A mutational hot-spot within an intron of the mouse beta 2-microglobulin gene.

beta 2-Microglobulin is the smaller, relatively non-polymorphic chain of class I major histocompatibility complex proteins. We have previously described a mutant mouse cell line which had been selected for loss of the class I thymus leukemia (TL) antigen and had concomitantly lost surface expression of H-2k antigens. Expression of class I antigens on the cell surface was restored by fusion to an antigenically distinct mouse lymphoma line, and the defect in the mutant was shown to be the loss of a functional beta 2-microglobulin gene. We now describe three additional mutants with the same phenotype, all selected for loss of TL but after different types of mutagenesis. All of these mutants have genomic rearrangements resulting in the absence of a functional beta 2-microglobulin gene. These data provide strong evidence for the requirement of beta 2-microglobulin for cell surface expression of the heavy chain of class I major histocompatibility complex proteins. We further show that the defects in at least one beta 2-microglobulin gene in each mutant cell line map to the same small DNA segment within the first intron. The breakpoints of these mutations define a hypermutable site within the mouse beta 2-microglobulin gene.     

The structure of manganese superoxide dismutase from Thermus thermophilus HB8 at 2.4-A resolution

An atomic model of tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been built into an electron density map at 2.4 A resolution, using chemical sequences of Mn dismutases from Thermus aquaticus and Bacillus stearothermophilus. The monomer fold is structurally very similar to the fold of iron dismutase and comprises two domains, each contributing two ligands to the metal. The Mn(III) ion is bound by protein ligands assigned as His 28, His 83, Asp 165, and His 169. Near neighbors in the metal-ligand environment include a series of hydrophobic residues, Phe 86, Trp 87, Trp 131, and Trp 167. The hydroxyl groups of two Tyr residues, at 36 and 182, are less than 7 A from the metal, as is His 32. Gln 150 forms a bridge between Tyr 36 and Trp 131. These ligands and nearby residues are strongly conserved in the known sequences of Mn dismutases. Only one of the two oxygens of Asp 165 has been assigned as a metal ligand, so that in the current model four protein atoms bind Mn(III). These ligand atoms form part of an approximate trigonal bipyramid in which water may occupy an axial position on the side opposite His 28. The conformation of the protein is unusual in the vicinity of the first ligand, His 28, as a consequence of the insertion of an extra residue in an alpha-helix. The distortion of the helix allows His 32 to stack against the ligand, His 169, and brings Tyr 36 close to the Mn ion. Across one of the dimer interfaces, the two Mn ions are separated by about 18 A, and active center residues from adjoining subunits interdigitate; Tyr 172 interacts with His 32 of the neighboring chain and Glu 168 with the backbone of 168 and with the ligand His 169 from the opposite subunit. Only one other dimer interface occurs in the tetramer; it involves residues 55-62 and sequences near 140 and 156. The center of the oligomeric molecule is filled with solvent.     

Requirement for direct association of ammonia-excreting Anabaena variabilis mutant (SA-1) with roots for maximal growth and yield of wheat

Direct association between wheat roots and an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis, strain SA-1, was required for maximal enhancement of growth of wheat plants in nitrogen (N)-free, hydroponic medium. Over 85% of the cyanobacterial mutant SA-1 inoculated to the roots were adsorbed under non-saturating conditions. The adsorption process of SA-1 to wheat roots was biphasic: an initial rapid adsorption was followed by a slow phase with about 10% of the initial adsorption rate. The maximal adsorption rate of filaments observed was 1.6 mg dry wt. SA-1 adsorbed·plant^–1·h^–1. Bypassing CO₂ fixation and sugar formation, the ¹⁴C label from [¹⁴C]sucrose was directly applied to leaf blades to study sugar translocation. The ¹⁴C label from this treatment appeared in the wheat culture medium within an hour. Nitrate-grown plants excreted about 30% of the ¹⁴C label into the medium, compared to only 10% excreted by wheat/Anabaena co-cultures. SA-1 assimilated 27% of all ¹⁴C translocated from [U-¹⁴C]sucrose applied to wheat leaves, and ¹⁴C label from this treatment was recovered from strain SA-1 after 30 min. Roots and cyanobacteria accounted for 51% of all radioactive label recovered in the plants co-cultured with SA-1 vs 20% for nitrate-grown plants. We studied the activity of -fructosidase (invertase) in wheat of variety Yecora rojo. Roots from nitrate-grown wheat plants produced high levels of invertase activity, which converted over 85% of 3 mm sucrose into glucose and fructose in 24 h. The rate of sucrose disappearance in the medium of co-cultures using A. variabilis SA-1, was 70% of that of nitrate-grown plants, but the levels of glucose and fructose in these cultures were always very low during sucrose conversion, suggesting hexose assimilation. To study the role of diffusible metabolites, a dialysis membrane was employed to separate the ammonia-excreting SA-1 from the wheat roots. Containing SA-1 in a dialysis bag away from direct root contact, severely limited leaf growth to less than one-third of the growth rate of nitrate control cultures. Ammonia produced by mutant SA-1 in dialysis bag cultures was excreted into the medium at 0.4 mm vs 1.2 mm in free-living cultures, but ammonia was not detectable in co-cultures with or without the dialysis bag containing the mutant. The nitrogenase activity derepressed in the mutant and responsible for ammonia excretion was always higher in the association co-cultures than in either free cells or in dialysis-bag cultures. The nitrogenase activity of strain SA-1 was highest (200 mol ethylene formed·mg^–1 Chl·h^–1) when the cyanobacterium was associated with the root tips. Dialysis membrane separation of plant and cyanobacterium severely inhibited growth of wheat during a complete growth cycle of 2 months. Total biomass and grain yield were very similar for control cultures without inorganic N or SA-1, and for diffusion cultures containing SA-1, kept in a dialysis bag around the roots. Total biomass of the association co-culture attained 75% of the biomass of the nitrate-grown control. It is proposed that wheat roots supplied fructose derived from sucrose for growth and nitrogen fixation of SA-1 in the light, and that ammonia excreted by SA-1 was utilized by the wheat plant for its own growth. Correspondence to: H. Spiller     

Identification and regional localization of a human IMPdehydrogenase-like locus (IMPDHL1) at 16p13.13

Sequence-tagged sites (STSs) are versatile chromosomalmarkers for a variety of genome mapping efforts. In this report, we describe a randomly generated STS (323F4) from human chromosome 16 genomic DNA that has 90.0% sequence identity to the type I human inosine-5′-monophosphate dehydrogenase (IMPDH1) gene and 72% identity to the type II human inosine-5′-monophosphate dehydrogenase (IMPDH2) gene. Additional sequencing by primer walking has provided a total of 1380 by of the human chromosome 16 sequence. The IMPDH-like sequence 323F4 was regionally localized by PCR analysis of a panel of somatic cell hybrids containing different portions of human chromosome 16 to 16p13.3-13.12, between the breakpoints found in hybrids CY196/CY197 and CY198. This regional mapping assignment was further refined to subband 16p13.13 by high-resolution fluorescence in situ hybridization using cosmid 323F4 as a probe. We conclude that a third, previously undescribed IMPDH locus, termed IMPDHL1, exists at human chromosome 16p13.13.     

Manganese and iron superoxide dismutases are structural homologs

The crystal structure of a tetrameric manganese superoxide dismutase from a thermophilic bacterium, Thermus thermophilus HB8, has been determined at 4.4-A resolution by local averaging of electron density maps calculated by isomorphous replacement. The spatial arrangement of the principal secondary structural features of iron superoxide dismutase is conserved in manganese dismutase. The structural homology is displayed by orienting the polypeptide chain of Escherichia coli Fe dismutase in the electron density map of Mn dismutase. Densities corresponding to bound Mn3+ occur at locations equivalent to the Fe3+ positions in iron dismutase, indicating one metal binding site per chain, or four sites per tetramer. The Mn tetramer, with 222 symmetry, is approximately rectangular in shape and appears to be constructed with only two unique interfaces. One set of interchain contacts closely resembles the dimer interface of Fe dismutase, but the other interface utilizes an inserted polypeptide segment that has no equivalent in Fe dismutase.     

Crystallographic studies of Escherichia coli citrate synthase

The citrate synthase from Escherichia coli B has been crystallized in a cubic space group with a unit cell spacing of 220 A. X-ray diffraction, electron microscopy, symmetry considerations, and low resolution projection Patterson syntheses are consistent with a model proposed in which 24 tetrameric molecules of Mr = 188,000 +/- 12,000 occupy the unit cell. The space group is apparently P23, although at low resolution the observed systematic absences in reflections are consistent with the space group P43n, a space group not allowed for asymmetric molecules. Estimates of VM suggest that in the true space group, P23, two tetrameric molecules occupy the asymmetric unit.     

Isometric force development, isotonic shortening, and elasticity measurements from Ca(2+)-activated ventricular muscle of the guinea pig

Isometric tension and isotonic shortening were measured at constant levels of calcium activation of varying magnitude in mechanically disrupted EGTA-treated ventricular bundles from guinea pigs. The results were as follows: (a) The effect of creatine phosphate (CP) on peak tension and rate of shortening saturated at a CP concentration more than 10 mM; below that level tension was increased and shortening velocity decreased. We interpreted this to mean that CP above 10 mM was sufficient to buffer MgATP(2-) intracellularly. (b) The activated bundles exhibited an exponential stress-strain relationship and the series elastic properties did not vary appreciably with degree of activation or creatine phosphate level. (c) At a muscle length 20 percent beyond just taut, peak tension increased with Ca(2+) concentration over the range slightly below 10(-6) to slightly above 10(-4)M. (d) By releasing the muscle length-active tension curves were constructed. Force declined to 20 percent peak tension with a decrease in muscle length (after the recoil) of only 11 percent at 10(-4)M Ca(2+) and 6 percent at 4x10(-6)M Ca(2+). (e) The rate of shortening after a release was greater at lower loads. At identical loads (relative to maximum force at a given Ca(2+) level), velocity at a given time after the release was less at lower Ca(2+) concentrations; at 10 M(-5), velocity was 72 percent of that at 10(-4)M, and at 4x10(-6)M, active shortening was usually delayed and was 40 percent of the velocity at 10(-4) M. Thus, under the conditions of these experiments, both velocity and peak tension depend on the level of Ca(2+) activation over a similar range of Ca(2+) concentration.     

Lifestyle modifications to prevent and control hypertension. 2. Recommendations on obesity and weight loss. Canadian Hypertension Society,Canadian Coalition for High Blood Pressure Prevention and Control,Laboratory Centre for Disease Control at Health Canada,Heart and Stroke Foundation of Canada

OBJECTIVE: To provide updated, evidence-based recommendations concerning the effects of weight loss and maintenance of healthy weight on the prevention and control of hypertension in otherwise healthy adults (except pregnant women). OPTIONS: The main options are to attain and maintain a healthy body weight (body mass index [BMI] 20-25 kg/m2) or not to do so. For those at risk for hypertension, weight loss and maintenance of healthy weight may prevent the condition. For those who have hypertension, weight loss and maintenance of healthy weight may reduce or obviate the need for antihypertensive medications. OUTCOMES: The health outcome considered was change in blood pressure. Because of insufficient evidence, no economic outcomes were considered. EVIDENCE: A MEDLINE search was conducted for the years 1992-1996 with the terms hypertension and obesity in combination and antihypertensive therapy and obesity in combination. Other relevant evidence was obtained from the reference lists of the articles identified, from the personal files of the authors and through contacts with experts. The articles were reviewed, classified according to study design and graded according to level of evidence. VALUES: A high value was placed on the avoidance of cardiovascular morbidity and premature death caused by untreated hypertension. BENEFITS, HARMS AND COSTS: Weight loss and the maintenance of healthy body weight reduces the blood pressure of both hypertensive and normotensive people. The indirect benefits of a health body weight are well known. The negative effects of weight loss are primarily the frustrations associated with attaining and maintaining a healthy weight. The costs associated with weight loss programs were not measured in the studies reviewed. RECOMMENDATIONS: (1) It is recommended that health care professionals determine weight (in kilograms), height (in metres) and BMI for all adults. (2) To reduce blood pressure in the population at large, it is recommended that Canadians attain and maintain a healthy BMI (20-25). (3) All overweight hypertensive patients (BMI greater than 25) should be advised to reduce their weight. VALIDATION: These recommendations are similar to those of the World Hypertension League, the National High Blood Pressure Education Program Working Group on Primary Prevention of Hypertension, the Canadian Hypertension Society and the Canadian Coalition for High Blood Pressure Prevention and Control. They have not been clinically tested. SPONSORS: The Canadian Hypertension Society, the Canadian Coalition for High Blood Pressure Prevention and Control, the Laboratory Centre for Disease Control at Health Canada, and the Heart and Stroke Foundation of Canada.     

Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression.

We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.