ABSTRACT
Calpains are intracellular calcium-activated cysteine proteases whose unregulated proteolysis following the loss of calcium homeostasis can lead to acute degeneration during ischemic episodes and trauma, as well as Alzheimer's disease and cataract formation. The determination of the crystal structure of the proteolytic core of mu-calpain (muI-II) in a calcium-bound active conformation has made structure-guided design of active site inhibitors feasible. We present here high-resolution crystal structures of rat muI-II complexed with two reversible calpain-specific inhibitors employing cyclic hemiacetal (SNJ-1715) and alpha-ketoamide (SNJ-1945) chemistries that reveal new details about the interactions of inhibitors with this enzyme. The SNJ-1715 complex confirms that the free aldehyde is the reactive species of the cornea-permeable cyclic hemiacetal. The alpha-ketoamide warhead of SNJ-1945 binds with the hydroxyl group of the tetrahedral adduct pointing toward the catalytic histidine rather than the oxyanion hole. The muI-II-SNJ-1945 complex shows residue Glu261 displaced from the S1' site by the inhibitor, resulting in an extended "open" conformation of the domain II gating loop and an unobstructed S1' site. This conformation offers an additional template for structure-based drug design extending to the primed subsites. An important role for the highly conserved Glu261 is proposed.
Subject(s)
Calpain/antagonists & inhibitors , Crystallography, X-Ray , Protease Inhibitors/chemistry , Amino Acid Sequence , Animals , Binding Sites , Calpain/chemistry , Calpain/genetics , Calpain/metabolism , Catalytic Domain , Conserved Sequence , Cysteine/chemistry , Hydrogen Bonding , Hydrogen-Ion Concentration , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Leupeptins/chemistry , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Molecular Structure , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Sequence Homology, Amino Acid , Solutions/chemistry , Water/chemistryABSTRACT
The endogenous calpain inhibitor, calpastatin, modulates some patho-physiological aspects of calpain signaling. Excess calpain can escape this inhibition and as well, many calpain isoforms and autolytically generated protease core fragments are not inhibited by calpastatin. There is a need, therefore, to develop specific, cell-permeable calpain inhibitors to block uncontrolled proteolysis and prevent tissue damage during brain and heart ischemia, spinal-cord injury and Alzheimer's diseases. Here, we report the first high-resolution crystal structures of rat mu-calpain protease core complexed with two traditional, low molecular mass inhibitors, leupeptin and E64. These structures show that access to a slightly deeper, but otherwise papain-like active site is gated by two flexible loops. These loops are divergent among the calpain isoforms giving a potential structural basis for substrate/inhibitor selectivity over other papain-like cysteine proteases and between members of the calpain family.
Subject(s)
Calpain/chemistry , Glycoproteins/chemistry , Leucine/analogs & derivatives , Leucine/chemistry , Leupeptins/chemistry , Amino Acid Sequence , Animals , Calpain/antagonists & inhibitors , Catalytic Domain , Cathepsin K , Cathepsins/chemistry , Cathepsins/metabolism , Crystallization , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , Glycoproteins/metabolism , Leucine/metabolism , Leupeptins/metabolism , Molecular Sequence Data , Papain/chemistry , Papain/metabolism , Protein Structure, Tertiary , RatsABSTRACT
The concept of mode locking in laser is applied to a two-photon state with frequency entanglement. Cavity enhanced parametric down conversion is found to produce exactly such a state. The mode-locked two-photon state exhibits a comblike correlation function. An unbalanced Hong-Ou-Mandel type interferometer is used to measure the correlation function. A revival of the typical interference dip is observed. We will discuss a scheme for engineering of quantum states in time domain.
ABSTRACT
True allergies to local anesthetics are rare. It is common for practitioners to misdiagnose a serious adverse event to local anesthetics as an allergic reaction. The most likely causes for an allergic response are the preservative, antioxidant, or metabolites and not the anesthetic itself. This case report illustrates the need for practitioners to understand the many potential allergens in local anesthetics and to correctly diagnose patients that are truly allergic to the local anesthetic.
Subject(s)
Anesthesia, Dental/adverse effects , Anesthesia, Local/adverse effects , Antioxidants/administration & dosage , Drug Hypersensitivity/etiology , Sulfites/adverse effects , Adult , Anesthetics, Local , Edema/etiology , Female , Humans , Lidocaine , Skin TestsABSTRACT
Severe hyperhidrosis palmaris represents a disabling problem for many patients. Thoracoscopic techniques that involve dissection and removal of the upper thoracic sympathetic chain are believed to result in the lowest incidence of recurrent symptoms. However, aside from an axillary incision, an additional upper anterior chest wall approach is usually required. Over the past 2 years, we have used a periareolar incision in eight patients to improve postoperative cosmesis for this benign condition.
Subject(s)
Hyperhidrosis/therapy , Sympathectomy/methods , Thoracoscopy , Adult , Female , Follow-Up Studies , Hand , Humans , Male , NipplesABSTRACT
Aggressive surgical and radiotherapeutic management of a patient with desmoplastic malignant melanoma arising from the mucosa of the oral cavity has resulted in disease-free survival of more than 2(1/2) years after diagnosis. This case represents only the tenth reported instance of desmoplastic malignant melanoma arising from the oral cavity and only the third for which survival has exceeded 2 years. Details of the clinical, histopathologic, and therapeutic features of the case are provided to augment the paucity of literature available to clinicians managing this rare disease.
Subject(s)
Melanoma/radiotherapy , Melanoma/surgery , Palatal Neoplasms/radiotherapy , Palatal Neoplasms/surgery , Alveolar Process/pathology , Alveolar Process/surgery , Biopsy , Disease-Free Survival , Female , Follow-Up Studies , Humans , Maxilla/surgery , Melanoma/diagnosis , Melanoma/pathology , Middle Aged , Mouth Mucosa/pathology , Mouth Mucosa/surgery , Palatal Neoplasms/diagnosis , Palatal Neoplasms/pathology , Palate/pathology , Palate/surgery , Radiotherapy, AdjuvantABSTRACT
Steroid hormones share a very similar structure, but they behave distinctly. We present structures of human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) complexes with dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), providing the first pictures to date of DHEA and DHT bound to a protein. Comparisons of these structures with that of the enzyme complexed with the most potent estrogen, estradiol, revealed the structural basis and general model for sex hormone recognition and discrimination. Although the binding cavity is almost entirely composed of hydrophobic residues that can make only nonspecific interactions, the arrangement of residues is highly complementary to that of the estrogenic substrate. Relatively small changes in the shape of the steroid hormone can significantly affect the binding affinity and specificity. The K(m) of estrone is more than 1000-fold lower than that of DHEA and the K(m) of estradiol is about 10 times lower than that of DHT. The structures suggest that Leu-149 is the primary contributor to the discrimination of C-19 steroids and estrogens by 17beta-HSD1. The critical role of Leu-149 has been well confirmed by site-directed mutagenesis experiments, as the Leu-149 --> Val variant showed a significantly decreased K(m) for C-19 steroids while losing discrimination between estrogens and C-19 steroids. The electron density of DHEA also revealed a distortion of its 17-ketone toward a beta-oriented form, which approaches the transition-state conformation for DHEA reduction.
Subject(s)
17-Hydroxysteroid Dehydrogenases/chemistry , 17-Hydroxysteroid Dehydrogenases/metabolism , Dehydroepiandrosterone/metabolism , Dihydrotestosterone/metabolism , Estradiol/metabolism , Amino Acid Sequence , Conserved Sequence , Crystallography, X-Ray , Dehydroepiandrosterone/chemistry , Dihydrotestosterone/chemistry , Estradiol/chemistry , Humans , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
The objective of this article is to present the recurrence pattern of olfactory groove meningiomas after surgical resection. Four patients, one female and three males, with surgically resected olfactory groove meningiomas presented with tumor recurrence. All patients underwent resection of an olfactory groove meningioma and later presented with recurrent tumors. The mean age at initial diagnosis was 47 years. All presented initially with vision changes, anosmia, memory dysfunction, and personality changes. Three patients had a preoperative MRI scan. All patients had a craniotomy, with gross total resection achieved in three, and 90% tumor removal achieved in the fourth. Involved dura was coagulated, but not resected, in all cases. Three patients were followed with routine head CT scans postoperatively, and none was followed with MRI scan. The mean time to recurrence was 6 years. Three patients presented with recurrent visual deterioration, and one presented with symptoms of nasal obstruction. Postoperative CT scans failed to document early tumor recurrence, whereas MRI documented tumor recurrence in all patients. Tumor resection and optic nerve decompression improved vision in two patients and stabilized vision in two. Complete resection was not possible because of extensive bony involvement around the anterior clinoid and inferior to the anterior cranial fossa in all cases. Evaluation of four patients with recurrent growth of olfactory groove meningiomas showed the epicenter of recurrence to be inferior to the anterior cranial fossa, with posterior extension involving the optic canals, leading to visual deterioration. This location led to a delay in diagnosis in patients who were followed only with routine CT scans. Initial surgical procedures should include removal of involved dura and bone, and follow-up evaluation should include formal ophthalmologic evaluations and routine head MRI scans.
ABSTRACT
Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17Beta-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41). To further study the transition state, two non-reactive ternary complexes, 17beta-HSD1-EM519-NADP+ and 17beta-HSD1-EM553-NADP+ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a = 155.59 A, b = 42.82 A, c = 121.15 A, beta = 128.5 degrees for 17beta-HSD1-EM519-NADP+, and a = 124.01 A, b = 45.16 A, c = 61.40 A, beta = 99.2 degrees for 17beta-HSD1-EM553-NADP+, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.
Subject(s)
17-Hydroxysteroid Dehydrogenases/chemistry , Enzyme Inhibitors/chemistry , Estradiol/analogs & derivatives , Estradiol/chemistry , Crystallization , Molecular Structure , NADP/chemistryABSTRACT
Human estrogenic dehydrogenase (17beta-HSD1) catalyses the last step in the biosynthesis of the active estrogens that stimulate the proliferation of breast cancer cells. While the primary substrate for the enzyme is estrone, the enzyme has some activity for the non-estrogenic substrates. To better understand the structure function relationships of 17beta-HSD1 and to provide a better ground for the design of inhibitors, we have determined the crystal structures of 17beta-HSD1 in complex with different steroids. The structure of the complex of estradiol with the enzyme determined previously (Azzi et al., Nature Structural Biology 3, 665-668) showed that the narrow active site was highly complementary to the substrate. The substrate specificity is due to a combination of hydrogen bonding and hydrophobic interactions between the steroid and the enzyme binding pocket. We have now determined structures of 17beta-HSD1 in complex with dihydrotestosterone and 20alpha-OH-progesterone. In the case of the C19 androgen, several residues within the enzyme active site make some small adjustments to accommodate the increased bulk of the substrate. In addition, the C19 steroids bind in a slightly different position from estradiol with shifts in positions of up to 1.4 A. The altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished). The known kinetic parameters for these substrates can be rationalized in light of the structures presented. These results give evidence for the structural basis of steroid recognition by 17beta-HSD1 and throw light on the design of new inhibitors for this pivotal steroid enzyme.
Subject(s)
17-Hydroxysteroid Dehydrogenases/chemistry , Dihydrotestosterone/metabolism , 17-Hydroxysteroid Dehydrogenases/metabolism , 20-alpha-Dihydroprogesterone/metabolism , Crystallization , Humans , Protein Conformation , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The technique of autologous iliac crest bone grafting is an important aspect in the treatment of patients with cleft lip, cleft palate, and other craniofacial disorders. In patients with cleft lip and palate, the alveolar bone graft creates a continuous maxillary arch, closes the oronasal fistula, provides bony support for facial soft tissue and teeth, and facilitates orthodontic movement of teeth. The anatomic and physiologic benefits of this and similar autologous bone graft procedures are apparent. However, pain at the donor site represents a significant source of postoperative morbidity. This study was conducted to evaluate postoperative pain and the ability to perform activities of daily living after bupivacaine infiltration to iliac crest donor sites. Thirty-four alveolar bone graft patients (18 females, 16 males) treated at two teaching hospitals were included in the study. Eleven of the patients received intraoperative bupivacaine at the iliac donor site and 23 did not. A questionnaire was returned by all participants, and telephone follow-up was obtained. Responses to postoperative pain, time period to ambulation, and ability to perform activities of daily living were evaluated. Patients who received postoperative bupivacaine experienced delayed onset of postoperative pain, earlier ambulation, and were able to return to normal daily activity in a shorter period of time than those patients who received no local anesthesia. The concept of preemptive analgesia and its application to craniofacial surgery is discussed.
Subject(s)
Anesthetics, Local/administration & dosage , Bone Transplantation/adverse effects , Bupivacaine/administration & dosage , Ilium/surgery , Pain, Postoperative/prevention & control , Activities of Daily Living , Adolescent , Anesthesia, Local , Child , Early Ambulation , Female , Humans , Ilium/physiopathology , Male , Pain Measurement , Premedication , Surveys and QuestionnairesABSTRACT
Older adults with heart failure have extensive and complex care needs. This article presents a framework for assessment and intervention with this fragile patient group. Comprehensive assessment of elders with heart failure includes gathering information about care, advocacy, and knowledge needs. Nursing interventions are focused on providing direct care, counseling, teaching, and advocacy. The unique role of the advanced practice nurse in discharge planning and home follow-up of elders with heart failure is emphasized.
Subject(s)
Geriatric Nursing , Heart Failure/nursing , Home Care Services , Patient Discharge , Aged , Humans , Nursing ProcessABSTRACT
Fifteen consecutive pediatric patients ranging from 3 to 5 years old were selected to receive one of three sedative/hypnotic techniques. Group 1 received oral chloral hydrate 50 mg/kg, and groups 2 and 3 received intramuscular ketamine 2 mg/kg and 3 mg/kg, respectively. In addition to ketamine, patients in groups 2 and 3 received transmucosal intramuscular injections of meperidine and promethazine into the masseter muscle. Sedation for the satisfactory completion of restorative dentistry was obtained for over 40 min on average in the chloral hydrate group, but completion of dental surgery longer than 40 min was achieved in groups 2 and 3 only by intravenous supplements of ketamine.
Subject(s)
Anesthesia, Dental/methods , Chloral Hydrate , Conscious Sedation/methods , Dental Care for Children , Hypnotics and Sedatives , Ketamine , Meperidine , Promethazine , Administration, Oral , Anesthetics, Dissociative/administration & dosage , Anesthetics, Dissociative/adverse effects , Child Behavior , Child, Preschool , Chloral Hydrate/administration & dosage , Dental Care for Children/methods , Drug Combinations , Female , Humans , Hypnotics and Sedatives/administration & dosage , Hypnotics and Sedatives/adverse effects , Injections, Intramuscular , Ketamine/administration & dosage , Male , Meperidine/administration & dosage , Promethazine/administration & dosage , Promethazine/adverse effectsABSTRACT
OBJECTIVES: The purpose of this study was to evaluate patients with hypertension or cardiovascular disease, or both, for myocardial ischemia and cardiac arrhythmias while undergoing minor oral surgery. STUDY DESIGN: Sixteen patients were studied with noninvasive monitoring including heart rate, systolic, diastolic, and mean arterial blood pressure to calculate rate-pressure product (systolic blood pressure multiplied by heart rate) and pressure-rate quotient (mean arterial pressure divided by heart rate). These calculated measures were compared with the incidence of cardiac arrhythmias and ST segment depression recorded on a continuous Holter monitoring system. RESULTS: Nine of 16 (56%) developed supraventricular or ventricular ectopy during dental extractions or minor preprosthetic surgery performed with local anesthesia 2% xylocaine with 1/100,000 epinephrine dilution. Three of the nine patients who experienced these arrhythmias had coincident abnormal rate pressure product and pressure-rate quotient values. None of these patients exhibited ST-T wave changes suggestive of myocardial ischemia. Atrial or ventricular ectopy suggests that myocardial irritability was more likely to occur than ischemia as measured by Holter monitoring and compared with abnormal rate pressure product and pressure-rate quotient values recorded.
Subject(s)
Cardiovascular Diseases , Dental Care for Chronically Ill , Electrocardiography, Ambulatory , Monitoring, Intraoperative/methods , Adult , Aged , Anesthesia, Dental , Arrhythmias, Cardiac/etiology , Blood Pressure , Electrocardiography , Follow-Up Studies , Heart Rate , Humans , Hypertension , Middle Aged , Myocardial Ischemia/etiology , Oral Surgical Procedures, Preprosthetic/adverse effects , Stress, Physiological , Tooth Extraction/adverse effectsABSTRACT
The equilibrium constants for complexation of C60 with naphthalene, phenanthrene and pyrene in toluene have been determined by UV visible spectroscopy. The magnitude of the equilibrium constants was found to increase with decreasing ionization potential of the donor. Values for complexation enthalpy have been determined for the first time for C60/aromatic hydrocarbons. Well-defined charge transfer (C-T) bands have been observed for complexes of C60 with a variety of aromatic hydrocarbons, with C-T band maxima moving to higher frequency with increasing donor ionization potential.
Subject(s)
Carbon/chemistry , Fullerenes , Polycyclic Aromatic Hydrocarbons/chemistry , Chemical Phenomena , Chemistry, Physical , Molecular Structure , Naphthalenes/chemistry , Phenanthrenes/chemistry , Pyrenes/chemistry , Spectrophotometry , Spectrophotometry, Ultraviolet , Temperature , ThermodynamicsABSTRACT
Two hundred geriatric patients ranging from age 65 to 92 yr (mean age 72 yr) were evaluated for office oral surgery and intravenous sedation. Surgical time ranged from 6 to 129 min. Monitored anesthesia care was utilized for the administration of fentanyl, midazolam or diazepam, and methohexital. No serious complications were seen and no patients were hospitalized.
Subject(s)
Ambulatory Surgical Procedures , Anesthesia, Dental/methods , Anesthesia, Intravenous/methods , Conscious Sedation/methods , Oral Surgical Procedures , Aged , Aged, 80 and over , Anesthesia Recovery Period , Humans , Monitoring, IntraoperativeABSTRACT
Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector. They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A. A complete native data set has been collected at 3.1 A resolution for these crystals.
Subject(s)
Arginine-tRNA Ligase/chemistry , Escherichia coli/enzymology , Chemical Precipitation , Crystallization , Crystallography, X-Ray , Hydrogen-Ion Concentration , Molecular Weight , Polyethylene GlycolsABSTRACT
The structure-function relationship of the estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), a pivotal enzyme in the synthesis of active sex hormones, has been studied via protein chemistry and crystallography. A highly active and homogeneous 17 beta-HSD1 was prepared with a rapid purification from human placenta. We then characterized the native and expressed enzyme, and concluded, for the first time, that 17 beta-HSD1 is formed by two identical subunits. The enzyme was also overproduced in insect cells with a baculovirus expression system. The highly active 17 beta-HSD1 preparation was successfully crystallized in the presence of NADP-, polyethylene glycol, beta-octylglucoside and glycerol, resulting in the first diffraction quality crystals of any steroid-converting enzyme from a human source. The three-dimensional structure of 17 beta-HSD1 was determined at 2.2 A resolution, showing that the overall structure of the enzyme is similar to the other enzymes in the short-chain dehydrogenase family, with a conserved Tyr-X-X-X-Lys sequence and a serine residue in the active site. It is distinguished from the other known structures reported for short-chain dehydrogenases by the insertion of two helix-turn-helix motifs that appear to govern membrane association and substrate specificity [corrected]. More recently, the complex of 17 beta-HSD1 with estradiol has been successfully crystallized and its structure determined. The latter demonstrates detailed information of the interactions between the substrate and residues Ser142, Tyr155, His221 and Glu282 of the enzyme. These interactions and the complementarity of the substrate with the hydrophobic binding pocket make critical contributions to the enzyme specificity. The above results provide a strong basis for the design of potent inhibitors of this pivotal steroid dehydrogenase.