Fungal colonies reproduce via asexual spores which differentiate on selected hyphae. At low magnification, the spores appear as a point set distribution. A practical method to empirically evaluate these spatial point sets is developed which is premised on finding the Minimal Spanning Tree (MST). This is a graph theoretic approach to solving the generalized ‘Travelling Salesman Problem’ - that is, how to connect a set of isolated points in the most efficient way. This paper applies a computerised method using the S-Plus object oriented programming language for cluster analysis of these spatial patterns. The MST returns a unique branching, continuously connected pattern which summarises the shortest distance path which connects all the spores. We can hypothesise that this pathway is one geometric representation of the minimum physiological connectedness needed for the coordinated structural development of the asexual reproduction mechanism in fungi. Sporulation is generally considered to be an adaptive response which allows epigenic control of growth in hostile conditions. The MST therefore provides empirical measurement of the spatial cluster-correlation of the pattern.

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To study the intracellular Ca²⁺ mobilization of MEG-01 cells, which are a megakaryoblastic leukemia cell line, we used thapsigargin, a specific inhibitor of intracellular Ca²⁺-pump. The stimulation of single, fura-2-loaded MEG-01 cells by thapsigargin resulted in a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that appears to be due to both initial discharge of Ca²⁺ from the intracellular stores and successive entry of Ca²⁺ across the plasma membrane. After removal of extracellular Ca²⁺, the elevated [Ca²⁺]_i produced by thapsigargin decreased with an initial rapid phase, followed by a gradual phase. lonomycin applied during the period of the gradual decrease elicited a transient increase in [Ca²⁺]_i while La³⁺, which blocks both inward and outward movement of Ca²⁺ across the plasma membrane, produced a sustained elevation as long as La³⁺ was present. Using a laser scanning confocal fluorescence imaging system, localization of cytosol Ca²⁺ was found in fluo-3-loaded cells in the gradual decrease phase. Heterogeneous pattern of cytosol high Ca²⁺ concentration seemed to correspond to location of mitochondria labeled with rhodamine 123. Inhibitors of mitochondrial electron transport, NaN₃ and antimycin A, eliminated the gradual decrease phase. These results suggest the presence of an unknown intracellular Ca²⁺ pool, possibly mitochondria, which could accumulate excessive intracellular Ca²⁺, and indicate that the [Ca²⁺]_i in the gradual decrease phase represents the Ca²⁺ concentration in the unknown pool.

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We succeeded in detecting fine three-dimensional structures of living neuronal terminals with at least 40-nm vertical information, and in observing the realtime dynamics of their terminals, such as attraction and repulsion with each other, using an atomic force microscope (AFM) for a liquid environment. The neurons in central ganglia from the pond snail, Lymnaea stagnalis, were primarily cultured in modified L-15 medium. They branched out neurites and formed a neural network within 2 days with the help of nerve growth factor. The AFM enabled us to measure the fine structures of living neuronal terminals with very uneven height (520—2010 nm) and a lot of small and short branches (tens of nm in height) in their connective field. We could also observe time-dependent changes in fine three-dimensional structures of lamellipodia and record movement of two growth-cone terminals in different neurons as, for example, they first approached each other and then separated or withdrew. The time-dependent fine dynamics of the neurons obtained here is beyond the resolution power of an optical microscope, and actually it has never been accomplished with an electron microscope that requires of fixing and staining. Our data, therefore, suggest that using AFM for observing neuronal terminals will be a powerful tool for acquiring new knowledge of functions in a neural network.

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The intracellular distribution of Ca²⁺/calmodulindependent protein kinase IV (CaMKIV) was investigated. Confocal microscopy revealed a nuclear localization of CaMKIV in transfected COS7 cells while Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was cytoplasmic. Calspermin, an alternative initiation product of CaMKIV identical to the carboxyl-terminal 169 amino acids of CaMKIV, was strongly targeted to the nucleus. However, deletion analysis of CaMKIV demonstrated that mutants lacking the carboxyl-terminal end corresponding to calspermin did not affect their nuclear localization. In contrast, a mutant lacking the first 132 amino acids of CaMKIV was no longer localized to the nucleus. Furthermore, truncated CaMKIV, containing the catalytic domain of the enzyme but lacking the regulatory domain, was still localized in the nucleus while truncated CaMKII was not. These results suggest that the nuclear localization mechanism of CaMKIV is entirely independent from that of calspermin although the coding sequence for calspermin is contained within the CaMKIV sequence. These data also suggested that there are significant differences in the amino-terminal part of the catalytic domain of CaMKIV and CaMKII, which may contribute to the nuclear localization of CaMKIV.

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The early work of Camillo Golgi and Santiago Ramón Cajal on the structure of the nervous system was based on silver staining of fixed specimens. We have used a new, real-time, scanning slit confocal microscope to visualize the innervation in the unstained, living human cornea. Both the central cornea and the midperipheral cornea at the level of the anterior stroma were studied. Both normal and anomalous structures on the nerve fiber bundles are shown. A technique is described to obtain real-time, reflected light confocal images of the nerve fiber bundles in the anterior stroma of the central and midperipheral human cornea.

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