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Facial nerve injury and facial nerve stimulation are rare but important risks of cochlear implant surgery. Careful preoperative and intraoperative techniques can help minimize the possibility of facial nerve involvement in cochlear implantation. Meanwhile, judicious postoperative treatment can help minimize the impact of these disorders if either facial nerve injury or stimulation does occur. [Copyright &y& Elsevier]

Endothelial progenitor cells (EPCs) play a role in the repair of ischemic or injured tissue. Because endothelial injury can be associated with apoptosis, we have investigated whether apoptotic bodies from mature endothelial cells (ECs) may affect growth and differentiation of EPCs in vitro. A 24-hour incubation of isolated human EPCs with apoptotic bodies-rich medium (ABRM) from ECs led to a significant increase in the number of spindle-shaped attached cells. EPCs were characterized by DiI-Ac-LDL/lectin staining and measurement of CD34 and kinase insert domain receptor (KDR) expression. The treatment with ABRM resulted in a 2-fold increase of DiI-Ac-LDL/lectin-positive cells and up-regulation of CD34 (22% ± 2% versus 13% ± 3%, P< .05 and KDR (49% ± 12% versus 19% ± 7%, P< .05). Fluorescence and confocal laser microscopy demonstrated the uptake of apoptotic bodies by the EPCs. Apoptotic bodies-depleted medium had no effect, whereas the incubation with suspension of apoptotic bodies induced effects similar to those of ABRM. Our results suggest that apoptotic bodies from ECs are taken up by EPCs, increasing their number and differentiation state. Such a mechanism may facilitate the repair of injured endothelium and may represent a new signaling pathway between progenitor and damaged somatic cells. (Blood. 2004;104:2761-2766)

ABSTRACTThe subunits of DNA gyrase and topoisomerase IV from Staphylococcus haemolyticuswere expressed in Escherichia coli, purified to homogeneity, and used to reconstitute active enzymes that were sensitive to known topoisomerase inhibitors. This represents the first description of a method for isolating type II topoisomerases of a coagulase-negative staphylococcal species.

The origin binding protein (OBP) encoded by the UL9 open reading frame of herpes simplex virus type 1 (HSV-1) plays an essential role in productive infection by promoting the initiation of viral DNA synthesis. In this study, OBP was inducibly expressed in Escherichiacoli and purified to homogeneity using a two-step chromatographic separation procedure. The properties of this recombinant OBP (rOBP) were found to be indistinguishable from those of the virus-encoded protein. Since rOBP was synthesized in bacterial cells, it lacked the posttranslational processing which normally occurs in OBP produced in HSV-1-infected mammalian cells and could therefore be exploited in experiments which addressed the effects of protein modification on OBP function. As an initial study, the impact of phosphorylation on enzymatic activity was examined using rOBP which had been treated with a panel of purified cellular kinases. rOBP was found to act as a substrate for nearly all of the kinases tested in 32P-labeled phosphate transfer assays. However, only phosphorylation by protein kinase A (PKA, or cAMP-dependent protein kinase) was shown to significantly alter the enzymatic properties of rOBP, as it increased by five- to eightfold the ATPase activity associated with this protein. Activation of this critical viral DNA replication enzyme by a cAMP-dependent kinase such as PKA may be of some relevance in the natural course of HSV-1 infections, since reactivation of latent virus is thought to involve both signal transduction events and the induction of viral DNA synthesis. Thus, the expression and purification strategy outlined in this work provides an economical source of unmodified HSV-1 OBP that should prove useful in future in vitrostudies.

Herpes simplex virus type 1 (HSV-1) encodes a deoxyribonuclease that is frequently referred to as alkaline nuclease (AN) because of its elevated pH optimum. Studies with recombinant viruses which contain deletions in the HSV-1 gene encoding AN have indicated that this enzyme is required for efficient virus replication and therefore represents a potential target for novel antiviral therapies. A simple colorimetric assay for deoxyribonuclease activity employing a DNA–methyl green substrate was adapted for use in a high-throughput screen to identify small molecule inhibitors of this enzyme. This screen identified 1,2-benzoisothiazolin-3-one as a specific inhibitor of AN, since it exhibited activity against AN but was completely inactive against bovine pancreatic DNaseI. Subsequent studies revealed that this compound most likely inhibited AN by forming disulfide linkages with one or more exposed cysteine residues on the surface of the enzyme and that AN was sensitive to sulfhydryl-group-modifying reagents in general. These results demonstrated the utility of this DNA–methyl green substrate-based assay in both the rapid identification and the characterization of novel small molecule inhibitors of the AN encoded by HSV-1 and other herpesviruses. Copyright 2001 Academic Press.

Membrane-associated proteins with specific binding properties to modified LDL were investigated in J774 macrophages and Mono Mac 6 sr cells. Ligand blotting of membrane proteins revealed a 54-kDa protein which bound oxidized and acetylated but not native LDL. The 54-kDa protein, isolated by 2D-PAGE, was identified as vimentin. 125I-AcLDL bound to purified vimentin and desmin in a saturable manner, with an approximate Kd of 1.7 × 10−7 M (89 μg/ml) and 8.0 × 10−8 M (41 μg/ml), respectively. Blots of vimentin mutant proteins with deletions in the positively charged N-terminal head domain showed that amino acids 26–39 are essential for the binding of AcLDL by vimentin. Taken together, our data indicate that vimentin binds modified LDL, but not native LDL, in a specific and saturable manner. Vimentin filaments extend throughout the cytoplasm as far as the inner surfaces of plasma and vesicular membranes. Vimentin may thus play a role in membrane-associated steps involved in the intracellular processing of oxidized LDL, contributing to its unregulated uptake and intracellular retention by cells of the atherogenic plaque.

A reliable imaging technique is needed for follow-up of patients with temporal and facial osteomyelitis. Clinical outcome in 20 patients with suspected osteomyelitis of the temporal/mastoid, calvarium, and mandible facial bones was evaluated with 30 combined In-WBC/Tc-99m MDP bone single photon emission computed tomographic (SPECT) scans and 27 computed tomographic scans. Simultaneous dual-tracer 25-minute SPECT scans were acquired 18 to 20 hours after radiotracer injection by use of a three-detector system. Diagnosis of the 20 patients (age range, 3 to 74 years) included 8 with facial osteomyelitis, 6 with malignant otitis externa, 3 with mandibular osteomyelitis, and 3 with calvarial osteomyelitis. Diagnosis was confirmed by biopsy/culture results in 18 patients and by endoscopic and clinical evaluation in 2 patients with initial negative scans. Of the 30 In-WBC/MDP scans, 15 were true-positive, 13 true-negative, 1 false-negative, and 1 equivocal. Of a total of 27 CT scans, 9 were true-positive, 5 false-negative, and 1 equivocal in patients with biopsy-proven osteomyelitis. Three computed tomographic scans were false-positive and 1 was equivocal in patients without osteomyelitis, because of concurrent postoperative bone abnormalities. Additionally, 8 computed tomographic scans were true-negative. These results suggest that dual In-WBC/Tc-99m MDP bone SPECT scintigraphy provides an accurate imaging modality for diagnosis and follow-up of temporal and facial osteomyelitis when existing clinical or postoperative bone changes make it difficult to detect active osteomyelitis by computed tomographic scan.

Idiopathic sudden sensorineural hearing loss is an enigmatic condition of unknown cause. Although the treatment for sudden sensorineural hearing loss is controversial, the evaluation for a cause should not be. All patients are evaluated with a complete history, physical examination, audiologic examination, and blood draw to evaluate complete blood count, general chemistry screen, thyroid function test results, erythrocyte sedimentation rate, and fluorescent treponemal antibody absorbance. Magnetic resonance imaging with gadolinium contrast is essential in the evaluation of idiopathic sudden sensorineural hearing loss, even if there is a complete response to either treatment or no treatment. During the last year we treated 16 patients for idiopathic sudden sensorineural hearing loss with our protocol of intravenous dextran/Hypaque or oral high-dose steroids. Fifteen patients were evaluated immediately before treatment with a magnetic resonance imaging scan. An additional patient had been treated successfully with high-dose steroids at an outside institution and came in for an evaluation. Of these 16 patients, 3 (18.75%) were found to have significant pathologic conditions on magnetic resonance imaging scan. The patient who had been treated successfully on the outside was noted to have a 5-mm intracanalicular acoustic neuroma, the second patient was found to have a multiple sclerosis lesion at the level of the superior olive, and the third patient, who had had a normal magnetic resonance imaging scan 18 months previously, was now found to have a large 4- to 5-cm meningioma in the cerebellopontine angle. We believe it is essential that all patients with idiopathic sudden sensorineural hearing loss be evaluated at some point during their treatment with a magnetic resonance imaging scan with gadolinium contrast. (Otolaryngol Head Neck Surg 1997;116:153-6.)

The low incidence of myocardial infarction in Greenland Eskimos has been related to their traditional marine diet rich in eicosapentaenoic acid. However, whether dietary eicosapentaenoic acid is indeed transformed in man to antiaggregatory PGI3and weakly proaggregatory TXA3has not been clarified. In our studies we ingested either cod liver oil or mackerel both rich in eicosapentaenoic acid. Formation of TXB3, the hydrolysis product of TXA3, in platelet-rich plasma stimulated ex vivowith collagen was traced by capillary GC/EIMS. Via external standard, TXB3formation in platelets was estimated to be 5–15% of TXB2formation. From urine we extracted dinor metabolites of PGI according to a selective method. We utilized ?17-2,3-dinor-6-keto-PGF1a(PGI3-M) as an index of total body production of PGI3in analogy to 2,3-dinor-6-keto-PGF1a(PGI2-M), the major urinary metabolite of PGI2. We separated PGI2-M and PGI3-M as the Me, MO, Me3Si derivatives by capillary gas chromatography and identified PGI3-M by EI mass spectrometry. Excretion of PGI3-M, which was not detectable under control conditions, was 83±25 ng/24 h (SD) after ingestion of cod liver oil and 134±38 ng/24 h, respectively. Our findings with diets rich in EPA show that it is possible in man to change in vivothe spectrum of biologically active prostanoids by nutritional means and alter it in a favourable direction.

We have studied the effects of two stable prostaglandin endoperoxide analogues on platelet lipid metabolism in relation to specific platelet functional changes. During platelet shape change, the endoperoxide analogues induce the formation of 1,2-diacylglycerol and phosphatidic acid, indicating the activation of a phosphoinositide-specific phospholipase C. In parallel, they stimulate the phosphorylation of a 40-kd and a 20-kd protein. During platelet shape change, arachidonic acid is released, but not metabolized by platelet cyclo-oxygenase or lipoxygenase. Phospholipase C activation and platelet shape change are independent of extracellular Ca++and Mg++arachidonate metabolism, and release of adenosine diphosphate (ADP). Activation of phospholipase C during platelet aggregation seems, however, to be mediated partly by release of ADP. We conclude that endoperoxide analogues initially stimulate in platelets the formation of products derived from phospholipase C activation, which might serve as intracellular messengers for phosphorylation of specific proteins related to platelet shape change.

OBJECTIVE: The findings of an initial evaluation of 202 patients treated for squamous cell carcinoma of the supraglottic larynx have been previously reported. Recurrent disease was observed at the primary site in four patients (2%), in the regional lymphatics in 39 patients (20%), and at distant sites in 24 patients (12%). Critical analysis of patients who demonstrated recurrent disease in the cervical lymphatics indicated that 38 of 39 recurrences developed in nonsurgically treated necks irrespective of the use of postoperative radiation therapy. Accordingly, all patients with supraglottic carcinoma have been treated with routine bilateral neck dissection since that time. We have reviewed our data to determine if bilateral neck dissections deter recurrence and improve survival. DESIGN: A retrospective review of the clinical course of 76 patients undergoing excision of supraglottic squamous carcinoma combined with bilateral neck dissection between 1980 and 1990 was performed to determine survival and recurrence rates. Surgery alone was used to treat 32 patients, while 44 patients were treated with surgery plus adjunctive therapy. SETTING: Department of Otolaryngology—Head and Neck Surgery, The Eye and Ear Institute, University of Pittsburgh (Pa) School of Medicine. RESULTS: Distant metastatic spread was the most frequent site of failure, occurring in nine (11.8%) of 76 patients. Local recurrence was experienced by two patients (1%). The incidence of cervical recurrence (seven patients, 9.2%) following treatment for squamous cell carcinoma of the supraglottic larynx has been reduced from 20% to 9% through the use of bilateral neck dissection. The 2-year survival rate increased from 72% to 76%. CONCLUSION: Routine bilateral neck dissection is beneficial in the surgical management of squamous cell carcinoma of the supraglottic larynx.(Arch Otolaryngol Head Neck Surg. 1994;120:703-706)

1. Plasma aldosterone, 18-hydroxycorticosterone (18-OH-B), 18-hydroxydeoxycorticosterone (18-OH-DOC), corticosterone, cortisol and prolactin levels were determined during an angiotensin II infusion at increasing rates both with and without a simultaneous infusion of dopamine in seven normotensive subjects, in ten patients with essential hypertension, and in ten patients with primary aldosteronism. In a second set of experiments, maximum increases of these plasma levels were determined after metoclopramide (10 mg intravenously) in all subgroups. 2. As compared with the other groups, an exaggerated angiotensin II-induced response of plasma aldosterone and 18-OH-B levels was observed in the five patients with low-renin essential hypertension (LREH) and in five patients with idiopathic hyperaldosteronism (IHA). 3. Dopamine reduced the maximal increase of aldosterone and of 18-OH-B after angiotensin II to 259 ± 48 (sem) pg/ml and 511 ± 152 pg/ml repectively in LREH (without dopamine: 515 ± 74 and 908 ± 201 respectively; P<0.05), and to 466 ± 197 and 741 ± 212 in IHA (without dopamine: 766 ± 193 and 1264 ± 337 respectively; P<0.05). 4. The maximal increases of plasma aldosterone, 18-OH-B, and prolactin after metoclopramide (10 mg intravenously) were higher (P<0.01) in patients with LREH and in patients with primary aldosteronism. Plasma levels of 18-OH-DOC, corticosterone and cortisol were not affected by the stimuli applied. 5. The exaggerated response to metoclopramide as well as to angiotensin II and its reversion only by pharmacological doses of dopamine are consistent with an increased but ineffective dopamine inhibition of aldosterone and 18-OH-B in LREH and IHA.

• Previously reported data from our institution has led us to perform bilateral neck dissections for therapeutic as well as staging advantages for horizontal supraglottic laryngectomies. Concern over the possibility of increased morbidity associated with simultaneous bilateral neck dissection prompted this retrospective review of patients with supraglottic laryngectomy who were treated with either unilateral (46 patients) or bilateral (23 patients) neck dissection. No significant differences were found in morbidity when patients were evaluated for transfusion rate, cervical wound drainage, need for tracheotomy, oral diet, or duration of hospitalization. Significant differences were noted in surgical operating time, eg, it took 100 minutes longer to perform bilateral dissections, and slight increases were noted in estimated blood loss and fluids given intravenously. No significant differences were noted in the percentage or type of postoperative complications. It seems that bilateral neck dissection in conjunction with supraglottic laryngectomy does not increase postoperative surgical morbidity and may actually avoid complications associated with postoperative radiation therapy in patients with supraglottic laryngectomy.(Arch Otolaryngol Head Neck Surg. 1993;119:61-64)

ABSTRACTStereotactic inoculation of a herpes simplex virus (HSV) gene transfer vector into the hippocampus and caudate of rat brain resulted in limited and transient viral replication and the establishment of latency. Virus attenuation was achieved by insertional inactivation of a viral gene, Us3. Insertion of a lacZreporter gene, under the control of the HSV glycoprotein C (gC) late gene promoter, allowed viral replication to be monitored in vivo. Unlike unattenuated virus, the Us3pgC-lacZrecombinant caused little apparent damage to normal hippocampal morphology. Transient lacZexpression was detected in a considerable population of neurons of the dentate gyrus following hippocampal injection, whereas few positively staining neurons were present within the caudate after injection at that site. Latency-associated transcripts, the hallmark of latent infection, were detected in the brain 10 months after injection. This recombinant virus may be useful as a gene transfer vector for long-term expression of foreign genes in the central nervous system.

The bacterial transposable element Tn5 was observed to undergo high-frequency sequence inversion when integrated into the herpes simplex virus type 1 (HSV-1) genome. Deletion analysis of the IS50 elements through which this recombination event occurred demonstrated the absence of cis-acting signals involved in the inversion process. Several observations suggested an intimate association of the recombination mechanism with HSV-1 DNA replication, including the ability of the seven viral genes that are essential for HSV-1 DNA synthesis to mediate Tn5 inversion in the absence of any other viral functions. Comparable results were obtained by using duplicate copies of the L-S junction of the HSV-1 genome. Thus inversion of the L and S components of the HSV-1 genome during productive infection does not appear to be a site-specific process, but rather is the result of generalized recombination mediated by the complex of gene products that replicate the viral DNA.

Summary: In 24 conscious newborn piglets the effects of 20 μg/kg body weight IV 1-deamino-8-d-arginine-vasopressin (DDAVP) in group 1,5 mg/kg PO indomethacin in group 2, and the combined effects of both drugs in group 3 were studied by measuring urinary flow rate, urinary osmolatity, creatinine clearance, total urinary and nephrogenous cyclic-adenosine 3':5'-monophosphate (cAMP) excretion, medullary cAMP content, and renal prostaglandin (PG)E2and PGF20, excretion. DDAVP alone had no significant effects on the above parameters, whereas indomethacin alone reduced only the PG excretion significantly. When both drugs were administered simultaneously, urinary concentration increased significantly (urinary flow rate decreased from 2.4 × 0.4 to 1.4 × 0.3 ml/hr (means × S.E.), and urinary osmolality increased from 444 × 29 to 552 × 33 mOsm/liter). Total urinary and nephrogenous cAMP excretion increased from 590 × 48 to 854 × 78 and 302 × 36 to 590 × 81 pmoles/hr/g kidney weight, respectively, whereas PGE2and PGF20decreased from 249 × 33 to 19 × 4 and 192 × 32 to 43 × 7 pg/hr/g kidney weight, respectively. In addition, medullary cAMP content was considerably higher in group 3 (2010 × 200 pmoles/g medulla) than that observed in the control (1187 × 137), DDAVP (1218 × 115), and indomethacin (1230 × 168) groups.Speculation: During the neonatal period, renal prostaglandins inhibit the antidiuretic hormone-dependent cyclic adenosine 3′:s′-monophosphate system.

Ten IU of ACTH (1–24) per day was infused for 34 h (starting at 7 a.m.) into 8 normal men on a constant diet containing 135 mmNa+per day. All subjects retained between 152 and 181 mmof sodium. Potassium balance was negative. Plasma renin activity (PRA) and plasmaangiotensin II (P-A II) started to rise in most subjects after 6 to 8 h of infusion, reached a maximum after 24 h and then tended to decline. As shown previously, the rise in PRA is not due to a rise in plasma renin substrate concentration. Systolic, but not diastolic blood pressure increased significantly on the second day of ACTH-infusion. Plasma cortisol (P-F) was continuously stimulated by ACTH. Plasma aldosterone (P-aldo) increased rapidly 1 h after ACTH administration, then tended to fall, and increased again in most subjects, roughly in parallel with PRA. No significant changes in electrolyte balance, PRA, P-A II, P-F and P-aldo occurred in 3 subjects receiving 'sham'-infusions. Additional experiments in subjects treated with propranolol or indomethacin allowed the conclusion that the effect of ACTH on PRA and P-A II is not mediated by renal beta-adrenergic receptors, but perhaps (partially?) by prostaglandins. Since the infusion rate of ACTH was not much higher than the secretion rate of ACTH in the early morning hours, it is possible that ACTH is physiologically involved in the regulation of renin secretion.

Greenland Eskimos who live on a traditional marine diet rich in long chain ω3-polyunsaturated fatty acids have a low incidence of cardiovascular disease and myocardial infarction1–3. In their plasma and platelet lipids, arachidonic acid, the precursor of dienoic prostanoids, is partly replaced by eicosapentaenoic acid (C20:5, ω3; EPA), the precursor of trienoic prostanoids4,5. Studies with an Eskimo diet6or a Western diet supplemented with sea fish or fish oil rich in EPA7–14resulted in an ‘Eskimo-like’ pattern of plasma and platelet lipids. Moreover, less reactive platelets, a reduced ex vivo formation of proaggregatory thromboxane A2and a blunted circulatory response to pressor hormones were reported. These favourable functional effects may be induced by a shift of prostanoid formation from the dienoic to the trienoic series14,15. We show here that the major urinary metabolite of endogenous prostaglandin I3is present in subjects that have ingested either cod liver oil (∼4 g EPA per day) or mackerel (∼10–15 g EPA per day). Our studies provide the first direct evidence for in vivo formation of prostaglandin I3in man.

ICP0R is the polypeptide product of an alternatively spliced transcript of the gene encoding the transactivator protein ICP0 of herpes simplex virus type 1 (HSV-1). Although it has been shown to act as a transrepressor of gene expression in transfection assays, overexpression of the ICP0R protein in the recombinant virus HSV–KST was previously found to have no detectable effect on virus replication, so that the role it plays in HSV-1 infection remains unclear. Analysis of HSV–KST-infected cell lysates by Western blotting revealed the presence of not only the 41-kDa ICP0R polypeptide but also a 64-kDa processed form of the protein. This processing event was the result of ubiquination of the ICP0R protein, as demonstrated by the reactivity of the 64-kDa species with antibody specific for the influenza virus hemagglutinin (HA) protein epitope in experiments where the gene encoding ICP0R was coexpressed with a gene encoding HA-tagged ubiquitin. Surprisingly, the 64-kDa form of ICP0R was found to be remarkably stable and persisted in infected cells for many hours after processing, despite the fact that ubiquitination normally functions as a means of tagging proteins for rapid degradation. Analyses of mutant polypeptides containing arginine substitutions at each of the lysine residues of ICP0R, which represent potential ubiquitin conjugation sites, revealed that a single lysine residue at codon 248 was both necessary and sufficient for the appearance of the 64-kDa processed form. However, a number of ICP0R mutants that retained the ubiquitination site at lysine 248 but contained disruptions of sequences at distant sites also lacked detectable 64-kDa protein, indicating that the integrity of the overall structure of ICP0R was an additional determinant for ubiquitination.

In 24 conscious newborn piglets the effects of 20 μg/kg body weight IV 1-deamino-8-d-arginine-vasopressin (DDAVP) in group 1,5 mg/kg PO indomethacin in group 2, and the combined effects of both drugs in group 3 were studied by measuring urinary flow rate, urinary osmolatity, creatinine clearance, total urinary and nephrogenous cyclic-adenosine 3':5'-monophosphate (cAMP) excretion, medullary cAMP content, and renal prostaglandin (PG)E2and PGF20, excretion. DDAVP alone had no significant effects on the above parameters, whereas indomethacin alone reduced only the PG excretion significantly. When both drugs were administered simultaneously, urinary concentration increased significantly (urinary flow rate decreased from 2.4 × 0.4 to 1.4 × 0.3 ml/hr (means × S.E.), and urinary osmolality increased from 444 × 29 to 552 × 33 mOsm/liter). Total urinary and nephrogenous cAMP excretion increased from 590 × 48 to 854 × 78 and 302 × 36 to 590 × 81 pmoles/hr/g kidney weight, respectively, whereas PGE2and PGF20decreased from 249 × 33 to 19 × 4 and 192 × 32 to 43 × 7 pg/hr/g kidney weight, respectively. In addition, medullary cAMP content was considerably higher in group 3 (2010 × 200 pmoles/g medulla) than that observed in the control (1187 × 137), DDAVP (1218 × 115), and indomethacin (1230 × 168) groups.

Thromboxane released from activated platelets and prostacyclin of the vessel wall may act as potent antagonistic modulators of platelet aggregability and coronary vascular tone. Therefore, urinary excretion of their major metabolites, 2,3-dinor-thromboxane B2and 2,3-dinor-6-ketoprostaglandin F1α, was studied in 16 patients presenting with prolonged angina at rest. The 10 patients whose condition did not improve under vigorous antianginal treatment within 48 hours exhibited higher thromboxane metabolite excretion than did the 6 patients who responded to therapy (2,208 ± 1,542 versus 609 ± 312 ng/g creatinine; p < 0.01). Elevated values were also found in four of eight patients with sustained postinfarction angina. Enhanced thromboxane metabolite excretion was frequently associated with angiographic evidence of thrombus formation.