Completion of the amino acid sequence of the alpha 1 chain from type I calf skin collagen. Amino acid sequence of alpha 1(I)B8.

The complete amino acid sequence of the 279-residue CNBr peptide CB8 from the alpha 1 chain of type I calf skin collagen is presented. It was determined by sequencing overlapping fragments of CB8 produced by Staphylococcus aureus V8 proteinase, trypsin, Endoproteinase Arg-C and hydroxylamine. Tryptic cleavages were also made specific for lysine by blocking arginine residues with cyclohexane-1,2-dione. This completes the amino acid sequence analysis of the 1054-residues-long alpha (I) chain of calf skin collagen.     

Purification and partial characterization of fibrillin, a cysteine-rich structural component of connective tissue microfibrils.

Fibrillin, a connective tissue macromolecule (Mr = 350,000) which is normally insoluble in its tissue form, has been purified from the medium of human skin fibroblast and ligament cells in culture. Analysis of the amino acid composition indicates that fibrillin contains approximately 14% cysteine, of which one-third appears to be in the free reactive sulfhydryl form. Electron microscopic images of fibrillin reveal an extended, flexible molecule approximately 148 nm long and 2.2 nm wide. These length measurements are consistent with shape calculations based upon velocity sedimentation data. It is likely that the material we have purified from cell culture medium represents monomeric fibrillin consisting of a single polypeptide chain. Additional ultrastructural immunohistochemical data presented here suggest a model for the parallel, head-to-tail alignment of fibrillin molecules in microfibrils.     

Unstable expression and amplification of a transfected oncogene in confluent and subconfluent cells.

NIH 3T3 cells were transfected with a plasmid containing the transforming gene, v-src, from Rous sarcoma virus. One of the transformed cell lines isolated reverted to a flat, nontransformed morphology after cloning through soft agar. This cell line did not express the src gene and could no longer grow in soft agar. When these cells were held at confluence, spontaneous foci appeared which eventually covered the dish. The appearance of foci correlated with an increase in v-src gene expression, ability to grow in soft agar, and tumorigenicity in mice. When these transformed cells were trypsinized and held at subconfluence, both v-src expression and the transformed phenotype were progressively lost. Whereas rearrangement of the transfected gene was not detected, the gene copy number in the transformed cells was markedly increased (greater than 50-fold). Confluence-dependent gene amplification and deamplification have been retained after several cycles of growth alternately at high and low density, in cells recloned through soft agar, and after cells had been maintained continuously at high or low density. The results suggest that, in this cell line, reversible gene amplification plays a central role in expression of the transfected gene.     

The effect of insulin deficiency, dietary protein intake, and plasma amino acid concentrations on brain amino acid levels in rats

The effect of diabetes (streptozotocin, 65 mg/kg ip), dietary protein intake (15-60%), and plasma amino acid concentrations on brain large neutral amino acid levels in rats was examined. After 20 days, the plasma concentrations of methionine and the branched chain amino acids (BCAA), valine, isoleucine, and leucine were increased in diabetic rats. In brain tissue, methionine and valine levels were increased but threonine, tyrosine, and tryptophan concentrations were depressed. Increased protein consumption promoted a diabetic-like plasma amino acid pattern in normal rats while enhancing that of diabetic animals. However, with the exception of threonine, glycine, valine, and tyrosine, there was little effect on brain amino acid levels. A good association was found between the calculated brain influx rate and the actual brain concentration of threonine, methionine, tyrosine, and tryptophan in diabetic animals. There was no correlation, however, between brain influx rate and brain BCAA levels. Thus, the brain amino acid pattern in diabetes represents the combined effects of insulin insufficiency and composition of the diet ingested on plasma amino acid levels as well as metabolic adaptation within the brain itself.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: II. Cloning and expression in Escherichia coli

The genes for the large and small subunits of anthranilate synthase (trpE and trpG, respectively) have been cloned from Pseudomonas aeruginosa PAC174 into E. coli by R-prime formation with the broad-host- range plasmid R68.44. Sequential subcloning into plasmid vectors reduced the active Pseudomonas DNA fragment to a length of 3.1 kb. We obtained evidence that this region contains the promoter for its own expression and retains a vestigial regulatory response to tryptophan scarcity or excess.      

Structure of mouse type IV collagen. Amino-acid sequence of the C-terminal 511-residue-long triple-helical segment of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain

The sequence of 511 residues from the C-terminal portion of the triple helix of mouse alpha 2(IV) chain was determined by using the pepsin fragment P2 of collagen IV and two cDNA clones selected from an Engelbreth-Holm-Swarm (EHS) tumor library. The sequence contains nine interruptions of the triplet repeat Gly-Xaa-Yaa ranging in size from single insertions or deletions up to stretches of eleven amino acid residues. Five of these interruptions match those present in the homologous segment of the alpha 1(IV) chain but are otherwise different in length and/or sequence. A low homology was found for the triplet regions of the alpha 1(IV) and alpha 2(IV) chain which constitute more than 90% of the sequence. The data indicate a remote evolutionary relationship of the triple-helical sequences of the two constituent chains of basement membrane collagen.     

In vivo and in vitro noncovalent association of excised alpha 1 (I) amino-terminal propeptides with mutant pN alpha 2(I) collagen chains in native mutant collagen in a case of Ehlers-Danlos syndrome, type VII

The cause of the Ehlers-Danlos syndrome Type VII (EDS VII) is considered to be defective removal of the amino-terminal propeptide (N-propeptide) of Type I procollagen due to deficiency of procollagen N-proteinase, the enzyme responsible for the normal proteolytic excision of this precursor-specific domain. Molecules retaining the N-propeptide (pN-collagen molecules) are thought to cause defective fibrillogenesis and cross-linking which eventuate in dramatic joint laxity and joint dislocations, the clinical hallmark of this variety of EDS. Recent studies demonstrate that some EDS VII patients harbor small deletions of either the pro-alpha 1(I) or pro-alpha 2(I) chain of Type I procollagen. We have found an 18-amino acid deletion (due to exon outsplicing) in a mutant pro-alpha 2(I) chain from such a patient. The deleted peptide is the junctional segment (N-telopeptide) linking the alpha 2(I) N-propeptide and major triple helical domains; loss of this short segment results in union of these latter domains and produces a shortened pN alpha 2(I) chain. Directly extracted tissue collagen and pepsin-digested fibroblast collagen contain this mutant pN alpha 2(I) chain and normal alpha 1(I) chains, but not pN alpha 1(I) chains, indicating that the relatively larger alpha 1(I) N-propeptide is excised from the related alpha 1(I) chains. The fate of this alpha 1(I) N-propeptide was unclear and therefore whether or not the intact N-propeptide was, in fact, retained in native mutant collagen was also unclear. In this paper, we describe morphologic, chemical, and immunochemical studies which indicate that the alpha 1(I) N-propeptide is retained in noncovalent association with the mutant pN alpha 2(I) chain in native mutant collagen molecules both in vivo and in vitro. In both instances, the alpha 1(I) N-propeptides are proteolytically cleaved from the related alpha 1(I) chains. These data suggest that retention of a partially cleaved, but essentially intact N-propeptide in mutant collagen may play a role in the pathogenesis of this disease.     

Sodium channel activation does not alter lipid metabolism in cultured neuroblastoma cells

The interaction of voltage-sensitive Na⁺-channels and membrane lipid metabolism was examined by incubating cultured neuroblastoma cells with neurotoxins which alter the voltage-dependent relationship between the closed and open conformation of the channel protein. Guanidinium flux rate, a measure of Na⁺-channel activation, was increased 10-fold by the combined action of veratridine (100 M) and scorpion venom (28 g/ml). This response was completely blocked by tetrodotoxin (1 M). Under the same experimental conditions, the toxins did not increase the efflux of [³H]arachidonic acid from prelabeled cell membrane lipids or stimulate uptake of exogenous [³H]arachidonic acid. In addition, altering membrane fatty acid composition by incubating cells for 24 hr in a medium containing 50 M arachidonic or oleic acid did not alter guanidinium flux rates relative to that of control cultures. When cells were pulsed with³²Pi for 60 min and stimulated by veratridine plus scorpion venom for an additional 30 min, uptake of³²Pi into phosphatidylinositol as reduced; stimulating cells with bradykinin, a receptor agonist which activates the inositol cycle, promoted a 3.8 fold increase. Polyphosphoinositide turnover was not affected by Na⁺-channel activation, but was stimulated by bradykinin. These results suggest that voltage-sensitive Na⁺-channel activation in cultured neuroblastoma cells can function independent of membrane phospholipid and fatty acid metabolism.     

Schiff and pseudo-Schiff reagents: the reactions and reagents of Hugo Schiff,including a classification of various kinds of histochemical reagents used to detect aldehydes

During the 1860’s, Hugo Schiff studied many reactions between amines and aldehydes, some of which have been used in histochemistry, at times without credit to Schiff. Much controversy has surrounded the chemical structures and reaction mechanisms of the compounds involved, but modern analytical techniques have clarified the picture. I review these reactions here. I used molecular modeling software to investigate dyes that contain primary amines representing eight chemical families. All dyes were known to perform satisfactorily for detecting aldehydes in tissue sections. The models verified the correct chemical structures at various points in their reactions and also determined how decolorization occurred in those with “leuco” forms. Decolorization in the presence of sulfurous acid can occur by either adduction or reduction depending on the dye. The final condensation product with aldehyde was determined to be either a C-sulfonic acid adduct on the carbonyl carbon atom or an aminal at the same atom. Based on the various outcomes, I have placed the dyes and their reactions into five categories. Because Hugo Schiff studied the reactions between aldehydes and amines with and without various acids or alcohol, it is only proper to call each of them Schiff reactions that used various types of Schiff reagents.