Monoclonal antibodies to squid neurofilament (aNFP) and intermediate filament (aIFA) proteins were used as probes for the biochemical and immunocytochemical analyses of neurofilament structure and distribution in the squid giant axon and stellate ganglion. On Western blots the aNFP antibody stained exclusively the 220 kDa and high-molecular-weight (HMW) components of neurofilaments in the giant axon, whereas the aIFA antibody primarily labeled the 60 kDa protein in the giant axon and the 60 and 65 kDa proteins in the stellate ganglion. Dephosphorylation of axoplasmic proteins by alkaline phosphatase resulted in a decrease in the molecular weights of both the 220 kDa and HMW neurofilament proteins and a concomitant loss of reactivity with the aNFP antibody on Western blots. This indicated that the aNFP antibody is specific for a phosphorylated epitope in the neurofilament. Increased dephosphorylation of the 220 kDa protein led to an enhanced immunostaining of the resultant 190 kDa polypeptide by the aIFA antibody, suggesting that the phosphorylated epitope may mask the conserved epitope recognized by aIFA. Light and electron microscopic immunocytochemical studies show intense labeling by the aNFP antibody in the giant axon. In contrast, the aIFA antibody labeled the glial cells around the giant axon intensely, while labeling of the giant axon itself was considerably less than that with the aNFP antibody. Since the 60 kDa protein in axoplasm is intensely stained by the aIFA antibody on Western blots, the relatively low amounts of labeling seen on semithin and thin sections of the giant axon by this antibody may be due to the masking of the 60 kDa protein by in situ fixed axoplasmic proteins. However, the aIFA antibody intensely labeled glial cells within the stellate ganglion and "islands" of filaments and nuclear membranes within ganglion cells. No reactivity for either antibody was seen in synapses. The aNFP antibody specifically labeled "beadlike" portions and cross-bridges on the axonal neurofilaments, suggesting that these components consist of the 220 kDa and HMW proteins. In contrast, the aIFA antibody labeled relatively smooth filaments in ganglion and glial cells. These data suggest that the 65 kDa protein represents the squid glial filament protein and that the 60 kDa protein found in axoplasm represents the low-molecular-weight subunit in the axonal neurofilament. The latter appears to be formed and/or organized in "islands" of filaments within ganglion cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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Lasek and Hoffman (7) and Gilbert (1) have shown that the two major subunits of squid neurofilament protein (NFP) have molecular weights by SDS-PAGE of 2.0 x 105 and 6.0 • 104 daltons. The subunits of NFP were identified by purifying the intact neurofilaments away from the other major constituents of extruded axoplasm. Neurofilaments were identified by their characteristic fine structure as seen with the electron microscope. The standard methods developed to purify

Changes in the concentrations of ovarian steroids and pituitary gonadotrophins were measured by radioimmunoassay in the jugular plasma of six Clun Forest ewes throughout the oestrous cycle. The concentration of oestradiol began to rise 12-14 h before the onset of oestrus from values of 11-2 +/- 0-36 (S.E.M.) pg/ml during the luteal phase to 21-1 +/- 2-01 pg/ml at -8 to 0 h (oestrus). There was no distinct increase during the luteal phase. Circulating progesterone varied in a cyclic manner with the highest values at the mid-luteal phase (3-70 +/- 0-28 ng/ml; n = 28). In five out of six ewes the concentration was still quite high (1-86 +/- 0-43 ng/ml) at 35 h before the onset of oestrus. The concentration declined rapidly thereafter, reaching minimum values about 12 h before oestrus coincident with the increase in oestradiol concentration. Plasma LH increased from very low values of 2-59 +/- 0-09 ng/ml during the luteal phase to 75-3 +/- 7-4 ng/ml about 9 h after the onset of oestrus. Two peaks of plasma FSH concentration were detected after the onset of oestrus. The first peak (171-0 +/- 35-5 ng/ml) coincided with the LH peak and the second (133-0 +/- 10-7 ng/ml) occurred about 24 h later at a time when LH values were low. The mean FSH concentration at other times during the cycle was 61-9 +/- 2-8 ng/ml.

Cyclin‐dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and τ protein phosphorylation. This report describes the characterization of a 62‐kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full‐length cDNA was characterized coding for a 67.5‐kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose‐dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino‐terminal peptide‐specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components.

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The identities and quantities of calcium-binding proteins were determined in axoplasm isolated from the squid giant axon. 45Ca-binding assays on nitrocellulose filters containing axoplasm proteins separated by SDS-polyacrylamide electrophoresis revealed 4 major calcium-binding bands. These included the high-molecular-weight (Mr greater than 330 and 220 X 10(3] neurofilament proteins, an unidentified protein band that migrated around Mr 55,000, and a diverse group of proteins that migrated together around Mr 17,000. The low-molecular-weight (Mr 17,000) calcium-binding proteins could be resolved into calmodulin (ca. 120 mumol/kg axoplasm), 2 other Mr 17,000 calcium-binding proteins, and a small amount of calcineurin B. It is estimated that these calcium-binding proteins in squid axoplasm could theoretically bind about 1 mmol Ca2+/kg axoplasm. 125I-Calmodulin overlay and Western blot analyses disclosed a number of calmodulin-binding proteins in axoplasm. These included fodrin, calcineurin A, and Ca2+/CaM protein kinase II subunits.

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