[Morphologic study of GABA-ergic neurons in dissociated cell culture of spinal cord and dorsal root ganglion].

gamma-Aminobutyric acid (GABA) is an important inhibitive neurotransmitter in central nervous system (CNS). Many studies have been made about the distribution of GABA-ergic neurons in the spinal cord (SC), but little is known about the morphology of GABA-ergic spinal cord neuron (SCN) in culture. Moreover, whether GA-BA-ergic neuron existed in dorsal root ganglion (DRG) or not is still under discussion. Considering together with the fact that the same neuron can synthesize different. kinds of neurotransmitter in different periods of development, we find that it is attractive to study the GABA immunoreactivity of cultured SCNs and DRG cells. The SC and DRG were dissected from 12-14 day old mouse (C57BL/6J) embryo and plated at a density of 1 x 10(6)-2 x 10(6) cells per dish. At 5 day in vitro (DIV), the cells reaggregate and form complicated neurite network. While SCNs varies in the morphology of cell body and neurite, DRG cells of different sizes can be easily discriminated by their round cell bodies and sharply defined nuclei and nucleoli, and their sizes do not undergo major change during the culture period. Immunoreaction was performed by using a polyclonal anti-GABA serum (rabbit) and PAP procedure. Two types of immunoreactive SCNs were observed: 1. SCNs with intensely positive reacted somata, nucleoli, and neurites. 2. SCNs with reactivity shown only on part or whole of neurites and cell membrane while the cell body is negative (to be considered as the result of GABA uptake).(ABSTRACT TRUNCATED AT 250 WORDS)

Conformation of the abortifacient protein pinellin: a circular dichroic study.

The conformation of pinellin was studied by circular dichroism, which showed a minimum at 223 nm and a double maximum at 198-200 nm. The protein was rich in beta-sheet (about 40%) with little alpha-helix, based on current CD analyses. It was stable between pH4 and 10 beyond which it unfolded reversibly, but in alkaline solution, prolongly stored at, say, pH 12, it became irreversibly denatured. Thermal denaturation indicated a transition between 55 degrees and 68 degrees C; the solution at 80 degrees C was partially renatured upon air-cooling back to room temperature. Addition of sodium dodecyl sulfate caused a sharp increase in alpha-helix, which leveled off at 0.25 mM surfactant.

Time-dependent ultraviolet absorption changes in proteins at high pH.

The effect of alkali on the ultraviolet absorption of several proteins was examined by difference spectrophotometry. In addition to the well known generation of phenolate ions from tyrosine, time-dependent changes occurred. These were relatively slow in water, but arose more quickly and to a greater degree in 6 M guanidine hydrochloride. These time-dependent changes were attributed to modification of the sulfur-containing amino acids, cystine and cysteine. The magnitude of the changes depended on the number and accessibility to solvent of the cystine, cysteine or derivatized species present. The increase in absorption at 295 nm typically reached a maximum value for disulfide containing proteins after ca. 1 h exposure to pH greater than 12 in 6 M guanidine hydrochloride; thereafter the changes were at least partially reversible. Taken in conjunction with amino acid analysis data, the results lend support to the beta-elimination mode of action of alkali on proteins. However the reaction mechanism appears to be complex and more than one chromophore, arising from more than one reaction pathway, seems to be involved in the alkaline degradation of the sulfur-containing amino acids. Particularly for proteins containing large amounts of cystine or cysteine, caution must be exercised when performing tyrosine titration experiments in order to recognize and minimize potential interference from other non-tyrosine related chromophores.

The antibacterial effect of attacins from the silk moth Hyalophora cecropia is directed against the outer membrane of Escherichia coli.

The attacins are antibacterial proteins which accumulate in the hemolymph of the giant silk moth, Hyalophora cecropia, in response to a bacterial infection. Here we show that the permeability barrier function of the outer membrane is affected shortly after addition of attacin to growing cultures of Escherichia coli. Specifically, the penetration through the outer membrane of beta-lactam antibiotics, chicken egg white lysozyme and the detergent Triton X-100 was found to be facilitated. The sensitivity of E. coli to cecropin B, another antibacterial protein present in the hemolymph of H. cecropia, was also found to be increased after treatment with attacin. The results suggest that the target of the attacins in E. coli is the outer membrane. Other effects of the attacins which have been observed are likely to be indirect consequences of the alteration in the properties of the outer membrane. These effects include changes in the cell shape, irregular patterns of cell division and lysis. The minimal concentration at which the attacins affected the growth of E. coli was 1 and 0.5 microM for the neutral (pI 7) and basic (pI 9) attacins, respectively, which corresponds to less than 2% of the concentration of the attacins in the hemolymph of infected pupae.

Stability and physicochemical properties of a trypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L)DC).

The stability and physicochemical properties of the major trypsin inhibitor from the winged bean seed (designated trypsin inhibitor 2) have been studied in solution. The purified inhibitor, which stoichiometrically inhibits bovine trypsin in the molar ratio 1:1, is stable over the pH range 3-11 at ambient temperatures. Only a slight decrease in inhibitory activity occurs down to pH 2, but a sharper decrease occurs at pH values above 11. The inhibitor is stable to heat up to 60 degrees C, but at higher temperatures (60-90 degrees C) it is more stable at pH than at pH 5.5 or pH 8.0. Trypsin inhibitor 2 retains its inhibitory activity in 8 M urea at pH 8.0, but is more susceptible to 8 M urea at pH 4.0. The stronger denaturant 6 M guanidine hydrochloride, however, abolishes the inhibitory activity at both pH 4.0 and pH 8.0. The inhibitor was not inactivated in 0.14 M beta-mercaptoethanol at either pH; however, reduction in the presence of 8 M urea or 6 M guanidine hydrochloride results in a loss of inhibitory activity. Circular dichroism and optical rotatory dispersion studies indicate that the inhibitor structure is characterized by beta-sheet and unordered forms and the absence of alpha-helix. The positive CD band centered at 227 nm has been used to follow conformation change as a function of temperature. In line with the stability studies, the inhibitor conformation was thermally most stable at pH 3.0 and changed increasingly as the pH was raised. This band showed little change at neutral pH up to 8 M guanidine hydrochloride. Tyrosine titration in aqueous solution indicates that 1 or 2 of the 11 tyrosines are difficult to titrate even at pH 13. A more normal titration curve is obtained in 6 M guanidine hydrochloride, although at least one tyrosine side-chain appears to be buried in the protein interior and resists complete titration at pH values in excess of 12. These data show that this inhibitor has a high degree of stability, typical of other known protein proteinase inhibitors.

Insect immunity. The primary structure of the antibacterial protein attacin F and its relation to two native attacins from Hyalophora cecropia.

The attacins are antibacterial proteins present in the hemolymph of the pupae of the silk moth Hyalophora cecropia after bacterial infection. We present the primary structure of one attacin, the F form. We show that this protein is derived by proteolysis from the native protein, attacin E. Using a method for rapid purification from the hemolymph of immunized pupae of the neutral attacin E and a basic attacin, both proteins were found in freshly collected immune hemolymph. We conclude that they are the native products of two attacin genes, the existence of which was inferred from the isolation of two cDNA clones as described in the accompanying paper. The two proteins, which differed in their pIs (7 and 9), were found to have similar mol. wts. (20 000) and closely related primary structures, displaying a total of 40 amino acid substitutions, 12 of which were of a non-conservative nature.