Transgenes and gene suppression: telling us something new?

Transgenes provide unique opportunities to assess the relationship between genotype and phenotype in an organism. In most cases, introduction and subsequent expression of a transgene will increase (with a sense RNA) or decrease (with an antisense RNA) the steady-state level of a specific gene product. However, a number of surprising observations have been made in the course of many transgenic studies. We develop a hypothesis that suggests that many examples of endogenous gene suppression by either antisense or sense transcripts are mediated by the same cellular mechanism.

Is the development of desalination compatible with sustainable development of the Arabian Gulf?

The development of desalination has been essential to the rapid economic development of the countries bordering the Arabian Gulf. The current production capacity of sea water desalination plants drawing water from Gulf is over 20 million m3 day-1, which may rise to 80 million m3 day-1 by 2050. Whilst supporting aspects of sustainable development related to water and sanitation, desalination impacts the marine environment through impingement and entrainment of organisms in intakes, and through thermal, brine and chemical discharges. This may compromise other objectives for sustainable development related to sustainable use of the oceans. Under business as usual scenarios, by 2050, the impact of individual desalination plants will combine causing a regional scale impact. Without mitigating actions to avoid the business as usual scenario, by 2050, desalination in combination with climate change, will elevate coastal water temperatures across more than 50% of the Gulf by at least 3 °C, and a volume of water equivalent to more than a third of the total volume of water between 0 and 10 m deep will pass through desalination plants each year. This will adversely impact the coastal ecosystem of the Gulf, with impacts on biodiversity, fisheries and coastal communities and may cause potential loss of species and habitats from the Gulf. Given the significant implications of these preliminary findings, and in light of the precautionary approach to management, it is recommended that mitigating options addressing behavioural, regulatory and technological change are rapidly evaluated and implemented to avoid the development of desalination in the region along a business as usual pathway, and multidisciplinary research studies should be conducted to reduce uncertainty in predictions of future impacts.

Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance.

Transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (TEV) coat protein or a form truncated at the N terminus of the TEV coat protein were initially susceptible to TEV infection, and typical systemic symptoms developed. However, 3 to 5 weeks after a TEV infection was established, transgenic plants "recovered" from the TEV infection, and new stem and leaf tissue emerged symptom and virus free. A TEV-resistant state was induced in the recovered tissue. The resistance was virus specific. Recovered plant tissue could not be infected with TEV, but was susceptible to the closely related virus, potato virus Y. The resistance phenotype was functional at the single-cell level because protoplasts from recovered transgenic tissue did not support TEV replication. Surprisingly, steady state transgene mRNA levels in recovered tissue were 12-to 22-fold less than transgene mRNA levels in uninoculated transgenic tissue of the same developmental stage. However, nuclear run-off studies suggested that transgene transcription rates in recovered and uninoculated plants were similar. We propose that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivation.

Effects of essential fatty acid deficiency and indomethacin on histologic, ultrastructural, and phagocytic responses of hepatic macrophages to glucan.

Glucan administration in the rat induces a hyperplasia and hypertrophy of the reticuloendothelial system (RES) and a concomitant leukocytosis. Increased phagocytic function and lysozymal immunoreactivity of macrophages are also characteristic of the glucan effect. The potential role of arachidonic acid metabolites in mediating this hepatic inflammatory response induced by the RES stimulant glucan was assessed in the present study by two experimental approaches. In one study, rats were depleted of arachidonic acid by rendering them deficient in essential fatty acids (EFA). In another study, rats were pretreated with the fatty acid cyclooxygenase inhibitor indomethacin. Both treatment interventions markedly attenuated the hepatic Kupffer cell proliferative and granulomatous response to glucan and the associated leukocytosis. Lysozyme immunoreactivity of the Kupffer cells and rates of colloidal carbon clearance (T/2), however, were enhanced by the above treatments. Supplementation of EFA-deficient rats with ethyl arachidonate restored their glucan response to an extent that was not significantly different from nondeficient rats. Marked hepatic proliferative responses were apparent only in those treatment groups characterized by leukocytosis, which suggests that extrahepatic recruitment is an important component of the glucan response in normal, nutritionally adequate rats. Collectively these data suggest that arachidonic acid metabolites may play a role in modulating this extrahepatic recruitment and the associated cellular proliferative and granulomatous responses following glucan administration of the rat.

Pathogen-derived resistance to a potyvirus: immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence.

Transgenic Nicotiana tabacum 'Burley 49' plants containing one of six different forms of the tobacco etch virus (TEV) coat protein (CP) nucleotide sequence have been generated. In whole plant studies, R1 and R2 progeny were inoculated mechanically with TEV, and the appearance and severity of symptoms were recorded. Symptom phenotype was altered, ranging from near wild type susceptibility to apparent immunity. Protoplasts derived from wild type and transgenic Burley 49 plant lines were transfected with TEV RNA. Protoplasts from transgenic plants expressing full-length or truncated forms of TEV CP supported virus replication. Protoplasts from certain transgenic plants, producing plus- or minus-sense CP transcripts but no CP, did not support virus replication at wild type levels. A model is proposed to account for these observations.

RNA-mediated resistance with nonstructural genes from the tobacco etch virus genome.

Sense RNA-mediated virus resistance has been described for transgenic plants expressing potyviral capsid protein sequences. This study was undertaken to determine if expression of other viral sequences could induce this type of virus resistance. Plants showing highly resistant or 'recovery' phenotypes were generated by expressing the tobacco etch virus (TEV) 6 kDa/21 kDa reading frames. Expression of translatable or untranslatable versions of this TEV sequence produced resistant lines. Highly resistant and recovery phenotype plants expressing TEV coat protein sequences. High transcription rates with low steady-state levels of the transgene transcript generally correlated with resistance. During recovery and induction of the resistance, RNA and protein steady-state levels decreased 5- to 20-fold, while transcription of the transgene continued at a similar level. A posttranscriptional, cellular system eliminating sequences contained in the transgene transcript and viral genome would be consistent with the results.

RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation.

Transgenic Nicotiana tabacum cv. Burley 49 plants were generated that express the 5' untranslated region of the tobacco etch potyvirus (TEV) genome ligated to a mutated version of the TEV coat protein gene sequence that rendered it untranslatable. Eight different transgenic plant lines were analyzed for transgene expression and for resistance to TEV. Three different responses were noted when the transgenic plant lines were inoculated with TEV: 1) some were highly resistant, and no virus replication occurred; 2) some were susceptible but able to recover from systemic TEV infection; and 3) some were susceptible to TEV infection. Plant tissue displaying the recovery phenotype was analyzed for virus replication and transgene expression. Recovered tissue could not be infected with TEV and had steady-state transgene RNA levels which were five- to eightfold lower than those of unchallenged transgenic plant tissue. Nuclear runoff assays suggested a post-transcriptional reduction in specific RNA levels. The highly resistant and recovery phenotypes associated with TEV challenge inoculation and the reduction of steady-state RNA levels in recovered transgenic leaf tissue may be manifestations of a common mechanism.

Second order auditory pathways in the chimpanzee.

Substantial portions of the dorsal, and almost the entire posteroventral and anteroventral (Av) cochlear nuclei were aspirated unilaterally in a chimpanzee. Axonal degeneration was studied by the Fink-Heimer method. The greatest amount of degeneration was followed medially from the region of Av into the lateral part of the trapezoid body. Degeneration also coursed around the superior surface of the restiform body and was traced into the dorsal and intermediate acoustic striae. Within the superior olivary complex, degeneration was distributed to: the ipsilateral lateral superior olive; laterally and medially oriented dendrites of the ipsilateral and contralateral medial superior olivary nuclei respectively (some perisomatic degeneration also was present bilaterally); the contralateral medial trapezoid nucleus; retro-olivary and preolivary cell groups bilaterally. Abundant degeneration passed into the contralateral lateral lemniscus and was distributed largely to its ventral nucleus. The contralateral central nucleus of the inferior colliculus was a major site of termination of ascending second order auditory fibers. The caudal tip of the ipsilateral ventral nucleus of the lateral lemniscus received abundant degeneration, but this diminished rostrally. The ipsilateral inferior colliculus contained a moderate amount of degeneration. A fair number of degenerated second order auditory fibers ascended in the contralateral brachium of the inferior colliculus and were distributed both to the principle and magnocellular divisions of the medial geniculate body. This pathway appears to represent a phylogenetic advance in the brain of the great ape.

High-frequency percussive ventilation improves oxygenation in patients with ARDS.

STUDY OBJECTIVES: To evaluate changes in respiratory and hemodynamic function of patients with ARDS and requiring high-frequency percussive ventilation (HFPV) after failure of conventional ventilation (CV). DESIGN: Retrospective case series. SETTING: Surgical ICU (SICU) and medical ICU (MICU) of an academic county facility. MEASUREMENTS AND RESULTS: Thirty-two consecutive patients with ARDS (20 from SICU, 12 from MICU) who were unresponsive to at least 48 h of CV and were switched to HFPV were studied. Data on respiratory and hemodynamic parameters were collected during the 48 h preceding and the 48 h after institution of HFPV and compared. Between the period of CV and the period of HFPV, the ratio of PaO2 to the fraction of inspired oxygen (F(IO2)) increased ([mean+/-SE] 130+/-8 vs. 172+/-17; p = 0.027), peak inspiratory pressure (PIP) decreased (39.5+/-1.7 vs. 32.5+/-1.9 mm Hg; p = 0.002), and mean airway pressure(MAP) increased (19.2+/-1.2 vs. 27.5+/-1.4 mm Hg; p<0.001). The rate of change of PaO2/F(IO2) per hour was also significantly improved between the two periods. The same changes in PaO2/F(IO2), PIP, and MAP were observed when the last value recorded while the patients were on CV was compared with the first value recorded after 1 h of HFPV. This improvement was sustained but not amplified during the hours of HFPV. The patterns of improvement in these three parameters were similar in SICU and MICU patients as well as in volume-control and pressure-control patients. There were no changes in hemodynamic parameters. CONCLUSION: The HFPV improves oxygenation by increasing MAP and decreasing PIP. This improvement is achieved soon after institution of HFPV and is maintained without affecting hemodynamics.

Multicenter study of noninvasive monitoring systems as alternatives to invasive monitoring of acutely ill emergency patients.

BACKGROUND: Recent reports showed lack of effectiveness of pulmonary artery catheterization in critically ill medical patients and relatively late-stage surgical patients with organ failure. Since invasive monitoring requires critical care environments, the early hemodynamic patterns may have been missed. Ideally, early noninvasive hemodynamic monitoring systems, if reliable, could be used as the "front end" of invasive monitoring to supply more complete descriptions of circulatory pathophysiology. OBJECTIVES: To evaluate the accuracy and reliability of noninvasive hemodynamic monitoring consisting of a new bioimpedance method for estimating cardiac output combined with arterial BP, pulse oximetry, and transcutaneous PO2 and PCO2; we compared this system of noninvasive monitoring with simultaneous invasive measurements to evaluate circulatory deficiencies in acutely ill patients shortly after hospital admission where invasive monitoring was not readily available. We also preliminarily explored early differences in temporal hemodynamic patterns of survivors and nonsurvivors. DESIGN AND SETTING: Prospective comparison of simultaneous invasive and noninvasive measurements of circulatory function with retrospective analysis of data in university-run county hospitals, university hospitals and affiliated teaching hospitals, and a community private hospital. PATIENTS: We studied 680 patients, including 139 severely injured or hemorrhaging patients in the emergency department (ED), 129 medical (nontrauma) patients on admission to the ED, 274 high-risk surgical patients intraoperatively, and 138 patients recently admitted to the ICU. RESULTS: A new noninvasive impedance device provided cardiac output estimations under conditions in which invasive thermodilution measurements were not usually applied. There were 2,192 simultaneous bioimpedance and thermodilution cardiac index measurements; the correlation coefficient, r = 0.85, r2 = 0.73, p < 0.001. The precision and bias was -0.124+/-0.75 L/min/m2. Both invasive and noninvasive monitoring systems provide similar information and identified episodes of hypotension, low cardiac index, arterial hemoglobin desaturation, low transcutaneous O2, high transcutaneous CO2, and low oxygen consumption before and during initial resuscitation. The limitations of noninvasive systems were described. CONCLUSIONS: Noninvasive monitoring systems gave continuous displays of physiologic data that provided information allowing early recognition of low flow and poor tissue perfusion that were more pronounced in the nonsurvivors. Noninvasive systems may be acceptable alternatives where invasive monitoring is not available.

The impact of a dedicated trauma program on outcome in severely injured patients.

BACKGROUND: In recent years, many trauma centers have been closing or scaling down their operations because of financial losses and lack of commitment by the relevant authorities. OBJECTIVE: To investigate the effect of commitment to trauma and the establishment of a dedicated trauma program on injury outcome. DESIGN: In 1992, a well-funded dedicated trauma program was implemented at the Los Angeles County--University of Southern California Medical Center, Los Angeles. We analyzed the outcome in severely injured patients (Injury Severity Score [ISS] > 15) before and after implementation of the program (1991 and 1993). SETTING: Large, urban, level 1 trauma center. PATIENTS: Patients with trauma and an ISS higher than 15. RESULTS: There were 737 patients with an ISS higher than 15 in 1991 and 812 patients with an ISS higher than 15 in 1993. The overall mortality rate was 30% in 1991 and 24.5% in 1993 (P = .018), which is a reduction by 18.3%. In patients with blunt trauma and an ISS higher than 15, mortality was reduced by 33% (mortality rate of 31.1% in 1991 vs 20.8% in 1993) (P < .002). Mortality in patients with penetrating trauma and an ISS higher than 30 was reduced by 42.7% (mortality rate of 59.3% in 1991 vs 34% in 1993) (P = .019). There was also a trend toward lower permanent disabilities among survivors with an ISS higher than 15 (14.7% in 1991 vs 11.3% in 1993). CONCLUSION: Commitment of financial and human resources for the establishment of a dedicated trauma program is a sound investment in terms of improved survival and fewer permanent disabilities in critically injured patients.

Detection of pea seedborne mosaic potyvirus by sequence specific enzymatic amplification.

The polymerase chain reaction (PCR) was used to detect pea seedborne mosaic potyvirus (PSbMV) pathotype P1 RNA after reverse transcription of total nucleic acid preparations from pea (Pisum sativum) tissues. Tissues assayed for PSbMV included leaves, roots, petals, seed parts, and pollen. Three oligonucleotide primers in appropriate combination yielded two products of the predicted size: 730 and 1200 bp. The described methodology allows for rapid pathotype-specific PSbMV detection with utmost sensitivity and wide applicability.

Misconceptions about posterior approach for renoureteral surgery.

Misconceptions about the posterior approach for renal and ureteral surgery are dispelled. A review of the pertinent lumbar anatomy and a few fine points of technique are emphasized. The advantages of easy access to the kidney, minimal postoperative pain, and absence of the occurrence of postoperative hernia warrant more frequent use of this procedure.

Left renal vein hypertension as a cause of persistent gross hematuria.

Left renal vein hypertension as a source of persistent gross hematuria has been described in the literature. This type of hematuria is postulated to result from an elevation of left renal vein pressure leading to calyceal-venous communications. We report a case of left renal vein hypertension in which surgical and radiologic findings identify the cause of the hypertension as an aberrant peripheral nerve. To our knowledge, this etiology has never been described. A review of the literature with emphasis on the pathophysiology of renal-vein hypertension is also presented.

The Bloch equations in high-gradient magnetic resonance force microscopy: theory and experiment.

We report theory and observations of paramagnetic resonance in a measured field gradient of 44,000 T per meter by the technique of magnetic resonance force microscopy (MRFM). Resonance was induced in a dilute solid solution of diphenylpicrylhydrazyl in polystyrene at 77 and 10 K by an amplitude-modulated microwave field. This modulated the force between resonant sample spins and a micrometer-scale SmCo magnetic tip on a force microscope cantilever. The force signals were typically of order 10 fN, and were detected above a thermal noise floor of 80 aN per root hertz at 10 K, equivalent to a magnetic moment noise of 200 micro(B) per root hertz of bandwidth. Resonance saturation was readily observed. Starting with the Bloch equations, we derived simple analytic expressions for the predicted cantilever signal amplitudes and T(1)-dependent phase lags, valid at low microwave power levels. For power levels below saturation, the data were in good agreement with the Bloch equation predictions, while above saturation the measured force increased more slowly with power than predicted. Several ESR mechanisms which might lead to non-Bloch dynamics in the MRFM environment are reviewed. Spin-relaxation mechanisms are also reviewed. A detailed description of the experimental apparatus is offered.

Radioimmunoassay of rhesus monkey chorionic gonadotropin.

A radioimmunoassay for rhesus monkey chorionic gonadotropin (mCG) employing an antiovine LH antiserum, 125I or 131I-ovine LH tracer, and mCG for standards was developed. Radioimmunoassay of serum levels of mCG during pregnancy indicated that mCG began increasing as early as day 12 after mating, reached a peak by day 25, and declined to nondetectable levels around day 35. The assay procedure was adapted for use as a rapid method for pregnancy diagnosis; the results were available as early as 12 hours after collection of serum samples. The method is capable of detecting a few pregnancies by day 12 and all pregnancies by day 17. Routine use of this method provided accurate pregnancy diagnosis four days earlier than was possible with the mouse uterine weight bioassay method previously used in this laboratory.

Duration of antibiotic prophylaxis in high-risk patients with penetrating abdominal trauma: a prospective randomized trial.

To evaluate the effect of varying durations of antibiotic prophylaxis in trauma patients with multiple risk factors for postoperative septic complications, a prospective randomized trial was undertaken at an urban level I trauma center. The inclusion criteria were full-thickness colon injury and one of the following: (1) Penetrating Abdominal Trauma Index > 25, (2) transfusion of 6 units or more of packed red blood cells, or (3) more than 4 hours from injury to operation. Patients were randomly assigned to a short course (24 hours) or a long course (5 days) of antibiotic therapy. All patients received 2 g cefoxitin en route to the operating room and 2 g intravenously piggyback every 6 hours for a total of 1 day vs. 5 days. Sixty-three patients were equally divided into short-course (n = 31) and long-course (n = 32) therapy. This was a high-risk patient population, as assessed by the mean Penetrating Abdominal Trauma Index (33), number of patients with multiple blood transfusions (51 of 63; 81%), number of patients with an Injury Severity Score greater than 15 (37 of 63; 59%), number of patients with destructive colon wounds requiring resection (27 of 63; 43%), and number of patients requiring postoperative critical care (37 of 63; 59%). Differences in intra-abdominal (1-day, 19%; 5-days, 38%) and extra-abdominal (1-day, 45%; 5-days, 25%) infection rates did not achieve statistical significance. There continues to be no evidence that extending antibiotic prophylaxis beyond 24 hours is of benefit, even among the highest risk patients with penetrating abdominal trauma. A large, multi-institutional trial will be necessary to condemn this common practice with statistical validity.

Cranioplasty with the Medpor porous polyethylene flexblock implant. Technical note.

The authors describe the use of a porous polyethylene Flexblock implant for cosmetic cranioplasty. The implant may be used to cover any small- or medium-sized (< 8 cm) cranial defect, offering similar cosmetic results to standard alloplast cranioplasty while decreasing operation time. The porous implant design permits ingrowth of soft tissue and bone to increase implant strength and decrease the risk of infection. The Flexblock alloplast has been utilized in 25 cases with excellent cosmetic results and no implant-related complications.

The complexities of managing severe burns with associated trauma.

A high incidence of severe inhalation injuries can be expected in the combined injury patient. The initial management remains attention to the ATLS priorities of airway, breathing, and circulation, with prompt and safe transfer to a regional center of excellence. The treatment of either the burn or the associated injuries may be compromised by their combined presence, and a team approach is essential to their optimal management. Circulatory management goals based on oxygen consumption and delivery allow greater understanding and control of the physiologic demands placed on the patient by the disease process. The management of inhalation injury and ARDS is at an exciting turning point in history, and we now have in hand and use many techniques that allow salvage of these mortal conditions. Pain management is essential to humane care and requires frequent assessment and patient control to be effective. Rehabilitation of the burn and trauma patient starts on the day of injury and requires team dedication to the areas of greatest morbidity early in the planning of surgical priorities and physical therapy.

Necrotizing fasciitis of the face.

Necrotizing fasciitis (NF) is a rapidly progressive soft tissue infection with high morbidity and mortality rates. Its occurrence in the head and neck region is uncommon, the majority of reported cases being limited to involvement of the neck, usually from infections of dental or pharyngeal origin. Involvement of the face from NF is rare; only 35 such cases have been reported in the literature since 1960. It is not only associated with a high mortality but can also result in severe disfigurement of the face, posing challenging reconstructive problems. Successful management of facial NF requires early diagnosis, prompt institution of broad spectrum antibiotics, aggressive surgical debridement to control the infection, and reconstruction of the resultant soft tissue defects. This report describes four additional cases of facial necrotizing fasciitis. One of the four patients died as a result of sepsis and multi-organ system failure. Two of the three surviving patients had significant facial disfigurement. A comprehensive review of the facial NF cases reported in the literature is also provided. Based on our experience with facial NF and the results of all previous case reports, the clinical manifestations, pathogenesis, and management of this disease are discussed.