Structural and functional variability among DQ β alleles of DR2 subtypes

Homozygous lymphoblastoid cell lines representing various Dw subtypes of DR2 were examined for polymorphism at the DQ locus by molecular and cellular techniques. The subtypes studied included Dw2, Dw12, and a group heterogenous by cellular typing that we shall refer to as non-Dw2/non-Dw12. Restriction fragment length polymorphism analysis of cell lines representing these subtypes revealed DQ -specific patterns consistent with cellular typing. Two-dimensional gel electrophoresis of DQ molecules from representative cell lines revealed a structural polymorphism of DQ among the three subtypes. The DQ chain migrated to a position that was unique to each subtype and was consistent among various representative cell lines of each subtype. Nucleotide sequence analysis of cDNA clones of DQ from Dw2, Dw12, and non-Dw2/non-Dw12 lines confirmed that the variability resided at the genetic level. Variability was found in the form of numerous scattered nucleotide substitutions throughout the first domain of these alleles. The DQ gene of the non-Dw2/non-Dw12 cell line AZH was further found to be almost identical with the DQ gene of a DR1 line (Bell et al. 1985b), implicating a common evolutionary origin of these alleles. The only difference between these two sequences was due to an apparent gene conversion event at amino acid 57. T-cell cloning experiments resulted in the derivation of Epstein-Barr virus-specific, DQw1-restricted clones that proliferated against only those cell lines that exhibited the DQ gene common to AZH and the DR1 cell line. Thus, the polymorphism among DQ alleles within DR2 results in subtype-specific restriction.     

Blidingia ramifera stat. nov. (Chlorophyta): a new marine alga for eastern North America

Blidingia minima var. ramifera is reported for the first time in eastern North America. It occurs in the Gulf of St. Lawrence, Nova Scotia and in Maine. In the estuary of the West and Rights Rivers (Antigonish Harbour, Nova Scotia) it is the most common intertidal alga and during its maximum growth period (June-August) covers 75–90% of the intertidal zone for several km of shoreline at the mouth of the Rights River. In culture, spore germination and early development were typical of the taxon as described from Europe. The taxon is raised to specific status as Blidingia ramifera stat. nov. Blidingia subsalsa is confirmed from New England based on observations of spore germination in plants from Maine and Connecticut.     

N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I-MIL also displays a high affinity for serotonin 5-HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I-MIL exhibits at least 60-fold higher affinity for serotonin 5-HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I-MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0 degree C. Autoradiographic experiments confirm the improved specificity of 125I-MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I-MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I-MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.     

In support of the trap hypothesis. Chymotrypsin is not rigidly held in its complex with human alpha 2-macroglobulin

Complexes (2:1) of chymotrypsin with human alpha 2-macroglobulin have been prepared in the presence of 200 mM methylamine such that 90% of the chymotrypsin remains noncovalently bound to the alpha 2-macroglobulin. Reaction of this complex with the active-site-directed spin-labeling reagent 4-[(ethoxyfluorophosphinyl)oxy]-2,2,6,6-tetramethylpiperidinyl+ ++-1-oxy results in nitroxide labeling of the active-site serine residue of the complexed chymotrypsin. Electron spin resonance (ESR) spectra of this complex were recorded at 275 K in buffer and at 263 K in 50% glycerol. At 263 K in 50% glycerol the spectrum is that expected for a rigid glass, whereas at room temperature the ESR spectrum shows that the chymotrypsin is only slightly immobilized compared with free spin-labeled chymotrypsin. These findings are discussed in relation to possible models of inhibition of protease activity by alpha 2-macroglobulin. It is concluded that the trap mechanism of Barrett and Starkey [Barrett, A. J., & Starkey, P. M. (1973) Biochem. J. 133, 709-724] is the only model currently considered that can account for the present findings.     

Models of proteolysis of oligomeric enzymes and their applications to the trypsinolysis of citrate synthases.

A simple statistical approach was used to generate predictive models of the proteolysis of multisubunit enzymes in order to correlate the loss of enzyme activity with the loss of native subunit. The models were applied to the trypsinolysis of the citrate synthases of pig heart, Bacillus megaterium and Escherichia coli. With the dimeric citrate synthases (pig heart and B. megaterium) trypsinolysis of one of the subunits appears to destroy the activity of the whole enzymic molecule. The hexameric E. coli citrate synthase behaves like a trimer of dimeric units, each of the dimers behaving similarly to the B. megaterium and pig heart enzymes. Palmitoyl-CoA is required for the trypsinolysis of pig heart citrate synthase, and at relatively high concentrations of this compound trypsinolysis of one subunit leaves the other subunit fully active. Palmitoyl-CoA is not required for the trypsinolysis of the other citrate synthases, and high concentrations of this metabolite do not affect the correlation of proteolysis with inactivation of these enzymes.     

Depletion of Brain Glutathione in Preweanling Mice by L-Buthionine Sulfoximine

Previous studies indicated that DL-buthionine sulfoximine (DL-BSO), an agent that inhibits the biosynthesis of GSH in liver and other peripheral organs, fails to suppress levels of GSH in the CNS. In the current study, preweanling mice responded to repeated injections of L-BSO with marked declines (79.6-86.5%) of GSH content in brain and spinal cord. In adult mice, the same treatment schedule produced only modest declines (17.8-29.2%) of GSH content in brain and a 55.9% decline in spinal cord. Pretreatment of preweanling mice with L-BSO represents a tool for studying the role of GSH in the CNS.     

The effect of dissolved oxygen on the growth of young-of-the-year winter flounder,Pseudopleuronectes americanus

Synopsis The effects of constant and diurnally fluctuating levels of dissolved oxygen on the growth of young-of-the-year winter flounder,Pseudopleuronectes americanus, were examined under controlled laboratory conditions. Fish were exposed for either 10 or 11 weeks to constant levels of 6.7 (high) and 2.2 (low) mg l^–1, and a diurnal fluctuation, ranging from 2.5 to 6.4 mg 0₂l^–1. Growth rates, calculated for both standard length and weight, for fish exposed to low and diurnally fluctuating levels were significantly reduced (p < 0.001) as compared to those for fish exposed to the high level. Growth rates of fish exposed to the high level were over twice those of fish held under low oxygen conditions. Under fluctuating conditions, fish grew at intermediate rates. Following these exposures, all fish were subsequently held at 7.2 mg Oz l^–1 for five weeks. Growth rates increased over two and a half times for fish previously exposed to the low oxygen level and were significantly (p < 0.001) higher than for the other two groups.     

Occurrence of two distinct succinate thiokinases in animal tissues

Although succinate thiokinase from mammalian sources has hitherto been described as showing substrate specificity for guanine nucleotide, a range of mammalian tissues has here been found to display succinate thiokinase activity with both guanine and adenine nucleotides as substrates. Evidence is presented for the existence of two distinct succinate thiokinases and this is confirmed by their separation by affinity chromatography. Each enzyme is specific for one nucleotide and is inhibited by the non-substrate nucleotide. The physiological roles of the two enzymes is yet to be established.