Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion.

The actin cortex both facilitates and hinders the exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca(2+) and PtdIns(4,5)P(2) lipid levels that in turn drive cyclic recruitment of N-WASP and cortical actin level oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca(2+) level oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin levels are high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca(2+) levels subsequently increase. Thus, cells employ oscillating levels of Ca(2+), PtdIns(4,5)P(2) and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion.

A novel PRNP-P105S mutation associated with atypical prion disease and a rare PrPSc conformation.

OBJECTIVE: To define the clinicopathologic, genetic, and pathogenic prion protein (PrP(Sc)) characteristics associated with a novel mutation of the prion protein gene (PRNP). METHODS: The coding segment of PRNP from the proband and family members was sequenced and the brain of the proband was histologically studied. The Western blot profile of the proteinase K (PK) resistant fraction of PrP(Sc), an approximation of its conformation, or "PrP(Sc)-type," was determined. RESULTS: We detected a novel mutation at codon 105 of PRNP that results in a serine (S) substitution of proline (P) (P105S), in a young woman who developed progressive aphasia, behavioral changes, dementia, and parkinsonism, lasting 10 years to her death. Histopathologic findings included an intense focus of multicentric PrP-plaques within the hippocampus, punctate plaques scattered throughout the cerebellum, and intense spongiform degeneration focally within the putamen, suggesting a variant of Gerstmann-Sträussler-Scheinker syndrome (GSS). However, PrP(Sc)-typing revealed two PK-resistant PrP(Sc) fragments (approximately 21 and 26 kDa), a pattern not previously detected in GSS. CONCLUSIONS: This mutation is the third sequence variation at codon 105 of PRNP. The unusual phenotype and PrP(Sc)-type distinguishes this genetic prion disease from typical Gerstmann-Sträussler-Scheinker syndrome and other codon 105 substitutions, suggesting that, in addition to the loss of proline at this position, the PrP(Sc) conformation and phenotype is dependent on the specific amino acid substitution.

Efficient chromosome capture requires a bias in the 'search-and-capture' process during mitotic-spindle assembly.

The mitotic spindle assembles into a bipolar, microtubule-based protein machine during prometaphase. One proposed mechanism for this process is "search-and-capture," in which dynamically unstable microtubules (MTs) search space to capture chromosomes. Although existing theoretical estimates suggest that dynamic instability is efficient enough to allow capture within characteristic mitotic timescales, they are limited in scope and do not address the capture times for realistic numbers of chromosomes. Here we used mathematical modeling to explore this issue. We show that without any bias toward the chromosomes, search-and-capture is not efficient enough to explain the typical observed duration of prometaphase. We further analyze search-and-capture in the presence of a spatial gradient of a stabilizing factor that biases MT dynamics toward the chromosomes. We show theoretically that such biased search-and-capture is efficient enough to account for chromosome capture. We also show that additional factors must contribute to accelerate the spindle assembly for cells with large nuclear volumes. We discuss the possibility that a RanGTP gradient introduces a spatial bias into microtubule dynamics and thus improves the efficiency of search-and-capture as a mechanism for spindle assembly.

Low grade gliomas treated with adjuvant radiation therapy in the modern imaging era.

The purpose of this study is to evaluate tumor control and failure patterns in patients with low grade gliomas treated with surgery and conventional adjuvant radiation therapy. Twenty-eight patients with low grade gliomas (7 grade I, 21 grade II) were retrospectively evaluated. Extent of resection was gross total (3), subtotal (17), and biopsy alone (8). All grade I tumors underwent subtotal resection. Median radiation therapy dose was 54 Gy delivered to localized fields. Tumor control and patterns of failure were determined from follow-up computed tomography and/or magnetic resonance scans. Median follow-up was 86 months (range, 2.4-177 months). Thirteen patients (46%) (four grade I, nine grade II) developed tumor progression. The 5-year actuarial progression-free survival rates for grade I and grade II patients were 86% and 51%, respectively. Corresponding 5-year actuarial survival rates were 100% and 70%. All recurrences were within the treated volume. Our results reveal that conventional adjuvant radiation therapy is associated with high rates of local tumor progression in both grade II and incompletely resected grade I low grade gliomas. Alternative strategies need to be explored in these patients in an effort to improve tumor control and outcome.

A novel tumor necrosis factor-alpha inhibitory protein, TIP-B1.

TIP-B1, a novel TNF inhibitory protein, has been identified, purified and characterized from cytosolic extracts of TNF-treated human fibroblasts, and a partial TIP-B1 cDNA clone has been obtained. The (27 kDa pI approximately 4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids), and the nucleotide sequence of the corresponding cDNA clone. TNF-sensitive cells, when exposed to TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Thus, this TIP-B1 treatment effectively makes these cells TNF-resistant. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. TIP-B1 does not interfere with the interactions between TNF and the TNF receptors based on flow cytometric analysis of the cellular binding of biotinylated TNF. These and other data indicate that TIP-B1 is not a soluble TNF receptor, nor an anti-TNF antibody, nor a protease that degrades TNF, yet TIP-B1 functions when added exogenously to cells. Thus, TIP-B1 is not one of the proteins previously reported to be involved in resistance to TNF. The fact that incubation of the newly discovered novel TIP-B1 with TNF-sensitive cells protects them from TNF-induced cell death, including TNF-mediated apoptosis, makes TIP-B1 a candidate for therapeutic modulation of TNF-induced effects.

Integrators of the cytoskeleton that stabilize microtubules.

Sensory neurodegeneration occurs in mice defective in BPAG1, a gene encoding cytoskeletal linker proteins capable of anchoring neuronal intermediate filaments to actin cytoskeleton. While BPAG1 null mice fail to anchor neurofilaments (NFs), BPAG1/NF null mice still degenerate in the absence of NFs. We report a novel neural splice form that lacks the actin-binding domain and instead binds and stabilizes microtubules. This interaction is functionally important; in mice and in vitro, neurons lacking BPAG1 display short, disorganized, and unstable microtubules defective in axonal transport. Ironically, BPAG1 neural isoforms represent microtubule-associated proteins that when absent lead to devastating consequences. Moreover, BPAG1 can functionally account for the extraordinary stability of axonal microtubules necessary for transport over long distances. Its isoforms interconnect all three cytoskeletal networks, a feature apparently central to neuronal survival.

Adenosine triphosphate causes vasospasm of the rat femoral artery.

OBJECTIVE: Adenosine 5'-triphosphate (ATP) causes vasoconstriction by activation of P2-purinoceptors on vascular smooth muscle cells. Erythrocytes contain ATP at a concentration (1.6 mmol/L) that contracts smooth muscle. Previous studies of hemoglobin solutions did not assess whether the vasoactivity was caused by ATP rather than or in addition to hemoglobin. It was hypothesized that the hemolysis of erythrocytes that occurs after subarachnoid hemorrhage releases ATP in concentrations that cause vasospasm. METHODS: Thirty-eight rats were randomly assigned to undergo placement of one of the following compounds in a silastic elastomer cuff around each femoral artery: 1) agarose gel (n = 8); 2) dog erythrocyte hemolysate (n = 8); 3) purified human hemoglobin (Hemolink; Hemosol, Inc., Toronto, Canada; n = 8); 4) ATP (n = 8); or 5) clotted autologous blood (n = 6). The amounts of hemoglobins and adenine nucleotides in the compounds were measured by spectrophotometry and high pressure liquid chromatography. Hemolysate, purified hemoglobin, and ATP were mixed with agarose gel to create an artificial clot. Rats were killed and fixed by perfusion at physiological blood pressure 7 days after perivascular cuff and spasmogen placement. Vasospasm was assessed by image analysis of cross sections of fixed femoral arteries. Arteries were assessed for histopathological changes on 3-point scales. RESULTS: There was significant variance in arterial diameters among groups (mean diameter +/- standard deviation: agarose gel, 0.29 +/- 0.06; purified hemoglobin, 0.28 +/- 0.04; hemolysate, 0.24 +/- 0.05; ATP, 0.25 +/- 0.05; clotted blood, 0.24 +/- 0.01; P < 0.05, analysis of variance, n = 11-20). Animals exposed to clotted blood, hemolysate that contained ATP, or ATP, developed vasospasm, whereas purified hemoglobin and agarose did not cause vasospasm. Endothelial proliferation and perivascular inflammation were more severe (P < 0.05) in arteries exposed to clotted blood, purified hemoglobin, and hemolysate. CONCLUSION: These results suggest that ATP may be a vasospastic substance released by erythrocyte hemolysis. The concentration of ATP in impure solutions of hemoglobin is too low to account for the vasoactivity of these solutions. The discrepancy between arterial narrowing and histopathological changes suggests that either histopathological changes may not be an important correlate of arterial vasospasm or that other substances are important in vasospasm.

Quantitative imaging study of extent of surgical resection and prognosis of malignant astrocytomas.

OBJECTIVE: This study used quantitative radiological imaging to determine the effect of surgical resection on postoperative survival of patients with malignant astrocytomas. Previous studies relied on the surgeons' impressions of the amount of tumor removed, which is a less reliable measure of the extent of resection. METHODS: Information concerning possible prognostic factors was collected for 75 patients undergoing magnetic resonance imaging or computed tomography preoperatively and within 10 days postoperatively. Image analysis of the neuroradiological studies was conducted to quantify pre- and postoperative total tumor volumes and enhancing volumes. Univariate and multivariate proportional hazards models were used to analyze the regression of survival regarding 22 covariates that might affect survival. The covariates that were entered included age, gender, tumor grade, cumulative radiation dose, chemotherapy, seizures as a first symptom, Karnofsky performance status at presentation, pre- and postoperative total and enhancing tumor volumes, ratio of pre- to postoperative total and enhancing tumor volumes, tumor location, surgeon's impression of the degree of resection, and subsequent surgery. RESULTS: There were 23 patients with anaplastic astrocytomas and 52 with glioblastomas multiforme. The estimated mean survival time was 27 months for patients undergoing gross total resection, 33 months for subtotal resection, and 13 months for open or stereotactic biopsy. Five factors that were significant predictors of survival in multivariate analysis were tumor grade, age, Karnofsky performance status, radiation dose, and postoperative complications (P < 0.05). In univariate analysis, tumor grade, radiation dose, age, Karnofsky status, complications, presence of enhancing tumor in postoperative imaging, and postoperative volume of enhancing tumor were significantly associated with survival (P < 0.05). CONCLUSION: We conclude that the most important prognostic factors affecting survival of patients with anaplastic astrocytomas and glioblastomas multiforme are tumor grade, age, preoperative performance status, and radiation therapy. Postoperative complications adversely affect survival. Aggressive surgical resection did not impart a significant increase in survival time. Surgical resection may improve survival, but its importance is less than that of other factors and may be demonstrable only by larger studies.

Nuclear magnetic resonance spectroscopy of mussel adhesive protein repeating peptide segment.

Mussel adhesive protein (MAP) is the adhesive agent used by the common blue sea mussel (Mytilus edulis) to attach the animal to various underwater surfaces. It is generally composed of 75 to 85 repeating decameric units with the reported primary sequence NH2-Ala(1)-Lyst(2)-Pro(3)-Ser(4)-Tyr(5)-Hyp(6)-Hyp(7)-Thr(8)-DOPA( 9)- Lys(10)-COOH. This study examines this peptide's solution-state conformation using proton nuclear magnetic resonance (NMR) spectroscopy. NMR and molecular modeling of the decamer before and after molecular dynamics calculations in water suggests a conformation that retains an overall bent helix.

First experimental transmission of fatal familial insomnia.

Originally described by Lugaresi et al. in 1986 (ref. 1), fatal familial insomnia (FFI) is a rare inherited neurological disease characterized by the subacute progression of intractable insomnia and other autonomic abnormalities, cerebellar and pyramidal signs, myoclonus and dementia; neuropathologically, the major feature is severe neuronal loss with associated gliosis in the ventral and mediodorsal thalamic nuclei. The disease has been related to the group of spongiform encephalopathies by virtue of the presence of low levels of proteinase-resistant amyloid protein (PrPres) in the brain, and of a pathogenic single-allele mutation at codon 178 of the PRNP gene that encodes PrPres (refs 2, 5). Here we report the successful transmission of the disease to experimental animals, placing FFI within the group of infectious cerebral amyloidoses.

Effect of epidermal growth factor on the growth and radiation sensitivity of human breast cancer cells in vitro.

PURPOSE: To study the effect of epidermal growth factor (EGF) on the radiation sensitivity of MCF-7 breast cancer cells. METHODS AND MATERIALS: Radiation dose survival curves were generated for MCF-7 cells under conditions of hormonal deprivation. Epidermal growth factor and/or a monoclonal antibody to its receptor (mAb-225) were added prior to irradiation. Cell cycle distribution was determined by flow cytometry and cellular glutathione (GSH) levels were measured by a glutathione reductase assay. RESULTS: Under hormonal deprivation (control), more than 90% of the MCF-7 cells were arrested in G0/G1 phase and the D(o) of their survival curve was 0.66 +/- .01 Gy. The addition of EGF resulted in (a) growth stimulation; (b) increased percentage of cells in the S-phase of the cell cycle; (c) increased radioresistance (D(o) = 0.81 +/- .04 Gy; p < .05, compared with controls); (d) increased cellular GSH level. The EGF effect on radiation response was observed in a time- and dose-dependent manner. The addition of mAb-225 blocked the ability of EGF to enhance growth and radioresistance (D(o) = 0.68 +/- .03 Gy). CONCLUSION: Epidermal growth factor stimulates the growth and when administered prior to irradiation increases the radioresistance of hormone-deprived MCF-7 cells. These effects are inhibited by a specific antibody to the EGF receptor. Epidermal growth factor concomitantly increased the fraction of S-phase cells and intracellular GSH levels. This system of growth factor-altered radiosensitivity in human breast cancer cells provides a useful model for the study of the radiation response mechanisms in human malignancy.

Neuromuscular transmission in amyotrophic lateral sclerosis.

The functional and structural characteristics of the neuromuscular junction were studied in anconeus muscle biopsies of 10 patients with amyotrophic lateral sclerosis (ALS). Intracellular recordings revealed decreased amplitudes of miniature endplate potentials (MEPPs). The MEPP frequencies were highly variable in ALS patients but the average MEPP frequency was not different from that of control patients. The mean quantal content of endplate potentials (m), the mean quanta available for immediate release (n), and the mean quantal stores (N) were all decreased. In contrast, the mean probability of quantal release (p) was normal and the mean probability of quantal store release (P) was surprisingly high at the majority of ALS endplates. Histologic evidence of denervation and small or absent nerve terminals were observed in all ALS patients. These functional and structural abnormalities of the neuromuscular junction may explain the fatigability and the electromyographic evidence of impaired neuromuscular transmission often encountered in ALS patients.

Neuromuscular transmission as a function of motor unit size in patients with prior poliomyelitis.

We studied neuromuscular transmission in 16 patients with prior poliomyelitis by measuring single fiber electromyographic (SFEMG) jitter. This was compared with 3 indirect methods of assessing reinnervation: SFEMG fiber density, macro EMG, and the presence of fiber type grouping on muscle biopsy. In patients with acute poliomyelitis before the age of 10, there was a positive correlation between the extent of neuromuscular transmission impairment, demonstrated by increased SFEMG jitter, and the enlargement of the motor unit, as indicated by increased fiber density, increased macro EMG signals, and fiber type grouping on muscle biopsy. However, there was no correlation between any of these parameters and the presence or absence of new symptoms of weakness. These findings suggest that impaired neuromuscular transmission is most common in patients with prior poliomyelitis whose motor units have been maximally enlarged by axonal sprouting, but is independent of the presence or absence of new symptoms of weakness.

A Monte Carlo simulation of hydration of xanthine-derivatives and their stacked forms.

Results on a Monte Carlo simulation of the hydration of monomer and possible stacked dimer forms of a purine alkaloid series in 200- and 400-water molecule clusters are presented. Investigation of different purine stacked dimers in a 200-water molecule cluster reveals that for caffeine there exists one, for theophylline two and for theobromine four dimers are energetically favorable. For caffeine, the same energetically favored stacked dimer form is observed in both the 200- and 400-water molecule cluster. The main factor stabilizing the preferred dimer stacks is the change in the interaction between water molecules of the monomer cluster and those water molecules in the dimer cluster.

"Instant-mix" whole brain photon with neutron boost radiotherapy for malignant gliomas.

From July 1985 through March 1987, 44 consecutive patients with supratentorial, nonmetastatic anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) were treated with whole brain photon irradiation with concomitant neutron boost at the University of Chicago. All patients had biopsy proven disease and surgery ranged from biopsy to total gross excision. Whole brain photon radiation was given at 1.5 Gy per fraction, 5 days weekly for a total dose of 45 Gy in 6 weeks. Neutron boost radiation was prescribed to a target minimum dose that included the pre-surgical CT tumor volume plus 1 cm margin. Neutrons were administered 5-20 minutes prior to photon radiation twice weekly and a total dose of 5.2 Gyn gamma was administered over 6 weeks. Median follow-up was 36 months. The median survival was 40.3 months for anaplastic astrocytoma (10 patients) and 11 months for glioblastoma multiforme (34 patients) and 12 months for the overall group. Variables that predicted longer median survival included histology (AA vs. GBM), age (less than or equal to 39 years vs. older), and extent of surgery (total gross or partial excision vs. biopsy) whereas tumor size and Karnofsky performance status did not have a significant influence. The median survival of the anaplastic astrocytoma group was better than expected compared to the RTOG 80-07 study (a dose-finding study of similar design to this study) and historical data. Reasons for this are discussed.

Distant angiogenesis in a patient with glioblastoma multiforme.

The case of a 42-year-old man with a cerebral glioblastoma multiforme associated with marked neovascularization of the arachnoid of the brain stem and spinal cord is reported. All of the neurological symptoms and signs were referrable to the glioblastoma and resultant craniotomies. The arachnoid contained a proliferation of well differentiated blood vessels. This neovascularization occurred in the absence of local tumor or inflammation. We suggest that the neovascularization resulted from release of a tumor angiogenesis factor into the cerebrospinal fluid.

Computed tomography of gliomatosis cerebri.

Gliomatosis cerebri, a rare diffusely infiltrating astrocytoma, was discovered on the postmortem examination of a 22-year-old woman with a 13 year history of seizures. Computed tomography of the brain revealed bifrontal white matter low density changes that were most consistent with a demyelinating or dysmyelinating disorder.

Fibrinolytic activity in experimental intracerebral hematoma.

In an attempt to investigate the role of tissue fibrinolytic activity in the resolution of intracerebral hematoma, an experimental model of intracerebral hematoma was developed in the rat. The fibrinolytic activity was studied using a histochemical fibrin slide technique. A total of 59 adult male rats were studied. Twenty-nine rats were used for developing the intracerebral hematoma model via injection of autologous whole blood into the left frontal lobe; in the remaining 30 rats, the intracerebral hematomas were studied sequentially. Intracerebral hematoma formation was unsuccessful in six (21%) of 29 rats. Four rats died in the immediate postoperative period and two showed no intraparenchymal clot. Intense fibrinolytic activity was demonstrated in the blood vessel walls of the normal brain, especially in the meninges, choroid plexus, and ependymal cell layer. In the initial stages of hematoma resolution, fibrinolytic activity was not seen in the hematoma or parenchyma except in the preexisting blood vessels. However, 3 to 5 days later, fibrinolytic activity was observed in the capillary buds surrounding the hematoma and among the infiltrating mononuclear cells. This activity increased for 7 to 10 days following formation of the hematoma and decreased after 21 to 28 days. It is concluded that tissue fibrinolytic activity associated with newly formed blood vessels appears to be important in lysis of intracerebral hematomas.

Neural cell adhesion molecule in normal, denervated, and myopathic human muscle.

The neural cell adhesion molecule (N-CAM) is a cell-surface glycoprotein that may mediate some intercellular adhesive interactions in the nervous system. In adult rat muscle, N-CAM is concentrated near neuromuscular junctions and on satellite cells, but is nearly undetectable in nonsynaptic portions of myofibers. However, N-CAM is abundant throughout myofibers in denervated and regenerating muscles. Using affinity-purified antibodies to N-CAM, we were able to demonstrate a similar distribution and regulation of N-CAM in human muscle. Myofiber N-CAM was not detectable immunohistochemically in any of 10 normal biopsies or in 4 biopsies that were abnormal but showed no evidence of fiber denervation or regeneration. N-CAM was present, however, at end plates, nerves, and satellite cells in normal human muscle. In contrast, myofiber N-CAM was detected in 16 of 16 patients with histological evidence of denervation and in 10 of 10 patients who had myopathy with degenerating/regenerating myofibers. In addition, 2 of 2 histologically nondiagnostic biopsies from patients with amyotrophic lateral sclerosis contained N-CAM-positive myofibers. Immunoblot analysis also detected N-CAM in denervated and myopathic, but not normal, human muscle. These results suggest that N-CAM may play a role in muscle reinnervation or regeneration and that N-CAM immunohistochemistry may complement conventional techniques in the diagnosis of neuromuscular disease.