Analysis of the human respiratory system in hypoxic and anoxic hypoxia by analogue computer simulation

The goal of this work is to synthesise a model of an adaptive system of respiration control in man, which could be used for the prognosis of human performance under extreme conditions. The model was partly based on 428 experimental results from hypoxic and anoxic hypoxia. Simulation of the oxygen regulatory circuit was realised on the NADAC 100 and MEDA T 80 analogue computers with satisfactory results. Equations constituting a new version of the model are presented in some detail, together with the results of the simulation of ventilatory mechanics.     

Measurement of Electromagnetic Field Immunity

A short survey is given of various methods proposed for the measurement of equipment immunity to extraneous electromagnetic fields. Their relative merits are discussed and progress in international standardization within the IEC is reported.     

A review of rapid microwave fixation technology: Its expanding niche in morphologic studies

Microwave (MW) fixation methods are important because excellent preservation of both cell structure and antigenicity can be attained several orders of magnitude faster than by routine chemical fixation methods. However, because of the limitations of commercial MW ovens, fixation results are often irreproducible. We present a standardization protocol for MW fixation in household MW ovens that emphasizes magnetron warm-up; the use of a water load during sample irradiation, of an agar/saline/Giemsa model to evaluate uniformity of irradiation within the MW cavity, and of specimen containers with one dimension less than 1.5 cm; and fast specimen handling to prevent conductive heating artifacts after irradiation. We describe a prototypic MW device that improves the precision of sample irradiation and fixes blocks of tissue and cells in suspension in milliseconds. The solutions used to immerse the specimen during irradiation influence the specimen morphology. Aldehyde- or osmium-containing solutions used simultaneously with MW irradiation resulted in the best morphologic preservation of specimens up to 1 cm³. Using MW fixation methods and a postembedding, ultrastructural immunogold-labeling approach, we have localized granule chymase and histamine in rat mast cells and amylase in rat parotid acinar cells.     

Clinical and technical assessment of the cervical spine

In analysis of the cervical and cervicobrachial syndrome with or without signs of compression of the nerve root or spinal cord, functional assessment of the cervical spine is of great importance. Comparisons between actively performed and passively induced motion can be verified by using standardized computer-assisted assessment allowing precise documentation of the range of motion and coupled motion. The age-related normal values should be considered. The neurological assessment includes not only the cranial nerves and upper extremities but also lower extremities to avoid overlooking the signs of cervical myelopathy. In patients with compression of nerve roots or the spinal cord neurophysiology might be helpful in identifying or verifying compression. In patients with suspected myelopathy sensory evoked potentials will allow assessment of the function of the ascending spinal pathways and motor evoked potentials, assessment of the function of the descending cortical spinal pathways.     

An integrated approach to the study of living cells by atomic force microscopy

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled-environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, long-term imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells.
The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady-state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.     

Immunocytochemical localization of chymase to cytoplasmic vesicles after rat peritoneal mast cell stimulation by compound 48/80

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1-10 sec after exposure to the secretogogue compound 48/80 (10 micrograms/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant (p < 0.01) increases in the area and number of chymase-immunoreactive vesicles per microns2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.     

In situ compositional analysis of acidocalcisomes in Trypanosoma cruzi

We measured the elemental content of different compartments in Trypanosoma cruzi epimastigotes using quick freezing, ultracryomicrotomy, and electron probe microanalysis. Vacuoles identified by high electron density contained (in units of mmol/kg dry weight +/- S.E.) large amounts of phosphorus (1390 +/- 13), magnesium (646 +/- 19), calcium (171 +/- 5), sodium (161 +/- 18), and zinc (148 +/- 6). No other compartment had appreciable calcium or zinc content. Iron (128 +/- 16 mmol/kg) was detected only in vacuoles distinct from the electron-dense vacuoles and other organelles. Incubation of cells for 70 min in culture medium in the presence of ionomycin plus nigericin led to a very significant 3- or 2-fold increase in potassium in the electron-dense vacuoles and the iron-rich vacuoles, respectively, with no significant change in the other elements investigated. This indicated the acidic nature of the vacuoles and demonstrated that the electron-dense vacuoles correspond to what were described previously as acidocalcisomes, i.e. acidic compartments rich in Ca2+. The acidocalcisomes were investigated by separation of epimastigote fractions on Percoll gradients in combination with Triton WR-1339 treatment. This detergent caused a rapid vacuolation; these vacuoles were shown by electron microscopy to be largely transparent, with a diffuse matrix. Percoll gradient fractionation demonstrated decreases in the density of various organelle markers in detergent-treated cells compared with controls. Large decreases in the density of the acidocalcisome and the mitochondrion were seen, as well as smaller decreases in the density of the other markers. Conventional electron microscopy of epimastigotes loaded with gold-labeled transferrin indicated that the endosomal system was separate from vacuoles that probably corresponded to the calcium-containing organelles detected by electron probe microanalysis. The combined results provide evidence that acidocalcisomes are organelles different from lysosomes or other organelles previously described in these parasites.     

Location of a VIPoma by iodine-123-vasoactive intestinal peptide scintigraphy

A major problem in patients with small endocrine tumors is the difficulty in localizing the primary tumor site. Many endocrine tumors possess larger amounts of high affinity vasoactive intestinal peptide (VIP) binding sites compared with normal tissue or blood cells. We used radiolabeled VIP to localize the tumor site in a patient with Verner-Morrison syndrome (VMS). Under octreotide therapy, the VIP levels had declined in this patient, but a tumor site could not be detected by conventional techniques or by radiolabeled octreotide. However, using 123I-VIP, the tumor was detectable in the pancreatic tail. Surgical resection of the tumor was followed by complete remission of the VMS. Expression of VIP binding sites in the tumor was confirmed by a radioreceptor assay and showed cross-competition between VIP and octreotide. The identity of the VIP binding site in the tumor was analyzed by Northern blotting and revealed the expression of somatostatin receptor subtype 3, which binds both somatostatin-14 and VIP with higher affinity than octreotide. Iodine-123-VIP scintigraphy would be an effective tracer to identity the tumor site in VMS patients.     

Vascular permeability factor/vascular endothelial growth factor-mediated signaling in mouse mesentery vascular endothelium

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine and growth factor that has important roles in both pathological and physiological angiogenesis. VPF/VEGF induces vascular hyperpermeability, cell division, and other activities by interacting with two specific receptor tyrosine kinases, KDR/Flk-1 and Flt-1, that are selectively expressed on vascular endothelium. The signaling cascade that follows VPF/VEGF interaction with cultured endothelium is only partially understood but is known to result in increased intracellular calcium, activation of protein kinase C, and tyrosine phosphorylations of both receptors, phospholipase C-gamma (PLC-gamma) and phosphatidylinositol 3'-kinase. For many reasons, signaling events elicited in cultured endothelium may not mimic mediator effects on intact normal or tumor-induced microvessels in vivo. Therefore, we developed a system that would allow measurement of VPF/VEGF-induced signaling on intact microvessels. We used mouse mesentery, a tissue whose numerous microvessels are highly responsive to VPF/VEGF and that we found to express Flk-1 and Flt-1 selectively. At intervals after injecting VPF/VEGF i.p., mesenteries were harvested, extracted, and immunoprecipitated. Immunoblots confirmed that VPF/VEGF induced tyrosine phosphorylation of several proteins in mesenteric microvessels as in cultured endothelium: Flk-1; PLC-gamma; and mitogen-activated protein kinase. Similar phosphorylations were observed when mesentery was exposed to VPF/VEGF in vitro, or when mesenteries were harvested from mice bearing the mouse ovarian tumor ascites tumor, which itself secretes abundant VPF/VEGF. Other experiments further elucidated the VPF/VEGF signaling pathway, demonstrating phosphorylation of both PYK2 and focal adhesion kinase, activation of c-jun-NH2-kinase with phosphorylation of c-Jun, and an association between Flk-1 and PLC-gamma. In addition, we demonstrated translocation of mitogen-activated protein kinase to the cell nucleus in cultured endothelium. Taken together, these experiments describe a new model system with the potential for investigating signaling events in response to diverse mediators on intact microvessels in vivo and have further elucidated the VPF/VEGF signaling cascade.