Calcium compartments and fluxes are affected by the src gene product of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus

Total cellular calcium content (determined by atomic absorption spectrometry) of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus decreases with cell density, but is found not significantly different at permissive and at non-permissive temperature. Kinetic analysis of 45Ca efflux from preloaded cells exhibits three separable pools of exchangeable calcium. The ratio of pool size of the fast-exchanging Ca-compartment (bound to cell surface) to pool size of the intermediate Ca-compartment (cytoplasmic) was found to decrease from 2.5 to 1.3 upon shift from non-permissive to permissive temperature. The slowly exchanging Ca-pool (presumably mitochondrial) did not change significantly upon temperature shift. These and further data demonstrate a close correlation between distribution of cellular Ca among different cellular compartments and characteristics of cellular proliferation, both attributable to the function(s) of a single oncogene.     

Whole-genome patenting

Gene patenting is now a familiar commercial practice, but there is little awareness that several patents claim ownership of the complete genome sequence of a prokaryote or virus. When these patents are analysed and compared to those for other biological entities, it becomes clear that genome patents seek to exploit the genome as an information base and are part of a broader shift towards intangible intellectual property in genomics.     

Shoot organogenesis and plant regeneration in <Emphasis Type="Italic">Pityopsis ruthii</Emphasis>

Pityopsis ruthii is an endangered herbaceous perennial species from the United States. In vitro multiplication of this species can be valuable for germplasm conservation. Flower receptacles of P. ruthii were cultured on Murashige and Skoog medium (MS) supplemented with 11.4 μM indole-3-acetic acid (IAA) in combination with 2.2, 4.4 or 8.8 μM 6-benzyladenine (BA). Shoots were visible within 14–28 days and three plants were successfully rooted on MS medium supplemented with 5.7 μM IAA. A two tailed t-test for paired-variates revealed that shoot regeneration on MS medium amended with 11.4 μM IAA and 2.2 μM BA was significantly higher (P < 0.05) than on other treatments. Leaf explants were also cultured on MS not supplemented with growth regulators or supplemented with 11.4 μM IAA in combination with 0, 2.2, 4.4 or 8.8 μM BA. Shoots were visible within 21–35 days and one plant was successfully rooted on MS medium supplemented with 5.4 μM NAA. Shoot regeneration on MS medium augmented with 11.4 μM IAA and 2.2 μM BA was significantly higher (P < 0.05) than the other treatments according to analysis of variance (ANOVA) with a rank transformation. Hyperhydricity and rooting of shoots was problematic for explants derived from flower receptacles and leaf tissue, but viable plants were regenerated using both explants sources indicating the potential role for micropropagation in the ex situ conservation of the species.     

Osmium tetroxide, used in the treatment of arthritic joints, is a fast mimic of superoxide dismutase

Aqueous solutions of osmium tetroxide (OsO4) have been injected into arthritic knees for the past 45 years to chemically destroy diseased tissue, in a procedure termed "chemical synovectomy." Arthritis is an inflammatory disease. The primary inflammatory chemical species are the superoxide anion radical (O2.-) and nitric oxide (.NO), which combine to form the peroxynitrite anion (ONOO-). Here we show that OsO4 does not react with ONOO- but very efficiently catalyzes the dismutation of O2.- to O2 and H2O2. Using the pulse-radiolysis technique, the catalytic rate constant has been determined to be (1.43+/-0.04) x 10(9) M-1 s-1, independent of the pH in the 5.1-8.7 range. This value is about half that for the natural Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Per unit mass, OsO4 is about 60 times more active than Cu,Zn-SOD. The catalytically active couple is OsVIII/OsVII, OsVIII oxidizing O2.- to O2 with a bimolecular rate constant of k=(2.6+/-0.1)x10(9) M-1 s-1 and OsVII reducing it to H2O2 with a bimolecular rate constant of (1.0+/-0.1)x10(9) M-1 s-1. Although lower valent osmium species are intrinsically poor catalysts, they are activated through oxidation by O2.- to the catalytic OsVIII/OsVII redox couple. The OsVIII/OsVII catalyst is stable to biochemicals other than proteins and peptides comprising histidine, cysteine, and dithiols.     

Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells

BACKGROUND: The Ral guanine nucleotide-exchange factors (RalGEFs) serve as key effectors for Ras oncogene transformation of immortalized human cells. RalGEFs are activators of the highly related RalA and RalB small GTPases, although only the former has been found to promote Ras-mediated growth transformation of human cells. In the present study, we determined whether RalA and RalB also had divergent roles in promoting the aberrant growth of pancreatic cancers, which are characterized by the highest occurrence of Ras mutations. RESULTS: We now show that inhibition of RalA but not RalB expression universally reduced the transformed and tumorigenic growth in a panel of ten genetically diverse human pancreatic cancer cell lines. Despite the apparent unimportant role of RalB in tumorigenic growth, it was nevertheless critical for invasion in seven of nine pancreatic cancer cell lines and for metastasis as assessed by tail-vein injection of three different tumorigenic cell lines tested. Moreover, both RalA and RalB were more commonly activated in pancreatic tumor tissue than other Ras effector pathways. CONCLUSIONS: RalA function is critical to tumor initiation, whereas RalB function is more important for tumor metastasis in the tested cell lines and thus argues for critical, but distinct, roles of Ral proteins during the dynamic progression of Ras-driven pancreatic cancers.     

A Critical Tryptophan and Ca2+ in Activation and Catalysis of TPPI,the Enzyme Deficient in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis

Background

Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca²⁺.

Methodology

Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme.

Principal Findings

NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI.

Conclusions/Significance

We propose that W542 and Ca²⁺ are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca²⁺ is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.     

Recruitment of histone modifications by USF proteins at a vertebrate barrier element

The chicken beta-globin 5'HS4 insulator element acts as a barrier to the encroachment of chromosomal silencing. Endogenous 5'HS4 sequences are highly enriched with histone acetylation and H3K4 methylation regardless of neighboring gene expression. We report here that 5'HS4 elements recruit these histone modifications when protecting a reporter transgene from chromosomal silencing. Deletion studies identified a single protein binding site within 5'HS4, footprint IV, that is necessary for the recruitment of histone modifications and for barrier activity. We have determined that USF proteins bind to footprint IV. USF1 is present in complexes with histone modifying enzymes in cell extracts, and these enzymes specifically interact with the endogenous 5'HS4 element. Knockdown of USF1 expression leads to a loss of histone modification recruitment and subsequent encroachment of H3K9 methylation. We propose that barrier activity requires the constitutive recruitment of H3K4 methylation and histone acetylation at multiple residues to counteract the propagation of condensed chromatin structures.     

<Emphasis Type="Italic">Daphnia galeata</Emphasis> in the deep hypolimnion: spatial differentiation of a “typical epilimnetic” species

One of the most serious threats to tropical mangrove ecosystems caused by shrimp farming activities is the poor management of pond waste materials. We hypothesise that mangroves can tolerate chemical residues discharged from shrimp farms and can be used as biofilters, but the capability of mangroves to cope with solid sediments dredged from shrimp ponds is limited. Our study in Pak Phanang, Thailand, confirmed that the excess sediments discharged from nearby shrimp ponds reduced mangrove growth rates and increased mortality rates. A series of transformed multi-temporal satellite images was used in combination with the field data to support this claim. In addition, a comparison between four dominant mangrove species revealed that Avicennia marina could tolerate sedimentation rates of >6 cm year⁻¹, while Bruguiera cylindrica tolerated sedimentation rates of 5 cm year⁻¹ (total sediment depth = 25 cm) before dying, while Excoecaria agallocha and Lumnitzera racemosa performed intermediate. This outcome implied that in our situation A. marina and to lesser extent E. agallocha and L. racemosa could be more effective as biofilters than B. cylindrica, as they may survive the sedimentation longer in the disposal areas. Further studies on the impact of sedimentation and chemical pollution of shrimp farm wastes on mangrove mortality and growth are required.