American and korean ginseng tissue cultures: Growth,chemical analysis,and plantlet production

Summary Roots, stems, or leaves of American (Panax quinquefolium) and Korean (Panax ginsing) ginseng were grown as callus or supension tissue cultures. Tissue cultures ofP. ginseng would occasionally form plantlets. The fundamental chemical composition, inorganic analysis, and saponin (panaquilin) content of American and Korean ginseng plants and tissue cultures were determined. The crude saponin content is very similar to, but approximately one-half (1.3%, fresh weight) of that present in ginseng roots. Two-dimensional thin layer chromatographic analysis revealed minor differences in the panaquilins present in American and Korean ginseng tissue cultures. The sapogenin, panaxadiol, was isolated from Korean ginseng callus.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.     

Haplotyping cryptic Adélie penguin taxa using low-cost,high-resolution melt curves

Distinguishing morphologically cryptic taxa, by definition, requires genetic data such as DNA sequences. However, DNA sequences may not be obtained easily for taxa from remote sites. Here we provide the details of a high-resolution melt-curve-based method using taxon-specific primers that can distinguish two taxa of Adélie penguins, and that will be usable in Antarctica when combined with some of the newly developed field-deployable thermal cyclers. We suggest that the wider adoption of field-deployable polymerase-chain-reaction-based techniques will enable faster assignation of haplotype to individuals in situ, and so allow the targeting of observations and sample collection to specimens relevant to the research question. Targeting individuals will also reduce the need to repeatedly handle animals and reduce the time and travel required to complete field work.     

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.     

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its Nα Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N^α acetylation of the initiator methionine of the α1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of α1 in H. volcanii cells. To further examine this, α1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP “gate” formed by the α1 N terminus were examined for their N^α acetylation. Both the “gate” deletion and Q2A substitution completely altered the N^α-acetylation pattern of α1, with the deletion rendering α1 unavailable for N^α acetylation and the Q2A modification apparently enhancing cleavage of α1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two α1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although α1 proteins with Q2D, Q2P, and Q2T substitutions were N^α acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of α1 as rings in N^α-acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these α1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N^α acetylation of α1 is important in CP assembly and activity, high levels of α1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP “gate” impairs the ability of cells to overcome salt stress.Proteolysis is important in regulation and protein quality control. Energy-dependent proteases are crucial to early stages of these proteolytic events and include proteasomes, multicatalytic proteases present in all eukaryotes and archaea and in some bacteria. The catalytic component of proteasomes, the 20S core particle (CP), consists of four heptameric rings of α- and β-type subunits stacked as a barrel in an α7β7β7α7 configuration and is essential for growth of archaeal and eukaryotic cells (39, 54). The active sites responsible for peptide bond hydrolysis are formed by N-terminal Thr residues of β-type subunits and are sequestered within the central chamber of the barrel-like structure. Energy-dependent triple-A ATPases, including regulatory particle triple-A ATPases (Rpt) in eukaryotes and proteasome-activating nucleotidases (PAN) in archaea, mediate the unfolding and translocation of substrate proteins through the α-rings for degradation within the CP (39, 40).One major difference between eukaryotic and prokaryotic proteasomal CPs is in the crystal structure of the channel opening formed by the α-rings. Due to partial disorder of the α-subunit N termini, the site of substrate entry appears open at the ends of the cylinders of archaeal and bacterial CPs (e.g., CPs of Thermoplasma acidophilum, Archaeoglobus fulgidus, and Mycobacterium tuberculosis) (13, 15, 27). In contrast, X-ray structures of the CPs of yeast (14) and bovine (45) do not contain this opening. Instead the extreme N termini of the α2, α3, and α4 subunits and the loop structure of α5 fill the central pore in a gate-like structure.Evidence suggests that all CPs are gated, and the major differences observed in the state of the α-ring gate in crystal structures are not physiological. For example, the N-terminal 11 amino acids of the A. fulgidus α subunit, which are not defined by electron density in the CP structure, are more ordered in the 16S “half” proteasome precursor (13). Furthermore, cryoelectron microscopy of the M. tuberculosis CP reveals closed ends that are dependent on the first eight residues of the α-subunit and which diminish peptidolytic activity. Consistent with this, deletion of the N-terminal α-helix (Δ2-12) of the T. acidophilum CP α-subunit abolishes the need for an ATPase (i.e., PAN) in the proteasome-mediated degradation of acid-denatured green fluorescent protein-SsrA or casein (4). In addition, the conserved YDR motif thought to be important in the sterics of α-ring gating is present in all archaeal α-type subunits to date (13). Thus, prokaryotes are thought to gate the α-ring aperture of their proteasomes; however, the physiological consequences of unregulated opening of this gate have not been examined.A gated CP channel formed by the N termini of α-rings may be a general mechanism for regulating the activity of proteasomes. The rate-limiting step in proteasome-mediated protein degradation is translocation of substrates through the α-rings to the active sites contained within the β-rings of the CP (24). Gating is supported by the finding that eukaryotic CPs have no peptidolytic activity in the absence of Rpt proteins or mild chaotropic agents such as sodium dodecyl sulfate (SDS) or heat treatment (9). Furthermore, peptidase activity of the yeast CP is blocked by the N-terminal regions of the α3 subunit. Deletion (Δ2-9) or single substitution (D9A) of N-terminal residues of α3 derepresses this peptidase activity (12).An additional gating mechanism could be employed by posttranslational modifications of the N termini of the α-type subunits. The α-type subunits of CPs are modified by N^α acetylation in several eukaryotes and haloarchaea, including Haloferax volcanii (10, 16, 20, 21, 44). In yeast, N-acetyltransferase 1 (NAT1), the catalytic component of NatA, is responsible for the N^α acetylation of five of the α-type subunits (α1, α2, α3, α4, and α7). Proteasomes purified from a nat1 mutant have twofold-higher chymotrypsin-like peptidase activity in the absence of SDS compared to the wild type, suggesting that N^α acetylation enhances closure of the α-gate (21). In H. volcanii, both α1 and α2 are N^α acetylated on their initiator methionine residue with a subset of α1 not acetylated and instead cleaved by an apparent methionine aminopeptidase (16). A large-scale proteomic survey reveals N^α acetylation is common to other proteasomal α-type proteins of the haloarchaea (10). In this previous survey, the ratios of N^α-acetylated and cleaved forms of the α-type proteins were quantified by spectral counting and estimated to be around 3:1 and 4:3 for Halobacterium salinarum and Natronomonas pharaonis, respectively (10). So far, this existence of these two unique forms of α subunit N termini in the cell simultaneously (initiator methionine N^α acetylated and methionine aminopeptidase [MAP] cleaved) has only been observed in the haloarchaea.In the present study, quantitative tandem mass spectrometry (MS/MS) was used to precisely determine the ratio of the N^α-acetylated to MAP-cleaved forms of the proteasomal α1 protein in H. volcanii. In addition, site-directed mutagenesis was used to examine how the N-terminal penultimate (second) residue and N-terminal α-helix of α1 influence its N^α-acetylated state, CP activity, and cell physiology. Alterations that either fully abolished N^α acetylation or enhanced MAP cleavage of α1 (i) resulted in an increase in CP peptidase activity and (ii) rendered the cells more sensitive to hypoosmotic stress than wild type. In contrast, site-directed changes that generated a mixed population of α1 proteins in various N^α-acetylated states, yet similar N^α-acetylation profiles in CPs to wild type, had profound consequences, including (i) a substantial increase in the levels of α1 protein as heptameric rings, (ii) higher growth rate and cell yield, and (iii) enhanced tolerance of cells to thermal and hypoosmotic stress.     

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.