Localization of anti-clathrin antibody in the sarcomere and sensitivity of myofibril structure to chloroquine suggest a role for clathrin in myofibril assembly

Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and alpha-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.     

Organization of macroalgal assemblages in the Northeast Pacific: the assumption of homogeneity and the illusion of generality

Evidence for the geographic generality of the causes of intertidal zonation and the indirect effects of a keystone predator, the sea otter, on subtidal kelp assemblages was examined. Most research on intertidal algal assemblages has been done at a few protected sites where zonation is distinct. Surveys of wave-exposed intertidal sites in central and northern California show that assemblage structure is highly variable. This indicates that our present understanding of assemblage organization, including the effects of mussel-algal interactions, may not be widely applicable. Surveys of kelp forest habitat along the entire coast of California suggest that deforestation by sea urchins is uncommon in the absence of sea otters. These examples indicate that the generality of commonly accepted causes of algal assemblage structure in the Northeast Pacific may be an illusion based on assumptions of environmental homogeneity.     

Sequence analysis and mapping of the Salmonella typhimurium LT2 umuDC operon.

In Escherichia coli, efficient mutagenesis by UV requires the umuDC operon. A deficiency in umuDC activity is believed to be responsible for the relatively weak UV mutability of Salmonella typhimurium LT2 compared with that of E. coli. To begin evaluating this hypothesis and the evolutionary relationships among umuDC-related sequences, we cloned and sequenced the S. typhimurium umuDC operon. S. typhimurium umuDC restored mutability to umuD and umuC mutants of E. coli. DNA sequence analysis of 2,497 base pairs (bp) identified two nonoverlapping open reading frames spanning 1,691 bp that were were 67 and 72% identical at the nucleotide sequence level to the umuD and umuC sequences, respectively, from E. coli. The sequences encoded proteins whose deduced primary structures were 73 and 84% identical to the E. coli umuD and umuC gene products, respectively. The two bacterial umuDC sequences were more similar to each other than to mucAB, a plasmid-borne umuDC homolog. The umuD product retained the Cys-24--Gly-25, Ser-60, and Lys-97 amino acid residues believed to be critical for RecA-mediated proteolytic activation of UmuD. The presence of a LexA box 17 bp upstream from the UmuD initiation codon suggests that this operon is a member of an SOS regulon. Mu d-P22 inserts were used to locate the S. typhimurium umuDC operon to a region between 35.9 and 40 min on the S. typhimurium chromosome. In E. coli, umuDC is located at 26 min. The umuDC locus in S. typhimurium thus appears to be near one end of a chromosomal inversion that distinguishes gene order in the 25- to 35-min regions of the E. coli and S. typhimurium chromosomes. It is likely, therefore, that the umuDC operon was present in a common ancestor before S. typhimurium and E. coli diverged approximately 150 million years ago. These results provide new information for investigating the structure, function, and evolutionary origins of umuDC and for exploring the genetic basis for the mutability differences between S. typhimurium and E. coli.     

Partial characterization of a metalloendopeptidase activity produced by cultured endothelial cells that removes the COOH-terminal tripeptide from 125I-atrial natriuretic factor

The presence of the COOH-terminal region of human atrial natriuretic factor-(99-126) (hANF) is necessary for the full expression of its biological activity. Here, we report on the partial characterization of a proteolytic activity in the conditioned medium from cultured bovine aortic endothelial cells that cleaves the Ser123-Phe124 bond of 125I-hANF generating the COOH-terminal tripeptide. The concentrated conditioned medium was fractionated by gel filtration high performance liquid chromatography and fractions were assayed for the ability to generate the COOH-terminal tripeptide from 125I-hANF. This analysis indicated that the protein responsible for this activity had an approximate molecular weight of 200,000 daltons. Of 16 protease inhibitors tested, only 1,10 phenanthroline, EDTA, EGTA and N-ethylmaleimide significantly inhibited the endopeptidase activity. Thus, we conclude that cultured bovine aortic endothelial cells produce a potentially novel phosphoramidon-insensitive metalloendopeptidase that removes the COOH-terminal tripeptide from 125I-hANF.     

Leukotrienes do not regulate interleukin 1 production by activated macrophages

The objective of this work was to investigate the role of leukotrienes in the production of IL-1 by activated human peripheral blood monocytes and mouse peritoneal macrophages. Using overnight adherent macrophages, stimulation with lipopolysaccharide or zymosan caused a time-dependent increase in IL-1 production. LTC4 was detected and preceded IL-1 production only in zymosan-treated macrophages. Lipopolysaccharide did not stimulate macrophages to produce LTC4. Zymosan-stimulated LTC4 production was inhibited by the lipoxygenase inhibitors, ICI207968 (3.20 microM), nordihydroguaiaretic acid (0.22 microM), phenidone (4.60 microM), REV5901 (0.20 microM), and the Merck 5-lipoxygenase "translocation inhibitor" MK886 (0.02 microM) with IC50 values as shown in parenthesis. However, none of these inhibitors reduced IL-1 production at concentrations which completely inhibited leukotriene synthesis. Taken together, these results do not support a role for leukotrienes in the production of IL-1 by zymosan-activated macrophages.     

Quenching of the amidolytic activity of one-chain tissue-type plasminogen activator by mutation of lysine-416

In contrast to most other serine proteases, tissue-type plasminogen activator (t-PA) possesses enzymatic activity as the one-chain zymogen form. The hypothesis that lysine residues 277 or 416 may be involved in stabilization of an active conformation of one-chain t-PA via salt-bridge formation with aspartic acid residue 477 was tested by site-directed mutagenesis. Four recombinant t-PA mutants were constructed. The amidolytic activities of these analogues were compared to that of authentic t-PA. Substitution of arginine-275 provided an analogue [( R275G]t-PA) resistant to plasmin cleavage. The amidolytic activity of [R275G]t-PA was comparable to that of authentic one-chain t-PA, and so was the activity of [R275L,K277L]t-PA, in which additional substitution of lysine residue 277 was carried out. This suggested that its presence was nonessential for obtaining one-chain t-PA activity. In contrast, substitution of lysine residue 416 to obtain [K416S]t-PA and [K416S,H417T]t-PA resulted in substantial quenching of amidolytic one-chain activity. As expected, the amidolytic activities of the two-chain forms were less affected by the substitution. Involvement of lysine residue 416 in one-chain t-PA activity was also indicated by decreased activities of [K416S]t-PA and [K416S,H417T]t-PA with plasminogen as the substrate. The one-chain activity of the lysine residue 416 substitution analogues was partially restored in the presence of fibrin. This could indicate that strong ligands such as fibrin might provide an alternative stabilization of the active conformation of one-chain t-PA.     

The use of bridging systems to increase genetic variability in compound chromosome strains for genetic control of Lucilia cuprina (Wiedemann)

Summary Genetic variability in the non-compound portion of the genomes of compound-chromosome (CC) strains intended for genetic control can be increased by the use of bridging strains which can be crossed to both CC and normal strains. Two bridging systems are described for chromosome-5 CC strains of Lucilia cuprina. The first system relies on the established viability and fertility of males trisomic for chromosome 5R. Males carrying the (5L.YL)23 half-translocation, a C(5R), and a normal chromosome 5 were crossed successfully to a CC strain and a normal strain. The second system uses a pair of reciprocal whole-arm 4;5 translocations to generate gametes disomic for 5R and nullosomic for 5L, which in combination with C(5L)-bearing gametes form viable near-euploid offspring with only small duplications and deficiencies. These offspring (C(5L); (4L.5R)357; (4R.5R)194; (4L.4R)) were crossed successfully with both CC and T(4;5)357/ + individuals. The latter were in turn crossed successfully with normal strains. The T(Y;5)23 system allows replacement of the non-CC genome with wild material more rapidly than the T(4;5)357/T(4;5)194 system, but unlike the latter does not allow replacement of the Y chromosome in the CC strain. The double translocation system is currently being used in L. cuprina.     

Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine

The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied. This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity. Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center. When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics. The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment. Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R. B., Rhee, S. G., Luterman, D. L., and Chock, P. B. (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme. The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity. The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.