The macquarie system for comprehensive management of complex ventral hernia.

BACKGROUND: Despite recent advances in the operative management of complex ventral hernia (CVH), significant challenges remain. Closure of large defects can have serious pathophysiological consequences due to chronic contraction and retraction of the lateral abdominal wall muscles. Certain features of CVH make repair technically demanding and time consuming, such as massive fascial defects, unusual hernia locations, involvement of other abdominal wall structures and previous tissue trauma. METHODS: Preoperative assessment with three-dimensional volume rendered CT (3DVR-CT) imaging and an illustrative series of clinical cases is introduced for repair of CVH using laparoscopic approach. RESULTS: CVH presented here include traumatic hernias involving extensive tissue trauma, massive ventral hernias with defects > 20 cm in width, hernias requiring additional procedures such as wiring of ribs, and hernias in difficult locations such as suprapubic and flank hernias. Specific techniques such as individually tailoring mesh and size, transfascial mesh straps fixation and transcutaneous defect closure will be discussed. All hernias in this series have been repaired laparoscopically (Lap) or laparoscopic-open-laparoscopic (LOL) technique with transcutaneous fascial closure. After hernia closure the mesh is placed in either an intra-peritoneal onlay mesh (IPOM) placement or modified Rives-Stoppa technique with pre-peritoneal mesh placement. CONCLUSION: CVH repair requires multidisciplinary planning with management tailored to each patient's clinical and surgical requirements. The surgeon must have a variety of surgical skills and strategies to address the multiple and/or atypical defects that affect these patients.

Contribution of metabolic disease to bone fragility in MAGP1-deficient mice.

Microfibril-associated glycoprotein-1 (MAGP1) is an extracellular matrix protein that interacts with fibrillin and is involved in regulating the bioavailability of signaling molecules such as TGFß. Mice with germline MAGP1 deficiency (Mfap2-/-) develop increased adiposity, hyperglycemia, insulin resistance, bone marrow adipose tissue expansion, reduced cancellous bone mass, cortical bone thinning and bone fragility. The goal of this study was to assess whether the Mfap2-/- bone phenotypes were due to loss of MAGP1 locally or secondary to a change in whole body physiology (metabolic dysfunction). To do this, mice with conditional deletion of MAGP1 in the limb skeleton were generated by crossing MAGP1-flox mice (Mfap2lox/lox) with Prx1-Cre mice. Mfap2Prx-/- mice did not show any changes in peripheral adiposity, hyperglycemia or insulin sensitivity, but did have increased bone length and cancellous bone loss that was comparable to the germline Mfap2-/- knockout. Unlike the germline knockout, marrow adiposity, cortical bone thickness and bone strength in Mfap2Prx-/- mice were normal. These findings implicate systemic metabolic dysfunction in the development of bone fragility in germline Mfap2-/- mice. An unexpected finding of this study was the detection of MAGP1 protein in the Mfap2Prx-/- hematopoietic bone marrow, despite the absence of MAGP1 protein in osseous bone matrix and absent Mfap2 transcript expression at both sites. This suggests MAGP1 from a secondary site may accumulate in the bone marrow, but not be incorporated into the bone matrix, during times of regional MAGP1 depletion.

Laparoscopic repair of complex ventral hernia facilitated by pre-operative chemical component relaxation using Botulinum Toxin A.

PURPOSE: The operative management of complex ventral hernia poses a formidable challenge, despite recent advances in surgical techniques. Recurrence rates after complex ventral hernia repair remain high, and increase with each failed attempt. This study examines the effect of pre-operative abdominal wall chemical component relaxation using Botulinum Toxin A (BTA) to induce temporary flaccid paralysis in order to facilitate laparoscopic repair of large complex ventral hernia. METHODS: This is a prospective evaluation of 27 patients from January 2013 to August 2015 who underwent ultrasound guided BTA injections to the lateral abdominal wall muscles prior to elective complex ventral hernia repair. Non-contrast serial CT imaging was obtained pre- and post-BTA injection to measure change in fascial defect size and abdominal wall muscle thickness and length. Fascial defects were closed and hernias repaired using laparoscopic or laparoscopic-assisted intra-peritoneal onlay mesh (IPOM) techniques. RESULTS: 27 patients received pre-operative BTA injections which were well tolerated with no complications. Comparison of pre-BTA and post-BTA CT imaging demonstrated a significant increase in mean length of the lateral abdominal wall from 15.7 cm pre-BTA to 19.9 cm post-BTA (p < 0.0001), with mean unstretched length gain of 4.2 cm/side (range 0-11.7 cm/side). All hernias were surgically reduced and repaired with mesh, with no early recurrences. CONCLUSION: Pre-operative administration of BTA is a safe and effective technique in the pre-operative preparation of patients undergoing elective complex ventral hernia repair. This technique lengthens and relaxes the laterally retracted abdominal muscles and enables laparoscopic closure of large complex ventral hernia.

Endoglin inhibits prostate cancer motility via activation of the ALK2-Smad1 pathway.

Endoglin is a transforming growth factor beta (TGFbeta) superfamily auxiliary receptor. We had previously shown that it suppressed prostate cancer (PCa) cell motility, and that its expression was lost during PCa progression. The mechanism by which endoglin inhibits PCa cell motility is unknown. Here we demonstrate that endoglin abrogates TGFbeta-mediated cell motility, but does not alter cell surface binding of TGFbeta. By measuring Smad-specific phosphorylation and Smad-responsive promoter activity, endoglin was shown to constitutively activate Smad1, with little-to-no effect upon Smad3. Knockdown of Smad1 increased motility and abrogated endoglin's effects. As type I activin receptor-like kinases (ALKs) are necessary for Smad activation, we went on to show that knockdown of ALK2, but not TGFbetaRI (ALK5), abrogated endoglin-mediated decreases in cell motility and constitutively active ALK2 was sufficient to restore a low-motility phenotype in endoglin deficient cells. These findings provide the first evidence that endoglin decreases PCa cell motility through activation of the ALK2-Smad1 pathway.

Lepeophtheirus salmonis: characterization of prostaglandin E(2) in secretory products of the salmon louse by RP-HPLC and mass spectrometry.

Lepeophtheirus salmonis is an ectoparasitic copepod that causes serious disease outbreaks in both wild and farmed salmonids. As the relationship between L. salmonis and its hosts is not well understood, the current investigation was undertaken to investigate whether any immunomodulatory compounds could be identified from secretions of L. salmonis. By incubating live L. salmonis adults with the neurotransmitter dopamine in seawater, we were able to obtain secretions from the parasite. These were analyzed by RP-HPLC column, as well as LC-MS. L. salmonis secretions contained a compound with the same retention time and mass of PGE(2). The identity of this compound as PGE(2) was confirmed by MS-in source dissociation. The concentrations of PGE(2) in L. salmonis secretions ranged from 0.2 to 12.3 ng/individual and varied with incubation temperature and time kept off the host. Prostaglandin E(2) is a potent vasodilator and thought to aid in parasite evasion from host immune responses. This is the first reported evidence of prostaglandin production in parasitic copepod secretions and its implications for the host-parasite relationship are discussed.

Species-specific differences in expression of G-protein-coupled receptor kinase (GRK) 7 and GRK1 in mammalian cone photoreceptor cells: implications for cone cell phototransduction.

Desensitization plays an important role in the rapid termination of G-protein signaling pathways. This process, which involves phosphorylation by a G-protein-coupled receptor kinase (GRK) followed by arrestin binding, has been studied extensively in the rod photoreceptor cell of the mammalian retina. In contrast, less is known regarding desensitization in cone photoreceptor cells, which occurs more rapidly than in rod cells. Recently, our laboratory has cloned a novel GRK family member, GRK7, from the retina of a cone-dominant mammal, the 13-lined ground squirrel. Here we report the cloning of GRK7 from rod-dominant pig and human retinas, suggesting that this kinase plays a role in human visual signaling. Because GRK1 (rhodopsin kinase), the GRK that mediates rhodopsin desensitization in the rod cell, is reportedly expressed in both rods and cones, a detailed comparison of the localization of the two kinases is a necessary step toward determining their potential roles in cone visual signaling. Immunocytochemical analysis using antibodies selective for these two GRKs unexpectedly demonstrated species-specific differences in GRK7 and GRK1 expression in cones. In pigs and dogs, cones express only GRK7, whereas in mice and rats, we detected only GRK1 in cones. These results suggest that either GRK7 or GRK1 may participate in cone opsin desensitization, depending on the expression pattern of the kinases in different species. In contrast, GRK7 and GRK1 are coexpressed in monkey and human cones, suggesting that coordinate regulation of desensitization by both kinases may occur in primates.

Modulation of CRX transactivation activity by phosducin isoforms.

Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucleotide protein (G protein) betagamma subunits (Gbetagamma), while Phd-like orphan proteins (PhLOPs) lack the major functional domain for the binding of Gbetagamma. A retina- and pineal gland-specific transcription factor, cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen using PhLOP1 as the bait. Direct protein-protein interactions between Phd or PhLOP1 and CRX were demonstrated using a beta-galactosidase quantitative assay in the yeast two-hybrid system and were confirmed by an in vitro binding assay and a glutathione S-transferase (GST) pull-down assay. To determine if the interaction with Phd or PhLOP1 affected CRX transactivation, a 120-bp interphotoreceptor retinoid binding protein (IRBP) promoter-luciferase reporter construct containing a CRX consensus element (GATTAA) was cotransfected into either COS-7 or retinoblastoma Weri-Rb-1 cells with expression constructs for CRX and either Phd or PhLOP1. Phd and PhLOP1 inhibited the transcriptional activation activity of CRX by 50% during transient cotransfection in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably transfected with CRX. Phd inhibited CRX transactivation in a dose-dependent manner. Whereas Phd is a cytoplasmic phosphoprotein, coexpression of Phd with CRX results in Phd being localized both in the cytoplasm and nucleus. By contrast, PhLOP1 is found in the nucleus even without CRX coexpression. To address the physiological relevance of these potential protein interacting partners, we identified immunoreactive proteins for Phd and CRX in retinal cytosolic and nuclear fractions. Immunohistochemical analysis of bovine retinas reveals colocalization of Phd isoforms with CRX predominantly in the inner segment of cone cells, with additional costaining in the outer nuclear layer and the synaptic region. Our findings demonstrate that both Phd and PhLOP1 interact directly with CRX and that each diminishes the transactivation activity of CRX on the IRBP promoter. A domain that interacts with CRX is found in the carboxyl terminus of the Phd isoforms. Phd antibody-immunoreactive peptides are seen in light-adapted mouse retinal cytosolic and nuclear extracts. Neither Phd nor PhLOP1 affected CRX binding to its consensus DNA element in electrophoretic mobility shift assays. A model that illustrates separate functional roles for interactions between Phd and either SUG1 or CRX is proposed. The model suggests further a mechanism by which Phd isoforms could inhibit CRX transcriptional activation.

Identification of specific histidine residues and the carboxyl terminus are essential for serotonin N-acetyltransferase enzymatic activity.

Melatonin is synthesized in pinealocytes of the pineal gland and in photoreceptors of the retina. Synthesis rate from serotonin to melatonin is controlled by the rapid and dramatic enzymatic increase in darkness of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, EC 2.3.1.87) and hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The primary structure of these critical indoleamine enzymes is now known and the regulation of the enzyme catalysis can be examined. As a first step, the conserved cysteine (C) and histidine (H) residues were targeted for site-directed mutagenesis as potential amino acid residues involved in the N-acetylation reaction of AA-NAT. Our studies concluded that among 6 histidine (H) to alanine (A) mutations, three residues (H110A, H118A, H120A) within the AA-NAT protein showed little or no enzymatic activity, whereas the others (H28A, H70A, H125A) retained enzymatic activity, compared to the unaltered AA-NAT protein. Cysteine to alanine mutations, C37A and C177A, had no significant effect on the AA-NAT enzymatic activity; however, C61A had a four-fold increase in K(m) for acetyl CoA and an altered sensitivity to the thiol modification chemical, N-ethylmaleimide (NEM), implying that C61 may participate in the acetyl CoA binding. Further studies examined the AA-NAT enzyme regulation of the highly conserved carboxyl terminus. When 12 terminal amino acid residues were deleted systematically from the carboxyl terminus of the 205 amino acid residue AA-NAT protein, enzyme activity was retained. However, further residue deletion resulted in enzyme activity plummeting, implicating that the essential information either for the correct structural folding into an active enzyme form or for enzyme stability is in the 193 residues. To test the relative importance of the AA-NAT carboxyl terminal region, a single leucine (L) was altered to alanine (A) or proline (P). Both mutants, either L193A or L193P, had a marked decrease in AA-NAT enzymatic activity and a decrease in thermal stability, suggesting the leucine, in addition to the cysteine and histidine residues, is involved in either enzyme catalysis or stability. In light of the recently reported three-dimensional structure of AA-NAT (17,18), the site-directed mutagenesis data demonstrate experimentally the importance of essential amino acid residues for acetyl CoA binding and AA-NAT activation.

The carboxyl terminal domain of phosducin functions as a transcriptional activator.

In previous work, we identified a set of phosducin (Phd) isoforms with unknown function including the phosducin (Phd)-like orphan protein 1 (PhLOP1), an amino terminal truncated isoform of the retinal Phd lacking the Gbetagamma binding domain. To investigate the potential biological function of PhLOP1, PhLOP1 was fused at its amino terminus with the DNA binding domain (BD) of the yeast transcriptional factor, GAL4, and used as bait in a yeast two-hybrid screen. Two potential functional protein partners were identified during the screen: SUG1, a subunit of the 26S proteasome and a putative transcriptional mediator, and CRX, a retina- and pineal-specific transcription factor. Upon localizing the interacting domain of PhLOP1 with one of the new partners, SUG1, we found that a domain of 40 amino acids at the carboxyl terminus of Phd and PhLOP1 had intrinsic transcriptional activation activity in yeast. The transactivation activity was further confirmed in mammalian cells. This region contains an acidic domain that has been shown to be involved in the function of several transcriptional activators. In addition, we showed that Phd is cytoplasmic while PhLOP1 is localized predominantly to the nucleus when fused to an enhanced green fluorescent protein (EGFP) and transiently expressed in transfected cells, suggesting that PhLOP1 may play a distinct functional role in transcriptional regulation independent of the known Phd interaction/regulation of Gbetagamma transduction.

The validity of self-reported diabetes quality of care measures.

OBJECTIVE: First, to test the validity of self-reported quality of care and treatment measures compared with medical records and administrative data for: eye examinations, hemoglobin A1C tests, and use of insulin and oral agents for adult patients with diabetes; and secondly to assess the consistency between medical record information and administrative data for the same measures plus microalbumin testing. DESIGN: Cross-sectional study using data from telephone survey, primary care medical and eye records, and administrative claims. SETTING: Statewide health maintenance organization in Minnesota, USA, 1995. STUDY PARTICIPANTS: Four hundred and forty adults with diabetes, aged 31-64 years. MAIN OUTCOME MEASURES: Validity++ of self-reported diabetes quality of care measures compared with a criterion standard combining information from primary care and eye records with information from administrative data; and reliability of medical record information compared with administrative data. RESULTS: Although the sensitivity of self-reported eye examination was high (89%), the specificity was low (65%). Self-report of hemoglobin A1C also had high sensitivity (99%) and a lower specificity than that of eye examination (28%). The two information sources (medical records and claims) used in the criterion standard each contained complementary and non-overlapping information. Reliability was highest for microalbumin testing (kappa, 0.75) and lowest for eye examination (kappa, 0.37). CONCLUSIONS: Quality of care measures for diabetes are often drawn from a variety of sources. To the extent that data sources are biased, the measures can be misleading. Self-report is likely to lead to an overestimate of eye screening and the measurement of hemoglobin A1C. Reported rates of quality of care should be inspected carefully. The 'same' rate taken from different sources may vary.

Biochemical characterization of recombinant serotonin N-acetyltransferase.

Pineal and retinal melatonin synthesis is controlled by the enzymatic activity of arylalkylamine N-acetyltransferase (AA-NAT, EC 2.3.1.87), which is regulated by light/dark signals and circadian factors. This enzyme converts serotonin to N-acetylserotonin by the transfer of an acetyl group from acetyl coenzyme A. Endogenous AA-NAT instability during routine purification has made enzyme characterization difficult, but now a stable recombinant protein for AA-NAT has been synthesized to investigate the intrinsic biochemical properties of AA-NAT from a rat pineal cDNA encoding a 205 amino acid, 23 kilodalton protein, by using a glutathione-S-transferase (GST) fusion protein system. Recombinant GST-AA-NAT showed substrate specificity for arylalkylamines and stability at 4 degrees C; however, the enzyme activity was reduced by 40% upon preincubation at 37 degrees C for 2 hr. GST-AA-NAT is preferentially phosphorylated by either cyclic AMP- or cyclic GMP-dependent kinases in vitro, but no detrimental effect was observed on AA-NAT enzymatic activity. Among the metal cations tested in this study, Ca2+, Mg2+, Mn2+, Fe2+, and Co2 showed little or no inhibitory potency, while either 1 mM Zn2+ or 0.1 mM Cu2+ nearly abolished the enzymatic activity. GST-AA-NAT enzyme activity is also inhibited by reagents that are known biochemically to modify thiol groups (N-ethylmaleimide, NEM) and histidine residues (p-chloromercuribenzoate, NBS and diethyl pyrocarbonate, DEPC), suggesting the presence of essential cysteine and histidine moieties. Moreover, preincubation of acetyl CoA completely protects the recombinant AA-NAT from inactivation by NEM and DEPC, indicating that specific cysteine and histidine residues may be at the acetylation site. The conclusion is that the biochemical properties of rat recombinant AA-NAT is similar to the endogenous pineal and retinal AA-NAT with respect to the sensitivity to temperature, metal cations, as well as the thiol modification reagents. These data also suggest that the phosphorylation status of the AA-NAT does not affect enzymatic activity directly, and histidine residues are potentially important residues required for high catalytic activity.

A conceptual model of feminine hardiness.

Comprehensive literature reviews of hardiness consistently reveal empirical ambiguities and conceptual limitations. The article describes a grounded theory study focused on the conceptual nature of hardiness. In a group of 13 women with breast cancer, two major characteristics of hardiness emerged: a strong sense of purpose and the ability to endure. These characteristics are described in terms of their internal dimensions, manifestations, and outcomes. The findings are compared with the original conceptualization of hardiness, specifically its attributes of commitment, control, and challenge. The similarities and differences expand, extend, and modify the original definition of hardiness.

OTX2 homeodomain protein binds a DNA element necessary for interphotoreceptor retinoid binding protein gene expression.

Transcription of the human interphotoreceptor retinoid binding protein (IRBP) gene is strictly tissue specific, being restricted to retinal photoreceptors and pinealocytes. We have previously demonstrated that a sequence named A element, in the IRBP promoter is essential for IRBP gene transcription in vivo. Here we demonstrate that the human homeodomain protein OTX2 is present in nuclear extracts of IRBP expressing cells and specifically interacts with the IRBP A promoter element in vitro. OTX2, as well as CRX, a homeodomain protein very similar to OTX2, activates the human IRBP promoter in co-transfection experiments.

Bovine arylalkylamine N-acetyltransferase activity correlated with mRNA expression in pineal and retina.

Arylalkylamine N-acetyltransferase (AA-NAT, E. C. 2.3.1.87) is the enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to serotonin to form N-acetylserotonin (NAS) in the indoleamine biosynthetic pathway. Bovine pineal AA-NAT, partially purified on an anion exchange column, displayed an 8-fold higher enzymatic activity in pineals from animals killed in early morning (0800) compared to an afternoon group (1430). Poly A(+) mRNA was isolated from early morning bovine pineals, used to construct a mammalian expression cDNA library (lambdaZAP Express), and then screened with a rat AA-NAT cDNA to isolate a 924 basepair cDNA that encodes the bovine pineal AA-NAT. The amino acid sequence alignment reveals that bovine AA-NAT shares 94.20%, 78.54%, 76.33% and 56.3% identity to ovine, rat, human and chicken sequences, respectively. Northern blot analysis demonstrates a 0.7-fold higher mRNA level in pineal glands taken from animals from the 0800 time-point compared with mRNA from the 1430 time-point. AA-NAT mRNA was expressed at high levels in pineal and retina, but the message was undetectable in adrenal, cerebellum, cortex, small intestine, testis and thyroid. Based on the significant identity of amino acid sequence and the similar mRNA expression pattern, these data suggest that the bovine AA-NAT is more analogous to the ovine rather than either the rat, human or chicken AA-NAT.

Hydroxyindole-O-methyltransferase is another target for L-glutamate-evoked inhibition of melatonin synthesis in rat pinealocytes.

Rat pinealocytes use L-glutamate as a modulator for melatonin synthesis. Upon binding of L-glutamate to the class II metabotropic glutamate receptor, norepinephrine (NE)-dependent formation of cAMP was inhibited, resulting in decreased serotonin-N-acetyltransferase (NAT) activity and melatonin output. Although L-glutamate at 1 mM caused 90% inhibition of melatonin synthesis, about 30% of the NAT activity remained, suggesting the presence of another target for L-glutamate. In this study, we found that L-glutamate also inhibits hydroxyindole-O-methyltransferase (HIOMT). The inhibition is reversible and dose-dependent: the maximal inhibition was obtained with more than 0.4 mM L-glutamate. Contrary to L-glutamate-evoked inhibition of NAT, agonists for class II metabotropic receptors such as (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) had no effect on HIOMT. Neither (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), an specific antagonist for class II mGluRs, nor dibutyryl cAMP restored the L-glutamate-evoked inhibition of HIOMT. Northern blot analyses revealed that L-glutamate significantly inhibits the expression of mRNA of NAT, but not that of HIOMT. These results indicated that HIOMT is an another target for L-glutamate due to its inhibition of melatonin synthesis, and the signaling pathway toward the inhibition is distinct from that of NAT.

PhLPs and PhLOPs in the phosducin family of G beta gamma binding proteins.

In this study, we identify new isoforms of the retinal phosducin and investigate the expression of the phosducin family, showing that an isoform, PhLP1, has sequence homology with Phd and Gbeta gamma binding capability, whereas two isoforms (phosducin-like orphan proteins, PhLOPs) share sequence homology with Phd but fail to bind Gbeta gamma. Original identification of PhLP1 and the PhLOPs was from a human retina cDNA library, using a PCR product for library hybridization screening that contained a predicted functional epitope domain. The screen identified Phd and three related, but distinct, recombinants (PhLP1, PhLOP1, and PhLOP2). By RT-PCR, all isoforms are expressed in either retina or forskolin-stimulated Y79 retinoblastoma cells; however, the new isoforms are below the level of detection on Northern blot analysis. The predicted amino acid translation of each homologue revealed major differences, arising from either splice variants or gene duplication of Phd. To test the functional interaction of all phosducin isoforms with Gbeta gamma in vitro, a glutathione S-transferase (GST) fusion protein was developed for each member. Biochemical interaction with purified retinal transducin Gbeta gamma was verified for GST-Phd and demonstrated for GST-PhLP1; however, neither GST-PhLOP1 nor GST-PhLOP2 bound Gbeta gamma. Comparable results were observed when the GST-phosducin fusion proteins selectively sequestered Gbeta gammas from retinal extracts or when functional Gbeta gamma interactions were assessed using surface plasmon resonance technology. Phosducin and its isoforms are widely distributed in body tissues where they may participate in signal transduction pathways. Phd and PhLP1 possess an 11-amino acid conserved epitope domain (TGPKGVINDWR) that controls the high-affinity binding of Gbeta gamma; these isoforms are implicated in the G-protein signaling pathway. The phosducin-like orphan proteins (PhLOPs) fail to bind Gbeta gamma, suggesting that the PhLOP isoforms may participate in still unidentified signaling pathways.

Interaction of phosducin and phosducin isoforms with a 26S proteasomal subunit, SUG1.

PURPOSE: Retinal phosducin (Phd) and phosducin-like protein 1 (PhLP1) selectively bind G-protein beta/gamma subunits (Gbetagamma). Our laboratory has recently identified two phosducin-like orphan proteins (PhLOP1 and PhLOP2) that lack the ability to interact with Gbetagamma. In search of potential functional protein partner(s) for these phosducin orphans, we examined their protein-protein interactions using a yeast two-hybrid screen. METHODS: A bovine retina yeast expression cDNA library was screened with the GAL4 DNA binding domain (BD) fusion of PhLOP1. Quantitative analysis of the selected positives with PhLOP1 and other Phd isoforms was assessed by growth and beta-galactosidase activity. Further molecular, biochemical, and immunological detection methods utilizing glutathione S-transferase (GST)-Phd isoform fusion proteins and the potential partner were also performed. RESULTS: A member of the superfamily of putative ATPases was selected in the yeast two hybrid screen. Further characterization identified a direct interaction of this putative ATPase with PhLOP1, as well as Phd and PhLP1, but not with PhLOP2. A database search verified this ATPase as a bovine orthologue of the yeast SUG1 (ySUG1), a putative transcriptional mediator and a subunit of the 26S proteasome complex. Our experiments reveal that the carboxy-terminus of PhLOP1 is essential for the protein-protein interaction with SUG1, but it alone is not sufficient to mediate SUG1 interaction. CONCLUSIONS: Based on these experimental results, Phd, PhLP1 and PhLOP1 have protein-protein interaction with SUG1. PhLOP1, a truncated amino-terminal splice variant of Phd, is the best candidate for the interaction with SUG1 among the four Phd isoforms studied, which suggests a potential function for PhLOP1.

Pneumococcal vaccine administration associated with splenectomy: missed opportunities.

BACKGROUND: The spleen defends against infection from encapsulated organisms. Patients who have had splenectomies are at risk for the development of overwhelming pneumococcal infections. Guidelines recommend that pneumococcal vaccine be given to all patients who have splenectomies. METHODS: This retrospective study was performed to evaluate compliance with the guidelines in patients from a large multispecialty group practice who had splenectomies between 1988 and 1991. Ninety-five patients were identified, and their clinic and hospital records were reviewed. RESULTS: Overall, 73.7% of patients who had splenectomies received the pneumococcal vaccine. No significant differences were found in the vaccination rates over time or among the surgeons. CONCLUSIONS: Improvement is needed in ensuring that patients who have splenectomies receive pneumococcal vaccine.

The effects of thyroid hormone level and action in developing brain: are these targets for the actions of polychlorinated biphenyls and dioxins?

Alterations in thyroid hormone level or responsivity to thyroid hormone have significant neurologic sequelae throughout the life cycle. During fetal and early neonatal periods, disorders of thyroid hormone may lead to the development of motor and cognitive disorders. During childhood and adult life, thyroid hormone is required for neuronal maintenance as well as normal metabolic function. Those with an underlying disorder of thyroid hormone homeostasis or mitochondrial function may be at greater risk for developing cognitive, motor, or metabolic dysfunction upon exposure to substances which alter thyroid hormone economy. Polychlorinated biphenyls (PCBs) and dioxins have been argued to interfere with thyroid hormone action and thus may affect the developing and mature brain. Animal models provide useful tools for studying the effects of thyroid hormone disorders and the effects of environmental endocrine disruptors. The congenitally hypothyroid, hyt/hyt, mouse exhibits abnormalities in both the cognitive and motor systems. In this mouse and other animal models of thyroid hormone disorders, delayed somatic and reflexive development are noted, as are permanent deficits in hearing and locomotor and adaptive motor behavior. This animal's behavioral abnormalities are predicated on anatomic abnormalities in the nervous system. In turn, these abnormalities are correlated with differences in neuronal structural proteins. In normal mice, the expression of mRNAs coding for these proteins occurs temporally with the onset of autonomous thyroid hormone production. The hyt/hyt mouse has a mutation in the thyroid stimulating hormone receptor (TSHr) gene which renders it incapable of transducing the TSH signal in the thyrocyte to produce thyroid hormone. Some behavioral and possibly some biochemical abnormalities in mice exposed to PCBs are similar to those seen in the hyt/hyt mouse. In addition to direct effects on brain development and neuronal maintenance, thyroid hormone is necessary for maintaining metabolic functioning through its influence on mitochondria. Because the brain is particularly sensitive to inadequate energy generation, disorders of thyroid hormone economy also indirectly impair brain functioning. Alterations in thyroid hormone level result in differing expression of mitochondrial genes. Mutations in these mitochondrial genes lead to well-recognized syndromes of encephalomyopathy, myopathy, and multisystem disorder. Hence, PCBs and dioxins, by possibly altering the thyroid hormone milieu, may alter the functioning of mitochondria in the generation of adenosine triphosphate (ATP). The use of animal models of thyroid hormone deficiency for behavioral, anatomic, histologic, and molecular comparison will help elucidate the mechanisms of action of these putative endocrine-disrupting compounds. The study of thyroid hormone disorders provides a template for relating thyroid hormone mediated effects on the brain to these compounds.

Validation of claims diagnoses and self-reported conditions compared with medical records for selected chronic diseases.

This article assesses the validity of ambulatory administrative and encounter data and patient self-reported information compared with information contained in the ambulatory medical record for 17 chronic diseases. Using a sample of 213 adults (18 to 64 years old) and seniors (65 years and older) enrolled in a health maintenance organization and receiving care at multispecialty group practice in Minneapolis, Minnesota, sensitivity and specificity for claims and self-report were calculated for each chronic condition using the medical record as the criterion standard. The analysis was performed first by blinded review and then repeated with an unblinded review.