Proteinase sensitivity of bacteriophage lambda tail proteins gpJ and pH in complexes with the lambda receptor.

Previous studies have shown that bacteriophage lambda initially binds to liposomes bearing its receptor protein by the tip of the tail fiber (type 1 complex). It then associates more directly so that the hollow tail tube is in direct contact with the membrane (type 2 complex). DNA can be injected across the lipid bilayer into the liposome from type 2 complexes. We show here that gpJ, the tail fiber protein, becomes more sensitive to proteolytic degradation in type 2 complexes, indicating that the tail fiber does not pass into the liposome and that the tail fiber may undergo a conformational change in type 2 complexes. Another bacteriophage protein, pH, is sensitive to proteolytic degradation in free bacteriophage, type 1 complexes, or type 2 complexes formed with free receptor, but is resistant to proteinases in type 2 complexes formed with liposomes. This finding suggests that pH associates with the membrane. We suggest that this association is part of the mechanism by which a transmembrane hole for DNA entry is formed.     

Rab5 association with the angiotensin II type 1A receptor promotes Rab5 GTP binding and vesicular fusion.

Previous studies have demonstrated that the internalization of the angiotensin II type 1A receptor (AT(1A)R) may be mediated by both beta-arrestin-sensitive and -insensitive mechanisms. Therefore, we have used the AT(1A)R carboxyl-terminal tail to screen a rat brain yeast two-hybrid expression library for novel AT(1A)R-interacting proteins that might contribute to the regulation of AT(1A)R internalization. We have identified Rab5a as an AT(1A)R-binding protein that selectively associates with the AT(1A)R and not with the beta2-adrenergic receptor. A Rab5a-S34N mutant defective in GTP binding does not prevent the internalization of the AT(1A)R but does prevent the trafficking of the AT(1A)R into larger hollow cored vesicular structures. Agonist activation of the AT(1A)R promotes both the formation of Rab5a.AT(1A)R protein complexes and Rab5a GTP binding. Rab5a interactions with the AT(1A)R are mediated in part by the last 10 amino acid residues of the AT(1A)R carboxyl-terminal tail, and although a mutant receptor lacking these residues internalizes normally, it does not redistribute into larger hollow vesicles. Our data suggest that AT(1A)R activation modulates Rab5a activity leading to the homotypic fusion of endocytic vesicles. These observations suggest that vesicular cargo proteins, such as the AT(1A)R, may control their targeting between intracellular compartments by directly regulating the activity of components of the intracellular trafficking machinery such as Rab5a.     

Fluorescence studies of the interaction of DNA with Hoechst 33258

Absorption and fluorescence measurements of DNA-Hoechst 33258 complexes at high molar ratio of DNA phosphate to dye are consistent with the existence of two types of bound species. One type (Type I) predominates at high ionic strength, whereas the other (Type II) occurs at low ionic strength. The fluorescence peak (lambda fmax) depends on the excitation wavelength (lambda ex); lambda fmax shifts toward longer wavelength with increasing lambda ex. Optical properties obtained are summarized in the following: for Type I, lambda amax (absorption) = 352 nm, lambda fmax at lambda ex of 335 nm = 460 nm, tau (fluorescence lifetime) = 2.0-2.5 ns; for Type II, lambda amax = 360 nm, lambda fmax at lambda ex of 335 nm = 470 nm, tau = 4.0-5.0 ns. This behavior is interpreted in terms of solvent-solute relaxation. Type I corresponds to less hydrated bound species, while Type II to more hydrated bound species.     

Structural linkage between ligand discrimination and receptor activation by type I interferons

Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.     

Lambda Interferon Renders Epithelial Cells of the Respiratory and Gastrointestinal Tracts Resistant to Viral Infections

Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-α, IFN-β, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-λ) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-λ plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-λ receptor defect. Careful analysis revealed that expression of functional IFN-λ receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-λ contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.The interferon (IFN) system represents a major element of the innate immune response against viral infections (10, 13, 14). Virus-induced IFN is a complex mixture of biologically active molecules, which includes type I and type III IFN. Type I IFN consists of 14 different IFN-α subtypes in the mouse as well as IFN-β, IFN-κ, IFN-ɛ, and limitin, which all signal through the same universally expressed cell surface receptor complex (IFNAR) (30). Type III IFN includes IFN-λ1, IFN-λ2, and IFN-λ3 (21, 28), of which only the latter two are encoded by genes that are expressed in the mouse (22). Type III IFN uses a distinct receptor complex (IL28R) for signaling (21, 28), which appears to be expressed on only a few cell types, including epithelial cells (29). Binding of type I IFN and type III IFN to their cognate receptor complexes triggers signaling cascades that result in the activation of a large number of genes, many of which encode antiviral proteins (10, 32). Type I IFN and type III IFN trigger highly similar gene expression profiles in responsive cells, suggesting that both IFN types might serve similar functions. However, it has to date been largely unclear to which extent IFN-λ might contribute to innate immunity.Using knockout mouse strains that lack receptors for type I IFN (IFNAR1^0/0), type III IFN (IL28Rα^0/0), or both (IFNAR1^0/0IL28Rα^0/0), we have recently shown that IFN-λ contributes to resistance against influenza A virus (FLUAV) (26). Here, we used the same mouse strains to investigate the relative contribution of IFN-λ in resistance against additional viral pathogens that infect the respiratory and gastrointestinal tract and to visualize IFN-λ-responsive cells. We found that the double-knockout mice showed enhanced susceptibility to various viruses that primarily replicate in lung epithelial cells. Our analysis further revealed that epithelial cells of both lung and gastrointestinal tracts can strongly respond to IFN-λ and that IFN-λ inhibited the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in both lung and gastrointestinal tracts.     

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.     

A three amino acid tail following the TM4 region of the N-methyl-D-aspartate receptor (NR) 2 subunits is sufficient to overcome endoplasmic reticulum retention of NR1-1a subunit

The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, we found that the three amino acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino acids following the TM4 region (NR2Delta2) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR2Delta2 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR2Delta2 subunits were cotransfected with either the NR1-4a or NR1-1a(AAA) mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, we found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2BDelta5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence-specific-dependent. Taken together, our results show that the C terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface.     

The elastic behavior of the urinary bladder for large deformations

The purpose of this paper is to investigate the theoretical basis for the pressure-distension behavior of the urinary bladder. A finite strain theory is developed for hollow spherical structures and it is shown that the Treloar model is a good prototype only for rubber balloons. The pressure-extension ratio relationship is inverted to lead a general form of strain energy function, and fitted by an empirical relation involving one exponential. The following form of strain energy function is derived: W(lambda, lambda, lambda -2) = C1 (P(1), a) + P(1)C2 (a, lambda)ea(lambda -1). Where C1(P(1), a) is a constant (N m-2), P(1) is the initial pressure, a is the rate of pressure increase and C2 (a, lambda) a third degree polynomial relation. P(1) and a are experimentally determined through volumetric pressure-distension data. It is verified that this type of energy function is also valid for uniaxial loading experiments by testing strips coming from the same bladder for which P(1) and a were computed. There is a good agreement between the experimental points and the theoretical stress-strain relation. Finally, the strain energy function is plotted as a function of the first strain invariant and appears to be of an exponential nature.     

Mutations That Affect the Efficiency of Translation of mRNA for the cII Gene of Coliphage Lambda

Starting with the lambda pRE-strain lambda ctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, lambda dya2 ctr1 cy3008 and lambda dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations in cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'. --The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII-mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A----G mutation four bases before the initial AUG codon, and cII3059, a GUU----GAU (Val2----Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA Polymerase I-Dependent Mutants of Coliphage Lambda

Mutagenized lysates of bacteriophage lambda were screened for mutants unable to plate on DNA polymerase I-deficient (polA(-)) hosts. The mutants obtained were all recombination deficient (red(-)). These mutants, like red(-) and gam(-) mutants previously isolated by others, grow more poorly than wild-type lambda even on polA(+) hosts (burst size 14 to 30% of wild-type lambda.) In a polA(-) host, the burst size of red(-) and gam(-) mutants is reduced an additional five- to tenfold, and lysis is delayed. Wild-type lambda grows normally in polA(-) hosts. Neither lambdaN(-)nin (which doesn't express red or gam) nor lambdabio phages (from which all or part of the red-gam region is deleted) form plaques on polA(-) hosts. Apparent revertants, able to plate on polA(-) hosts, have been selected from both lambdaN(-)nin and lambdabio. Those derived from N(-)nin seem to be N(-)nin cro(-) mutants; whereas those coming from lambda bio have a new bypass mutation (pas) that lies between genes P and Q.     

The photophore morphology of Selenoteuthis scintillans Voss and other lycoteuthids (Cephalopoda: Lycoteuthidae)

Females and juveniles of Selenoteuthis scintillans have photophores of several structural types, distributed on the tentacles and eyeballs, and within the mantle cavity and tail. Three distinct photophore types can be recognized on the basis of their accessory structures, though their photocytes are identical. The tail and some tentacular photophores (Type 1) lack any accessory optical structures; other tentacular and abdominal photophores (Type 2) have collagenous diffusing fibres; the anal and ocular photophores (Type 3) have a variety of iridosomes but no collagen. The distal tentacular organ is a double structure composed of a unit each of Type 1 and Type 2. Ocular photophores 1 and 5 are also double structures, composed of two Type 3 units. The photophores closely resemble in structure those of Lycoteuthis diadema. The photocytes have a marked fluorescence and luminesce on treatment with dilute hydrogen peroxide. The bio-luminescence intensity of the tail organ may be modified by chromatophore movements and has a blue-green spectral emission.
The photophores of juvenile Lampadioteuthis megaleia are similar in structure to those of Selenoteuthis but somewhat less complex. A comparison between the morphology of the photophores of lycoteuthid and enoploteuthid squids emphasizes the close similarity between the two families. At the ultrastructural level, certain photophores of both families have very characteristic microvillous blood vessels associated with the photocytes.     

Blood group-active carbohydrate chains on the receptor for epidermal growth factor of A431 cells

The antigens expressed on the carbohydrate chains of the receptor for epidermal growth factor of A431 cells were studied by immunoblotting with monoclonal antibodies. Blood group A and the Type 1 based blood group ALeb and Lea antigens were detected as well as antigens associated with unsubstituted, monofucosylated and difucosylated Type 2 blood group chains. The Lea and the difucosylated Type 2 antigen activities were abolished by treating the blotted receptor with endo-beta-galactosidase, indicating that they are expressed on backbone structures of poly-lacto/neolacto type. (The term 'poly-lacto/neolacto' is used here to describe oligosaccharide backbone structures consisting of repeating Type 1, Gal beta 1-3GlcNAc (lacto) or Type 2, Gal beta 1-4GlcNAc (neolacto) sequences.) The glycosidic linkage of oligosaccharides to protein was investigated using Pronase digests of the receptor biosynthetically labelled with [3H]glucosamine or [3H]fucose. The oligosaccharides were alkali-resistant, consistent with N- rather than O-glycosidically linked chains. A proportion of [3H]fucose-labelled glycopeptides was susceptible to endo-beta-galactosidase, confirming the immunoblotting experiment using antibodies against the Lea and the difucosylated Type 2 antigenic determinants. Oligosaccharides were released from the [3H]fucose- and [3H]-glucosamine-labelled glycopeptides by hydrazinolysis. Chromatography of the oligosaccharides on Bio-Gel P6 and Concanavalin A columns indicated a spectrum of oligosaccharides which include those of high mannose type labelled with [3H]glucosamine, and a mixture of oligosaccharides labelled with [3H]fucose and [3H]glucosamine of bi- and multiantennary complex types of which a subpopulation is susceptible to digestion with endo-beta-galactosidase.     

Lambda integrase: armed for recombination

Bacteriophage lambda moves its viral genome into and out of the bacterial chromosome using site-specific recombination. Crystal structures of reaction intermediates in this recombination pathway provide exciting new snapshots of full length lambda integrase interacting with both core and regulatory DNA elements.     

Lambda phagemids and their transducing properties

Two recombinant lambda DNAs, lambda gt::pMB9 and lambda NM::pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized. Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies. Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids. The efficiency of transduction for nonlysogenic E. coli strains with lambda gt::pMB9 phage producing lambda repressor cIts ranges from 10(-7) to 10(-2) transductant colonies per input phage, depending on the temperature and strain used, while lambda NM::pBR322 phage carrying imm21 transduces with a frequency of up to 1. This means that each lambda NM::pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony. The maximal level of transduction with lambda gt::pMB9 was obtained when E. coli cells lysogenic for lambda were used. Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant. In addition, we have found that all lambda gt::pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from lambda NM::pBR322 has not been observed.     

Variations in the unstructured C-terminal tail of interferons contribute to differential receptor binding and biological activity

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunomodulatory properties in cells. All of them bind to the same receptor proteins, IFNAR1 and IFNAR2, with different affinities. While the 13 known IFNalphas are highly conserved, the C-terminal unstructured tail was found to have large variation in its net charge, from neutral to +4. This led us to speculate that the tail may have a role in modulation of the IFN biological activity, through fine-tuning the binding to IFNAR2. To evaluate this hypothesis, we replaced the tail of IFNalpha2 with that of IFNalpha8 and IFNbeta tails, or deleted the last five residues of this segment. Mutations to the more positively charged tail of IFNalpha8 resulted in a 20-fold higher affinity to IFNAR2, which results in a higher antiviral and antiproliferative activity. Double and multiple mutant cycle analysis placed the tail near a negatively charged loop on IFNAR2, comprising of residues Glu 132-134. Deleting the tail resulted in only twofold reduction in binding compared to the wild-type. Next, we modeled the location of the tail using a two-step procedure: first we generated 200 models of the tail docked on IFNAR2 using HADDOCK, second the models were scored according to the fit between experimentally determined rates of association of nine mutant complexes, and their calculated rates using the PARE software. From the results we suggest that the unstructured tail of IFNalpha is gaining a specific structure in the bound state, binding to a groove below the 132-134 loop in IFNAR2.     

Lambda integrase cleaves DNA in cis.

In the Int family of site-specific recombinases, DNA cleavage is accomplished by nucleophilic attack on the activated scissile phosphodiester bond by a specific tyrosine residue. It has been proposed that this tyrosine is contributed by a protomer bound to a site other than the one being cleaved ('trans' cleavage). To test this hypothesis, the difference in DNA binding specificity between closely related integrases (Ints) from phages lambda and HK022 was exploited to direct wild type Ints and cleavage- or activation-defective mutants to particular sites on bispecific substrates. Analysis of Int cleavage at individual sites strongly indicates that DNA cleavage is catalyzed by the Int bound to the cleaved site ('cis' cleavage). This conclusion contrasts with those from previous experiments with two members of the Int family, FLP and lambda Int, that supported the hypothesis of trans cleavage. We suggest explanations for this difference and discuss the implications of the surprising finding that Int-family recombinases appear capable of both cis and trans mechanisms of DNA cleavage.     

NMR Structure of the Amino-Terminal Domain of the Lambda Integrase Protein in Complex with DNA: Immobilization of a Flexible Tail Facilitates Beta-Sheet Recognition of the Major Groove

The integrase protein (Int) from bacteriophage lambda is the archetypal member of the tyrosine recombinase family, a large group of enzymes that rearrange DNA in all domains of life. Int catalyzes the insertion and excision of the viral genome into and out of the Escherichia coli chromosome. Recombination transpires within higher-order nucleoprotein complexes that form when its amino-terminal domain binds to arm-type DNA sequences that are located distal to the site of strand exchange. Arm-site binding by Int is essential for catalysis, as it promotes Int-mediated bridge structures that stabilize the recombination machinery. We have elucidated how Int is able to sequence specifically recognize the arm-type site sequence by determining the solution structure of its amino-terminal domain (Int^N, residues Met1 to Leu64) in complex with its P′2 DNA binding site. Previous studies have shown that Int^N adopts a rare monomeric DNA binding fold that consists of a three-stranded antiparallel beta-sheet that is packed against a carboxy-terminal alpha helix. A low-resolution crystal structure of the full-length protein also revealed that the sheet is inserted into the major groove of the arm-type site. The solution structure presented here reveals how Int^N specifically recognizes the arm-type site sequence. A novel feature of the new solution structure is the use of an 11-residue tail that is located at the amino terminus. DNA binding induces the folding of a 3₁₀ helix in the tail that projects the amino terminus of the protein deep into the minor groove for stabilizing DNA contacts. This finding reveals the structural basis for the observation that the “unstructured” amino terminus is required for recombination.     

Lambda bacteriophage-mediated transduction of ColE1 deoxyribonucleic acid having a lambda bacteriophage-cohesive end site: selection of packageable-length deoxyribonucleic acid.

An in vitro recombinant ColE1-cos lambda deoxyribonucleic acid (DNA) molecule, pKY96, has 70% of the length of lambda phage DNA. The process of lambda phage-mediated transduction of pKY96 generated a small amount of transducing phage particles containing ColE1-cos lambda DNA molecules of 80 or 101% of the length of lambda phage DNA, in addition to those containing original pKY96 DNA molecules. The newly isolated larger plasmid DNAs were transduced 100 times more efficiently than pKY96 DNA. Their structures were compared with that of a prototype pKY96 DNA, and the mechanism of the formation of these molecules is discussed.