Antibodies to junctional sarcoplasmic reticulum proteins: probes for the Ca2+-release channel.

The junctional face membrane plays a key role in excitation-contraction coupling in skeletal muscle. A protein of 350 kDa, tentatively identified as a component of the junctional feet, connects transverse tubules to terminal cisternae of sarcoplasmic reticulum [Kawamoto, Brunschwig, Kim & Caswell (1986) J. Cell Biol. 103, 1405-1414]. The membrane topology and protein composition of sarcoplasmic reticulum Ca2+-release channels of rabbit skeletal muscle were investigated using an immunological approach, with anti-(junctional face membrane) and anti-(350 kDa protein) polyclonal antibodies. Upon preincubation of the terminal cisternae with anti-(junctional face membrane) antibodies, Ca2+-ATPase and Ca2+-loading activities were not affected, whereas anti-(350 kDa protein) antibodies stimulated Ca2+-ATPase activity by 25% and inhibited Ca2+-loading activity by 50% (at an antibody/terminal cisternae protein ratio of 1:1). Specific photolabelling of terminal cisternae proteins with [14C]doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Stimulation of Ca2+ release by doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Half-maximal inhibition was obtained at an antibody/terminal cisternae protein ratio of 1:1. Kinetic measurements of Ca2+ release indicated that anti-(350 kDa protein) antibodies prevented Ca2+-induced Ca2+ release, whereas the ATP-stimulation and the inhibition by Mg2+ were not affected. These results suggest that: (i) Ca2+- and doxorubicin-induced Ca2+ release is mediated by Ca2+ channels which are selectively localized in the junctional face membrane; (ii) the 350 kDa protein is a component of the Ca2+-release channel in native terminal cisternae vesicles; and (iii) the Ca2+-activating site of the channel is separate from other allosteric sites.     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.     

Developmental changes in galactosyltransferase activity in the rat small intestine

During postnatal development, UDP-Gal: GlcNAc(beta 1-4)-galactosyltransferase (4 beta-GT) and UDP-Gal:GalNAc(beta 1-3)-galactosyltransferase (3 beta-GT) activities were increased by 17- and 24-fold, respectively, in the rat small intestine. The injection of cortisone into suckling rats resulted in precocious induction of distal 4 beta- and 3 beta-GT activities by 2.7- and 1.8-fold, respectively. Injection of phorbol-12-myristate-13-acetate (PMA) resulted in precocious induction of distal 3 beta-GT by 2.7-fold. These results suggest that intestinal galactosyltransferase activities are under developmental regulation and can be modified by cortisone and PMA.     

Molecular dynamics of antitumor ether-linked phospholipids in model membranes: a spin-label study

We have synthesized a spin-labeled derivative of ET-18-OCH3, a known antitumor ether-linked phospholipid. The spin-labeled analog was shown to be as potent as ET-18-OCH3 in inhibiting 3H-thymidine uptake of HL60 leukemic cells. Electron spin resonance (ESR) studies showed that the mobility of this ether-linked phospholipid in the membrane is more restricted when compared to its ester-linked counterparts. It is probable that the absence of the bulky carbonyl oxygens allows closer packing of the two alkyl chains in the ether-linked phospholipid, thereby reducing the angular amplitude of the motion of the alkyl chains. These findings may be of importance in elucidating mechanisms by which the antitumor ether-linked phospholipids perturb the structure of cellular membranes.     

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the lelel of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5-triphosphate and uridine 5-phosphate.     

P1 nuclease defines a subpopulation of active SV40 chromatin--a new nuclease hypersensitivity assay.

Under exhaustive digestion conditions P1 nuclease was found to cleave a subpopulation of intracellular SV40 chromatin only once. The major P1 cleavage site in SV40 DNA was mapped at the origin of DNA replication, and the two minor sites at the SV40 enhancers. The P1-sensitive SV40 chromatin subpopulation was found to have higher superhelical density than the bulk of the intracellular SV40 chromatin. Furthermore, pulse labeled SV40 DNA which had higher superhelical density than that of the steady state viral DNA (S.S.Chen and M.T.Hsu, J.Virol 51:14-19, 1984) was also found to be preferentially cleaved by P1 nuclease. These results are consistent with a supercoil-dependent alteration of chromatin conformation near the regulatory region of the viral genome that can be recognized by P1 nuclease. Since P1 nuclease cleaves the subpopulation of SV40 chromatin only once without further degradation, this nuclease can be used as a general tool to define viral or cellular chromatin fraction with altered chromatin conformation and to map nuclease hypersensitive sites. Preliminary studies indicate that P1 makes limited double stranded cleavages in cellular chromatin to generate large DNA fragments.     

Effects of methotrexate-carcinoembryonic-antigen-antibody immunoconjugates on GW-39 human tumors in nude mice

Summary Methotrexate (MTX) was conjugated to an anti-carcinoembryonic antigen monoclonal antibody (NP2) by using amino-dextran as an intermediate carrier. The drug was chemically linked to amino-dextran (averageM _r = 40000), and the resulting MTX-dextran was then site-specifically attached to the carbohydrate moiety of the antibody. Athymic nude mice that carried human colonic GW-39 tumors (s. c.) were treated with the immunoconjugate. In this study, the specific conjugate caused a greater inhibition of the tumor growth than either free MTX or its conjugate with dextran and an irrelevant antibody. The intermediate MTX-dextran and the unlinked mixture of MTX-dextran with NP2 were both relatively ineffective in inhibiting tumor growth. The greatly reduced host toxicity permitted the use of the MTX-dextran-NP2 in a high-dose therapy of this tumor system.Supported in part by U.S.P.H.S. grant CA39 841 from the NIH     

Mapping of Col3a1 and Col6a3 to proximal murine chromosome 1 identifies conserved linkage of structural protein genes between murine chromosome 1 and human chromosome 2q

We have investigated the degree of synteny between the long arm (q) of human chromosome 2 and the proximal portion of mouse chromosome 1. To define the limits of synteny, we have determined whether mouse homologs of seven human genes mapping to chromosome 2q cosegregated with anchor loci on mouse chromosome 1. The loci investigated were NEB/Neb, ELN/Eln, COL3A1/Col3a1, CRYG/Len-2, FN1/Fn-1, VIL/Vil, and COL6A3/Col6a3. Ren-1,2 and Acrg were included as two proximal mouse chromosome 1 anchor loci. The segregation of restriction fragment length polymorphisms at these loci was analyzed in the progeny of Mus spretus x C57BL/6J hybrids backcrossed to the C57BL/6J inbred strain. We found that five of the structural protein loci and the two anchor loci form a linkage group on proximal murine chromosome 1. The proposed gene order of this group of linked markers is centromere - Col3a1 - Len-2-Fn-1-Vil-Acrg-Col6a3-Ren1,2. Neb and Eln are linked neither to each other nor to any other marker on proximal mouse chromosome 1. Therefore, the mouse loci Col3a1 and Col6a3 are identified as flanking markers of the linkage group of structural protein loci. The estimated genetic map distances are Col3a1-13.3 cM-Len-2-3.4 cM-Fn-1-3.8 cM-Vil-9.6 cM-Acrg-2.1 cM-Col6a3-18.3 cM-Ren1,2. The available map information for human chromosome 2q markers and mouse chromosome 1 markers presented here tentatively identifies Col3a1 and Col6a3 as the border markers that define the limits of the syntenic chromosome segment. The order of mouse genes on chromosome 1 and their human homologs on chromosome 2q also appears to be conserved, suggesting that mapping of murine genes on the conserved segment may be useful to predict gene order in man.     

Extraction of amino acids by aqueous two-phase partition

Summary Amino acids, including lysine, glutamic acid, and phenylalanine, in pure solution or in fermentation broth, were extracted with the aqueous two-phase system consisting of polyethylene glycol and salts, giving a very sharp separation. The partition is influenced by the type and the amount of salts used, pH and components of the broth.