Positive selection of Caenorhabditis elegans mutants with increased stress resistance and longevity

We developed selective conditions for long-lived mutants of the nematode Caenorhabditis elegans by subjecting the first larval stage (L1) to thermal stress at 30 degrees for 7 days. The surviving larvae developed to fertile adults after the temperature was shifted to 15 degrees. A total of one million F(2) progeny and a half million F(3) progeny of ethyl-methanesulfonate-mutagenized animals were treated in three separate experiments. Among the 81 putative mutants that recovered and matured to the reproductive adult, 63 retested as thermotolerant and 49 (80%) exhibited a >15% increase in mean life span. All the known classes of dauer formation (Daf) mutant that affect longevity were found, including six new alleles of daf-2, and a unique temperature-sensitive, dauer-constitutive allele of age-1. Alleles of dyf-2 and unc-13 were isolated, and mutants of unc-18, a gene that interacts with unc-13, were also found to be long lived. Thirteen additional mutations define at least four new genes.     

Ploidy race distributions since the Last Glacial Maximum in the North American desert shrub, Larrea tridentata

• 1 A classic biogeographic pattern is the alignment of diploid, tetraploid and hexaploid races of creosote bush (Larrea tridentata) across the Chihuahuan, Sonoran and Mohave Deserts of western North America. We used statistically robust differences in guard cell size of modern plants and fossil leaves from packrat middens to map current and past distributions of these ploidy races since the Last Glacial Maximum (LGM).
• 2 Glacial/early Holocene (26–10 ¹⁴C kyr bp or thousands of radiocarbon years before present) populations included diploids along the lower Rio Grande of west Texas, 650 km removed from sympatric diploids and tetraploids in the lower Colorado River Basin of south‐eastern California/south‐western Arizona. Diploids migrated slowly from lower Rio Grande refugia with expansion into the northern Chihuahuan Desert sites forestalled until after ~4.0 ¹⁴C kyr bp . Tetraploids expanded from the lower Colorado River Basin into the northern limits of the Sonoran Desert in central Arizona by 6.4 ¹⁴C kyr bp . Hexaploids appeared by 8.5 ¹⁴C kyr bp in the lower Colorado River Basin, reaching their northernmost limits (~37°N) in the Mohave Desert between 5.6 and 3.9 ¹⁴C kyr bp .
• 3 Modern diploid isolates may have resulted from both vicariant and dispersal events. In central Baja California and the lower Colorado River Basin, modern diploids probably originated from relict populations near glacial refugia. Founder events in the middle and late Holocene established diploid outposts on isolated limestone outcrops in areas of central and southern Arizona dominated by tetraploid populations.
• 4 Geographic alignment of the three ploidy races along the modern gradient of increasingly drier and hotter summers is clearly a postglacial phenomenon, but evolution of both higher ploidy races must have happened before the Holocene. The exact timing and mechanism of polyploidy evolution in creosote bush remains a matter of conjecture.

     

Recombinant carbazole-degrading strains for enhanced petroleum processing

Biotechnological upgrading of fossil fuels is of increasing interest as remaining stocks of petroleum show increasing levels of contaminants such as heavy metals, sulfur and nitrogen-containing heteroaromatic compounds. Carbazole is of particular interest as a major petroleum component known to reduce refining yields through catalyst poisoning. In this study, the biotransformation of carbazole was successfully demonstrated in a liquid two-phase system, when solubilized in either 1-methylnaphthalene or in diesel fuel. The effects of solvent toxicity were investigated by expressing the carbazole-transformation genes from MB1332, a rifampicin-resistant derivative of Pseudomonas sp. LD2, in a solvent-resistant heterologous host, P. putida Idaho [1]. This solvent-resistant strain successfully degraded carbazole solubilized in 1-methylnaphthalene and in the presence of 10 vol% xylenes similar to the non-recombinant strain Pseudomonas sp. LD2. Identification of a suitable recombinant host, however, was essential for further investigations of partial pathway transformations. Recombinant P. putida Idaho expressing only the initial dioxygenase enzymes transformed carbazole to an intermediate well retained in the oil phase. Partial carbazole transformation converts carbazole to non-aromatic species; their effect is unknown on refinery catalyst poisoning, but would allow almost complete retention of carbon content and fuel value. Electronic Publication     

Shaping and stretching life by autophagy

In a recent study published in Science, demonstrate that genes required for autophagy act downstream of insulin-like signaling, and are involved in the expression of major life history traits, including dauer larva development and adult life span.     

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.     

Relationships between uterine culture, cytology and pregnancy rates in a Thoroughbred practice

Endometrial cytology and culture specimens (n=2123) were collected concurrently with a guarded uterine culture instrument from 970 mares (738 barren, 1230 foaling and 155 maiden mares) during three breeding seasons (2001-2004). Results were compared to the 28-d pregnancy rate for the cycle from which the samples were taken. Cytological smears were evaluated for inflammation at x100 and graded as: not inflammatory (0-2 neutrophils/field), moderate inflammation (2-5 neutrophils/field), severe inflammation (>5 neutrophils/field), or hypocellular (scant epithelial cells and no neutrophils). Uterine culture swabs were plated within 6h, incubated for 72 h and results determined at 24, 48, and 72 h. Approximately, 20% (n=423) cytology samples were positive for inflammation (>2 neutrophils), whereas approximately 11% (n=231) of cultures had microorganisms recovered. A majority (64%) of the positive cultures (147/231) had inflammation on cytology smears. Streptococcus equi subsp. zooepidemicus was associated with more positive cytology results than coliforms (P<0.01). Mares with positive cytology or culture had lower pregnancy rates than mares with normal findings (P<0.01). Lowest pregnancy rates were recorded for mares with severe endometrial inflammation (21%, versus moderate inflammation 48%). Isolation of a microorganism from mares with endometrial inflammation was not associated with a further reduction in pregnancy rates. In barren, foaling and maiden mares, cytology was positive in 28, 17, and 5%, respectively, and culture was positive in 12.2, 11.1, and 3.2%. Foaling and maiden mares had higher pregnancy rates than barren mares (62, 69, and 44%, respectively, P<0.001). In conclusion, a positive cytology was twice as common as a positive culture, and isolation of microorganisms was associated with reduced pregnancy rates, even in the apparent absence of inflammation.     

Large-scale generation of highly enriched neural stem-cell-derived oligodendroglial cultures: maturation-dependent differences in insulin-like growth factor-mediated signal transduction

Multipotent neural stem cells (NSCs) are competent for commitment to the oligodendrocyte (OL) lineage both in vitro and in vivo. We exploited this property to develop a rat neurospheres (NS)/oligospheres (OS)-based culture system to generate large numbers of highly enriched late OL progenitors (preOLs) and mature OLs (MatOLs). CNS neuroblastoma cell line B104-derived conditioned medium promoted the generation of nearly pure populations of preOLs from dissociated OS. The subsequent culture of preOLs with ciliary neurotrophic factor (CNTF) and 3,3',5'-triiodo-L-thyronine (T(3)) generated nearly pure populations of MatOLs. OL lineage specificity was confirmed by immunocytochemistry, quantitative RT-PCR and gene expression profiling, which demonstrated large differences between preOLs and MatOLs. The insulin-like growth factors (IGFs) are potent neuro-protective agents required for OL survival. We used this system to systematically define maturation-dependent changes in IGF signaling during the course of OL differentiation. The IGF-I and insulin receptors, insulin receptor substrate-1 (IRS-1) and IRS-2, protein kinase B (PKB)/Akt and Janus kinase (JNK) were expressed at higher levels in NS and preOLs compared with OS and MatOLs. Erk expression increased markedly from NS to OS, decreased only partially upon commitment to preOLs, and, in MatOLs, returned to a low level similar to NS. IGF activation of the generally proliferative Erk pathway was gradually acquired during NSC differentiation, whereas IGF activation of the generally pro-survival, anti-apoptotic PI3K/PKB pathway was consistently robust at each developmental stage.     

Osteoblast Menin Regulates Bone Mass in Vivo

Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1^f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1^f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance.