Conditional Maximum Likelihood Estimation Following a Group Sequential Test

We consider estimation after a group sequential test. An estimator that is unbiased or has small bias may have substantial conditional bias (Troendle and Yu, 1999, Coburger and Wassmer, 2001). In this paper we derive the conditional maximum likelihood estimators of both the primary parameter and a secondary parameter, and investigate their properties within a conditional inference framework. The method applies to both the usual and adaptive group sequential test designs. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Active compounds in Populus nigra L. wilted leaves responsible for attracting Helicoverpa armigera (Hübner) (Lep., Noctuidae) and new agaropectin formulation

Abstract:  The leaf extracts of Populus nigra were collected and identified by steam distillation, air entrainment and gas chromatographic–mass spectrometric analysis. Electroantennograms were recorded from Helicoverpa armigera adults in response to the chemicals identified. Both aromatic compounds and green-leaf volatiles elicited strong responses. Field experiments revealed that the active compounds responsible for attracting H. armigera moths are mainly short-side-chain aromatic alcohols and aldehydes. We, for the first time, used agaropectin as the controlled-release matrix of insect attractants. A five-component lure containing all the aromatics without phenolics, mixed in the proportions as found in the steam distillate of the leaves collected in August, produced the best trap catch. The results showed that the volatiles of wilted leaves of P. nigra can attract H. armigera adults by feeding attraction.     

Two integral membrane proteins located in the cis-middle and trans-part of the Golgi system acquire sialylated N-linked carbohydrates and display different turnovers and sensitivity to cAMP-dependent phosphorylation

The localization and chemical characteristics of two Golgi integral membrane proteins (GIMPs) have been studied using monoclonal antibodies. The two proteins are segregated in different parts of the Golgi system and whereas GIMPc(130 kD) is located in the cis and medial cisternae, GIMPt (100 kD) is confined in the trans-most cisterna and trans-tubular network. Both GIMPs are glycoproteins that contain N- and O-linked carbohydrates. The N-linked carbohydrates were exclusively of the complex type. Although excluded from the trans-side of the Golgi system, where sialylation is believed to occur, GIMPc acquires sialic acid in both its N- and O-linked carbohydrates. Sialic acid was also detected in the N-linked carbohydrates of GIMPt. GIMPc is apparently phosphorylated in the luminal domain in vivo. Phosphorylation occurred exclusively on serine and was stimulated by dibutyryl cyclic AMP. GIMPc and GIMPt displayed half-lives of 20 and 9 h, respectively.     

The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations

High-performance electrophoresis chromatography (HPEC) is a recent development that features continuous-elution gel electrophoresis for isolating proteins or peptides in range of 1 to 300 microgram quantities. Column gel electrophoresis is conducted under thermostated conditions, and the field voltage can be varied within a run with a programmable power supply. Applications of this apparatus in protein purification are presented to demonstrate the utility of the (Model 230A) HPEC. These examples include on-line detection with direct analyte recovery of highly purified sample, which mimics high-performance liquid chromatography, for subsequent structure-function characterization. A method to remove salts from sodium dodecyl sulfate electrophoresed samples for subsequent sequencing or amino acid analysis is described. This desalting procedure recovers from 90%-95% of the sample and employs a low molecular weight cut-off membrane during sample centrifugation onto a polyvinylidene difluoride membrane. Subsequent washings are performed to efficiently remove salts, free amino acids and detergents that are known to interfere with sequence analysis. Sequence information such as initial recovery, repetitive yields and chromatogram comparisons are presented to demonstrate the utility of this procedure when used following isolation of sample with HPEC.     

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.

Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFA's effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.     

Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.     

Biosynthesis, glycosylation, movement through the Golgi system, and transport to lysosomes by an N-linked carbohydrate-independent mechanism of three lysosomal integral membrane proteins

The biosynthesis, glycosylation, movement through the Golgi system, transport to lysosomes, and turnover of three lysosomal integral membrane proteins (LIMPSs) have been studied in normal rat kidney cells using specific anti-LIMP monoclonal antibodies. Immunoelectron microscopy studies revealed the presence of LIMPs in secondary lysosomes, Golgi cisterna, and coated and uncoated vesicles located in the trans-Golgi cisterna, area. Pulse-chase experiments recorded LIMP precursors of 27 (LIMP I), 72 (LIMP II), and 86 kDa (LIMP III) and mature LIMPs of 35-50 (LIMP I), 74 (LIMP II), and 90-100 kDa (LIMP III). Time course studies on the acquisition of endoglycosidase H resistance by LIMPs indicated that all three LIMPs moved from the site of their synthesis in the endoplasmic reticulum to the medial Golgi within 30-60 min after their synthesis. All three LIMPs were fully glycosylated before leaving the Golgi system, the process during which LIMP I was retained in the trans side of the organelle. LIMP I reached the lysosomes with a halftime of 2 h and LIMPs II and III with half-times of 1 h after their synthesis by a mechanism that was independent of N-linked carbohydrates. LIMPs free of N-linked carbohydrates displayed much shorter half-lives than fully glycosylated LIMPs, suggesting an important role of the sugars in protecting LIMPs against proteolytic degradation. Double immunofluorescence microscopy experiments showed that LIMP I, LIMP II, and LIMP III are localized in the same lysosomes.     

Dual action of anti-sporozoite antibodies in vitro

With the use of a double staining technique that permits localization of the sporozoite during the process of entering a host cell, we studied the biologic effects of three mAb directed against determinants contained in the circumsporozoite of Plasmodium yoelii. These mAb, which included one IgM and two IgG3, were studied in primary cultures of rodent hepatocytes inoculated with sporozoites of P. yoelii. These results confirm previous reports of the extended action of antibodies on Plasmodium falciparum after entering hepatocytes by producing a strong intrahepatocyte inhibitory effect in addition to the inhibitory effect on sporozoite entry. As with P. falciparum the intracellular effects on P. yoelii liver stages are only observed when the antibodies are present at the time the sporozoite enters the cell. While carrying out experiments on this phenomenon, it was discovered that, at lowered antibody concentrations, an increase in number of maturing liver schizonts occurs, with the increase or enhancement of infection reaching up to 150% of that of controls. It was also observed that there was an inverse relationship between the antibody concentration that was inhibitory and that which enhanced parasite infectivity.     

山东莒南发现的石制品

本文报道的128件人工石制品分别采自山东莒南县的石莲子乡烟敦岭和扁山乡九顶莲花山两个地点。石制品常见类型有边刮器,端刮器和砍斫器等,另有少量的雕刻器和尖状器,内含典型细石器。就石制品的组合及工艺水平而言,其时代可能属于旧石器时代末期。