Size fractionation of Trypanosoma brucei DNA: localization of the 177-bp repeat satellite DNA and a variant surface glycoprotein gene in a mini-chromosomal DNA fraction

We have size-fractionated intact DNA from Trypanosoma brucei into a major large DNA fraction (greater than 350S) and minor middle-sized (60-250S) and small (less than 60S) DNA fractions. Large DNA contains the rRNA genes, the basic copy genes for several variant surface glycoproteins (VSGs), including one which lies near a telomer, and the expression-linked copies of the two VSG genes. The middle-sized DNA contains at least one VSG gene, but the hybridization of this fraction with probes for the conserved repetitive sequences that mark the edges of the transposed segments of VSG genes, suggests that it may contain many VSG genes. The 177-bp repeat satellite DNA is also exclusively found in this fraction.     

Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous α-amylase

The ups45 gene encodes the major extracellular protein from Lactococcus lactis. The deduced sequence of the 27 residue leader peptide revealed the tripartite characteristics of a signal peptide. This leader peptide directed the efficient secretion of the homologous proteinase (PrtP) in L. lactis, indicating that the putative signal peptide of PrtP can be replaced by the 27 residue Usp45 leader peptide. In addition, the 27 residue leader peptide could be used to secrete the Bacillus stearothermophilus α-amylase, encoded by the amyS gene. Fusion of the usp45 promoter region and various parts of the leader sequence to an amyS gene devoid of its signal sequence, showed that in Escherichia coli the first 19, 20, and 27 residues of the Usp45 leader are able to direct α-amylase secretion. In L. lactis the shorter signal peptides did not result in secretion of α-amylase, providing experimental evidence for the hypothesis that gram-positive bacteria require a longer signal peptide for secretion than gram-negative organisms.     

The effect of surface charge density on valinomycin-K+ complex formation in model membranes

The model membrane approach was used to investigate the surface charge effect on the ion-antibiotic complexation process. Mixed monolayers of valinomycin and lipids were spread on subphases containing K⁺ or Na⁺. The surface charge density was modified by spreading ionizable valinomycin analogs on aqueous subphases of different pH or by changing the nature of the lipid (neutral, negatively charged) in the mixed film. Surface pressure and surface potential measurements demonstrated that a neutral lipid (phosphatidylcholine) or positively charged valinomycin analogs didn't enhance the antibiotic complexing capacity. However, a maximal complexation is reached for a critical lipid concentration in the valinomycin-phosphatidylserine mixed film. The role of the surface charge on the valinomycin complexing properties was examined in terms of the Gouy-Chapman theory. As a consequence of the negative charge of the lipid monolayer, the K⁺ concentration near the surface is larger than the bulk concentration, by a Boltzmann factor. A good agreement was observed between the experimental results and the theoretical predictions. Conductance measurements of asymmetric bilayers containing a neutral lipid (egg lecithin) on one side and a negatively charged lipid (phosphatidylserine) on the other, confirm the role of the surface charge. Indeed, addition of K⁺ to the neutral side of the bilayer containing valinomycin had no effect on the conductance whereas addition of K⁺ to the charged side of the bilayer caused a 80-fold conductance increase.     

The pelagic system in the photic zone of the tropical Atlantic

Summary A multidisciplinary study of the carbon budget in the upper 300 meter of permanently stratified waters was started by two NECTAR-expeditions (North Equatorial Current Trans Atlantic Research) with HMS TYDEMAN in 1977 and 1978 (BAARS, ZIJLSTRA and TIJSSEN, 1979). In 1979 additional measurements were performed during the Gulf of Guinea-expedition with MS TYRO.Primary production in the nutrient depleted mixed layer of the North Equatorial Current estimated from the diurnal cycle in the O₂ concentration (TIJSSEN, 1979) and in POC (POSTMA and ROMMETS, 1979) revealed values 4–10 times higher than the data from the¹⁴C method in the literature: 800–2000 against ca. 200 mg C/m²/day. Moreover,¹⁴C incubations performed in bottle volumes of 4 liter and over 2 hour periods, instead of the recommended 12 hours for oligotrophic waters, gave 5–15 times higher values as incubations in the commonly used 300 ml (or smaller) bottles (GIESKES, KRAAY and BAARS, 1979). In the latter bottles a dramatic decrease of chlorophyll concentrations was observed, suggesting either heavy damage to fragile microflagellates by glasswall contacts and/or insufficient nutrient recycling by the lack of zooplankton in small samples. This then could account for the phenomena of low production and decreasing algal stock in long incubations with small bottles. These results suggest that today's picture of the primary production in the world's oceans (DE VOOIJS, 1979) needs probably a thorough reexamination. On the other hand, experiments in the North Sea and in the Gulf of Guinea did not indicate an effect of bottle size on¹⁴C incorporation and the¹⁴C method gave comparable estimates of primary production as the high precision oxygen method (photometric endpoint detection in the Winkler titration,cf. TIJSSEN, 1979). However, in these waters nutrient concentrations are always clearly above detection levels so that all previous data from somewhat richer areas may have been correct. In relation to the large oligotrophic parts of the oceans a question remains about the fate of the probably high primary production. Is it consumed by the algae themselves by night (POSTMA, 1980), are bacteria and microzooplankton more important consumers than formerly thought, or is the daily ration of zooplankters much higher as expected from extrapolation of filtration experiments in more eutrophic waters? We hope to clarify this topic during the coming NECTAR'82 expedition.Another main objective of the programme concerns the character of the deep chlorophyll maximum in permanently stratified waters. This layer is in the North Equatorial Current located at the depth of the nitratocline and at ca. 1% of the incident light at the surface (SPITZER and WERNAND, 1981). Primary production in the layer seems to be negligible while detailed vertical profiles of zooplankton, obtained with a Longhurst-Hardy Plankton Recorder, revealed no obvious concentrations in the layer. Chlorophyll analysis by thin-layer-chromatography (GIESKES, KRAAY and TIJSSEN, 1978) demonstrated that more than half of the chlorophyll a in the layer consists of an isomer which bleaches rapidly when transferred to higher light levels. The hypothesis was formulated that the chlorophyll maximum layer, at least in this region of the Atlantic, is an accumulation of chlorophyll breakdown products with a quite long turnover time at low light levels. In contrast to these findings, chlorophyll maxima near West-Africa and in the Gulf of Guinea were located at 5–10% light and contained more living algal cells (most of them as small as 1–3 m) than the nutrientpoor mixed layer above it. Primary production profiles had therefore a bimodal shape, with peaks at 30–50% light and at the chlorophyll maximum. Chlorophyll isomers, now analysed with High Pressure Liquid Chromatography, were also in these waters present at nearly all stations and comprised 30–70% of total chlorophyll comparable to the situation in the North Equatorial Current (GIESKES and KRAAY, 1981). It could be shown that these isomers are not involved in primary production, so that well established relationships between chlorophyll, light and primary production, found in oceanographic literature, have to be reconsidered.     

Synthesis of protected oligo (γ-methyl-L-glutamates) and study of their conformation in solution and at the air–water interface

Oligopeptides having the general formula N-carbobenzoxy(γ-methyl-L -glutamyl)_n-dimethyl-L -glutamate (with n = 1, 3, 5, 7, 11) were prepared, using both solid-phase synthesis and conventional peptide couplings in solution. The appearance of an ordered structure with increasing chain length was studied in organic solvents and at the air–water interface. The results obtained by thin-layer chromatography were interpreted. All the studies have demonstrated a very similar behaviour of this type of oligopeptide in solution and in monolayers.     

Loss of Multi-Epitope Specificity in Memory CD4+ T Cell Responses to B. Pertussis with Age

Pertussis is still occurring in highly vaccinated populations, affecting individuals of all ages. Long-lived Th1 CD4⁺ T cells are essential for protective immunity against pertussis. For better understanding of the limited immunological memory to Bordetella pertussis, we used a panel of Pertactin and Pertussis toxin specific peptides to interrogate CD4⁺ T cell responses at the epitope level in a unique cohort of symptomatic pertussis patients of different ages, at various time intervals after infection. Our study showed that pertussis epitope-specific T cell responses contained Th1 and Th2 components irrespective of the epitope studied, time after infection, or age. In contrast, the breadth of the pertussis-directed CD4⁺ T cell response seemed dependent on age and closeness to infection. Multi-epitope specificity long-term after infection was lost in older age groups. Detailed knowledge on pertussis specific immune mechanisms and their insufficiencies is important for understanding resurgence of pertussis in highly vaccinated populations.     

Signatures of Diversifying Selection in European Pig Breeds

Following domestication, livestock breeds have experienced intense selection pressures for the development of desirable traits. This has resulted in a large diversity of breeds that display variation in many phenotypic traits, such as coat colour, muscle composition, early maturity, growth rate, body size, reproduction, and behaviour. To better understand the relationship between genomic composition and phenotypic diversity arising from breed development, the genomes of 13 traditional and commercial European pig breeds were scanned for signatures of diversifying selection using the Porcine60K SNP chip, applying a between-population (differentiation) approach. Signatures of diversifying selection between breeds were found in genomic regions associated with traits related to breed standard criteria, such as coat colour and ear morphology. Amino acid differences in the EDNRB gene appear to be associated with one of these signatures, and variation in the KITLG gene may be associated with another. Other selection signals were found in genomic regions including QTLs and genes associated with production traits such as reproduction, growth, and fat deposition. Some selection signatures were associated with regions showing evidence of introgression from Asian breeds. When the European breeds were compared with wild boar, genomic regions with high levels of differentiation harboured genes related to bone formation, growth, and fat deposition.     

Intranasal Mesenchymal Stem Cell Treatment for Neonatal Brain Damage: Long-Term Cognitive and Sensorimotor Improvement

Mesenchymal stem cell (MSC) administration via the intranasal route could become an effective therapy to treat neonatal hypoxic-ischemic (HI) brain damage. We analyzed long-term effects of intranasal MSC treatment on lesion size, sensorimotor and cognitive behavior, and determined the therapeutic window and dose response relationships. Furthermore, the appearance of MSCs at the lesion site in relation to the therapeutic window was examined. Nine-day-old mice were subjected to unilateral carotid artery occlusion and hypoxia. MSCs were administered intranasally at 3, 10 or 17 days after hypoxia-ischemia (HI). Motor, cognitive and histological outcome was investigated. PKH-26 labeled cells were used to localize MSCs in the brain. We identified 0.5×10⁶ MSCs as the minimal effective dose with a therapeutic window of at least 10 days but less than 17 days post-HI. A single dose was sufficient for a marked beneficial effect. MSCs reach the lesion site within 24 h when given 3 or 10 days after injury. However, no MSCs were detected in the lesion when administered 17 days following HI. We also show for the first time that intranasal MSC treatment after HI improves cognitive function. Improvement of sensorimotor function and histological outcome was maintained until at least 9 weeks post-HI. The capacity of MSCs to reach the lesion site within 24 h after intranasal administration at 10 days but not at 17 days post-HI indicates a therapeutic window of at least 10 days. Our data strongly indicate that intranasal MSC treatment may become a promising non-invasive therapeutic tool to effectively reduce neonatal encephalopathy.