Electron microscopic histochemistry of acetylcholinesterase of rat brain by Karnovsky's method

Summary Karnovsky's electron microscopic acetylcholinesterase method was successfully applied to rat brain fixed by vascular perfusion with either 2% glutataldehyde or 4% formaldehyde. 2% glutaraldehyde showed better fine structure but worse preservation of the enzyme than 4% formaldehyde.In the neuropil of the caudate nucleus, locus coeruleus and dorsal nucleus of the vagus, AChE activity was most intensely demonstrated on the plasma membranes of preterminal axons and somewhat less strongly on those of axon terminals and contacting dendritic branches. The axoplasm and synaptic vesicles were usually negative, while the cytoplasm and neurotubules of the dendritic branches showed some activity. In the nodule and uvula of the cerebellum moderate activity was exhibited on the synaptic contacts between the mossy fiber endings and granule cell dendrites. In the hypothalamus and other autonomic regions the characteristic coexistence of AChE and granulated vesicles of axon terminals could be demonstrated.In the perikaryon of positive nerve cells, AChE was observed strongly in the cytoplasm, disseminated irregularly or attached to the endoplasmic reticulum, while it was absent in the mitochondria and lysosomal dense bodies.     

Observations on the ultrastructure of nerve cells in the brain of the planarian,Dugesia gonocephala

Summary The submicroscopic structure of the nerve cells in the planarian brain was studied. Close similarities with neurons of other invertebrates were noted. In the cytoplasm of the planarian nerve cells there are at least three types of vesicular inclusions: 1) Clear vesicles (200–800 Å in epon embedded tissue) similar in morphological appearance to classical synaptic vesicles. These have generally some content of extremely low density but occasionally a dense core. 2) Dense vesicles (400–1,200 Å in epon embedded tissue) containing highly osmiophilic granules. Between the limiting membrane of the vesicle and the granule there is always a clear rim of variable width. These vesicles closely resemble synaptic vesicles described in vertebrate adrenergic endings. 3) Neurosecretory vesicles (600–1,300 Å in Vestopal embedded tissue) similar to elementary granules observed in neurosecretory systems in vertebrates and invertebrates. All three vesicle types have the same mode of origin from the Golgi membranes. All are present in the nerve cell processes of the neuropil as well as in the perikarya. Any given perikaryon or axon contains only one of the three vesicle types. All of these vesicles are considered to be discharged into the axons from their site of origin within the perikaryon.     

Three separate mitochondrial DNA sequences are contiguous in human genomic DNA

We isolated from a HeLa genomic library 38 plaques that hybridized to total mitochondrial (mt) DNA isolated from human placenta. One clone (HLmt-17.8) hybridized to a 740 base-pair (12 S ribosomal RNA gene and displacement loop) mtDNA probe and was characterized in more detail. Within its 17.8 x 10(3) base-pair insert a 1.6 x 10(3) base-pair mtDNA fragment was similar to three non-sequential coding genes of human mtDNA, including a part of the 12 S ribosomal RNA (684-971), the cytochrome oxidase I (6553-7302), and two NADH dehydrogenase [ND4L/ND4] (10,606-11,159). The similarity to human mtDNA sequences was 92.0%, 92.3% and 92.4%, respectively, the highest degree of similarity to human mtDNA so far reported. This is also the first report of several adjacent mtDNA-like sequences in cellular chromosomes. The mtDNA-like sequences in HLmt-17.8 was found in the DNAs of human placenta, freshly isolated human leukocytes, foreskin and several human cell lines; but it was not present in other primates or lower organisms. The HLmt-17.8 mtDNA-like region appears to be a pseudogene that transferred into the nucleus in humans more recently than nine million years ago.     

The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting in Escherichia coli DNA topoisomerase I deletion mutant.

Plasmid pBR322 DNA isolated from Escherichia coli DNA topoisomerase I deletion mutant DM800 is estimated to contain about 10% of the knotted forms (Shishido et al., 1987). These knotted DNA species were shown to have the same primary structure as usual, unknotted pBR322 DNA. Analysis of the knotting level of deletion, insertion and sequence-rearranged derivatives of pBR322 in DM800 showed that the presence of the region on pBR322 encoding resistance to tetracycline (tet) is required for high levels of plasmid knotting. When the entire tet region is present in a native orientation, the level of knotting is highest. Inactivating the tet promoter is manifested by a middle level of knotting. For deletion derivatives lacking various portions of the tet region, the level of knotting ranges from lowest to high depending on the site and length of the tet gene remaining. Inverting the orientation of tet region on the pBR322 genome results in a middle level of knotting. Deleting the ampicillin-resistance (bla)gene outside of its second promoter does not affect the level of knotting, if the entire tet gene remains. A possible mechanism of regulation of plasmid knotting is discussed.     

Purification and characterization of multicatalytic proteinase from eggs of the ascidian Halocynthia roretzi

A multicatalytic (high-molecular-weight) proteinase has been purified from eggs of the ascidian Halocynthia roretzi by a procedure including column chromatographies on DEAE-cellulose and hydroxylapatite and gel filtration on Sepharose 6B. The purified enzyme seemed to be homogeneous, as judged by disc-polyacrylamide gel electrophoresis, isoelectrofocusing, sedimentation velocity, and gel filtration. The molecular weight of the enzyme was estimated to be 610,000 by gel filtration. The isoelectric point and the sedimentation coefficient (S20,w) were 6.2 and 22.8S, respectively. The enzyme showed several protein bands with molecular weight ranging from 25,000 to 33,000 on SDS-polyacrylamide gel electrophoresis and a cylindrical or ring-like structure composed of several subunits under the electron microscope, indicating that the enzyme exists as a large molecule consisting of several protein components. The enzyme exhibited chymotrypsin-like and trypsin-like activities whose pH optima were both 7.0. Chymostatin and its analog, calpain inhibitor I, and elastatinal inhibited both activities, whereas leupeptin and antipain only inhibited the latter. The former activity was stimulated by a low concentration of SDS or fatty acid, whereas the latter was not. Thus, the properties of the enzyme purified from ascidian eggs are similar to those of multicatalytic proteinases from mammalian tissues.     

Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus.

The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.     

Primary structure of rat insulin-like growth factor-I and its biological activities

Rat insulin-like growth factor-I (IGF-I), a serum polypeptide with growth promoting activity, was isolated from rat serum by a combination of acid/ethanol extraction, affinity chromatography, and a series of reversed phase high performance liquid chromatography, cation exchange, and reversed phase. All peptide fragments produced by chymotrypsin digestion of reduced and carboxymethylated rat IGF-I were amino acid sequenced and compared with the sequence of human IGF-I. Three out of 70 of the rat amino acid residues differed from those of human IGF-I as follows: Asp20----Pro, Ser35----Ile and Ala67----Thr. Purified rat IGF-I cross-reacted with polyclonal anti-human IGF-I antibody 75% as compared to human IGF-I, but it cross-reacted only 3% with monoclonal anti-human IGF-I antibody. Thus, it is possible to monitor the metabolic fate of human IGF-I, when injected into rats, without interference by endogenous rat IGF-I. Rat IGF-I showed 65% activity in the radioreceptor, 28.6% activity in the lipogenesis and 22.5% activity in the free fatty acid release inhibition assays as compared to human IGF-I on a protein quantity basis.     

Strain-dependent differences in susceptibility of mice to experimental Angiostrongylus costaricensis infection

Experimental Angiostrongylus costaricensis infection was carried out in inbred strains of mice (C57BL/6 BALB/c, DBA/2 and C3H/He). All strains became infected with this parasite. Marked differences in mortality and in worm burden were found among inbred strains of mice tested. A significant reduction was shown in worm length from mice compared to that from cotton rats.     

Characterization of Cysteine Proteases Functioning in Degradation of Dynorphin in Neuroblastoma Cells: Evidence for the Presence of a Novel Enzyme with Strict Specificity Toward Paired Basic Residues

Two dynorphin-degrading cysteine proteases, I and II, were extracted with Triton X-100 from neuroblastoma cell membrane, isolated from accompanying dynorphin-degrading trypsin-like enzyme by affinity chromatography on columns of soybean trypsin inhibitor-immobilized Sepharose and p-mercuribenzoate-Sepharose, and separated by ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose and TSK gel DEAE-5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes, were inhibited by p-chloromercuribenzoate, N-ethylmaleimide, and high-molecular-weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E-64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1-17) at the Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1-17) at the Lys11-Leu12 bond and the Leu12-Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg-Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme.     

Procalpain I in cytoplasm is translocated onto plasma and granule membranes during platelet stimulation with thrombin and then activated on the membranes.

Thrombin stimulation of platelets resulted in changes in the subcellular localization of calpain I, with a concomitant alteration of its molecular weight as measured by immunoblotting. Calpain I in resting platelets was distributed as procalpain I, an 80 kDa form which does not exhibit the enzyme activity, and 83% of the total antigen was localized in the cytosol fraction. When platelets were stimulated with thrombin, the total content of calpain I antigen was not significantly changed as compared with that of the resting platelets, though a decrease in the cytosolic distribution of 80 kDa form (from 83% to 47% of the total antigen) was observed with concomitant appearance of the active 76 kDa and intermediate 78 kDa forms of calpain I and increase in the 80 kDa form in the granule and membrane fractions. These results indicated that calpain I was translocated from the cytosol to both the plasma and granule membranes as procalpain I and then activated on the membranes during platelet stimulation with thrombin.