Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

A quinazoline-derivative DOTA-type gallium(III) complex for targeting epidermal growth factor receptors: synthesis, characterisation and biological studies

The novel DOTA-like chelator 1,4,7,10-tetraazacyclododecane-1-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}propionamide-4,7,10-triacetic acid (H₃L) was synthesised by alkylation of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl acetate) with N-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}-3-bromopropionamide, followed by hydrolysis of the ester groups with trifluoracetic acid. H₃L has been fully characterised by multinuclear NMR spectroscopy, mass spectrometry and high-performance liquid chromatography (HPLC). Five protonation constants, log K _Hi , of H₃L were determined by potentiometry and UV–vis spectrophotometry and the values found are 10.47, 9.18, 5.24, 4.00 and 2.23. These methods, complemented with variable-pH ⁷¹Ga NMR studies, allowed us to ascertain the stability constant of the Ga(III) complex of L. GaL has a remarkably high thermodynamic stability constant (log K _ML = 24.5). The radioactive complex ⁶⁷GaL was prepared in high yield and high radiochemical purity. Its HPLC chromatogram is identical to that obtained for the GaL complex prepared at the macroscopic level. At pH 7.4, ⁶⁷GaL has an overall neutral charge, is highly hydrophilic (log D = −1.02 ± 0.03) and presents high in vitro stability in physiological media and in the presence of an excess of diethylenetriaminepentaethanoic acid . In vitro studies indicated that H₃L and GaL do not inhibit the cell growth of epidermal growth factor receptor expressing cell lines, such as A431 cervical carcinoma cells, a result which agrees with the very low cell internalisation found for ⁶⁷GaL in the same cell line. Biodistribution studies in mice indicated high in vivo stability for ⁶⁷GaL, a high total excretion rate and a relatively slow blood clearance, in full accordance with its hydrophilic character and the relatively important protein binding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 A resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep.4, 704-709) and have now solved the structure of Glc-Cerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.     

The NF-κB RelB Protein Is an Oncogenic Driver of Mesenchymal Glioma

High-grade gliomas, such as glioblastomas (GBMs), are very aggressive, invasive brain tumors with low patient survival rates. The recent identification of distinct glioma tumor subtypes offers the potential for understanding disease pathogenesis, responses to treatment and identification of molecular targets for personalized cancer therapies. However, the key alterations that drive tumorigenesis within each subtype are still poorly understood. Although aberrant NF-κB activity has been implicated in glioma, the roles of specific members of this protein family in tumorigenesis and pathogenesis have not been elucidated. In this study, we show that the NF-κB protein RelB is expressed in a particularly aggressive mesenchymal subtype of glioma, and loss of RelB significantly attenuated glioma cell survival, motility and invasion. We find that RelB promotes the expression of mesenchymal genes including YKL-40, a marker of the MES glioma subtype. Additionally, RelB regulates expression of Olig2, a regulator of cancer stem cell proliferation and a candidate marker for the cell of origin in glioma. Furthermore, loss of RelB in glioma cells significantly diminished tumor growth in orthotopic mouse xenografts. The relevance of our studies for human disease was confirmed by analysis of a human GBM genome database, which revealed that high RelB expression strongly correlates with rapid tumor progression and poor patient survival rates. Thus, our findings demonstrate that RelB is an oncogenic driver of mesenchymal glioma tumor growth and invasion, highlighting the therapeutic potential of inhibiting the noncanonical NF-κB (RelB-mediated) pathway to treat these deadly tumors.     

Mortality,Morbidity, and Developmental Outcomes in Infants Born to Women Who Received Either Mefloquine or Sulfadoxine-Pyrimethamine as Intermittent Preventive Treatment of Malaria in Pregnancy: A Cohort Study

Background

Little is known about the effects of intermittent preventive treatment of malaria in pregnancy (IPTp) on the health of sub-Saharan African infants. We have evaluated the safety of IPTp with mefloquine (MQ) compared to sulfadoxine-pyrimethamine (SP) for important infant health and developmental outcomes.

Methods and Findings

In the context of a multicenter randomized controlled trial evaluating the safety and efficacy of IPTp with MQ compared to SP in pregnancy carried out in four sub-Saharan countries (Mozambique, Benin, Gabon, and Tanzania), 4,247 newborns, 2,815 born to women who received MQ and 1,432 born to women who received SP for IPTp, were followed up until 12 mo of age. Anthropometric parameters and psychomotor development were assessed at 1, 9, and 12 mo of age, and the incidence of malaria, anemia, hospital admissions, outpatient visits, and mortality were determined until 12 mo of age. No significant differences were found in the proportion of infants with stunting, underweight, wasting, and severe acute malnutrition at 1, 9, and 12 mo of age between infants born to women who were on IPTp with MQ versus SP. Except for three items evaluated at 9 mo of age, no significant differences were observed in the psychomotor development milestones assessed. Incidence of malaria, anemia, hospital admissions, outpatient visits, and mortality were similar between the two groups. Information on the outcomes at 12 mo of age was unavailable in 26% of the infants, 761 (27%) from the MQ group and 377 (26%) from the SP group. Reasons for not completing the study were death (4% of total study population), study withdrawal (6%), migration (8%), and loss to follow-up (9%).

Conclusions

No significant differences were found between IPTp with MQ and SP administered in pregnancy on infant mortality, morbidity, and nutritional outcomes. The poorer performance on certain psychomotor development milestones at 9 mo of age in children born to women in the MQ group compared to those in the SP group may deserve further studies.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00811421","term_id":"NCT00811421"}}NCT00811421     

Lasuén's pterodactyl: An early use of a pterosaur in plastic arts

Among the sculptures adorning the former building of the Faculties of Medicine and Sciences (1893) in Saragossa (Spain) is one that represents a pterosaur. It is a work by D. Lasuén based on one of the first, but little known, restorations of these animals, drawn and engraved by T. Susemihl more than half a century before. Although it seems out of place, this sculpture was simply seen as a symbol of zoology. We suggest that it may have several significations. Besides embodying the animal kingdom, it may have been a surrogate of the dragon and thus a reference to the House of Aragon through its dragon-slaying holy patron, Saint George.     

Monozygotic and dizygotic twins' retrospective and current bereavement-related behaviors: an evolutionary perspective.

The present study compared bereavement responses of 325 monozygotic (MZ) and 176 dizygotic (DZ) adolescent and adult twins following the loss of their co-twins. A subset of twins completed the Grief Experience Inventory using a retrospective time frame, while a second subset completed it using a current time frame. It was hypothesized that MZ twins (in both retrospective and current groups) would report higher levels of grief-related behavior than DZ twins, consistent with Hamilton's (1964) concept of inclusive fitness. Discriminant function and profile analyses yielded supportive findings, but only for the retrospective MZ and DZ twin comparisons. Females in both groups expressed higher levels of bereavement-related behavior than males. Findings are discussed with reference to theoretical aspects of grief and mourning.     

Photoinhibition and recovery in a herbicide-resistant mutant from<Emphasis Type="Italic"> Glycine max</Emphasis> (L.) Merr. cell cultures deficient in fatty acid unsaturation

Photoinhibition and recovery were studied in two photosynthetic cell suspensions from soybean (Glycine max L. Merr): the wild type (WT) and the herbicide-resistant D1 mutant STR7. This mutant also showed an increase in saturated fatty acids from thylakoid lipids. STR7 was more sensitive to photoinhibition under culture conditions. In vivo photoinhibition experiments in the presence of chloramphenicol, in vitro studies in isolated thylakoid membranes, and immunoblot analysis indicated that the process of light-induced degradation of the D1 protein was not involved in the response of STR7 to light. At growth temperature (24°C), the recovery rate of photoinhibited photosystem II (PSII) was slower in STR7 relative to WT. Photoinhibition and recovery were differentially affected by temperature in both cell lines. The rates of photoinhibition were faster in STR7 at any temperature below 27°C. The rates of PSII recovery from STR7 were more severely affected than those of WT at temperatures lower than 24°C. The photoinhibition and recovery rates of WT at 17°C mimicked those of STR7 at 24°C. In organelle translation studies indicated that synthesis and elongation of D1 were substantially similar in both cell lines. However, sucrose gradient fractionation of chloroplast membranes demonstrated that D1 and also other PSII proteins such as D2, OEE33, and LCHII had a reduced capability to incorporate into PSII to yield a mature assembled complex in STR7. This effect may become the rate-limiting step during the recovery of photoinhibited PSII and may explain the increased sensitivity to high light found in STR7. Our data may hint at a possible role of fatty acids from membrane lipids in the assembly and dynamics of PSII.Abbreviations DCBQ 2,6-Dichloro p-benzoquinone - DM Dodecyl--d-maltoside - DTT Dithiothreitol - HL High light - LHCII Light-harvesting complex II - LL Low light - OEE Oxygen-evolving extrinsic proteins - PAGE Polyacrylamide gel electrophoresis - PG Phosphatidylglycerol - PSII Photosystem II - Q_A and Q_B Secondary quinone electron acceptors from PSII - RC Reaction center - SDS Sodium dodecyl sulfate - WT Wild type