Unrestricted recognition of a nonpeptide antigen by CD8+ cytolytic T lymphocytes

CD8+ murine CTL that are specific for an unusual nonpeptide Ag, the heme moiety of hemoglobin, have been derived by in vitro stimulation of spleen cells with hemin. Such CTL demonstrate a requirement for the expression of class I Ag on target cells, yet appear to be unrestricted to the extent that both syngeneic and allogeneic targets precoated with hemin are sensitive to lysis. A series of CTL clones with specificity for hemin was derived from C57BL/6 mice. They exhibited the same type of promiscuous recognition that was observed in CTL populations from a number of different strains. The possibility that hemin acts as a nonspecific mediator of lysis by CTL was ruled out by the fact that a variety of CTL populations and clones specific for different Ag did not exhibit hemin-specific lysis. Some explanations offered to explain these results include 1) the possibility that hemin is recognized by binding to a site on the MHC other than the Ag-binding groove, and 2) the possibility that TCR recognition of a rigid molecule, such as hemin, may be less sensitive to polymorphic variation in the MHC than is recognition of a conventional peptide Ag whose conformation may differ significantly when bound to MHC molecules whose sequences differ within the Ag-binding groove.     

Maturation in Larch : II. Effects of Age on Photosynthesis and Gene Expression in Developing Foliage

The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor- and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO₂ per square millimeter per second) increases with age on indoor- but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturation-related differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbsS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark-grown seedlings (~2% of light-grown seedlings). Steady-state cab mRNA levels are relatively higher (~40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions reported previously (MS Greenwood, CA Hopper, KW Hutchison [1989] Plant Physiol 90: 406-412) is not due to increased NPS or cab expression. Long shoot foliage is the dominant foliar type on young trees and its lower specific leaf weight will permit production of more photosynthetic surface area per unit of leaf biomass.     

A new transthyretin mutation associated with amyloid cardiomyopathy.

In transthyretin (TTR) a new mutation (TTR-Thr45) has been identified in a patient with familial amyloidosis characterized clinically by prominent cardiomyopathy and the absence of peripheral neuropathy. Comparative peptide mapping by high-performance liquid chromatography of the patient's plasma TTR together with normal TTR showed the presence of an abnormal tryptic peptide in the patient's TTR. The sequence of this peptide (peptide 6, residues 36-48) demonstrated the presence of a threonine-for-alanine substitution at position 45. This change can be explained by a single base change of adenine for guanine in the Ala-45 codon and was demonstrated directly by DNA sequence analysis of PCR-amplified exon 2 of the TTR gene; allele-specific oligonucleotide hybridization both in the patient and in fixed heart tissue from his aunt confirmed the base change. The TTR-Thr45 mutation is a new variant TTR found associated with cardiomyopathy.     

A new method for stoichiometric analysis of proteins in complex mixture--reevaluation of the stoichiometry of E. coli ribosomal proteins

A novel way is presented for determination of the stoichiometry of ribosomal proteins in the ribosome. The 70S E. coli r-proteins, completely separated on a two-dimensional gel system, were used throughout our experiments. The method is based on our previous observation that the amount of Coomassie R bound to a protein molecule is directly proportional to the number of positive charges on that protein. By plotting the amount of bound Coomassie as a function of the number of positive charges of each r-protein, and relating the experimental amount of the dye bound to each r-protein to the value obtained from the linear regression line based on all (a total of some 50 proteins), one can obtain the molar concentration of every protein in the ribosome. A parallel experiment can be carried out, which relates the radioactivity contributed by 3H-labeled amino acid in each r-protein to its amino acid content in that molecule. The two sets of data, which are completely independent of each other, are well correlated. Further verification of the validity of our procedure is provided by the fact that we found the known proportions of four copies of L7/L12 and one copy of S6 per ribosome. The rationale behind the present study was our finding that recalculation of Hardy's data (Hardy, S.J.S. (1975) Mol. Gen. Genet. 140, 253-274), with the accurate molecular weight value of the r-proteins provided by Giri et al. (Adv. Protein Chem. (1984) 36, 1-78), raises some doubt with regard to his experimental results, although we agree with his final conclusion that E. coli ribosome is homogeneous with respect to its proteins.     

Longitudinal study of a heteroplasmic 3460 Leber hereditary optic neuropathy family by multiplexed primer-extension analysis and nucleotide sequencing.

Nucleotide-sequencing and multiplexed primer-extension assays have been used to quantitate the mutant-allele frequency in 14 maternal relatives, spanning three generations, from a family that is heteroplasmic for the primary Leber hereditary optic neuropathy (LHON) mutation at nucleotide 3460 of the mitochondrial genome. There was excellent agreement between the values that were obtained with the two different methods. The longitudinal study shows that the mutant-allele frequency was constant within individual family members over a sampling period of 3.5 years. Second, although there was an overall increase in the mutant-allele frequency in successive generations, segregation in the direction of the mutant allele was not invariant, and there was one instance in which there was a significant decrease in the frequency from parent to offspring. From these two sets of results, and from previous studies of heteroplasmic LHON families, we conclude that there is no evidence for a marked selective pressure that determines the replication, segregation, or transmission of primary LHON mutations to white blood cells and platelets. Instead, the mtDNA molecules are most likely to replicate and segregate under conditions of random drift at the cellular level. Finally, the pattern of transmission in this maternal lineage is compatible with a developmental bottleneck model in which the number of mitochondrial units of segregation in the female germ line is relatively small in relation to the number of mtDNA molecules within a cell. However, this is not an invariant pattern for humans, and simple models of mitochondrial gene transmission are inappropriate at the present time.     

Enhanced stability in vivo of a thermodynamically stable mutant form of yeast iso-1-cytochrome c

Previous work has established that the N57I amino acid replacement in iso-1-cytochrome c from the yeast Saccharomyces cerevisiae causes an unprecedented increase in thermodynamic stability of the protein in vitro, whereas the N57G replacement diminishes stability. Spectrophotometric measurements of intact cells revealed that the N57I iso-l-cytochrome c is present at higher than normal levels in vivo. Although iso-1-cytochrome c turnover is negligible during aerobic growth, transfer of fully derepressed, aerobically grown cells to anaerobic growth conditions leads to reduction in the levels of all of the cytochromes. Pulsechase experiments carried out under these anaerobic conditions demonstrated that the N57I iso-l-cytochrome c has a longer half-life than the normal protein. This is the first report of enhanced stability in vivo of a mutant form of a protein that has an enhanced thermodynamic stability in vitro. Although the N57I protein concentration is higher than the normal level, reduced growth in lactate medium indicated that the specific activity of this iso-l-cytochrome c in vivo is diminished relative to wild-type. On the other hand, the level of the thermodynamically labile N57G iso-1-cytochrome c was below normal. The in vivo levels of the N57I and N57G iso-l-cytochrome c suggest that proteins in the mitochondrial intermembrane space can be subjected to degradation, and that this degradation may play a role in controlling their normal levels.     

Common mutations in the phosphofructokinase-M gene in Ashkenazi Jewish patients with glycogenesis VII--and their population frequency.

Phosphofructokinase (PFK) catalyzes the rate-limiting step of glycolysis. Deficiency of the muscle enzyme is manifested by exercise intolerance and a compensated hemolytic anemia. Case reports of this autosomal recessive disease suggest a predominance in Ashkenazi Jews in the United States. We have explored the genetic basis for this illness in nine affected families and surveyed the normal Ashkenazi population for the mutations we have found. Genomic DNA was amplified using PCR, and denaturing gradient-gel electrophoresis was used to localize exons with possible mutations. The polymorphic exons were sequenced or digested with restriction enzymes. A previously described splicing mutation, delta 5, accounted for 11 (61%) of 18 abnormal alleles in the nine families. A single base deletion leading to a frameshift mutation in exon 22 (delta C-22) was found in six of seven alleles. A third mutation, resulting in a nonconservative amino acid substitution in exon 4, accounted for the remaining allele. Thus, three mutations could account for all illness in this group, and two mutations could account for 17 of 18 alleles. In screening 250 normal Ashkenazi individuals for all three mutations, we found only one delta 5 allele. Clinical data revealed no correlation between the particular mutations and symptoms, but male patients were more symptomatic than females, and only males had frank hemolysis and hyperuricemia. Because PFK deficiency in Ashkenazi Jews is caused by a limited number of mutations, screening genomic DNA from peripheral blood for the described mutations in this population should enable rapid diagnosis without muscle biopsy.