BACKGROUND: A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles. METHODS: In the present study a system was developed in which genetic reshuffling during the retroviral life cycle is exploited, allowing reconstitution of functional expression cassettes from separate elements exclusively in transduced target cells. For construction of these murine leukaemia virus (MLV)-based reconstituting viral vectors (ReCon), a promoterless inverted enhanced green fluorescent protein (EGFP) reporter gene cassette was inserted in place of the U3 region of the 3' LTR. Subsequently, the human ubiquitin promoter was inserted in the inverse orientation into the R/U5 border of the 5' LTR of the vector. RESULTS: PA317 packaging cells stably transfected with ReCon vectors were established and EGFP expression was analysed by fluorescence-activated cell sorting (FACS). After detection of low-level background expression, an additional polyadenylation signal was introduced in antisense orientation into the 3' LTR at the R/U5 border to prevent accidental read-through transcription from neighbouring cellular promoters. Virus-containing cell culture supernatants were then used to infect NIH3T3 target cells. EGFP expression, recloning and sequencing of integrated proviruses demonstrated the correct reassembly of the transduced ubiquitin/EGFP transcription unit in these infected cells. CONCLUSIONS: This facile and convenient system should allow production of retroviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector production if expressed in the retroviral packaging cell.

3T3 Cells↗

PubMedDate not supplied

Xenoreactive natural antibodies and induced antibodies--their effects of beating cardiomyocytes as a model of a xenograft.

Xenotransplantation has been complicated by hyperacute rejection reactions, which are supposedly triggered by preformed natural antibodies (PNAb) of the recipient organism, whereas the role of antibodies specifically induced by previous antigen contact (IAb) is less clear. Primary cultures of spontaneously beating neonatal rat cardiomyocytes were used as a model of the heart to elaborate the effects of both PNAb and IAb from xenogeneic species and to investigate into their mechanisms of action. An experimental setup allowing for rapid medium exchange under continuous observation was employed. Sera containing PNAb reproducibly bring about a stereotype pattern of altered contractility including an initial increase in beating frequency followed by a temporary cessation of beating within the first minutes after administration. After recovery of spontaneous contractions, the cells within the monolayer exhibited a dissociation of the synchronicity of the beating persisting for several hours. The temporary pause in beating was prevented by a very high extracellular calcium concentration, but not by extracellular electrical stimulation sufficient to trigger contractions in control cells. Electrophysiological measurements carried out in adult ventricular guinea pig heart muscle cells under the same experimental conditions revealed an increase of the excitation threshold of the cells after application of sera containing PNAb due to an enhanced input resistance. These results indicate that the effect of PNAb is the consequence rather of a generally reduced excitability of the cell than of the inhibition of a singular ionic conductance. After specific absorption of PNAb directed against rat antigens beating of neonatal rate cardiomyocytes ensued without interruption. Sera specimens devoid of complement produced similar effects on contractility, although the duration of the standstill period was significantly shorter. The increase in input resistance visualized in guinea pig myocytes was absent after removing PNAb against guinea pig antigens but not after absorbing PNAb directed against rat epitopes. Signs of a permanent cytotoxicity after the administration of PNAb were lacking in all experiments. IAb against rat heart tissue raised in rabbits stopped the contractions of neonatal rat cardiomyocytes within 30 min after administration irreversibly and lead concentration-dependently to a destruction of the cells.

Animals↗

PubMedDate not supplied

Metallothionein induction by nonsteroidal antiinflammatory drugs.

In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).

Animals↗

PubMedDate not supplied

Inducible expression of p21WAF-1/CIP-1/SDI-1 from a promoter conversion retroviral vector.

Constitutive, high-level expression of the potentially therapeutic WAF-1/CIP-1/SDI-1 gene is incompatible with cell growth. A promoter conversion retroviral vector carrying the WAF-1/CIP-1/SDI-1 gene under the transcriptional control of the glucocorticoid inducible promoter of mouse mammary tumor virus was used to infect human bladder carcinoma or feline kidney cells. Reduced cell growth due to a greater proportion of cells being in the G0/G1 phase of the cell cycle was observed when WAF-1/CIP-1/SDI-1 expression was activated by addition of glucocorticoid hormone. This system demonstrates the potential long-term therapeutic use of WAF-1/CIP-1/SDI-1 delivered by retroviral vectors for inhibiting the growth of rapidly proliferating cells. Moreover, the conditional expression of genes such as WAF-1/CIP-1/SDI-1 from such retroviral vectors may facilitate analysis of their function.

Animals↗

PubMedDate not supplied

Genetic and epidemiological investigations on pigmentary degeneration of the retina and allied disorders in Switzerland.

Explore the source record for details and available documents.

Adult↗

PubMedDate not supplied

The syndrome of Laurence-Moon-Bardet-Biedl and allied diseases in Switzerland. Clinical, genetic and epidemiological studies.

Explore the source record for details and available documents.

Adolescent↗

PubMedDate not supplied

Protection of islets in culture by delivery of oxygen binding neuroglobin via protein transduction.

Islet transplantation has become an accepted method to treat type 1 diabetes. To succeed and achieve normal levels of glucose in transplant recipients, the quality of the transplanted islets is of the utmost importance. Lack of oxygen during organ procurement, islet isolation, and subsequent culture triggers apoptosis or necrosis and loss of islet function, causing the yield and quality to diminish. A promising candidate for cytoprotection against oxygen deprivation is neuroglobin (Ngb). Ngb is a recently described member of globin family and is expressed in neurons, retina, and pancreatic islets. To overexpress this protein in the islets and study its ability to protect them, we utilized protein transduction. Protein transduction is achieved by fusing Ngb to the TAT/PTD transduction domain, a peptide originated from the HIV transcriptional transactivator protein. Our study proved that TAT-Ngb is an efficient fusion protein capable of protecting the human islets in culture from loss of cell mass and function, thus increasing the quality of transplantable islets. If the islets could be cultured for a longer period of time without suffering harmful effects, it would be possible to precondition the recipient and there would be more time to assess their quality and function before transplantation.

Automation↗

PubMedDate not supplied

Psychiatric disorders in patients with fibromyalgia. A multicenter investigation.

The authors conducted an investigation in four tertiary-care centers to determine if psychiatric comorbidity and psychological variables were predictive of functional impairment in patients with fibromyalgia syndrome (FMS). Seventy-three individuals were administered the Structured Clinical Interview for DSM-III-R, the Rand 36-item Health Survey (SF-36), and multiple self-report measures. The patients with FMS were found to have a high lifetime and current prevalence of major depression and panic disorder. The most common disorders were major depression (lifetime [L] = 68%, current [C] = 22%); dysthymia (10% [C only]); panic disorder (L = 16%, C = 7%); and simple phobia (L = 16%, C = 12%). The self-report scales revealed significant elevations in depression, anxiety, neuroticism, and hypochondriasis. Functional impairment on all measures of the SF-36 was severe (e.g., physical functioning = 45.5 and role limitations due to physical problems = 20.0). Stepwise multiple-regression analysis revealed that current anxiety was the only variable that predicted a significant proportion of the variance (29%) in SF-36 physical functioning. Thus, in this multicenter study, the persons with FMS exhibited marked functional impairment, high levels of some lifetime and current psychiatric disorders, and significant current psychological distress. Current anxiety level appears to be an important correlate of functional impairment in individuals with FMS.

Adult↗

PubMedDate not supplied

Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay.

A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.

Animals↗

PubMedDate not supplied

[Ultrasonographic measurement of leg length and leg length differences].

A new diagnostic method for measuring the leg length and the leg length difference by means of ultrasound is presented. A special device for holding and moving the ultrasound transducer was constructed. The measuring points on the hip, knee and upper ankle can be visualised by means of a 5- or 7.5-MHz linear scanner. The measuring device gives the distance of the points in cm so that the difference correspond to the length of the leg, femur and tibia. Tests conducted on corpes and clinical examples show that ultrasound in combination with our special device is an ideal method for determining the exact lengths of the leg. The ultrasound measuring of the leg length offers a reliable, non invasive and easily performed method. Because ultrasound is not limited by radiation hazards, our technique can be used for clinical screening.

Adolescent↗

PubMedDate not supplied

[An opto-electronic procedure for the analysis of movement and posture].

The described procedure has been developed for the quantitative estimation of human postures and motions. The system is based upon the detection of the components of the force vector between the foot and the ground and also the position of the marked pints of rotation of the joints or of the markes above the spine. The course of this vector of force in conjunction with the distance of the pints of rotation permits conclusions to be drawn about stress of joints during walking or different posture.

Biomechanical Phenomena↗

PubMedDate not supplied

[Etiology of hip dislocation in children with cerebral movement disorders and possibilities of conservative treatment using rotating and spreading plates and electrostimulation].

Dislocations of the hip associated with spastic paralysis are almost always found in severely disabled children. The onset is in infancy. Two methods, electrostimulation of the minor gluteal muscles and treatment with simple leg casts to correct the malposition, are described. Follow-up examinations showed that the treatment had had a positive effect on the hip condition in 33 of the 42 children treated with casts and 26 of the 32 children treated by electrotherapy. Electrotherapy is an innovation, since in the past it was considered contraindicated in these patients. However, it is based on the fact that the gluteal muscles do not normally show any spasticity. Irrespective of this, the treatment is long-term, and has to be continued for many years, a fact which makes greater demands on the cooperativeness of the family.

Adolescent↗

PubMedDate not supplied

Perspectives of pregnant substance-using women: findings from the California Perinatal Needs Assessment.

This article reports on selected major findings of a study on alcohol and drug use patterns and problems and unmet service needs, drawing on interview data from a sample of substance-involved pregnant women who were clients at public health, social service, and criminal justice agencies in two California counties. Based on screening rates, a sizable group of pregnant agency clients regularly used alcohol and other drugs, particularly marijuana and cocaine, and most of these users had not been in substance abuse treatment. Reported use of alcohol and other drugs diminished considerably during pregnancy. Negative consequences of alcohol and drug use, problems with family and friends' substance use, neighborhood alcohol and drug problems, and substance-related violence were widely experienced. The greatest service needs include job placement, educational programs and housing. Prenatal health care was widely received, although often inconsistently or late in the pregnancy. Despite high treatment satisfaction among those in recovery programs, relatively few women desired formal treatment. These findings, which are consistent with those of other recent studies, suggest the complexity of problems and needs among substance-involved, low-income pregnant and parenting women.

Adolescent↗