Correction to: Three­dimensional topography of scapular nutrient foramina.

Correction to: Surgical and Radiologic Anatomy.

Three-dimensional topography of scapular nutrient foramina.

PURPOSE: The aim of this study is to describe the number and location of the nutrient foramina in human scapulae which can minimize blood loss during surgery. METHODS: 30 cadaveric scapulae were macerated to denude the skeletal tissue. The nutrient foramina of 0.51 mm and larger were identified and labeled by adhering glass beads. CT scans of these scapulae were segmented resulting in a surface model of each scapula and the location of the labeled nutrient foramina. All scapulae were scaled to the same size projecting the nutrient foramina onto one representative scapular model. RESULTS: Average number of nutrient foramina per scapula was 5.3 (0-10). The most common location was in the supraspinous fossa (29.7%). On the costal surface of the scapula, most nutrient foramina were found directly inferior to the suprascapular notch. On the posterior surface, the nutrient foramina were identified under the spine of the scapula in a somewhat similar fashion as those on the costal surface. Nutrient foramina were least present in the peri-glenoid area. CONCLUSION: Ninety percent of scapulae have more than one nutrient foramen. They are located in specific areas, on both the posterior and costal surface.

Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation.

BACKGROUND: Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation. METHODS: Combining chemical synthesis, biological evaluation, and structure-activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone. RESULTS: The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives. CONCLUSION: By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.

Discovery of entry inhibitors for HIV-1 via a new de novo protein design framework.

A new (to our knowledge) de novo design framework with a ranking metric based on approximate binding affinity calculations is introduced and applied to the discovery of what we believe are novel HIV-1 entry inhibitors. The framework consists of two stages: a sequence selection stage and a validation stage. The sequence selection stage produces a rank-ordered list of amino-acid sequences by solving an integer programming sequence selection model. The validation stage consists of fold specificity and approximate binding affinity calculations. The designed peptidic inhibitors are 12-amino-acids-long and target the hydrophobic core of gp41. A number of the best-predicted sequences were synthesized and their inhibition of HIV-1 was tested in cell culture. All peptides examined showed inhibitory activity when compared with no drug present, and the novel peptide sequences outperformed the native template sequence used for the design. The best sequence showed micromolar inhibition, which is a 3-15-fold improvement over the native sequence, depending on the donor. In addition, the best sequence equally inhibited wild-type and Enfuvirtide-resistant virus strains.

Kinase chips hit the proteomics era.

Protein kinase chips, in which kinases are tested for their ability to phosphorylate immobilized substrates, have been developed and used to evaluate the protein kinases encoded by the yeast genome. This new technology promises to be a valuable addition to the biochemists' and cell biologists' arsenal for evaluating the substrate selectivity and function of protein kinases in cell signaling.

Chromatin-dependent cooperativity between constitutive and inducible activation domains in CREB.

The cyclic AMP (cAMP)-responsive factor CREB induces target gene expression via constitutive (Q2) and inducible (KID, for kinase-inducible domain) activation domains that function synergistically in response to cellular signals. KID stimulates transcription via a phospho (Ser133)-dependent interaction with the coactivator paralogs CREB binding protein and p300, whereas Q2 recruits the TFIID complex via a direct association with hTAF(II)130. Here we investigate the mechanism underlying cooperativity between the Q2 domain and KID in CREB by in vitro transcription assay with naked DNA and chromatin templates containing the cAMP-responsive somatostatin promoter. The Q2 domain was highly active on a naked DNA template, and Ser133 phosphorylation had no additional effect on transcriptional initiation in crude extracts. Q2 activity was repressed on a chromatin template, however, and this repression was relieved by the phospho (Ser133) KID-dependent recruitment of p300 histone acetyltransferase activity to the promoter. In chromatin immunoprecipitation assays of NIH 3T3 cells, cAMP-dependent recruitment of p300 to the somatostatin promoter stimulated acetylation of histone H4. Correspondingly, overexpression of hTAFII130 potentiated CREB activity in cells exposed to cAMP, but had no effect on reporter gene expression in unstimulated cells. We propose that cooperativity between the KID and Q2 domains proceeds via a chromatin-dependent mechanism in which recruitment of p300 facilitates subsequent interaction of CREB with TFIID.

LSD1 Histone Demethylase Assays and Inhibition.

The lysine-specific demethylase (LSD1) is a flavin-dependent amine oxidase that selectively removes one or two methyl groups from histone H3 at the Lys4 position. Along with histone deacetylases 1 and 2, LSD1 is involved in epigenetically silencing gene expression. LSD1 has been implicated as a potential therapeutic target in cancer and other diseases. In this chapter, we discuss several approaches to measure LSD1 demethylase activity and their relative strengths and limitations for inhibitor discovery and mechanistic characterization. In addition, we review the principal established chemical functional groups derived from monoamine oxidase inhibitors that have been investigated in the context of LSD1 as demethylase inhibitors. Finally, we highlight a few examples of recently developed LSD1 mechanism-based inactivators and their biomedical applications.

The epigenetic regulators CBP and p300 facilitate leukemogenesis and represent therapeutic targets in acute myeloid leukemia.

Growing evidence links abnormal epigenetic control to the development of hematological malignancies. Accordingly, inhibition of epigenetic regulators is emerging as a promising therapeutic strategy. The acetylation status of lysine residues in histone tails is one of a number of epigenetic post-translational modifications that alter DNA-templated processes, such as transcription, to facilitate malignant transformation. Although histone deacetylases are already being clinically targeted, the role of histone lysine acetyltransferases (KAT) in malignancy is less well characterized. We chose to study this question in the context of acute myeloid leukemia (AML), where, using in vitro and in vivo genetic ablation and knockdown experiments in murine models, we demonstrate a role for the epigenetic regulators CBP and p300 in the induction and maintenance of AML. Furthermore, using selective small molecule inhibitors of their lysine acetyltransferase activity, we validate CBP/p300 as therapeutic targets in vitro across a wide range of human AML subtypes. We proceed to show that growth retardation occurs through the induction of transcriptional changes that induce apoptosis and cell-cycle arrest in leukemia cells and finally demonstrate the efficacy of the KAT inhibitors in decreasing clonogenic growth of primary AML patient samples. Taken together, these data suggest that CBP/p300 are promising therapeutic targets across multiple subtypes in AML.

Chemical approaches to the study of protein tyrosine kinases and their implications for mechanism and inhibitor design.

Protein tyrosine kinases are critical enzymes for signal transduction. Using C-terminal Src kinase (Csk) as a model system, we discuss progress in three main areas. First, we describe our efforts to measure the transition state of the reaction using peptide substrates containing fluorotyrosine analogs. It is shown that the Brønsted nucleophile coefficient for the reaction is near zero (similar to the nonenzymatic reaction) and the required nucleophile is the neutral phenol (rather than the more chemically reactive phenoxide anion). By studying the kinase reaction in the reverse direction, a Brønsted leaving group coefficient of -0.3 was measured, indicative of protonation of the departing phenol in the transition state. Taken together, these results strongly support a dissociative transition state mechanism for the kinase. These findings set constraints on the design of transition state analog inhibitors. Second, we describe efforts toward defining the specificity of Csk for peptide and protein substrates. The main findings are that local amino acids surrounding a phosphorylated tyrosine can influence recognition, but that long-range interactions probably are more important in a physiologic protein substrate. These findings underscore the complexities in how protein kinases select protein substrates. Third, we describe a new method in protein engineering that has been applied to the study of protein kinases. The method, expressed protein ligation, allows a general approach for ligating synthetic peptides to recombinant proteins. Using expressed protein ligation, obtaining site-specifically phosphorylated proteins and proteins with the incorporation of biophysical probes becomes relatively straightforward. We have used this method to generate a tail phosphorylated, conformationally altered Csk that showed an unexpected increase in kinase activity.

Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.

Mechanistic studies on the alkyltransferase activity of serotonin N-acetyltransferase.

BACKGROUND: Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) catalyzes the first, rate-limiting step in the biosynthesis of the circadian hormone melatonin (5-methoxy-N-acetyltryptamine) from serotonin. Our recent discovery that, in addition to catalyzing the acetyl transfer from acetyl-coenzyme A (acetyl-CoASH) to serotonin, AANAT is also a robust catalyst for the alkyl transfer reaction between CoASH and N-bromoacetyltryptamine has not only opened up a new way to develop cell-permeable AANAT acetyltransferase inhibitors that are valuable in vivo tools in helping elucidate melatonin's (patho)physiological roles, but has also raised a question - how does AANAT accelerate the alkyl transfer reaction? In this study, mechanistic aspects of the AANAT-catalyzed alkyl transfer reaction were explored by employing CoASH and a series of N-haloacetyltryptamines that were also evaluated for their AANAT acetyltransferase inhibitory activities. RESULTS: Investigation of various N-haloacetyltryptamine analogs showed a similar leaving group effect on the enzymatic and non-enzymatic reaction rates. Steady-state kinetic analyses demonstrated that AANAT alkyltransferase obeys a sequential, ternary complex mechanism, with random substrate binding. Rate versus pH profiles revealed the catalytic importance of an ionizable group with pK(a) of approximately 7. All those N-haloacetyltryptamines that serve as substrates of AANAT alkyltransferase are also potent (low micromolar) in vitro inhibitors against AANAT acetyltransferase activity. In particular, N-chloroacetyltryptamine was also shown to be a potent inhibitor of intracellular melatonin production in a pineal cell culture assay. CONCLUSIONS: This is the first detailed investigation of the alkyltransferase activity associated with an acetyltransferase. Our results indicate that AANAT does not accelerate the alkyl transfer reaction by simple approximation effect as previously proposed for the similar alkyl transfer reaction catalyzed by other acyltransferases. This study has general implications for developing novel inhibitors by taking advantage of the promiscuous alkyltransferase activity associated with several acyltransferases.

Ghrelin.

BACKGROUND: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. SCOPE OF REVIEW: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. MAJOR CONCLUSIONS: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

Bisubstrate ketone analogues as serotonin N-acetyltransferase inhibitors.

Serotonin N-acetyltransferase, also called the melatonin rhythm enzyme, is thought to play an important regulatory role in circadian rhythm in animals and people. A series of analogues were synthesized in which indole and coenzyme A were linked via ketone tethers as designed inhibitors of this enzyme. These compounds were tested against purified serotonin N-acetyltransferase. The parent ketone compound was found to be as potent as an amide linked compound studied previously, suggesting that there are no key hydrogen bonds to the nitrogen atom of the corresponding substrate necessary for tight inhibition. Reduction of the parent ketone afforded the diastereomeric carbinol mixture which showed reduced inhibitory potency, arguing against tetrahedral analogue mimicry as an important inhibitory theme. Several conformationally constrained ketone analogues were synthesized and investigated, and the results indicated that directing the orientation of the two substrates within the bisubstrate system could be used to maximize enzyme inhibition.

Protein C activity, stage of disease, and vascular thrombosis in colon carcinoma.

To determine the etiology of the increased incidence of postoperative deep venous thrombosis (DVT) in patients with carcinoma of the colon, serum levels of protein C were measured preoperatively in 65 patients with colorectal adenocarcinoma. Noninvasive lower-extremity Doppler studies were performed on all patients prior to discharge to assess patency of the deep veins. Six patients (9%) were found to have DVT. The protein C level was considered elevated if it was greater than 125% of control values and reduced if less than 75% of control values. The development of DVT was found to be independent of the serum carcinoembryonic antigen, albumin, total protein, hemoglobin, hematocrit, platelet count, prothrombin time, partial thromboplastin time, and the patient's age and percentage of ideal body weight. There was an inverse relationship between the protein C level (p less than 0.001), Dukes stage of the tumor (p less than 0.001), and the development of DVT. Linear regression analysis revealed that only the tumor stage and the protein C level could be used to predict the development of DVT. The data show that for these patients with colorectal malignancy, the development of DVT may be related to decreased levels of protein C.

New developments in rehabilitation of neuropathic pain syndromes.

The common medical treatments of neuropathic pain, medication and nerve blocks, are often only partially effective in providing significant and long-term pain relief. Patients suffering chronic pain often fall prey to associated emotional suffering, functional impairment, and difficulties in multiple areas of their lives, including family disruption, social withdrawal, and vocational disability. An interdisciplinary approach to pain management draws on the skills of physical and occupational therapists, pain psychologists, biofeedback specialists, and vocational counselors. It focuses on both pain management and functional restoration, and should be considered standard treatment for chronically painful conditions. Interdisciplinary pain management views the patient as an active agent, responsible for learning and applying self-management techniques for controlling pain, with the staff assuming a teaching and consulting role. Although much more labor intensive, interdisciplinary pain management is more effective over time in managing chronic pain, in preventing unnecessary emotional and physical impairment, and in controlling overall medical costs.

Triplication of the gallbladder: review of literature and report of a case.

A case of triplication of the gallbladder is presented. This is the ninth case to be reported in the world literature. The triple gallbladder with a common cystic duct was found on single dose oral cholecystogram during the gastrointestinal evaluation of a man with hematemesis. No surgery was performed due to the absence of biliary tract symptoms and absence of pathology on roentgenograms. The embryology, anatomy, pathology, and clinical significance of vesica fellea triplex are discussed and the previous eight case reports are reviewed.

Feasibility of obtaining sexual risk and STD history in the context of a drinking drivers' program.

OBJECTIVES: This study was designed (a) to assess the feasibility of obtaining data about sexually transmitted diseases and sexual risk behavior in an alternative-to-incarceration program for convicted drinking drivers and (b) to determine whether asking health history and sexual risk questions using an anonymous questionnaire, anonymous interviews, or confidential interviews affected the willingness of people to participate. METHODS: The same survey instrument was used across three data collection modes to collect information on sexually transmitted diseases and sexual risk behavior. RESULTS: Overall, there were no differences across modes in self-reports of STDs and details of sexual history. Although the difference in refusal rates between the anonymous questionnaire and the anonymous interview was not significant, the refusal rate for the anonymous questionnaire was significantly higher than the rate for the confidential interview. Those answering the self-administered questionnaire were more likely than those receiving face-to-face interviews to refuse to answer questions about having sex while high and condom use. CONCLUSIONS: A drinking driver intervention program may be an appropriate site for health screenings and prevention activities for an at-risk population.

Enhancing substance abuse treatment with case management. Its impact on employment.

This article examines the effectiveness of strengths-based case management in assisting persons with substance abuse problems improve employment-related functioning. In a study of 632 veterans seeking treatment for substance abuse problems, Wright State University's Enhanced Treatment Project found that veterans in substance abuse treatment had improved in several areas of employment functioning, including number of days employed. Among clients who expressed interest in receiving assistance with employment-related issues, those who received strengths-based case management demonstrated additional improvement in employment functioning including more days employed, fewer employment problems and being less troubled about their employment situation. Correlations between improved employment functioning and improved functioning in other life areas further support the value of case management. The implications of these findings for the inclusion of case management services in substance abuse treatment programs will be discussed.

Pediatric chance fractures from lapbelts: unique case report of three in one accident.

In 1948, G. Q. Chance described a traumatic spinal injury as a "horizontal splitting of the spine," which has since come to be known as the Chance fracture. In 1965, the first such fracture was described by Howland et al. in a passenger as a result of a lap seatbelt during a motor vehicle accident. Until 1980, there were 36 such injuries reported, but the number of reports has since risen with the advent of mandatory seatbelt laws. We report three cases occurring in a single accident when a popular 4-wheel drive vehicle moving at only approximately 25 mph struck a tree, causing flexion-distraction fractures in all three children wearing lapbelts while seated in the rear seat. All three had a different Chance fracture variant and associated intraabdominal injuries. One child was rendered paraplegic. The purpose of this report is to promote awareness of the associated injuries, and to encourage appropriate use and development of passenger restraints for children.

The strengths perspective of case management: a promising inpatient substance abuse treatment enhancement.

The medical model or disease concept approach to substance abuse treatment is practiced in most residential substance abuse treatment programs. Despite wide acceptance, many of the factors inherent in this approach may actually increase patient noncompliance with treatment regimens. These factors are related to the pathology-based nature of substance abuse treatment, an overemphasis on patient denial, and the paternalistic role that treatment staff often take in the treatment of substance abusers. The Strengths Perspective of Case Management/Advocacy has demonstrated usefulness as an adjunct to treatment that remedies these detrimental factors and improves compliance with, and retention in, treatment. The specific activities that lead to this improvement are discussed.