Xenopus Kielin: A dorsalizing factor containing multiple chordin-type repeats secreted from the embryonic midline

The midline tissues are important inductive centers of early vertebrate embryos. By signal peptide selection screening, we isolated a secreted factor, Kielin, which contains multiple cys-rich repeats similar to those in chordin (Chd). Expression of Kielin starts at midgastrula stages in the notochord and is detected in the floor plate of neurula embryos. Kielin is induced in mesoderm and in ectoderm by nodal-related genes. Chd is sufficient to activate Kielin expression in mesoderm whereas Shh or HNF-3β in addition to Chd is required for induction in ectoderm. Kielin has a distinct biological activity from that of Chd. Injection of Kielin mRNA causes dorsalization of ventral marginal zone explants and expansion of MyoD expression in neurula embryos. Unlike Chd, Kielin does not efficiently induce neural differentiation of animal cap ectoderm, suggesting that the activity of Kielin is not simply caused by BMP4 blockade. Kielin is a signaling molecule that mediates inductive activities of the embryonic midline.

Characterization of a replication-incompetent adenovirus type 5 mutant deleted for the preterminal protein gene

An adenovirus type 5 mutant deleted for the preterminal protein (pTP) gene was constructed using cell lines that express pTP. The pTP deletion mutant virus is incapable of replicating in the absence of complementation and does not express detectable levels of viral mRNAs that are expressed only after the onset of replication. Accumulation of early-region mRNAs, including that for E1A, exhibits a lag relative to that observed from the wild-type virus. However, E1A mRNA accumulation attains a steady-state level similar to the level of expression during the early phase of infection with the wild-type virus. In 293-pTP cells (human embryonic kidney cells that express pTP in addition to high levels of adenovirus E1A and E1B proteins), the pTP deletion mutant virus replicates efficiently and yields infectious titers within 5-fold of that of the wild-type virus. The deletion of 1.2 kb of pTP-encoding sequence increases the size of foreign DNA that can be introduced into the virus and, with an absolute block to replication, makes this virus an important tool for gene therapy.

Early expression of antiinsulin autoantibodies of humans and the NOD mouse: Evidence for early determination of subsequent diabetes

With the development of an insulin autoantibody (IAA) assay performed in 96-well filtration plates, we have evaluated prospectively the development of IAA in NOD mice (from 4 weeks of age) and children (from 7 to 10 months of age) at genetic risk for the development of type 1 diabetes. NOD mice had heterogeneous expression of IAA despite being inbred. IAA reached a peak between 8 and 16 weeks and then declined. IAA expression by NOD mice at 8 weeks of age was strongly associated with early development of diabetes, which occurred at 16–18 weeks of age (NOD mice IAA+ at 8 weeks: 83% (5/6) diabetic by 18 weeks versus 11% (1/9) of IAA negative at 8 weeks; P < .01). In man, IAA was frequently present as early as 9 months of age, the first sampling time. Of five children found to have persistent IAA before 1 year of age, four have progressed to diabetes (all before 3.5 years of age) and the fifth is currently less than age 2. Of the 929 children not expressing persistent IAA before age 1, only one has progressed to diabetes to date (age onset 3), and this child expressed IAA at his second visit (age 1.1). In new onset patients, the highest levels of IAA correlated with an earlier age of diabetes onset. Our data suggest that the program for developing diabetes of NOD mice and humans is relatively “fixed” early in life and, for NOD mice, a high risk of early development of diabetes is often determined by 8 weeks of age. With such early determination of high risk of progression to diabetes, immunologic therapies in humans may need to be tested in children before the development of IAA for maximal efficacy.

Adhesion to fibronectin stimulates proliferation of wild-type and bcr/abl-transfected murine hematopoietic cells

Cells of most tissues require adhesion to a surface to grow. However, for hematopoietic cells, both stimulation and inhibition of proliferation by adhesion to extracellular matrix components have been described. Furthermore, it has been suggested that progenitor cells from chronic myelogenous leukemia show decreased β1 integrin-mediated adhesion to fibronectin, resulting in increased proliferation and abnormal trafficking. However, we show here that the chronic myelogenous leukemia-specific fusion protein p210bcr/abl stimulates the expression of α5β1 integrins and induces adhesion to fibronectin when expressed in the myeloid cell line 32D. Moreover, proliferation of both p210bcr/abl-transfected 32D (32Dp210) cells and untransfected 32D cells is stimulated by immobilized fibronectin. Cell cycle analysis revealed that nonadherent 32D and 32Dp210 cells are arrested in late G1 or early S phase, whereas the adherent fractions continue cycling. Although both adherent and nonadherent p210bcr/abl-transfected and parental 32D cells express equal amounts of cyclin A, a protein necessary for cell cycle progression at the G1/S boundary, cyclin A complexes immunoprecipitated from 32D cells cultured on immobilized fibronectin were found to be catalytically inactive in nonadherent but not in adherent cells. In addition, as compared with untransfected 32D cells, cyclin A immunoprecipitates from 32Dp210 cells exhibited a greatly elevated kinase activity and remained partially active irrespective of the adhesion status. The lack of cyclin A/cyclin-dependent kinase (CDK) 2 activity in nonadherent 32D cells appeared to result from increased expression and cyclin A complex formation of the CDK inhibitor p27Kip1. Taken together, our results indicate that adhesion stimulates cell cycle progression of hematopoietic cells by down-regulation of p27Kip1, resulting in activation of cyclin A/CDK2 complexes and subsequent transition through the G1/S adhesion checkpoint.

A distinctive clade B HIV type 1 is heterosexually transmitted in Trinidad and Tobago

HIV-1 transmission worldwide is predominantly associated with heterosexual activity, and non-clade B viruses account for the most spread. The HIV-1 epidemic in Trinidad/Tobago and the Caribbean shares many features with such heterosexual epidemics, including a prominent role for coincident sexually transmitted diseases. This study evaluates the molecular epidemiology of HIV-1 in Trinidad/Tobago during a period when abrupt transition from homosexual to heterosexual transmission occurred in the absence of injecting drug use, concomitant with a rapid rise in HIV-1 prevalence in the heterosexual population. Of 31 viral isolates studied during 1987–1995, all cluster with subtype B reference strains. In the analysis of full env genes from 22 early seroconverters, the Trinidad isolates constitute a significant subcluster within the B subtype. The Trinidad V3 consensus sequence differs by a single amino acid from the prototype B V3 consensus and demonstrates stability over the decade of this study. In the majority of isolates, the V3 loop of env contains a signature threonine deletion that marks the lineage of the Trinidad HIV-1 clade B epidemic from pre-1984. No phenotypic features, including syncitium induction, neutralization profiles, and chemokine receptor usage, distinguish this virus population from other subtype B viruses. Thus, although the subtype B HIV-1 viruses being transmitted in Trinidad are genetically distinguishable from other subtype B viruses, this is probably the result of a strong founder effect in a geographically circumscribed population rather than genetic selection for heterosexual transmission. These results demonstrate that canonical clade B HIV-1 can generate a typical heterosexual epidemic.

Contribution of Salmonella typhimurium type III secretion components to needle complex formation

The prgHIJK operon encodes components of the Salmonella typhimurium pathogenicity island 1 type III secretion system (TTSS). Previously, prgH and prgK were shown to be required for formation of the supramolecular type III secretion needle complex (NC) [Kubori, T., et al. (1998) Science 280, 602–605]. This work indicates that all prg operon genes are required for NC formation. PrgH multimerizes into a distinct tetrameric-shaped structure that may be an early intermediate of NC assembly and may provide the structural foundation required for PrgK oligomerization. PrgH and PrgK, in the absence of other TTSS components, oligomerize into ring-shaped structures identical in appearance and size to the base of the NC, indicating that they are likely the major inner membrane structural components required for secretion. PrgI and PrgJ cofractionate with the NC and are secreted into the culture supernatant. NC from prgI and prgJ mutants have an identical morphology to the envelope-spanning (basal body) NC components, but are missing the external needle, indicating that PrgI and PrgJ are required for full NC assembly and are likely components of the external needle. Therefore, PrgI and PrgJ are secreted through the NC basal body, composed in part of PrgH/K and InvG/H rings, to participate in assembly of the more distal components of the NC.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Murine cytomegalovirus M78 protein, a G protein-coupled receptor homologue, is a constituent of the virion and facilitates accumulation of immediate-early viral mRNA

The M78 protein of murine cytomegalovirus exhibits sequence features of a G protein-coupled receptor. It is synthesized with early kinetics, it becomes partially colocalized with Golgi markers, and it is incorporated into viral particles. We have constructed a viral substitution mutant, SMsubM78, which lacks most of the M78 ORF. The mutant produces a reduced yield in cultured 10.1 fibroblast and IC21 macrophage cell lines. The defect is multiplicity dependent and greater in the macrophage cell line. Consistent with its growth defect in cultured cells, the mutant exhibits reduced pathogenicity in mice, generating less infectious progeny than wild-type virus in all organs assayed. SMsubM78 fails to efficiently activate accumulation of the viral m123 immediate-early mRNA in infected macrophages. M78 facilitates the accumulation of the immediate-early mRNA in cycloheximide-treated cells, arguing that it acts in the absence of de novo protein synthesis. We conclude that the M78 G protein-coupled receptor homologue is delivered to cells as a constituent of the virion, and it acts to facilitate the accumulation of immediate-early mRNA.

Profilin I is essential for cell survival and cell division in early mouse development

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1ko) allele in mice. Homozygous pfn1ko/ko mice are not viable. Pfn1ko/ko embryos died as early as the two-cell stage, and no pfn1ko/ko blastocysts were detectable. Adult pfn1ko/wt mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1ko/wt embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.

γ-Aminobutyric acid type A receptors modulate cAMP-mediated long-term potentiation and long-term depression at monosynaptic CA3–CA1 synapses

cAMP induces a protein-synthesis-dependent late phase of long-term potentiation (LTP) at CA3–CA1 synapses in acute hippocampal slices. Herein we report cAMP-mediated LTP and long-term depression (LTD) at monosynaptic CA3–CA1 cell pairs in organotypic hippocampal slice cultures. After bath application of the membrane-permeable cAMP analog adenosine 3′,5′-cyclic monophosphorothioate, Sp isomer (Sp-cAMPS), synaptic transmission was enhanced for at least 2 h. Consistent with previous findings, the late phase of LTP requires activation of cAMP-dependent protein kinase A and protein synthesis. There is also an early phase of LTP induced by cAMP; the early phase depends on protein kinase A but, in contrast to the later phase, does not require protein synthesis. In addition, the cAMP-induced LTP is associated with a reduction of paired-pulse facilitation, suggesting that presynaptic modification may be involved. Furthermore, we found that Sp-cAMPS induced LTD in slices pretreated with picrotoxin, a γ-aminobutyric acid type A (GABAA) receptor antagonist. This form of LTD depends on protein synthesis and protein phosphatase(s) and is accompanied by an increased ratio of failed synaptic transmission. These results suggest that GABAA receptors can modulate the effect of cAMP on synaptic transmission and thus determine the direction of synaptic plasticity.

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-γ production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-α (TNF-α), IL-6, IL-12, and IFN-γ mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-α mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-α/β. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

X-ray emission from clusters and groups of galaxies

Recent major advances in x-ray imaging and spectroscopy of clusters have allowed the determination of their mass and mass profile out to ≈1/2 the virial radius. In rich clusters, most of the baryonic mass is in the gas phase, and the ratio of mass in gas/stars varies by a factor of 2–4. The baryonic fractions vary by a factor of ≈3 from cluster to cluster and almost always exceed 0.09 h50−[3/2] and thus are in fundamental conflict with the assumption of Ω = 1 and the results of big bang nucleosynthesis. The derived Fe abundances are 0.2–0.45 solar, and the abundances of O and Si for low redshift systems are 0.6–1.0 solar. This distribution is consistent with an origin in pure type II supernova. The amount of light and energy produced by these supernovae is very large, indicating their importance in influencing the formation of clusters and galaxies. The lack of evolution of Fe to a redshift of z ≈ 0.4 argues for very early enrichment of the cluster gas. Groups show a wide range of abundances, 0.1–0.5 solar. The results of an x-ray survey indicate that the contribution of groups to the mass density of the universe is likely to be larger than 0.1 h50−2. Many of the very poor groups have large x-ray halos and are filled with small galaxies whose velocity dispersion is a good match to the x-ray temperatures.

Timing of metamorphosis and the onset of the negative feedback loop between the thyroid gland and the pituitary is controlled by type II iodothyronine deiodinase in Xenopus laevis

Two important features of amphibian metamorphosis are the sequential response of tissues to different concentrations of thyroid hormone (TH) and the development of the negative feedback loop between the pituitary and the thyroid gland that regulates TH synthesis by the thyroid gland. At the climax of metamorphosis in Xenopus laevis (when the TH level is highest), the ratio of the circulating precursor thyroxine (T4) to the active form 3,5,3′-triiodothyronine (T3) in the blood is many times higher than it is in tissues. This difference is because of the conversion of T4 to T3 in target cells of the tadpole catalyzed by the enzyme type II iodothyronine deiodinase (D2) and the local effect (cell autonomy) of this activity. Limb buds and tails express D2 early and late in metamorphosis, respectively, correlating with the time that these organs undergo TH-induced change. T3 is required to complete metamorphosis because the peak concentration of T4 that is reached at metamorphic climax cannot induce the final morphological changes. At the climax of metamorphosis, D2 expression is activated specifically in the anterior pituitary cells that express the genes for thyroid-stimulating hormone but not in the cells that express proopiomelanocortin. Physiological concentrations of T3 but not T4 can suppress thyrotropin subunit β gene expression. The timing and the remarkable specificity of D2 expression in the thyrotrophs of the anterior pituitary coupled with the requirement for locally synthesized T3 strongly support a role for D2 in the onset of the negative feedback loop at the climax of metamorphosis.

Initial Steps in the Assembly of the Vacuole-Type H+-ATPase1

The plant vacuole is acidified by a complex multimeric enzyme, the vacuole-type H+-ATPase (V-ATPase). The initial association of ATPase subunits on membranes was studied using an in vitro assembly assay. The V-ATPase assembled onto microsomes when V-ATPase subunits were supplied. However, when the A or B subunit or the proteolipid were supplied individually, only the proteolipid associated with membranes. By using poly(A+) RNA depleted in the B subunit and proteolipid subunit mRNA, we demonstrated A subunit association with membranes at substoichiometric amounts of the B subunit or the 16-kD proteolipid. These data suggest that poly(A+) RNA-encoded proteins are required to catalyze the A subunit membrane assembly. Initial events were further studied by in vivo protein labeling. Consistent with a temporal ordering of V-ATPase assembly, membranes contained only the A subunit at early times; at later times both the A and B subunits were found on the membranes. A large-mass ATPase complex was not efficiently formed in the absence of membranes. Together, these data support a model whereby the A subunit is first assembled onto the membrane, followed by the B subunit.

The type I BMP receptor BmprIB is essential for female reproductive function

Maintenance of female reproductive competence depends on the actions of several hormones and signaling factors. Recent reports suggest roles for bone morphogenetic proteins (BMPs) in early stages of folliculogenesis. A role for the type I BMP receptor BmprIB as a regulator of ovulation rates in sheep has been described recently, but little is known about the roles of BMP signaling pathways in other aspects of reproductive function. We report here that BMPRIB is essential for multiple aspects of female fertility. Mice deficient in BmprIB exhibit irregular estrous cycles and an impaired pseudopregnancy response. BmprIB mutants produce oocytes that can be fertilized in vitro, but defects in cumulus expansion prevent fertilization in vivo. This defect is associated with decreased levels of aromatase production in granulosa cells. Unexpectedly, levels of mRNA for cyclooxygenase 2, an enzyme required for cumulus expansion, are increased. BmprIB mutants also exhibit a failure in endometrial gland formation. The expression of BmprIB in uterine linings suggests that these defects are a direct consequence of loss of BMP signaling in this tissue. In summary, these studies demonstrate the importance of BMP signaling pathways for estrus cyclicity, estradiol biosynthesis, and cumulus cell expansion in vivo and reveal sites of action for BMP signaling pathways in reproductive tissues.