Autographa californica M nucleopolyhedrovirus ProV-CATH is activated during infected cell death.

V-CATH, a cathepsin L-like cysteine protease encoded by the baculovirus Autographa californica M nucleopolyhedrovirus, has been shown to play an essential role in host liquefaction. Similar to cellular cathepsin L, V-CATH is synthesized as an inactive proenzyme and is activated by cleavage of the propeptide. Previous studies indicated that removal of the propeptide was rapid, occurring as soon as the protein could be detected by Western blot, 22 h postinfection. We found, however, that these results reflected artifactual processing of the proenzyme. When the protease inhibitor E-64 was used to prevent this aberration, we found that proV-CATH accumulated in infected cells and activation did not begin until the onset of cell death, at approximately 80 h postinfection. Western blot analysis of fractions of live and dead cells isolated by fluorescence-activated cell sorting revealed that mature V-CATH was found only in dead cells. The regulation of activation of proV-CATH, therefore, was quite different from that of cellular cathepsins. Acridine orange staining revealed that lysosome integrity was lost in dead cells, an occurrence that could lead to the activation of proV-CATH by lysosomal proteases.

Central role of hemocytes in Autographa californica M nucleopolyhedrovirus pathogenesis in Heliothis virescens and Helicoverpa zea.

Autographa californica M nucleopolyhedrovirus (AcMNPV) can infect and kill a wide range of larval lepidopteran hosts, but the dosage required to achieve mortal infection varies greatly. Using a reporter gene construct, we identified key differences between AcMNPV pathogenesis in Heliothis virescens and Helicoverpa zea, a fully permissive and a semipermissive host, respectively. Even though there was more than a 1,000-fold difference in the susceptibilities of these two species to mortal infection, there was no significant difference in their susceptibilities to primary infections in the midgut or secondary infections in the tracheal epidermis. Foci of infection within the tracheal epidermis of H. zea, however, were melanized and encapsulated by 48 h after oral inoculation, a host response not observed in H. virescens. Further, H. zea hemocytes, unlike those of H. virescens, were highly resistant to AcMNPV infection; reporter gene expression was observed only rarely even though virus was taken up readily, and nucleocapsids were transported to the nucleus. Collectively, these results demonstrated that hemocytes-by removing virus from the hemolymph instead of amplifying it and by participating in the encapsulation of infection foci-together with the host's melanization response, formed the basis of H. zea's resistance to fatal infection by AcMNPV.

Characterization of two novel Microplitis demolitor polydnavirus mRNAs expressed in Pseudoplusia includens haemocytes.

The braconid wasp Microplitis demolitor carries M. demolitor polydnavirus (MdPDV) and parasitizes the larval stage of the moth Pseudoplusia includens. M. demolitor injects MdPDV into P. includens larvae when it lays an egg and the virus infects various cells including haemocytes. Two new MdPDV transcripts expressed in host haemocytes were characterized in this study. Screening of an MdPDV-infected haemocyte cDNA library identified a 0.4 kb cDNA encoding a predicted protein of 103 amino acids which was named Egf0. 4. This protein contained a cysteine-rich epidermal growth factor (EGF)-like motif at its N terminus that was similar to the EGF-like domains in the previously identified MdPDV genes egf1.5 and egf1.0. Sequencing of the genomic clone pMd-10 indicated that it contained the egf0.4 gene, which consisted of two introns and three exons. This gene was located on MdPDV segment O and appeared to exist in multiple copies. A nucleic acid and expression screen identified a 1. 8 kb cDNA encoding a predicted protein of 515 amino acids designated Glc1.8. This protein consisted of a heavily glycosylated central core of six tandemly arranged repeats flanked by hydrophobic N- and C-terminal domains. Northern blotting and in situ hybridization studies indicated that both egf0.4 and glc1.8 were expressed in MdPDV-infected host haemocytes. Immunocytochemical studies also indicated that Glc1.8 localized to the cell surface.

[Perceptions, adaptation and health care strategies of the elderly family caregivers from the standpoint of the shift to ambulatory care]. / Perceptions, stratégies adaptatives et attentes des femmes agées aidantes familiales dans la perspective du virage ambulatoire.

This article presents the results of an exploratory study funded by the Social Sciences and Humanities Research Council of Canada. The objectives of this research were to explore (a) the perception by elderly women caregivers of the shift to ambulatory care; (b) their use of coping behaviours in the face of this major change within the health care system; and (c) their perceptions of health care services. In a theoretical framework of stress and coping, and using standardized questionnaires and open-ended questions, 40 home interviews were initially conducted with elderly women, each of whom was caring for her spouse after his discharge from hospital. Additional data were collected through focus groups. Quantitative and qualitative analyses revealed that elderly women caregivers perceive ambulatory care in a positive manner and as a change involving challenges only when they feel in control of the health care situation and when the care does not necessitate specialized abilities. When the care to be provided is complex and requires continued involvement and specific competence on the caregiver's part, the situation results in insecurity for elderly women. Caregivers use cognitive coping strategies to deal with their problems and are not inclined to use formal services in their social support network. Participants in this study wished to be better informed about the health care system in general ("how the system works") so that they could acquire a sense of predictability. Other expectations of elderly women caregivers concerned the importance of continuity in health services delivery, better preparation for the transition from hospital to home and availability of psychological support for themselves. These results suggest a critical analysis of the present paradigm guiding health care services, in which family caregivers are considered as resources or coworkers instead of coclients.

Quantitative EEG findings associated with chronic stimulant and cannabis abuse and ADHD in an adult male substance use disorder population.

QEEG was studied in a population of chronic male PSUD/ADHD (psychoactive substance use disorder/attention deficit hyperactivity disorder) subjects vs. a matched sample of non-ADHD subjects with PSUD. Our first interest in conducting this study was to determine if the Thatcher University of Maryland database and complex demodulation method could replicate the specific QEEG findings reported for cocaine and cannabis using the John-NYU database and Fourier Transform method. The effects of cannabis and stimulants were also studied both separately and together to see if there were interactions and to see if the QEEG changes associated with chronic stimulant dependence were predicted by childhood ADHD status. Eyes-closed QEEGs were obtained and two independent artifacted 60 second samples were compared for reliability. The Thatcher database was used to analyze QEEG data from 56 subjects with mixed substance use disorder. Results showed that the Thatcher database replicates the John database for chronic stimulant dependence findings. Because of confounding variables of alcohol and polysubstance abuse, the findings related to cannabis and stimulant interaction were difficult to assess. Cannabis and stimulant dependence together produced more QEEG changes than either alone. More right temporal abnormalities were observed with stimulant dependence. In the absence of stimulant use, the QEEG effects of cannabis were relatively small; however, sample selection and methods used precluded comparison to previous studies. The persistent QEEG abnormalities associated with chronic stimulant dependence were independent of ADHD status in this sample using the methods of this study. Further research is needed to clarify the relationship of stimulant dependence with QEEG changes and ADHD status, and to clarify the interactions of chronic stimulant and cannabis abuse on QEEG.

Limitations of the American Academy of Neurology and American Clinical Neurophysiology Society paper on QEEG.

The AAN/ACNS report is misleadingly negative regarding the current status of quantitative EEG and tends to discourage its development and use with other related clinical problems. There have been many excellent studies showing that QEEG can be useful for the evaluation and understanding of mild traumatic brain injury, learning disabilities, attention deficit disorders, alcoholism, depression, and other types of substance abuse. In fact, Hughes and John recently provided in this Journal an extensive and detailed review of the use of QEEG in psychiatric disorders. The bias of the AAN/ACNS report is also evident when contrasted to the outstanding review of the clinical utility of QEEG by the American Medical EEG Association, which clearly articulates the opposite points in many cases and concludes that QEEG has reached maturity. At present, the most one can say is that there are legitimate scientific debate and differences of opinion concerning the utility of QEEG, as there are in many other areas of medicine. The AAN/ACNS article should not be considered the definitive opinion. Too many implications for health care are at stake. The debate and research may continue without withholding valuable help from the public. We hope that revised guidelines will be drafted in such a way as to encourage the development of quantitative EEG and brain mapping rather than discourage future research support and use of QEEG with patients. Furthermore, we strongly feel that this technology should be available to, and be explored and used by, nonphysicians who are properly trained and certified.

Findings of mild traumatic brain injury in combat veterans with PTSD and a history of blast concussion.

Veterans with chronic posttraumatic stress disorder were evaluated for a history of blast concussion, controlling for confounding conditions. Electroencephalograms were analyzed by discriminant function for traumatic brain injury. A difference was found in discriminant scores between veterans with and without blast concussion. More members of the blast group had attentional symptoms and attentional dysfunction. Combat veterans with a remote history of blast injury have persistent electroencephalographic features of traumatic brain injury as well as attentional problems. The authors hypothesize that these constitute a type of chronic postconcussive syndrome that has cognitive and mood symptoms overlapping those of posttraumatic stress disorder.

A limited role in parasitism for Microplitis demolitor polydnavirus.

Spodoptera frugiperda larvae stung by Microplitis demolitor undergo physiological alterations characteristic of parasitism. However, despite these physiological modifications, parasitized S. frugiperda larvae never yield adult wasps. Our original hypothesis that unsuccessful parasitism was due to a transcriptionally inactive polydnavirus proved untrue. Microplitis demolitor polydnavirus (MdPDV) successfully infected and expressed, albeit transiently, in S. frugiperda hemocytes. MdPDV expression was most abundant in the first three days of parasitism, then sharply declined on Day 4 post-parasitization and continued to decline for the remainder of the study. During the period of MdPDV expression, S. frugiperda hemocytes were non-adherent, incapable of spreading in vitro and did not encapsulate M. demolitor eggs in vivo. Concurrent with diminishing viral expresssion, S. frugiperda hemocytes regained their ability to adhere and spread in vitro and encapsulated M. demolitor eggs in vivo. Although MdPDV disrupted S. frugiperda's encapsulation response for the first three days post-parasitization, M. demolitor was unable to develop in this noctuid species. Failure to develop was independant of viral activity, all M. demolitor eggs oviposited in S. frugiperda larvae failed to complete embryogenesis and died within 24 hour of oviposition. S. frugiperda larvae infected with MdPDV exhibited alterations in development very similar to other lepidopterans that are permissive hosts for M. demolitor. In addition, MdPDV DNA persisted in Spodoptera frugiperda hemocytes in the absence of viral expression.

Two Microplitis demolitor polydnavirus mRNAs expressed in hemocytes of Pseudoplusia includens contain a common cysteine-rich domain.

Microplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. A previous study indicated that M. demolitor polydnavirus (MdPDV) infects primarily hemocytes in parasitized hosts. Thereafter, several alterations that compromise the immune response of P. includens toward the developing parasitoid occur in hemocytes. In this study, we identified two MdPDV mRNAs (1.0 and 1.5 kb) expressed in P. includens hemocytes that have homology to the viral genomic clone pMd-2. Corresponding 1.0- and 1.5-kb cDNA clones (MdPi455 and MdPi59) were isolated from an MdPDV-infected hemocyte cDNA library. Nucleotide sequence analysis of the cDNA clones confirmed that the 1.5- and 1.0-kb mRNAs have significant regions of homology. Sequence alignment revealed that the gene, OMd1.0, encoding the 1.0-kb mRNA is present in pMd-2. This gene contains two introns and three exons that agree with the sequence for MdPi455. In contrast, the 1.5-kb mRNA is likely encoded by a related gene located on the same MdPDV genomic DNA as is OMd1.0. The predicted peptide sequences for the 1.0- and 1.5-kb transcripts contain a cysteine-rich region at their 5' ends that have some similarity with epidermal growth factor-like motifs. Hybridization studies revealed that both mRNAs are expressed in granular cells and plasmatocytes, the primary classes of hemocytes involved in defense against M. demolitor and other parasites.

Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66.

A gene encoding the extracellular domain of the human erythropoietin receptor (EPO-R) was constructed using oligonucleotides, with a view to maintaining preferred codon usage for the Streptomycetes. The gene was subcloned into a multicopy Streptomyces-Escherichia coli shuttle vector, pCAN46 (derived from pIJ680), containing a strong constitutive promoter from the S. fradiae aph gene, a signal peptide coding region derived from the protease B gene of S. griseus, and a transcription terminator sequence also derived from the S. fradiae aph gene. Extracellular expression of authentic EPO-R by S. lividans was demonstrated using SDS-PAGE and Western blot analysis, followed by direct amino terminal sequencing of the purified product. Specific binding of S. lividans-expressed EPO-R to recombinant human glycosylated EPO was demonstrated using BIAcore (surface plasmon resonance) analysis and native gel shift assays.

Efficacy of smoking cessation strategies in a treatment program.

Efficacy of a total smoking ban on an inpatient drug and alcohol rehab program was assessed by urine cotinine levels and indicated a continued smoking rate of 70%. In spite of these results, however, some staff were concerned that removal of the smoking ban would lead to a dramatic increase in patients' smoking. However, other staff were concerned over the apparent dishonesty undermining the objectives of the program. Therefore, a change in policy was initiated. Under this new approach, where smoking was no longer punished, cotinine levels were 60% positive. Although this was not a statistically significant reduction, it was clinically significant. Patients were openly encouraged to discuss their smoking, and therapies were designed to match patients' level of motivation. A significant reduction in number of cigarettes smoked (mean = 26 pretreatment, mean = 17 posttreatment; p < 0.01), increased interest to stop smoking (on a linear scale from 1 to 5, mean = 1.86 pretreatment and mean = 2.61 posttreatment; p < 0.01), and increased patient satisfaction was noted in a subsequent sample. Nonprohibitive counseling approaches were just as effective as prohibition of smoking. Change in smoking behavior and motivation were demonstrable with programming that emphasized behavior change and motivational counseling. Relationships between nicotine dependence, depression and other substance use disorders are discussed.

Separation and behavior in vitro of hemocytes from the moth, Pseudoplusia includens.

Hemocytes collected from larvae of Pseudoplusia includens (Lepidoptera:Noctuidae) were separated by centrifugation on Percoll cushions. The procedure resulted in 95% purity of plasmatocytes and greater than 99% purity of granular and spherule cells. Medium supplemented with chicken serum enhanced cell viability and promoted spreading of plasmatocytes. Cell-free plasma and medium preconditioned by plasmatocytes or granular cells stabilized cells in vitro and also accelerated spreading of plasmatocytes relative to medium supplemented with chicken serum. Oenocytoids were the only morphotype that exhibited endogenous phenoloxidase activity, while granular cells and plasmatocytes were the only cells that endocytosed fluorescent beads in vitro. Granular cells and plasmatocytes ingested fluorescently labelled beads, both in mixed populations of hemocytes and after separation. Plasmatocytes were the only morphotype that encapsulated large foreign targets in vitro following separation. Separated granular cells attached and spread on the surface of foreign targets but never formed a multilayered capsule.

Clonazepam prescribing patterns and abuse by methadone patients in a medical center setting.

Methadone patients who require inhospital detoxification from self-administered clonazepam (CZM) were interviewed. Patients enrolled in drug dependency (DDTP), combined addiction psychiatry (CAPP) and methadone maintenance (MM) were surveyed. Twenty out of 20 DDTP and 15 out of 30 CAPP patients responded indicating no knowledge of CZM as a drug of abuse. MM patients were reluctant to respond, but the 18 out of 72 who did, indicated knowledge of CZM abuse. Ten identified CZM as a "downer," 11 indicated its use to "get high," 10 knew other people who abused, and 6 believed abuse to be common. Six identified a street value as $1-5 per mg and 5 identified the amount to get high as 2 mg or more. A 90 day monitoring of prescription writing by all physicians at the same institution used a threshold of benzodiazepine potency equivalent to 40 mg diazepam daily with unmonitored refills. Using this arbitrary descriptor of potentially hazardous prescribing, CZM was the most commonly "excessively" prescribed benzodiazepine. (Fifty cases out of 194 prescriptions as compared to 14 cases of all other benzodiazepines combined.) Psychiatrists wrote 68%, neurologists 15% and internists 14%. As a result of this information, physicians are cautioned and prescription monitoring mechanisms are recommended.