New developments in enzymatic peptide synthesis.

This review article describes new enzymatic methods developed for the efficient and irreversible synthesis of peptides based on native and modified proteases, and for the synthesis of polypeptides containing D- and/or unnatural amino acids. Potential opportunities for future developments in the field based on new enzymes, tailor-made catalytic antibodies, and on the technique of in vitro mutagenesis are also described.     

Single neurones can initiate synchronized population discharge in the hippocampus

The synchronized firing of neuronal populations is frequently observed in the mammalian central nervous system. The generation of motor activities such as locomotion and respiration requires the simultaneous activation of many neurones and synchronous firing also underlies the cortical alpha rhythm and the hippocampal theta rhythm. However the influence that single neurones may have on such neuronal population discharges is not clear. We have examined this question using small isolated segments of the CA3 region of the guinea pig hippocampus. We report here that in the presence of picrotoxin, a gamma-aminobutyric acid (GABA) antagonist, these segments spontaneously generate synchronized rhythmic bursts comparable with the interictal epileptiform discharges observed in the hippocampus and neocortex in the presence of penicillin. The activation of some individual neurones by intracellular current injection can partially entrain and reset the rhythm. The probability that a synchronized burst will follow stimulation of a single cell increases with time after a spontaneous synchronized discharge, suggesting that each population discharge is followed by a period of relative population refractoriness. A delay of 40-200 ms elapses between the activation of a single neurone and the synchronized discharge. We suggest that during this time activity elicited in one neurone spreads to other neurones through multisynaptic excitatory pathways and leads eventually to the participation of the whole population in a synchronous burst.     

Further studies on the effect of red and far red light on rat retinal development

Summary The influence on the development of the outer segments of the rat retina of far red and red light in different sequences was investigated. The far red treatment appeared to be dominating, and for animals treated with far red light first, further treatment of red light could not bring the outer segment growth back to normal. The treatments also initiated different dopamine uptakes in the retinas.     

Genome-wide analysis of single-nucleotide polymorphisms in human expressed sequences

Single-nucleotide polymorphisms (SNPs) have been explored as a high-resolution marker set for accelerating the mapping of disease genes. Here we report 48,196 candidate SNPs detected by statistical analysis of human expressed sequence tags (ESTs), associated primarily with coding regions of genes. We used Bayesian inference to weigh evidence for true polymorphism versus sequencing error, misalignment or ambiguity, misclustering or chimaeric EST sequences, assessing data such as raw chromatogram height, sharpness, overlap and spacing, sequencing error rates, context-sensitivity and cDNA library origin. Three separate validations-comparison with 54 genes screened for SNPs independently, verification of HLA-A polymorphisms and restriction fragment length polymorphism (RFLP) testing-verified 70%, 89% and 71% of our predicted SNPs, respectively. Our method detects tenfold more true HLA-A SNPs than previous analyses of the EST data. We found SNPs in a large fraction of known disease genes, including some disease-causing mutations (for example, the HbS sickle-cell mutation). Our comprehensive analysis of human coding region polymorphism provides a public resource for mapping of disease genes (available at http://www.bioinformatics.ucla.edu/snp).     

Leukaemia inhibitory factor is identical to the myeloid growth factor human interleukin for DA cells

Leukaemia inhibitory factor (LIF) is a cytokine that induces macrophage differentiation of the murine M1 myeloid leukaemia cell line. We have isolated a cDNA clone encoding a novel human haemopoietic growth factor, human interleukin for DA cells (HILDA) that supports the proliferation of the murine interleukin-3-dependent leukaemic cell line, DA-la (refs 3-5). HILDA proved to be identical to LIF. The demonstration that the differentiation factor LIF will also serve as a growth factor for at least one myeloid leukaemic cell line provides further evidence that the distinction between growth-promoting and differentiation-inducing activities are largely determined by the target cell type.     

Deletion of genes on chromosome 1 in endocrine neoplasia

Recent studies have identified normal cellular DNA sequences which are lost in the development of embryonal and adult tumours. These tumours are thought to arise after a primary mutation in one allele of such a sequence is followed by loss of its normal homologue. In familial cases, the primary mutation is transmitted in the germ line. The secondary mutation may involve a substantial loss of chromosomal material and thus lead to identification of the site of the inherited mutation. We have examined constitutional and tumour genotypes of medullary thyroid carcinomas and phaeochromocytomas which develop in the dominantly inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2) to locate the predisposing gene in this syndrome. We observed deletion of a hypervariable region of DNA on the short arm of chromosome 1 in seven out of fourteen tumours. Analysis of the parental origin of the deleted allele in two families showed that it was derived from the affected parent in one case, which suggests that the deletion does not reflect the site of the inherited mutation in MEN2. The deleted region is distal to the breakpoint commonly detected in neuroblastomas, which share with the tumours of MEN2 embryological origin from neuroectoderm.     

Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis

Papillon-Lefèvre syndrome, or keratosis palmoplantaris with periodontopathia (PLS, MIM 245000), is an autosomal recessive disorder that is mainly ascertained by dentists because of the severe periodontitis that afflicts patients. Both the deciduous and permanent dentitions are affected, resulting in premature tooth loss. Palmoplantar keratosis, varying from mild psoriasiform scaly skin to overt hyperkeratosis, typically develops within the first three years of life. Keratosis also affects other sites such as elbows and knees. Most PLS patients display both periodontitis and hyperkeratosis. Some patients have only palmoplantar keratosis or periodontitis, and in rare individuals the periodontitis is mild and of late onset. The PLS locus has been mapped to chromosome 11q14-q21 (refs 7, 8, 9). Using homozygosity mapping in eight small consanguineous families, we have narrowed the candidate region to a 1.2-cM interval between D11S4082 and D11S931. The gene (CTSC) encoding the lysosomal protease cathepsin C (or dipeptidyl aminopeptidase I) lies within this interval. We defined the genomic structure of CTSC and found mutations in all eight families. In two of these families we used a functional assay to demonstrate an almost total loss of cathepsin C activity in PLS patients and reduced activity in obligate carriers.