Inducible control of gene expression: prospects for gene therapy.

A number of drug-related gene expression systems are available for controlling target gene transcription through the use of small-molecule inducing compounds. While the utility of such systems has been demonstrated in vitro and in transgenic mice, recent improvements are likely to make these systems more amenable for use in a therapeutic context, such as gene therapy. These improvements include further optimization of the antiprogestin-regulated gene switch, rendering it more sensitive to RU486, and the synthesis of nonimmunosuppressive rapamycin analogs for use in dimerization-based strategies of gene regulation.

A method for measuring rate of neutrophil phagocytosis of Staphylococcus epidermidis or Candida guilliermondii using uptake of tritiated uridine.

A method is described for measuring the rate of neutrophil phagocytosis, together with the total and intracellular kill at 20 min of 2 different organisms. The technique is sensitive over the early stages of phagocytosis as it distinguishes between adherent and engulfed organisms and is totally objective. It therefore allows detection of defects in phagocytosis which are missed if only the end point is assessed. Both total and intracellular killing are measured under the same conditions as those used to assess phagocytosis and results obtained on the same day the assay is performed.

QUNTITATIVE ION DISTRIBUTION WITHIN ROOT CELLS OF SALT-SENSITIVE AND SALT-TOLERANT MAIZE VARIETIES.

Significant varietal differences were apparent in the survival of seedlings of maize in saline conditions but only at relatively high external concentrations (200 mol m-3 NaCl), where there was a range from 0 to 66% survival, 25 d after salinization. For the varieties examined there was a strong negative correlation between Na concentrations in the third leaf and survival. Two resistant varieties (Across 8024 and Protador) and one salt-sensitive variety (LG11 ) were identified. The characteristics of ion accumulation were clearly different in salt-tolerant and salt-sensitive types, the difference becoming more pronounced with plant age. The distribution of ions, particular those of Na, K and Cl, was determined within subcellular compartments of roots cells using X-ray microanalysis of freeze-substituted tissue. Salinity induced a greater increase (about 1.7 times) in cytoplasmic Na concentration in the salt-sensitive variety (LG11 ) than in resistant varieties (Across 8024 or Protador). The mean K:Na ratio in the cytoplasm of the root cortical cells in the salt-resistant varieties grown for 15 d in saline conditions (100 mol m-3 NaCl) was twice that found for LG11 . Sodium and Cl concentrations in the vacuoles decreased radially inwards from the epidermal cells in salt-treated roots of LG11 .

Transient hypocalcemic reperfusion does not improve postischemic recovery in the rat heart after preservation with St. Thomas' Hospital cardioplegic solution.

We used the isolated perfused working rat heart to investigate the effects of transient hypocalcemic reperfusion after cardioplegic arrest with the St. Thomas' Hospital cardioplegic solution and 25 minutes of global normothermic (37 degrees C) ischemia. Hearts were reperfused (Langendorff mode) transiently (20 minutes) with solutions containing various concentrations of calcium; this was followed by 30 minutes of reperfusion with standard (1.4 mmol/L, the physiologic concentration) calcium buffer (10 minutes in the Langendorff mode and 20 minutes in the working mode). Recovery of cardiac output in control hearts (calcium concentration 1.4 mmol/L throughout) was 51.7% +/- 4.6%; in hearts transiently reperfused with hypocalcemic buffer (0.25, 0.5, 0.75, or 1.0 mmol/L) the recoveries of cardiac output were 49.3% +/- 6.4%, 52.2% +/- 7.2%, 58.7% +/- 3.2%, and 47.2 +/- 4.7%, respectively (all not significant), whereas recovery was only 14.7% +/- 2.8% (p less than 0.05) in hearts transiently reperfused with calcium 0.1 mmol/L. Creatine kinase leakage was significantly (p less than 0.05) greater in the group reperfused with calcium 0.1 mmol/L, but it did not vary significantly between the other groups. Tissue high-energy phosphate content was similar and in the normal range in all groups except for the group reperfused with calcium 0.1 mmol/L. In further experiments, the duration of hypocalcemic (0.5 mmol/L) reperfusion was varied (0, 5, 10, 15, 20, or 30 minutes). No significant differences in recovery of cardiac output were observed (58.2% +/- 5.0%, 52.3% +/- 5.7%, 52.0% +/- 8.2%, 61.2% +/- 5.0%, 62.2% +/- 4.3%, and 66.2% +/- 3.2%, respectively). In additional studies, the standard calcium concentration (1.4 mmol/L) used before and after ischemia was replaced by hypercalcemic solution (2.5 mmol/L). Despite this, transient (10 minutes) hypocalcemic (0.5 mmol/L) reperfusion did not improve recovery. Finally, studies were undertaken with a longer duration of ischemia (40 minutes), and although recovery of cardiac output in the hypocalcemic group (0.5 mmol/L for 10 minutes) tended to be higher than in the control group (29.7% +/- 4.8% versus 18.5% +/- 4.9%, respectively), statistical significance was not achieved. We conclude that in these studies transient hypocalcemic reperfusion did not afford any additional protection over and above that afforded by cardioplegia alone.

Instant center of rotation estimation using the Reuleaux technique and a Lateral Extrapolation technique.

A mathematical model of a rolling wheel was used to investigate the errors encountered when the Reuleaux technique is employed to estimate planar instant centers of rotation (ICRs). The investigation showed that large errors can result when this pole measurement technique is applied to objects rotating more than 12 degrees. The investigation also showed that these errors can be substantially reduced by applying a new Lateral Extrapolation technique to the pole data. When the Reuleaux technique is applied to marks on a 10cm radius wheel, the resulting offset errors from the ICR are 3.96cm for a 45 degrees roll and 1cm for a 12 degrees roll. Following lateral extrapolation, these offset errors reduce to 0.52cm for the 45 degrees roll and less than 0.04cm for the 12 degrees roll. Thus, the extrapolation technique is over seven times more accurate for a 45 degrees roll, and over 25 times more accurate for a 12 degrees roll. The extrapolation technique has been validated with the model for joints that exhibit both slip and roll, such as the knee. As joint ICR pathway measurement can be used to detect pathology, these accuracy improvements offer potential benefits for clinical applications.

A surface-marker imaging system to measure a moving knee's rotational axis pathway in the sagittal plane.

This paper describes a new surface-marker imaging system designed to measure the rotational axis pathway (RAP) of a moving knee in the sagittal plane. Measurement of this parameter can provide important information about a knee's slipping and rolling action that can aid clinical assessment. Seated subjects are video recorded as they actively extend their legs. A series of stills is then captured and analyzed to extract the coordinates of markers placed on the subjects upper and lower legs. These coordinates are then processed to deduce an instant center of rotation (ICR) for each still. These ICRs are then plotted to derive the joint's RAP. The system has been validated with a mechanical model and tested in a clinical study of ten patients with unilateral anterior cruciate ligament (ACL) ruptures. The study found that the system could consistently measure differences between a patients normal and injured knees. Leg extension caused the normal knees ICRs to displace anteriorly with a mean value of 17.4 mm, whereas the injured knees had a mean displacement of 7.5 mm. This loss of roll in the ACL-deficient knees is consistent with their abnormal biomechanical arrangement.

Effect of feverfew on phagocytosis and killing of Candida guilliermondii by neutrophils.

The in vitro effect of the herbal remedy feverfew on neutrophil function was examined. It was shown that addition of feverfew extract inhibited phagocytosis of Candida guilliermondii and its overall killing. However, intracellular killing was not affected, suggesting that the apparent defect in total killing merely reflected the failure of uptake. The implications for the in vivo effects of feverfew are discussed.

Measurement of body surface topography using an automated imaging system.

An automatic computer imaging system for recording body surface topography has been developed on a microcomputer-based image processing system. The computer processes fringe patterns generated on the surface of the trunk and reconstructs the complete 3-dimensional form of the surface. From the topographic reconstruction, clinical parameters of scoliotic deformity such as Angle of Trunk Inclination are calculated at a number of levels from the upper thoracic to the sacral region. These multiple level measurements illustrate the change in deformity over the trunk and correspond to measurements obtained using conventional tactile devices on patients.

Management of venous and cardiovascular thrombosis: enoxaparin.

Enoxaparin has strong clinical evidence that supports its license in a broad spectrum of therapeutic indications, including thromboprophylaxis in surgical patients, medical patients bedridden because of acute illness, the once-daily treatment of venous thromboembolism and the treatment of unstable angina and non-Q wave myocardial infarction.

Freeze-substitution.

Freeze-substitution is a technique suitable for the preparation of unicellular and multi-cellular plant and animal specimens for conventional light microscopy, TEM and SEM. It is also widely used as a means of preparing animal and plant tissues for the localization of water soluble substances by analytical electron microscopy, autoradiography or visual detection of precipitates. The technical requirements of preparation, together with an evaluation of the procedures, are presented for various applications. Careful selection and evaluation of freezing technique, substitution solvent and regime are required for meaningful results.

The effects of salinity on ion concentrations within the root cells of Zea mays L.

Zea mays is a salt-sensitive crop species which in saline (≦100 mol m(-3) NaCl) conditions suffers considerable growth reduction correlated with elevated Na(+) and Cl(-) concentration within the leaves. To increase understanding of the regulation of ion uptake and transport by the roots in saline conditions, ion concentrations within individual root cortical cells were determined by X-ray microanalysis. There was variation in Na(+), K(+) and Cl(-) distributions among individual cells, which could not be correlated with their spatial position in the roots. Generally, however, in response to saline growth conditions (100 mol m(3) NaCl) Na(+) and Cl(-) were mostly localized in the vacuoles, although their concentrations were also sometimes increased in the cytoplasm and cell walls. The concentration of K(+) in the cytoplasm was usually maintained at a level (mean 79 mol m(-3)) compatible with the biochemical functions ascribed to this ion.

p53 alteration is a common event in the spontaneous immortalization of primary BALB/c murine embryo fibroblasts.

It has been shown previously that mutant p53 can act as an immortalizing gene when cotransfected into primary rat embryo fibroblasts along with a selectable marker. To determine whether a mutation at the p53 locus is a common event in the pathways leading to spontaneous cellular immortalization, 11 clonally derived BALB/c murine embryo fibroblast lines were established by passage on a 3T3 schedule and examined for p53 alterations. By the following criteria, all 11 independently established lines contain at least one mutant allele of p53. Seven of these lines have a PAb240-reactive p53 species and exhibit an extended p53 half-life as determined by pulse-chase analysis. The p53 protein species in a subset of these lines is also capable of complex formation with the constitutive heat shock protein hsc70. p53 cytoplasmic DNAs (cDNAs) from several of these lines have been cloned by reverse transcription of cytoplasmic RNA followed by PCR amplification, and the mutations have been mapped by DNA sequence analysis. Point mutation in conserved domains of p53 appears to be a common alteration in these lines, although one established line carries a 24-bp in-frame deletion of p53. The remaining four cell lines do not express detectable p53 protein. For each line there is a different molecular event underlying the lack of p53 expression: (1) deletion of at least the first 6 exons of both p53 alleles; (2) expression of a single p53 mRNA encoding a stop codon at amino acid position 173; (3) no detectable p53 mRNA; and (4) greatly diminished expression of p53 mRNA. These findings indicate that p53 alteration commonly occurs in spontaneously immortalized BALB/c mouse embryo fibroblasts passaged on a 3T3 schedule and, therefore, may be an important event for the immortalization process.

Localization of kainate receptors to the presynaptic active zone of the rod photoreceptor in primate retina.

Visual information is encoded at the photoreceptor synapse by modulation of the tonic release of glutamate from one or more electron-dense ribbons. This release is highest in the dark, when photoreceptors are depolarized, and decreases in grades when photoreceptors hyperpolarize with increasing light. Functional diversity between neurons postsynaptic at the synaptic ribbon arises in part from differential expression of both metabotropic (G-protein-gated) and ionotropic (ligand-gated) glutamate receptor. In the brain, different subunits also modulate the presynaptic active zone. In hippocampus, ionotropic kainate receptors localize to the presynaptic membrane of glutamatergic axon terminals and facilitate depolarization of the synapse (e.g. Lauri et al., 2001). Such facilitation may be helpful in the retina, where consistent depolarization of the photoreceptor axon terminal is necessary to maintain glutamate release in the dark. We investigated whether such a mechanism could be present in primate retina by using electron microscopy to examine the localization of the kainate subunits GluR6/7 at the rod axon terminal, where only a single ribbon synapse mediates glutamate release. We scored 54 rod axon terminals whose postsynaptic space contained one or more GluR6/7-labeled processes and traced these processes through serial sections to determine their identity. Of 68 labeled processes, 63% originated from narrow "fingers" of cytoplasm extending from the presynaptic axon terminal into the postsynaptic cleft. Each rod terminal typically inserts 4-6 presynaptic fingers, and we scored several instances where multiple fingers contained label. Such consistency suggests that each presynaptic finger expresses GluR6/7. The physiological properties of kainate receptors and the geometry of the rod axon terminal suggest that presynaptic GluR6/7 could provide a steady inward current to maintain consistent depolarization of the rod synapse in the long intervals between photons in the dark.

Improvement of quantitation of biological X-ray microanalysis.

Absolute measurements of elemental concentrations within thin biological samples are often made by reference to a series of standards which resemble the samples in chemical and physical properties and the linear relationship between (p-b)/c and concentration. This principle requires that the chemical and physical properties of the matrix remain constant throughout a series of standards with different elemental contents and throughout different regions of the samples. Some of the changes undergone by specimens during X-ray microanalysis, e.g. loss of elements or organic mass loss, are also influenced by the composition of the matrix. A simple empirical modification to the linear (p-b)/c versus concentration relationship is presented to account for some of these effects and therefore improve quantitation of analyses.

Evidence for the cytoplasmic localization of betaine in leaf cells of Suaeda maritima.

An attempt has been made to localize glycinebetaine in shoots of Suaeda maritima L. Dum. using a technique based on the formation of an iodoplatinate precipitate. Deposits were largely restricted to the cytoplasm of salt-grown plants and were analysed by transmission analytical electron microscopy. The results are considered to support the hypothesis that glycinebetaine acts as a cytoplasmic osmoticum to balance high vacuolar salt levels in certain halophytes.

Neutrophil function in patients on continuous ambulatory peritoneal dialysis.

Frequent and recurrent episodes of peritonitis are a major cause of morbidity in patients on continuous ambulatory peritoneal dialysis (CAPD). One factor contributing to this problem may be an abnormality of neutrophil function in these patients. We have therefore quantified phagocytosis and killing by circulating and peritoneal neutrophils from patients on CAPD with and without peritonitis. Circulating neutrophils from uninfected patients showed reduced phagocytosis of both Staphylococcus epidermidis and Candida guilliermondii because of an opsonic defect in CAPD serum and because of a defect of the neutrophils themselves. In contrast, phagocytosis by circulating and peritoneal neutrophils from patients with peritonitis was normal. Intracellular killing of C. guilliermondii was normal in all groups of neutrophils but killing of S. epidermidis, the organism most commonly isolated in CAPD peritonitis, was reduced. The possible mechanisms for the enhanced neutrophil activity seen in peritonitis, and for the decreased killing of S. epidermidis in contrast to normal killing of C. guilliermondii are discussed. A defect in killing of S. epidermidis may explain why peritonitis caused by this organism can be difficult to erradicate.

Effect of dialysate fluids on phagocytosis and killing by normal neutrophils.

Inadequate host defenses may partly explain the problem of recurrent peritonitis in patients on continuous ambulatory peritoneal dialysis. It has been suggested that these defenses may be adversely affected by the fluids used for dialysis, and so we examined the effects of unused, effluent, and infected peritoneal dialysis fluids on phagocytosis and killing by normal neutrophils. We used a clinical isolate of Staphylococcus epidermidis as the test organism, as this organism is the most commonly cultured in continuous ambulatory peritoneal dialysis peritonitis; we also used a fungal species, Candida guilliermondii. There was no phagocytosis of either organism in unused dialysate because of lack of opsonins and low pH. Phagocytosis in effluent dialysate did not occur because of inadequate opsonin levels and was variable in infected effluents, depending on quantities of immunoglobulins present. Intracellular killing of both test organisms was normal in unused dialysate in the presence of 5% normal serum, but was reduced in effluent and infected dialysates because of factors inhibiting killing by neutrophils. These factors adversely affected the killing of S. epidermidis more than that of C. guilliermondii. These results may explain why peritonitis recurs, particularly peritonitis due to S. epidermidis, because organisms could be sequestered within the neutrophils and thus be protected from antibiotic action. Reinfection of the peritoneal cavity would then take place following neutrophil breakdown, causing a clinical relapse.

Allopurinol-enhanced myocardial protection does not involve xanthine oxidase inhibition or purine salvage.

Isolated working rat hearts were subjected to aerobic perfusion (25 min), cardioplegic infusion (3 min), global ischemia (30 min at 37 degrees C) and reperfusion (35 min). Measurements of myocardial xanthine oxidase and dehydrogenase activity, together with various adenine nucleotides and metabolites, were made at defined stages of the protocol (n = 6/group). Allopurinol pretreatment (20 mg/kg body wt/day for 3 days) improved the postischemic recovery of cardiac function; thus, aortic flow (a representative index) recovered to 68.8 +/- 4.2% compared with 53.2 +/- 2.3% in untreated controls (p less than 0.05). In fresh tissue, allopurinol pretreatment inhibited xanthine dehydrogenase activity by 73.1% (from 11.9 +/- 0.5 to 3.2 +/- 0.8 mIU/g wet wt: p less than 0.05) and xanthine oxidase activity by 95.2% (from 8.3 +/- 1.2 to 0.4 +/- 0.2 mIU/g wet wt: p less than 0.05); however, this inhibition was not maintained during perfusion. During reperfusion, myocardial xanthine dehydrogenase and oxidase activity was reduced by 40-60% (p less than 0.05) in both allopurinol pretreated and control hearts. Tissue content of creatine phosphate, adenosine triphosphate and catabolites, NAD and inorganic phosphate were not different in allopurinol pretreated or control hearts during either ischemia or reperfusion. This study does not support the concept that allopurinol protects the rat heart during ischemia and reperfusion by inhibition of xanthine oxidase activity or by conservation of purines. It appears that allopurinol achieves its protective effects by some, as yet undefined, mechanism.

Quantitative ion localization within Suaeda maritima leaf mesophyll cells.

Grown under saline conditions, Suaeda maritima accumulates Na(+) and Cl(-) into its leaves, where individual mesophyll cells behave differently in their compartmentation of these ions. Measurements of ion concentrations within selected subcellular compartments show that freeze-substitution with dry sectioning is a valuable preparative technique for analytical electron microscopy of highly vacuolate plant material. Using this approach, absolute estimates were made of Na(+), K(+) and Cl(-) concentrations in the cytoplasm, cell walls, chloroplasts and vacuoles of leaf mesophyll cells.