Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its function as a carcinoembryonic antigen receptor

Elevated concentrations of carcinoembryonic antigen (CEA) in the blood are associated with the development of hepatic metastases from colorectal cancers. Clearance of circulating CEA occurs through endocytosis by liver macrophages, Kupffer cells. Previously we identified heterogeneous nuclear ribonucleoproteins M4 (hnRNP M4) as a receptor (CEAR) for CEA. HnRNP M4 has two isoform proteins (p80, p76), the full-length hnRNP M4 (CEARL) and a truncated form (CEARS) with a deletion of 39 amino acids between RNA binding domains 1 and 2, generated by alternative splicing. The present study was undertaken to clarify any isoform-specific differences in terms of their function as CEA receptor and localization. We develop anti-CEAR isoform-specific antibodies and show that both CEAR splicing isoforms are expressed on the surface of Kupffer cells and can function as CEA receptor. Alternatively, in P388D1 macrophages CEARS protein has nuclear and CEARL has cytoplasmic localization. In MIP101 colon cancer and HeLa cells the CEARS protein is localized to the nucleus and CEARL to the cytoplasm. These findings imply that different functions are assigned to CEAR isoforms depending on the cell type. The search of 39 amino acids deleted region against the Prosite data base revealed the presence of N-myristylation signal PGGPGMITIP that may be involved in protein targeting to the plasma membrane. Overall, this report demonstrates that the cellular distribution, level of expression, and relative amount of CEARL and CEARS isoforms determine specificity for CEA binding and the expression of alternative spliced forms of CEAR is regulated in a tissue-specific manner.     

Proton conduction through the M2 protein of the influenza A virus; a quantitative,mechanistic analysis of experimental data

The M2 proton channel from influenza A virus forms proton-selective ion channels, which are the target of the drug amantadine. Here, existing experimental data are quantitatively examined for insights into mechanisms to account for the pH- and voltage-dependences of M2 proton conduction. The analysis shows that a model involving protonation equilibria of His37, including pH-dependent changes in the relative rates of diffusion on either side of the pore, is quantitatively able to account for recently reported electrophysiological data examining the pH- and voltage-dependences of Rostock and Weybridge strain M2 proton conduction.     

Genetic Variation of Microsatellite Loci in the Major Histocompatibility Complex (MHC) Region in the Southeast Asian House Mouse (<Emphasis Type="Italic">Mus musculus castaneus</Emphasis>)

Major histocompatibility complex (MHC) genes are the most polymorphic loci known for vertebrates. Here we employed five microsatellite loci closely linked to the MHC region in an attempt to study the amount of genetic variation in 19 populations of the southeast Asian house mouse (Mus musculus castaneus) in Taiwan. The overall polymorphism at the five loci was high (H_e = 0.713), and the level of polymorphism varied from locus to locus. Furthermore, in order to investigate if selection is operating on MHC genes in natural mouse populations, we compared the extent and pattern of genetic variation for the MHC-linked microsatellite loci (the MHC loci) with those for the microsatellite loci located outside the MHC region (the non-MHC loci). The number of alleles and the logarithm of variance in repeat number were significantly higher for the MHC loci than for the non-MHC loci, presumably reflecting linkage to a locus under balancing selection. Although three statistical tests used do not provide support for selection, their lack of support may be due to low statistical power of the tests, to weakness of selection, or to a profound effect of genetic drift reducing the signature of balancing selection. Our results also suggested that the populations in the central and the southwestern regions of Taiwan might be one part of a metapopulation structure.     

Functional analysis of BamHI DNA cytosine-N4 methyltransferase

We show that the kinetic mechanism of the DNA (cytosine-N(4)-)-methyltransferase M.BamHI, which modifies the underlined cytosine (GGATCC), differs from cytosine C(5) methyltransferases, and is similar to that observed with adenine N(6) methyltransferases. This suggests that the obligate order of ternary complex assembly and disassembly depends on the type of methylation reaction. In contrast, the single-turnover rate of catalysis for M.BamHI (0.10s(-1)) is closer to the DNA (cytosine-C(5)-)-methyltransferases (0.14s(-1)) than the DNA (adenine-N(6)-)-methyltransferases (>200s(-1)). The nucleotide flipping transition dominates the single-turnover constant for adenine N(6) methyltransferases, and, since the disruption of the guanine-cytosine base-pair is essential for both types of cytosine DNA methyltransferases, this transition may be a common, rate-limiting step for methylation for these two enzyme subclasses. The similar overall rate of catalysis by M.BamHI and other DNA methyltransferases is consistent with a common rate-limiting catalytic step of product dissociation. Our analyses of M.BamHI provide functional insights into the relationship between the three different classes of DNA methyltransferases that complement both prior structural and evolutionary insights.     

Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.     

Effect of environmental conditions on proteins secreted by enterohemorrhagic Escherichia coli O26:H11

Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal diseases in humans, and these bacteria have recently emerged as a leading cause of renal failure and encephalitis in children and the aged. In this study, we examined the environment-dependent production of proteins secreted from a strain of STEC O26:H11 by trichloroacetic acid precipitation, SDS-PAGE, Western blotting and N-terminal amino acid sequence analysis. Growth of bacteria in essential minimum medium (M9) led to the detection of secreted proteins of 104, 80,40, 37 and 25 kDa (P104, P80, P40, P37 and P25, respectively). When grown in serum-free MEM, only P104, P40, P37 and P25 were observed in supernatant fluids. Growth of the bacteria in Luria-Bertani broth (LB) enhanced the expression of P104, but the productions of the other proteins were remarkably reduced. CO2 increased the secretion of P80 and P37, but reduced the production of P104. N-terminal amino acid sequencing revealed that P104 was EspP of STEC, which was homologous to EspC of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a subclass of the IgA protease family. P80, which was identified as EspE of STEC, was homologous to Tir of EPEC. P40, P37 and P25 were found to be highly homologous to the similarly sized EspD, EspB and EspA proteins, previously detected in culture supernatants of EPEC. Those proteins are thought to be STEC virulence factors. Sera were obtained from two patients, one with colitis and another with hemolytic uremic syndrome (HUS), caused by STEC O157:H7, to study immune response to secreted proteins. Our results suggested that Tir caused immune response following STEC disease.     

Chlorophyll isolation,structure and function: major landmarks of the early history of research in the Russian Empire and the Soviet Union

This paper covers major events of the early history of chlorophyll research in the Russian Empire and the Soviet Union from 1771 until 1952, when the modern period of studies on photosynthesis began in full swing. Short biographical sketches of key scientists, reviews of their major research contributions and some selected photographs are included. This revised version was published online in August 2006 with corrections to the Cover Date.     

Membrane Peptides and their Role in Protobiological Evolution

How simple membrane peptides performed such essential protocellular functions as transport of ions and organic matter across membranes separatingthe interior of the cell from the environment, capture and utilization ofenergy, and transduction of environmental signals, is a key question inprotobiological evolution.On the basis of detailed, molecular-level computer simulations we explainhow these peptides fold at water-membrane interfaces, insert intomembranes, self-assemble into higher-order structures and acquire functions.We have investigated the interfacial behavior and folding of several peptides builtof leucine and glutamine residues and have demonstrated that many of them tendto adopt ordered structures. Further, we have studied the insertion of an -helical peptide containing leucine (L) and serine (S) of the form (LSLLLSL)₃ into a model membrane. The transmembranestate is metastable, and approximately 15 kcal mol^-1 is required to insert the peptide into the membrane.Investigations of dimers formed by (LSLLLSL)₃ and glycophorin A demonstratehow the favorable free energy of helix association can offset the unfavorable free energy of insertion, leading to self-assembly of peptidehelices in the membrane. An example of a self-assembled structure is thetetrameric transmembrane pore of the influenza virus M2 protein, which isan efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism and provide guidelines how to re-engineer thechannel to act as a simple proton pump. In general, emergence of integralmembrane proteins appears to be quite feasible and may be easierto envision than the emergence of water-soluble proteins.     

Resistance to carbosulfan in Anopheles gambiae from Ivory Coast,based on reduced sensitivity of acetylcholinesterase

Resistance to carbosulfan, a carbamate insecticide, was detected in field populations of the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) from two ecologically contrasted localities near Bouaké, Ivory Coast: rural M'bé with predominantly M form of An. gambiae susceptible to pyrethroids; suburban Yaokoffikro with predominantly S form of An. gambiae highly resistant to pyrethroids (96% kdr). The discriminating concentration of 0.4% carbosulfan (i.e. double the LC100) was determined from bioassays with the susceptible An. gambiae Kisumu strain. Following exposure to the diagnostic dosage (0.4% carbosulfan for 1 h), mortality rates of female An. gambiae adults (reared from larvae collected from ricefields) were 62% and 29% of those from M'bé and Yaokoffikro, respectively, 24 h post-exposure. Exposure for 3 min to netting impregnated with the operational dosage of carbosulfan 200 mg/m2 gave mortality rates of 88% of those from M'bé and only 12.2% for Yaokoffikro. In each case the control untreated mortality rate was insignificant. Biochemical assays to detect possible resistance mechanism(s) revealed the presence of insensitive AChE in populations of An. gambiae at both localities, more prevalent in the S form at Yaokoffikro than in M form at M'bé, as expected from bioassays results. Our study demonstrates the need to monitor carbamate resistance among populations of the An. gambiae complex in Africa, to determine its spread and anticipate vector control failure if these insecticides are employed.     

Pyrethroid resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae s.s. malaria vectors in Nigeria and Ghana

Resistance to pyrethroid insecticides and DDT caused by the kdr gene in the malaria vector Anopheles gambiae Giles s.s. (Diptera: Culicidae) has been reported in several West African countries. To test for pyrethroid resistance in two more countries, we sampled populations of the An. gambiae complex from south-western Ghana and from urban and rural localities in Ogun State, south-west Nigeria. Adult mosquitoes, reared from field-collected larvae, were exposed to the WHO-recommended discriminating dosage of exposure for 1 h to DDT 4%, deltamethrin 0.05% or permethrin 0.75% and mortality was recorded 24 h post-exposure. Susceptibility of An. gambiae s.l. to DDT was 94-100% in Ghana and 72-100% in Nigeria, indicating low levels of DDT resistance. Deltamethrin gave the highest mortality rates: 97-100% in Ghana, 95-100% in Nigeria. Ghanaian samples of An. gambiae s.l. were fully susceptible to permethrin, whereas some resistance to permethrin was detected at 4/5 Nigerian localities (percentage mortalities 75, 82, 88, 90 and 100%), with survivors including both An. arabiensis Patton and An. gambiae s.s. identified by PCR assay. Even so, the mean knockdown time was not significantly different from a susceptible reference strain, indicating absence or low frequency of kdr-type resistance. Such low levels of pyrethroid resistance are unlikely to impair the effectiveness of pyrethroid-impregnated bednets against malaria transmission. Among Nigerian samples of An. gambiae s.l., the majority from two urban localities were identified as An. arabiensis, whereas the majority from rural localities were An. gambiae s.s. These findings are consistent with those of M. Coluzzi et al. (1979). Differences of ecological distribution between molecular forms of An. gambiae s.s. were also found, with rural samples almost exclusively of the S-form, whereas the M-form predominated in urban samples. It is suggested that 'urban island' populations of An. arabiensis and of An. gambiae s.s. M-form in the rainforest belt of West Africa might be appropriate targets for elimination of these malaria vectors by the sterile insect technique.