Impact of orientation and flexibility of peptide linkers on <Emphasis Type="Italic">T. maritima</Emphasis> lipase Tm1350 displayed on <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spores surface using CotB as fusion partner

Fusion protein construction often requires peptide linkers for prolonged conformation, extended stability and enzyme activity. In this study a series of fusion between Thermotoga maritima lipase Tm1350 and Bacillus subtillis coat protein CotB, comprising of several peptide linkers, with different length, flexibility and orientations were constructed. Effects of temperature, pH and chemicals were examined, on the activity of displayed enzyme. The fusion protein with longer flexible linkers L9 [(GGGGS)₄] and L7 (GGGGS-GGGGS-EAAAK-EAAAK-GGGGS-GGGGS) possess 1.29 and 1.16-fold higher activity than the original, under optimum temperature and pH respectively. Moreover, spore surface displaying Tm1350 with L3 (EAAAK-GGGGS) and L9 ((GGGGS)₄) showed extended thermostably, maintaining 1.40 and 1.35-fold higher activity than the original respectively, at 80 °C after 5 h of incubation. The enzyme activity of linkers with different orientation, including L5, L6 and L7 was determined, where L7 maintained 1.05 and 1.27-fold higher activity than L5 and L6. Effect of 0.1% proteinase K, bromelain, 20% ethanol and 30% methanol was investigated. Linkers with appropriate Glycine residues (flexible) showed higher activity than Alanine residues (rigid). The activity of the displayed enzyme can be improved by maintaining orientation and flexibility of peptide linkers, to evaluate high activity and stability in industrial processes.     

The abundance of an invasive freshwater snail Tarebia granifera (Lamarck, 1822) in the Nseleni River,South Africa

The invasive freshwater snail Tarebia granifera (Lamarck, 1822) was first reported in South Africa in 1999 and it has become widespread across the country, with some evidence to suggest that it reduces benthic macroinvertebrate biodiversity. The current study aimed to identify the primary abiotic drivers behind abundance patterns of T. granifera, by comparing the current abundance of the snail in three different regions, and at three depths, of the highly modified Nseleni River in KwaZulu-Natal, South Africa. Tarebia granifera was well established throughout the Nseleni River system, with an overall preference for shallow waters and seasonal temporal patterns of abundance. Although it is uncertain what the ecological impacts of the snail in this system are, its high abundances suggest that it should be controlled where possible and prevented from invading other systems in the region.     

Fetal death persists through recurrent pregnancies in mice following Ljungan virus infection

OBJECTIVES: Laboratory mice infected with Ljungan virus (LV) early in pregnancy suffer from perinatal death. Here we investigate the persistence of that effect through the outcome of consecutive pregnancies in LV‐infected mice. STUDY DESIGN: CD‐1 mice were infected while pregnant and their adult female offspring were followed in parallel with uninfected control mice during repeated pregnancies. Three mating attempts resulted in two or three pregnancies per dam. The outcome of the last pregnancy was carefully monitored. RESULTS: Both the dams infected as adults and their adult female offspring suffered perinatal deaths during the last pregnancy which occurred approximately 6 months after the original LV exposure and acute infection. The non‐infected control animals experienced no perinatal death. CONCLUSIONS: Perinatal death persists across recurrent pregnancies in this mouse model of LV infection, both in animals infected as adults and in females exposed to the virus in utero. This implies that LV persists in mice long after intial infection, and is maintained in a quiescent state but can remain pathogenic in later pregnancies. Birth Defects Res (Part B) 83:507‐510, 2008. © 2008 Wiley‐Liss, Inc.     

Implementation of an insecticide-treated net subsidy scheme under a public-private partnership for malaria control in Tanzania – challenges in implementation

Background

In the past decade there has been increasing visibility of malaria control efforts at the national and international levels. The factors that have enhanced this scenario are the availability of proven interventions such as artemisinin-based combination therapy, the wide scale use of insecticide-treated nets (ITNs) and a renewed emphasis in indoor residual house-spraying. Concurrently, there has been a window of opportunity of financial commitments from organizations such as the Global Fund for HIV/AIDS, Tuberculosis and Malaria (GFATM), the President's Malaria Initiative and the World Bank Booster programme.

Methods

The case study uses the health policy analysis framework to analyse the implementation of a public-private partnership approach embarked upon by the government of Tanzania in malaria control – 'The Tanzania National Voucher Scheme'- and in this synthesis, emphasis is on the challenges faced by the scheme during the pre-implementation (2001 – 2004) and implementation phases (2004 – 2005). Qualitative research tools used include: document review, interview with key informants, stakeholder's analysis, force-field analysis, time line of events, policy characteristic analysis and focus group discussions. The study is also complemented by a cross-sectional survey, which was conducted at the Rufiji Health Demographic Surveillance Site, where a cohort of women of child-bearing age were followed up regarding access and use of ITNs.

Results

The major challenges observed include: the re-introduction of taxes on mosquito nets and related products, procurement and tendering procedures in the implementation of the GFATM, and organizational arrangements and free delivery of mosquito nets through a Presidential initiative.

Conclusion

The lessons gleaned from this synthesis include: (a) the consistency of the stakeholders with a common vision, was an important strength in overcoming obstacles, (b) senior politicians often steered the policy agenda when the policy in question was a 'crisis event', the stakes and the visibility were high, (c) national stakeholders in policy making have an advantage in strengthening alliances with international organizations, where the latter can become extremely influential in solving bottlenecks as the need arises, and (d) conflict can be turned into an opportunity, for example the Presidential initiative has inadvertently provided Tanzania with important lessons in the organization of 'catch-up' campaigns.     

Demonstration of long-range interactions in a PDZ domain by NMR, kinetics, and protein engineering

Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.     

Lipid interacting regions in phosphate stress glycosyltransferase atDGD2 from Arabidopsis thaliana

Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227-245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130-148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227-245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.     

Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) and KO mice were fed 3H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested 3H-SM in the intestinal content increased by 6-fold and the formation of 3H-ceramide decreased markedly, resulting in 98% reduction of 3H-ceramide/3H-SM ratio 1 h after gavage. The absorbed 3H-palmitic acid portion was decreased by 95%. After 3 h, a small increase in 3H-ceramide was identified in distal intestine in KO mice. In feces, 3H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.