Preparation and characterization of 'heparinocytes': erythrocytes with covalently bound low molecular weight heparin

In an attempt to create the possibility of stable, long acting, intravascular anticoagulation, low molecular weight heparin was modified by introducing a sulfhydryl group into the molecule (LMWH-SH). Human erythrocytes were covalently grafted with LMWH-SH by the use of a heterobifunctional coupling reagent which reacts with the SH group of LMWH-SH and surface exposed amino groups of erythrocytes now called 'heparinocytes' (HC). HC were morphologically indistinguishable from untreated erythrocytes and displayed identical osmotic resistance. The functionality of HC was analyzed by classical coagulation tests in which they dose dependently inhibited clot formation. HC were also functional in recalcified whole blood inhibiting thrombin formation as assessed by the cleavage of the chromogenic substrate S-2238. The system appears applicable as a potential autologous, long-term anticoagulant treatment or prophylaxis.     

Presence of di- and polyamines covalently bound to protein in rat liver

Acid hydrolysis of trichloroacetic acid precipitate from rat tissue (liver, kidney and testis) homogenate released significant amounts of acid-insoluble putrescine, spermidine and spermine. Following incubation of liver homogenate with [1,4-¹⁴C]putrescine, 1.4% of total radioactivity and 1.0% of labelled diamine were recovered in the acid-insoluble fraction. Exhaustive digestion of acid-precipitable material with proteinases (Pronase, aminopeptidase M, carboxipeptidase A, B and Y) revealed the presence of di- and polyamines and of N¹-(γ-glutamyl)spermidine, N¹-(γ-glutamyl)sperminine and N¹, N¹²-bis(γ-glutamyl)spermine. These derivatives were identified both by chromatographic analysis and by enzymatic digestion with purified γ-glutamylamine cyclotransferase. The finding of di- and polyamine γ-glutamyl derivatives in the proteinase-digested acid-insoluble fraction of homogenate may be considered as a proof of the in vivo transglutaminase-catalyzed binding of polyamines to proteins. This evidence suggests that di- and polyamines might have an important role in mammalian tissues through covalent binding to proteins by either one or both the primary amino groups.     

Identification of a covalently bound flavoprotein in rat liver mitochondria with sarcosine dehydrogenase.

A covalently bound flavoprotein having highest molecular weight among four covalently bound flavoproteins found in rat liver mitochondria was partially purified and characterized. Its subunit molecular weight was estimated to be 94,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its absorption maxima were observed at 353 and 460 nm. Since this flavoprotein was reduced by either sarcosine or dimethylglycine and oxidized by phenazine methosulfate, it was identified with sarcosine dehydrogenase.     

Noncovalently and covalently bound lipid on the receptor for immunoglobulin E

We describe experiments which indicate that lipids interact with the receptor for immunoglobulin E (IgE) in several ways. Evidence for loosely bound lipid comes from observations on the special conditions that are required in order to oxidatively iodinate the alpha chain of the receptor in IgE-receptor complexes. Evidence for tightly, but still noncovalently, bound lipids comes from studies on the lipids required to inhibit dissociation of the subunits of the receptor in micellar detergent. Finally, biosynthetic studies indicate that the subunits of the receptor contain ester-linked fatty acids. The latter modification appears to take place on receptors that have already been inserted into the plasma membrane.     

Effect of relaxin on bound cAMP in rat uterus

Relaxin was able to elevate uterine bound cAMP in the presence of 3-isobutyl-1-xanthine in buffers of low or high (0.5 M NaCl) ionic strength. Significant elevation was observed 10 min after exposure to the hormone. The peak in bound cAMP preceded that in total cAMP (15 vs 20 min, respectively, under isometric conditions). The response was concentration-dependent, with maximal elevation in bound cAMP observed at 0.1-0.4 microgram/ml relaxin. The effect of relaxin on bound cAMP represented a change in a sizable proportion of the total available binding sites. However, relaxin did not elevate bound cAMP in the absence of phosphodiesterase inhibitors under conditions where it clearly inhibited contractile activity. These data suggest that although relaxin can alter the level of bound cAMP in the rat uterus under some conditions, these effects may not be involved in the acute changes elicited by this hormone which result in the rapid inhibition of spontaneous contractile activity.     

Localization and identification of covalently bound flavoproteins in rat liver mitochondria by prelabeling of their flavin moiety

The covalently bound flavoproteins in rat liver mitochondria were prelabeled by injecting [14C]riboflavin into a rat, then liver mitochondria were obtained and further labeled with [3H]pargyline, a suicide inhibitor of monoamine oxidase. When the mitochondria were subjected to osmotic lysis, two covalently bound flavoproteins having molecular weights of 110,000 and 94,000 were found in the supernatant. These proteins were identified as sarcosine dehydrogenases. Upon treatment of the membranous fraction with 1% Triton X-100, succinate dehydrogenase with a molecular weight of 70,000 was found in the soluble fraction, while two well-separated proteins doubly-labeled with 14C and 3H were found in the insoluble fraction. Their molecular weights were 61,000 and 57,000. By isoelectric focusing, two 3H peaks were observed with pI values of 8.3 and 8.4. The former corresponded to the 61,000-dalton protein, and the latter, to the 57,000 one. From the data obtained by using selective inhibitors, deprenyl and clorgyline, the [3H]pargyline-binding proteins with molecular weights of 61,000 and 57,000 were assigned to proteins of monoamine oxidases of type A and type B, respectively.     

Distribution of covalently bound and non-covalently bound secretory component on sbuclasses of rabbit secretory IgA.

The distribution of non-covalently bound secretory component (SC) on the two subclasses, IgA-f and IgA-g of rabbit secretory IgA (sIgA) was determined; the two subclasses were separated from each other by the use of antibody-immunosorbent columns and were subjected to SDS polyacrylamide gel electrophoresis. No SC appeared to be dissociated from the IgA-f molecules from each of 11 different rabbits; the IgA-g molecules, however, did have SC which was dissociated by SDS. Thus, all of the noncovalently bound SC on rabbit sIgA resides on the IgA-g subclass molecules.     

Microscopic studies on the transition between the sigmoid sinus, the superior bulb of the jugular vein and the first portion of the internal jugular vein

The author studied the structure of the tissue components of the tunicae of the terminal segment of the sigmoid sinus, particularly at the level of the transition between the sigmoid sinus, the superior bulb of the jugular vein and the first portion of the human internal jugular vein; it was established that the transition between the sigmoid sinus and the first portion of the internal jugular vein occupies the whole extension of the superior bulb of the jugular vein up to the inferior third of the first portion of this vessel. These vascular walls exhibit a structure similar to that of the dura, i.e. the tunica adventitia is formed by fascicles of collagenic fibers which describe discontinuous spirals, more open proximal to the beginning of the first portion of the internal jugular vein. Approximately in the inferior third of the first portion of the internal jugular vein, there appear fascicles of smooth muscle fibers which are arranged similarly to those of the venous walls. The tunica intima of these vascular segments exhibits an endothelium resting on a network of elastic fibers which may play the role of an internal elastic lamina. From the bony border of the jugular foramen there originates a connective system whose fascicles of collagenic and elastic fibers incorporate to the wall of the internal jugular vein after describing a stretch in spiral around the vascular lumen.     

Non-surgical catheterization of the jugular vein in young pigs

Current methods utilized for serial blood collection in the young pig are limited due to the stress and/or discomfort to which the pig is exposed. Thus, we have developed a non-surgical, minimally invasive cannulation technique which allows jugular vein catheter placement in the young pig without causing extended discomfort or stress. The procedure described is rapid (approximately 8 min/pig) and relatively simple, requiring only minimal anaesthesia for immobilization of the pig during the procedure. Routinely, 2-week-old piglets are standing in their pens within 15-20 min from initiation of the procedure. Piglets recover rapidly from the procedure and display no clinical indications of pain or discomfort. Serum concentrations of cortisol, a standard indicator of stress and/or discomfort, are asymptomatic within 2 h of completing the procedure (k = 26.14+/-3.03 ng/ml). Stress is limited to the initial immobilization of the piglets. With this technique of cannulation, we routinely maintain catheter patency for 2 days, and often for as long as 5 days.     

Norepinephrine, monoamine oxidase, and acetylcholinesterase in the rat jugular vein compared with other blood vessels

The observation that the rat jugular vein relaxed in response to norepinephrine but not to field stimulation prompted us to evaluate the extent of innervation in this tissue. The norepinephrine concentration in the jugular vein was about 10% of that in the mesenteric artery and vein. The low levels of norepinephrine were not due to higher monoamine oxidase activity relative to the enzyme activity in other blood vessels. In the jugular vein, as in heart and brain, serotonin was preferred substrate for monoamine oxidase whereas in the femoral vein, mesenteric vein, and mesenteric artery, phenylethylamine oxidation was greater. Based on kinetic and inhibitory studies with LY51641, a selective type A inhibitor, monoamine oxidase activity was not found to be uniform throughout the cardiovascular system. In addition to low levels of norepinephrine, acetylcholinesterase activity in the jugular vein was only 5 and 13% of the activity in the portal vein and mesenteric artery, respectively. Thus, we provide strong evidence that our inability to generate a response to field stimulation in the rat jugular vein results from the lack of functional innervation in this tissue. This information adds to the usefulness of this preparation for comparative studies of agents acting on the smooth muscle without the added complication of neuronal uptake mechanisms.     

Effect of various types of sports on the anatomy of the common carotid artery and the internal jugular vein

By means of ultrasonography the common carotid artery and the internal jugular vein at certain sports have been investigated. In swimmers the internal jugular vein is essentially dilated as compared to the common carotid artery. In high jumpers there is not any noticeable differences between the width of the artery and the vein. These data should be taken into account at training sportsmen. They are also important for development of investigations in the ultrasound anatomy.     

Comparative characteristics of chymotrypsin covalently bound on chemically and enzymatically oxidized agaroses

Summary The enzymatically activated agaroses compared with chemically activated show 25 fold lower amount of generated aldehyde groups, 33 fold lower binding capacity for chymotrypsin, 3 fold lower proteolytic as well as amidolytic activity toward AntAlaAlaPheNA of the corresponding fixed enzyme. Trans-cinnamoylimidazole titration data demonstrate 100% active bound enzyme in the case of enzymatically oxidized agaroses and 57% for chemically oxidized. The enzymic activation offers a small number of sites for ligand attachment in a unique microenvironment. The chemical activation yields a suitable matrix for effective chymotrypsin immobilization.     

Presence in the composition of bacterial DNA of covalently bound RNA fragments

The nucleotide composition of chromosome and plasmid DNA free of hybrid RNA isolated from resting Escherichia coli cells preliminary cultivated with the help of [14C] uracil has been studied. It has been established that DNA contains [14C]uracil side by side with adenine, thymine, guanine and cytosine. It confirms the presence of RNA fragments in the composition of bacterial DNA which are connected with it covalently.