Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34+ hematopoietic progenitors in presence of GM-CSF+TNF alpha for 12 d. The present study demonstrates that cord blood CD34+ HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CD1a and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CD80, CD83, CD86, CD58, high HLA class II). CD1a+ precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14+ progenitors mature into CD1a+ DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIIIa described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA+ naive T cells. Interestingly, the CD14+ precursors, but not the CD1a+ precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T cells. Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14(+)-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.

TLRs are involved in innate cell activation by conserved structures expressed by microorganisms. Human T cells express the mRNA encoding most of TLRs. Therefore, we tested whether some TLR ligands may modulate the function of highly purified human CD4+ T lymphocytes. We report that, in the absence of APCs, flagellin (a TLR5 ligand) and R-848 (a TLR7/8 ligand) synergized with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent stimuli (anti-CD2 mAbs or IL-2) to up-regulate proliferation and IFN-gamma, IL-8, and IL-10 but not IL-4 production by human CD4+ T cells. No effect of poly(I:C) and LPS, ligands for TLR3 and TLR4, respectively, was detected. We also observed that CD4+CD45RO+ memory T cell responses to TLR ligands were more potent than those observed with CD4+CD45RA+ naive T cells. Moreover, among the memory T cells, CCR7- effector cells were more sensitive to TLR ligands than CCR7+ central memory cells. These data demonstrate for the first time a direct effect of TLR5 and TLR7/8 ligands on human T cells, and highlight an innate arm in T cell functions. They also suggest that some components from invading microorganisms may directly stimulate effector memory T cells located in tissues by up-regulating cytokine and chemokine production.

A direct quantitative and phenotypic cytofluorographic analysis of TCR-gamma/delta+ lymphocytes as well as an immunohistologic study of their tissue distribution and microanatomy was made possible by the availability of two mAbs (anti-TCR-delta 1 and anti-C gamma M1) specific for framework determinants on human TCR gamma and delta chains, respectively. TCR-gamma/delta+ lymphocytes, ranging between greater than 0.5 and 16% of CD3+ cells, were found in fetal and postnatal thymus, fetal and adult peripheral lymphoid organs, and adult peripheral blood. While TCR-gamma/delta+ lymphocytes comprised a small subpopulation of T cells (mean, approximately 4%) occasionally greater than 10-16% of CD3+ cells expressed TCR-gamma/delta. Virtually all TCR-gamma/delta+ thymocytes/lymphocytes expressed CD7, <em>CD2</em>, and CD5 but were heterogeneous with respect to their expression of CD1, CD4, CD8, <em>CD2</em>8, CD11b, CD16, and Leu-7. Human TCR-gamma/delta+ cells populate both organized lymphoid tissues (thymus, tonsil, lymphnode, and spleen) as well as the gut- and skin-associated lymphoid systems at similar frequencies without obvious tropism for epithelial microenvironments. TCR-gamma/delta+ lymphocytes tend to be located within a given organ wherever TCR-alpha/beta+ lymphocytes are found. This study shows that TCR-gamma/delta+ lymphocytes constitute a small but numerically important, phenotypically diverse T cell population distributed throughout the body. These results support the concept that TCR-gamma/delta+ cells comprise a distinct, functionally heterogeneous, mature T cell sublineage that may substantially broaden the T cell repertoire at all immunologically relevant sites.

The intracellular calcium ([Ca2+]i) transient in adult rat heart cells was examined using the fluorescent calcium indicator fluo-3 and a laser scanning confocal microscope. We find that the electrically evoked [Ca2+]i transient does not rise at a uniform rate at all points within the cell during the [Ca2+]i transient. These spatial non-uniformities in [Ca2+]i are observed immediately upon depolarization and largely disappear by the time the peak of the [Ca2+]i transient occurs. Importantly, some of the spatial non-uniformity in [Ca2+]i varies randomly in location from beat to beat. Analysis of the spatial character of the non-uniformities suggests that they arise from the stochastic nature of the activation of SR calcium-release channels. The non-uniformities in [Ca2+]i are markedly enhanced by low concentrations of Cd2+, suggesting that activation of L-type calcium channels is the primary source of activator calcium for the calcium transient. In addition, the pattern of calcium release in these conditions was very similar to the spontaneous calcium sparks that are observed under resting conditions and which are due to spontaneous calcium release from the SR. The spatial non-uniformity in the evoked [Ca2+]i transient under normal conditions can be explained by the temporal and spatial summation of a large number of calcium sparks whose activation is a stochastic process. The results are discussed with respect to a stochastic local control model for excitation-contraction (E-C) coupling, and it is proposed that the fundamental unit of E-C coupling consists of one dihydropyridine receptor activating a small group of ryanodine receptors (possibly four) in a square packing model.

ATF6 is an endoplasmic reticulum (ER) transmembrane transcription factor that is activated by the ER stress/unfolded protein response by cleavage of its N-terminal half from the membrane. We find that ER stress induces ATF6 to move from the ER to the Golgi, where it is cut in its luminal domain by site 1 protease. ATF6 contains a single transmembrane domain with 272 amino acids oriented in the lumen of the ER. We found that this luminal domain is required for the translocation of ATF6 to the Golgi and its subsequent cleavage, and we have mapped regions required for these properties. These results suggest that the conserved CD1 region is required for translocation, whereas the CD2 region is required for site 1 protease cleavage. We also find that ATF6's luminal domain is sufficient to sense ER stress and cause translocation to the Golgi when fused to LZIP, another ER transmembrane protein. These results show that ATF6 has a mechanism to sense ER stress and respond by translocation to the Golgi.

Ca2+ inward currents evoked by membrane depolarization have been studied by the intracellular dialysis technique in the somatic membrane of isolated dorsal root ganglion neurones of new-born rats. In about 20% of the investigated cells a hump has been detected on the descending branch of the current-voltage curve, indicating the presence of two populations of Ca2+ channels differing in their potential-dependent characteristics. An initial less regular component of the Ca2+ current was activated at membrane potentials from -75 to -70 mV. Its amplitude reached 0.2-0.9 nA at 14.6 mM-extracellular Ca2+. The activation kinetics of this component could be approximated by the Hodgkin-Huxley equation using the square of the m variable. tau m varied in the range from 8 to 1 ms at potentials between -60 and -25 mV ('fast' Ca2+ current). The second component of the Ca2+ current was activated at membrane depolarizations to between -55 and -50 mV. It could be recorded in all cells investigated and reached a maximum value of 1-7 nA at the same extracellular Ca2+ concentration. This component decreased rapidly during cell dialysis with saline solutions. The decrease could be slowed down by cooling and accelerated by warming the extracellular solution. Intracellular introduction of 3',5'-cAMP together with ATP and Mg2+ not only prevented the decrease but often restored the maximal current amplitude to its initial level. The activation kinetics of this component could also be approximated by a square function, tau m being in the range 16-2.5 ms at membrane potentials between -20 and +3 mV ('slow' Ca2+ current). The fast Ca2+ current inactivated exponentially at sustained depolarizations in a potential-dependent manner, tau h varying from 76 to 35 ms at potentials between -50 and -30 mV. The inactivation of the slow Ca2+ current studied in double-pulse experiments was current-dependent and developed very slowly (time constant of several hundreds of milliseconds). It slowed down even more at low temperature or after substitution of Ba2+ for Ca2+ in the extracellular solution. Both currents could also be carried by Ba2+ and Sr2+, although the ion-selecting properties of the two types of channels showed quantitative differences. Specific blockers of Ca2+ channels (Co2+, Mn2+, Cd2+, Ni2+ or verapamil) exerted similar effects on them. The existence of metabolically dependent and metabolically independent Ca2+ channels in the neuronal membrane and their possible functional role are discussed.

Proteolytic cleavage of von Willebrand factor (vWF) takes place in the circulating blood of healthy subjects and is increased in some patients with von Willebrand disease type 2A. The hemostatically active large vWF multimers are degraded to smaller less active forms. It has been suggested that the polypeptide subunit of vWF is cleaved at the peptide bond 842Tyr-843Met. We purified (approximately 10,000-fold) from human plasma a vWF-degrading protease, using chelating Sepharose, hydrophobic interaction chromatography, and gel filtration. The enzyme was found to be virtually absent in the platelet lysates obtained by repeated freezing and thawing. The proteolytic activity was associated with a high molecular weight protein (approximately 300 kD) as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. vWF was resistant against the protease in a neutral buffer at physiological ionic strength but became degraded at low salt concentration or in the presence of 1 mol/L urea. No degradation of human fibrinogen, bovine serum albumin, of calf skin collagen by the purified protease was noted under the same experimental conditions. Proteolytic activity showed a pH optimum at 8 to 9 and was strongly inhibited by chelating agents, whereas only slow inhibition was observed with N-ethylmaleimide. There was no inhibition by iodoacetamide, leupeptin, or serine protease inhibitors. The best peptidyl diazomethyl ketone inhibitor was Z-Phe-Phe-CHN2. Activation by divalent metal ions was found to increase in the following order: Zn2+ approximately Cu2+ approximately <em>CD2</em>+ approximately Ni2+ approximately Co2+ <Mn2+ <Mg2+ <Ca2+ <Sr2+ <Ba2+. The observed properties of the vWF-degrading enzyme differ from those of all other hitherto described proteases. Purified vWF was incubated with the protease, and the degraded material subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis after disulfide reduction. The size, amino acid composition, and amino terminal sequence of the reduced fragments confirmed that the peptide bond 842Tyr-843Met had been cleaved, ie, the same bond that has been proposed to be cleaved in vivo.

Tat, the transactivating protein from HIV, forms a metal-linked dimer with metal ions bridging cysteine-rich regions from each monomer. This novel arrangement is distinct from the "zinc finger" domain observed in other eukaryotic regulatory proteins. Ultraviolet absorption spectra show that Tat binds two Zn2+ or two Cd2+ ions per monomer, and electrophoresis of the Tat-metal complexes demonstrates that the protein forms metal-linked dimers. Partial proteolysis and circular dichroism spectra suggest that metal binding has its primary effects in the cysteine-rich region and relatively little effect on the folding of other regions. These results suggest new directions for biological studies and new approaches to drug design.

The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.

The direct effects of IL-10 on the proliferation and lymphokine production of human peripheral blood T cells and CD4+ T cell clones representing the Th0, Th1-like, and Th2-like Th cell subsets were investigated in the absence of professional APC. IL-10 partially inhibited the proliferative responses of CD4+ human T cell clones induced by anti-<em>CD2</em> or anti-CD3 mAb cross-linked on CD32 (Fc gamma RII)-transfected mouse L cells. Transfection of ICAM-1 or LFA-3 in CD32+ L cells resulted in enhanced proliferative responses of CD4+ T cell clones after activation by anti-CD3 mAb, whereas transfection of B7 in CD32+ L cells enhanced proliferative responses of CD4+ T cell clones after activation by anti-<em>CD2</em> mAb. In addition, B7 expression on CD32+ L cells was required for activation of small resting T cells by anti-CD3 or anti-<em>CD2</em> mAb. IL-10 inhibited the proliferation of T cell clones induced by anti-<em>CD2</em> or anti-CD3 mAb on CD32+ L cells expressing these accessory molecules, indicating that interactions of LFA-3, ICAM-1, and B7 with their ligands on T cells did not overcome the inhibitory effects of IL-10. Inhibition of proliferation of T cell clones by IL-10 was in all instances completely neutralized by relatively low concentrations of IL-2, whereas IL-4 was ineffective. IL-10 did not affect the expression of the TCR/CD3 complex, <em>CD2</em>, LFA-1, <em>CD2</em>8, or IL-2R alpha- or beta-chains, nor did it inhibit the induction of the latter two molecules on T cells after activation. Inhibition of proliferation was found to be the result of specific inhibition of IL-2 production by the responding T cell subsets, which occurred at the mRNA level. The production and mRNA levels of IL-4, IL-5, IFN-gamma, and granulocyte/macrophage-CSF were not affected by IL-10. Taken together, these results indicate that IL-10/IL-10R interaction on CD4+ T cell clones and peripheral blood T cells results in signaling pathways that specifically interfere with activation processes leading to IL-2 production. These direct inhibitory effects on IL-2 production by activated T cells may contribute to the general immunosuppressive activities of IL-10.

A single-electrode voltage-clamp technique was employed on in vitro hippocampal slices to examine the membrane current responsible for the slow afterhyperpolarization (AHP) in CA1 pyramidal cells. This was achieved by using conventional procedures to evoke an AHP in current clamp, followed rapidly by a switch into voltage clamp (hybrid clamp). The AHP current showed a dependence on extracellular K+, which was close to that predicted for a K+ current by the Nernst equation. The AHP current could be blocked by Cd2+ or norepinephrine. Although the AHP current showed a requirement for voltage-dependent Ca2+ entry, the current did not show any clear intrinsic voltage dependence. Once activated, AHP current is not turned off by hyperpolarizing the membrane potential. The effects of norepinephrine, Cd2+, and tetraethylammonium (TEA) were used to identify an AHP current component to the outward current evoked by depolarizing voltage commands from holding potentials that approximate to the resting potential for these cells. The AHP current can contribute significantly to the outward current during the depolarizing command. Upon repolarization it is evident as a slow outward tail current. This slow tail current had the same time constant as AHP currents evoked by hybrid clamp. Fast components to the tail currents were also observed. These were sensitive to Cd2+ and TEA. They probably represent a voltage-sensitive gKCa, sometimes termed C-current. The strong sensitivity to voltage and TEA displayed by the conventionally described gKCa (IC) are properties inconsistent with the AHP. It seems likely that the AHP current (IAHP) represents a Ca2+-activated K+ current separate from IC and that these two currents coexist in the same cell.

Membrane currents were recorded from voltage-clamped, EGTA-loaded muscle fibres under conditions where currents through ordinary Na+, K+ and Cl- channels were prevented by drugs or by absence of permeant ions (K+, and Cl-). At 10 mM-external [Ca2+], substitution of Na+ for the large and presumably impermeant organic cations tetramethyl- (TMA+) or tetraethylammonium (TEA+) failed to increase peak inward current. Hence the Ca2+ channel was not significantly permeable to Na+ under these conditions. When external [Ca2+] was reduced to levels below 1 microM in the presence of external Na+, step depolarizations to negative potentials produced tetrodotoxin-resistant inward currents. At -20 mV, they rose to a peak of 30-200 microA/cm2 within 150 ms and declined thereafter. Ca2+ and several other divalent cations reversibly blocked this inward current. The sequence of blocking potencies was Ca2+ greater than Sr2+ greater than or equal to Co2+ greater than Mn2+ congruent to Cd2+ greater than Ni2+ congruent to Mg2+. Large inward currents may be carried by Li+, Na+, K+, Rb+ and Cs+ but not by TMA+ and TEA+. The effect of external Ca2+ ([Ca2+]o) was explored over a 10(8)-fold range in concentrations. Na+ was present at a fixed concentration. When [Ca2+]o was gradually increased from 10(-10) to 10(-2) M, inward current first diminished 10-fold, reached a minimum at [Ca2+]o = 60 microM and then increased again as [Ca2+]o was increased further and Ca2+ itself became a current carrier. Block of inward current at [Ca2+]o less than 10(-5) M could be described by binding of a single Ca2+ to a site, with a dissociation constant of the order of 0.7 microM at -20 mV.

This paper provides a brief overview of the diversity of voltage-gated Ca2+ channels and our recent work on neuronal Ca2+ channels with novel pharmacological and biophysical properties that distinguish them from L, N, P or T-type channels. The Ca2+ channel alpha 1 subunit known as alpha 1A or BI [Mori Y., Friedrich T., Kim M.-S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T., Mikoshiba K., Imoto K., Tanabe T. and Numa S. (1991) Nature 350, 398-402] is generally assumed to encode the P-type Ca2+ channel. However, we find that alpha 1A expressed in Xenopus oocytes differs from P-type channels in its kinetics of inactivation and its degree of sensitivity to block by the peptide toxins omega-Aga-IVA and omega-CTx-MVIIC [Sather W. A., Tanabe T., Zhang J.-F., Mori Y., Adams M. E. and Tsien R. W. (1993) Neuron 11, 291-303]. Thus, alpha 1A is capable of generating a Ca2+ channel with characteristics quite distinct from P-type channels. Doe-1, recently cloned from the forebrain of a marine ray, is another alpha 1 subunit which exemplifies a different branch of the Ca2+ channel family tree [Horne W. A., Ellinor P. T., Inman I., Zhou M., Tsien R. W. and Schwarz T. L. (1993) Proc. Natn. Acad. Sci. U.S.A. 90, 3787-3791]. When expressed in Xenopus oocytes, doe-1 forms a high voltage-activated (HVA) Ca2+ channel [Ellinor P. T., Zhang J.-F., Randall A. D., Zhou M., Schwarz T. L., Tsien R. W. and Horne W. (1993) Nature 363, 455-458]. It inactivates more rapidly than any previously expressed calcium channel and is not blocked by dihydropyridine antagonists or omega-Aga-IVA. Doe-1 current is reduced by omega-CTx-GVIA, but the inhibition is readily reversible and requires micromolar toxin, in contrast to this toxin's potent and irreversible block of N-type channels. Doe-1 shows considerable sensitivity to block by Ni2+ or Cd2+. We have identified components of Ca2+ channel current in rat cerebellar granule neurons with kinetic and pharmacological features similar to alpha 1A and doe-1 in oocytes [Randall A. D., Wendland B., Schweizer F., Miljanich G., Adams M. E. and Tsien R. W. (1993) Soc. Neurosci. Abstr. 19, 1478]. The doe-1-like component (R-type current) inactivates much more quickly than L, N or P-type channels, and also differs significantly in its pharmacology.(ABSTRACT TRUNCATED AT 400 WORDS)

Addition of membrane vesicles prepared from transverse tubule (T-tubule) membranes of rabbit skeletal muscle to the aqueous phase of a planar lipid bilayer induces a stepwise increase in conductance. This conductance is both voltage and Ca2+ dependent. At 1 mM Ca2+, the steady-state conductance is maximal at voltages higher than +20 mV and decreases for more negative voltages. (Voltages refer to the side to which the vesicles are added, cis) Decreasing the Ca2+ concentration reversibly shifts the conductance-voltage curve toward the right along the voltage axis. Furthermore, Ca2+ can activate the conductance only if added to the cis compartment. Neither Mg2+, Ba2+, nor Cd2+ can activate the conductance induced by T-tubule vesicles. Addition of 5 mM tetraethylammonium ion to the trans, but not the cis, side abolishes the T-tubule-induced conductance. The Ca2+-dependent conductance appears as a consequence of ionic channel formation. Single-channel activity appears in bursts followed by periods of time in which the channel remains "silent". The conductance of the open channel averages 226 pS in 0.1 M KC1 and is voltage and Ca2+ independent. However, the fraction of time that the channel remains in the open state is voltage and Ca2+ dependent in a manner that parallels the voltage and Ca2+ dependence of the multichannel membrane. The channel is 6.6 times more permeable to K+ than to Na+ and is impermeable to C1-.

Sequence similarity between alpha B-crystallin and small heat shock proteins (HSPs) has prompted us to investigate whether alpha B-crystallin expression is induced by heat shock. Indeed, accumulation of alpha B-crystallin was detected immunologically in NIH 3T3 cells after incubation at elevated temperatures and after addition of Cd2+ or sodium arsenite to these cells. Two-dimensional gel electrophoresis revealed identity between alpha B-crystallin from eye lenses and from heat-treated fibroblasts. The promoter of the alpha B-crystallin gene was fused to the bacterial chloramphenicol acetyltransferase gene and was shown to confer heat inducibility on this reporter gene in transient transfection assays. A perfect heat shock element within the promoter region is likely to mediate this response. Small HSPs and alpha B-crystallin were shown to share the following two physical properties: (i) they form supramolecular structures with sedimentation values around 17 S and (ii) they are associated with the nucleus at high temperatures and are localized in the cytoplasm under normal conditions. We conclude that alpha B-crystallin has to be considered a member of the class of small HSPs.

We present an analysis of the complete genome of African swine fever virus (ASFV) strain BA71V, including 80 kbp of novel sequence and 90 kbp previously reported by several authors. The viral DNA is 170,101 nucleotides long and contains 151 open reading frames. Structural and/or functional information is available on 113 viral proteins. ASFV encodes five multigene families, putative membrane and secreted proteins, and enzymes involved in nucleotide and nucleic acid metabolism (including DNA repair) and protein modification. Database comparisons have provided clues about genes that may modulate the virus-host interaction, thus, possibly controlling ASFV virulence and persistence. The virus possesses genes similar to CD2, IkappaB, C-type lectins, MyD116/gadd34/gamma, 34.5, bcl-2/bax, iap, NifS, and ERV1, which may allow a viral regulation of cell adhesion, apoptosis, and redox metabolism, as well as of the host immune response against ASFV infection. The proteins encoded by different ASFV isolates are highly similar, the most variable ones being those belonging to multigene families, some membrane proteins, and those containing tandem repeats. DNA sequence data confirm the intermediate characteristics of ASFV between poxviruses and iridoviruses, supporting the notion that ASFV belongs to an independent virus family.

We have localized a set of T cell-specific DNAase I hypersensitive sites in the 3'-flanking region of the human CD2 gene. A 5.5 kb BamHI-XbaI fragment containing these DNAase I hypersensitive sites conferred efficient, copy number-dependent, T cell-specific expression of a linked human CD2 minigene, independent of the position of integration in the transgenic mouse genome. When linked to the mouse Thy-1.1 gene or the human beta-globin gene, this fragment conferred the same T cell-specific expression, independent of its orientation. These results suggest that this flanking region is both necessary and sufficient for full tissue-specific activation of homologous and heterologous genes in transgenic mice.

The structurally related T cell surface molecules CD2CD2CD2CD2CD2> or = 1.6 and>> or = 0.43 s-1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate-dynamic T cell-APC contacts and to facilitate scanning of APC for antigen.

Experiments in vitro suggest that although interleukin 5 (IL-5) stimulates the late stages of eosinophil differentiation, other cytokines are required for the generation of eosinophil progenitor cells. In this study transgenic mice constitutively expressing the IL-5 gene were established using a genomic fragment of the IL-5 gene coupled to the dominant control region from the gene encoding human CD2. Four independent eosinophilic transgenic lines have thus far been established, two of which with 8 and 49 transgene copies, are described in detail. These mice appeared macroscopically normal apart from splenomegaly. Eosinophils were at least 65- and 265-fold higher in blood from transgenics, relative to normal littermates, and approximately two- or sevenfold more numerous relative to blood from mice infected with the helminth Mesocestoides corti. Much more modest increases in blood neutrophil, lymphocyte, and monocyte numbers were noted in transgenics, relative to normal littermates (less than threefold). Thus IL-5 in vivo is relatively specific for the eosinophil lineage. Large numbers of eosinophils were present in spleen, bone marrow, and peritoneal exudate, and were highest in the line with the greatest transgene copy number. Eosinophilia was also noted in histological sections of transgenic lungs, Peyer's patches, mesenteric lymph nodes, and gut lamina propria but not in other tissues examined. IL-5 was detected in the sera of transgenics at levels comparable to those seen in sera from parasite-infected animals. IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were not found. IL-5 mRNA was detected in transgenic thymus, Peyer's patches, and superficial lymph nodes, but not in heart, liver, brain, or skeletal muscle or in any tissues from nontransgenics. Bone marrow from transgenic mice was rich in IL-5-dependent eosinophil precursors. These data indicate that induction of the IL-5 gene is sufficient for production of eosinophilia, and that IL-5 can induce the full pathway of eosinophil differentiation. IL-5 may therefore not be restricted in action to the later stages of eosinophil differentiation, as suggested by earlier in vitro studies.

Calcium current, Ica, was studied in isolated nerve cell bodies of Helix aspersa after suppression of Na+ and K+ currents. The suction pipette method described in the preceding paper was used. Ica rises to a peak value and then subsides exponentially and has a null potential of 150 mV or more and a relationship with [Ca2+]o that is hyperbolic over a small range of [Ca2+]o's. When [Ca2+]i is increased, Ica is reduced disproportionately, but the effect is not hyperbolic. Ica is blocked by extracellular Ni2+, La3+, Cd2+, and Co2+ and is greater when Ba2+ and Sr2+ carry the current. Saturation and blockage are described by a Langmuir adsorption relationship similar to that found in Balanus. Thus, the calcium conductance probably contains a site which binds the ions referred to. The site also appears to be voltage-dependent. Activation and inactivation of Ica are described by first order kinetics, and there is evidence that the processes are coupled. For example, inactivation is delayed slightly in its onset and tau inactivation depends upon the method of study. However, the currents are described equally well by either a noncoupled Hodgkin-Huxley mh scheme or a coupled reaction. Facilitation of Ica by prepulses was not observed. For times up to 50 ms, currents even at small depolarizations were accounted for by suitable adjustment of the activation and inactivation rate constants.

The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded beta-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2 (ref. 5). A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous 'substrate groove' around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte-mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined.

On the basis of similarities in sequence and structure, the protein domains that form the immunoglobulin superfamily have been divided into three sets: one with variable-like domains, the V set, and two with different variants of the constant-like domains, the C1 and C2 sets. Examination of a muscle member of the immunoglobulin superfamily, telokin, shows that its structure is closely related to those of the variable domains found in antibodies, CD2, CD4 and CD8. However, it also contains structural features that, previously, have only been found in constant domains. Telokin represents a new structural set in the superfamily which we call the I set. Using the structures of telokin, and variable domains from antibodies, CD4 and CD8, we constructed a profile that describes the sequence characteristics of the structural core common to those proteins. This sequence profile makes a good match to the sequences of many of the immunoglobulin superfamily domains that form the cell adhesion molecules and surface receptors. This match implies that these domains also have structures that belong to the I set.

Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3+ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR = 2.13, P = 0.0008). An immune response gene expression signature and presence of TILs and CD3+ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging.