Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nα‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side‐chain and backbone anchoring strategies,and compatible protection for N‐terminal cysteine*,†

Abstract: Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C‐terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N^α‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N^α‐t‐butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side‐chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N‐terminal cysteine residue have been developed: (i) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐(N‐methyl‐N‐phenylcarbamoyl)sulfenylcysteine [Fmoc‐Cys(Snm)‐OH], allowing creation of an otherwise fully protected resin‐bound intermediate with N‐terminal free Cys; (ii) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐triphenylmethylcysteine [Fmoc‐Cys(Trt)‐OH], generating a stable Fmoc‐Cys(H)‐peptide upon acidolytic cleavage; and (iii) incorporation of N^α‐t‐butyloxycarbonyl‐S‐fluorenylmethylcysteine [Boc‐Cys(Fm)‐OH], generating a stable H‐Cys(Fm)‐peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C‐termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on‐resin, as well as in solution after cleavage/purification.     

Use of a posterior calf fasciocutaueous flap with distal pedicle to reconstruct a huge ankle defect

Summary A distally based posterior calf fasciocutaneous flap was successfully used to reconstruct a major ankle defect with exposed bones and joints. The flap, which reaches easily this region, can also be used as a free flap for coverage of heel and ankle when the forefoot is not involved in the injury.     

Long-term hypertransaminasemia disclosing a muscular disease

Prolonged increase in serum transaminase activity is usually seen in chronic liver disease. Evaluation in one patient with such a prolonged increase having no evidence of liver disease including histological examination, revealed a limb girdle muscular dystrophy. This case suggests that a muscular disease should be suspected in all children with persistent increase in serum transaminase activity when the liver is apparently normal.     

In-vitro oocyte maturation: some questions concerning the initiation and prevention of this process in humans

Human oocytes isolated from healthy non-preovulatory antralfollicles which had not been stimulated spontaneously resumedmeiosis at a low rate (31%) when compared with those from atreticfollicles (73%). Human follicular fluid did not inhibit spontaneousmaturation of rat oocytes in vitro, regardless of the qualityor maturity of the follicle or the treatment of the patient.In the human, oocyte maturation may involve a stimulatory processas well as a removal of inhibition.     

T-lymphocyte subsets in the embryonic spleen undergoing a graft-versus-host reaction.

Allogeneic immunocompetent T cells injected into chicken embryos induce a graft-versus-host reaction (GVHR) whose most prominent manifestation is splenic hyperplasia. The highly inbred CC and CB strains of chickens used here are, respectively, homozygous for the B4 or B12 MHC haplotypes. By means of a panel of immunological reagents, including alloantisera and monoclonal antibodies against public domains of the T-cell receptor, CD4, CD8, and the inducible interleukin-2-receptor light chain (CD25), it is shown that the bulk of cells in the enlarged spleen are of host origin and do not express markers typical of mature T or B lymphocytes. Among recipient splenocytes, the quantitatively most important population consists of TCR alpha beta-TCR gamma delta- CD4-CD8+CD25+ (TCR0) lymphocytes. Donor cells encountered in the spleen prevalently exhibit a TCR alpha beta+CD4+CD8-CD25+ phenotype and proliferate in vivo. The data demonstrate that nonspecific host and potentially specific donor-derived cellular elements contribute to splenomegaly.     

Molecular characterization of Cryptosporidium isolates obtained from humans in France

Cryptosporidium parvum is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification of Cryptosporidium species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whom Cryptosporidium oocysts were recognized by clinical laboratories. A fragment of the Cryptosporidium 18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes of Cryptosporidium parvum. However, a number of immunocompromised patients were infected with C. meleagridis (3 of 57), C. felis (6 of 57), or a new genotype of C. muris (1 of 57). This is the first report of the last three species of Cryptosporidium in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes.     

What triggers catch-up saccades during visual tracking?

When tracking moving visual stimuli, primates orient their visual axis by combining two kinds of eye movements, smooth pursuit and saccades, that have very different dynamics. Yet, the mechanisms that govern the decision to switch from one type of eye movement to the other are still poorly understood, even though they could bring a significant contribution to the understanding of how the CNS combines different kinds of control strategies to achieve a common motor and sensory goal. In this study, we investigated the oculomotor responses to a large range of different combinations of position error and velocity error during visual tracking of moving stimuli in humans. We found that the oculomotor system uses a prediction of the time at which the eye trajectory will cross the target, defined as the "eye crossing time" (T(XE)). The eye crossing time, which depends on both position error and velocity error, is the criterion used to switch between smooth and saccadic pursuit, i.e., to trigger catch-up saccades. On average, for T(XE) between 40 and 180 ms, no saccade is triggered and target tracking remains purely smooth. Conversely, when T(XE) becomes smaller than 40 ms or larger than 180 ms, a saccade is triggered after a short latency (around 125 ms).     

Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1.

We report the identification of mutations in lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) genes in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17. Linkage disequilibrium analysis of six families affected by NCIE permitted us to reduce a recently reported interval of 8.4 cM on chromosome 17p13.1 to a 600 kb region around the marker D17S1796, which contains LOX genes. LOX products have long been implicated in skin disorders. Two point mutations and one deletion were found in ALOXE3 and three point mutations were found in ALOX12B in these consanguineous families from the Mediterranean basin. ALOXE3 and ALOX12B are two genes which are physically linked and functionally related. They are separated by 38 kb, have one more exon than the other LOX genes and are mainly expressed in epithelial cells including keratinocytes. Although the main substrate(s) of the two enzymes is (are) still unknown, the products of ALOX12B obtained in experimental systems have been demonstrated to be of R-chirality. It seems likely that the product of one of these enzymes may be the substrate of the other, and that they belong to the same metabolic pathway.     

Evidence for high-velocity smooth pursuit in the trained cat

It is generally accepted that in cats smooth pursuit velocity of the eye never exceeds a few degrees per second. This is in contrast with observations in primates, where smooth pursuit velocity can reach values as high as 100°/s. Cats were trained to fixate and pursue spots of light appearing on a translucent screen. Spots were moved in the horizontal and vertical planes at different constant velocities up to 80°/s. Eye position was recorded with the sclerai search coil technique. Naive cats did not pursue moving targets with high efficiency. Smooth eye movement velocity saturated at 5°/s. After a few days of training, smooth-pursuit eye velocity increased with target velocity and saturated at 25°/s on average. However, velocities twice as high have been observed frequently. When the target was unexpectedly extinguished, smooth eye movement velocity dropped to values close to 0°/s in approximately 350 ms. After a short training period (usually 5 times the same target presentation), the eye continued to move smoothly until the target reappeared. These data suggest that smooth pursuit eye movements of the cat are qualitatively similar to those of primates, but reach lower velocities and are more variable in their characteristics.