Long-term survival in multiple myeloma is associated with a distinct immunological profile, which includes proliferative cytotoxic T-cell clones and a favourable Treg/Th17 balance.

Despite improved outcomes in multiple myeloma (MM), a cure remains elusive. However, even before the current therapeutic era, 5% of patients survived >10 years and we propose that immune factors contribute to this longer survival. We identified patients attending our clinic, who had survived >10 years (n=20) and analysed their blood for the presence of T-cell clones, T-regulatory cells (Tregs) and T helper 17 (Th17) cells. These results were compared with MM patients with shorter follow-up and age-matched healthy control donors. The frequency of cytotoxic T-cell clonal expansions in patients with <10 years follow-up (MM patients) was 54% (n=144), whereas it was 100% (n=19/19) in the long-survivors (LTS-MM). T-cell clones from MM patients proliferated poorly in vitro, whereas those from LTS-MM patients proliferated readily (median proliferations 6.1% and 61.5%, respectively (P<0.0001)). In addition, we found significantly higher Th17 cells and lower Tregs in the LTS-MM group when compared with the MM group. These results indicate that long-term survival in MM is associated with a distinct immunological profile, which is consistent with decreased immune suppression.

Functional and metabolic remodelling in GLUT4-deficient hearts confers hyper-responsiveness to substrate intervention.

Impaired glucose uptake is associated with both cardiac hypertrophy and contractile dysfunction, but whether there are common underlying mechanisms linking these conditions is yet to be determined. Using a 'gene dose' Cre-Lox GLUT4-deficient murine model, we examined the effect of suppressed glucose availability on global myocardial gene expression and glycolysis substrate bypass on the function of isolated perfused hearts. Performance of hearts from 22- to 60-week-old male GLUT4 knockout (KO, >95% reduction in GLUT4), GLUT4 knockdown (KD, 85% reduction in cardiac GLUT4) and C57Bl/6 wild-type (WT) controls was measured ex vivo in Langendorff mode perfusion. DNA microarray was used to profile mRNA expression differences between GLUT4-KO and GLUT4-KD hearts. At 22 weeks, GLUT4-KO hearts exhibited cardiac hypertrophy and impaired contractile function ex vivo, characterized by a 40% decrease in developed pressure. At 60 weeks, dysfunction was accentuated in GLUT4-KO hearts and evident in GLUT4-KD hearts. Exogenous pyruvate (5 mM) restored systolic pressure to a level equivalent to WT (GLUT4-KO, 176.8+/-13.2 mmHg vs. WT, 146.4+/-9.56 mmHg) in 22-week-old GLUT4-KO hearts but not in 60-week-old GLUT4-KO hearts. In GLUT4-KO, DNA microarray analysis detected downregulation of a number of genes centrally involved in mitochondrial oxidation and upregulation of other genes indicative of a shift to cytosolic beta-oxidation of long chain fatty acids. A direct link between cardiomyocyte GLUT4 deficiency, hypertrophy and contractile dysfunction is demonstrated. These data provide mechanistic insight into the myocardial metabolic adaptations associated with short and long-term insulin resistance and indicate a window of opportunity for substrate intervention and functional 'rescue'.

Increased nicotinamide nucleotide transhydrogenase levels predispose to insulin hypersecretion in a mouse strain susceptible to diabetes.

AIMS/HYPOTHESIS: Insulin hypersecretion may be an independent predictor of progression to type 2 diabetes. Identifying genes affecting insulin hypersecretion are important in understanding disease progression. We have previously shown that diabetes-susceptible DBA/2 mice congenitally display high insulin secretion. We studied this model to map and identify the gene(s) responsible for this trait. METHODS: Intravenous glucose tolerance tests followed by a genome-wide scan were performed on 171 (C57BL/6 x DBA/2) x C57BL/6 backcross mice. RESULTS: A quantitative trait locus, designated hyperinsulin production-1 (Hip1), was mapped with a logarithm of odds score of 7.7 to a region on chromosome 13. Production of congenic mice confirmed that Hip1 influenced the insulin hypersecretion trait. By studying appropriate recombinant inbred mouse strains, the Hip1 locus was further localised to a 2 Mb interval, which contained only nine genes. Expression analysis showed that the only gene differentially expressed in islets isolated from the parental strains was Nnt, which encodes the mitochondrial proton pump, nicotinamide nucleotide transhydrogenase (NNT). We also found in five mouse strains a positive correlation (r2 = 0.90, p < 0.01) between NNT activity and first-phase insulin secretion, emphasising the importance of this enzyme in beta cell function. Furthermore, of these five strains, only those with high NNT activity are known to exhibit severe diabetes after becoming obese. CONCLUSIONS/INTERPRETATION: Insulin hypersecretion is associated with increased Nnt expression. We suggest that NNT must play an important role in beta cell function and that its effect on the high insulin secretory capacity of the DBA/2 mouse may predispose beta cells of these mice to failure.

The antioxidant tempol inhibits cardiac hypertrophy in the insulin-resistant GLUT4-deficient mouse in vivo.

Reactive oxygen species such as superoxide are implicated in cardiac hypertrophy, but their contribution to the cardiac complications of insulin resistance is unresolved. We tested the hypothesis that the antioxidant tempol attenuates cardiac hypertrophy in insulin-resistant mice. Mice with cardiac GLUT4 deletion (GLUT4-knockout), superimposed on global GLUT4 suppression (GLUT4-knockdown) were administered tempol for 4 weeks. Age-matched GLUT4-knockdown littermates were used as controls (14 mice/group). GLUT4-knockout mice exhibited marked cardiac hypertrophy: heart to body weight ratio was increased 61+/-7% and expression of the hypertrophic genes beta-myosin heavy chain and B-type natriuretic peptide (BNP) were elevated 5.5+/-0.7- and 6.2+/-1.5-fold relative to control, respectively. Pro-fibrotic pro-collagen III expression was also higher (3.8+/-0.7-fold) in the GLUT4-knockout myocardium (all p<0.001). Both gp91(phox) and Nox1 subunits of NADPH oxidase were also upregulated, 4.9+/-1.2- and 9.3+/-2.8-fold (both p<0.01). Tempol treatment significantly attenuated all of these abnormalities in GLUT4-knockout mice. Heart to body weight ratio was decreased, as was fold expression of beta-myosin heavy chain (to 3.8+/-0.8), BNP (to 2.5+/-0.5), pro-collagen III (to 1.9+/-0.4), gp91(phox) (to 0.9+/-0.3) and Nox1 (to 2.3+/-0.1, all p<0.05 versus untreated GLUT4-knockout mice). In addition, tempol upregulated ventricular expression of both thioredoxin-2 (confirming an antioxidant action) and glycogen synthase kinase-3beta (GSK-3beta). Tempol did not elicit any other significant changes in control mice. Cardiac superoxide generation, however, was not altered by GLUT4-knockout or tempol. In conclusion, tempol treatment reduced morphological and molecular evidence of cardiac hypertrophy in the GLUT4-knockout insulin-resistant mouse in vivo, even at doses insufficient to lower cardiac superoxide. Parallel reductions in pro-collagen III and NADPH oxidase have important implications for our understanding of the molecular basis of cardiac hypertrophy in the setting of insulin resistance. Antioxidants may offer new alternatives in this disorder.

Threshold effects of glucose transporter-4 (GLUT4) deficiency on cardiac glucose uptake and development of hypertrophy.

The aim of this study was to investigate the metabolic and structural consequences of a decrease in glucose transporter-4 (GLUT4) levels on the heart. The CreLoxP system was utilised to delete GLUT4 in muscle tIssue including heart. The presence of the PGK-neoR cassette in the GLUT4-Lox mice resulted in reduced expression in all tIssues to levels 15-30% of wild-type control mice. In mice expressing Cre recombinase, there was a further reduction of GLUT4 in cardiac tIssue to almost undetectable levels. Cardiac glucose uptake was measured basally and during a euglycaemic/hyperinsulinaemic clamp using 2-deoxy-[1-(14)C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy.

Aberrant cryptic responsiveness of the pCAT 3- and pGL3-promoter reporter vectors.

Transfection analyses are an informative method to assess the activity of specific promoter or enhancer elements in mammalian cells. Commercially available reporter vectors can be extremely useful investigative tools for such studies. This study reports that the pCAT 3- and pGL3-promoter vectors display cryptic responsiveness to androgens when they contain a DNA insert, while the empty vector, a commonly used negative control, is nonresponsive. Our studies initially aimed to characterize novel androgen-responsive DNA sequences in human genomic DNA through transactivational analyses. An isolated DNA fragment, designated ARC-3, contained three putative androgen response element "half-sites" and was androgen-responsive when cloned into the pCAT3-promoter vector. While we originally believed this to be a novel enhancer element, subsequent analyses of this clone revealed that this vector displays cryptic activity in the presence of an androgen. This was confirmed by cloning several unrelated DNA fragments that did not contain any known classic response elements into the pCAT3-promoter vector, all of which were found to be responsive. The empty vector (negative control) was again nonresponsive. The ARC-3 DNA fragment was also weakly responsive to stimulation when cloned into the pGL3-promotor vector, which is identical to the pCAT3-promoter vector, with the exception of an intron located 5' of the chloramphenicol acetyltransferase gene, and the reporter genes. This work demonstrates that both the pCAT3- and pGL3-promoter vectors are inappropriate to assess androgen-responsive enhancers and emphasizes the importance of the careful selection of reporter vectors and controls when conducting transactivational analysis.

Peripheral insulin resistance develops in transgenic rats overexpressing phosphoenolpyruvate carboxykinase in the kidney.

AIMS/HYPOTHESIS: To study the secondary consequences of impaired suppression of endogenous glucose production (EGP) we have created a transgenic rat overexpressing the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in the kidney. The aim of this study was to determine whether peripheral insulin resistance develops in these transgenic rats. METHODS: Whole body rate of glucose disappearance (R(d)) and endogenous glucose production were measured basally and during a euglycaemic/hyperinsulinaemic clamp in phosphoenolpyruvate carboxykinase transgenic and control rats using [6-(3)H]-glucose. Glucose uptake into individual tissues was measured in vivo using 2-[1-(14)C]-deoxyglucose. RESULTS: Phosphoenolpyruvate carboxykinase transgenic rats were heavier and had increased gonadal and infrarenal fat pad weights. Under basal conditions, endogenous glucose production was similar in phosphoenolpyruvate carboxykinase transgenic and control rats (37.4+/-1.1 vs 34.6+/-2.6 micromol/kg/min). Moderate hyperinsulinaemia (810 pmol/l) completely suppressed EGP in control rats (-0.6+/-5.5 micromol/kg/min, p<0.05) while there was no suppression in phosphoenolpyruvate carboxykinase rats (45.2+/-7.9 micromol/kg/min). Basal R(d) was comparable between PEPCK transgenic and control rats (37.4+/-1.1 vs 34.6+/-2.6 micromol/kg/min) but under insulin-stimulated conditions the increase in R(d) was greater in control compared to phosphoenolpyruvate carboxykinase transgenic rats indicative of insulin resistance (73.4+/-11.2 vs 112.0+/-8.0 micromol/kg/min, p<0.05). Basal glucose uptake was reduced in white and brown adipose tissue, heart and soleus while insulin-stimulated transport was reduced in white and brown adipose tissue, white quadriceps, white gastrocnemius and soleus in phosphoenolpyruvate carboxykinase transgenic compared to control rats. The impairment in both white and brown adipose tissue glucose uptake in phosphoenolpyruvate carboxykinase transgenic rats was associated with a decrease in GLUT4 protein content. In contrast, muscle GLUT4 protein, triglyceride and long-chain acylCoA levels were comparable between PEPCK transgenic and control rats. CONCLUSIONS/INTERPRETATION: A primary defect in suppression of EGP caused adipose tissue and muscle insulin resistance.

Low-intensity pulsed ultrasound stimulates a bone-forming response in UMR-106 cells.

Low-intensity (<100 mW/cm(2)) pulsed ultrasound (US) is an established therapy for fracture repair. In both animal and human trials, such US has been shown to facilitate fresh fracture repair and initiate healing in fractures with repair defects. However, the mechanism by which US achieves these outcomes is not clear. One possible mechanism is the direct stimulation of bone formation. To investigate this hypothesis, the current study investigated the mRNA response of isolated bone-forming cells (UMR-106 cells) to a single 20-min dose of low-intensity pulsed US. Using a novel US-cell coupling method, US was found to stimulate expression of the immediate-early response genes c-fos and COX-2 and elevate mRNA levels for the bone matrix proteins ALP and OC. These findings suggest that low-intensity pulsed US has a direct effect on bone formation. This may contribute to the beneficial effect of low-intensity pulsed US on fracture repair.

The identification of a potent imidazoline-based vascular K(ATP) channel antagonist.

In this study the activity of a number of novel imidazoline-based compounds (IMID series) was assessed by functional and binding studies to determine their actions at K(ATP) channels. The novel compounds, which we synthesised, were methoxy-, methyl-, butyl- and fluorophenyl derivatives of clonidine. In functional studies we determined the potency (by calculating a pK(B) value) of the IMID compounds to antagonise levcromakalim responses in segments of isolated pig coronary artery. The most potent compounds identified (laboratory codes: IMID-1M, IMID-26F and IMID-4F) had apparent pK(B) values of approximately 7 which is similar to that for the sulphonylurea, glibenclamide and the lipophilic quaternary ion, tetraphenylphosphonium. This inhibitory action was specific for levcromakalim since the imidazoline antagonist IMID-1M failed to effect vasorelaxation response-curves to the non-KATP channel opener, sodium nitroprusside. In the spontaneously beating rat right atrium preparation the majority of the compounds were able to cause slowing of heart rate, but with low EC50 values (approximately 10-30 microM). In binding studies, the compounds were unable to displace binding of [3H]P1075 to bovine aortic smooth muscle preparations nor [3H]glibenclamide binding to rat cerebral cortex membranes. These studies show that some imidazoline-based compounds are potent antagonists of levcromakalim-mediated vasorelaxation responses in the pig coronary artery. The compounds displayed only minimal bradycardic activity. The site of action of the imidazoline compounds does not appear to be the same as that used by K(ATP) channel openers or sulphonylurea-based antagonists. It is likely that these compounds interact with the K(ATP) channel pore itself.

Effect of the androgen receptor CAG repeat polymorphism on transcriptional activity: specificity in prostate and non-prostate cell lines.

The action of androgens is essential for the development of benign prostatic hyperplasia and carcinoma of the prostate. The androgen receptor is a ligand-dependent nuclear transcription factor. The transcriptional activation domain of the androgen receptor gene contains a polymorphic CAG repeat sequence. A shorter CAG repeat sequence within the normal range has been reported to be associated with increased risk of prostate cancer and symptomatic benign prostatic hyperplasia. Here, we examine the in vitro transcriptional activity of the androgen receptor (AR) with different numbers of CAG repeats within the normal range in a number of different cell lines of prostatic (LNCaP, PC3) and non-prostatic (COS-1, MCF7) origin. We utilize a luciferase reporter driven by the rat probasin promoter (-286/+28) containing two androgen receptor binding sites. Transcriptional activation of the androgen responsive reporter was observed to be greater with the AR containing 15 vs 31 CAG repeats in COS-1 cells (123.2+/-16.6 vs 78.2+/-10.9, P value 0.01) and the well differentiated prostate cancer cell line LNCaP (103.4+/-17.7 vs 81.4+/-7.7, P value 0.045). No difference was observed in the poorly differentiated prostate cancer cell line, PC3 (106.9+/-21.9 vs 109. 6+/-21.4, P>0.5) or the breast cancer cell line MCF7 (120.4+/-39.4 vs 103.1+/-23.1, P value >0.5). Dose-response experiments with varying quantities of ligand (0.01, 0.1, 1 and 10 nM dihydrotestosterone) or AR cDNA did not demonstrate significant differences in transactivation of the androgen responsive reporter in PC3 cells by the different AR constructs. This suggests that the lack of influence of CAG number in this prostatic cell line is not related to dose of ligand or quantity of androgen receptor. Western immunoblot analysis of androgen receptor protein in transiently transfected COS-1 cells did not demonstrate a difference in the expression of the androgen receptor protein with different numbers of CAG repeats following incubation in the presence or absence of androgen. Gel shift assay did not demonstrate increased DNA binding by androgen receptor with a shorter CAG repeat sequence. These experiments using a relatively androgen- and prostate-specific reporter provide evidence for an inverse relationship between androgen receptor transcriptional activity and the number of CAG repeats in the transcriptional activation domain. The effect of CAG repeat number was cell specific suggesting the involvement of accessory factors expressed differentially between different cell lines.

Functional and electrophysiological effects of a novel imidazoline-based K(ATP) channel blocker, IMID-4F.

1. The functional and electrophysiological effects of IMID-4F (2-[N-(2, 6-dichlorophenyl)-N-(4-flurorobenzyl)amino]imidazoline), a fluoro-benzyl derivative of clonidine, on vascular K(ATP) channels were investigated. In pig coronary artery, IMID-4F inhibited the vasorelaxation response to the K(ATP) channel opener levcromakalim with a pK(B) value of approximately 7.1. IMID-4F (30 microM) did not affect the vasorelaxation response to sodium nitroprusside (SNP). 2. In rat mesenteric artery smooth muscle cells IMID-4F (1 - 10 microM) caused a concentration-dependent depolarization of membrane potential. IMID-4F (10 microM) abolished the hyperpolarizing effects of levcromakalim (10 microM). 3. In patch clamp experiments using rat mesenteric artery smooth muscle cells, K(ATP) channel currents induced by levcromakalim (10 microM) were inhibited by IMID-4F (0.3 - 3 microM) in a concentration-dependent manner. The calculated IC(50) for IMID-4F inhibiting K(ATP) channel current was approximately 0.8 microM. 4. Radioligand binding studies using bovine aortic smooth muscle cell membranes showed that IMID-4F (30 microM) did not displace binding to the K(ATP) channel opener [(3)H]-P1075. However, both levcromakalim (10 microM) and glibenclamide (10 microM) caused significant displacement of [(3)H]-P1075. 5. These studies show that the imidazoline compound IMID-4F is one of the most potent antagonists of arterial K(ATP) channels identified. Vasorelaxation, hyperpolarization and K(+) currents through K(ATP) channels were all inhibited by IMID-4F at micromolar concentrations. Radioligand binding studies indicate that IMID-4F does not bind to the same site as levcromakalim or as glibenclamide. Considering other evidence, it is likely that IMID-4F acts by interacting directly with the pore of the K(IR) channel, rather than through the sulphonylurea subunit of the K(ATP) channel complex.

Rabbit mononuclear leukocytes cause contraction of isolated aorta by the release of serotonin.

OBJECTIVE: The aim of this study was to examine the vasoactive effects of rabbit isolated mononuclear leukocytes, and to identify the mediators responsible for those vasoactive effects. METHODS: Mononuclear leukocytes (MNLs) were isolated from New Zealand White rabbit whole blood, suspended at 5 x 10(7) cells/ml and incubated for 30 min at 37 degrees C. This cell suspension, or the cell-free supernatant from this suspension, were then examined for vasoactive effects in rabbit isolated thoracic aorta. RESULTS: Both the MNL suspension and the cell-free supernatant from this suspension caused endothelium-independent contraction of aortic rings, both from resting tension and when pre-contracted. The MNL suspension caused a significantly greater contraction than the MNL supernatant under all conditions. The contractions to the MNL product were significantly inhibited by the 5-HT2 receptor antagonist ketanserin (0.1 microM), but not by the alpha 1-adrenoceptor antagonist prazosin (10 microM). High-performance liquid chromatography (HPLC) analysis showed that the MNL supernatant contained serotonin (5-hydroxytryptamine, 5-HT) at an average concentration of 5 microM. CONCLUSIONS: We conclude that MNLs cause contraction of rabbit isolated aortic rings by the release of 5-HT.

Rabbit polymorphonuclear leukocytes release a factor that causes constriction of the coronary vasculature.

Polymorphonuclear leukocytes (PMN) have been shown to have numerous vasoactive effects, particularly in large artery bioassays. This study shows that rabbit PMN passively release a contractile factor that constricts the coronary vasculature of isolated, Langendorff-perfused rabbit hearts. The mechanism of action of this factor does not involve inhibition of nitric oxide (NO), production of cyclooxygenase metabolites, 5-hydroxytryptamine, or endothelin, or the activation of alpha-adrenoceptors but is a Ca2+-dependent process, because the constriction is inhibited by the Ca2+-channel blocker amlodipine. The activity of this factor is significantly inhibited if it is pretreated with trypsin or heated to 90 degreesC for 10 min, and the active factor is concentrated in the retentate of 100-kDa cutoff centrifuge filters, indicating that the factor is a protein >100 kDa in size. This study shows that rabbit PMN spontaneously release a protein factor that causes constriction of isolated, perfused rabbit hearts by a NO-independent but Ca2+-dependent mechanism.

A novel alternate secretory pathway for the export of Plasmodium proteins into the host erythrocyte.

The malarial parasite dramatically alters its host cell by exporting and targeting proteins to specific locations within the erythrocyte. Little is known about the mechanisms by which the parasite is able to carry out this extraparasite transport. The fungal metabolite brefeldin A (BFA) has been used to study the secretory pathway in eukaryotes. BFA treatment of infected erythrocytes inhibits protein export and results in the accumulation of exported Plasmodium proteins into a compartment that is at the parasite periphery. Parasite proteins that are normally localized to the erythrocyte membrane, to nonmembrane bound inclusions in the erythrocyte cytoplasm, or to the parasitophorous vacuolar membrane accumulate in this BFA-induced compartment. A single BFA-induced compartment is detected per parasite and the various exported proteins colocalize to this compartment regardless of their final destinations. Parasite membrane proteins do not accumulate in this novel compartment, but accumulate in the endoplasmic reticulum (ER), suggesting that the parasite has two secretory pathways. This alternate secretory pathway is established immediately after merozoite invasion and at least some dense granule proteins also use the alternate pathway. The BFA-induced compartment exhibits properties that are similar to the ER, but it is clearly distinct from the ER. We propose to call this new organelle the secondary ER of apicomplexa. This ER-like organelle is an early, if not the first, step in the export of Plasmodium proteins into the host erythrocyte.

Molecular variation in a novel polymorphic antigen associated with Plasmodium falciparum merozoites.

A cDNA clone encoding part of a novel polymorphic merozoite antigen from Plasmodium falciparum was isolated by screening a cDNA library with human immune serum from Papua New Guinea. Immunofluorescence microscopy and immunoblotting with affinity-purified antibodies recognized a highly polymorphic antigen, Ag956, present in schizonts and merozoites. Biosynthetic labeling and immunoprecipitation experiments demonstrated that Ag956 is proteolytically cleaved during merozoite maturation. The complete genomic sequence of Ag956 from the D10 clone of P. falciparum isolate FC27 encodes a secreted protein of calculated molecular mass 43,243 that is very hydrophilic and contains a region of unusual heptad repeats of the general structure AXXAXXX. This antigen has been named the secreted polymorphic antigen associated with merozoites (SPAM). The sequence of a second SPAM allele from the 3D7 clone of isolate NF54 reveals that the alanine heptad repeats and the hydrophilic C-terminal half of the protein are conserved. Variation among SPAM alleles is the result of deletions and amino acid substitutions in non-repetitive sequences within and flanking the alanine heptad-repeat domain. Heptad repeats in which the a and d position contain hydrophobic residues generate amphipathic alpha-helices which give rise to helical bundles or coiled-coil structures in proteins. Thus, SPAM is the first example of a P. falciparum antigen in which a repetitive sequence has features characteristic of a well-defined structural element.

Sequence diversity of the erythrocyte membrane antigen 1 in various strains of Plasmodium chabaudi.

We have determined the complete genomic sequence of the Plasmodium chabaudi erythrocyte membrane antigen (PcEMA1) in 4 different parasite strains. The gene structure consisted of a short region encoding a signal sequence separated from the main coding region by an intervening sequence. The overall identity of the three P. chabaudi adami deduced protein sequences to their consensus was 100%, 99.8% and 88% for 556KA, DK and DS respectively, with a general pattern of increasing divergence from the N- to the C-terminus. The P. chabaudi chabaudi strain CB was 72% homologous to the P. chabaudi adami consensus sequence. A gene related to PcEMA1, designated PcEMA1-R, has been identified in the genome of P. chabaudi adami but not in P. chabaudi chabaudi. The partial sequence for this gene in P. chabaudi adami strain DS predicts that it could encode a truncated form of PcEMA1, but its status as a pseudogene or an independent, expressed gene has not been resolved.

A Plasmodium chabaudi antigen located in the parasitophorous vacuole membrane.

In order to study antigens from the rodent malaria Plasmodium chabaudi, clones reacting with mouse hyperimmune serum were selected from an expression library of blood-stage P. chabaudi cDNAs in the vector pGEX-2T. Sixty-four such clones were shown to derive from 19 different P. chabaudi antigens. One of these, Antigen 3008 (Ag3008), has a predicted size of 17.5 kDa and an observed size of 24 kDa. It is located in the parasitophorous vacuole membrane of maturing parasites and in the dense granules of merozoites. The cDNA sequence predicts a highly charged molecule with an N-terminal signal sequence and a central transmembrane domain, but no tandem repeats. The sequence reported for Pc24, a previously identified but uncharacterized P. chabaudi protein, corresponds to part of the 3' untranslated region in the Ag3008 mRNA. Ag3008 has many features in common with the P. falciparum circumsporozoite protein-related antigen (CRA).

Putative glycophorin-binding protein is secreted from schizonts of Plasmodium falciparum.

A cDNA clone expressing an antigen of Plasmodium falciparum, selected by screening an expression library cloned in Escherichia coli, encodes a portion of the protein identified as a glycophorin-binding protein [Kochan et al. (1986) Cell 44, 689-696]. Human antibodies affinity-purified on extracts from this clone were used to characterize the antigen by immunoblotting. This protein was present in all isolates tested, restricted to mature trophozoites and schizonts. It was abundant in culture supernatants at the time of merozoite release but present in minor amounts if at all in merozoites. The pattern of antigen distribution over schizont-infected cells observed by immunoelectron microscopy differed from that of the precursor of the major merozoite surface antigens in that most of the antigen appeared to be located over the erythrocyte cytoplasm without any obvious association with organelles. It thus appears unlikely that this antigen is present on the merozoite surface prior to schizont rupture.