Synthesis of N-hydroxysuccinimide esters of phosphatidylethanolamine and some properties of liposomes containing these derivatives

The N-hydroxysuccinimide (NHS) ester of N-suberyl-dimyristoylphosphatidylethanolamine (sub-DMPE) was synthesized by reaction of DMPE with disuccinimidyl suberate, and isolated by preparative plate chromatography. Liposomes, which contain NHS-sub-DMPE, can covalently bind compounds that possess a free amino group such as ε-dinitrophenyl-lysine. The extent of DNP-lysine binding is influenced by the time and temperature of incubation, the amount of NHS-sub-DMPE incorporated into the liposomes, and the initial concentration of DNP-lysine. Binding occurs as a consequence of the formation of a new dinitrophenylated compound which has been characterized. Although NHS-sub-DMPE is stable to storage in organic solvents, preformed liposomes rapidly lose their ability to bind DNP-lysine due to hydrolysis of the N-hydroxysuccinimide ester bond. These findings bear on the future applicability of liposomes, containing N-hydroxysuccinimide esters of PE, as illustrated by the preparation of immunogenic liposomes.     

Decadienal afforestation and risks to montane grassland amphibians in the Udzungwa Plateau,Tanzania

Human modification of the environment has drastically changed ecosystems around the globe. While forest-loss (deforestation) has been well studied for its effects on ecosystems, afforestation also has major impacts. The Udzungwa Plateau in Tanzania is naturally a mix of forests and grasslands. However, non-native pine plantations have recently increased in the area displacing the native grasslands of the montane plateau. In this study, we explored the effects of land use change on amphibian communities by comparing amphibian surveys before non-native plantations were introduced (~2000) and after (2017–2019). To put these amphibian surveys into wider context across the plateau and to distinguish local population loss compared to plateau-wide extinctions, we sample diversity at new sampling locations and locations with multiple surveys through time. We then assessed landscape change through remotely sensed data and ground surveys. Amphibian encounter surveys found approximately the same species diversity across all sampling locations, but local extinctions appear likely. Remaining amphibians appear to be limited to small refugia in non-pine wetlands. Approximately half (45%) of the species found have limited distributional ranges (<72,000 km²) and are described as habitat specialists by the IUCN Red List. Monitoring pine plantation growth using remote sensing techniques suggested only limited extensions of plantations between 2000 through to 2013–2016, but ground surveys found small pines (<3 years old) throughout almost all areas identified as grasslands on satellite imagery. Our study highlights the plight of this narrowing biome on the African continent and calls for more research on the impact of non-native plantations on natural communities. Abstract in Swahili is available with online material.     

Isolation and characterization of two Brassica napus embryo acyl-ACP thioesterase cDNA clones

Acyl-ACP thioesterases are involved in regulating chain termination of fatty acid biosynthesis in plant systems. Previously, acyl-ACP thioesterase purified from Brassica napus seed tissue has been shown to have a high preference for hydrolysing oleoyl-ACP. Here, oligonucleotides derived from B. napus oleoyl-ACP thioesterase protein sequence data have been used to isolate two acyl-ACP thioesterase clones from a B. napus embryo cDNA library. The two clones, pNL2 and pNL3, contain 1642 bp and 1523 bp respectively and differ in the length of their 3 non-coding regions. Both cDNAs contain open reading frames of 366 amino acids which encode for 42 kDa polypeptides. Mature rape thioesterase has an apparent molecular weight of 38 kDa on SDS-PAGE and these cDNAs therefore encode for precursor forms of the enzyme. This latter finding is consistent with the expected plastidial location of fatty acid synthase enzymes. Northern blot analysis shows thioesterase mRNA size to be ca. 1.6 kb and for the thioesterase genes to be highly expressed in seed tissue coincident with the most active phase of storage lipid synthesis. There is some sequence heterogeneity between the two cDNA clones, but overall they are highly homologous sharing 95.7% identity at the DNA level and 98.4% identity at the amino acid level. Some sequence heterogeneity was also observed between the deduced and directly determined thioesterase protein sequences. Consistent with the observed sequence heterogeneity was Southern blot data showing B. napus thioesterase to be encoded by a small multi-gene family.     

Refuge use by fish as a function of body length-related metabolic expenditure and predation risks

Theory predicts that staying in a refuge has benefits in terms of predator avoidance and costs in terms of lost feeding opportunities. In this study, we investigated how the relative importance of these costs and benefits changes with increasing body length. This is of particular interest in animals such as fish, which show continuous growth throughout their lives. Our results suggest that larger fish are subject to lower predation risks and are less affected by food deprivation than small fish, with fish decreasing their responses to food-deprivation treatments more strongly with increasing body length than to predation treatments. This may explain our observation that large fish emerged later from a refuge than small ones and spent shorter times outside the refuge. The key role of differential responses to food deprivation was further illustrated by the finding that the relative weight loss of individual fish was strongly correlated with a reduction in hiding time even in the absence of body length differences. The importance of inter-individual differences in metabolic rates for the decision-making behaviour of animals is discussed.     

Cystic fibrosis carrier population screening in the primary care setting.

To determine the receptivity of prenatal care providers and their patients to carrier testing for cystic fibrosis (CF), we offered free carrier screening, followed by genetic counseling of carriers, to all prenatal care providers in Rochester, NY, for all their female patients of reproductive age, pregnant or not. Of 124 prenatal care providers, only 37 elected to participate, but many of these offered screening only to pregnant women. The acceptance rate among pregnant women was approximately 57%. The most common reasons for accepting screening were to obtain reassurance (50.7%) and to avoid having a child with CF (27.8 %). The most common reasons for declining screening were not intending to terminate a pregnancy for CF (32.4%) and believing that the chance of having a CF child was very low (32.2%). Compared with decliners, acceptors were more likely to have no children, regarded having a child with CF as more serious, believed themselves more susceptible to having such a child, knew more about CF, would be more likely to terminate a pregnancy if the fetus were shown to have CF, and more strongly supported offering CF screening to women of reproductive age. Of 4,879 women on whom results were obtained, 124 were found to be carriers. Of these 124 carriers, the partners of 106 were tested. Of the five at-risk couples, four requested prenatal diagnosis and one requested neonatal diagnosis. No woman found to be a carrier whose partner tested negative requested prenatal diagnosis. Except for the imperfect knowledge of those testing negative, none of the adverse outcomes predicted for CF carrier testing in the general population were observed in this study.     

Prenatal hemoglobinopathy screening. IV. Follow-up of women at risk for a child with a clinically significant hemoglobinopathy.

To determine the benefits and burdens of prenatal hemoglobinopathy carrier identification and genetic counseling and its impact on subsequent reproductive behavior, we recontacted women whom we had previously identified as at risk for having a child with a clinically significant hemoglobinopathy, regardless of whether they had accepted the offer of prenatal diagnosis. Of the 46 such women, 31 were available for interview. These 31 women had received offers of prenatal diagnosis in 47 pregnancies. Seventeen had been accepted, and 30 had been declined. The proportion of patients accepting the offer of prenatal diagnosis was higher for the index pregnancy (50%) than for subsequent pregnancies (22%). The mean interval between the initial counseling of the patient and the follow-up interview was 43 mo (standard error +/- 2.7 mo). Ninety-four percent of those interviewed recalled having received information from the screening program; 74% recalled the name of their condition; 90% knew that trait did not affect their health; 84% recalled the name of the condition for which their fetus had been at risk; and 77% could state at least one symptom of the disease. Of the 29 women asked whether they intended to use prenatal diagnosis in future pregnancies, 13 said yes and 16 said no. Of the 26 patients asked about satisfaction with their previous decision about prenatal diagnosis, all were satisfied with their decision. Eighteen said they would make the same decision in their next pregnancy, but seven patients said they would not, and one was undecided.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prenatal screening for hemoglobinopathies. II. Evaluation of counseling.

Learning during genetic counseling is often below expectations, especially in the context of genetic screening. In this report we describe learning as a result of genetic counseling of 298 pregnant women identified as hemoglobinopathy carriers, 234 with sickle cell trait and 64 with beta-thalassemia trait. Counseling was designed to provide the information needed in a simple, clear, and nondirective manner. A special videotape produced for this purpose provided dramatization and a role model illustrating an appropriate response. After viewing the videotape the counselee had an opportunity to question the counselor and to have any misconceptions corrected. Questionnaires revealed significantly increased knowledge as a result of counseling in each of the three hemoglobinopathy subject areas tested-namely, clinical manifestations, genetics, and prenatal diagnosis. Five factors correlated with higher knowledge scores after counseling-namely, a younger patient age, more years of education, knowledge of having trait before this identification, knowledge of the baby's father having trait before counseling, and having no prior children.     

Two groups control light-induced schiff base deprotonation and the proton affinity of asp(85) in the Arg(82)His mutant of bacteriorhodopsin

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.