Adoptive transfer of the entire gld (generalized lymphoproliferative disease) syndrome in nude beige mice by a single gld thymocyte graft.

C57BL/6 nude beige mice (B6 nubq; no T cell, no NK activity) were used as recipients for the adoptive transfer of thymocytes from B6 gld mice (generalized lymphoproliferative disease) which are a model of systemic lupus erythematous. The [gld----nubg] chimeras showed several similarities with gld control mice including the T cell disorders (lymphoproliferation and Con A-response deficiency of splenocytes) and B cell disorders (hyperglobulinemia and elevated anti-single-stranded DNA antibody titers). This suggests that the gld lymphoproliferative disorder has a thymic origin (and does not result from an abnormally extrathymic T cell development) and that the gld T cells have an essential role for the emergence of the disorders of both the T and B cells.     

Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

A chemically synthesized radiolabeled signal peptide: design, preparation, and biological evaluation of an iodinated analog of preproparathyroid hormone

The chemically synthesized signal peptide (native-sequence signal peptide) of preproparathyroid hormone exhibits signal sequence-like activity by inhibiting the translocation/processing of precursor proteins to their mature forms in an in vitro translation system. In order to prepare a biologically functional radiolabeled form of this peptide, we undertook structure-function studies of the native-sequence signal peptide. Since conventional iodination of peptides is performed under oxidizing conditions, chemical design efforts were focused on the oxidation-labile residues, methionine and cysteine, present in the native sequence. Substitution of the three methionines with norleucine and the single cysteine with alanine yielded a surfur-free analog, [Nle-(-25), Nle-(-21),Nle-(-18),Ala-(-14),D-Tyr-(+1)]pre-proPTH-(-29-+1)amide, which is resistant to oxidation and active in the inhibition of processing assay. An interaction between the signal region and one of the components of the intracellular secretory apparatus, signal recognition particle (SRP), was demonstrated: iodinated sulfur-free analog was cross-linked (using the homo-bifunctional reagent disuccinimidyl suberate) to the 54 kilodalton (kDa) subunit of SRP. The 68 kDa and 72 kDa subunits of SRP were also labeled, but to a lesser extent, by the iodinated peptide.     

Heterogeneity in cblG: differential retention of cobalamin on methionine synthase.

Cultured fibroblasts from patients with functional methionine synthase deficiency have been shown to belong to two complementation classes, cblE and cblG. Both are associated with decreased intracellular levels of methylcobalamin (MeCbl) and decreased incorporation of label from 5-methyltetrahydrofolate into macromolecules. Methionine synthase specific activity is normal or near normal in cell extracts from cblE patients under standard reducing conditions, whereas specific activity is low in cblG extracts. Seven of 10 cblG cell lines accumulated [57Co]CN-Cbl equivalent to control cells and showed similar proportions of label associated with the two intracellular cobalamin binders, methionine synthase and methylmalonyl-CoA mutase. The remaining three cblG lines showed reduced accumulation of labeled Cbl and virtually none associated with methionine synthase. The specific activity of methionine synthase was decreased in cell extracts from both cblG subgroups, being almost undetectable in extracts from the latter three lines. Incorporation of label from [14C]MeTHF into either macromolecules or into methionine was decreased in both cblG groups, but was paradoxically higher in the three lines with very low in vitro methionine synthase activity. These results demonstrate further heterogeneity within cblG and suggest that the defect in the three variant lines affects the ability of methionine synthase to retain Cbl.     

Biological activity of parathyroid hormone antagonists substituted at position 13.

Lysine occupies position 13 in the parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2. Acylation of the epsilon-amino group in lysine 13 by a hydrophobic moiety is well tolerated in terms of bioactivity: the analog [Nle8,18, D-Trp12,Lys 13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(7-34)NH2 is equivalent to the parent peptide in its affinity for PTH receptors and its ability to inhibit PTH-stimulated adenylate cyclase in both kidney- and bone-based assays. Truncation of this peptide by deletion of phenylalanyl7 with concomitant removal of the amino-terminal alpha-amino group yielded the analog desamino[Nle8,18,D-Trp12,Lys13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(8-34)NH2, an antagonist of high potency in vitro (Kb = 4 and 9 nM, Ki = 73 and 3.5 nM in kidney- and bone-based assays, respectively). Also this analog is potentially stable to aminopeptidases present in many biological systems.     

Response to low-dose pulsatile cortisol in Addison's disease with suspected corticotropinoma

A 65 year old woman with long-standing Addison's disease treated with oral glucocorticoid and mineralocorticoid replacement had persistently high ACTH levels, inadequate suppression of ACTH on low-dose dexamethasone, sellar enlargement, and pigmentation, and thus resembled patients alleged to develop corticotropinomas while on oral replacement for adrenal insufficiency. Since animal studies suggested that rapid rises of corticosteroids within the physiologic range can inhibit ACTH release, we administered brief infusions of cortisol every three hours with total daily dose equal to her chronic dose. Prompt suppression of ACTH and immunoreactive beta-endorphin occurred during each cortisol dose profiled, suggesting a role for ultradian cortisol fluctuations in tonic inhibition of ACTH secretion in humans, and a possible therapeutic benefit of mimicking ultradian cortisol rhythms during replacement therapy.