Structural analysis of an epidermal growth factor/transforming growth factor-alpha chimera with unique ErbB binding specificity

Various chimeras of the ErbB1-specific ligands epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) display an enlarged repertoire as activators of ErbB2.ErbB3 heterodimers. Mutational analysis indicated that particularly residues in the N terminus and B-loop region of these ligands are involved in the broadened receptor specificity. In order to understand the receptor specificity of T1E, a chimeric ligand constructed by the introduction of the linear N-terminal region of TGFalpha into EGF, we determined in this study the solution structure and dynamics of T1E by multidimensional NMR analysis. Subsequently, we studied the structural characteristics of T1E binding to both ErbB1 and ErbB3 by superposition modeling of its structure on the known crystal structures of ErbB3 and liganded ErbB1 complexes. The results show that the overall structure of T1E in solution is very similar to that of native EGF and TGFalpha but that its N terminus shows an extended structure that is appropriately positioned to form a triple beta-sheet with the large antiparallel beta-sheet in the B-loop region. This conformational effect of the N terminus together with the large overall flexibility of T1E, as determined by 15N NMR relaxation analysis, may be a facilitative property for its broad receptor specificity. The structural superposition models indicate that hydrophobic and electrostatic interactions of the N terminus and B-loop of T1E are particularly important for its binding to ErbB3.     

Cathepsins F and S block HDL3-induced cholesterol efflux from macrophage foam cells

In atherosclerosis, accumulation of cholesterol in macrophages may partially depend on its defective removal by high-density lipoproteins (HDL). We studied the proteolytic effect of cathepsins F, S, and K on HDL(3) and on lipid-free apoA-I, and its consequence on their function as inductors of cholesterol efflux from cholesterol-filled mouse peritoneal macrophages in vitro. Incubation of HDL(3) with cathepsin F or S, but not with cathepsin K, led to rapid loss of prebeta-HDL, and reduced cholesterol efflux by 50% in only 1min. Cathepsins F or K partially degraded lipid-free apoA-I and reduced its ability to induce cholesterol efflux, whereas cathepsin S totally degraded apoA-I, leading to complete loss of apoA-I cholesterol acceptor function. These results suggest that cathepsin-secreting cells induce rapid depletion of lipid-poor (prebeta-HDL) and lipid-free apoA-I and inhibit cellular cholesterol efflux, so tending to promote the formation and maintenance of foam cells in atherosclerotic lesions.     

Experimental prediction of the natural evolution of antibiotic resistance

The TEM family of beta-lactamases has evolved to confer resistance to most of the beta-lactam antibiotics, but not to cefepime. To determine whether the TEM beta-lactamases have the potential to evolve cefepime resistance, we evolved the ancestral TEM allele, TEM-1, in vitro and selected for cefepime resistance. After four rounds of mutagenesis and selection for increased cefepime resistance each of eight independent populations reached a level equivalent to clinical resistance. All eight evolved alleles increased the level of cefepime resistance by a factor of at least 32, and the best allele improved by a factor of 512. Sequencing showed that alleles contained from two to six amino acid substitutions, many of which were shared among alleles, and that the best allele contained only three substitutions.     

Mechanical strain regulation of the chicken glypican-4 gene expression in the avian eggshell gland

Comparison of RNA fingerprinting of the avian eggshell gland (ESG) without and with an egg revealed upregulation of a 382-bp cDNA fragment that showed high homology to the mammalian glypican 4 (GPC-4). The gene sequence revealed a conserved glypican signature, a glycosyl phosphatidyl inositol-anchorage site, and cystein residues, most of which were conserved. GPC-4 was expressed in the ESG in a circadian fashion only during the period of eggshell calcification, when maximal mechanical strain was imposed. Removal of the egg just before to its entry into the ESG, with consequent elimination of the mechanical strain, caused reduction in the gene expression. Artificial application of the mechanical strain induced expression of the GPC-4 gene that was related to the level of the strain. GPC-4 expression was strain dependent in other parts of the oviduct. In the ESG, GPC-4 was expressed exclusively by the glandular epithelium and not by the pseudostratified epithelium facing the lumen. In summary, we cloned the avian homologue of GPC-4, established its pattern of expression in the avian ESG, and demonstrated for the first time that this gene is regulated by mechanical strain.     

Skin cancer after X-ray treatment for scalp ringworm

Some 2,224 children given X-ray therapy for tinea capitis (ringworm of the scalp) have been followed for up to 50 years to determine cancer incidence, along with a control group of 1,380 tinea capitis patients given only topical medications. The study found a relative risk (RR) of 3.6 (95% confidence interval, 2.3-5.9) for basal cell skin cancer (BCC) of the head and neck among irradiated Caucasians (124 irradiated cases and 21 control cases), in response to a scalp dose of about 4.8 Gy. No melanomas of the head and neck have been seen, and only a few squamous cell carcinomas. About 40% of irradiated cases have had multiple BCCs, for a total of 328 BCCs. Although 25% of both the irradiated and control groups are African-American, only 3 skin cancers have been seen among them, all in the irradiated group, indicating the importance of susceptibility to UV radiation as a cofactor. Light complexion, severe sunburning and North European ancestry were predictive of BCC risk in the irradiated group, but chronic sun exposure was not. Children irradiated at young ages had the highest BCC risk. The RR for BCC risk is approximately constant with time since exposure, suggesting that risk will probably last for a lifetime.     

New evidence for hominid presence in the Lower Pleistocene of the Southern Levant

The site of 'Ubeidiya is located in the Jordan Valley, Israel and has been biochronologically dated to 1.5 m.y.a. It exhibits large lithic and faunal assemblages. Previous published hominid material includes a molar (UB 1701) and I(2) (UB 1700). A recent review of the faunal material from previous excavations has revealed a highly worn hominid right lateral lower incisor (UB 335). The tooth was found in situ in the Lower Pleistocene deposits of stratum I-26a, which is comprised of sand and conglomerates of flint, limestone and basalt indicative of a pebbled lakeshore environment. Taphonomic analysis of the macromammal assemblage indicates high-energy fluvial transport. Paleoecological reconstruction suggests a large woodland fauna with a small percent of open steppe species.UB 335 did not differ significantly from the Lower Pleistocene hominid and modern populations but did differ significantly from all other fossil populations. Two-tailed Student t -test and single classification Model II ANOVA of the buccolingual diameter did not distinguish between Lower Pleistocene species: Homo habilis, H. ergaster and H. cf. erectus. Thus, UB 335 can be identified as a Lower Pleistocene hominid although it cannot be securely assigned to any particular species within that time frame. The current date of the 'Ubeidiya deposits and the location of the site within the Levantine corridor suggests a tenative identification as H. ergaster.     

Nesprin-1alpha self-associates and binds directly to emerin and lamin A in vitro

The mitogen-stimulated protein kinase p70(s6k)/p85(s6k) (S6K) plays an essential role in cell proliferation and growth, with inhibitors of the S6K signalling pathway showing promise as anti-tumour therapeutics. Here, we report that the bisindolylmaleimide derivative Ro 31-6045, previously reported to be inactive as a kinase inhibitor, inhibited S6K activity in vivo with an IC50=8 microM. Structure/function analysis using mutant forms of S6K indicates that Ro 31-6045 inhibition is independent of the upstream activator mTOR. Ro 31-6045 will prove useful in elucidating the complex activation mechanism of S6K and its independence from mTOR will allow confirmation of functional data obtained using the mTOR inhibitor rapamycin.     

Phylogenetic analysis shows that the OXA beta-lactamase genes have been on plasmids for millions of years

The OXA genes encode a class of b-lactamases that confer resistance to a wide range of b-lactam antibiotics. To determine whether the diversity of the OXA b-lactamases is the result of recent or ancient events, and to determine whether mobilization of the OXA genes from chromosomes to plasmids occurred recently or long ago, we have constructed a Bayesian phylogeny of the OXA b-lactamase genes. Analysis of that phylogeny shows that much of the diversity is the result of ancient events and that the OXA genes were mobilized from chromosomes to plasmids on at least two independent occasions that occurred millions of years ago. That observation contradicts the commonly held impression that mobilization of antibiotic resistance genes is strictly the result of modern use of antibiotics.