Heterologous expression of the invertebrate FMRFamide-gated sodium channel as a mechanism to selectively activate mammalian neurons.

Considerable effort has been directed toward the development of methods to selectively activate specific subtypes of neurons. Focus has been placed on the heterologous expression of proteins that are capable of exciting neurons in which they are expressed. Here we describe the heterologous expression of the invertebrate FMRFamide (H-phenylalanine-methionine-arginine-phenylalanine-NH2) -gated sodium channel from Helix aspersa (HaFaNaC) in hippocampal slice cultures. HaFaNaC was co-expressed with a fluorescent protein (green fluorescent protein (GFP), red fluorescent protein from Discosoma sp (dsRed) or mutated form of red fluorescent protein from Discosoma sp (tdTomato)) in CA3 pyramidal neurons of rat hippocampal slice cultures using single cell electroporation. Pressure application of the agonist FMRFamide to HaFaNaC-expressing neuronal somata produced large prolonged depolarizations and bursts of action potentials (APs). FMRFamide responses were inhibited by amiloride (100 microM). In contrast, pressure application of FMRFamide to the axons of neurons expressing HaFaNaC produced no response. Fusion of GFP to the N-terminus of HaFaNaC showed that GFP-HaFaNaC was absent from axons. Bath application of FMRFamide produced persistent AP firing in HaFaNaC-expressing neurons. This FMRFamide-induced increase in the frequency of APs was dose-dependent. The concentrations of FMRFamide required to activate HaFaNaC-expressing neurons were below that required to activate the homologous acid sensing ion channel normally found in mammalian neurons. Furthermore, the mammalian neuropeptides neuropeptide FF and RFamide-related peptide-1, which have amidated RF C-termini, did not affect HaFaNaC-expressing neurons. Antagonists of NPFF receptors (BIBP3226) also had no effect on HaFaNaC. Therefore, we suggest that heterologous-expression of HaFaNaC in mammalian neurons could be a useful method to selectively and persistently excite specific subtypes of neurons in intact nervous tissue.

Refined diagnostic criteria for implants associated with ovarian atypical proliferative serous tumors (borderline) and micropapillary serous carcinomas.

Characterization of invasive peritoneal implants from patients with noninvasive serous ovarian tumors has important prognostic and treatment implications, but the criteria for distinguishing invasive and noninvasive implants vary among investigators and can be difficult to apply. The authors studied 148 implants from 60 patients, 33 with primary atypical proliferative serous tumor, and 27 with primary noninvasive micropapillary serous carcinoma, with a mean follow-up of 62 months (median follow-up, 52 months). Previously reported and newly proposed histologic features for implant classification were evaluated and correlated with clinical outcome. Three criteria were applied for the diagnosis of "invasive" implants: invasion of underlying normal tissue, micropapillary architecture, and solid epithelial nests surrounded by clefts. Implants displaying any one of these three features were classified as "invasive," whereas those lacking all three features were classified as "noninvasive." Sixty-six implants were invasive and 82 were noninvasive. Of the 31 patients with invasive implants, six were dead of disease (DOD), 13 were alive with progressive disease (AWPD), and 12 were alive with no evidence of disease (NED). Of the 29 patients with noninvasive implants, two were DOD, one was dead of uncertain causes, one was AWPD, and 25 were alive with NED. Eighty-nine percent of invasive implants had a micropapillary architecture and 83% had solid epithelial nests surrounded by clefts. A minority of invasive implants (14% of those with underlying normal tissue) demonstrated invasion of normal underlying tissue. Nuclear atypia, mitoses, calcification, necrosis, and identification of individual cells "infiltrating" the stroma did not correlate with implant type. The proposed criteria permitted recognition of implants that correlated strongly with adverse outcome. Sixty-one percent of patients with implants displaying any one of the three features used to diagnose invasive implants were AWPD or DOD compared with 10% of patients whose implants lacked these features (p = 0.00001). Because implants associated with an adverse outcome can be identified before they invade underlying normal tissue, the term invasive implant to describe them is inaccurate and misleading. These implants resemble patterns of growth in micropapillary serous carcinoma of the ovary and the recurrent tumor that is obvious carcinoma. Accordingly, we propose that these extraovarian lesions be designated "well-differentiated serous carcinoma."

Diagnosis of aneuploidy in archival, paraffin-embedded pregnancy-loss tissues by comparative genomic hybridization.

OBJECTIVE: To evaluate the detection of aneuploidy in archival tissues from miscarriages by a method that uses microdissection and DNA extraction of villus cells from paraffin blocks, followed by universal DNA amplification and comparative genomic hybridization (CGH). DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENT(S): Nine archival tissues from cases of spontaneous abortion with trisomy 16 (two cases), trisomy 21 (three cases), trisomy 22 (two cases), triploidy (one case), and monosomy X (one case). INTERVENTION(S): Villus DNA was extracted from microdissected, formalin-fixed, paraffin-embedded tissues. Aneuploidy was detected by CGH after universal amplification of the DNA with the use of degenerate oligonucleotide-primed polymerase chain reaction. MAIN OUTCOME MEASURE(S): Detection of aneuploidy in archival pregnancy-loss tissues using CGH. RESULT(S): In all nine cases, DNA was successfully extracted from the microdissected tissues and was of sufficient quantity and quality for evaluation by CGH. In six of nine cases, the chromosomal abnormality detected by conventional cytogenetic analysis was identified by CGH: trisomy 16 (2/2), trisomy 21 (3/3), and trisomy 22 (1/2). One case of each of the following was not detectable: triploidy (1/1), monosomy X (1/1), and trisomy 22 (1/2). CONCLUSION(S): We propose CGH as a method for determination of aneuploidy in pregnancy-loss archival tissues when conventional cytogenetic analysis is unsuccessful or when it was not performed when fresh tissue was available.

A clinicopathologic analysis of atypical proliferative (borderline) tumors and well-differentiated endometrioid adenocarcinomas of the ovary.

Atypical proliferative (borderline) endometrioid tumors (APTs) and well-differentiated endometrioid carcinomas of the ovary constitute a spectrum of morphologically diverse proliferative tumors. There is currently no agreement on the criteria for distinguishing them. We report the clinicopathologic features of 56 proliferative endometrioid tumors focusing on the criteria for invasion, the clinical significance of microinvasion and cytologic atypia, and prognosis. Endometriomas, adenofibromas, adenosarcomas and moderately to poorly differentiated carcinomas were excluded, as were patients with concurrent endometrioid carcinoma of the endometrium. The tumors were classified as atypical proliferative tumor (APT) (33 tumors), APT with intraepithelial carcinoma (high-grade cytology in a tumor lacking stromal invasion) (three tumors), APT with microinvasion (invasion <5 mm) (five tumors), and invasive carcinoma (invasion > or = 5 mm) ( 15 tumors). All tumors were confined to the ovary (stage I). In 50 patients, the tumor involved one ovary, and in three patients, the tumors were bilateral. The predominant growth pattern was adenofibromatous in 29 tumors and glandular or papillary in 27 tumors. In 8 (24%) of 41 APTs, areas of benign adenofibroma were identified, and in 13 (87%) of 15 carcinomas, areas of associated APT were identified. Stromal invasion was manifested by confluent glandular growth in all 15 invasive carcinomas and all tumors with microinvasion. Destructive infiltrative growth was also present in 2 (13%) of 15 carcinomas. Confluent glandular growth was the most common manifestation of stromal invasion and therefore served as the best criterion for the diagnosis of carcinoma. Squamous differentiation was observed in 24 tumors, and mucinous differentiation was seen in 20 tumors and was most often seen in APTs. Endometriosis was present in 14 patients with APTs and one patient with carcinoma. Four patients had hyperplasia or atypical hyperplasia of the endometrium. One patient with an APT had a concurrent peritoneal serous neoplasm. Twenty-one patients had available clinical follow-up. Twenty (95%) of 21 patients, including six with invasive carcinoma, two with microinvasion, one with intraepithelial carcinoma, and 11 with APT were alive with no evidence of disease with a mean follow-up of 47 months. One patient with carcinoma had recurrent tumor after 46 months and was alive 40 months after resection of the recurrent tumor. In this large series of proliferative endometrioid tumors, all were stage I and only one patient had a recurrence. Most carcinomas contained evidence of a precursor APT, and in some APTs, an associated benign adenofibroma was identified. Microinvasion or intraepithelial carcinoma occurred in 19% of APTs. This finding likely reflects the various stages of endometrioid carcinogenesis in the ovary. For clinical management, we suggest that these tumors be divided into two categories-APTs and well-differentiated carcinoma-because based on the available data, cytologic atypia and microinvasion appear not to affect the prognosis.

Minimal uterine serous carcinoma: diagnosis and clinicopathologic correlation.

The clinicopathologic features of uterine serous carcinoma (USC) lacking myometrial invasion, including its putative precursor lesion endometrial intraepithelial carcinoma (EIC), have not been studied extensively. Some USCs may prove fatal even when myometrial invasion is apparently absent, whereas others may be cured with surgery alone. Accordingly, the authors studied eight cases of pure EIC (no invasion identified) and 13 superficial serous carcinomas (SSCs) in which invasion was limited to the endometrial stroma to clarify the behavior of these lesions. The review demonstrated that the most important feature in assessing prognosis is the presence or absence of extrauterine disease at presentation. Thirteen of 14 patients (93%) with EIC or SSC confined to the uterus (stage I or IIA) were disease free and one was dead of unrelated causes at 52 months, whereas seven women who presented with extrauterine disease, even if only microscopic, were either dead of disease or alive with recurrences. Accordingly, patients with EIC or SSC must undergo meticulous surgical staging at the time of hysterectomy. Because the distinction between EIC and SSC based on the identification of stromal invasion is difficult and these lesions share a unique pattern of clinical behavior, the authors regard EIC and SSC measuring 1 cm or less as "minimal uterine serous carcinoma."

Molecular genetic testing from paraffin-embedded tissue distinguishes nonmolar hydropic abortion from hydatidiform mole.

BACKGROUND: Diagnosis of hydatidiform mole by histology and ploidy analysis is limited by overlap of criteria for nonmolar hydropic abortion, complete mole, and partial mole. With early presentation, diagnosis is difficult due to limited tissue and lack of clinical features. Accurate diagnosis of these entities is important for both prognosis and patient management. We assessed a polymerase chain reaction (PCR) assay for polymorphic short tandem repeats (STR) for discrimination between nonmolar hydropic abortion, complete mole, and partial mole based on the genetic composition of molar pregnancies. METHODS: Seventeen cases of products of conception (POC) diagnosed by histology and flow cytometry ploidy analysis were studied retrospectively. PCR was performed using maternal and chorionic villus DNA extracted from microdissected, formalin-fixed, paraffin-embedded tissue sections. Allelic patterns for up to eight well-characterized polymorphic STR loci were determined using the GenePrint Fluorescent STR System (Promega Corporation, Madison, WI). The presence of three villus alleles at a single locus was interpreted as partial mole. Detection of only one allele in the villi, different from all maternal allele(s) at the same locus, was interpreted as a complete mole. RESULTS: This technique identified eight complete moles previously diagnosed as complete mole (3), hydatidiform mole, otherwise unspecified (1), hydropic villi (2), hydropic villi versus partial mole (1), and partial mole (1). The diagnoses of five partial moles by the molecular assay were consistent with the diagnoses by histology and flow cytometry. One nonmolar gestation was identified, which had been diagnosed previously as hydropic villi. In three cases, maternal DNA amplification was insufficient for definitive diagnosis. CONCLUSION: Molecular genetic testing of POC from paraffin-embedded tissue accurately distinguishes complete mole, partial mole, and nonmolar hydropic abortion. Identification of triploidy by flow cytometry can confirm a histological impression of partial mole. Histological and ploidy analysis of POC results in underdiagnosis of complete moles.

Cytogenetic diagnosis of "normal 46,XX" karyotypes in spontaneous abortions frequently may be misleading.

OBJECTIVE: To use the molecular identification of Y chromosome material in products of conception cytogenetically diagnosed as "46,XX" to confirm the occurrence of inaccurate cytogenetic test results most likely attributable to maternal cell contamination. DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENT(S): Thirty-four archival tissues from cases of spontaneous abortion with a "46,XX" karyotype based on cytogenetic analysis. INTERVENTION(S): Maternal and villus DNA were extracted from microdissected, formalin-fixed, paraffin-embedded archival tissues. The presence of the X and Y chromosomes was detected with the use of polymerase chain reaction assays and confirmed with fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): Accuracy of cytogenetic evaluation of products of conception. RESULT(S): Four (29%) of 14 first trimester and 1 (5%) of 20 second trimester "46,XX" pregnancy losses contained Y chromosome-specific DNA and demonstrated a single X chromosome-specific allele by polymerase chain reaction analysis consistent with an "XY" karyotype. Fluorescence in situ hybridization was confirmatory in 4 of 5 samples that demonstrated single X and Y signals in villus cells. CONCLUSION(S): Inaccuracy exists in the cytogenetic analysis of early products of conception that most likely is due to maternal cell contamination. In the absence of confirmatory testing, such as with a "DNA fingerprinting" assay, reports of a "46,XX" karyotype should be used cautiously in patient counseling and management.

Germline mutations of the BRCA1 and BRCA2 genes in a breast and ovarian cancer patient.

A patient with breast carcinoma diagnosed at the age of 30 years and ovarian carcinoma diagnosed at the age of 41 years was found to have germline mutations in both the BRCA1 and the BRCA2 genes. The patient was of Ashkenazi Jewish descent and the BRCA2 mutation was 6174delT, known to be very common in this population. The BRCA1 mutation, however, was 3888delGA, a mutation not previously reported in this ethnic group. The patient's breast cancer exhibited loss of heterozygosity (LOH) at the BRCA1 locus but not at BRCA2, and her ovarian cancer sustained LOH at BRCA1 and BRCA2. The BRCA1 mutation originated from patient's father, who had no personal or family history of cancer. The patient's mother, who was found to carry the BRCA2 mutation, was affected by late-onset breast cancer and her tumor exhibited LOH at BRCA2. These findings indicate that compound heterozygotes for germline mutations of BRCA1 and BRCA2 exist and may be expected to develop normally and that either gene may contribute to breast or ovarian cancer development in such individuals. The implications of this case in regard to genetic testing and counseling are also substantial.

Pulmonary venous aneurysm presenting as a middle mediastinal mass.

A large mediastinal mass in a 43-year-old man was proven at thoracotomy to comprise a right superior pulmonary vein aneurysm. Intraoperative transesophageal echocardiography was useful in defining the abnormality. Pulmonary venous aneurysm appears to represent an extremely rare but surgically correctable addition to the differential diagnosis of middle mediastinal masses.

The added gradient echo pulse sequence technique: application to imaging of fluid in the temporomandibular joint.

PURPOSE: To assess the value of an added gradient echo in the same pulse sequence with a T1-weighted spin echo for determining the presence of an abnormal fluid collection in the temporomandibular joint with no additional imaging time. MATERIALS AND METHODS: Using a standard T1-weighted sequence used in cine temporomandibular joint imaging, a readout gradient reversal was added and the resulting gradient echo collected. This image was compared with standard T1- and T2-weighted sequences, a short inversion recovery imaging sequence, and a small flip angle fast low-angle shot gradient-echo sequence. RESULTS: The T1-weighted spin echo preceding the added gradient echo is not affected by the gradient reversal, but the additional gradient echo adds T2* contrast information that displays fluid as bright as and compares favorably with other fluid detection sequences. CONCLUSION: The added gradient-echo technique adds sensitivity for the detection of an abnormal increase in fluid in the temporomandibular joint without adding to the overall imaging time of a routine T1-weighted sequence.

Cine magnetic resonance imaging of the temporomandibular joint.

Magnetic resonance imaging (MRI) provides a significant amount of information about both the position of the meniscoligamentous complex and the osseous structures in evaluation of internal derangements of the temporomandibular joint (TMJ). With the advent of computer-driven cine displays, dynamic information is now available, providing cost-effective diagnostic evaluation similar to cine fluoroscopy with arthrography, but without the ionizing radiation exposure or discomfort. Using a bite block, a series of sagittal images are obtained with the patient "posing" at different degrees of jaw opening. The subsequent cine MRIs with "posed" motion are striking and very helpful in evaluating various types of TMJ dysfunction. At the authors' institution, 205 patients were examined with a history of TMJ arthralgia. Correlation of imaging studies and surgical findings reveals a sensitivity (98%) and specificity (96%) for identifying the meniscoligamentous complex.

Direct and correlated responses to artificial selection on lipid and glycogen contents in Drosophila melanogaster.

A large outbred population of Drosophila melanogaster was subjected to artificial selection on lipid and glycogen storage. In three separate experiments, two replicates underwent sib selection for both increased and decreased storage. In the first study, flies were selected on the basis of total triacylglycerol for ten generations. This experiment resulted in no significant direct response, but there was a significant change in total body weight, underscoring the importance of concern for the allometric relationship between body weight and lipid content. In the second study, selection was performed for 15 generations on the percentage of body composition that was triacylglycerol. A significant direct response was obtained, and the two replicates revealed heritability estimates of 0.40 and 0.43. The third study selected glycogen content for 15 generations, and produced a significant response with heritabilities of 0.25 and 0.31. A series of 12 biochemical and enzyme kinetic traits was examined at five generation intervals in all experiments, and a number of correlated responses were detected. The results are interpreted with respect to the evolutionary constraints on energy storage evolution and the genetic basis of the allometric relationship between body weight and fat content.

Pneumocephalus associated with a frontoethmoidal osteoma.

A 51-year-old man complained of a headache of 2-months duration. Computerized tomography revealed pneumocephalus and an osteoma at the confluence of the frontal and ethmoid sinuses on the right. Craniotomy confirmed an osteoma with a spur as the cause of the pneumocephalus.

Transient cortical blindness following cerebral angiography.

Transient cortical blindness following cerebral angiography was previously assumed to result from embolism or other factors impairing cortical perfusion during angiography. Contrast medium-induced disruption of the blood-brain barrier and a direct neurotoxic effect by contrast media have been proposed as a potential mechanism of neurotoxicity. Support of this concept has been provided by reports that demonstrate blood-brain barrier damage with postangiography computerized tomography in patients experiencing various acute neurologic sequelae of cerebral angiography including one case of transient cortical blindness. We report a case of transient cortical blindness following cerebral angiography in which computerized tomography documents blood-brain barrier disruption limited to the occipital lobes.

Distribution of phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive neurons in the avian brain.

The distribution of neurons immunoreactive to tyrosine hydroxylase and phenylethanolamine-N-methyltransferase (PNMT) were described in adjacent sections of the avian medulla oblongata. PNMT-positive neurons were found in two bilaterally symmetrical columns in the ventrolateral and dorsomedial medulla. Within the ventrolateral column, PNMT cells were centered in and around the lateral paragigantocellular and lateral reticular nuclei. In the dorsomedial medulla, PNMT neurons were concentrated within and around the nucleus of the tractus solitarius. The distribution of PNMT-immunoreactive neurons in the avian medulla is similar to those observed in mammals, except there appears to be a greater number of PNMT-positive cells in the bird.

Metastatic renal cell carcinoma presenting as an intrasellar mass on computerized tomography.

We report a case of renal cell carcinoma metastatic to the pituitary gland. A review of the literature indicated breast carcinoma to be the most frequent primary tumor metastatic to this site, while renal cell carcinoma metastasis has not been reported previously. This case emphasizes the capricious nature of renal cell carcinoma, particularly in a patient presenting with no evidence of disseminated disease.

Characterization of a reduced-eye mutant of the grasshopper, Melanoplus sanguinipes.

A reduced-eye (re) mutant grasshopper of Melanoplus sanguinipes has been characterized by small flat compound eyes lacking facets, no lateral ocelli and only a remnant of the median ocellus. The re grasshoppers walk, jump, fly and feed in a normal manner, but do not respond to visual and auditory stimuli, suggesting they may be blind and deaf. Extracellular recordings from the ventral nerve cord of re mutants verified the lack of neural activity in response to visual and auditory inputs, yet the mutants detected mechanical and tactile stimuli. Electroretinograms implied that a visual deficit may be within the photoreceptors of the compound eye. Histological examination of the compound eyes and ocelli indicated that the cells of the mutant compound eye incompletely differentiate. The optic lamina underlying the retina is missing, as is the outer optic chiasma. The medulla and lobula of the mutant optic lobe are present, however, the neuropil of the medulla lacks the characteristic axonal projection patterns of wild-type grasshoppers. The re grasshopper also lacks all ocellar nerves. Ocellar nerves are normally formed from processes of second order ocellar neurons (SONs), suggesting that if the mutant SONs are present within the protocerebrum, their morphology is drastically altered. Comparison of embryos and juvenile nymphs supports the suggestion that the alterations in the re visual system are the result of abnormal differentiation during development. Even though there is clear evidence of morphological alterations in second and third order optic lobe interneurons, one higher order visual interneuron of the midbrain, the descending contralateral movement detector (DCMD), has the same morphology as the DCMD in a wild-type brain. In this instance, the complete deprivation of the primary sensory input does not appear to alter cellular development.