Optimizing the stability of single-chain proteins by linker length and composition mutagenesis

Linker length and composition were varied in libraries of single-chain Arc repressor, resulting in proteins with effective concentrations ranging over six orders of magnitude (10 μM–10 M). Linkers of 11 residues or more were required for biological activity. Equilibrium stability varied substantially with linker length, reaching a maximum for glycine-rich linkers containing 19 residues. The effects of linker length on equilibrium stability arise from significant and sometimes opposing changes in folding and unfolding kinetics. By fixing the linker length at 19 residues and varying the ratio of Ala/Gly or Ser/Gly in a 16-residue-randomized region, the effects of linker flexibility were examined. In these libraries, composition rather than sequence appears to determine stability. Maximum stability in the Ala/Gly library was observed for a protein containing 11 alanines and five glycines in the randomized region of the linker. In the Ser/Gly library, the most stable protein had seven serines and nine glycines in this region. Analysis of folding and unfolding rates suggests that alanine acts largely by accelerating folding, whereas serine acts predominantly to slow unfolding. These results demonstrate an important role for linker design in determining the stability and folding kinetics of single-chain proteins and suggest strategies for optimizing these parameters.     

Forensic anthropology and the concept of race: if races don't exist, why are forensic anthropologists so good at identifying them?

Most anthropologists have abandoned the concept of race as a research tool and as a valid representation of human biological diversity. Yet, race identification continues to be one of the central foci of forensic anthropological casework and research. It is maintained in this paper that the successful assignment of race to a skeletal specimen is not a vindication of the race concept, but rather a prediction that an individual, while alive was assigned to a particular socially constructed 'racial' category. A specimen may display features that point to African ancestry. In this country that person is likely to have been labeled Black regardless of whether or not such a race actually exists in nature.     

Graft-versus-Host-Erkrankung

Graft-versus-host disease (GvHD) is the most important cause for transplant-related morbidity and mortality after hematopoietic stem cell transplantation. Its incidence and severity depend on several factors, such as HLA matching, donor age, source of the graft, and graft manipulation. Significant progress has been made in understanding the pathophysiology of this complication and some of these findings have been implemented in prophylactic or therapeutic approaches that are currently under clinical evaluation. This review summarizes important diagnostic features of GvHD, gives and overview of prophylactic concepts, outlines effective treatment concepts and comments briefly on some innovative therapeutic approaches for patients who fail first line therapy.     

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.     

Localization of the human membrane-type 2 matrix metalloproteinase gene (MMP15) to 16q12.1 near DNA elements that are part of centromeric and non-centromeric heterochromatin of 11 human chromosomes

We have localized a second gene for membrane-type matrix metalloproteinases, MT2-MMP, to chromosome 16q12 by in situ hybridization. FISH experiments using a genomic PAC clone containing the MT2-MMP gene resulted in an unusual hybridization pattern detecting centromeric and non-centromeric heterochromatin regions or its flanking sequences in 11 human chromosomes in addition to the MT2-MMP locus on chromosome 16q12. The detailed analysis of this hybridization pattern using molecular cytogenetic methods together with the specific hybridization of the MT2-MMP cDNA allowed a refined mapping of the gene to 16q12.1, directly adjacent to the 16q heterochromatin. Our findings may give some insights into the evolution of the MMP gene family.     

Congenital bone anomalies associated with lipomas

Joint pain and swelling in patients on fluoride therapy are generally attributed to rheumatic phenomena; however, their occurrence exclusively in the lower limbs suggests a mechanical cause. Eight patients receiving daily doses of sodium fluoride 1.09 mg/kg, elemental calcium 1 gm, and vitamin D 1000–2800 units for osteoporosis spontaneously developed 17 episodes of periarticular pain and swelling in the lower limbs. Radiographs taken within two weeks of the onset of pain were negative, but when repeated 4–6 weeks later showed features of healing stress fractures in the periarticular cancellous bone at the following sites: distal femur (2) proximal tibia (3), distal tibia (6), calcaneum (6). Bone scintigraphy was positive on five occasions, two before radiographic signs had appeared.     

Lokal rezidiviertes und metastasiertes malignes Melanom Langzeitergebnisse und Prognosefaktoren nach perkutaner Radiotherapie

PURPOSE: Radiotherapy (RT) is used as last resort for patients with advanced cutaneous malignant melanoma (MM). Herein our 20-year clinical experience is presented analyzing different endpoints and prognostic factors in patients with locally advanced, recurrent or metastatic MM. PATIENTS AND METHODS: From 1977 to 1995, 2,917 consecutive patients were entered in the MM registry of our university hospital. RT was indicated in 121 patients (56 females, 65 males) for palliation in locally advanced recurrent and metastatic MM stages UICC IIB to IV. At the time of RT initiation, 11 patients had primary or recurrent lesions which were either not eligible for surgery or had residual disease (R2) after resection of a primary or recurrent MM lesion (UICC IIB); 57 patients had lymph node (n = 33) or in-transit metastases (n = 24) (UICC III), and 53 had distant organ metastases (7 M1a, 46 M1b) (UICC IV). The time from first diagnosis to on-study RT averaged overall 19 months (median: 18; range: 3 to 186 months). In 77 patients conventional RT and in 44 patients hypofractionted RT was applied with 2 to 6 Gy fractions up to a mean total RT dose of 45 (median: 48; range: 20 to 66) Gy. RESULTS: At 3 months follow-up, complete response (CR) was achieved in 7 (64%), overall response (CR + PR) in all (100%) UICC IIB patients, in 25 (44%) and 44 (77%) of 57 UICC III patients, and in 9 (17%) and 26 (49%) of 53 UICC IV patients. Tumor progression during RT occurred in 25 (21%) patients. Patients with CR survived longer (median: 40 months) than those without CR (median 10 months) (p < 0.01). At the time of evaluation and last FU (December 31, 1996), 26 patients were still alive: 6 (55%) stage UICC IIB, 17 (30%) stage UICC III, and 3 (6%) stage UICC IV patients (p < 0.01). Univariate analysis revealed following prognostic factors for CR and long-term survival: UICC stage (p < 0.001), primary location in the head and neck, total RT dose > 40 Gy (all p < 0.05), while age, gender and primary histological subtype had no impact. In multivariate analysis, UICC stage was the only independent favorable prognostic factor for achievement of CR and long-term survival (p < 0.001). CONCLUSION: External RT provides effective palliation in advanced UICC stages. The UICC staging system is a good predictor of initial and long-term tumor response in metastatic MM. Prospective randomized trials using RT with or without adjuvant therapy for advanced MM are justified.