Genetic suppression of seizure susceptibility in Drosophila.

Despite the frequency of seizure disorders in the human population, the genetic and physiological basis for these defects has been difficult to resolve. Although many genetic defects that cause seizure susceptibility have been identified, the defects involve disparate biological processes, many of which are not neural specific. The large number and heterogeneous nature of the genes involved makes it difficult to understand the complex factors underlying the etiology of seizure disorders. Examining the effect known genetic mutations have on seizure susceptibility is one approach that may prove fruitful. This approach may be helpful both in understanding how different physiological processes affect seizure susceptibility and in identifying novel therapeutic treatments. In this study, we have taken advantage of Drosophila, a genetically tractable system, to identify factors that suppress seizure susceptibility. Of particular interest has been a group of Drosophila mutants, the bang-sensitive (BS) mutants, which are much more susceptible to seizures than wild type. The BS phenotypic class includes at least eight genes, including three examined in this study, bss, eas, and sda. Through the generation of double-mutant combinations with other well-characterized Drosophila mutants, the BS mutants are particularly useful for identifying genetic factors that suppress susceptibility to seizures. We have found that mutants affecting Na+ channels, mle(napts) and para, K+ channels, Sh, and electrical synapses, shak-B(2), can suppress seizures in the BS mutants. This is the first demonstration that these types of mutations can suppress the development of seizures in any organism. Reduced neuronal excitability may contribute to seizure suppression. The best suppressor, mle(napts), causes an increased stimulation threshold for the giant fiber (GF) consistent with a reduction in single neuron excitability that could underlie suppression of seizures. For some other double mutants with para and Sh(KS133), there are no GF threshold changes, but reduced excitability may also be indicated by a reduction in GF following frequency. These results demonstrate the utility of Drosophila as a model system for studying seizure susceptibility and identify physiological processes that modify seizure susceptibility.

The characteristics of 'new users' of cocaine and heroin unknown to treatment agencies: results from the Swiss Hidden Population Study.

AIMS: To examine the characteristics of 'new users' (i.e. first use not more than 5 years before interview) of heroin or cocaine currently unknown to treatment agencies in Switzerland, compared to 'new users' who are in treatment, as well as to 'experienced users' (i.e. first use more than 5 years before interview). METHOD: A sample of 917 users of heroin and/or cocaine users were recruited outside treatment settings by 31 privileged access interviewers and interviewed face to face using a standardized questionnaire. The study group was composed of 95 'new users' of heroin and/or cocaine who had never been in treatment. It was compared to four control groups: control group 1 (n = 48) consisting of 'new users' known to treatment agencies; control group 2 (n = 443) of 'experienced users' who reported having never had any contact with treatment agencies; control group 3 (n = 53) of 'experienced users' with some history of treatment but not in contact at the time of interview; and control group 4 (n = 166) of 'experienced users' in contact with treatment agencies. FINDINGS: Compared with 'new' and 'experienced' users in treatment (control groups 1 and 4), 'new users' unknown to treatment agencies have a less problematic profile of drug use: they inject less, use drugs less frequently, more often report the impression of controlling their drug use, have fewer health problems and fewer social difficulties. However, compared to experienced users not in contact with treatment agencies (control groups 2 and 3), the profile of drug use in the study group is more problematic. Injection-related HIV-risk behaviour is lower in the study group than in all the control groups. The analysis showed no influence of duration of drug use, gender or education over getting in touch with treatment agencies in the first five years of drug use. CONCLUSIONS: Some of the 'new users' unknown to treatment agencies can be considered as being in need of assistance. Efforts to enhance the treatment on offer should be continued. Drug users should be provided with better knowledge of how to identify signs for problem drug use and need for assistance.

Modifications of seizure susceptibility in Drosophila.

In a given population, certain individuals are much more likely to have seizures than others. This increase in seizure susceptibility can lead to spontaneous seizures, such as seen in idiopathic epilepsy, or to symptomatic seizures that occur after insults to the nervous system. Despite the frequency of these seizure disorders in the human population, the genetic and physiological basis for these defects remains unclear. The present study makes use of Drosophila as a potentially powerful model for understanding seizure susceptibility in humans. In addition to the genetic and molecular advantages of using Drosophila, it has been found that seizures in Drosophila share much in common with seizures seen in humans. However, the most powerful aspect of this model lies in the ability to accurately measure seizure susceptibility across genotypes and over time. In the current study seizure susceptibility was quantified in a variety of mutant and wild-type strains, and it was found that genetic mutations can modulate susceptibility over an extremely wide range. This genetic modulation of seizure susceptibility apparently occurs without affecting the threshold of individual neurons. Seizure susceptibility also varied depending on the experience of the fly, decreasing immediately after a seizure and then gradually increasing over time. A novel phenomenon was also identified in which seizures are suppressed after certain high-intensity stimuli. These results demonstrate the utility of Drosophila as a model system for studying human seizure disorders and provide insights into the possible mechanisms by which seizure susceptibility is modified.

Functional analysis of the DNA-packaging/terminase protein gp17 from bacteriophage T4.

In bacteriophage T4, the terminase complex constituted by the large subunit gp17 (69 kDa) and the small subunit gp16 (18 kDa) is a critical component of the ATP-driven DNA-packaging pump that translocates DNA into an empty capsid shell. Evidence suggests that the large subunit gp17 is the critical component and consists of a number of the functional sites required for DNA-packaging. It exhibits a terminase activity that introduces non-specific cuts into DNA, a portal vertex binding site that allows linkage of cleaved DNA to an empty prohead, an in vitro DNA-packaging activity, and an ATPase activity. In addition, a consensus metal-binding motif and two consensus ATP-binding sites have been identified by sequence analysis. In order to understand the mechanism of action of the multifunctional gp17, we developed an expression-based selection strategy to select for mutants that are defective in terminase function. Characterization of one of the mutants revealed a unique phenotype in which a single H436R mutation resulted in a dramatic loss of both the terminase and the DNA-packaging functions. Indeed, in vivo substitution of H436 with any of the 12 amino acids for which a suppressor is available was lethal to T4 development. According to one hypothesis, H436 is part of a metal-binding motif that is essential for gp17 function. This hypothesis was tested by introducing mutations at each of the three histidine pairs, the H382-X2-H385 pair, the H411-X2-H414 pair and the H430-X5-H436 pair, which constitute the histidine-rich region near the C terminus of gp17. A mutation at either the H411 pair or the H430 pair resulted in a loss of gp17 function, whereas a mutation at the H382 pair had no effect. In addition to the putative metal-binding motif, substitutions at residue K166 within the putative N terminus-proximal ATP-binding site also resulted in a loss of gp17 function. We propose that a metal-binding motif involving the histidine residues within the sequence H411-X2-H414-X15-H430-X5-H436 is essential for gp17 function. Metal-terminase interactions may be required for structural alignment and stabilization of functional sites in phage T4 terminase and other double-stranded DNA phage terminases.

The Swiss Hidden Population Study: practical and methodological aspects of data collection by privileged access interviewers.

In order to recruit heroin and/or cocaine users outside treatment settings, recruitment of subjects through Privileged Access Interviewers (PAI) was tested and implemented in the Swiss Hidden Population Study. This article discusses practical aspects of the PAI method as well as issues of reliability and validity. From June 1994 to June 1995, 31 Privileged Access Interviewers were recruited in the main regions of Switzerland. They conducted 943 standardized interviews altogether, of which 917 could be considered valid. Fifty-four per cent of the respondents correspond to the criteria of the target population. The PAI method collects reliable data in a relatively short amount of time, given adequate means of control. Analysis of the age distribution and of the patterns of drug use in our sample shows that the question of validity is mainly linked to the diversity of the milieus from which PAIs recruit the respondents. Encouraging PAIs to do as many interviews as possible did not skew the data. Hence, well-founded inferences on a PAI generated database relies on the analysis of qualitative information on the ways in which the Privileged Access Interviewers have recruited their respondents.

Peritoneal fluid cytology in advanced mucinous cystadenocarcinoma of the pancreas.

OBJECTIVE: To assess the role of peritoneal cytology in the management of mucinous cystadenocarcinoma. STUDY DESIGN: A review of the peritoneal fluid cytologic findings in eight selected cases of pancreatic mucinous cystadenocarcinomas. RESULTS: Four of eight cases (50%) were positive for malignant cells. All four patients with positive peritoneal cytology had unresectable tumors and extensive intraabdominal metastases. Of the four patients with negative cytology, three had unresectable, locally invasive tumors without metastases, and one had a resectable tumor confined to the pancreas. CONCLUSION: Peritoneal cytology is frequently positive in unresectable mucinous cystadenocarcinomas with intraabdominal metastases, but cytology may often be negative in unresectable cases. Therefore, negative cytologic findings in these cases do not reliably ensure resectability, while positive findings clearly correlate with the presence of intraabdominal metastases. A larger study of unselected patients, including more with resectable tumors, will be required to define the full utility of this technique in preoperative assessment.

Activation of cell growth inhibitor by ectoprotein kinase-mediated phosphorylation in transformed mouse fibroblasts.

Our previous studies have shown that exogenous ATP induces cell growth inhibition in transformed mouse fibroblasts, 3T6 cells, whereas the growth of their nontransformed counterparts, Swiss 3T3 cells, is only slightly affected. In this study a similar selective, ATP-induced growth inhibition was found in Balb/c SV40-3T3 cells and in primary cultures of adenovirus-transformed murine fibroblasts. The inhibitory activity was found in the conditioned media of ATP-treated cultures. Several lines of evidence have shown that ectoprotein kinase (ecto-PK) plays a major role in the ATP-induced growth inhibition. (a) There is a good correlation between the activity of ecto-PK and the ability of ATP to induce cell growth inhibition. (b) The removal of the ecto-PK from the cell surface prevents the ATP-induced growth inhibition. (c) Addition of the removed enzyme to the cell culture reconstitutes the ability of ATP to induced growth inhibition. (d) Serum-containing, or serum-free, conditioned media from untreated cultures gain an inhibitory activity after their phosphorylation, and dephosphorylation of conditioned media from ATP-treated cultures results in the loss of the inhibitory activity. (e) Growth medium by itself does not inhibit cell proliferation after its phosphorylation. The findings described in d and e indicate, as well, that the ATP-induced growth inhibitor is produced by the cells. The putative inhibitor was found to be a protein, with an apparent molecular mass of 13 kDa. The selectivity of the inhibition for transformed cells is due to the higher level of ecto-PK in these cells, as well as to their higher susceptibility to the inhibitor, as compared with their non-transformed counterparts.

Squamous epithelial proliferative lesions associated with rhesus Epstein-Barr virus in simian immunodeficiency virus-infected rhesus monkeys.

Proliferative lesions were found on the squamous epithelium of the tongue, esophagus, or penis or haired skin of the lip, hand, or thorax of 8 simian immunodeficiency virus-infected rhesus monkeys that died of simian AIDS. The lesions were focal and consisted of hyperkeratosis, parakeratosis, and acanthosis in the skin, with additional ballooning degeneration in the tongue, esophagus, and penis. The epithelial surfaces were frequently colonized by Candida species or gram-positive cocci. Intranuclear inclusion bodies were seen in cells in the middle and superficial layers. Herpesvirus virions were found in inclusion-bearing cells by transmission electron microscopy. An Epstein-Barr-like virus was identified in inclusion-bearing cells by immunohistochemistry and in situ hybridization. No virus was detectable in basal layers of the epithelium. These lesions resemble oral hairy leukoplakia in AIDS patients and may thus provide a useful primate model to study permissive epithelial infection by Epstein-Barr-like viruses.

Distribution of SIV in lymph nodes of serially sacrificed rhesus monkeys.

Rhesus monkeys were inoculated with SIVDeltaB670 and sacrificed 2, 4, 8, and 24 weeks after inoculation or when moribund. Two monkeys predicted to have a rapid disease course and two predicted to have a slower disease course were sacrificed at each time point. Lymph nodes were studied by histopathology, immunohistochemistry, in situ hybridization, electron microscopy, flow cytometry for lymphocyte subsets, and mitogen responsiveness. A greater selective decrease in peripheral CD4+CD29+ (helper-inducer/memory) T cells occurred in monkeys with high antigenemia. Although the percentage of CD8+ lymphocytes was increased and the CD4+/CD8+ ratio decreased in all infected groups, there were no consistent differences between monkeys with high or low antigenemia in lymph node lymphocyte subsets. Blastogenic responses of lymph node lymphocytes to PHA, ConA, or PWM were not significantly altered in infected monkeys. A reticular pattern typical of antigen deposition within germinal center follicular dendritic cells was seen in three monkeys with atrophic lymph nodes, high serum antigenemia, and a low percentage of circulating CD4+/CD29+ cells. More individually stained cells were in monkeys with high serum antigen and in moribund animals. By in situ hybridization, most monkeys had signal in a reticular pattern of germinal centers. Animals with higher levels of serum antigenemia tended to have more infected cells and a more intense signal. Extracellular virions were found between the FDC foot processes in the germinal centers of lymph nodes. Disease course was already established 2 weeks after inoculation.

Lentivirus-induced pulmonary lesions in rhesus monkeys (Macaca mulatta) infected with simian immunodeficiency virus.

Necropsy reports from 28 rhesus monkeys that had been experimentally infected with simian immunodeficiency virus (SIV) and that were free of cytomegalovirus were reviewed. Lung sections from 24 of these monkeys that had no etiologic agent other than SIV detected in the lung were studied in detail by histopathologic, immunohistochemical, and electron microscopic examination and by in situ hybridization. Fourteen of the monkeys were part of a serial euthanasia study, while others were euthanatized after they became moribund. The following lesions were detected: perivascular inflammation, vasculitis, interstitial pneumonia, syncytial cells, hemorrhage, fibrin exudation, and pleural fibrosis. Perivascular inflammation was the most frequent lesion and occurred as early as 2 weeks after inoculation. Severe pneumonia and numerous syncytial cells were seen only in animals euthanatized because they had become moribund. The lesions appeared to be directly due to SIV infection. SIV antigens, RNA, and virions were detected in syncytial cells and macrophages by immunohistochemical examination, in situ hybridization, and transmission electron microscopic examination, respectively. The amount of virus present was correlated with the severity of the lesions. The SIV-induced lesions were different from those of the lymphocytic interstitial pneumonia, which occurs in human immunodeficiency virus-infected children and in ovine lentivirus-infected sheep and goats.

Thymus in simian immunodeficiency virus-infected rhesus monkeys.

The thymuses from 20 simian immunodeficiency virus (SIV)-infected and 4 uninfected rhesus monkeys were examined at intervals after infection to determine whether there were specific SIV-induced lesions, to document the serial distribution of SIV antigens, mRNA, and DNA, to quantitate the number of infected cells, and to correlate thymic changes with other parameters of infection. The following techniques were used: gross pathology, histopathology, immunohistochemistry, electron microscopy, in situ hybridization, polymerase chain reaction, and limiting dilution culture. Thymic involution due to loss of lymphocytes was apparent 8 weeks after inoculation. No epithelial damage or loss of Hassall's corpuscles was observed. Culture was the most sensitive technique for detecting SIV, being positive in 19 of 20 inoculated monkeys. The polymerase chain reaction was negative in one thymus that was positive at a low level by culture. In situ hybridization was positive in 14 of 19 thymuses examined, with a few macrophages in the cortex having a strong signal and numerous lymphocytes in the medulla having a weak signal. Mature viral particles and viral budding could not be demonstrated by electron microscopy. The number of cells positive for viral RNA by in situ hybridization correlated with the level of serum antigenemia. These observations suggest that thymic macrophages and lymphocytes are infected with SIV within 2 weeks after inoculation. SIV apparently directly causes loss of thymic lymphocytes and immunodeficiency without infecting or damaging the thymic epithelium. No specific SIV-induced lesions were recognized. The number of cells in the thymic medulla expressing SIV RNA correlates with the level of serum antigen, which has been previously shown to be correlated with disease progression.

Serial pathogenesis study of SIV brain infection.

A serial study of early SIV brain infection revealed initial involvement of leptomeninges, followed by perivascular infection of brain parenchyma. Severity of SIV encephalitis correlated with severity of systemic disease rather than with length of infection. Diffuse white matter disease was not observed, and there was little evidence of SIV infection of brain endothelial cells. SIV infection of leptomeninges is separate from infection of the brain, which appears to be due to transvascular spread of infected monocytes/macrophages.

Cytologic features of endometrial papillary serous carcinoma.

Endometrial papillary serous carcinoma (EPSC) is an uncommon variant of endometrial carcinoma that histologically resembles ovarian serous carcinoma and has an aggressive clinical course. The cytomorphologic features of 17 patients with histologically confirmed EPSC of the endometrium were reviewed and compared with those of 20 patients with histologically typical endometrial adenocarcinoma (TEC). Preoperative cervicovaginal Papanicolaou smear results were available from 14 of the 17 patients with EPSC; 10 (71%) were positive, 1 (7%) was suspicious and 3 (21%) were negative for malignancy. Initial cervicovaginal smear results were available from all 20 patients with TEC; 7 (35%) were positive, 4 (20%) were atypical or suspicious and 9 (45%) were negative for malignancy. Twelve patients with EPSC had peritoneal washings or fluids examined; seven were positive and five negative. Twelve patients with TEC had peritoneal washings or fluids examined; two (17%) were positive and ten (83%) were negative. The cervicovaginal smears from patients with EPSC revealed numerous large tumor cells (with prominent nucleoli) frequently arranged in papillary clusters with background necrosis and, in two cases, amorphous material suggestive of psammoma bodies. In contrast, the smears of patients with TEC showed small to medium-sized cells with extensive phagocytosis and many background histiocytes. The diagnosis of EPSC should be considered when the cervicovaginal smear contains numerous papillary groups of large tumor cells with macronucleoli but without prominent phagocytosis, especially when structures suggestive of psammoma bodies are present. The peritoneal fluids in these patients are more often positive than in patients with TEC, a finding consistent with the propensity of EPSC to involve peritoneal surfaces.