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Biomedical subjects

E C Lee

Publications and source records attributed to E C Lee.

At least 19 recordsLinked to original sources
PubMed2000

An efficient recombination system for chromosome engineering in Escherichia coli.

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

Bacteriophage lambda↗
PubMed2000

Diverticulitis and polycystic kidney disease.

Patients with adult polycystic kidney disease (PKD) have previously been shown to have an increased incidence of complicated diverticulitis after renal transplantation. The purpose of this study was to assess the risk of diverticulitis in the PKD population. We retrospectively reviewed patients with advanced PKD, defined as end-stage renal failure requiring dialysis. Patients were obtained from a single nephrology group practice between January 1985 and January 1997, or from all patients being evaluated or actively considered for renal transplantation at our institution as of May 1997. The incidence and severity of diverticulitis in these patients was compared with that observed in a similar cohort of patients with end-stage renal disease due to other etiologies. The study population consisted of 184 renal failure patients, 59 with PKD and 125 with other causes of end-stage renal disease. Twelve (20%) patients with PKD had a history of active diverticulitis, whereas only 4 (3%) of the non-PKD controls had diverticulitis (P = 0.0003, Fisher's exact test). Six of the 12 PKD patients required surgical intervention. Patients with renal failure due to PKD experience a significantly higher rate of diverticulitis than do other patients with end-stage renal disease. Furthermore, diverticulitis is frequently severe in PKD patients, with 50 per cent requiring surgical intervention. These data suggest that diverticular disease may be an extrarenal manifestation of polycystic kidney disease.

Case-Control Studies↗
PubMed1998

Complicated diverticulitis following renal transplantation.

PURPOSE: Colonic perforations in renal transplant recipients have historically been associated with mortality rates as high as 50 to 100 percent. However, these previous series generally predate the use of cyclosporine-based immunosuppressive protocols. METHODS: We retrospectively reviewed all patients who had undergone renal transplant from our institution and who developed complicated diverticulitis. Complicated diverticulitis was defined as diverticulitis involving free perforation, abscess, phlegmon, or fistula. Factors analyzed included the time interval since transplantation, use of cyclosporine, living-related vs. cadaveric donor, cause of renal failure, and presenting signs and symptoms. RESULTS: Between August 1969 and September 1996, 1,211 kidney transplants were performed in 1,137 patients. The first 388 patients (1969-1984) received prednisone and azathioprine, with cyclosporine added to the immunosuppressive regimen for the subsequent 823 recipients (1984-1996). Thirteen (1.1 percent) patients had episodes of complicated diverticulitis, occurring from 25 days to 14 years after transplant; all required surgical therapy. Clinical presentation was highly variable, ranging from asymptomatic pneumoperitoneum (2 patients) to generalized peritonitis. There was one perioperative mortality (7.7 percent). Patients with polycystic kidney disease as the cause of renal failure had a significantly higher rate of complicated diverticulitis. Specifically, patients with polycystic kidney disease (9 percent of the total transplant population) accounted for 46 percent of the cases of diverticulitis (P < 0.001, Fisher's exact probability test). Neither treatment with cyclosporine nor donor source had a significant effect on the rate of diverticular complications (P = 0.36 and P = 0.99, respectively, Fisher's exact probability test). CONCLUSION: Complicated diverticulitis following renal transplantation is rare, and the clinical presentation may be atypical in the immunosuppressed transplant recipient. Patients with polycystic kidney disease experience a significantly higher rate of complicated diverticulitis than do other transplant patients and, therefore, warrant aggressive diagnostic evaluation of even vague abdominal symptoms. In addition, pretransplant screening and prophylactic sigmoid resection in this high-risk population deserve consideration and further study.

Adult↗
PubMed1998

Functional analysis of the fibrinogen-related scabrous gene from Drosophila melanogaster identifies potential effector and stimulatory protein domains.

The scabrous (sca) gene encodes a secreted dimeric glycoprotein with putative coiled-coil domains N-terminally and a C-terminal region related to the blood clot protein fibrinogen. Homozygous sca mutants have extra bristle organs and rough eyes. We describe a GAL4-based expression system for testing rescue of the sca mutant phenotype by altered SCA proteins and for misexpression. We find that deletion of the fibrinogen-related domain (FReD) greatly decreases SCA function, confirming the importance of this conserved region. SCA function could not be restored by FReDs from human fibrinogen chain genes. However, proteins lacking any FReD still showed some function in both rescue and misexpression experiments, suggesting that putative effector-binding regions lie outside this domain. Consistent with this, proteins expressing only the FReD had no rescuing activity but were recessive negative; i.e., they enhanced the phenotype of sca mutations but had no phenotype in the presence of a wild-type sca allele. This suggests that the FReD contributes to SCA function by binding to other components of the bristle determination pathway, increasing the activity of the linked N-terminal region.

Amino Acid Sequence↗
PubMed1997

Intraoperative colonic lavage in nonelective surgery for diverticular disease.

BACKGROUND: Staged resection of the sigmoid colon has been the traditional strategy for treating patients who require nonelective surgery to manage complications of diverticular disease. Resection and primary anastomosis has not generally been recommended when the clinical setting is compromised by contiguous inflammation or inadequate mechanical cleansing of the colon because of concerns regarding the potential risk of anastomotic dehiscence. Although many reports have confirmed that intraoperative colonic lavage (ICL) is a safe method for relieving fecal loading of the colon to facilitate primary intestinal anastomosis in patients with mechanical obstruction of the distal colon, there is very limited experience with the use of this technique in treating acute inflammatory disorders of the colon. In this report, we present our results with ICL in the nonelective treatment of patients with complications of diverticulitis. METHODS: Records of all patients undergoing urgent operations at the Lahey Clinic to treat complications of diverticular disease from July 1987 to January 1996 were reviewed. RESULTS: Of 62 patients who required nonelective operations, 33 underwent ICL in an attempt to perform primary anastomosis. In five patients, the operation included creation of a colostomy. The indication for surgery was obstruction in 13 patients (39 percent), persistent abscess or phlegmon in 13 (39 percent), perforation in 6 patients (18 percent), and hemorrhage in 1 patient (3 percent). According to Hinchey's classification system, 18 patients had Stage I disease, 10 had Stage II, and 5 patients had Stage III disease. There were no patients with Stage IV disease. The single anastomotic complication in the series was responsible for the sole operative mortality. The morbidity rate of 42 percent, included three intraoperative complications (2 splenic injuries and 1 ureteral laceration), two intra-abdominal abscesses (6 percent), and six wound infections (18 percent). CONCLUSION: In our experience, ICL has proven to be a safe method for accomplishing single-stage resection of the colon in selected patients with diverticulitis who require an urgent operation. When there is no evidence of diffuse purulent or feculent peritonitis, we believe this is the preferred method for treating patients who are hemodynamically stable.

Abdominal Abscess↗
PubMed1996

gp300sca is not a high affinity Notch ligand.

The Notch protooncogene encodes a receptor important for determination and differentiation of many cell types, and is conserved between vertebrates and invertebrates. It has been suggested that the secreted protein scabrous (sca) might be a Notch ligand acting in the peripheral nervous system. The sca protein was purified and a cell line expressing 18,000 Notch molecules per cell surface used to test sca binding by coimmunoprecipitation, cell adhesion assays, and binding with labeled sca. No interaction was detected between gp300sca and Notch or the related protein Delta, suggesting that sca acts through a distinct mechanism.

Animals↗
PubMed1996

Inpatient vs. outpatient bowel preparation for elective colorectal surgery.

BACKGROUND: Recent pressures to decrease the cost of medical care have mandated preoperative outpatient bowel preparation (OBP) for elective colorectal surgery without any data documenting equivalent quality of care. This study examined the safety and efficacy of OBP compared with inpatient bowel preparation (IBP). METHODS: Records of all patients who underwent OBP for elective colorectal resection since the inception of the OBP program from July 1993 to June 1994 were compared with records of all patients who received IBP for elective procedures from January to June 1993. RESULTS: The two groups, 90 patients who underwent OBP and 98 patients who had IBP, were well matched for age, sex, diagnosis, and operations performed. The OBP group had a shorter length of hospital stay (median, 7 vs. 9 days; P < 0.0001; chi-squared analysis), whereas the complication rate was similar (19 percent in the OBP group vs. 18 percent in the IBP group), including infectious complications (10 percent in the OBP group vs. 7 percent in the IBP group). Although operating time was similar (mean, 199 vs. 213 minutes) and estimated blood loss (mean, 528 vs. 536 ml), the OBP group had significantly higher perioperative fluid requirements: intraoperative fluids (median, 4300 vs. 3700 ml; P < 0.05; Student's t-test), intraoperative colloid administration (48 vs. 29 percent; P < 0.0002; chi-squared), 24-hour postoperative fluids (3224 vs. 2700 ml; P < 0.0001; Student's t-test), and postoperative fluid challenges (50 vs. 20 percent; P < 0.0001; chi-squared analysis). CONCLUSION: Outpatient bowel preparation for elective colorectal surgery is safe and effective. It offers shorter hospital stay, and, therefore, potentially reduces medical care cost. Patients with multiple medical problems may not tolerate extensive fluid shifts; therefore, other preoperative arrangements, such as inpatient or outpatient intravenous fluid therapy, need to be considered to minimize complications that may outweigh potential cost savings.

Ambulatory Care↗
PubMed1996

The scabrous gene encodes a secreted glycoprotein dimer and regulates proneural development in Drosophila eyes.

R8 photoreceptor cells play a primary role in the differentiation of Drosophila eyes. In scabrous (sca) mutants, the pattern of R8 photoreceptor differentiation is altered. The sca gene is predicted to encode a secreted protein related in part to fibrinogen and tenascins. Using expression in Drosophila Schneider cells, we showed that sca encoded a dimeric glycoprotein which was secreted and found in soluble form in the tissue culture medium. The sca protein contained both N- and O-linked carbohydrates and interacted with heparin. This Schneider cell protein was similar to protein detected in embryos. We showed that sca mutations, along with conditional alleles of Notch (N) and Delta (Dl), each affected the pattern of cells expressing atonal (ato), the proneural gene required for R8 differentiation. In normal development, about 1 cell in 20 differentiates into an R8 cell; in the others, ato is repressed. N and Dl were required to repress ato in the vicinity of R8 cells, whereas sca had effects over several cell diameters. Certain antibodies detected uptake of sca protein several cells away from its source. The overall growth factor-like structure of sca protein, its solubility, and its range of effects in vivo are consistent with a diffusible role that complements mechanisms involving direct cell contact. We propose that as the morphogenic furrow advances, cell secreting sca protein control the pattern of the next ommatidial column.

Amino Acid Sequence↗
PubMed1995

Molecular analysis of scabrous mutant alleles from Drosophila melanogaster indicates a secreted protein with two functional domains.

Mutations at the scabrous locus (sca) affect cell-cell signaling during neural development. Twenty-one mutant alleles of scabrous have been analyzed. Many synthesize no sca protein. In others, a defective protein is arrested intracellularly. Two mutants in which protein is not arrested must affect sca protein function outside the cell. Both affect the fibrinogen related domain (FReD), a 200-amino acid segment conserved in fibrinogen, tenascins, and other proteins. In fibrinogen, this region is involved in protein interactions and is altered in human mutations affecting blood clotting. In sca(UM2), an invariant Asp residue is replaced by Asn. In sca(MSKF) allele has dominant negative properties, indicating that the truncated amino-terminal portion interferes with the function of so me other gene product. These mutations show that the conserved FReD is essential for wild-type sca function, but suggest that the amino-terminal domain also interacts with other proteins, but other neural mutations were without effect. Models for the role of a two-domain protein in neural development are discussed.

Alleles↗
PubMed1994

General practice screening clinic for Bangladeshi families.

AIM: A screening clinic for Bangladeshi families was established in order to improve the health care provided by one general practice to its Bangladeshi patients. METHOD: The clinic was run by a general practitioner, a health visitor and a Bangladeshi health worker. Patients were invited to attend household by household. The composition of each household was recorded using a genogram. Details of family illness, housing and employment were noted. A medical history was taken from each individual. Every adult was screened for diabetes and risk factors for coronary heart disease; cervical cytology was offered to women. The immunization status of all patients was recorded and adults were immunized. Children were referred to the child health clinic for immunizations. The clinic concluded with a health education session focusing on smoking, exercise and diet. RESULTS: Over a two year period, 58% of the Bangladeshi families registered with the practice attended the clinic, a total of 207 people. Meeting Bangladeshi patients household by household was an effective and apparently efficient way of providing basic screening and health education. It allowed the practice to learn about the structure of its Bangladeshi families, the social problems faced by their community, and the areas in which their health care could be improved. CONCLUSION: This clinic provides a model which could be adapted for use with other ethnic or 'hard-to-reach' groups. It may also prove an effective way of screening all families in general practice.

Adolescent↗
PubMed1993

Decreased expression of Mac-2 (carbohydrate binding protein 35) and loss of its nuclear localization are associated with the neoplastic progression of colon carcinoma.

The Mac-2 lectin (carbohydrate binding protein 35) is a soluble, 32- to 35-kDa phosphoprotein that binds galactose-containing glycoconjugates. We report here that the colonic epithelium is a major site of Mac-2 expression in vivo based on immunohistochemistry of human tissue specimens. In this epithelium, proliferating cells at the base of the crypts do not express Mac-2 but its expression increases with differentiation along the crypt-to-surface axis. Mac-2 expression is concentrated in the nuclei of these differentiated epithelial cells. The progression from normal mucosa to adenoma to carcinoma is associated with significant changes in Mac-2 nuclear localization and expression. In all adenomas (9/9) and carcinomas (13/13) examined, Mac-2 was not present in the nucleus but was localized in the cytoplasm. Sequencing of Mac-2 cDNAs from normal mucosa and carcinoma revealed no specific mutations that could account for this loss of nuclear localization. We also observed a 5- to 10-fold decrease in Mac-2 mRNA levels in cancer compared to normal mucosa as well as a significant reduction in the amount of Mac-2 protein expressed. These observations suggest that Mac-2 exclusion from the nucleus and its decreased expression may be related to the neoplastic progression of colon cancer.

Adenocarcinoma↗
PubMed1993

An EGF-pseudomonas exotoxin A recombinant protein with a deletion in toxin binding domain specifically kills EGF receptor bearing cells.

We constructed two chimeric toxins; one composed of epidermal growth factor (EGF) and pseudomonas exotoxin A (PE), designated EGF-PE and the other composed of EGF and PE with a deletion of the Ia domain (cell-binding domain), designated EGF-PE (delta Ia). Both chimeric toxins reacted with anti-EGF and anti-PE antibodies. The cell-killing experiments showed that EGF-PE, but not EGF-PE (delta Ia), was cytotoxic to the murine fibroblast cell line NR6, which carried the PE receptor, but not the EGF receptor. However, after NR6 was transfected with DNA for the expression of human EGF receptor, the transfected cell line, designated NRHER5, over-expressed human EGF receptors and became sensitive to EGF-PE(delta IA). The cytotoxicity of EGF-PE(delta Ia), but not EGF-PE, to NRHER5 can be completely blocked by an excess amount of EGF. To completely reverse the cytotoxicity of EGF-PE on NRHER5, both the EGF receptor pathway and the PE receptor pathway need to be blocked. These results suggest that EGF-PE exhibits both EGF and PE binding activities, while EGF-PE(delta IA) possesses only EGF binding activity. Thus, EGF-PE(delta Ia) may be a better chimeric toxin than EGF-PE in terms of target specificity to EGF receptor bearing cells. We, therefore, examined the cytotoxicity of EGF-PE(delta Ia) to various human cancer cell lines. We find that human cancer cells containing more EGF receptors are more sensitive to EGF-PE(delta Ia).

ADP Ribose Transferases↗
PubMed1992

The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities.

The integration host factor (IHF) of Escherichia coli is a small, basic protein that is required for lambda site-specific recombination and a variety of cellular processes. It is composed of two subunits, alpha and beta, that are encoded by the himA and hip (himD) genes, respectively. IHF is a sequence-specific DNA-binding protein and bends the DNA when it binds. We have used the bacteriophage P22-based challenge phage selection to isolate suppressor mutants with altered, expanded DNA binding specificities. The suppressors were isolated by selecting mutants that recognize variants of the phage lambda H'IHF recognition site. Two of the mutants recognize both the wild-type and a single variant site and contain amino acid substitutions at positions 64 (Pro to Leu) or 65 (Lys to Ser) of the alpha subunit. These substitutions are in a region of the protein that is predicted to contain a flexible arm that interacts with DNA. Three other mutants, which recognize the wild-type and a different variant site, contain amino acid substitutions at position 44 (Glu to Lys, Val or Gly) of the beta subunit. These substitutions are in the middle of a predicted beta-strand of the subunit. We discuss the possible mechanisms of suppression by the mutants in terms of a model of the IHF-DNA complex proposed by Yang and Nash [Cell, 57, 869-880 (1989)].

Amino Acid Sequence↗
PubMed1992

Time-dependent regulation of rat adipose tissue glucose transporter (GLUT4) mRNA and protein by insulin in streptozocin-diabetic and normal rats.

Streptozocin (STZ) administration (125 mg/kg) to normal rats resulted in a rapid (24-hour) decrease in circulating insulin levels, marked hyperglycemia, and weight loss. Adipose tissue glucose transporter 4 (GLUT4) mRNA levels decreased approximately eightfold, whereas GLUT4 protein levels were unchanged. However, GLUT4 protein levels decreased approximately 30% by 48 hours and fivefold by 72 hours of insulin deficiency. Although GLUT4 mRNA levels were rapidly restored by insulin therapy (twofold above control levels within 12 hours), GLUT4 protein levels increased only gradually, reaching peak values of 1.5-fold control levels following 7 to 10 days of insulin treatment. Insulin treatment in normal rats increased adipose GLUT4 mRNA levels nearly 100% by 24 hours, while GLUT4 protein levels increased in a more gradual fashion. The delay in GLUT4 protein induction relative to its mRNA was shorter in normal rats treated with insulin than in insulin-treated diabetic rats. These data demonstrate that insulin-induced changes in adipose GLUT4 protein are considerably delayed relative to its mRNA, and that the diabetic state enhances this difference. The known in vivo time-dependent effects of insulin treatment on adipocyte glucose transport activity can be at least partly explained by altered specific expression of GLUT4 protein.

Animals↗
PubMed1992

Localization of two genes for Usher syndrome type I to chromosome 11.

The Usher syndromes (USH) are autosomal recessive diseases characterized by congenital sensorineural hearing loss and progressive pigmentary retinopathy. While relatively rare in the general population, collectively they account for approximately 6% of the congenitally deaf population. Usher syndrome type II (USH2) has been mapped to chromosome 1q (W. J. Kimberling, M. D. Weston, C. Möller, et al., 1990, Genomics 7: 245-249; R. A. Lewis, B. Otterud, D. Stauffer, et al., 1990, Genomics 7: 250-256), and one form of Usher syndrome type I (USH1) has been mapped to chromosome 14q (J. Kaplan, S. Gerber, D. Bonneau, J. Rozet, M. Briord, J. Dufier, A. Munnich, and J. Frezal, 1990. Cytogenet. Cell Genet. 58: 1988). These loci have been excluded as regions of USH genes in our data set, which is composed of 8 French-Acadian USH1 families and 11 British USH1 families. Both of these sets of families show linkage to loci on chromosome 11. Linkage analysis demonstrates locus heterogeneity between these sets of families, with the French-Acadian families showing linkage to D11S419 (Z = 4.20, theta = 0) and the British families showing linkage to D11S527 (Z = 6.03, theta = 0). Genetic heterogeneity of the data set was confirmed using HOMOG and the M test (log likelihood ratio > 10(5)). These results confirm the presence of two distinct USH1 loci on chromosome 11.

Chromosome Mapping↗