Proteolysis of GluR1 Subunits of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate Receptors in Rat Brain

Abstract: Previous results have indicated that GluR1 subunits of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are targets of calpain. In the present study, we determined the effects of calpain treatment of synaptic membranes on GluR1 subunits using western blots with antibodies directed against the C-terminal (C-Ab) and the N-terminal (N-Ab) domains of the proteins, and compared them with the effects of calcium treatment of frozen-thawed brain sections. Calpain treatment of synaptic membranes resulted in a large decrease in the GluR1 band (105 kDa) labeled with C-Ab and in the formation of a doublet band labeled with N-Ab due to the appearance of a new species of GluR1 (98 kDa). These effects were blocked almost completely by calpain inhibitors. Calpain-induced changes in GluR1 immunological properties were not associated with modifications of [³H]AMPA or 6-cyano-7-[³H]nitroquinoxaline-2,3-dione ([³H]CNQX) binding. Treatment of frozen-thawed brain sections with concentrations of calcium as low as 0.2 m M resulted in a large decrease in the 105-kDa GluR1 band and in the concurrent appearance of the 98-kDa band. This treatment was associated with increased [³H]AMPA and [³H]CNQX binding. These results suggest that there exist several types/states of GluR1 subunits exhibiting different sensitivities to calpain. Our data also indicate the existence of additional calcium-dependent processes regulating the characteristics of receptors in intact tissues.     

Amine fluorescamine compounds inhibit oxidative phosphorylation in rat liver mitochondria

The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism. VI. Enzymatic studies of two mutants unable to convert inosinic acid to adenylic acid

Ade^–H and ade^–I are two auxotrophic mutants of Chinese hamster ovary (CHO-K1) cells which specifically require adenine as the purine source to grow. The enzymatic defects of these mutants were examined in cell-free extracts. It was found that ade^–H did not have any detectable adenylosuccinate synthetase activity and ade^–I was defective in the adenylosuccinate lyase enzyme. The relevance of adenine-requiring mutants to the study of the regulation of purine metabolism in mammalian cells is discussed.This work was supported by research grants from the National Institute of Aging (AG00029) and the National Foundation, March of Dimes (1-423), and by a contract from the Center for Toxicological Research, Food and Drug Administration (72-213). David Patterson is a recipient of a Research Career Development Award from the National Institute of Arthritis, Metabolic and Digestive Diseases (AM00044).Contribution (No. 218) from the Eleanor Roosevelt Institute for Cancer Research.     

The transsulfuration pathway in Tetrahymena pyriformis.

Four enzymes necessary for the metabolism of methionine by the trans-sulfuration pathway, methionine adenosyltransferase (EC 2.5.1.6), adenosylhomocysteinase (EC 3.3.1.1), cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-lyase (EC 4.4.1.1) were identified in Tetrahymean pyriformis. The ability of these cells to transfer 35S from E135S]methionine to form [35S] cysteine was also observed and taken as direct evidence for the functional existence of this pathway in Tetrahymena. An intermediate in the pathway and an active methyl donor, S-adenosylmethionine, was qualitatively identified in Tetrahymena and its concentration was found to be greater in late stationary phase cells than in early stationary phase cells.     

State of water in extremely halophilic bacteria: Heat of dilution ofHalobacterium halobium

Summary Heat of dilution ofHalobacterium halobium was measured when thick pastes of the bacteria, harvested throughout a complete growth cycle, were lysed by mixing with 40 times their volume of water in a microcalorimeter. A series of comparative measurements was made with pastes of bacteria previously disrupted by freezing and thawing but otherwise identical to the pastes of whole bacteria. The frozen-thawed pastes gave endothermic values some 18% greater than those obtained with intact bacteria; the difference was highly significant. Evidence was obtained that the mechanical component of bursting did not contribute to the difference between whole and lysed bacteria. On the other hand, when a correction was applied for heat of mixing of intracellular salts with extracellular NaCl, such as occurs when the bacteria lyse, the difference between whole and disrupted organisms was largely eliminated from exponential phase halobacteria but not from those harvested in stationary phase. It is concluded that there is no evidence, as reflected in heat of dilution, of abnormal solution properties of the cytosol of young halobacteria, which are rich in potassium. On the other hand, and paradoxically, some doubt remains about stationary phase organisms whose cytosol has a much higher Na⁺ content (and Na/K⁺ ratio) than the cytosol of exponential phase bacteria.     

Conformational analysis of a snake neurotoxin by prediction from sequence, circular dichroism, and raman spectroscopy.

The secondary structure of the major neurotoxin from the sea snake Lapemis hardwickii was investigated by several methods of conformational analysis: structure prediction, circular dichroism, and laser Raman spectroscopy. From the primary structure, secondary structure prediction yielded two regions of β-sheet structure at residues 1–7 and 41–45. β-Turns were predicted at residues 14–17, 20–23, 30–33, 37–40, and 46–49. From the predictions, the toxin appears to be composed of approximately 20% β-sheet and 33% β-turn. The CD spectrum of the native toxin appears to be a hybrid of model spectra for β-sheet and β-turn proteins. The pH perturbation studies on the toxin observed by CD demonstrated that the toxin is a very stable molecule except at extremely high or low pH values. The Raman data indicated that the toxin contains both antiparallel β-sheet and β-turn structure. Using two methods of secondary structure quantitation from Raman spectra the molecule was calculated to contain 35% β-sheet from one method and 27% from the other. Overall, the various methods demonstrate that the toxin is composed of β-sheet and β-turn structure with little or no α-helix present. From the comparison of these different techniques appreciation can be gained for the necessity of several methods when identifying and quantitating secondary structure.     

Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases.

We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.