Molecular monitoring in malaria vaccine trials

Molecular techniques offer new approaches for malaria field trials, particularly PCR techniques, which facilitate accurate diagnosis of Plasmodium infections and increase the power of estimates of vaccine effects on malaria prevalence or incidence. Molecular methods also help to assess selective effects of vaccines. Longitudinal genotyping data can be used to initiate novel analyses of parasite dynamics, including estimates of incidence of infection with individual parasite clones and duration of infections. In addition, high-throughput methods can be used to apply these techniques routinely in randomized controlled trials, as well as programme-based evaluations of malaria control.     

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.     

Motilin effects on the proximal stomach in patients with functional dyspepsia and healthy volunteers

This study investigates motilin effects on the proximal stomach in patients with functional dyspepsia (FD) and healthy volunteers. Eight healthy volunteers and 12 patients with FD were infused with synthetic motilin or placebo. Proximal gastric volume was measured with a barostat at constant pressure and during isobaric distensions. Abdominal symptoms were scored by visual analog scales. Plasma motilin concentrations were measured by radioimmunoassay. Motilin concentrations and baseline gastric volumes were similar for patients and healthy volunteers. Motilin, compared with placebo, reduced gastric volume by 112 ml [F(29,195); confidence interval (CI) 95%] in patients and by 96 ml [F(-7,200); CI 95%] in healthy volunteers. In patients, motilin decreased compliance by 76 ml/mmHg [F(9,143); CI 95%] compared with placebo, which was similar in volunteers [66 ml/mmHg; F(11,120); CI 95%]. Patients were more nauseous during motilin compared with placebo (P = 0.04), whereas healthy volunteers did not experience nausea. We conclude that in a fasted condition, FD patients have a similar proximal gastric motor response to motilin as healthy volunteers, but experience an exaggerated sensation of nausea.     

Systemic poly(ADP-ribose) polymerase-1 activation,chronic inflammation,and oxidative stress in COPD patients

Oxidative stress and systemic inflammation in chronic obstructive pulmonary disease (COPD) strongly suggest a role for the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1, E.C.2.4.2.30) in the disease pathophysiology. PARP-1 is highly activated by reactive oxygen species-induced DNA strand breaks, upon which it forms extensive poly(ADP-ribose) (PAR) polymers from its substrate NAD(+). We hypothesized that in COPD, chronic inflammation and oxidative stress would lead to systemic PARP-1 activation and to a reduced NAD(+) status. In a patient-control study, systemic PARP-1 activation was assessed by immunofluorescent detection of PAR polymers in peripheral blood lymphocytes. The percentage of PAR polymer-positive lymphocytes appeared to be higher in COPD patients (27 +/- 3%) than in healthy age-matched controls (17 +/- 2%, p <.05). Trolox equivalent antioxidant capacity (TEAC) of deproteinized plasma (p <.001), plasma uric acid (p <.05), as well as blood NAD(+) (p <.01) of stable COPD patients were significantly reduced when compared to controls. In addition, levels of proinflammatory cytokines IL-6, IL-8, and sICAM-1 were increased (p <.005) in COPD patients. In this study, evidence was found for the presence of systemic inflammation, chronic oxidative stress, and systemic PARP-1 activation in stable COPD patients. These data support a contribution of oxidative stress-induced PARP-1 activation to the pathophysiology of COPD.     

Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila

We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.     

A new Schizosaccharomyces pombe base excision repair mutant,nth1, reveals overlapping pathways for repair of DNA base damage

Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or gamma-irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3-methyladenine and 7-methylguanine whereas methyl-formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1-like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears to be their lack of AP lyase activity. The nth1 mutant also exhibited elevated frequencies of spontaneous mitotic intrachromosomal recombination, which is a phenotype shared by the MMS-hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.     

Drebrin particles: components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia

Drebrin, an actin-binding 70-kDa protein with an unusually slow SDS-PAGE mobility corresponding to approximately 120 kDa, containing a proline-rich, profilin-binding motif, had originally been reported from neuronal cells, but recently has also been found in diverse other kinds of tissues and cell lines. In biochemical analyses of various cells and tissues, employing gel filtration, sucrose gradient centrifugation, immunoprecipitation and -blotting, we have identified distinct states of soluble drebrin: a approximately 4S monomer, an 8S, ca. 217-kDa putative trimer, a 13S and a > 20S oligomer. In the 8S particles only [35S]methionine-labelled drebrin but no other actin-binding protein has been detected in stoichiometric amounts. By immunofluorescence and immunoelectron microscopy, drebrin-positive material often appeared as "granules" up to 400 nm in diameter, in some cell types clustered near the Golgi apparatus or in lamellipodia, particularly at leading edges, or in dense-packed submembranous masses at tips (acropodia) or ruffles of leading edges, in filopodia and at plaques of adhering junctions. We conclude that these drebrin complexes and drebrin-rich structures allow the build-up and maintenance of high local drebrin concentrations in strategic positions for the regulation of actin filament assembly, thereby contributing to cell motility and morphology, in particular local changes of plasticity and the formation of protrusions.     

Glycine,Serine, and Leucine Metabolism in Different Regions of Rat Central Nervous System

We have investigated the glycine, serine and leucine metabolism in slices of various rat brain regions of 14-day-old or adult rats, using [1-¹⁴C]glycine, [2-¹⁴C]glycine, L-[3-¹⁴C]serine and L-[U-¹⁴C]leucine. We showed that the [1-¹⁴C]glycine oxidation to CO₂ in all regions studied occurs almost exclusively through its cleavage system (GCS) in brains of both 14-day-old and adults rats. In 14-day-old rats, the highest oxidation of [1-¹⁴C]glycine was in cerebellum and the lowest in medulla oblongata. In these animals, the L-[U-¹⁴C]leucine oxidation was lower than the [1-¹⁴C]glycine oxidation, except in medulla oblongata where both oxidations were the same. Serine was the amino acid that showed lowest oxidation to CO₂ in all structure studied. In adult rats brains, the highest oxidation of [1-¹⁴C]glycine was in cerebral cortex and the lowest in medulla oblongata. We have not seen difference in the lipid synthesis from both glycine labeled, neither in 14-day-old rats nor in adult ones, indicating that the lipids formed from glycine were not neutral. Lipid synthesis from serine was significantly high than lipid synthesis and from all other amino acids studied in all studied structures. Protein synthesis from L-[U-¹⁴C]leucine was significantly higher than that from glycine in all regions and ages studied.     

Plant Effects on Spatial and Temporal Patterns of Nitrogen Cycling in Shortgrass Steppe

Because of the water-limited nature and discontinuous plant cover of shortgrass steppe, spatial patterns in ecosystem properties are influenced more by the presence or absence of plants than by plant type. However, plant type may influence temporal patterns of nutrient cycling between plant and soil. Plants having the carbon-3 (C₃) or carbon-4 (C₄) photosynthetic pathway differ in phenology as well as other attributes that affect nitrogen (N) cycling. We estimated net N mineralization rates and traced nitrogen-15 (¹⁵N) additions among plant and soil components during May, July, and September of 1995 in native plots of C₃ plants, C₄ plants, or mixtures of C₃ and C₄. Net N mineralization was significantly greater in C₃ plots than in C₄ plots during both July and September. C₃ plots retained significantly more ¹⁵N in May than did mixed and C₄ plots; these differences in ¹⁵N retention were due to greater ¹⁵N uptake by C₃ plants than by C₄ plants during May. There were no significant differences in total ¹⁵N retention among plant communities for July and September. Soil ¹⁵N was influenced more by presence or absence of plants than by type of plant; greater quantities of ¹⁵N remained in soil interspaces between plants than in soil directly under plants for July and September. Our results indicate that plant functional type (C₃ versus C₄) can affect both the spatial and the temporal patterns of N cycling in shortgrass steppe. Further research is necessary to determine how these intraseasonal differences translate to longer-term and coarser-scale effects of plants on N cycling, retention, and storage. Received 8 December 1997; accepted 6 May 1998.