Vanadate selectively inhibits the K0+-activated Na+ efflux in squid axons

The effects of internally applied 1 mM vanadate on the Na⁺ efflux in dialysed squid axons were found to depend on the presence of external K⁺. In K⁺-free artificial sea water, vanadate did not produce any change in the rate of Na⁺ efflux, whereas in the presence of 10 mM K⁺ the Na⁺ efflux was reduced to values even lower than those observed in the absence of K⁺ (inversion of the K⁺-free effect). In vanadate-poisoned axons, K⁺ and NH₄⁺ at low concentrations activated Na⁺ efflux, but at high concentrations both cations were inhibitory. However, NH₄⁺ was always a better activator and a poorer inhibitor than K⁺.     

Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage

Chondroitin sulfate E (CS-E), a chondroitin sulfate isomer containing GlcAbeta1-3GalNAc(4,6-SO(4)) repeating unit, was found in various mammalian cells in addition to squid cartilage and is predicted to have several physiological functions in various mammalian systems such as mast cell maturation, regulation of procoagulant activity of monocytes, and binding to midkine or chemokines. To clarify the physiological functions of GalNAc(4,6-SO(4)) repeating unit, preparation of CS-E with a defined content of GalNAc(4,6-SO(4)) residues is important. We report here the in vitro synthesis of CS-E from chondrotin sulfate A (CS-A) by the purified squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) which catalyzed transfer of sulfate from 3(')-phosphoadenosine-5(')-phosphosulfate to position 6 of GalNAc(4SO(4)) residues of CS-A and dermatan sulfate (DS). When CS-A was used as an acceptor, about half of GalNAc(4SO(4)) residues, on average, were converted to GalNAc(4,6-SO(4)) residues. Anion exchange chromatography of the CS-E synthesized in vitro showed marked heterogeneity in negative charge; the proportion of GalNAc(4,6-SO(4)) in the most negative fraction exceeded 70% of the total sulfated repeating units. GalNAc4S-6ST also catalyzed the synthesis of oversulfated DS with GalNAc(4,6-SO(4)) residues from DS. Squid GalNAc4S-6ST thus should provide a useful tool for preparing CS-E and oversulfated DS with a defined proportion of GalNAc(4,6-SO(4)) residues.     

Specific photoisomerization of retinal in squid rhodopsin and metarhodopsin

The composition of retinal isomers in the photosteady-state mixtures formed from squid rhodopsin and metarhodopsin was determined by high-pressure liquid chromatography. A large amount of 9-cis-retinal was obtained at liquid N₂ temperature when rhodopsin was irradiated with orange light, but only small quantities of 9-cis-retinal were obtained at 15°C. Scarcely any 9-cis-retinal was produced from metarhodopsin by irradiation at liquid N₂ temperature. A large quantity of 7-cis-retinal was found in the photoproduct of rhodopsin irradiated at solid carbon dioxide temperature, but not at 15°C and liquid N₂ temperature. 7-cis-Retinal was not produced from metarhodopsin at any temperatures. These results indicate that the photoisomerization of retinal is regulated by the structure of the retinal-binding site of this protein. The formation of 9-cis- and 7-cis-retinals is forbidden in the metarhodopsin protein.     

Analysis of the acid polysaccharides from squid cranial cartilage and examination of a novel polysaccharide

The polysaccharides of cranical cartilge were isolated by ethanol precipitation after papain digestion and β-elimination procedures and were fractionated chromatographically on CPC-cellulose. In addition to the previously described, heavily oversulphated chondroitin sulphate, the tissue contained small amounts of hyaluronic acid, which, however, co-eluted with the chondroitin sulphate from the CPC-cellulose. Approx. 20% of the isolated polysaccharides consisted of an acidic polysaccharide which to our knowledge is not previously described. This polysaccharide consists mainly of glucuronic acid, galactose and mannose in a molar ratio of 1:2:1. Gel chromatography of the preparation indicated a polydisperse molecule with an apparent average molecular weight of 39200 on weight basis $\text{(}\text{M}{}_{\mathrm{<mtext>w</mtext>}}\text{)}$ and 31400 on number basis $\text{(}\text{M}{}_{\mathrm{<mtext>n</mtext>}}\text{)}$.     

An ATP-dependent Na/Mg countertransport is the only mechanism for Mg extrusion in squid axons

The components of magnesium efflux in squid axons have been studied under internal dialysis and voltage clamp conditions. The present report rules out the existence of an ATP-dependent, Na₀- and Mg₀-independent Mg²⁺ efflux (ATP-dependent Mg²⁺ pump) leaving the Mg²⁺---Na⁺ exchange system as the only mechanism for Mg²⁺ extrusion. The main features of the Mg²⁺ efflux are: (1) The efflux is completely dependent on ATP. (2) The efflux can be activated either by external Na⁺ (forward Mg²⁺---Na⁺ exchange) or external Mg²⁺ (Mg²⁺---Mg²⁺ exchange). (3) The mobility of the Mg²⁺ exchanger in the Na₀⁺-loaded form is greater than that in the Mg²⁺-loaded one. (4) In variance with the Na⁺---Ca²⁺ exchange mechanism, Mg²⁺---Mg²⁺ exchange is not activated by external monovalent cations. (5) ATPγS replaces ATP in activating Mg²⁺---Na⁺ exchange suggesting that a phosphorylation/dephosphorylation process regulates this transport mechanism.     

The effect of temperature on veratridine action in squid giant axons

Resting membrane potential and intracellular sodium and potassium concentrations were determined at 5 and 21°C in normal and veratridine-treated axons of the squid Doryteuthis plei. 300 μM veratridine produced an increase in the intracellular sodium concentration, which changed from 52 to 284 mM in 10 min of exposure at 21°C, and from 76 to 260 mM at 5°C. Under the same treatment the intracellular potassium concentration changed from 357 to 221 mM (21°C) and from 334 to 194 mM (5°C). All the changes could be prevented by adding 1 μM tetrodotoxin. Veratridine (30, 100 and 300 μM) increased the resting sodium permeability of the giant axon, and the effect was greater at 21°C. The affinity of the membrane for veratridine increases when the nerves are cooled, the three concentrations tested produce maximum activation of the sodium channels at 5°C. But only the higher two concentrations are saturating at 21°C.     

Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons

Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na⁺ efflux goes through the Na⁺ pump. We found that (i) internal K⁺ had a strong inhibitory effect on Na⁺ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na⁺, (ii) a reduction in ATP levels from 3 mM to 50 μM greatly increased the apparent affinity for external K⁺, but reduced its effectiveness compared with other monovalent cations, as an activator of Na⁺ efflux, and (iii) the relative effectiveness of different K⁺ congeners as external activator of the Na⁺ efflux, though affected by the ATP concentration, was not affected by the Na⁺/⁺ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na⁺ + K⁺)-ATPase can be reached by interaction with external K⁺ after phosphorylation and with internal K⁺ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.     

Neutral protease inhibitors from human intervertebral disc and femoral head articular cartilage

Assays of several proteases, incorporating guanidinium chloride extracts of human femoral head cartilage and intervertebral disc, demonstrated that both tissues contain inhibitors of certain serine proteases. Trypsin, chymotrypsin and a granule extract of human polymorphonuclear leukocyte containing elastase and cathepsin G activities, were inhibited by low molecular weight fractions prepared by Sephadex G-75 chromatography. Using a radioassay, it was further shown that these fractions inhibit proteolysis of cartilage proteoglycan. The inhibitor in intervertebral disc is concentrated in the nucleus pulposus, with a decreasing gradient to the periphery of the annulus fibrosus.It is proposed that these inhibitors confer at least partial protection against pathological proteolysis of the proteoglycans in human articular cartilage and nucleus pulposus.     

Collagen type IX: Evidence for covalent linkages to type II collagen in cartilage

A major site of pyridinoline cross-linking in bovine type IX collagen was traced to a tryptic peptide derived from one of the molecule's HMW chains. This peptide gave two amino acid sequences (in 2/1 ratio) consistent with it being a three-chained structure. The major sequence matched exactly that of the C-telopeptide of type II collagen from the same tissue. A second HMW chain that contained pyridinoline cross-links also gave two amino-terminal sequences, one from its own amino terminus, the other matching exactly the N-telopeptide cross-linking sequence of type II collagen. We conclude that type IX collagen molecules are covalently cross-linked in cartilage to molecules of type II collagen, probably at fibril surfaces.     

An oligosaccharide component in proteoglycans of articular cartilage

Two types of sialic acid-containing component are released from articular cartilage proteoglycan monomer (D1) treated with 0.05 M NaOH containing 1 M NaBH₄. The smaller component, which has not been described before, contains galactosamine, glucosamine, galactose and sialic acid (Molar ratio 1:1:1:2). It is eluted from ECTEOLA-cellulose with low molarity (0.4 M) sodium formate and has a K_av of 0.70 on Bio-gel P30. Its presence on the proteoglycan monomer was demonstrated at all stages of foetal and adult life.     

放线菌来源的糖肽类抗生素的药学机制研究进展

糖肽类抗生素具有较好的抑制革兰氏阳性细菌生长的活性,临床上广泛用于治疗革兰氏阳性细菌导致的严重感染性疾病,也被认为是对抗这类顽固性病原菌的最后一道防线。随着耐药菌的不断涌现,糖肽类抗生素的应用越来越受到限制。本文针对糖肽类抗生素的结构特征与药效关系、生物学活性和病原菌对于它们的耐药机制,以及糖肽类抗生素的生物合成机制及其结构的合成生物学改造等方面进行了概述。最后,对糖肽类抗生素在应用中面临的问题进行了展望。     

Characterization of the (Na+ + K+)-ATPase in a membranous preparation from the optic ganglion of the squid (Loligo pealei)

(1) A membrane fraction enriched in $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ specific activity of approx. 2 units/mg and was $\text{>95\%}$ ouabain-sensitive. (2) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ had a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of $\text{0.42 ± 0.04}\text{mM}$ and a pH optimum of 7.0. It was inhibited by ouabain with a $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of $\text{0.32 ± 0.04 μ}\text{M}$. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with $\text{NaCl + KCl}\text{= 300}\text{mM}$. (4) The Mg²⁺ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was $\text{0.86 ± 0.10}\text{mM}$, and at 6 mM ATP, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was $\text{1.86 ± 0.44}\text{mM}$. High levels of Mg²⁺ caused inhibition of hydrolysis. (5) The interactions of Na⁺ and K⁺ were examined over a range of conditions. K⁺ levels caused modulations in the Na⁺ dependence in the range of 1–150 mM. (6) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.     

Plasmin degradation of cartilage proteoglycan

Employing agarose gel electrophoresis, physiological concentrations of plasmin have been shown to degrade purified proteoglycan monomers and aggregates isolated from bovine articular cartilage. Proteoglycan degradation was (1) proportional to plasmin concentration, (2) dependent on the conversion of plasminogen to plasmin by plasminogen activator, (3) not displayed by plasminogen or plasminogen activator alone, and (4) inhibited by a serine proteinase inhibitor. These results, coupled with other findings, provide further support for a possible role of plasmin/plasminogen activator in cartilage destruction associated with rheumatoid arthritis.     

Ribosomal RNAs synthesized by isolated squid nerves and ganglia differ from native ribosomal RNAs

The large rRNA of the squid comprises two chains that may be dissociated by heating at 65 degrees C. A single chain constitutes the small rRNA. Surprisingly, the RNAs synthesized by dissected squid fin nerves and stellate nerves and ganglia differed in size from native rRNAs and did not manifest thermal instability. Nonetheless, they resembled native rRNAs in relative abundance, subcellular distribution, lack of poly(A), and metabolic stability. In addition, newly synthesized RNA was localized in nerve and glial cells, as shown by autoradiographic analysis, and was assembled into 80S ribosomes, which supported the synthesis of neuron-specific neurofilament proteins. Following incubation of nerves and ganglia for >10 h, native rRNAs started to disappear, while two major newly synthesized RNAs progressively accumulated. As a result, after 20 h, native rRNAs were substituted by the two novel RNAs. With use of 32P-cDNA synthesized from the latter RNAs as a probe, the novel RNAs demonstrated a considerable degree of homology with native rRNA in northern analysis. Taken together, the data suggest that in dissected squid nerves and ganglia, the synthesis of native rRNAs is gradually terminated while two novel rRNAs are being synthesized, presumably as a correlate of reactive gliosis and/or neuronal degeneration/regeneration.     

Na---Ca exchange in squid optic nerve membrane vesicles is activated by internal calcium

The role of intracellular Ca²⁺ as essential activator of the Na⁺---Ca²⁺ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 μM free calcium exhibited a 2-fold increase in the initial rate of Na_i⁺-dependent Ca²⁺ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca²⁺ on the reverse mode of Na⁺---Ca²⁺ exchange (i.e. Na_i⁺---Ca₀²⁺ exchange) is saturated at about 100 μM Ca_i²⁺ and displays an apparent K_1/2 of 12 μM. Intravesicular Ca²⁺ produced activation of Na_i⁺---Ca₀²⁺ exchange activity rather than an increase in Ca²⁺ uptake due to Ca²⁺---Ca²⁺ exchange. The presence of Ca_i²⁺ was essential for the Na_i⁺-dependent Na⁺ influx, a partial reaction of the Na⁺---Ca²⁺ exchanger. In fact, the Na⁺ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca_i²⁺ an additional Na⁺---Na⁺ exchange was measured. The results suggest that in nerve membrane vesicles Ca²⁺ at the inner aspect of the membrane acts as an activator of the Na⁺---Ca²⁺ exchange system.     

vanA-mediated high-level glycopeptide resistance in Enterococcus faecium from animal husbandry

Abstract Glycopeptide-resistant Enterococcus faecium strains were isolated from a pig farm and a poultry farm both using avoparcin as a food additive. Such organisms were not isolated in a hen's eggs-producing farm not using avoparcin. Glycopeptide-resistant enterococci were also detected in broiler chicken carcasses that were delivered to a hospital's kitchen. The resistance was determined by the vanA gene as indicated by the detection of the inducible 39-kDa cytoplasmic membrane protein and of a vanA -specific DNA sequence amplified by polymerase chain reaction. Genomic DNA fragment patterns of strains from animal sources were different from each other and also from those of strains isolated in hospitals and from sewage treatment plants. This findings suggest the dissemination of the vanA determinant among different enterococcal strains of distinct ecological origin.     

Purification and properties of a diisopropyl-fluorophosphatase from squid Todarodes pacificus steenstrup

A diisopropyl-fluorophosphatase (DFPase) was purified from brain and ganglia of squid Todarodes pacificus steenstrup. The DFPase had a preference in hydrolysis toward diisopropylphosphorofluoridate (DFP). It also was able to hydrolyze O-1,2,2-trimethylpropyl methylphosphofluoridate (soman) and O-isopropyl methylphosphonofluoridate (sarin) at nearly equal hydrolytic rates but only 1/10 that of DFP. The hydrolytic activity toward diethyl-p-nitrophenylphosphate (paraoxon) was very low compared with DFP, so man, and sarin. The DFPase was purified 330-fold to a specific activity of 18,300 n mol/min/mg protein. Its molecular weight was 34,000 dalton determined by gel-filtration chromatography. Mn²⁺ stimulation of the DFPase was not observed when DFP and soman were the substrates, but with sarin, the rate increased onefold in the presence of 1.0 mM of Mn²⁺. Ethylenediamine tetraacetic acid disodium (EDTA-Na₂) at 0.05 M inhibited the DFPase activity about 30%. It could be concluded that this DFPase belongs to the squid-type DFPase.     

Purification and reconstitution of potassium channel proteins from squid axon membranes

Voltage-dependent K⁺ channels are responsible for repolarization of the cell membrane during the late phase of the action potential. Here we report the purification of proteins from squid axon membranes which bind the K⁺-channel blocker noxiustoxin (NTX), and their subsequent functional reconstitution in planar bilayers. The NXT-affinity purified proteins had M_r values of 60000 ± 6000, 160000 ± 15000 and 220000 ± 20000. Their incorporation into bilayers resulted in single-channel currents with three conductances, the most frequent one of 11 pS in 300/100 mM KCl (cis/trans). The voltage dependence, reversal potential and bursting behavior suggest that these are the K⁺ channels involved in the squid axon action potential.     

Synthesis of fibronectin in normal and osteoarthritic articular cartilage

The content and the biosynthesis of fibronectin was examined in disease-free articular cartilage and in articular cartilage from osteoarthritic canine joints. Fibronectin content was increased in extracts of cartilage from osteoarthritic joints. Incubation of cartilage in vitro with [³H]phenylalanine and subsequent isolation of [³H]fibronectin from a gelatin affinity column and characterization by SDS-polyacrylamide gel electrophoresis and by immunoprecipitation indicated that disease-free and osteoarthritic cartilage explants synthesized fibronectin. About 50% of the [³H]fibronectin was recovered in the incubation medium. The osteoarthritic cartilage synthesized and accumulated up to 5-fold more [³H]fibronectin than disease-free cartilage.     

The effects of vanadate on calcium transport in dialyzed squid axons Sidedness of vanadate-cation interactions

(1) Vanadate (VO₃^?) fully inhibits the ATP-dependent uncoupled Ca efflux (Ca pump) in dialyzed squid axons. (2) Vanadate inhibits with high affinity. The mean apparent affinity ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) obtained was 7 μM. (3) Inhibition by vanadate is dependent on Ca_o. External Ca lead to a release of the inhibitory effect. ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{mM}$). This antagonic effect can be reverted by increasing the vanadate concentration. Internal K⁺ increases the affinity of the intracellular vanadate binding site. External K⁺ has no effect on the inhibition. (4) Vanadate has no effect on the Na_o-dependent Ca efflux component (forward Na-Ca exchange) in the absence of ATP. In axons containing ATP vanadate modified this component.