Plasmodium falciparum: an intervening sequence in the GBP 130/96 tR gene

The 96 tR antigen is a heat stable protein produced during the late stages of the intraerythrocytic development of the malaria parasite Plasmodium falciparum and is released into the culture supernatant or the sera of infected patients at the time of schizont rupture. This antigen, identified as a putative protective antigen, was shown to be identical to the glycophorin-binding protein GBP 130 (Perkins 1988, Bonnefoy et al. 1988). We report here that the gene contains a small undescribed intervening sequence located immediately after the sequence coding for the signal sequence. This shows that in P. falciparum, all the genes described so far coding for proteins exported outside the parasitophorous vacuole share a common organization.     

A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro

The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.     

Plasma GBP2 promoter methylation is associated with advanced stages in breast cancer

Blood methylated cell-free DNA (cfDNA) as a minimally invasive cancer biomarker has great importance in cancer management. Guanylate binding protein 2 (GBP2) has been considered as a possible controlling factor in tumor development. GBP2 gene expression and its promoter methylation status in both plasma cfDNA and tumor tissues of ductal carcinoma breast cancer patients were analyzed using SYBR green comparative Real-Time RT-PCR and, Methyl-specific PCR techniques, respectively in order to find a possible cancer-related marker. The results revealed that GBP2 gene expression and promoter methylation were inversely associated. GBP2 was down-regulated in tumors with emphasis on triple negative status, nodal involvement and higher cancer stages (p<0.0001). GBP2 promoter methylation on both cfDNA and tumor tissues were positively correlated and was detected in about 88% of breast cancer patients mostly in (Lymph node positive) LN+ and higher stages. Data provided shreds of evidence that GBP2 promoter methylation in circulating DNA may be considered as a possible effective non-invasive molecular marker in poor prognostic breast cancer patients with the evidence of its relation to disease stage and lymph node metastasis. However further studies need to evaluate the involvement of GBP2 promoter methylation in progression-free survival or overall survival of the patients.     

Separate elements cause lineage restriction and specify boundaries of Hox-1.1 expression.

The Hox genes are a class of putative developmental control genes that are thought to be involved in the specification of positional identity along the anteroposterior axis of the vertebrate embryo. It is apparent from their expression pattern that their regulation is dependent upon positional information. In a previous analysis of the Hox-1.1 promoter in transgenic mice, we identified sequences that were sufficient to establish transgene expression in a specific region of the embryo. The construct used, however, did not contain enough regulatory sequences to reproduce all aspects of Hox-1.1 expression. In particular, neither a posterior boundary nor a restriction of expression to prevertebrae was achieved. Here we show correct regulation by Hox-1.1 sequences in transgenic mice and identify the elements responsible for different levels of control. Concomitant with the subdivision of mesodermal cells into different lineages during gastrulation and organogenesis, Hox-1.1 expression is restricted to successively smaller sets of cells. Distinct elements are required at different stages of development to execute this developmental programme. One position-responsive element (130 bp nontranslated leader) was shown to be crucial for the restriction of expression not only along the anteroposterior axis of the embryo, setting the posterior border, but also along the dorsoventral axis of the neural tube and to the lineage giving rise to the prevertebrae. Thus, Hox-1.1 expression is established in a specific region of the embryo and in a specific lineage of the mesoderm by restricting the activity of the promoter by the combined effect of several regulatory elements.     

N-terminal mutational analysis of the interaction between growth-blocking peptide (GBP) and receptor of insect immune cells

GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The 1H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core beta-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn2. Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.     

Molecular factors responsible for host cell recognition and invasion in Plasmodium falciparum.

In Plasmodium falciparum, the rhoptries involved in the invasion process are a pair of flask-shaped organelles located at the apical tip of invading stages. They, along with the more numerous micronemes and dense granules, constitute the apical complex in Plasmodium and other members of the phylum Apicomplexa. Several proteins of varying molecular weight have been identified in P. falciparum rhoptries. These include the 225-, 140/130/110-, 80/60/40-, RAP-1 80-, AMA-1 80-, QF3 80-, and 55-kDa proteins. Some of these proteins are lost during schizont rupture and release of merozoites. Others such as the 140/130/110-kDa complex are transferred to the erythrocyte membrane during invasion. The ring-infected surface antigen (RESA), a 155-kDa polypeptide located in dense granules also associates with the erythrocyte membrane during invasion. Erythrocyte-binding studies have demonstrated that both the 140/130/110-kDa rhoptry complex and RESA bind to inside-out-vesicles (IOVs) prepared from human erythrocytes. The 140/130/110-kDa complex also binds to erythrocyte membranes prepared by hypotonic lysis. These proteins, however, do not bind to intact human erythrocytes. In a heterologous erythrocyte model, both the 140/130/110-kDa complex and RESA are shown to bind directly to mouse erythrocytes. Other studies have shown that RESA associates with spectrin in the erythrocyte cytoskeleton. We have recently developed a liposome-binding assay to demonstrate the lipophilic binding properties of the P. falciparum rhoptry complex of 140/130/110 kDa. The rhoptry complex binds to liposomes containing neutrally, positively, and negatively charged phospholipids. However, liposomes containing phosphatidylethanolamine compete effectively for rhoptry protein binding to mouse erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organization of the gene for gelatin-binding protein (GBP28)

GBP28 is a novel human plasma gelatin-binding protein that is encoded by apM1 mRNA, expressed specifically in adipose tissue. Three overlapping clones (two lambda clones and one BAC clone) containing the human plasma gelatin-binding protein (GBP28) gene were isolated and characterized. The GBP28 gene spans 16kb and is composed of three exons from 18bp to 4277bp in size with consensus splice sites. The sizes of the two introns were 0.8 and 12kb, respectively. The gene's regulatory sequences contain putative promoter elements, but no typical TATA box.The third exon of this gene contains a long 3'-untranslated sequence containing three Alu repeats. The exon-intron organization of this gene was very similar to that of obese gene, encoding leptin. We also report the chromosome mapping of this gene by fluorescence in situ hybridization (FISH) using a genomic DNA fragment as a probe. The GBP28 gene was located on human chromosome 3q27. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers ABO12163, ABO12164 or ABO12165.     

A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head

The suboesophageal body of insects was identified over a century ago in the silkworm embryo, but its biological function is still unknown. We discovered that this tissue is differentiated in the earliest embryonic stages of the cabbage armyworm and secretes the insect cytokine, growth-blocking peptide (GBP), transiently from 24 to 60 h after oviposition when gastrulation is in progress. Over-expression of GBP, achieved by microinjection of the GBP gene driven by a cytomegalovirus (CMV) constitutive promoter, resulted in complex deformities of the procephalon (embryonic head). Severe abnormal phenotypes of the head structure were produced by silencing the GBP expression in the embryo by treating with GBP double-stranded RNA: the procephalon-containing optic lobes diminished and completely separated into bilateral halves. This indicates that GBP secreted from the suboesophageal body plays an essential role in the formation of the procephalic domain during early embryogenesis. The cytokine-induced fusion of bilateral procephalic lobes is thought to be evolutionarily conserved at least in insects, because of the widespread occurrence of the suboesophageal body in insect embryos.     

Structure and activity of the insect cytokine growth-blocking peptide. Essential regions for mitogenic and hemocyte-stimulating activities are separate.

Growth-blocking peptide (GBP) is a 25-amino acid insect cytokine found in Lepidopteran insects that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells (plasmatocytes). The tertiary structure of GBP consists of a structured core that contains a disulfide bridge and a short antiparallel beta-sheet (Tyr(11)-Arg(13) and Cys(19)-Pro(21)) and flexible N and C termini (Glu(1)-Gly(6) and Phe(23)-Gln(25)). In this study, deletion and point mutation analogs of GBP were synthesized to investigate the relationship between the structure of GBP and its mitogenic and plasmatocyte spreading activity. The results indicated that deletion of the N-terminal residue, Glu(1), eliminated all plasmatocyte spreading activity but did not reduce mitogenic activity. In contrast, deletion of Phe(23) along with the remainder of the C terminus destroyed all mitogenic activity but only slightly reduced plasmatocyte spreading activity. Therefore, the minimal structure of GBP containing mitogenic activity is 2-23 GBP, whereas that with plasmatocyte spreading activity is 1-22 GBP. NMR analysis indicated that these N- and C-terminal deletion mutants retained a similar core structure to wild-type GBP. Replacement of Asp(16) with either a Glu, Leu, or Asn residue similarly did not alter the core structure of GBP. However, these mutants had no mitogenic activity, although they retained about 50% of their plasmatocyte spreading activity. We conclude that specific residues in the unstructured and structured domains of GBP differentially affect the biological activities of GBP, which suggests the possibility that multifunctional properties of this peptide may be mediated by different forms of a GBP receptor.