Biomedical subjects

L M Smith

Publications and source records attributed to L M Smith.

At least 19 recordsLinked to original sources

PubMed2001

Which patients with microscopic disease and rhabdomyosarcoma experience relapse after therapy? A report from the soft tissue sarcoma committee of the children's oncology group.

PURPOSE: To identify which patients with rhabdomyosarcoma and microscopic residual disease (group II) are likely to not respond to therapy. PATIENTS AND METHODS: Six hundred ninety-five patients with group II tumors received chemotherapy and 90% received radiation therapy on Intergroup Rhabdomyosarcoma Study (IRS)-I to IRS-IV (1972 to 1997). Tumors were subgrouped depending on the presence of microscopic residual disease only (subgroup IIa), resected positive regional lymph nodes, (subgroup IIb), or microscopic residual disease and resected positive regional lymph nodes (subgroup IIc). RESULTS: Overall, the 5-year failure-free survival rate (FFSR) was 73%, and patients with embryonal rhabdomyosarcoma treated on IRS-IV fared especially well (5-year FFSR, 93%; n = 90). Five-year FFSRs differed significantly by subgroup (IIa, 75% and n = 506; IIb, 74% and n = 101; IIc, 58% and n = 88; P = .0037) and treatment (IRS-I, 68%; IRS-II, 67%; IRS-III, 75%; IRS-IV, 87%; P < .001). Multivariate analysis revealed positive associations between primary site (favorable), histology (embryonal), subgroup IIa or IIb, treatment (IRS-III/IV), and better FFSRs. Patterns of treatment failure revealed local failure to be 8%, regional failure, 4%, and distant failure, 14%. The relapse pattern noted over the course of IRS-I to IRS-IV shows a decrease in the systemic relapse rates, particularly for patients with embryonal histology, suggesting that improvement in FFSRs is primarily a result of improved chemotherapy. CONCLUSION: Group II rhabdomyosarcoma has an excellent prognosis with contemporary therapy as used in IRS-III/IV, and those less likely to respond can be identified using prognostic factors: histology, subgroup, and primary site. Patients with embryonal rhabdomyosarcoma are generally cured, although patients with alveolar rhabdomyosarcoma or undifferentiated sarcoma, particularly subgroup IIc at unfavorable sites, continue to need better therapy.

Adolescent↗

PubMed2001

Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions.

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5'-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).

Alleles↗

PubMed2001

Brain proton magnetic resonance spectroscopy in children exposed to methamphetamine in utero.

OBJECTIVE: To examine the possible neurotoxic effects of prenatal methamphetamine exposure on the developing brain using 1H-MRS. METHODS: Methamphetamine-exposed children (n = 12) and age-matched unexposed control subjects (n = 14) were evaluated with MRI, localized 1H-MRS, and a Child Behavior Checklist. Metabolite concentrations of N-acetyl-containing compounds (NA), total creatine (Cr), choline-containing compounds, myoinositol, and glutamate + glutamine were measured in the frontal white matter and striatum. RESULTS: Despite an absence of visible structural abnormalities in either group, children exposed to methamphetamine in utero had higher [Cr] (+10%, p = 0.02) in the striatum. [NA], primarily a measure of N-acetylaspartate, was normal in both regions, which suggests no significant neuronal loss or damage in the two brain regions examined. There were no differences in reported behavior problems among the methamphetamine-exposed children relative to the unexposed group. CONCLUSIONS: The authors found increased [Cr] in the striatum with relatively normal [NA] in children exposed to methamphetamine. These findings suggest an abnormality in energy metabolism in the brains of children exposed to methamphetamine in utero.

Brain↗

PubMed2001

Species identity: conserved inverted LINE repeat clusters (ILRC) in the vertebrate genome as indicators of population boundaries.

The avian LINE CR1 generates multiply-superimposed insertions, resulting in apparently fortuitous inverted LINE repeat clusters (ILRCs). These loci display size micro-heterogeneity within populations, with few or no presence/absence polymorphisms, and yet only very closely related species share loci. Whilst the CR1 sequences that the ILRC loci are derived from are not species-specific, the loci themselves appear to be species-characteristic if not totally species specific. The mammalian LINE L1 is shown to act similarly to CR1 and also forms ILRCs. It is proposed that whilst the formation of these loci may be from a near-random process of super-insertion certain of them are in some way functional, explaining their conservation and rapid spread to population boundaries, whilst non-functional or inactive loci are quickly lost. ILRCs appear to decay from the element as formed by the accumulation of point mutations. ILRCs appear to an unusual example of non-polymorphic sequences being younger than polymorphic sequences with no obvious selective reason.

Animals↗

PubMed2001

Breast cancer after mantle irradiation for Hodgkin's disease: correlation of clinical, pathologic, and molecular features including loss of heterozygosity at BRCA1 and BRCA2.

PURPOSE: Hodgkin's disease patients who receive mantle irradiation have an age-dependent increased risk of developing breast cancer. To determine if genetic factors predispose these patients to develop breast cancer, we evaluated breast cancer specimens for loss of heterozygosity (LOH) at regions where BRCA1 and BRCA2, two breast cancer tumor suppressor genes, are located. We also evaluated whether breast cancers in patients who were previously treated with radiation have a more aggressive phenotype, and whether the clinical course differed from a sporadic group of breast cancer patients. METHODS AND MATERIALS: All females with Hodgkin's disease who were subsequently diagnosed with breast cancer and for whom tissue blocks were available were included. Using a case-control design, case patients (previously treated with radiation therapy) were matched with sporadic control breast cancer patients for age, breast cancer stage, and date of breast cancer diagnosis. After microdissection of tumor and normal tissue from paraffin-embedded tissue blocks, DNA was extracted and samples were examined for LOH at chromosomal segments encompassing BRCA1 and BRCA2. Breast cancer specimens were also evaluated in a blinded fashion for tumor grade and immunoreactivity to estrogen and progesterone receptors, p53, her2-neu, and topoisomerase II alpha. Comparisons were made between the case and control populations using chi2 analysis, and a paired Student's t test. Survival differences were evaluated using a log-rank test. RESULTS: From January 1960 to December 1983, 917 patients were diagnosed with Hodgkin's disease. Twelve patients were subsequently diagnosed with breast cancer and tissue blocks were available on 10 cases. No statistical difference was observed between the case and control populations for LOH at BRCA1 or BRCA2. In the Hodgkin's disease group, LOH was observed in 30% of tumors at BRCA1 and 10% of tumors at BRCA2 vs. 10% and 0% of tumors in the control group at BRCA1 and BRCA2, respectively. Breast tumors from patients who received radiation therapy for Hodgkin's disease displayed greater nuclear pleomorphism (p < 0.02), and an increase in topoisomerase II alpha expression (p < 0.05) vs. the control population. Five of 10 patients were pregnant at the time of their Hodgkin's treatment, and those patients had a shorter time interval to the development of breast cancer compared with the patients who were not pregnant (12.4 years compared with 18.6 years). There was no significant difference in disease-free survival; however, overall survival was inferior in the population previously treated with radiation therapy for Hodgkin's disease (p = 0.01). 80% of patients with a previous Hodgkin's diagnosis died of breast cancer or treatment related effects vs. 30% in the control group. CONCLUSION: We were unable to find statistical evidence for LOH at BRCA1 and BRCA2 in breast cancers from patients previously irradiated for Hodgkin's disease. Breast cancer diagnosed after mantle irradiation may be more biologically aggressive based on the greater nuclear pleomorphism and increase in topoisomerase II alpha staining. This did not translate into a statistical difference in breast cancer disease-free survival; however, overall survival was significantly inferior in the Hodgkin's disease patients.

Adolescent↗

PubMed2001

Prenatal cocaine exposure and cranial sonographic findings in preterm infants.

PURPOSE: Prenatal cocaine exposure has been linked with subependymal hemorrhage and the formation of cysts that are detectable on cranial sonography in neonates born at term. We sought to determine if prenatal cocaine exposure increases the incidence of subependymal cysts in preterm infants. METHODS: We retrospectively reviewed the medical records and cranial sonograms obtained during a 1-year period on 122 premature (< 36 weeks of gestation) infants. Infants were categorized into 1 of 2 groups: those exposed to cocaine and those not exposed to cocaine. Infants were assigned to the cocaine-exposed group if there was a maternal history of cocaine abuse during pregnancy or if maternal or neonatal urine toxicology results were positive at the time of delivery. RESULTS: Five of the 122 infants were excluded from the study because of insufficient medical and drug histories. The incidence of subependymal cysts in the 117 remaining infants was 14% (16 of 117). The incidence of subependymal cysts in infants exposed to cocaine prenatally was 44% (8 of 18) compared with 8% (8 of 99) in the unexposed group (p < 0.01). CONCLUSIONS: We found an increased incidence of subependymal cyst formation in preterm infants who were exposed to cocaine prenatally. This result is consistent with results of similar studies in term infants.

Brain Diseases↗

PubMed2001

A cylindrical capacitor ionization source: droplet generation and controlled charge reduction for mass spectrometry.

A cylindrical capacitor ionization source was used in conjunction with corona discharge charge reduction for generation of singly charged ions for mass spectrometric analysis. The source consists of a fused-silica capillary threaded with a platinum wire and placed inside a stainless steel tube. Application of an electric potential to the wire results in the production of a linear stream of charged droplets when an aqueous solution is pumped through the capillary. Subsequent solvent evaporation yields ions, providing a continuous ion source for mass spectrometry. Passage of the ions through a corona discharge charge reduction chamber permits reduction of the charge state to predominantly singly charged species, facilitating analysis of DNA and protein mixtures. The change from production of multiply charged ions to production of singly charged ions is extremely simple, requiring only modulation of the voltage applied to the corona discharge electrode. A simple technique for construction of the ionization source is reported.

Animals↗

PubMed2001

Exogenous interleukin-12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice.

Experimental infection of BALB/c- or C57BL/6-gamma-interferon-knockout (GKO) mice with Cryptosporidium parvum results in infection in both strains with different outcomes of disease. The BALB/c-GKO mice recover from infection, whereas the C57BL/6-GKO mice succumb to infection in less than 2 weeks. Differences in cytokine mRNA expression suggested that recovery may involve other cytokines. To determine whether the addition of either a Th1 or Th2 cytokine could alter the outcome of infection, we treated GKO mice with either recombinant (r)IL-4 or rIL-12 1 day before infection (DBI) or daily. No effect on the oocyst shedding patterns in either strain nor an increase in survival of the C57BL/6-GKO mice was observed in the rIL-4-treated mice. Whereas one dose of 0.5 microg rIL-12 given 1 DBI had no effect on oocyst shedding, we found that daily doses of rIL-12 administered intraperitoneally exacerbated C. parvum infection in both animal models. Administration of rIL-12 shortened the survival time in the C57BL/6-GKO mice and prevented BALB/c-GKO mice from recovering from infection. Specific proliferation of T cells to cryptosporidial antigen and Th1 and Th2 mRNA cytokine expression was markedly decreased in rIL-12-treated mice. Nitric oxide (NO) may have played a minor role in the decreased proliferation observed since levels of NO present in the splenocyte cultures from rIL-12-treated mice in response to parasite antigen stimulation were higher than those observed in controls. Thus, we propose that resistance to and recovery from C. parvum infections involves a fine balance in the amount and timing of Th1 and Th2 cytokines.

Animals↗

PubMed2001

The role of p27 in endometrial carcinoma.

OBJECTIVE: The cyclin-dependent kinase inhibitor p27 has been shown to mediate cell growth arrest in response to various environmental stimuli. p27 protein levels have shown prognostic value in several different types of cancer. We examined the prognostic value of p27 protein expression in endometrial cancer, the most common gynecologic malignancy. METHODS: A total of 95 paraffin-embedded tumor blocks were obtained and stained via immunohistochemical techniques with a monoclonal antibody against p27. Ten high-power fields were evaluated per slide with at least 1000 cells per slide and two slides per specimen evaluated by two reviewers for nuclear and cytoplasmic staining. The specimens were evaluated for associations with age, stage, grade, and histology. Statistical analysis was performed using the Student t test, chi(2) Kaplan-Meier, and likelihood ratios to assess the data and to generate P values. RESULTS: A total of 91 patients met inclusion criteria for statistical analysis. Fifty-three patients were stage I, 13 stage II, 14 stage III and 11 stage IV with a positive stain (>50% of cells) for p27 obtained in 32.1, 23.1, 35.7, and 36.4%, respectively (Student t test P = 0.77). Survival data were available on 24 advanced stage patients. p27 protein immunostaining showed no association with patient survival. We also found no association of p27 staining with age or histology. Notably, we found a trend in increasing staining with increase in grade, particularly with stage I patients. Also, there was an association of the nuclear and cytoplasmic staining and stage (P = 0.05), but it had no correlation with patient survival. CONCLUSION: Our study showed decreased p27 protein staining in endometrial cancers compared to normal endometrial cells. We found that p27 protein staining shows no association with stage, age, or histology and is not prognostic for survival in advanced endometrial cancers. However, there may be a trend associated with increased p27 protein staining with advanced grades of tumors.

Cell Cycle Proteins↗

PubMed2001

Brain proton magnetic resonance spectroscopy and imaging in children exposed to cocaine in utero.

OBJECTIVE: The effects of prenatal cocaine exposure have been examined using neurobehavioral and brain structural evaluations; however, no study has examined the effects of prenatal cocaine on brain metabolism. Proton magnetic resonance spectroscopy ((1)H-MRS) is a noninvasive method to examine the biochemistry of various brain regions. The purpose of this study was to examine the possible neurotoxic effects of prenatal cocaine exposure on the developing brain using (1)H-MRS. METHODS: Cocaine-exposed children (n = 14) and age-matched unexposed control participants (n = 12) were evaluated with MRI and localized (1)H-MRS. Metabolite concentrations of N-acetyl-containing compounds (NA), total creatine (Cr), choline-containing compounds, myoinositol, and glutamate + glutamine were measured in the frontal white matter and striatum. RESULTS: Despite an absence of structural abnormalities in either group, children exposed to cocaine in utero had significantly higher Cr (+13%) in the frontal white matter. NA, primarily a measure of N-acetyl aspartate and neuronal content, was normal in both regions examined by (1)H-MRS. Normal NA suggests no significant neuronal loss or damage in the 2 brain regions examined in children exposed to cocaine prenatally. CONCLUSIONS: Consistent with findings in abstinent adult cocaine users, we found increased Cr in the frontal white matter, with normal NA in children exposed to cocaine. These findings suggest the need to investigate further possible abnormalities of energy metabolism in the brain of children exposed to cocaine in utero. In addition, this study demonstrates the feasibility of using (1)H-MRS to investigate the effects of prenatal drug exposure on the developing brain.

Aspartic Acid↗

PubMed2001

Human T and B cell immunoreactivity to a recombinant 23-kDa Cryptosporidium parvum antigen.

Cryptosporidial infection in humans results in parasite-specific IgG, IgM, and IgA antibody responses, but little is known of the cell-mediated immune responses to cryptosporidial antigens. In a convenience sample of 35 Haitian residents, there was a high level of cryptosporidial exposure (>90%) as determined by immunoblot reactivity of serum against cryptosporidial antigens. An attempt was made to determine if there was a relationship between antibody and T cell-mediated responses to recombinant Cp23 antigen and how this correlated with reactivity to crude sporozoite antigen preparations (SAg). T cell reactivity was greater against SAg (57%) than to Cp23 (34.3%) as measured by [3H]thymidine incorporation. Proliferative responses to Cp23 were significantly correlated with SAg responses. By enzyme-linked immunosorbent assay, most persons had IgG responses to both SAg (91.4%) and to recombinant Cp23 (88.5%). Antibody responses were greater among persons who exhibited T cell responses to SAg and Cp23. This study demonstrates that recombinant Cp23 antigen could be a useful antigen for detection of both antibody and cell-mediated responses in epidemiologic studies.

Adolescent↗

PubMed2000

Corona discharge in charge reduction electrospray mass spectrometry.

Corona discharge is applied to charge reduction electrospray mass spectrometry for the analysis of complex mixtures of biological molecules. Recent work has described a method of charge reduction (reducing the charge states of analyte ions generated by the electrospray process) employing the radioactive isotope 210Po to produce neutralizing species. A variation to this approach is presented, in which charge neutralization is mediated by ions produced in a corona discharge. Varying the corona discharge voltage controls the current and the degree of charge reduction, providing predominantly singly charged ions that are detected by a commercial electrospray time-of-flight mass spectrometer. This technique provides charge reduction for the simplification of ESI spectra, without need for any radioactive material.

Cytochrome c Group↗

PubMed2000

Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces.

A recently described reaction for the UV-mediated attachment of alkenes to silicon surfaces is utilized as the basis for the preparation of functionalized silicon surfaces. UV light mediates the reaction of t-butyloxycarbonyl (t-BOC) protected omega-unsaturated aminoalkane (10-aminodec-1-ene) with hydrogen-terminated silicon (001). Removal of the t-BOC protecting group yields an aminodecane-modified silicon surface. The resultant amino groups can be coupled to thiol-modified oligodeoxyribonucleotides using a heterobifunctional crosslinker, permitting the preparation of DNA arrays. Two methods for controlling the surface density of oligodeoxyribonucleotides were explored: in the first, binary mixtures of 10-aminodec-1-ene and dodecene were utilized in the initial UV-mediated coupling reaction; a linear relationship was found between the mole fraction of aminodecene and the density of DNA hybridization sites. In the second, only a portion of the t-BOC protecting groups was removed from the surface by limiting the time allowed for the deprotection reaction. The oligodeoxyribonucleotide-modified surfaces were extremely stable and performed well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of nucleic acid biomolecular recognition elements to semiconductor materials.

Alkanes↗

PubMed2000

Genetic identification by mass spectrometric analysis of single-nucleotide polymorphisms: ternary encoding of genotypes.

An approach to genetic identification using biallelic single-nucleotide polymorphism (SNP) genetic markers is described in which the three possible genotypes, AA, Aa, or aa, where "A" and "a" represent the two SNP alleles, are assigned a ternary (base 3) digit of 0, 1, or 2, respectively. Genotyping an individual over a panel of separate SNP markers produces a composite ternary genetic code that can be converted to an easily stored, decimal (base 10) genetic identification number. The unambiguous identification of 11 individuals is demonstrated using ternary genetic codes generated from MALDI-TOF mass spectrometric genotyping data from 7 different SNP markers.

Alleles↗

PubMed2000

Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption.

Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study, we investigated the role that the iNOS pathway plays in mediating the bone-resorbing effects of IL-1 by studying mice with targeted disruption of the iNOS gene. Studies in vitro and in vivo showed that iNOS-deficient mice exhibited profound defects of IL-1-induced osteoclastic bone resorption but responded normally to calciotropic hormones such as 1,25 dihydroxyvitamin D3 and parathyroid hormone. Immunohistochemical studies and electrophoretic mobility shift assays performed on bone marrow cocultures from iNOS-deficient mice showed abnormalities in IL-1-induced nuclear translocation of the p65 component of NFkappaB and in NFkappaB-DNA binding, which were reversed by treatment with the NO donor S-nitroso-acetyl penicillamine. These results show that the iNOS pathway is essential for IL-1-induced bone resorption and suggest that the effects of NO may be mediated by modulating IL-1-induced nuclear activation of NFkappaB in osteoclast precursors.

Animals↗

PubMed2000

The alpha 1-subunit of smooth muscle Ca(2+) channel preserves multiple open states induced by depolarization.

The cloned alpha 1-subunits of the smooth muscle Ca(2+) channel (alpha (1C-b)) from rabbit lung were expressed in Chinese hamster ovary cells. The effect of large depolarizations was examined using cell-attached patch clamp techniques. After large, long-duration depolarizations (to +80 mV, 4 s), the cloned smooth muscle Ca(2+) channels were still open, and also showed slow channel closure upon repolarization. The sum of unitary channel currents revealed that the tail current seen after large conditioning depolarizations had a slower deactivation time constant compared to that seen when the cell membrane was depolarized briefly with a test step (to +40 mV), suggesting that large depolarizations transform the conformation of the Ca(2+) channels to a second open state. The decay time course of the tail current induced by large conditioning depolarizations was prolonged by reducing the negativity of the repolarization step, and vice versa. Using the slow deactivating characteristic, the current-voltage relationship was directly measured by applying a ramp pulse after a large depolarization. Its slope conductance was approximately 26 pS. Since the patch pipettes contained Ca(2+) agonists, the transition of the Ca(2+) channel conformation to the second, long open state during a large depolarization was distinct from that caused by Ca(2+) agonists, suggesting that the cloned alpha 1-subunits of smooth muscle Ca(2+) channels preserve the characteristic features seen in native smooth muscle Ca(2+) channels. In addition, when skeletal muscle beta-subunits were coexpressed with the alpha 1-subunits, the long channel openings after large, long-duration depolarizations were frequently suppressed. This phenomenon could be explained if the skeletal muscle beta-subunits increased the inactivation rate during the preconditioning depolarization.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2000

Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification.

Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications.

Amino Acid Sequence↗

PubMed2000

Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production.

Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.

Amino Acid Substitution↗