Biochemical analysis of related,independently arising histocompatibility mutants: bm17 and KB-98 enlarge the “bg series” of H-2Kb mutants

The “bg” series of MHC mutations is the most prevalent type of mutations of K^b in C57BL/6 mice screened by reciprocal tail skin grafting. The basis for identification of this series of mutations is the incompatibility of grafts between the parental B6 and the mutant. This series takes the longest to reciprocally reject the skin grafts. The series can be subdivided into “bg 1” and “bg 2” groups based on K^b-restricted recognition of virus-infected mutant target cells. The biochemical basis for these mutations are amino acid substitutions at residues 116 and 121 of the K^b transplantation antigen. These substitutions do not alter monoclonal antibody binding sites. The structural basis of MAb binding and the genetic basis of the mutation are discussed. This study was supported in part by USPHS Grants AI-07289, AI-10702, NCI P30-CA-13330, American Cancer Society Grant IM-236, and American Cancer Society Fellowship PF-2126. Stanley G. Nathenson is a member of the Irvington House Institute for Medical Research.     

Effects of Physicochemical Factors on the Adhesion to Cellulose Avicel of the Ruminal Bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes subsp. succinogenes

Ruminococcus flavefaciens adhered instantly to cellulose, while Fibrobacter succinogenes had the highest percentage of adherent cells after about 25 min of contact between bacteria and cellulose. Adhesion of R. flavefaciens was unaffected by high concentrations of sugars (5%), temperature, pH, oxygen, metabolic inhibitors, and lack of Na⁺. In contrast, the attachment was affected by the removal of divalent cations (Mg²⁺ and Ca²⁺), the presence of cellulose derivatives (methylcellulose and hydroxyethylcellulose), and cystine. Adhesion of F. succinogenes was sensitive to low and high temperatures, high concentrations of glucose and cellobiose (5%), hydroxyethylcellulose (0.1%), redox potential, pH, lack of monovalent cations, and the presence of an inhibitor of membrane ATPases or lasalocid and monensin. Cells of F. succinogenes heated at 100°C no longer were adherent. On the other hand, adhesion was insensitive to the lack of divalent cations (Mg²⁺ and Ca²⁺), the presence of 2,4-dinitrophenol, tetrachlorosalicylanilide, or inhibitors of the electron transfer chains. Adhesion of F. succinogenes seems to be related to the metabolic functions of the cell. External proteins and/or cellulases themselves might play a part in the attachment process. Several mechanisms are probably involved in the adhesion of R. flavefaciens, the main one being the interaction between the large glycocalyx and the divalent cations Ca²⁺ and Mg²⁺. Hydrophobic bonds and enzymes may also be involved.     

Sorbitol dehydrogenase genetics in the mouse: A ';null' mutant in a ';European' C57BL strain

A ';null' activity variant phenotype for sorbitol dehydrogenase (SDH) was observed in C57BL/LiA mice and used to examine the genetics of this enzyme. Linkage studies of the locus ( Sdh-1 ) with non-agouti (a) and a biochemical Iocus encoding liver L-α-hydroxyacid oxidase ( Hao-1 ) demonstrated that it is coincident with or closely linked to the structural locus, previously localized on chromosome 2. Alcohol dehydrogenase (ADH) isozymes were also examined, since the liver A₂ isozyme exhibited some activity as a sorbitol dehydrogenase on cellulose acetate zymograms. It is apparent that SDH activity is not ';essential' in this mouse strain.     

CHANGES IN EPILOBIUM PHYLLOTAXY INDUCED BY N-1-NAPHTHYLPHTHALAMIC ACID AND α-4-CHLOROPHENOXYISOBUTYRIC ACID

Preliminary studies establishing relationships between leaf plastochron index and Epilobium hirsutum L. shoot growth provide a method for rigorous selection of plants utilized in experiments designed to test the working hypothesis that endogenous auxin gradient interactions are factors of phyllotactic control in this species. Application of N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, to one of the youngest bijugate primordia on the shoot meristem results in increased growth of the treated primordium. Fasciation between the treated primordium and one of the next primordia to be initiated alters relative vertical spacing of primordia. Angular shifts between subsequent primordia result in spiral transformation of Epilobium bijugate phyllotaxy. Application of α-4-chlorophenoxyisobutyric acid (CPIB), an auxin antagonist, to one of the youngest bijugate primordia on the shoot meristem results in decreased growth of the treated primordium that alters both radial and vertical spacing of primordia. This is followed by angular shifts between subsequent primordia resulting in spiral transformation of the bijugate phyllotaxy. Changes in the growth parameters of NPA- and CPIB-treated shoots are similar. Relative plastochron rates of radial and vertical shoot growth of induced spiral shoots are about half those of lanolin paste control shoots, as are the plastochrons and relative plastochron rates of leaf elongation. Treated shoot meristems have eccentricities of 0.5 as compared to bijugate control meristem eccentricities of 0.7. No significant difference is apparent between basal transverse areas of treated and control shoot meristems. The relative chronological rates of growth of treated shoots are not significantly different from those rates of control shoots. Spiral transformation results from changes in relative positions of leaf primordia insertion on the shoot meristem, not from changes in growth of treated shoots. These changes are accompanied by an increased rate of leaf initiation on a more circular shoot meristem. Existing theoretical models of phyllotaxy are discussed in relation to these chemically induced changes of Epilobium leaf arrangement.     

Oligosaccharides accumulated in the kidney of a goat with β-mannosidosis: Mass spectrometry of intact permethylated derivatives

Two oligosaccharides accumulate in the kidney of a goat with β-mannosidosis. These oligosaccharides were isolated and purified from kidney extracts by Bio-Gel P2 gel permeation column chromatography. Their structures were characterized as Manβ1 → 4GlcNAc and Manβ1 → 4G1cNAcβ1 → 4G1cNAc by mass spectrometry of the permethylated intact oligosaccharide alcohols and permethylated native oligosaccharides. Carbohydrate composition analysis, methylation linkage studies, and enzymatic hydrolysis were also performed. Stored in 1 g of kidney were 1.6 μmol of disaccharide and 7.6 μmol of trisaccharide, which was three times that found in the brain of this affected animal (M. Z. Jones and R. A. Laine, 1981, J. Biol. Chem., 256, 5181–5184). In both the brain and kidney of the affected goat, oligosaccharide accumulation was evidently represented by membrane-bound, electron-lucent vacuoles in numerous cell types. While lesions in the brain were associated with profound neurological deficits, functional impairment of the kidney was not apparent. Similar oligosaccharides excreted in urine may be derived from those stored in the kidney. The mass spectrometric methods utilized in this investigation will facilitate comparison of oligosaccharide composition in different tissues and biological samples in β-mannosidosis and other disorders of glycoprotein catabolism.     

Regional Amino Acid Distribution in Relation to Function in Insulin Hypoglycaemia

The amino acids glutamate, aspartate, gamma-aminobutyric acid (GABA), and glutamine were measured as their dansyl derivatives in whole brain and specific brain regions by a sensitive double-labelling technique at various times during the development of hypoglycaemic encephalopathy. Hypoglycaemia was induced by administration of insulin (100 i.u./kg) to 24-h fasted rats. No significant changes in glutamate, GABA, or glutamine were detected in whole brain at any time up to and including the onset of hypoglycaemic convulsions. In cerebral cortex, however, GABA levels were reduced to 65% or normal prior to the appearance of neurological symptoms of hypoglycaemia. Onset of symptoms (severe catalepsy and loss of righting reflex, but before the onset of convulsions) was accompanied by marked decreases of glutamate and glutamine in striatum and hippocampus. These regions, in addition to cerebral cortex, show the greatest vulnerability to hypoglycaemic insult, according to previous anatomical studies. Aspartate levels were significantly increased (p less than 0.01) in the cerebral cortex of convulsing animals, confirming a previous report. No changes were detectable in any of the amino acids studied in medulla-pons at any time during the progression of hypoglycaemia. Cerebral cortex and striatum showed a selective net loss of amino acids (2.2 and 3.5 mumol/g. respectively) prior to the onset of insulin-hypoglycaemic convulsions.     

Thermal Denaturation of Native Striatal Tyrosine Hydroxylase: Increased Thermolability of the Phosphorylated Form of the Enzyme

Tyrosine hydroxylase was purified from bovine corpus striatum. The native enzyme had a half-life of 15 +/- 3 min at 50 degrees C. Phosphorylation of tyrosine hydroxylase with protein kinase purified from both corpus striatum and heart activated the enzyme, but activity was rapidly lost with additional preincubation of the enzyme at 30 degrees C. Thermal denaturation studies indicated that phosphorylated tyrosine hydroxylase had a half-life of 5 +/- 2 min at 50 degrees C     

Regulation of Adenosine-Sensitive Adenylate Cyclase from Rat Brain Striatum

An adenosine-sensitive adenylate cyclase has been characterized from rat brain striatum. In whole homogenates as well as in particulate fractions, N⁶-phenylisopropyl adenosine (PIA), 2-chloroadenosine, and adenosine N′-oxide were equipotent in stimulating adenylate cyclase. Although GTP inhibited basal as well as PIA-stimulated activity of whole homogenates, the enzyme showed an absolute dependency on GTP for stimulation by PIA, dopamine, epinephrine, and norepinephrine in a particulate fraction derived from discontinuous sucrose gradient centrifugation. Adenosine exerts two effects on this adenylate cyclase, stimulation at low concentrations and inhibition at high concentrations, suggesting the presence of two adenosine binding sites. The stimulation of adenylate cyclase by PIA was dependent on the concentration of Mg^2-. The degree of stimulation by PIA was greater at a low concentration of Mg²⁺, which suggests that stimulation by PIA was accompanied by increasing the apparent affinity for Mg²⁺. Activation of adenylate cyclase by PIA was blocked by theophylline or 3-isobutyl- 1-methylxanthine (IBMX). The pH optimum for basal or (PIA + GTP)-stimulated activities was broad, with a peak between 8.5 and 9.5. In the presence of GTP, stimulation by an optimal concentration of PIA was additive, with maximal stimulation by the catecholamines. Phospholipase A₂ treatment at a concentration of 1 U/ml for 5 min completely abolished the stimulatory effect of dopamine, whereas PIA-stimulated activity remained unaltered. These data suggest that rat brain striatum either has a single adenylate cyclase, which is stimulated by catecholamines and adenosine by distinct mechanisms, or has different cyclase populations, stimulated by either adenosine or catecholamines.     

Temperature Coefficients for the Oxidative Metabolic Responses to Electrical Stimulation in Cerebral Cortex

Temperature coefficients of both cat and toad brain have been calculated for the active metabolic state induced by electrical stimulation. Values are higher than most of the values previously reported for "rest" metabolism, whether calculated from Arrhenius plots or from linear graphs. Relative rates of oxidative metabolism were obtained by measuring the time course of the transient changes in NADH fluorescence and cytochrome aa3 absorption by reflectance techniques directly from the surface of the exposed cat cerebral cortex in vivo and from the isolated intact toad brain mounted in a cuvet. These findings demonstrate that such optical methods accurately record metabolic processes.