Identification of six phosphorylation sites in the COOH-terminal tail region of the rat neurofilament protein M.

The COOH-terminal tail domain of the neurofilament polypeptide M from rat nervous tissue contains approximately six molecules of phosphate. We report here that protein kinases in a crude cytoskeleton preparation of rat nervous tissue phosphorylated a set of tryptic peptides of M similar (but not identical) to those phosphorylated by living dorsal root ganglion cells in culture. Using these phosphopeptides as markers, we purified these same peptides from rat spinal cord and identified six specific phosphorylation sites in M by enzymatic and chemical criteria. These sites, serines 502, 506, 536, 606, 608, and 666, are all located in the COOH-terminal tail domain. Four are embedded in the repeated motif KSP whereas two are within variants of this motif, KSD and ESP. All of the sites that were preceded by lysine were resistant to alkaline phosphatase prior to modification of the lysine with citraconic anhydride. The identification of these sites should aid in investigations of the function of the phosphorylation of this protein and provides criteria for identifying the relevant kinases.     

Enhancement of cellular immune function during cold adaptation of BALB/c inbred mice.

The cell-mediated immune function of cold-adapted BALB/c inbred mice was studied in experiments of splenic lymphocyte blastogenesis, indicated by tritium-labeled deoxythymidine incorporation and SDS-PAGE autoradiography of synthetic proteins in lymphocytes. Male BALB/c inbred mice were randomly divided into two groups: control (living at 25 degrees C) and cold-exposed (living at 2 degrees C). Results are as follows: in contrast with the control group, there was an obvious fluctuation of cell-mediated immune function in the cold-exposed group at initial cold exposure because of transient stress to cold; then cell-mediated immune function gradually recovered to control level. From Day 15, the cell-mediated immune function of the cold-exposed group was remarkably enhanced. On Day 15, the lymphocyte blastogenesis rate was increased by 20.66% (P less than 0.05), which implies the onset of cold adaptation; on Days 21 and 31, the rates increased by 80.15% (P less than 0.05) and 40.36% (P less than 0.05), respectively. Two to six months later, with continuing cold exposure, the murine lymphocyte blastogenesis rate in the cold-exposed group remained higher than that in the control group. The lymphocyte protein synthesis of the cold-exposed group, indicated by tritium-labeled leucine incorporation, apparently increased on Day 15 and the stimulated rate was 101.47% (P less than 0.05). SDS-PAGE autoradiography of synthetic proteins in lymphocytes demonstrated that after 2 weeks of cold exposure, protein bands were enriched in both quantity and quality. These results are identical to the results obtained from lymphocyte blastogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of Co(II)bleomycin with dioxygen.

The reaction of Co(II)bleomycin with dioxygen has been investigated. Dioxygen binds to the Co(II) complex within the time of mixing according to electron spin resonance and uv-visible spectroscopy and dioxygen analysis. Then, two dioxygenated cobalt centers react, releasing 1 mol of O2 and forming an intermediate characterized by a few highly shifted 1H NMR resonances and loss of the ESR spectrum. This is thought to be a dioxygen-bridged dimer of cobalt bleomycin molecules. Time-dependent absorbance and dioxygen measurements yield the same second order rate constant for this step of the reaction. According to uv-visible and NMR spectral analysis, the intermediate decays into diamagnetic products in a first order rate process. High performance liquid chromatography and 1H NMR studies demonstrate that the product contains two bleomycin species of equal concentration. One component is Co(III)bleomycin, designated Form II. The other is the peroxide adduct of Co(III)bleomycin, Form I, as determined by direct determination of hydrogen peroxide, which is slowly released from the product at low pH. In contrast, hydrogen peroxide is readily detected during the reaction of Co(II)Blm with O2. In isolation, Form I is unstable at pH 7 and is converted within 24 h into a mixture of Form I and Form II.     

Growth and morphological changes in the stomach of newborn pigs during the first three days after birth.

Growth and morphological changes in the stomach of newborn pigs were examined during the first 3 days after birth. The stomach grew disproportionately faster than the body as a whole during this period. The growth was due to hyperplasia and hypertrophy during the first day and mainly to hyperplasia thereafter as gastric DNA content increased progressively after birth, and the protein:DNA and RNA:DNA ratios increased only on the first day. Histological and morphometric analyses revealed that the growth was more pronounced in the gastric body region than in the cardiac and pyloric regions, and more pronounced in the mucosal layer than in other layers. The percentage of mucosal volume occupied by parietal cells (volume density) and the number of parietal cells per unit volume of gastric mucosa (numerical density) increased significantly 3 days after birth in the cardiac and body regions, but not in the pyloric region, of the stomach. The observed morphological changes coincide with the known pattern of functional maturation during the immediate postnatal period. It is suggested that a high level of circulating gastrin and oral ingestion of milk-derived growth factors in the newborn pig contribute to these changes.     

吲(口朶)乙酸和苄基腺嘌呤对菊芋再生新皮组织分化的影响

根据前人的研究,植物激素对部分茎段剥皮后新皮再生有一定的影响。草本植物菊芋(Helianthus tuberosus L.)在正常情况下环剥后,其维管组织的分化过程与杜仲、茄子很不一样,对于一些外源激素的刺激反应可能也有差异,为此,有必要应用一些外源激素对菊芋环剥后再生新皮的组织分化进行试验研究。     

A simple and efficient method for the separation and detection of small DNA fragments by electrophoresis in formamide containing agarose gels and Southern blotting to DBM-paper.

We show in this report that DNA fragments smaller than 300 bp are separated with high resolution by electrophoresis in concentrated (up to 7%) agarose gels containing 50% formamide. The separated DNA fragments can subsequently be quantitatively transferred to DBM-paper [Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA (1977) 74, 5350-54] using the Southern technique [Southern, E.M., J. Mol. Biol. (1975) 98, 503-517], while preserving the sharpness of the original gel pattern. Since thin (0.2-0.4 mm) and thick (up to 5 mm) agarose slab gels can be easily handled in vertical or horizontal apparatus, this method should prove to be a very useful extention of the Southern technique, applicable to a variety of analytical and preparative purposes.     

Cryo-EM structure of the fatty acid reductase LuxC–LuxE complex provides insights into bacterial bioluminescence

The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying bacterial luminescence have been studied for more than 60 years, the structure of the bacterial fatty acid reductase complex remains unclear. Here, we report the cryo-EM structure of the Photobacterium phosphoreum fatty acid reductase complex LuxC–LuxE to a resolution of 2.79 Å. We show that the active site Lys238/Arg355 pair of LuxE is >30 Å from the active site Cys296 of LuxC, implying that catalysis relies on a large conformational change. Furthermore, mutagenesis and biochemical experiments support that the L-shaped cleft inside LuxC plays an important role in substrate binding and reaction. We obtained a series of mutants with significantly improved activity as measured by in vitro bioluminescence assays and demonstrated that the double mutant W111A/F483K displayed the highest activity (370% of the WT). Our results indicated that the activity of LuxC significantly affects the bacterial bioluminescence reaction. Finally, we expressed this mutated lux operon in Escherichia coli but observed that the in vivo concentrations of ATP and NADPH limited the enzyme activity; thus, we conclude that the luminous intensity mainly depends on the level of metabolic energy.