Pre-pubertal diet restriction reduces reactive oxygen species and restores fertility in male WNIN/Obese rat.

Obesity is a multifactorial disorder associated with increased body adiposity, chronic oxidative stress which contributes to impaired fertility in males. Diet restriction and anti-oxidant supplementations are known to protect obese subjects from oxidative stress and improves fertility. However, the role of oxidative stress and the age of intervention in restoring male fertility are poorly understood. This study was aimed to assess the effect of diet restriction on fertility with respect to the age of intervention, body composition and oxidative stress using WNIN/Ob obese mutant rat strain. Unlike lean and carrier phenotypes, obese rats are hyperphagic, hyperlipaemic and infertile. Male obese rats aged for 35, 60 and 90 days were fed either ad libitum or diet restricted for 6 weeks. Upon diet restriction mean body weight, total body fat percentage, circulatory lipids and oxidative stress markers were significantly reduced and it follows the order as 35 < 60 < 90 days. Diet-restricted males of the three age groups were allowed to mate with female carrier rats, and fertility was restored only in 35-day group. Diet restriction in male obese WNIN/Ob rats lowered the rate of body weight gain, with reduced oxidative stress overall and fertility restoration in groups intervened at pre-pubertal stages.

Effect of Early Orthodontic Treatment on Long-Term Stability of Class II Malocclusions.

Background: Class II malocclusions are a common orthodontic problem, often requiring comprehensive treatment to achieve proper occlusion and facial harmony. Early orthodontic intervention in the mixed dentition phase has been advocated to address these issues. Materials and Methods: A retrospective analysis was conducted on a cohort of 150 patients with class II malocclusions who underwent early orthodontic treatment between the ages of 7 and 10 years. The treatment included fixed or removable appliances, headgear, and functional appliances, depending on individual needs. Records of their initial malocclusion severity, treatment modalities, and long-term follow-up data (mean follow-up duration of 10 years) were collected and analyzed. Stability was assessed by evaluating overjet and overbite changes from post-treatment to the long-term follow-up. Results: The initial mean overjet and overbite values were 8.5 mm and 4.0 mm, respectively. Following early orthodontic intervention, these values were significantly reduced to 3.0 mm and 1.5 mm, respectively (P < 0.001). At the long-term follow-up, the mean overjet and overbite remained stable at 3.2 mm and 1.6 mm, respectively. Analysis revealed that 85% of patients maintained their corrected class II occlusion within clinically acceptable limits, while 15% experienced minor relapse requiring minimal additional treatment. Conclusion: Early orthodontic treatment in class II malocclusions can lead to significant improvements in overjet and overbite, and these corrections tend to remain stable over the long term.

UV-B induced differential effect on growth and nitrogen metabolism in two cyanobacteria under copper toxicity.

In the present study, impact of low (UV-B(L): 0.1 µmol m(-2) s(-1)) and high (UV-BH: 1.0 µmol m(-2) s(-1)) fluence rates of ultraviolet-B on growth and nitrogen metabolism in two cyanobacteria: Phormidium foveolarum and Nostoc muscorum under copper toxicity (2 and 5 µM) was investigated after 24 and 72 h of experiments. Copper and UV-BH treatment suppressed growth but more in N. muscorum which was accompanied by significant accumulation of Cu. Nitrate and nitrite uptake rates and activities of nitrogen assimilating enzymes i.e. nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS) and glutamate synthase (GOGAT) except glutamate dehydrogenase activity (GDH; aminating) were decreased following treatments of Cu and UV-BH, and under combined treatments the effect was greater. On contrary, UV-BL declined Cu toxicity significantly. The study concludes that Cu and UV-BH suppressed the activity of NR, NiR, GS and GOGAT (except GDH) hence decreased growth. However, UV-BL showed cross tolerance in test organisms against Cu toxicity up to certain extent. Phormidium foveolarum is comparatively less sensitive against UV-BH and excess Cu, a situation likely exists in nature, hence it may be used as a biofertilizer for sustainable agriculture.

Ethylene and hydrogen sulphide are essential for mitigating hexavalent chromium stress in two pulse crops.

Chromium toxicity to crops is a major scientific problem of the present time. Thus, scientific attempts have been made for reducing chromium toxicity to crop plants. In this study, we examined the potential of ethylene (ET, 25 µM) and hydrogen sulphide (H2 S, 10 µM) to alleviate hexavalent chromium [Cr(VI), 50 µM] stress in two pulse crops, black bean and mung bean, by assessing physiological and biochemical attributes. Cr(VI) reduced shoot and root length in black bean and mung bean in comparison to the control. Plants had increased accumulation of oxidative stress markers, i.e. superoxide radicals (SOR), hydrogen peroxide (H2 O2 ) and lipid peroxidation (as malondialdehyde, MDA). The addition of AVG (an inhibitor of ET biosynthesis) and PAG (an inhibitor of H2 S biosynthesis) to Cr(VI)-treated plants further increased Cr(VI) toxicity, suggesting their endogenous levels are important for tolerating Cr(VI) toxicity. However, supplementation with either ET or H2 S alleviated Cr(VI) toxicity. Interestingly, ET did not rescue negative effects of PAG under Cr(VI) stress but NaHS rescued negative effect of AVG. Overall, results indicate that, although both ET and H2 S alleviate Cr(VI) stress, endogenous H2 S is better. Furthermore, H2 S appears to be a downstream signal for ET in alleviating Cr(VI) stress in these two pulse crops.

Hydrogen sulphide ameliorates hexavalent chromium toxicity in two cereal crops: Role of antioxidant enzymes and proline metabolism.

Chromium pollution in soils is a major threat as it reduces crop yields. Hence, researchers seek methods/strategies which could curtail such losses. We report the role of H2 S in alleviating hexavalent chromium [Cr(VI)] stress in two cereals crops, i.e. wheat and rice seedlings, by estimating various physiological attributes. Cr(VI) reduced shoot and root length in both cereals through increased accumulation of Cr(VI) in root tips and increased in oxidative stress markers, i.e. superoxide radicals (SOR), H2 O2 and lipid peroxidation (as MDA equivalent). Supplementation with H2 S alleviated Cr(VI) toxicity in both cereal crops. Application of H2 S increased tolerance to Cr(VI) stress by protecting photosynthesis and enhancing activity of antioxidant enzymes, particularly glutathione-S-transferase and content of proline. Rice was more resistant to Cr(VI) than wheat seedlings.

Role of the (acyloxy)methyl moiety in eliciting the adrenergic beta-blocking activity of 3-(acyloxy)propanolamines.

Some totally aliphatic 3-(acyloxy)propanolamines were synthesized with the aim of testing whether beta-blocking activity could be obtained from this class of drugs, even in the absence of an aromatic group. The significant and, in most cases, competitive beta-blocking activity shown by the compounds under examination, together with the results of a theoretical study in which their reactivity was compared with that of other adrenergic beta-blocking drugs, seems to confirm a hypothesis previously advanced on the basis of knowledge about the action mechanism of adrenergic beta-blocking drugs and of the results of structural studies. It was also possible to suggest some considerations about the role played by the (acyloxy)methyl portion of 3-(acyloxy)propanolamines in eliciting their adrenergic beta-blocking activity.

The role of cardioplegia induction temperature and amino acid enrichment in neonatal myocardial protection.

BACKGROUND: Warm cardioplegic induction improves the ischemically "stressed" adult heart. However, it is rarely used in infants, despite the fact that many newborn hearts are stressed by other factors such as hypoxia. The need for amino acids as well as their mechanism of action has also not been studied. METHODS: We first assessed the role of cardioplegic induction temperature in 10 nonhypoxic neonatal piglets undergoing 70 minutes of multidose blood cardioplegic arrest. Five piglets (group 1) received a cold (4 degrees C) induction, and 5 (group 2) a warm (37 degrees C) induction. Twenty-six other piglets underwent ventilator hypoxia (fraction of inspired oxygen, 8% to 10%) for 60 minutes before cardiopulmonary bypass (stress). Six piglets (group 3) then underwent 70 minutes of cardiopulmonary bypass without ischemia (hypoxia controls), and 20 underwent 70 minutes of cardioplegic arrest. Five of these (group 4) received cold cardioplegic induction, and 15 received warm induction; in 5 of these (group 5), the warm cardioplegic solution contained amino acids, in 5 others (group 6), it was unsupplemented, and in the remaining 5 (group 7), nitroglycerin was added to determine the role of vasodilation. Myocardial function was assessed by pressure-volume loops (expressed as a percent of control), and coronary vascular resistance was measured with cardioplegic infusions. RESULTS: In nonhypoxic (normal) piglets, cold (group 1) and warm (group 2) induction completely preserved systolic function (end-systolic elastance, 100% versus 104%) and preload recruitable stroke work (100% versus 102%), with minimal increase in diastolic compliance (162% versus 156%). Hypoxia-reoxygenation alone (group 3) depressed systolic function (end-systolic elastance, 51%+/-2%) and preload recruitable stroke work (54%+/-3%), and raised diastolic stiffness (260%+/-15%). The detrimental effects of reoxygenation persisted (unchanged from reoxygenation alone) with cold induction (group 4) or warm induction without amino acids (groups 6 and 7). In contrast, warm induction with amino acids (group 5) restored systolic function (end-systolic elastance, 105%+/-3%; p < 0.001 versus groups 3, 4, 6, and 7) and preload recruitable stroke work (103%+/-2%; p < 0.001 versus groups 3, 4, 6, and 7), and decreased diastolic stiffness (154%+/-7%; p < 0.001 versus groups 3, 4, 6, and 7). However, there was no difference in myocardial oxygen consumption in hypoxic hearts receiving a warm induction (6.9 versus 6.5 versus 7.3 mL/g per 5 minutes) (groups 5, 6, 7), and coronary vascular resistance was lowest with nitroglycerin (group 7). CONCLUSIONS: Cardioplegic induction can be given either warm or cold in nonhypoxic neonatal hearts. In contrast, only warm induction with amino acids repairs the hypoxic injury, but the primary mechanism of action is not related to increased metabolic activity or vasodilation.

Initial prospective multicenter clinical trial of robotically-assisted coronary artery bypass grafting.

BACKGROUND: This multicenter prospective trial was designed to assess the safety and efficacy of using a robotically-assisted microsurgical system to create endoscopic coronary anastomoses. METHODS: . Thirty-two patients scheduled for elective primary coronary surgery underwent endoscopic anastomosis of the left internal thoracic artery (LITA) to the left anterior descending (LAD) artery. Three thoracic ports (two for instruments and one for a camera) were placed, and a robotic system was used to perform the LITA-LAD graft. Conventional techniques were used to perform the other grafts. Thirty-one patients underwent median sternotomy and 1 patient underwent a limited anterior thoracotomy. RESULTS: . Graft flow was measured in the operating room and averaged 37 +/- 19 mL/min. Mean anastomosis time was 24 +/- 9 minutes. There were three intraoperative revisions (9%). Two were for inadequate flow and one for an inadvertent injury. Each of these grafts was successfully revised by hand. There were no technical failures of the robotic system. Average postoperative length of stay was 5.5 +/- 2.7 days. There were three reoperations for bleeding, but none of these were related to the LAD anastomosis. Two months following the operation, selective angiography revealed a graft patency of 93%. The patients have been followed for 16 +/- 4 months. CONCLUSIONS: This initial prospective multicenter trial documents the feasibility of robotically-assisted coronary bypass grafting. Further trials are warranted to establish the safety and efficacy of this new technology.

Follow-up study of survivors of severe protein energy manlnutrition.

OBJECTIVE: To evaluate the nutritional profile of survivors of severe protein energy malnutrition on follow-up at 1-3 years and 5-7 years. DESIGN: Prospective and Cross-sectional point prevalence. METHODS: Group I comprised 50 severely malnourished children dischared 5-7 years from nutritional rehabilitation center and Group II comprised 50 children discharged 1-3 years ago. The nutritional status of these 100 children was compared to the nutritional status of 35 siblings who had not suffered from severe malnutrition in their earlier life (Group III). RESULTS: All the malnourished children showed significant improvement in weight for age. More children in Group I had better height for age compared to Group II(p>0.05). Analysis of weight for height showed that study children has better nutritional status than their siblings who had not suffered from significant malnutrition. CONCLUSION: Significant improvement in nutritional status occurs with nutritional rehabilitation. However, improvement in height for age is more difficult to obtain. Interestingly, rehabilitated malnourished children on follow up had better nutritional status as compared to their siblings.

Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation.

OBJECTIVE: The maintenance of pluripotency of human embryonic stem cells (hESCs) requires a high efficiency of self-renewal. During in vitro propagation, however, hESCs have a propensity to differentiate spontaneously. In this study, we assessed the nature of hESC responses to hypoxic conditions. MATERIALS AND METHODS: Human embryonic stem cells were grown in normoxic and hypoxic conditions, and the cells expressing Oct4 and stage-specific embryonic antigen-1 were identified by indirect immunofluorescence. The transcriptional expression of Nanog, Notch1, and Oct4 was determined by a real-time reverse transcription-polymerase chain reaction, and the inhibition of Notch-mediated signalling was achieved with a gamma-secretase inhibitor. RESULTS: In contrast to culture at 21% oxygen, where the colonies displayed a marked degree of differentiation, we found that during exposure to 5% oxygen, the hESC colonies displayed a homogenous and flat morphology that was consistent with the presence of Oct4-positive phenotype, indicating no spontaneous differentiation. When cultured at 5% oxygen for either 4 weeks or up to 18 months, high levels of Nanog and Notch1 transcriptional expression were detected, albeit the expression was significantly lower during longer exposure. The suppression of differentiation was rapidly reversed on transfer of the hypoxic cultures to normoxic conditions. Looking into the molecular mechanisms of the maintenance of self-renewal at low oxygen tensions, we found that inhibition of Notch signalling fully abrogated the hypoxic induction of undifferentiated phenotype. CONCLUSION: Our data, thus, indicate that hypoxic exposure has the capacity to sustain long-term self-renewal of hESCs and that this effect is mediated through activation of Notch.

Structure of (+-)-threo-9,10,16-trihydroxypalmitic acid.

9,10,16-Trihydroxyhexadecanoic acid, C16H32O5, Mr = 304.4, triclinic, P1, a = 4.871 (1), b = 8.801 (1), c = 21.121 (2) alpha = 89.54 (1), beta = 84.88 (2), gamma = 76.35 (2) degrees, V = 876.14 (4) A3, Z = 2, Dx = 1.154 g cm-3, Mo K alpha, lambda = 0.71069 A, mu = 0.26 cm-1, F(000) = 336, T = 295 K. Final R = 0.055 for 1556 reflections with I greater than or equal to 3 sigma(I). The 16 C atoms form an all-trans zigzag chain. Each molecule is linked to seven neighboring molecules via eight possible hydrogen bonds with O...O distances varying from 2.591 (6) to 2.673 (3) A. Each carboxyl group is potentially hydrogen-bonded to the carboxyl and C16-OH groups of other molecules with O...O distances of 2.591 (6) and 2.644 (4) A. The conformation around the C9-C10 bond is gauche with an O3-C9-C10-O4 torsion angle of 66.4 (5) degrees.

Structure of N',N'-dibenzylbenzohydrazide.

C21H20N2O, Mr = 316.4, monoclinic, P2(1)/c, a = 9.985 (2), b = 17.070 (3), c = 10.119 (2) A, beta = 95.99 (1) degree, V = 1715.3 A3, Z = 4, Dx = 1.225 g cm-3, Mo K alpha (lambda = 0.71069 A), mu = 0.82 cm-1, F(000) = 672, T = 295 K. Final R = 0.043, wR = 0.042 for 1139 reflections with I greater than 3 sigma(I). There is one hydrogen bond in the structure with N-H...O = 2.05 (4) and N...O = 2.923 (5) A.

Chloramphenicol-erythromycin resistance mutations in a 23S rRNA gene of Escherichia coli.

Two chloramphenicol resistance mutations were isolated in an Escherichia coli rRNA operon (rrnH) located on a multicopy plasmid. Both mutations also confer resistance to 14-atom lactone ring macrolide antibiotics, but they do not confer resistance to 16-atom lactone ring macrolide antibiotics or other inhibitors of the large ribosomal subunit. Classic genetic and recombinant DNA methods were used to map the two mutations to 154-base-pair regions of the 23S RNA genes. DNA sequencing of these regions revealed that chloramphenicol-erythromycin resistance results from a guanine-to-adenine transition at position 2057 of the 23S RNA genes of both independently isolated mutants. These mutations affect a region of 23S RNA strongly implicated in peptidyl transfer and known to interact with a variety of peptidyl transferase inhibitors.

Antitermination by both the promoter and the leader regions of an Escherichia coli ribosomal RNA operon.

RNA polymerase initiating at Escherichia coli ribosomal RNA promoter-leader regions can efficiently read through factor rho-dependent termination signals. Dissection of the promoter-leader region reveals that the ability to read through termination signals is conferred independently by both promoter and leader regions. Events in the leader also affect the transcription rate of structural genes downstream of the leader. When cells are grown in rich medium, the rrnC leader reduces transcription by a factor of approximately 4 when downstream of the rrnC promoters and by a factor of 2 when downstream of the lac promoter.

Structure of RG-12561 dichloromethane solvate and a diastereomer.

(III): [4 alpha,6 beta(E)]-(+-)-6-(2-[2-(4-Fluoro-3-methylphenyl)- 4,4,6,6-tetramethyl-1-cyclohexen-1-yl]ethenyl)-4-hydroxytetrahy dro pyran-2-one (RG-12561) dichloromethane solvate, 2C24H31FO3.CH2-Cl2, Mr = 857.94, triclinic, Pl, a = 11.7413 (5), b = 13.0279 (5), c = 16.2332 (9) A, alpha = 99.456 (4), beta = 94.217 (4), gamma = 101.893 (4) degrees, V = 2381.9 (4) A3, Z = 4 [four molecules of (III)+two molecules of solvent per unit cell], Dx = 1.195 g cm-3, Cu K alpha, lambda = 1.54178 A, mu = 16.69 cm-1, F(000) = 912, T = 293 K, final R = 0.053, wR = 0.060 for 4031 reflections with I greater than 3 sigma(I). (IV): [4 beta,6 alpha(E)]-(+-)-6-(2-[2- (4-Fluoro-3-methylphenyl)-4,4,6,6-tetramethyl-1-cyclo-hexen-1-yl]e thenyl)-4- hydroxytetrahydropyran-2-one, C24H31FO3, Mr = 386.51, triclinic, Pl, a = 6.054 (2), b = 12.931 (2), c = 14.838 (3) A, alpha = 67.70 (2), beta = 85.75 (2), gamma = 82.85 (2) degrees, V = 1066.0 (8) A3, Z = 2, Dx = 1.203 g cm-3, Cu K alpha, lambda = 1.54178 A, mu = 6.86 cm-1, F(000) = 414, T = 293 K, final R = 0.073, wR = 0.081 for 1588 reflections with I greater than 3 sigma(I). (III) is a potent HMG-CoA reductase inhibitor and has the potential to function as a superior hypocholesterolemic agent; (IV) lacks this activity. (III) and (IV) have different conformations and molecular-model calculations suggest that crystal-packing effects are primarily responsible for the overall conformation of (IV). The principal intermolecular contacts are hydrogen bonds of the type O-H...O = C.

Structure of thialysine hydrochloride.

2-Ammonio-3-[(2-ammonioethyl)thio]propionate chloride, C5H13N2O2S+.Cl-, Mr = 200.7, monoclinic, P2(1), a = 5.1275 (4), b = 7.897 (1), c = 11.208 (1) A, beta = 104.129 (8) degrees, V = 440.1 A3, Z = 2, Dx = 1.519 g cm-3, Mo K alpha, lambda = 0.71069 A, mu = 6.12 cm-1, F(000) = 212, T = 295 K, final R = 0.021, wR = 0.027 for 784 reflections with I greater than 3 sigma (I). The two ammonium cations form seven hydrogen bonds: alpha-NH3+ acts as a donor for three carboxylate O atoms and one Cl- ion whereas epsilon-NH3+ is linked to three Cl- ions.

Structure of 3-methyl-1,2,3,4,5,6-hexahydro-1,6-methano-3-benzazocinium hydrogen oxalate.

C13H18N+.C2HO4-, Mr = 277.3, orthorhombic, P2(1)2(1)2(1), a = 5.752 (1), b = 15.502 (2), c = 15.910 (2) A, V = 1418.6 (6) A3, Z = 4, Dx = 1.298 g cm-3, Mo K alpha (lambda = 0.71069 A), mu = 1.01 cm-1, F(000) = 592, T = 295 K. Final R = 0.035, wR = 0.042 for 1003 reflections with I greater than 3 sigma(I). The compound is an ammonium hydrogen oxalate. Both O atoms of the ionized carboxyl group act as acceptors in the formation of two hydrogen bonds: (a) an N--H...O bond linking the cation and anion with N...O and H...O distances of 2.722 (4) and 1.78 (5) A and (b) a strong O--H...O bond linking a chain of oxalate ions along a with O...O and H...O distances of 2.578 (4) and 1.59 (6) A. The relative stereochemistry of the heterocyclic and aromatic rings in the cation is similar to that in morphine.

Identification of a carbohydrate recognition domain in filamentous hemagglutinin from Bordetella pertussis.

The adherence of Bordetella pertussis to ciliated cells and macrophages is critical to colonization and infection of the respiratory tract. Adherence to both types of cells involves the recognition of eukaryotic carbohydrates by the bacterial adhesin filamentous hemagglutinin (Fha). The carbohydrate recognition domain (CRD) of Fha is considered an important antigen for subcomponent vaccines to maximize the generation of antiadherence antibodies capable of protecting against colonization. For identification of the CRD of Fha, a bank of eight monoclonal antibodies (MAbs) that mapped to four contiguous regions were tested for their ability to block Fha binding to lactosylceramide or to block bacterial binding to ciliated cells. Only MAb 12.5A9, which maps to amino acid residues 1141 to 1279, blocked both Fha binding to lactosylceramide and bacterial binding to ciliated cells. An 18-kDa polypeptide corresponding to this region was expressed in Escherichia coli. Cell lysates containing this protein bound to lactosylceramide in a manner identical to that of native Fha. Mutant strains of B. pertussis that contained an in-frame deletion of the coding sequence for this region produced a truncated Fha that showed negligible cross-reactivity with MAb 12.5A9. In an adherence assay, these mutant strains failed to bind efficiently to either ciliated cells or macrophages. The numbers of adherent bacteria for these strains were reduced to the number obtained with a nonadherent strain. We conclude that the region defined by residues 1141 to 1279 of Fha constitutes a CRD critical for bacterial adherence and represents a potential candidate for a subcomponent vaccine.

Prospective clinical trial of robotically assisted endoscopic coronary grafting with 1-year follow-up.

OBJECTIVE: To follow up in prospective fashion patients with coronary artery anastomoses completed endoscopically with robotic assistance. The robotic system was evaluated for safety and its effectiveness in completing microsurgical coronary anastomoses. SUMMARY BACKGROUND DATA: Recently there has been an interest in using robotics and computers to enhance the surgeon's ability to perform endoscopic cardiac surgery. This interest has stemmed from the rapid advancement of technology and the desire to make cardiac surgery less invasive. Using traditional endoscopic instruments, it has not been possible to perform coronary surgery. METHODS: Nineteen patients underwent robotically assisted endoscopic coronary artery bypass grafting of the left internal thoracic artery (LITA) to the left anterior descending artery (LAD). Two robotic instruments and one endoscopic camera were placed through three 5-mm ports. A robotic system was used to construct the LITA-LAD anastomosis. All other required grafts were completed by conventional techniques. RESULTS: Seventeen LITA-LAD grafts (89%) had adequate intraoperative flow. The mean LITA-LAD graft flow was 38.5 +/- 5 mL/min. At 8 weeks, LITA-LAD grafts were assessed by angiography and showed 100% patency with thrombolysis in myocardial infarction (TIMI) I flow. At a mean follow-up of 17 +/- 4.2 months, all patients were NYHA class I and there were no adverse cardiac events. CONCLUSIONS: The results from the first prospective clinical trial of robotically assisted endoscopic coronary bypass surgery in the United States showed favorable short-term outcomes with no adverse events. Robotic assistance is an enabling technology allowing the performance of endoscopic coronary anastomoses.

Structure and function of the xenobiotic substrate binding site of a glutathione S-transferase as revealed by X-ray crystallographic analysis of product complexes with the diastereomers of 9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene.

The three-dimensional structures of isoenzyme 3-3 of glutathione (GSH) transferase complexed with (9R,10R)- and (9S,10S)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene [(9R,10R)-2 and (9S,10S)-2], which are the products of the addition of GSH to phenanthrene 9,10-oxide, have been determined at resolutions of 1.9 and 1.8 A, respectively. The structures indicate that the xenobiotic substrate binding site is a hydrophobic cavity defined by the side chains of Y6, W7, V9, and L12 from domain I (the GSH binding domain) and I111, Y115, F208, and S209 in domain II of the protein. All of these residues are located in variable-sequence regions of the primary structure of class mu isoenzymes. Three of the eight residues (V9, I111, and S209) of isoenzyme 3-3 that are in direct van der Waals contact with the dihydrophenanthrenyl portion of the products are mutated (V9I, I111A, and S209A) in the related isoenzyme 4-4. These three residues are implicated in control of the stereoselectivity of the class mu isoenzymes. The hydroxyl group of Y115 is found to be hydrogen-bonded to the 10-hydroxyl group of (9S,10S)-2, a fact suggesting that this residue could act as an electrophile to stabilize the transition state for the addition of GSH to epoxides. The Y115F mutant isoenzyme 3-3 is about 100-fold less efficient than the native enzyme in catalyzing the addition of GSH to phenanthrene 9,10-oxide and about 50-fold less efficient in the Michael addition of GSH to 4-phenyl-3-buten-2-one. The side chain of Y115 is positioned so as to act as a general-acid catalytic group for two types of reactions that would benefit from electrophilic assistance. The results are consistent with the notion that domain II, which harbors most of the variability in primary structure, plays a crucial role in defining the substrate specificity of class mu isoenzymes.