Effect of substrate rigidity in tissue culture on the function of insulin-secreting INS-1E cells.

Insulin-secreting INS-1E cells are a useful tool in diabetes research. However, during permanent culture the cells tend to lose their ß cell phenotype, with resultant loss of insulin-secretory responsiveness. This can be at least partially attributed to inappropriate cell culture conditions. One of the important causative factors is the rigidity of the extracellular matrix. We have therefore systematically studied the performance of INS-1E insulin-secreting cells cultured on polyacrylamide gels of different stiffnesses and analysed changes in insulin content and secretion, glucokinase enzyme activity, gene expression of ß cell transcription factors and cell death and proliferation rates. INS-1E cells were cultured on polyacrylamide gels with a wide range of rigidities, including the one that simulates the stiffness of the pancreas. We detected changes in insulin content and the insulin-secretory response to glucose stimulation in parallel to the increasing stiffness of the polyacrylamide gels in the range 1700-111 000 Pa. On substrates with the highest and lowest rigidities, 322 and 111 000 Pa, the cells mainly formed pseudo-islets, while at rigidities of 1700-64800 Pa, including the rigidity of native pancreas tissue (3100 Pa), cells grew as a monolayer attached to the polyacrylamide gel surface. These observations provide evidence for an apparent mechanosensitivity of insulin-secreting INS-1E cells affecting morphology and cellular functions. The results can also provide practical advice regarding a selection of the materials appropriate for successful cell culture of insulin-secreting cells. Copyright © 2014 John Wiley & Sons, Ltd.

A new algorithm for N-dimensional Hilbert scanning.

There have been many applications of the Hilbert curve, such as image processing, image compression, computer hologram, etc. The Hilbert curve is a one-to-one mapping between N-dimensional space and one-dimensional (l-D) space which preserves point neighborhoods as much as possible. There are several algorithms for N-dimensional Hilbert scanning, such as the Butz algorithm and the Quinqueton algorithm. The Butz algorithm is a mapping function using several bit operations such as shifting, exclusive OR, etc. On the other hand, the Quinqueton algorithm computes all addresses of this curve using recursive functions, but takes time to compute a one to-one mapping correspondence. Both algorithms are complex to compute and both are difficult to implement in hardware. In this paper, we propose a new, simple, nonrecursive algorithm for N-dimensional Hilbert scanning using look-up tables. The merit of our algorithm is that the computation is fast and the implementation is much easier than previous ones.

Effect of neurotransmitters on axoplasmic transport: how adrenaline affects superior cervical ganglion cells.

The effect of adrenaline on the axoplasmic transport of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Adrenaline increased the axoplasmic transport reversibly in both anterograde and retrograde directions. A beta 2-antagonist, butoxamine, antagonized the increasing effects of adrenaline, but alpha-antagonists and beta 1-antagonists did not. A beta 2-agonist, albuterol, mimicked the adrenaline effect, but beta 1-, alpha 1-, alpha 2-agonists did not. The adrenaline receptor may be a beta 2-receptor. Dibutyryl cyclic AMP and forskolin increased the axoplasmic transport. Therefore, adrenaline increases the axoplasmic transport by raising the cyclic AMP level. In light of our former report that acetylcholine suppresses the axoplasmic transport, neurotransmitters control axoplasmic transport and this neurotransmitter control reflects the activity of the nerve cell.

Immunohistochemical coexistence of calcitonin gene-related peptide and substance P in the nerve fibers of the internal gills of bullfrog (Rana catesbeiana) larvae.

A double-labelling immunofluorescence study of the internal gills of larval bullfrogs revealed coexistence of calcitonin gene-related peptide (CGRP) and substance P (SP) in the same nerve fibers in the gill tufts. More than about 95% of CGRP fibers showed coexistence of SP, although some fibers contained CGRP without SP. In the branchial muscle, all CGRP fibers demonstrated coexistence of SP. These findings suggest that both CGRP- and SP-containing fibers may use both peptides as neuromodulators of active ion transport and gill movements.

Mechanism of inhibitory action of capsaicin on particulate axoplasmic transport in sensory neurons in culture.

The inhibitory effect of capsaicin on axoplasmic transport in cultured dorsal root ganglion cells was analyzed by video-enhanced contrast microscopy. Capsaicin inhibited particle transports in a dose-dependent manner, irrespective of the diameter of axons. The effect of capsaicin was reversible at low concentrations. Capsaicin affected both the anterograde and retrograde transport. Large organelles were more sensitive to capsaicin than small ones in the retrograde transport. An experiment using calcium-sensitive dye, Fura 2, indicated that capsaicin raised the intraneuronal free calcium concentration preceding the inhibition of the transport. Electron microscopy revealed that microtubules and neurofilaments are disorganized and disoriented by capsaicin. We reached a conclusion that capsaicin inhibits fast axoplasmic transport of both anterograde and retrograde directions in all types of somatosensory neurons in culture by disorganizing intraaxonal cytoskeletal structures, through the elevated intracellular Ca2+ concentration.

Coexistence of substance P and calcitonin gene-related peptide in the nerve fibers of the carotid labyrinth of the bullfrog, Rana catesbeiana.

Double immunohistochemical staining with fluorescein isothiocyanate (FITC)- and rhodamine-conjugated antisera revealed the coexistence of substance P (SP) and calcitonin gene-related peptide (CGRP) in most nerve fibers in the intervascular stroma of the carotid labyrinth of the bullfrog, Rana catesbeiana. A few fibers contained SP without CGRP. The results suggest that the vascular regulatory function, which is one of the possible functions of the carotid labyrinth, may be controlled in part by the interaction of SP and CGRP.

Organelle transport in cultured smooth muscle cells of the rabbit basilar artery.

Organelle transport in cultured smooth muscle cell from the rabbit basilar artery was observed. The organelles transported in the cells were moving in both centrifugal and centripetal directions. The instantaneous velocities of organelles were 0.84 +/- 0.36 micron/s in the centrifugal direction and 0.86 +/- 0.48 micron/s in the centripetal direction. The transported organelles consisted mainly of lysosomes (67.5%) and mitochondria (29.0%).

How neurotransmitters affect axoplasmic transport?

The effects of acetylcholine (ACh) and adrenaline on fast axoplasmic transport of cultured superior cervical ganglion cells were analyzed with a computer-assisted video-enhanced differential interference contrast microscopic system. ACh suppressed the transport reversibly in both anterograde and retrograde directions, and adrenaline increased the transport reversibly. These effects are related to the amount of c-AMP. This amount of c-AMP in connection with neurotransmitters controls the axoplasmic transport, which in turn is related to the activity of the neuron.

Effect of neurotransmitters on axoplasmic transport: acetylcholine effect on superior cervical ganglion cells.

The effect of acetylcholine (ACh) on particle movements along axons of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. ACh suppressed the axoplasmic transport reversibly in both anterograde and retrograde directions. A muscarinic agonist, arecoline, mimicked the ACh effect, but nicotine did not. An experiment with the Ca(2+)-indicator dye, fura-2, revealed that ACh suppressed the transport without any change of intracellular Ca2+ concentration. ACh also suppressed the axoplasmic transport in Ca(2+)-free medium. Islet-activating protein (IAP), pertussis toxin, blocked the ACh effect. These results indicate that ACh activates muscarinic receptors and suppresses fast axoplasmic transport through the activation of IAP-sensitive GTP-binding protein, irrespective of Ca2+ ions.

Low responsiveness of lymphocytes from normal individuals against the stimulation with pokeweed mitogen.

The responsiveness of peripheral blood lymphocytes stimulated with pokeweed mitogen (PWM) and purified protein derivative of tuberculin (PPD) as B cell activator was studied in adult healthy subjects by immunoglobulin synthesis system in culture. We have shown that lymphocytes from some healthy subjects have low responsiveness in the polyclonal antibodies secretion following PWM stimulation but not PPD stimulation by Protein A-reverse hemolytic assay. In this system, when irradiated T cells (CD4+) as helper T cell were added to B cells from autologous or allogeneic subjects, helper T cell function of immunoglobulin synthesis on the low response groups to PWM significantly decreased. Whereas normal responses to PPD stimulation were observed in the presence of helper T cells (CD4+). The low responsiveness of lymphocytes stimulated with PWM but not PPD may be related to reduced functions of helper T cell populations. Thus the healthy subjects were classified into high, normal and low response groups with PWM on the basis of antibody production.

[Measurement of small quantities of alpha 2-macroglobulin in bronchoalveolar lavage fluid by enzyme-labelled immunoassay, and its clinical implication in pulmonary disease].

We established an enzyme labelled immunoassay for the determination of alpha 2 macroglobulin (alpha 2M). The assay range was from 2 to 140 ng/ml and the within-assay coefficient of variation (CV) were 5.2% at 31.2 ng/ml and 6.4% at 62.5 ng/ml. Between-day CV ranged from 6.9% to 15.4%. Using this method, alpha 2 M was determined in the bronchoalveolar lavage fluid (BALF) from patients with interstitial lung diseases. Those diseases were active and inactive sarcoidosis, hypersensitivity pneumonitis and idiopathic pulmonary fibrosis (IPF, including collagen disease). We divided the IPF patients into two groups, 'acute type' and 'chronic type', judging from the prognosis. alpha 2 M/Albumin ratio in BALF in the active sarcoidosis and acute type IPF groups is significantly higher than that in the inactive sarcoidosis and chronic type IPF. These findings suggest that alpha 2 M in BALF can be a sensitive marker of the interstitial lung disease.