Experimental studies on the effects of tension on intraneural microcirculation in sutured peripheral nerves.

Tension on an end-to-end nerve suture causes injury to the intraneural vessels. Impairment of the transverse anastomoses occurs first, followed by degeneration of the intrafascicular vessels. The the transverse anastomoses undergo complete degeneration, and the extrafascicular vessels also sustain severe damage. This series of circulatory disturbances is an important factor to consider when determining the advisability of an end-to-end nerve suture.     

Resonance Raman spectra of riboflavin and its derivatives in the bound state with egg riboflavin binding proteins

The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH3 RF), 3-carboxymethyl (3-CH2COOH RF), and 7,8-dichlororiboflavins (7,8-Cl RF), in H2O and D2O were observed in the 700-1700 cm-1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm-1 line of RF was shifted to ca. 1300 cm-1 for 3-D RF, 3-CH3 RF, and 3-CH2COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm-1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C = O stretching mode was observed in the present resonance Raman spectra.     

NMR relaxation characteristics of rubidium-87 in perfused rat salivary glands.

Rubidium is a good substitute for potassium in many biological systems, and it has been suggested that rubidium-87 nuclear magnetic resonance (87Rb-NMR) spectroscopy could be used to measure K+ fluxes across membranes in intact tissues. To evaluate this possibility, isolated rat mandibular salivary glands were perfused with solutions containing Rb+ in place of K+. The 87Rb signals arising from the intra- and extracellular compartments were first separated by spectral subtraction and then subjected to line-shape analysis. The narrow extracellular signal was a single Lorentzian (line-width 156 Hz), whereas the broader intracellular signal consisted of two Lorentzian components (ca. 530 and 3080 Hz). Double-quantum filtering of the 87Rb signal from the glands revealed two components of transverse relaxation in antiphase (rate constants 1.8 and 13.3 ms-1), showing the probable involvement of quadrupolar interactions in the relaxation of intracellular Rb+. We conclude, therefore, that both line-shape analysis and double-quantum filtering could provide a basis for the measurement of unidirectional K+ fluxes in intact tissues.     

A Novel Glial Growth Inhibitory Factor, Gliostatin, Derived from Neurofibroma

Neurofibroma tissue was investigated for the presence of glial growth modulators that would suppress the proliferation of glial cells. A novel endogenous polypeptide inhibitor of proliferation and DNA synthesis in glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising dye and anion-exchange column chromatography, and HPLC. A monoclonal antibody raised against partially purified gliostatin showed no cross-reactivity with known cytokines, but adsorbed the growth inhibitory activity of gliostatin and immunochemically visualized the putative gliostatin bands on western blot analyses. Although the product showed an apparent M(r) of 100,000 accompanied by an inhibitory activity on gel filtration column chromatography, it migrated at a lower apparent M(r) of 50,000 under the reducing conditions on western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50-kDa subcomponents. Gliostatin was a potent growth inhibitor acting at nanomolar concentrations against all glial tumor cells and glia maturation factor-stimulated astroblasts, but not neuronal cells.     

NMR characteristics of intracellular K in the rat salivary gland: a 39K NMR study using double-quantum filtering

Intracellular K of the perfused rat mandibular salivary gland was measured by 39K NMR spectroscopy at 8.45 T. Multiple-quantum NMR arising from multiple-exponential decay was used to eliminate the resonance due to extracellular K in the perfused gland at 25 degrees C. The resonance due to intracellular K consisted of two Lorentzian signals stemming from the [spin 1/2 to -1/2] coherence (sharp resonance) and the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences (broad resonance). The transverse relaxation time (T2) corresponding to the [spin 1/2 to -1/2] coherence was ca. 2.5 ms, and that corresponding to the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences was ca. 0.4 ms. The relaxation time of the double-quantum coherence of rank 3 (originating from product operators like Ix2Iz) was determined to be ca. 0.2 ms. These results suggest the possibility of the presence of a single homogeneous population of intracellular K with a correlation time of ca. 2.5 x 10(-8) s and a quadrupolar coupling constant of ca. 1.4 MHz.     

Measurement of intracellular Na in the rat salivary gland: a 23Na-NMR study using double quantum filtering

23Na in the prefused rat mandibular salivary gland was measured by spin-echo double quantum filter 23Na-NMR spectroscopy at 8.45 T. Resonances due to the intracellular 23Na and the interstitial 23Na were observed in the perfused gland at 25 degrees C. The resonance due to intracellular 23Na consisted of two Lorentzian signals stemming from the [1/2 mean value of -1/2[ coherence (sharp resonance) and the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences (broad resonance). The transverse relaxation rate constant corresponding to the [1/2 mean value of -1/2[ coherence was 95 +/- 4 s-1 and that corresponding to the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences was 1360 +/- 75 s-1 (mean +/- S.E., n = 5). The resonance due to the interstitial 32Na had longer relaxation rate constants, and disappeared upon administration of dysprosium triethylenetetramine-N,N',N",N",N"'-hexaacetic acid.     

Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin E1

BL191, a newly developed phosphodiesterase inhibitor, markedly potentiated a differentiation of neuroblastoma cell clones (Neuro2a, NS-20Y, and N1E115) induced by dibutyryl cyclic adensoine 3′:5′-monophosphate(dibutyryl cAMP) and prostaglandin E₁ (PGE₁). BL191 (1 mM) inhibited DNA synthesis more strongly when used together with PGE₁ (0.5 μg/ml) and dibutyryl cAMP (0.5 mM) than papaverine (1.6 μg/ml) alone did. The inhibition rates of DNA synthesis were 72.5% for N1E-115, 75.3% for Neuro2a, and 82.5% for NS-20Y. After the treatment with BL191. PGE₁, and dibutyryl cAMP for 48 h all of three cell lines became enlarged and flattened, and extended long processes. The specific activities of choline acetyl transferase (EC 2.3.1.9) of NS-20Y and dopamine β-hydroxylase (EC 1.14.17.1) of N1E-115 increased about 3-fold as compared to the controls. The tumorigenicities of Neuro2a and N1E-115 cells were decreased, but not of NS-20Y. These data suggest the heterogenous responsiveness in neuroblastoma cells to drug treatment.     

Resonance Raman study of D-amino acid oxidase-inhibitor complexes

The resonance Raman (RR) spectra of the complexes of D-amino acid oxidase (DAO) with benzoate derivatives were measured. The RR spectra of complexes of DAO with benzoate derivatives excited at 514.5 nm are similar to one another and also similar to that of oxidized flavin. In the cases of DAO-o-NH2-benzoate and DAO-o-OH-benzoate complexes, however, the line at 568 or 565 cm-1, derived from the benzoate derivative, was intensified. In the case of DAO-o-NH2-benzoate complex, which has an intense charge-transfer absorption band, the resonance enhancement of the Raman lines at 1583 and 568 cm-1 in the RR spectrum excited at 632.8 nm is striking. The former line is known to involve the vibrational displacements of the N(5) and C(4a) atoms of isoalloxazine and the latter is considered to be derived from a ring deformation mode of o-NH2-benzoate. This suggests that the o-NH2-benzoate molecule lies along the N(5)-C(4a) bond and parallel to the flavin face. A Raman line derived from o-OH-benzoate in the RR spectrum of DAO-o-OH-benzoate complex excited at 514.5 nm was detected. This result supports the view that the complex has a charge-transfer band, as has been pointed out by Massey and Ganther. Also, the spectrum of quasi-DAO-o-OH-benzoate complex is identical with that of the complex of DAO, suggesting that the active sites of these two enzymes have similar structures.     

A study of flavin-protein and flavoprotein-ligand interactions. Binding aspects and spectral properties of D-amino acid oxidase and riboflavin binding protein.

To study flavin-protein and flavoprotein-ligand interaction, the absorption, CD and MCD spectra of riboflavin, FAD, roseoflavin, the complexes of riboflavin and roseoflavin with riboflavin binding protein(RBP),D-amino acid oxidase(D-AO) and its complexes with ligands were observed in the spectral region of 310-600 nm and the binding properties of D-AO with di-substituted benzoate derivatives and of RBP with roseoflavin were also measured. The dimer of D-amino acid oxidase has a higher affinity for di-substituted benzoate derivatives than the monomer. The change in the absorption of FAD in D-AO caused by the binding of the first ligand to the dimer, which can bind two ligands, was similar to that caused by the binding of the second ligand. Roseoflavin could bind to RBP in a 1 : 1 ratio and the dissociation constant was 3.8 x 10(-8)M. The protein fluorescence of RBP was quenched by about 86% due to complex formation with roseoflavin. The MCD spectra showed similar patterns for all molecular complexes of riboflavin and FAD, with two negative extrema of ellipticity which probably correspond to the Faraday B-term, but the Faraday A-term could not be observed, suggesting that there was no degeneracy in the excited state of flavins. It is also suggested, based on a comparison of the absorption, CD and MCD spectra, that the vibronic structure of flavin was modified differently by each flavin-protein or flavoprotein-ligand interaction. Comparison of the absorption, CD and MCD spectra(310-600 nm) for roseoflavin and the roseoflavin-RBP complex revealed that there were five spectral components around 320, 340, 400, 500, and 550 nm in roseoflavin.