A simple and traceless solid phase method simplifies the assembly of large peptides and the access to challenging proteins.

Chemical protein synthesis gives access to well-defined native or modified proteins that are useful for studying protein structure and function. The majority of proteins synthesized up to now have been produced using native chemical ligation (NCL) in solution. Although there are significant advantages to assembling large peptides or proteins by solid phase ligation, reports of such approaches are rare. We report a novel solid phase method for protein synthesis which relies on the chemistry of the acetoacetyl group and ketoxime ligation for the attachment of the peptide to the solid support, and on a tandem transoximation/rearrangement process for the detachment of the target protein. Importantly, we show that the combination of solid phase and solution ligation techniques facilitates the production of a challenging and biologically active protein made of 180 amino acids. We show also that the solid phase method enables the purification of complex peptide segments through a chemoselective solid phase capture/release approach.

Correction: A simple and traceless solid phase method simplifies the assembly of large peptides and the access to challenging proteins.

[This corrects the article DOI: 10.1039/C7SC01912B.].

Subclinical hepatic encephalopathy: role of tryptophan binding to albumin and the competition with indole-3-acetic acid.

BACKGROUND: The role of tryptophan (TRY) and its metabolites in the pathogenesis of hepatic encephalopathy is conflicting. The aim of the present study is to investigate in posthepatitis cirrhotic patients with encephalopathy the serum levels of TRY and those of its metabolite indole-3-acetic acid, as well as TRY binding curve to serum albumin and the competition with indole-3-acetic acid. The presence of a relationship between encephalopathy severity and circulating free TRY was also investigated. METHODS: Serum TRY and indole-3-acetic acid were analyzed by HPLC; binding of TRY to serum albumin and the competition with indole-3-acetic acid was studied by equilibrium dialysis. RESULTS: Serum-free TRY was significantly higher in cirrhotic patients (43.33 +/- 14.70 vs 28.87 +/- 8.77 mumol/L, P = 0.02). The binding capacity of albumin was reduced in cirrhotics and further decreased by the addition of indole-3-acetic (K = 6.63 +/- 0.97 x 10(3) mol/L-1, gamma = 1.16 +/- 0.45 x 10(2) mol/L-1 in normal sera vs K = 1.04 +/- 0.20 x 10(3) mol/L-1, gamma = 1.91 +/- 0.92 10(2) mol/L-1 in cirrhotic sera). A multivariate analysis showed that among the psychometric tests the only independent predictor of serum levels of free TRY was the Block Design (R2 = 0.94, B = 0.16 +/- 0.01, beta = 0.97; P < 0.0001). CONCLUSIONS: A high percent ratio of free/total TRY and indoleacetic acid (IAA) was found in cirrhotic patients with hepatic encephalopathy. The concentrations of serum-free IAA and TRY correlated with the degree of subclinical encephalopathy, suggesting a role of these compounds in the development of mental derangement in liver cirrhosis.

A family of insertion mutations between codons 67 and 70 of human immunodeficiency virus type 1 reverse transcriptase confer multinucleoside analog resistance.

To investigate the occurrence of multinucleoside analog resistance during therapy failure, we surveyed the drug susceptibilities and genotypes of nearly 900 human immunodeficiency virus type 1 (HIV-1) samples. For 302 of these, the 50% inhibitory concentrations of at least four of the approved nucleoside analogs had fourfold-or-greater increases. Genotypic analysis of the reverse transcriptase (RT)-coding regions from these samples revealed complex mutational patterns, including the previously recognized codon 151 multidrug resistance cluster. Surprisingly, high-level multinucleoside resistance was associated with a diverse family of amino acid insertions in addition to "conventional" point mutations. These insertions were found between RT codons 67 and 70 and were commonly 69Ser-(Ser-Ser) or 69Ser-(Ser-Gly). Treatment history information showed that a common factor for the development of these variants was AZT (3'-azido-3'-deoxythymidine, zidovudine) therapy in combination with 2',3'-dideoxyinosine or 2',3'-dideoxycytidine, although treatment patterns varied considerably. Site-directed mutagenesis studies confirmed that 69Ser-(Ser-Ser) in an AZT resistance mutational background conferred simultaneous resistance to multiple nucleoside analogs. The insertions are located in the "fingers" domain of RT. Modelling the 69Ser-(Ser-Ser) insertion into the RT structure demonstrated the profound direct effect that this change is likely to have in the nucleoside triphosphate binding site of the enzyme. Our data highlight the increasing problem of HIV-1 multidrug resistance and underline the importance of continued resistance surveillance with appropriate, sufficiently versatile genotyping technology and phenotypic drug susceptibility analysis.

Prospective randomized study comparing human serum albumin with fetal cord serum as protein supplement in culture medium for in-vitro fertilization.

The use of human serum albumin (HSA) instead of fetal cord serum (FCoS) as protein supplement highly simplifies the preparation of culture medium for human in-vitro fertilization (IVF) but whether they are equivalent in sustaining embryo development is still controversial. We performed a prospective randomized study of patients undergoing IVF or intracytoplasmic sperm injection (ICSI) where embryos were cultured in Earle's balanced salt solution containing either 8% (v/v) FCoS or 0.4% (w/v) HSA as protein source. Fertilization rates, morphological embryonic quality and pregnancy rates were compared. A total of 2189 oocytes from 210 cycles were cultured in medium supplemented with HSA in patient group 1 and 2109 oocytes from 203 cycles in medium supplemented with FCoS in patient group 2. The fertilization rate, defined as the presence of two nuclei, for microinjected oocytes was similar in both patient groups (77.4 and 76.7%, respectively). The fertilization rate for inseminated oocyte-cumulus complexes was significantly higher in the HSA group than in the FCoS group (62.9 versus 53.8%, P < 0.025). The embryonic quality was significantly better after culture in medium supplemented with HSA than with FCoS (13.7 versus 9.9% morphologically excellent embryos, P < 0.001). Implantation rates per transferred embryo were not significantly different (22.5 versus 18.2%), but there was a significantly higher pregnancy rate per embryo transfer in the HSA group (45.7 versus 35.9%, P < 0.05, respectively). Non-evolutive pregnancy rates were significantly different (27.4 and 16.7%). Our data demonstrate that the use of human serum albumin as a protein supplement for culture medium in human IVF programmes is associated with improved embryonic quality and significantly higher pregnancy rates. For this reason as well as the additional benefits of being virus-free and being purified, HSA is preferable to FCoS for the preparation of culture media in human IVF.

Cytogenetic analysis of human oocytes parthenogenetically activated by puromycin.

PURPOSE: Treatment of aged human oocytes by puromycin allows a high rate of parthenogenetic activation and development until the first cleavage division. This technique was used for the study of the chromosome complement of oocytes which remained unfertilized after in vitro fertilization. Three hundred four unfertilized oocytes were treated with 10 micrograms/ml puromycin for 6-8 hr and further cultured for 12-15 hr. RESULTS: Activation occurred in 90.5% of the oocytes. Heterozygous diploids with two pronuclei predominated (61%), which is in contrast to the mouse, where the majority of oocytes activated by puromycin are uniform haploids (89%). CONCLUSIONS: Therefore we conclude that puromycin treatment induces retention of the second polar body in human oocytes, unlike in mouse oocytes treated in the same way. Chromosome analysis performed on 182 oocytes suggested a nondisjunction (ND) rate for the second meiotic division of 12.7%. This is a low figure considering the fact that puromycin itself has been reported to induce nondisjunction. For the first meiotic division a ND rate of only 5.6% was found. This rate is lower than the one found in metaphase II arrested oocytes and we believe that this difference is due to the technical differences between the study of meiotic and that of mitotic chromosomes.

Hexachlorobenzene and 1,1-di(4-chlorophenyl)-2,2-dichloroethene in serum of uremic patients and healthy persons: determination by capillary gas chromatography and electron capture detection.

The concentrations of organochlorine pesticides in serum have been determined by GC and electron capture detection. Studies were performed in 7 nondialyzed, 10 dialyzed and 6 healthy persons. Only hexachlorobenzene (HCB) and 1,1-di(4-chlorophenyl)-2,2-dichloroethene (p,p'-DDE) were consistently present. No interference by other pesticides or polychlorinated biphenyls was found by checking with GC-MS. For the nondialyzed uremic patients the average HCB concentration was 16.2 nmol/l (sigma = 6.7, n = 7), and the p,p'-DDE level 26.4 nmol/l (sigma = 31.4, n = 7). For the dialyzed uremic patients the average HCB level was 15.5 nmol/l (sigma = 11.2, n = 10) before dialysis and 17.2 nmol/l (sigma = 14.4, n = 8) after dialysis, the concentration of p,p'-DDE was 27.0 nmol/l (sigma = 38.4, n = 10) before dialysis and 28.0 nmol/l (sigma = 37.4, n = 8) after dialysis. For the healthy persons the average concentration of HCB was 7.7 nmol/l (sigma = 1.8, n = 6) and the concentration of p,p'-DDE was 20.1 nmol/l (sigma = 10.4, n = 6). HCB concentrations were significantly higher in serum of dialyzed and nondialyzed uremic patients than in controls (Wilcoxon's test).

[Prostate hypertrophy. Surgical experience in Belgium. Multi-center study of 1,140 patients of 80 years and older]. / Prostaathypertrofie. Chirurgische ervaring in België. Multicenter studie over 1.140 patiënten 80 jaar en ouder.

An inquiry organized by the Belgian Society of Surgery brought data on the mortality and morbidity of surgery on patients of 80 years and older for benign prostatic hypertrophy in a total number of 1,140 operations. Consensus exist to see this surgery as planned surgery. The mortality was 73 (6.3%) without statistical difference between endoscopic and open surgery. The morbidity of 824 patients was 165. This last figure relates to the big series in the literature. The mortality is in direct relation to the age of the patients. Evaluation of cardio-pulmonary risk (70.8% of the mortality), operation on indication at earlier diagnosis and prospective studies on the risk of operation in this population group are indicated.