Fork gratings based on ferroelectric liquid crystals.

In this article, we disclose a fork grating (FG) based on the photo-aligned ferroelectric liquid crystal (FLC). The Digital Micro-mirror Device based system is used as a dynamic photomask to generated different holograms. Because of controlled anchoring energy, the photo alignment process offers optimal conditions for the multi-domain FLC alignment. Two different electro-optical modes namely DIFF/TRANS and DIFF/OFF switchable modes have been proposed where the diffraction can be switched either to no diffraction or to a completely black state, respectively. The FLC FG shows high diffraction efficiency and fast response time of 50µs that is relatively faster than existing technologies. Thus, the FLC FG may pave a good foundation toward optical vertices generation and manipulation that could find applications in a variety of devices.

Protein phosphorylation affects binding of the Escherichia coli transcription activator UhpA to the uhpT promoter.

Expression of the Escherichia coli sugar phosphate transporter UhpT is induced by extracellular glucose 6-phosphate through a transmembrane signaling process dependent on the sensor kinase UhpB and the UhpT homolog, UhpC. These proteins are thought to regulate the phosphorylation of the transcription activator, UhpA. To examine the effect of protein phosphorylation on the binding of UhpA to target sequences in the uhpT promoter region, the UhpA protein was overexpressed and purified. Purified UhpA was phosphorylated by acetyl phosphate in a reaction that was dependent on Mg2+ and on the presence of aspartate 54, the site of phosphorylation in homologous response regulators. Gel electrophoretic mobility shift and DNase I and hydroxyl radical protection assays showed that UhpA bound specifically to the region of the uhpT promoter extending from -80 to -50 bp, relative to the transcription start site. At higher concentrations of UhpA, binding was extended to the -32 region. Binding to the -64 element exhibited positive cooperativity and was stimulated severalfold by phosphorylation of UhpA, whereas extension to the downstream region was more strongly affected by phosphorylation. The consensus sequences for the high affinity UhpA-binding sites in the -64 element and for the downstream, low affinity sites are proposed. The pattern of in vitro binding by UhpA agreed with the in vivo observations that phosphorylation-independent assembly of the transcription initiation complex can occur at elevated concentrations of UhpA.

Sequences of the Escherichia coli BtuB protein essential for its insertion and function in the outer membrane.

The Escherichia coli btuB gene encodes the outer membrane transporter for vitamin B12, the E colicins, colicin A, and bacteriophage BF23. Several series of mutant forms of BtuB resulting from the insertion of dipeptide sequences and from overlapping in-frame deletions and duplications were constructed. Strains expressing the variant genes in single and multiple copy numbers were analyzed for BtuB function, for the level of BtuB polypeptide in the outer membrane, and for changes in the outer membrane permeability barrier. Most dipeptide insertions had normal transport function and assembly in the membrane. Only 2 of the 27 deletions spanning residues 5 and 514 possessed transport function, and most of the remainder were not stably inserted in the membrane. Most duplications (19 of 21) retained transport function and were inserted in the outer membrane, although some were subject to proteolysis. Even long duplications containing as many as 340 repeated amino-terminal residues retained function, suggesting considerable plasticity in the sequence requirements for membrane insertion of BtuB. Expression of many deletion and duplication proteins conferred increased susceptibility to structurally unrelated inhibitors that are normally excluded by the outer membrane. These results could be consistent with the mutational disruption of extracellular loops or transmembrane segments of BtuB that constitute a gated channel, but the finding that alterations throughout the length of BtuB affect membrane permeability properties suggests that the altered proteins might perturb the outer membrane structure itself.

Substitution of class II alpha chain polymorphic residues defines location of A alpha k serologic epitopes and alters association between alpha beta and Ii polypeptides.

Structure-function studies of the MHC class II alpha chain have been performed by constructing a panel of A alpha k cDNA genes with one or more d allele substitutions at each polymorphic residue of the alpha 1 domain. The altered genes (A alpha k*) were transfected into a B lymphoma cell line (BKO), which is deficient in A alpha mRNA but retains constitutive wild-type A beta k mRNA expression. Cytofluorometric analysis of cell surface A alpha k* Ak beta molecules was used to map the polymorphic alpha chain residues comprising four serologic epitopes. A alpha k-reactive mAbs 1E9, 2A2, and 3F12 recognize an epitope that includes the polymorphic residue 44 of the A alpha k polypeptide, and the A alpha k-reactive antibody, K24-199, recognizes a conformationally determined epitope influenced by residues from all three polymorphic regions. In addition, we confirmed previous studies demonstrating that la.19 and la.2 mAbs bind to epitopes adjacent to residue 75 in A alpha k. A cell surface negative expression variant also was identified in the panel of mutant cell lines and biochemically characterized. Substitution of six d allele polymorphic residues at positions 11, 14, 28, 69, 70, and 75 in the A alpha k polypeptide (T.EG A alpha k* A beta k*) results in an A alpha k* polypeptide that associates with the A alpha k polypeptide but does not associate with the li polypeptide. This defect in li-A alpha k A beta k interaction is associated with a conformational change in the alpha 1 beta 1 domain that was identified by altered reactivity of the T.EG complex with conformationally dependent anti-alpha and anti-beta mAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of MHC class II A alpha k transgene. Pairing with endogenous beta-chain is dependent upon affinity and competition.

Studies on cell lines transfected with MHC class II genes have revealed important limitations on the assembly of haplotype-mismatched A alpha/A beta complexes. We have investigated pairing restriction in vivo by analyzing transgenic mice that carry an A alpha k gene on several H-2 haplotypes. Previously, we reported the A alpha k transgenic expression on the cell surface of H-2b and H-2u strains. Further transcomplementation studies performed with high A alpha k-expressing transgenic lines revealed that A alpha k chain can pair with A beta b,s,f,d,v, or u chains but not with A beta q. Homozygosity for the A alpha k transgene increased the expression of mixed heterodimers. The inefficient assembly of haplotype-mismatched class II polypeptides results from their inability to compete with the matched pairs. The higher affinity between the matched pairs can be overcome by increased synthesis of the mismatched pair. Thus, there is no "strict" sequence-based restrictions on pairing.

Triterpenoid glycosides from Decaisnea fargesii.

Five new triterpenoid glycosides designated decaisosides A-E together with five known ones were isolated from the stems of Decaisnea fargesii. Their structures were established by means of chemical and spectroscopic evidence.

Conserved structural and regulatory regions in the Salmonella typhimurium btuB gene for the outer membrane vitamin B12 transport protein.

The Salmonella typhimurium btuB gene encodes an outer membrane protein that is necessary for the transport of vitamin B12 and the uptake of the E colicins and bacteriophage BF23. The sequence of this gene showed 87% identity of the deduced polypeptide to its Escherichia coli homolog, and its product was found to function in transport as effectively in cells of E. coli as did the native protein. The extensive sequence conservation within the first 300 transcribed nucleotides, which include the leader and early part of the coding sequence, supports the proposed role of this region in the regulation of btuB gene expression.

Structure and function of the uhp genes for the sugar phosphate transport system in Escherichia coli and Salmonella typhimurium.

Expression of the Escherichia coli sugar phosphate transport system, encoded by the uhpT gene, is regulated by external glucose 6-phosphate through the action of three linked regulatory genes, uhpABC. The nucleotide sequence of the uhp region cloned from Salmonella typhimurium was determined. The deduced Uhp polypeptide sequences from the two organisms are highly related. Comparison with the corrected sequence from E. coli revealed that the four uhp genes are closely spaced, with minimal intergenic distances, and that uhpC is nearly identical in length to uhpT, both of which have substantial sequence relatedness along their entire lengths. To facilitate analysis of uhp gene function, we isolated insertions of a kanamycin resistance (Km) cassette throughout the uhp region. In-frame deletions that removed almost the entire coding region of individual or multiple uhp genes were generated by use of restriction sites at the ends of the Km cassette. The phenotypes of the Km insertions and the in-frame deletions confirmed that all three regulatory genes are required for Uhp function. Whereas the deletion of uhpA completely abolished the expression of a uhpT-lacZ reporter gene, the deletion of uhpB or uhpC resulted in a partially elevated basal level of expression that was not further inducible. These results indicated that UhpB and perhaps UhpC play both positive and negative roles in the control of uhpT transcription. Translational fusions of the uhpBCT genes to topological reporter gene phoA were generated by making use of restriction sites provided by the Km cassette or with transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with models predicting that UhpC and UhpT have identical transmembrane topologies, with 10 to 12 transmembrane segments, and that UhpB has 4 to 8 amino-terminal transmembrane segments that anchor the polar carboxyl-terminal half of the protein to the cytoplasmic side of the inner membrane.

Host dependence of naturally occurring temperature-sensitive influenza A viruses and location of their genetic lesions.

Naturally occurring temperature-sensitive (ts) strains have been found in large number in human influenza A viruses of all subtypes (J. Virol. 41 (1982) 353). Further studies have demonstrated that the ts phenotype of these viruses is host-dependent in that they are highly ts in chick embryos and chick embryonic cells, but are ts+ in MDCK cells. Previous studies have located by complementation tests the ts lesion of two H3N2 viruses (HK/8/68 and Ningxia/01/72, also known as Xia-ts) on the NP gene and that of two H1N1 viruses (Tianjin/78/77 and Beijing/1/79) on the M protein gene. By recombination and polyacrylamide electrophoresis migration of the RNA segments of its ts+ recombinant with PR8, the ts lesion of a later H3N2 virus A/Qi/39/79 has now been located on the M protein gene. The possibility for Qi/39/79 of acquiring the M gene lesion by reassortment with concurrently circulating Tianjin/78/77-like (H1N1) virus which also has ts lesion on the M gene was investigated. In contrast to Tianjin/78/77 (H1N1), however, Qi/39/79 complemented well with ts 51, a WSN ts strain with a single M gene lesion. Qi/39/79 and Tianjin/78/77 also complemented each other. Thus, there are two intra-segmental complementation groups of the M gene: Qi/39/79 belongs to one complementation group, while WSN ts 51 and Tianjin/78/77 belong to another. At present, there is no evidence of reassortment involving the genes concerned in the ts lesions of H3N2 and H1N1 viruses under natural conditions.

Local structure of a peptide contact site on Ak alpha.

We have sought to determine how much amino acid diversity is tolerable at position 69 of the Ak alpha chain, a position previously implicated as a peptide contact site. Slot-machine mutagenesis was used to create a set of 11 mutant Ak alpha cDNAs, each specifying a different amino acid at position 69. These cDNAs were individually expressed in L cells together with a wild-type Ak beta cDNA to produce a panel of mutant antigen-presenting cell lines. The ability of each member of this panel to present a hen egg lysozyme and a bovine ribonuclease peptide to various T hybridomas was assessed. We found that a surprising degree of amino acid diversity is tolerable at Ak alpha position 69: even charged (Glu, Arg) or bulky (Trp, Tyr) residues can be accommodated without abrogating cell-surface expression of Ak, peptide binding to it, or T cell recognition of it. We discuss the implications of these findings for models of T cell recognition of the class II molecule/antigen duplex.

Functional effects of N-linked oligosaccharides located on the external domain of murine class II molecules.

To evaluate the potential functional role of the alpha- and beta-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II Ak alpha and Ak beta genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The alpha 1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The beta 1 domain acceptor site at positions 19 to 21 was deleted by substituting Gln for Asn at position 19 or Ala for Thr at position 21. The mutant genes (Ak alpha* or Ak beta*) were transfected either individually (mutants T.19, T.21, and T.82) or together (mutant T.82-21) into class II cell surface negative B lymphoma cell lines. Quantitative immunofluorescence with a panel of Ak beta- or Ak alpha- reactive mAb demonstrated that although the oligosaccharide-deleted Ak alpha Ak beta molecules were serologically wild type, the Ad alpha serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 Ak beta* Ad alpha molecules. Cloned cell lines expressing the T.19 or T.21 Ak beta* Ak alpha molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 Ak alpha* Ak beta molecules exhibited defects in Ag presentation function to nine of the ten T hybridomas tested. Surprisingly, cells expressing the mutant T.82-21 class II molecule stimulated a response that was equal to the wild-type response from three of the nine T hybrids and a response that was significantly greater than that of wild-type cells from five of nine T hybridomas. These functional and serological analyses also indicate that some of the observed Ag presentation defects may be due to altered secondary structure caused by either deletion of the oligosaccharide or the amino acid substitution used to delete the N-linked oligosaccharide acceptor site.

A single amino acid substitution in the Ak molecule fortuitously provokes an alloresponse.

We discovered by chance that the R28 T cell hybridoma has dual specificity. It responds to a peptide derived from ribonuclease presented by cells displaying Ak molecules and it reacts, in the absence of added antigen, to cells expressing Ak complexes with a single amino acid substitution at position 69 of the alpha chain. Modelling and functional studies suggest that residue 69 is a peptide contact residue, prompting the hypothesis that R28's alloreactivity is a cross-reactive response to an unknown peptide bound in the 'groove' of the mutant Ak complex. In this report, we employ a competition assay to confirm that this alloresponse involves a groove-binding peptide, demonstrate that this peptide derives from or depends on fetal calf serum and exploit a panel of antigen-presenting cell lines--each displaying an Ak complex with a different position 69 substitution--to establish that the alloresponse is not just a heteroclitic response to ribonuclease, itself. We speculate that much of the alloreactivity against murine class II molecules that is revealed in vitro may prove to be directed at bovine serum-derived peptides, suggesting that in this context, alloreactivity is a misnomer.

Expression and function of mutant Ia antigen in transgenic mice.

Cell surface expression of Ia antigens requires the assembly of alpha and beta heterodimers. We have produced a double transgenic mouse with a wild form Ak alpha gene and a mutant Ak beta (Ak beta MB) gene with d-allele substitution at positions 63 and 65-67. Initial studies indicated that the Ak alpha and Ak beta MB transgenes are not expressed on the surface of lymphoid cells of the transgenic mice. However, when spleen cells were stimulated with LPS prior to FACS analyses, Ak/Ak MB assembly and subsequent surface expression was induced. The tail skins from transgenic founder mice were rejected by the parental mice indicating a role for the mutant antigen on the allograft. In addition, the Ak transgenic mice on H-2q/q background can partially delete V beta 6+ T cells, suggesting the presence of the transgene product in the thymus.

Expression of I-A alpha k and allelic restriction on A alpha/A beta pairing in transgenic mice.

Cell surface expression of class 11 major histocompatibility complex encoded (1a) molecules depends on association of the component alpha and beta chain into a stable heterodimer. Studies on cell lines transfected with MHC class II genes have revealed important limitations on the assembly of haplotype-mismatched A alpha:A beta complex. In order to study alpha:beta chain pairing restrictions in vivo a number of lines of transgenic mice carrying the A alpha k gene were generated. The transgene was studied in the context of H-2b, H-2s, H-2q, H-2u, H-2f and H-2v haplotypes. Initial FACS analysis of spleen and peripheral blood cells showed no A alpha kappa expression. Spleen cells stimulated with LPS and analysed by FACS showed A alpha k expression in three different H-2b strains as well as H-2u, but not in others. These data indicate that A alpha k can associate with A beta b and A alpha k, but not with A beta s, A beta q, A beta f, A beta v.

Cloning and expression of mosquito larvicidal protein gene from a highly toxic local strain of Bacillus sphaericus.

Bacillus sphaericus strain 10 (BS10), isolated from Jiangsu Province of China, is highly toxic to the mosquito larvae. During sporulation, BS10 produces a parasporal crystalline protein which is toxic to the larvae of a number of mosquito species, and extremely toxic against the larvae of Culex pipiens. Using the Escherichia coli cloning vector pAT153, two clones which hybridized with the synthesizing 18-bases oligonucleotides probe have been obtained. One of the recombinants, TG1 (pFL37), contains a 4.0 kb HindIII DNA fragment from BS10, and produces the 43 kd larvicidal toxin protein. Western blotting analysis and biological assay of mosquito larvicidal activity confirm that larvicidal toxin gene of BS10 is expressed in E. coli.

Restriction map of large plasmid pFW1 in Bacillus sphaericus strain 10.

Some strains of Bacillus sphaericus have been found to be toxic to mosquito larvae (1). They differ from Bacillus thuringiensis var. israelensis (BTI) (2,3) in the following aspects: BTI is relatively ineffective in polluted water, and its residual activity in most habitats is limited to a few days after treatment, whereas BS isolates are effective in such habitats, particularly against species of Culex; BS also remains effective longer, as a result of either persistence or recycling. B. sphaericus strain 10 (BS10) was isolated by the Lixiahe Institute of Agricultural Sciences in Jiangsu Province. Its larvicidal activity is much higher than that of BS1593, which was recommended by the World Health Organization for widespread use as a standard strain (1). The mosquito-larvicide gene of BS1593 has been cloned and expressed in E. coli and B. subtilis (4, 5). In the present study, BS10 was investigated to establish whether it contains the plasmid and whether homology exists between the mosquito-larvicide gene in BS and that in BTI.

Immunosuppression of collagen-induced arthritis in mice with an anti-IL-2 receptor antibody.

The use of an anti-IL-2R antibody, 7D4, was investigated in vivo in the suppression of collagen-induced arthritis in mice. 7D4 was shown to reduce the incidence of arthritis in a group of mice immunized with type II collagen as compared with a group similarly immunized with collagen and treated with a control isotype matched anti-Forsmann antibody. 7D4 was also shown to reduce the severity of arthritis in the treated mice as compared to the mice in the group treated with the control antibody. Anti-IL-2R antibodies may be useful in the treatment of autoimmune diseases by selectively suppressing activated T cells.

Suppression of the murine anti-trimellityl (TM) IgE response: induction of B cell tolerance by conjugates of TM and polyvinyl alcohol.

It was previously shown that administration of conjugates of trimellitic anhydride with polyvinyl alcohol (TM-PVA) could suppress the IgE anti-TM immune response of mice sensitized with a TM-ovalbumin conjugate. In the present study, the existence of TM-specific B cell tolerance was shown by cell transfer experiments in which splenic B cells from mice treated with TM-PVA failed to interact with either the helper T (Th) cells of carrier primed recipients or with Th cells derived from carrier-primed donors. In contrast to previous findings from this laboratory indicating that tolerogenic conjugates of PVA and the 2,4-dinitrophenyl group led to both B cell tolerance and activation of suppressor T (Ts) cells, no evidence was obtained for the induction of Ts cells by TM-PVA. Thus, the induction of demonstrable Ts cells by hapten-PVA conjugates may depend on some property conferred by the haptenic group.