Detection of the Antiviral Drug Oseltamivir in Aquatic Environments

Oseltamivir (Tamiflu®) is the most important antiviral drug available and a cornerstone in the defence against a future influenza pandemic. Recent publications have shown that the active metabolite, oseltamivir carboxylate (OC), is not degraded in sewage treatment plants and is also persistent in aquatic environments. This implies that OC will be present in aquatic environments in areas where oseltamivir is prescribed to patients for therapeutic use. The country where oseltamivir is used most is Japan, where it is used to treat seasonal flu. We measured the levels of OC in water samples from the Yodo River system in the Kyoto and Osaka prefectures, Japan, taken before and during the flu-season 2007/8. No OC was detected before the flu-season but 2–58 ng L⁻¹ was detected in the samples taken during the flu season. This study shows, for the first time, that low levels of oseltamivir can be found in the aquatic environment. Therefore the natural reservoir of influenza virus, dabbling ducks, is exposed to oseltamivir, which could promote the evolution of viral resistance.     

Risks of hormonally active pharmaceuticals to amphibians: a growing concern regarding progestagens

Most amphibians breed in water, including the terrestrial species, and may therefore be exposed to water-borne pharmaceuticals during critical phases of the reproductive cycle, i.e. sex differentiation and gamete maturation. The objectives of this paper were to (i) review available literature regarding adverse effects of hormonally active pharmaceuticals on amphibians, with special reference to environmentally relevant exposure levels and (ii) expand the knowledge on toxicity of progestagens in amphibians by determining effects of norethindrone (NET) and progesterone (P) exposure to 0, 1, 10 or 100 ng l⁻¹ (nominal) on oogenesis in the test species Xenopus tropicalis. Very little information was found on toxicity of environmentally relevant concentrations of pharmaceuticals on amphibians. Research has shown that environmental concentrations (1.8 ng l⁻¹) of the pharmaceutical oestrogen ethinylestradiol (EE₂) cause developmental reproductive toxicity involving impaired spermatogenesis in frogs. Recently, it was found that the progestagen levonorgestrel (LNG) inhibited oogenesis in frogs by interrupting the formation of vitellogenic oocytes at an environmentally relevant concentration (1.3 ng l⁻¹). Results from the present study revealed that 1 ng NET l⁻¹ and 10 ng P l⁻¹ caused reduced proportions of vitellogenic oocytes and increased proportions of previtellogenic oocytes compared with the controls, thereby indicating inhibited vitellogenesis. Hence, the available literature shows that the oestrogen EE₂ and the progestagens LNG, NET and P impair reproductive functions in amphibians at environmentally relevant exposure concentrations. The progestagens are of particular concern given their prevalence, the range of compounds and that several of them (LNG, NET and P) share the same target (oogenesis) at environmental exposure concentrations, indicating a risk for adverse effects on fertility in exposed wild amphibians.     

Dynamics of submersed aquatic vegetation on shallow soft bottoms in the Baltic Sea

Abstract. Persistence and colonization of submersed aquatic plant species were studied in permanent plots (20 cm × 20 cm) at three shallow sites adjacent to Askö island, in the northern Baltic Sea. The study started in 1991 at two of the sites, in 1992 at a third site and continued until 1997. Two major weather‐induced disturbances occurred during the study: a long, stormy period during a mild winter and a cold winter with extreme low water levels. The stormy period caused a large loss of species from plots (95 ± 5% SE) at the most wave‐exposed site, resulting in a low species number per plot (0.8 ± 0.2) the following summer. During the three following years the mean species number increased to 3.6 ± 0.2. The cold winter caused high species turnover and increased species number per plot at the two most wave‐exposed sites. The species turnover at the sheltered site was highest in the two years with low water level in May, possibly due to increased waterfowl herbivory. Annuals, loosely anchored and highly reproductive species had significantly higher plot colonization rates and lower persistence than perennials, well‐anchored species and species with none or low reproduction. The extent of lateral growth had no significant effect on colonization or persistence. Although these disturbances have a large effect on the dynamics, species mobility was also high in other years. Relative to other, similar, studies in terrestrial vegetation mean persistence in plots was remarkably low and mean mobility and species turnover rates were very high.     

Comparison between RFLP and MIRU-VNTR Genotyping of Mycobacterium tuberculosis Strains Isolated in Stockholm 2009 to 2011

Our aim was to analyze the difference between methods for genotyping of Mycobacterium tuberculosis complex isolates. We collected genotyping results from Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetitive Units - Variable Numbers of Tandem Repeat (MIRU-VNTR) in a geographically limited area (Stockholm) during a period of three years. The number and proportion of isolates belonging to clusters was reduced by 45 and 35% respectively when combining the two methods compared with using RFLP or MIRU-VNTR only. The mean size of the clusters was smaller when combining methods and smaller with RFLP compared to MIRU-VNTR. In clusters with confirmed epidemiological links RFLP coincided slightly better than MIRU-VNTR but where there was a difference, the variation in MIRU-VNTR pattern was only in a single locus. In isolates with few IS6110 bands in RFLP, MIRU-VNTR differentiated the isolates more, dividing the RFLP clusters. Since MIRU-VNTR is faster and less labour-intensive it is the method of choice for routine genotyping. In most cases it will be sufficient for epidemiological purposes but true clustering might still be considered if there are epidemiological links and the MIRU-VNTR results differ in only one of its 24 loci.     

Isolation of the epidermal growth factor from the shrew submaxillary gland

Entomocidal crystals produced by Bacillus thuringiensis ssp. israelensis are formed by two proteins with molecular masses of 130 and 28 kDa, whereas the protein with a molecular mass of 70 kDa appears as a result of 130 kDa protein limited proteolysis by admixtures of bacterial proteinases in the course of its dissolution. The comparison of the N-terminal sequences of the protein with molecular mass of 70 kDa (Met-Glu-Asn-Xaa-Pro-Leu-Asp-Thr-Leu-Ser-Ile-Val-Asn-Glu-Thr-Asp) and that of 28 kDa (Met-Glu-Asn-Leu-Asn-[Phe]-[Trp]-Pro-Leu-Gln-Asp-Ile-Lys-Val-Asn-Pro) reveals only marginal similarity between them (only 4 identical residues among 16 aligned). Both B. thuringiensis israelensis crystal-forming proteins appear hardly related to those contained in the crystal produced by other B. thuringiensis subspecies, e.g. kurstaki. It might be concluded that at least some of the entomocidal proteins found in the crystalline inclusion bodies of various B. thuringiensis subspecies revealed rather strong variations in their primary structures that facilitate their adaptation to different hosts.

Bacillus thuringiensis ssp. israelensis δ-Endotoxin Entomocidal crystal Insecticide Mosquito     

Immobilized metal ion affinity chromatography.

The introduction of immobilized metal ion affinity chromatography, directed toward specific protein side chains, has opened a new dimension in protein purification. This review covers the principles and practice of IMAC that can be performed under very mild, nondenaturing conditions. IMAC is particularly suitable for preparative group fractionation of complex extracts and biofluids, but can also be used in high-performance mode: "HP-IMAC." Single-step purifications of 1000-fold or more may allow isolation of a particular protein from crude extracts on a milligram or gram scale. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography, and the immobilized metal ion ligand complexes are more likely to withstand wear and tear than are antibodies or enzymes. The enormous potential of IMAC and related metal affinity techniques is only in the initial stages of being explored and exploited. Synthesis of IMA adsorbents, and various modes of performing IMAC are discussed and exemplified with selected applications. Advantages and disadvantages are listed. Effective means of counteracting the few undesirable effects that can occur are suggested.     

Purification of antibodies to protein hormones by affinity chromatography on divinylsulfonyl sepharose

Antibodies to human growth hormone and ovine interstitial cell stimulating hormone have been purified from rabbit antisera by affinity chromatography using a newly developed divinylsulfonyl activated agarose. Elution of the antibodies was accomplished by neutral solutions containing chaotropic ions.     

Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography

High-performance immobilized metal ion affinity chromatography was utilized to evaluate the adsorption properties of 67 synthetic, biologically active, peptides ranging in size from 5 to 42 residues. The metal ions, Cu(II), Ni(II) and Zn(II), were immobilized by iminodiacetic acid (IDA) coupled to TSK gel 5PW (10 microns). Two types of gradient elution (imidazole and pH) were used to evaluate peptide retention by the metal ions. A decreasing pH gradient and an increasing imidazole gradient eluted the peptides in similar order. IDA-Cu(II) and IDA-Zn(II) showed very similar selectivities for the peptides analyzed; however, IDA-Zn(II) displayed a weaker affinity for the peptides. IDA-Ni(II) showed a slightly different pattern of selectivity. Peptide adsorption effects contributed by the metal-free gel matrix were found to be relatively minor. The concentration and type of salt included in the mobile phase could affect the relative affinities of the peptides for the immobilized metal ions. Retention coefficients were assigned to individual amino acid residues by multiple linear regression analysis. Histidine showed the largest positive correlation with retention, followed by aromatic amino acid residues. Modified N-terminal residues resulted in negative contributions to retention. Analyses of peptide amino acid composition alone allowed prediction of peptide retention behavior on immobilized metal ion affinity columns.     

Purification of human serum amyloid P component (SAP) by calcium affinity chromatography

Serum amyloid P component (SAP) has been purified from human serum by means of immobilized metal ion affinity chromatography (IMAC). It was selectively concentrated on carboxymethylated aspartic acid agarose (CM-Asp-agarose) loaded with calcium and, employing very mild conditions, purified to electrophoretical and immunological homogeneity in a single step amounting to about 1900-fold purification. As a purification method our procedure thus compares well with bio-specific affinity chromatography.