Glutamate-Treated Rat Cortical Neuronal Cultures Die in a Way Different from the Classical Apoptosis Induced by Staurosporine

The alkaloid protein kinase inhibitor staurosporine induced neuronal cell death with both the morphological and the biochemical characteristics of apoptosis. The punctate chromatin associated with apoptosis with retention of plasma membrane integrity was observed in neurons identified by colocalization of NeuN staining. Such cells had DNA fragmentation visualized byin situend-labeling which was seen as a laddered pattern upon gel electrophoresis. In contrast cells treated with glutamate did not exhibit either of these morphological or biochemical hallmarks of apoptosis. Instead a much smaller and more compact pyknotic structure was observed associated with smeared DNA fragmentation patterns. A confocal time-lapse study of the appearance of the morphological changes in individual nuclei after staurosporine treatment showed collapse into punctate chromatin over a period of 10 min. In contrast, the collapse into small pyknotic nuclei after glutamate treatment was at least 10 times slower. It is concluded that excitotoxicity produced by glutamate did not induce cell death by an apoptotic mechanism in cultured cortical neurons.     

Kinetics of formation of fibrin oligomers. III. Ligation kinetics concurrent with and subsequent to oligomer assembly

Human fibrinogen was treated with thrombin in the presence of fibrinoligase (Factor XIIIa) and calcium ion at pH 8.5, ionic strength 0.45, and the ensuing polymerization was interrupted at various time intervals (t) both before and after the clotting time (t_c) by solubilization with a solution of sodium dodecyl sulfate and urea. Aliquots of the solubilized protein were subjected to gel electrophoresis on polyacrylamide gels after disulfide reduction by dithiothreitol and on agarose gels without reduction. The degree of γ-γ ligation was determined from the former. The latter provided the size distribution of ligated end-to-end sequences produced by splitting the ligated staggered overlapped oligomers down the middle, for degrees of polymerization, x, from 1 to 10. Addition of fibrinoligase (in which the activating thrombin had been inhibited by p-nitrophenyl-p′-guanidinobenzoate, NPGB) to Kabi fibrinogen showed the presence of small amounts of ligatable oligomers. Addition of fibrinoligase to a polymerizing mixture in which the action of thrombin had been stopped before clotting by NPGB produced the same distribution of ligated end-to-end sequences that was obtained when fibrinoligase was originally present, at least for reaction times up to 0.7 of the clotting time. The kinetics of γ-γ ligation by fibrinoligase acting on a polymerized mixture stabilized by NPGB were followed. The reaction was first order in the concentration of ligatable γ-γ junctions and the initial velocity was proportional to the enzyme concentration. The time evolution of size distribution of ligated end-to-end sequences agreed with a theory based on random ligation of ligatable junctions.     

Comparison of Two Liquid Blood Culture Media Containing Sodium Polyanetholesulfonate: Tryptic Soy and Columbia

In a comparison of tryptic soy broth and Columbia broth, two blood culture media containing sodium polyanetholesulfonate, there were 589 positive cultures (excluding presumed contaminants). The two media were equivalent in performance except for lower detection rates for Staphylococcus aureus (P < 0.01) and Pseudomonas aeruginosa (P = 0.05) and a higher detection rate for Bacillus (P < 0.01) in Columbia broth. No significant differences were noted in time intervals to detection of positivity. Routine subcultures on the 1st and 5th days of incubation provided the initial detection of 18.1% of the positive cultures.     

Human NPC1L1 and NPC1 can functionally substitute for the ncr genes to promote reproductive development in C. elegans

The NPC1 and NPC1L1 are related genes whose general role is in cholesterol trafficking. However, reduction of activity of these genes results in very different phenotypes. Niemann-Pick C disease type 1 is a neurodegenerative disease with no current treatment, where cholesterol accumulates in lysosomes. The disease arises due to autosomal recessive mutations in the NPC1 gene. The NPC1L1 gene has recently been identified as the target for the drug ezetimibe (Zetia), a cholesterol absorption inhibitor, and has been shown to be an intestinal cholesterol transporter. We demonstrate that human NPC1L1, as well as human NPC1, can functionally substitute for the Caenorhabditis elegans genes ncr-1 and/or ncr-2. These genes are known to play a role in the process of dauer formation, a process which can be modulated by cholesterol in sensitized genetic backgrounds. Our results demonstrate that these human proteins retain some functional conservation, though their biological roles are vastly different.     

Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca²⁺ionophore, ionomycin, suggesting the involvement of extracellular Ca²⁺ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca²⁺ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca²⁺ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143–153, 1998. © 1998 Wiley-Liss, Inc.     

Preliminary investigation of morphological differences between ten breeds of horses suggests selection for paedomorphosis

Paedomorphosis is the retention of juvenile morphology at maturity and is important in generating evolutionary change in domestic species and species in the wild. When comparing dogs with the wolf, this preliminary study saw paedomorphosis in their physical and behavioral traits (Goodwin, Bradshaw, & Wickens, 1997). This preliminary study compared morphological characteristics of 10 breeds from northern regions (Shetland ponies) and southern regions (Arabians) with the Exmoor pony. Twenty-three respondents from the United Kingdom and Australia rated the breeds for 7 physical traits. As evidenced by low standard deviations, the respondents demonstrated a high degree of agreement. The study ranked breeds from the most similar (Highland pony) to the least similar (Arabian) to the Exmoor pony. The least similar breeds had physical traits suggestive of paedomorphosis: small heads, long legs, and a low head-to-body ratio. This preliminary study suggests that morphological, behavioral, and physiological differences between the breeds-plus morphometric comparisons of extant breeds and faunal remains of predomestication horses-warrant further study.     

Organic matter accumulation in western boreal saline wetlands: A comparison of undisturbed and oil sands wetlands

Reconstructing landscapes after open pit mining of the Canadian oil sands presents enormous challenges. Freshwater peatlands dominate the pre-disturbance landscape; however, elevated salinity in the post-disturbance landscape will exclude the use of many freshwater vegetation species for reclamation. Successful reclamation will require plants to grow and accumulate peat despite elevated salinity. We evaluated the potential of salt-tolerant plants to accumulate peat by integrating plant production and decomposition rates in natural and oil sands wetlands across a salinity gradient. These wetlands were dominated by marsh-like vegetation with relatively rapid decomposition, especially of the belowground plant material. Aboveground production was high enough to compensate for rapid decomposition, resulting in mean annual organic matter accumulation of 307 g m^?2. Thus, both natural wetlands (which despite the elevated salinity had peat deposits >35 cm) and the oil sands wetlands accumulated organic matter during the study. There is potential for peat to accumulate in future oil sands wetlands, although long-term accumulation rates may be slower than in undisturbed freshwater fens and bogs. A reliable water supply and a host of other factors will be required for wetlands to accumulate organic matter, and eventually peat, in the post-mining landscape.     

Discovery of 13-oxa prostaglandin analogs as antiglaucoma agents: Synthesis and biological activity

FP-Class prostaglandin analogs have demonstrated utility for the treatment of glaucoma and ocular hypertension. A series of novel FP prostaglandin analogs was designed to optimize topical ocular activity and reduce ocular side-effects by replacing 13-carbon with oxygen. A facile synthesis was successfully developed for synthesis of the 13-oxa prostaglandins from the commercially available Corey aldehyde benzoate. Among the compounds synthesized, AL-16082 was the most potent prostaglandin FP agonist in vitro. In a prostaglandin FP receptor-linked second-messenger assay, phosphoinositide (PI) turnover, it exhibited a potency value (EC₅₀) of 1.9 nM (78% max. response relative to fluprostenol). The isopropyl ester of AL-16082, compound AL-16049, significantly lowered intraocular pressure (IOP) in the ocular hypertensive monkey eyes by 30%. In the study of acute ocular irritation response in New Zealand albino rabbits, AL-16049 produced lower incidence of hyperemia, swelling, and discharge than PGF_2α (1 μg), and a similar incidence of hyperemia, swelling, and discharge to latanoprost (1.8 μg). AL-16049 also produced no signs of ocular irritation or discomfort in the cat at the doses evaluated.     

DEAR1 Is a Dominant Regulator of Acinar Morphogenesis and an Independent Predictor of Local Recurrence-Free Survival in Early-Onset Breast Cancer

Background

Breast cancer in young women tends to have a natural history of aggressive disease for which rates of recurrence are higher than in breast cancers detected later in life. Little is known about the genetic pathways that underlie early-onset breast cancer. Here we report the discovery of DEAR1 (ductal epithelium–associated RING Chromosome 1), a novel gene encoding a member of the TRIM (tripartite motif) subfamily of RING finger proteins, and provide evidence for its role as a dominant regulator of acinar morphogenesis in the mammary gland and as an independent predictor of local recurrence-free survival in early-onset breast cancer.

Methods and Findings

Suppression subtractive hybridization identified DEAR1 as a novel gene mapping to a region of high-frequency loss of heterozygosity (LOH) in a number of histologically diverse human cancers within Chromosome 1p35.1. In the breast epithelium, DEAR1 expression is limited to the ductal and glandular epithelium and is down-regulated in transition to ductal carcinoma in situ (DCIS), an early histologic stage in breast tumorigenesis. DEAR1 missense mutations and homozygous deletion (HD) were discovered in breast cancer cell lines and tumor samples. Introduction of the DEAR1 wild type and not the missense mutant alleles to complement a mutation in a breast cancer cell line, derived from a 36-year-old female with invasive breast cancer, initiated acinar morphogenesis in three-dimensional (3D) basement membrane culture and restored tissue architecture reminiscent of normal acinar structures in the mammary gland in vivo. Stable knockdown of DEAR1 in immortalized human mammary epithelial cells (HMECs) recapitulated the growth in 3D culture of breast cancer cell lines containing mutated DEAR1, in that shDEAR1 clones demonstrated disruption of tissue architecture, loss of apical basal polarity, diffuse apoptosis, and failure of lumen formation. Furthermore, immunohistochemical staining of a tissue microarray from a cohort of 123 young female breast cancer patients with a 20-year follow-up indicated that in early-onset breast cancer, DEAR1 expression serves as an independent predictor of local recurrence-free survival and correlates significantly with strong family history of breast cancer and the triple-negative phenotype (ER⁻, PR⁻, HER-2⁻) of breast cancers with poor prognosis.

Conclusions

Our data provide compelling evidence for the genetic alteration and loss of expression of DEAR1 in breast cancer, for the functional role of DEAR1 in the dominant regulation of acinar morphogenesis in 3D culture, and for the potential utility of an immunohistochemical assay for DEAR1 expression as an independent prognostic marker for stratification of early-onset disease.