Optimization of enzyme-mediated peptide bond formation

Enzyme-catalyzed peptide bond formation requires thorough examination and optimization of each coupling step. In order to identify factors influencing the selectivity between aminolysis and hydrolysis, a systematic study was carried out for the kinetically controlled peptide synthesis. The reaction temperature, the type of C-terminal protecting group, and different organic cosolvents showed little influence on the selectivity. The enzyme, excess nucleophile, pH, N-terminal protecting group, and ionic strength of the solution were identified as major factors controlling the selectivity and, therefore, the yield of the dipeptide synthesis. Under optimized conditions, the selectivity of the chymotrypsin-catalyzed synthesis of PheSer could be increased from 35 to 100%.     

Methods for determining the proliferation kinetics of cells by means of 5-bromodeoxyuridine.

After treatment of cells with 5-bromodeoxyuridine (BUdR), the percentage of completely BUdR-labelled interphase nuclei is greater the longer the BUdR treatment. The labelling effect is visible after staining with the fluorochrome 33258 Hoechst and with Giemsa. Various formulae and a nomogram are presented by means of which the percentage of cells in S period, duration of the S period and the whole cell cycle can be determined by examination of a single preparation or by comparison of several preparations. The methods are tested using cell cultures of Microtus agrestis and compared with autoradiographic methods after labeling with 3H-thymidine.     

Increase in the number of atrial natriuretic hormone receptors in regenerating rat liver.

Forty-eight hours after partial (approximately 67%) hepatectomy the activity of the particulate guanylate cyclase was increased by 2-fold in the regenerating rat liver. This increase was not an artifact of membrane isolation procedures, and as determined by 125I-labeled Tyr-28 atrial natriuretic hormone-(1-28) ANF binding, was accompanied by a 2-fold increase in the number of ANF receptors. The Kd of the receptors in membranes of regenerating livers was not significantly different from the Kd of the receptors in livers of sham-operated rats. The linear synthetic descysteine analog of ANF, analog I, which binds only to the 66-kDa receptors, displaced approximately 40% of the specifically bound 125I-ANF in liver membranes from both hepatectomized and sham-operated (control) animals. Affinity cross-linking studies with 125I-ANF confirmed the increase in the 116-kDa ANF receptor in membranes of regenerating livers. In perfused livers derived from control and hepatectomized animals, the basal rates of cGMP production were not significantly different. However, atriopeptin II-stimulated cGMP production was twice as great in regenerating livers as compared with controls. These data demonstrate that the increase in particulate guanylate cyclase activity observed during liver regeneration is due to an increase in the 116-kDa ANF receptor-associated activity. Additionally, our data demonstrate that the regenerating rat liver may be a valuable model with which to study the role of the hepatic ANF receptor/particulate guanylate cyclase.     

A two-step enzymatic synthesis of dipeptides

A simple system is introduced to produce dipeptides continuously by enzyme catalyzed condensation of amino acid esters and amino acid amides. Synthesis of N-terminal free dipeptide-amides is achieved by means of carboxypeptidase Y. The peptide-amide is deamidated utilizing a newly isolated peptide-amide is deamidated utilizing a newly isolated peptide-amidase. Separation of substrates and products is accomplished by anion-exchange chromatography. Modeling of the reactions shows that the two reactions have to be carried out in a cascade of two reactors in order to prevent hydrolysis of the peptide by the carboxypeptidase. Continuous production of Kyotorphin (H-TyrArg-OH) with a space-time yield of 257 g/L . d shows the feasibility of this concept.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Studies of insulin release and rat pancreatic islet metabolism with diastereomers of D-glucose

Studies of insulin release with diastereomers and other analogues of D-glucose demonstrated that only sugars which undergo oxidation to CO₂ stimulate insulin release by the pancreatic islet. None of the non-metabolizable diastereomers of glucose stimulated insulin release in the presence of a substimulatory concentration of glucose for fuel. Although 5.5 mM glucose formed 77% as much CO₂ as 16.7 mM mannose and twice that of 16.7 mM fructose, 5.5 mM glucose did not stimulate insulin release whereas 16.7 mM mannose and fructose did stimulate insulin release. These results indicate that the important stimulus for glucose-induced insulin release involves metabolism of glucose, but that the stimulus does not involve solely a fuel function of glucose.     

Myelosarcomatosis of the skin preceding leukemic generalization of acute myelomonocytic leukemia

A case report of a patient with myelosarcomatosis of the skin six months preceding leukemic generalization of acute myelomonocytic leukemia is presented. To the best of our knowledge this is the first case of a myelosarcoma with generalized skin involvement diagnosed before development of an overt myeloproliferative disease.     

The effectiveness of flower strips and hedgerows on pest control,pollination services and crop yield: a quantitative synthesis

Floral plantings are promoted to foster ecological intensification of agriculture through provisioning of ecosystem services. However, a comprehensive assessment of the effectiveness of different floral plantings, their characteristics and consequences for crop yield is lacking. Here we quantified the impacts of flower strips and hedgerows on pest control (18 studies) and pollination services (17 studies) in adjacent crops in North America, Europe and New Zealand. Flower strips, but not hedgerows, enhanced pest control services in adjacent fields by 16% on average. However, effects on crop pollination and yield were more variable. Our synthesis identifies several important drivers of variability in effectiveness of plantings: pollination services declined exponentially with distance from plantings, and perennial and older flower strips with higher flowering plant diversity enhanced pollination more effectively. These findings provide promising pathways to optimise floral plantings to more effectively contribute to ecosystem service delivery and ecological intensification of agriculture in the future.     

Index hopping on the Illumina HiseqX platform and its consequences for ancient DNA studies

The high‐throughput capacities of the Illumina sequencing platforms and the possibility to label samples individually have encouraged wide use of sample multiplexing. However, this practice results in read misassignment (usually <1%) across samples sequenced on the same lane. Alarmingly high rates of read misassignment of up to 10% were reported for lllumina sequencing machines with exclusion amplification chemistry. This may make use of these platforms prohibitive, particularly in studies that rely on low‐quantity and low‐quality samples, such as historical and archaeological specimens. Here, we use barcodes, short sequences that are ligated to both ends of the DNA insert, to directly quantify the rate of index hopping in 100‐year old museum‐preserved gorilla (Gorilla beringei) samples. Correcting for multiple sources of noise, we identify on average 0.470% of reads containing a hopped index. We show that sample‐specific quantity of misassigned reads depends on the number of reads that any given sample contributes to the total sequencing pool, so that samples with few sequenced reads receive the greatest proportion of misassigned reads. This particularly affects ancient DNA samples, as these frequently differ in their DNA quantity and endogenous content. Through simulations we show that even low rates of index hopping, as reported here, can lead to biases in ancient DNA studies when multiplexing samples with vastly different quantities of endogenous material.