Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation

The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).     

Construction of recombination-deficient strains of Pseudomonas aeruginosa

Summary The rec-102 mutation had pleiotropic effects in Pseudomonas aeruginosa: low recombination proficiency in conjugation and transduction; high UV sensitivity; inability to induce pyocin R2 by mitomycin C; and increased susceptibility to mitomycin C and nalidixic acid. The rec-102 locus was mapped by R68.45-mediated conjugation in the 45 min region of the PAO chromosome, between argF and thr-9001. By selection for a marker in this region, rec-102 can be introduced into a P. aeruginosa strain of interest using an R68.45 rec-102 donor. The recombination-deficient strains constructed in this way were phenotypically similar to Escherichia coli recA mutants.     

The role of temperature in determining distributions and coexistence of three species of Plestiodon

The Common Five-Lined Skink (Plestiodon fasciatus), Southeastern Five-Lined Skink (P. inexpectatus), and Broadheaded Skink (P. laticeps) are all found in forested ecosystems of the southeastern United States and occur in apparent sympatry. No aspect of their ecology has been quantitatively shown to be different. Differences in their natural history, such as northern extent of geographic range, observations of differences in habitat preference, and differing physiological responses to temperature, implicate the thermal environment as a potential niche dimension by which these organisms partition resources. Here we show that the northernmost extent of each species' range can be adequately discriminated by temperature extremes and that their preferred habitats differ in canopy cover, mean temperature, and daily maximum temperatures. These differences are supported by observations in the field. Based upon these findings and observations, we conclude that these species may be able to exist in such close proximity due to differences in their thermal niche, particularly the two like-sized species, P. fasciatus and P. inexpectatus. P. laticeps is a much larger lizard as an adult and functionally coexists with its congeners, potentially by partitioning resources along a different axis.     

Efficient use and recycling of the micronutrient iodide in mammals

Daily ingestion of iodide alone is not adequate to sustain production of the thyroid hormones, tri- and tetraiodothyronine. Proper maintenance of iodide in vivo also requires its active transport into the thyroid and its salvage from mono- and diiodotyrosine that are formed in excess during hormone biosynthesis. The enzyme iodotyrosine deiodinase responsible for this salvage is unusual in its ability to catalyze a reductive dehalogenation reaction dependent on a flavin cofactor, FMN. Initial characterization of this enzyme was limited by its membrane association, difficult purification and poor stability. The deiodinase became amenable to detailed analysis only after identification and heterologous expression of its gene. Site-directed mutagenesis recently demonstrated that cysteine residues are not necessary for enzymatic activity in contrast to precedence set by other reductive dehalogenases. Truncation of the N-terminal membrane anchor of the deiodinase has provided a soluble and stable source of enzyme sufficient for crystallographic studies. The structure of an enzyme·substrate co-crystal has become invaluable for understanding the origins of substrate selectivity and the mutations causing thyroid disease in humans.     

Both soluble and membrane-bound forms of Flt3 ligand enhance tumor immunity following “suicide” gene therapy in a murine colon carcinoma model

In prodrug-activated (suicide) gene therapy, tumor cells are transfected with the gene for an enzyme that converts an inactive prodrug, such as ganciclovir (GCV), to a toxic compound. Transfected cells are killed on administration of GCV, as also are untransfected bystander cells. The ability of the dendritic cell stimulatory cytokine Flt3 ligand (Flt3-L) to modulate prodrug-activated gene therapy has been investigated. Transfectants of the murine colon carcinoma MC26 were generated expressing soluble (FLS) and membrane-bound forms of Flt3-L. They were inoculated together with wild-type MC26 cells and cells expressing herpes simplex virus-1 (HSV1) thymidine kinase into BALB/c mice, which were then administered GCV. Expression of Flt3-L or FLS prevented regrowth of tumor in most mice, which was comparable to the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), while tumors recurred in all mice receiving suicide gene therapy alone. Recurring tumor cells were resistant to direct killing by GCV but sensitive to bystander killing in vitro. Mice without tumor recurrence were rechallenged with unmodified MC26 cells. Of those mice given transfectants expressing GM-CSF, Flt3-L, or FLS, approximately 50% were immune to rechallenge. These mice also showed cytotoxic and proliferative responses to MC26 cells. These experiments show that both soluble and membrane-bound forms of Flt3-L were able to induce a protective immune response to colon carcinoma cells in a fashion similar to GM-CSF.Abdul-Razzak Alsheikhly and Jehad Zweiri contributed equally to this work and are listed in alphabetical order.     

Antigenic proteins of Mycobacterium leprae. Complete sequence of the gene for the 18-kDa protein

Recombinant clones expressing antigenic determinants of the 18-kDa protein antigen from Mycobacterium leprae recognized by the L5 monoclonal antibody were isolated from a lambda gt11 expression library and their nucleotide sequences determined. All clones expressed the M. leprae-specific determinant as part of a large fusion protein with Escherichia coli beta-galactosidase. The deduced amino acid sequence of the coding region indicated that all the lambda gt11 recombinant clones contained an incomplete M. leprae gene sequence representing the carboxy-terminal two-thirds (111 amino acids) of the 18-kDa gene and coding for a peptide of m.w. 12,432. Subsequent isolation and sequencing of a 3.2kb BamHI-PstI DNA fragment from a genomic M. leprae cosmid library permitted the deduction of the complete 148 amino acid sequence with a predicted m.w. of 16,607. A second open reading frame 560 bases downstream from the 18-kDa coding sequence was found to code for a putative protein of 137 amino acids (m.w. = 15,196). Neither this nor the 18-kDa amino acid sequence displayed any significant homologies with any proteins in the GENBANK, EMBL, or NBRF data bases. Crude lysates from recombinant lambda gt11 clones expressing part of the 18-kDa protein have been reported to stimulate the proliferation of some M. leprae-specific helper T cell clones. Thus, it is significant that the complete 18-kDa sequence contains five short peptides predicted to be possible helper T cell antigenic epitopes based on their propensity to form amphipathic helices. Although three of these occur within the 111 amino acid carboxy-terminal peptide expressed by lambda gt11 clones, the most highly amphipathic peptide is found in the amino-terminal region not present in the lambda gt11 recombinants.     

Further segregation analysis of the fragile X syndrome with special reference to transmitting males

Summary A new series of 96 pedigrees with the fra(X) syndrome was analysed using complex segregation analysis with pointers, defining affection as any degree of mental impairment. These families were found to exhibit the same segregation pattern as the first series of 110 pedigrees (Sherman et al. 1984). The best estimate for penetrance of mental impairment in males was 79% and in females was 35% for the combined data. Again, there was little evidence for sporadic cases among affected males.Many more intellectually normal transmitting males have been observed since the existence of such males and the concomitant need to investigate the paternal side of pedigrees was recognized. On further investigation of all 206 pedigrees from the old and new data sets, the sibships of nonexpressing males appeared to be different from those of expressing males. Our analysis, using mental impairment as the phenotype, suggested that obligate carrier mothers and daughters of intellectually normal transmitting males are rarely, if ever, mentally impaired and that the sibs of transmitting males are much less likely to be retarded than the sibs of mentally impaired males. Though mothers and daughters of transmitting males are similar in phenotype, the expression of the gene in their offspring appears to be different: the penetrance of mental impairment is higher in offspring of intellectually normal daughters of transmitting males than in offspring of intellectually normal mothers of transmitting males. The implications of these observations for genetic counseling and for genetic models of the fra(X) syndrome are discussed.