Post-translational modification and stability of low molecular weight cyclin E.

Our laboratory has previously described the presence of five tumor-specific low molecular weight isoforms of cyclin E in both tumor cell lines and breast cancer patient biopsies. We have also shown that one of these low forms arises from an alternate start site, whereas the other four appear as two sets of doublets following cleavage through an elastase-like enzyme. However, the origin of both sets of doublets was unknown. Here, we demonstrate that the larger isoform of each doublet is the result of phosphorylation at a key degradation site. Through site-directed mutagenesis of different phosphorylation sites within the cyclin E protein, we discovered that phosphorylation of threonine 395 is responsible for generating the larger isoform of each doublet. Because phosphorylation of threonine 395 has been linked to the proteasome-mediated degradation of full length cyclin E, we examined the stability of T395A phospho-mutants in both non-tumorigenic mammary epithelial cells and tumor cells. The results revealed that the low molecular weight isoforms appear to be stable in both a tumor cell line and a non-tumor forming cell line regardless of the presence of this critical phosphorylation site. The stability of low molecular weight cyclin E may have implications for both tumorigenesis and treatment of tumors expressing them.

Transcranial magnetic stimulation of left prefrontal cortex impairs working memory.

OBJECTIVES: Several lines of evidence suggest that the prefrontal cortex is involved in working memory. Our goal was to determine whether transient functional disruption of the dorsolateral prefrontal cortex (DLPFC) would impair performance in a sequential-letter working memory task. METHODS: Subjects were shown sequences of letters and asked to state whether the letter just displayed was the same as the one presented 3-back. Single-pulse transcranial magnetic stimulation (TMS) was applied over the DLPFC between letter presentations. RESULTS: TMS applied over the left DLPFC resulted in increased errors relative to no TMS controls. TMS over the right DLPFC did not alter working memory performance. CONCLUSION: Our results indicate that the left prefrontal cortex has a crucial role in at least one type of working memory.

Standard and alternative methods of stretcher carriage: performance, human factors, and cardiorespiratory responses.

Transporting a casualty on a stretcher is a common task for medical and military personnel. Stretchers are usually carried by hand, but distributing the load to other parts of the body may have advantages. To examine alternative carriage methods, 11 soldiers walked on a treadmill at 4.8 km/h while performing two-person carries of a stretcher containing an 80-kg manikin. In separate trials, soldiers carried the stretcher using: (1) hand carriage, (2) shoulder straps, (3) a specially designed harness that allowed load shifting between the hips and shoulders (hip-shoulder system), and (4) a clip that fitted on the belt of standard military load carrying equipment (LCE) and placed the stretcher mass mainly on the hips. With each system, subjects walked until volitional fatigue or 30 min. While walking, expired gases and heart rates were obtained and subjects rated their perceived exertion (Borg Scale). At the conclusion of all four trials, subjects rated each system on a number of subjective measures. Results showed that average (+/- SD) carriage times were 2.7+/-1.4, 14.5+/-8.3, 25.4+/-8.1, and 21.7+/-9.9 min with the hand, shoulder, hip-shoulder and LCE systems respectively (p<0.01). Hand carriage resulted in considerably more cardiorespiratory stress (higher heart rate and minute ventilation, p<0.05) than the other three systems, but there were few consistent differences among the other three systems. Perceived exertion in the upper body was less with the hip-shoulder and LCE systems than with the other two systems (p<0.05). Subjects preferred the hip-shoulder and LCE systems overall and for specific subjective characteristics such as comfort, ease of use and stability (p<0.01). These data indicate that moving the stretcher load from the hands and placing that load on the shoulders and/or hips results in improved performance, reduced cardiorespiratory stress and favourable subjective evaluations. Further developmental work should focus on the hip-shoulder and LCE systems.

Anticipation and execution of a simple reading task enhance corticospinal excitability.

OBJECTIVE: Electromyographic responses (EMG) evoked in the right hand by transcranial magnetic stimulation (TMS) of the left motor cortex are enhanced during continuous reading. This enhancement is the result of increased excitability of the motor cortex. We proposed that anticipation and reading of single words would also enhance corticospinal excitability. We studied the temporal course of corticospinal excitability changes following left and right hemisphere TMS. METHODS: Ten normal volunteers were studied. A warning stimulus (S1) was followed by an imperative stimulus (S2) whereupon a word was presented. Subjects responded by reading the word aloud or reading it silently. In other conditions, no word was displayed and the subjects responded to S2 by saying the word 'Cat', pursing their lips, or doing nothing. EMG was recorded over the contralateral hand following a TMS pulse over the motor cortex during and after the S1-S2 period. RESULTS: Enhancement of EMG amplitudes was significantly greater following left hemisphere TMS. The enhancement in the S1-S2 period and that following S2 had a time course similar to several event-related brain potentials. CONCLUSIONS: There may be a common mechanism underlying both corticospinal excitability and the contingent negative variation, readiness potential and N400.

Hepatocyte gene therapy in a large animal: a neonatal bovine model of citrullinemia.

The development of gene-replacement therapy for inborn errors of metabolism has been hindered by the limited number of suitable large-animal models of these diseases and by inadequate methods of assessing the efficacy of treatment. Such methods should provide sensitive detection of expression in vivo and should be unaffected by concurrent pharmacologic and dietary regimens. We present the results of studies in a neonatal bovine model of citrullinemia, an inborn error of urea-cycle metabolism characterized by deficiency of argininosuccinate synthetase and consequent life-threatening hyperammonemia. Measurements of the flux of nitrogen from orally administered 15NH4 to [15N]urea were used to determine urea-cycle activity in vivo. In control animals, these isotopic measurements proved to be unaffected by pharmacologic treatments. Systemic administration of a first-generation E1-deleted adenoviral vector expressing human argininosuccinate synthetase resulted in transduction of hepatocytes and partial correction of the enzyme defect. The isotopic method showed significant restoration of urea synthesis. Moreover, the calves showed clinical improvement and normalization of plasma glutamine levels after treatment. The results show the clinical efficacy of treating a large-animal model of an inborn error of hepatocyte metabolism in conjunction with a method for sensitively measuring correction in vivo. These studies will be applicable to human trials of the treatment of this disorder and other related urea-cycle disorders.

Increased excitability of the human corticospinal system with hyperventilation.

OBJECTIVES: Hyperventilation is effective in inducing generalized spike-wave discharges in patients with absence seizures and improves visual function and normalizes visual function in patients with multiple sclerosis. Hyperventilation increases the excitability of cutaneous and motor axons. In experimental animals, hyperventilation increases excitability of hippocampal neurons. There is however no direct evidence of a hyperventilation-induced increase in neuronal excitability within the central nervous system in humans. In this study we determined the effects of hyperventilation on the human corticospinal system. METHODS: We studied the effects of hyperventilation on (1) motor evoked potentials (MEPs) induced by transcranial magnetic pulse stimulation of the motor cortex and (2) F-wave responses. Six subjects were studied. RESULTS: Hyperventilation resulting in an end-tidal pCO2 of 15 mm Hg or less enhanced the amplitude of the MEP and resulted in a shortened onset latency. F-wave amplitudes were enhanced without any change in onset latency. CONCLUSIONS: These findings indicate that hyperventilation increases the excitability of the human corticospinal system. A hyperventilation-induced increase in excitability within the central nervous system may account for clinical phenomena such as facilitation of spike-wave discharges.

Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing alpha1-antitrypsin after systemic delivery.

Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1. To test these hypotheses further, we have developed an E2a-deleted adenoviral vector expressing human alpha1-antitrypsin (hAAT). Toxicity of first-generation and E2a-deleted vectors, as determined by hematological indices, liver function tests, and histological analyses, was evaluated in C3H mice for 21 days after vector administration at increasing doses starting at 1 x 10(12) particles/kg. Both vectors induced dose-dependent abnormalities including transient thrombocytopenia, elevated ALT levels in serum, and increased hepatocyte proliferation followed by inflammation and then hypertrophy. Differences in the ratio of particles to plaque-forming units among vector preparations led to differences in hAAT expression at similar particle doses. There were no differences in toxicity between the two vectors when measured at matching levels of hAAT expression. However, the E2a-deleted vector was demonstrated to have slightly reduced hepatocyte toxicity at an intermediate particle dose. This suggests that hepatocyte toxicity is related primarily to viral entry and expression, rather than to the presence of noninfectious particles, and implies that vectors with complete elimination of viral gene expression, such as vectors with all viral coding sequences deleted, are likely to have substantial advantages in terms of safety and toxicity.

Evaluation of gene therapy for citrullinaemia using murine and bovine models.

Citrullinaemia is an autosomal recessive disorder caused by the deficiency of argininosuccinate synthase. The deficiency of this enzyme results in an interruption in the urea cycle and the inability to dispose of excess ammonia derived from the metabolism of protein. The only treatment for this disorder has been dietary restriction of protein and supplementation with medications allowing for alternative excretion of excess nitrogen. Gene therapy offers the possibility of a long-term cure for disorders like citrullinaemia by expressing the deficient gene in the target organ. We have explored the use of adenoviral vectors as a treatment modality for citrullinaemia in two animal models, a naturally occurring bovine model and a murine model created by molecular mutagenesis. Mice treated with adenoviral vectors expressing argininosuccinate synthase lived significantly longer than untreated animals (11 days vs 1 day; however, the animals did not exhibit normal weight gain during the experiment, indicating that the therapeutic effectiveness of the transducing virus was suboptimal. It is speculated that part of the failure to observe better clinical outcome might be due to the deficiency of arginine. In the bovine model, the use of adenoviral vectors did not result in any change in the clinical condition of the animals or in the level of plasma ammonia. However, the use of 15N isotopic ammonia allowed us to assess the flux of nitrogen through the urea cycle during the experiment. These studies revealed a significant increase in the flux through the urea cycle following administration of adenoviral vectors expressing argininosuccinate synthase. We conclude that the use of adenoviral vectors in the treatment of citrullinaemia is a viable approach to therapy but that it will be necessary to increase the level of transduction and to increase the level of enzyme produced from the recombinant viral vector. Future experiments will be designed to address these issues.

Recombinant human interleukin-3 to dose-intensify carboplatin and cyclophosphamide chemotherapy in epithelial ovarian cancer: a phase I trial.

PURPOSE: To define the optimal dose of recombinant human interleukin-3 (rhIL-3) required to intensify the dose of carboplatin and cyclophosphamide for advanced epithelial ovarian cancer. PATIENTS AND METHODS: Seventeen patients were treated on day 1 with carboplatin (dose adjusted for creatinine clearance: range, 257 to 385 mg/m2; median, 300 mg/m2) and cyclophosphamide (750 mg/m2). rhIL-3 5 micrograms/kg/d (n = 10) or 10 micrograms/kg/d (n = 7) was administered subcutaneously (SC) on days 2 through 11. Carboplatin dose was escalated if no postponement of cycles 1 to 3 had occurred. RESULTS: A 3-week interval was achieved in 62% of cycles and a 4-week interval in 81%, with no difference between the rhIL-3 doses. A neutrophil nadir less than 0.5 x 10(9)/L occurred in 35% of the cycles at 5 micrograms/kg/d and in 52% at 10 micrograms/kg/d of rhIL-3 (nonsignificant difference). The mean platelet nadir in cycle 1 was 173 +/- 78 x 10(9)/L at 5 micrograms/kg/d and 340 +/- 152 x 10(9)/L at 10 micrograms/kg/d of rhIL-3 (P < .05), with a faster recovery of platelets at 10 micrograms/kg/d (P < .05). Progressive myelotoxicity occurred for leukocytes and platelets at both rhIL-3 doses and required chemotherapy postponement in later cycles. The planned six cycles were completed by 41% of patients. Fever (> or = 38.5 degrees C) occurred in 38% of cycles at 5 micrograms/kg/d and in 97% at 10 micrograms/kg/d (P < .0005); headache and myalgias occurred in 30% and 44%, respectively. After two cycles, diffuse erythema, facial edema, and urticaria were observed in two patients at 5 micrograms/kg/d and in five patients at 10 micrograms/kg/d of rhIL-3. This resolved after discontinuation of rhIL-3 and administration of corticosteroids and antihistamines. CONCLUSION: A dose of 5 micrograms/kg/d of rhIL-3 proved to be optimal to intensify the carboplatin and cyclophosphamide regimen. It permitted the administration of carboplatin and cyclophosphamide combination therapy every 3 weeks in 62% of cycles.

Tolerance of sequential or simultaneous administration of IL-3 and G-CSF in improving peripheral blood stem cells harvesting following multi-agent chemotherapy: a pilot study.

A pilot study was devised to assess tolerance of combined administration of interleukin-3 (IL-3) and granulocyte-colony stimulating factor (G-CSF) given after chemotherapy to mobilize peripheral blood progenitors cells (PBPC). Eight patients with advanced malignancies received 1 week courses of both IL-3 and G-CSF in one of three schedules: simultaneous 7 days administration (3 patients), sequential administration (3 patients) or partial (3 days) overlap of the two growth factors (2 patients). IL-3 (7.5 micrograms/kg/day) and G-CSF (5 micrograms/kg/day for the simultaneous schedule and 12 micrograms/kg/day for the partial overlapping and sequential schedules) were administered subcutaneously. Side-effects during cytokine administration included WHO grade I-II fever in 6 of 8 patients, flu-like symptoms (including myalgias and arthralgias) in 4 of 8, WHO grade I-II headache in 2 of 8 and WHO grade II nausea and vomiting in 1 of 8. Overall, side-effects appeared similar during combined administration of IL-3 and G-CSF to those observed during administration of IL-3 alone. No fever was observed when G-CSF was administered alone. Two leukaphereses were performed following the treatment with cytokines. Only the seven patients who received cytokines following chemotherapy were analyzed for PBPC mobilization. The median collection of CFU-GM/kg per patient in the seven analyzed patients was 1.3 x 10(5) (range 5.7 x 10(2)-3.6 x 10(5)). In two patients, a second cycle of mobilization with either granulocyte macrophage-colony stimulating factor (GM-CSF) or G-CSF was administered to allow safe engraftment.(ABSTRACT TRUNCATED AT 250 WORDS)

Successful treatment of metastatic renal cell carcinoma with a biologically active dose of recombinant interferon-gamma.

We tested the clinical efficacy of a biologically active dose (BAD) of interferon (IFN)-gamma for treatment of progressive renal cell carcinoma (RCC). Twenty-two RCC patients with disease progression subsequent to nephrectomy were entered on a phase II clinical trial. During an initial dose-finding phase, biochemical responses to repeated once-weekly subcutaneous injections of 10, 100, or 500 micrograms of recombinant IFN-gamma were tested in 16 patients. Results indicated that 100 micrograms IFN-gamma applied once weekly was biologically active with induction of serum beta 2-microglobulin and neopterin. Such a dose induced a nearly maximum response of both markers lasting more than 4 days. This dose was also associated with minimal side effects. A dose of 100 micrograms IFN-gamma given once weekly was, therefore, subsequently given weekly for long-term treatment. During a median time of therapy of 10 months (range, 2 to 32 months) two complete (CR; 20+, 20+ months) and four partial tumor responses (PR; 6+, 7+, 8+, 24+ months) were seen (30% CR plus PR; 95% confidence limits, 12% to 54%) among 20 patients evaluable for response. Patients with refractory disease had significantly lower IFN-gamma-induced increments of serum beta 2-microglobulin than those who achieved clinical remission or stable disease.

In-vitro and in-vivo studies on the induction of neopterin biosynthesis by cytokines, alloantigens and lipopolysaccharide (LPS).

Recently we presented evidence that cellular immune responses are associated with increased in-vitro and in-vivo excretion of neopterin (Huber et al., 1983) and that, in vitro at least, macrophages and IFN-gamma play a key role in the induction of this phenomenon (Huber et al., 1984). Although this marker is increasingly applied for monitoring of human disease, there is limited knowledge about the mechanism(s) responsible for its increased biosynthesis during inflammatory states. To further elucidate this question we evaluated neopterin and IFN-levels in culture supernatants of human blood cells and in patients' sera. Cells or patients were exposed to a panel of recombinant cytokines, alloantigens or lipopolysaccharide. To investigate indirect stimulation by induction of production of endogenous IFNs, the impact of neutralization of IFNs by addition of specific antibodies was also studied. The data confirm our previous results which identified the monocyte/macrophage as the main producer cell among human blood cells. They further demonstrate that, at least in vitro, IFN-gamma, IFN-alpha and LPS can all stimulate neopterin release independently from each other. Thirdly, they indicate that stimuli such as alloantigens or TNF-alpha can indirectly enhance neopterin release by their capacity to induce production of endogenous IFN-gamma. On the basis of these data we conclude that enhanced neopterin biosynthesis does not necessarily relate to activation of T cells but can also be caused by non-immune stimuli.

Enhanced serum levels of beta-2-microglobulin, neopterin, and interferon-gamma in patients treated with recombinant tumor necrosis factor-alpha.

The effect of different doses of recombinant human tumor necrosis factor-alpha (rTNF-alpha) on serum levels of neopterin, beta-2-microglobulin and interferon-gamma (IFN-gamma) was investigated in tumor patients. Twelve patients with advanced malignant disease were treated and received single doses of either 1, 10, or 100 micrograms/m2 rTNF-alpha on days 0 and 7. Neopterin, beta-2-microglobulin and IFN-gamma serum levels were measured from day -2 to day 12 of the study. Application of rTNF-alpha leads to a marked and dose-dependent increase of neopterin and beta-2-microglobulin levels; no rTNF-alpha-dependent changes were observed after 1 microgram/m2, and maximum increments were seen in patients receiving 100 micrograms/m2. Serum levels of both parameters peaked after 2 days and returned to baseline values within 1 week. IFN-gamma levels were also elevated after application of rTNF-alpha. We failed, however, to demonstrate a clear correlation between the serum levels of IFN-gamma, beta-2-microglobulin, and neopterin because of the wide range of pre- and posttreatment levels of IFN-gamma. We conclude that neopterin and beta-2-microglobulin represent useful markers for monitoring biological response to treatment with rTNF-alpha.