Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK. From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as d-galacturonic acid, d-talose, l-altrose, and l-glucose, all of which failed as wild-type GalK substrates. Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme. Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme. In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.     

Social values and decision making: a generation in the lifeboat

The author introduced the 'Lifeboat'exercise as a death-and-dying teaching technique in an article that appeared in the first volume of Death Studies (J. A.Thorson, 1978). It is a classroom exercise designed to illustrate the varying levels of social value placed on terminally ill patients. Using 'Lifeboat'with different groups in succeeding years, the author has seen changes in the ways that individuals playing different roles are perceived by the audience. These reactions may indicate changes in social values.     

The effects of news stories that put crime and violence into context: testing the public health model of reporting

The purpose of this study is to examine whether changing the way newspaper stories report crime and violence can induce shifts in readers' perceptions of the problem. Using an experiment that manipulates the framing and graphic presentation of newspaper stories on crime and violence, we seek to discover whether the public health model that calls for news stories to incorporate information on context, risk factors, and prevention strategies will help readers learn more about the context in which crime and violence occurs, endorse prevention strategies in addition to punishment, and become more attuned to societal risk factors and causes of crime and violence.     

Analysis of graft-versus-host disease (GVHD) and graft rejection using MHC class I-deficient mice

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, β₂-microglobulin-deficient mice (β₂m-/-) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. β₂m-/- mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all β₂m-/- recipients were predominantly CD3⁺ αβ TCR⁺ CD4⁺ cells (15–20% of all splenocytes). In contrast, CD8⁺ cells engrafted in very small numbers (1–5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4⁺ T cells induced and maintained GVHD in mH-mismatched β₂m-/- mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic β₂m-/- cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Patient and surgeon ranking of the severity of symptoms associated with fecal incontinence

PURPOSE: The purpose of this research was to develop and evaluate a severity rating score for fecal incontinence, the Fecal Incontinence Severity Index. METHODS: The Fecal Incontinence Severity Index is based on a type × frequency matrix. The matrix includes four types of leakage commonly found in the fecal incontinent population: gas, mucus, and liquid and solid stool and five frequencies: one to three times per month, once per week, twice per week, once per day, and twice per day. The Fecal Incontinence Severity Index was developed using both colon and rectal surgeons and patient input for the specification of the weighting scores. RESULTS: Surgeons and patients had very similar weightings for each of the type × frequency combinations; significant differences occurred for only 3 of the 20 different weights. The Fecal Incontinence Severity Index score of a group of patients with fecal incontinence (N = 118) demonstrated significant correlations with three of the four scales found in a fecal incontinence quality-of-life scale. CONCLUSIONS: Evaluation of the Fecal Incontinence Severity Index indicates that the index is a tool that can be used to assess severity of fecal incontinence. Overall, patient and surgeon ratings of severity are similar, with minor differences associated with the accidental loss of solid stool.Supported by a contract between the University of Minnesota Clinical Outcomes Research Center and The American Society of Colon and Rectal Surgery and the Minnesota Colon and Rectal Foundation.     

Histologic comparison of hereditary nonpolyposis colorectal cancer associated with MSH2 and MLH1 and colorectal cancer from the general population

PURPOSE: Hereditary nonpolyposis colorectal cancer is reported to have special histologic features. This study compares the histologic features of hereditary nonpolyposis colorectal cancer to colorectal cancers from the general population when hereditary nonpolyposis colorectal cancer cases are restricted to families with known MSH2 and MLH1 mutations. METHODS: Thirty-seven cancers from kindreds carrying MSH2 mutations, 27 cancers from kindreds carrying MLH1 mutations, and 37 colorectal cancers from the general population were reviewed by a pathologist blinded to hereditary nonpolyposis colorectal cancer gene status. Tumor grade, growth pattern, Crohn's-like lymphoid reaction, mucin production, extent of disease in the bowel wall, and lymph node status were evaluated. RESULTS: Poor differentiation and Crohn's-like reaction were a feature of 44 and 49 percent of hereditary nonpolyposis colorectal cancer compared with 14 percent (P=0.002) and 27 percent (P=0.049) of colorectal cancers from the general population, respectively. There was no difference in growth pattern, mucin production, lymph node involvement, or local extent of disease between hereditary nonpolyposis colorectal cancer and colorectal cancers from the general population. Poor differentiation and lymph node metastases were found in 57 and 49 percent of MSH2 compared with 26 percent (P=0.002) and 10 percent (P=0.03) of MLH1-associated cancers, respectively. There was no difference in growth pattern, mucin production, Crohn's-like lymphoid reaction, or local extent of disease between subgroups of hereditary nonpolyposis colorectal cancer. CONCLUSIONS: Poor differentiation and Crohn's-like reaction are more common in hereditary nonpolyposis colorectal cancer than colorectal cancers from general population. Poor differentiation and lymph node metastases are more commonly seen in MSH2-associated cancers than MLH1. Evaluation of the natural history, pathogenesis, and prognosis of colorectal cancer in hereditary nonpolyposis colorectal cancer should include consideration of which mismatch repair genes are mutated and what the specific mutations are.Poster presentation at the meeting of The American Society of Colon and Rectal Surgeons, Philadelphia, Pennsylvania, June 22 to 27, 1997.     

The glycan-rich outer layer of the cell wall of Mycobacterium tuberculosis acts as an antiphagocytic capsule limiting the association of the bacterium with macrophages

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that infects macrophages and other host cells. We show that sonication of M. tuberculosis results in the removal of material from the surface capsule-like layer of the bacteria, resulting in an enhanced propensity of the bacteria to bind to macrophages. This effect is observed with disparate murine and human macrophage populations though, interestingly, not with freshly explanted alveolar macrophages. Enhanced binding to macrophages following sonication is significantly greater within members of the M. tuberculosis family (pathogens) than within the Mycobacterium avium complex (opportunistic pathogens) or for Mycobacterium smegmatis (saprophyte). Sonication does not affect the viability or the surface hydrophobicity of M. tuberculosis but does result in changes in surface charge and in the binding of mannose-specific lectins to the bacterial surface. The increased binding of sonicated M. tuberculosis was not mediated through complement receptor 3. These results provide evidence that the surface capsule on members of the M. tuberculosis family may be an important virulence factor involved in the survival of M. tuberculosis in the mammalian host. They also question the view that M. tuberculosis is readily ingested by any macrophage it encounters and support the contention that M. tuberculosis, like many other microbial pathogens, has an antiphagocytic capsule that limits and controls the interaction of the bacterium with macrophages.     

The role of the tissue adhesive fibrin seal (FS) in esophageal anastomoses

Following Surgical Removal Of Esophageal Tumors, Leakage And Medistinitis Is A Frequent And Often Fatal Complication. A New Method Has Been Developed To Seal Suture Lines In The Esophagus With Preparations Containing Fibrinogen, Cold Insoluble Globulin, Factor Xiii, Antiplasmin, Platelet Growth Factor, Thrombin, And Calcium Chloride. In Experimental Animals Operated On By Standard Methods, Esophageal Leakage Developed In 50% Of The Animals And Death In 40%. By Contrast, In Treated Animals, Esophageal Leak And Death Developed In Only 20%. More Adhesions Were Found In Treated Animals Than In Control Animals.