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Biomedical subjects

K Rose

Publications and source records attributed to K Rose.

At least 91 records · Page 5Linked to original sources
PubMed1995

In vitro and in vivo comparison of a randomly coupled antibody fragment-enzyme conjugate with a site-specific conjugate.

Two antibody fragment-enzyme conjugates, one obtained by random coupling of the two protein component, the other by site-specific ligation of the same component, were compared in vitro and in vivo for their usefulness in antibody directed enzyme prodrug therapy (ADEPT). The in vitro studies have shown that the site-specific conjugate has a higher antigen binding capacity, while both conjugates had similar specific enzymic activities. In vivo, the site-specific conjugate was cleared more rapidly. When correction was made for this faster clearance, both conjugates showed similar antitumor efficacy in a mouse xenograft system upon administration of a prodrug.

Animals↗
PubMed1994

Preparation and characterization of novel substrates of insulin proteinase (EC 3.4.99.45).

The specificity of insulin proteinase (EC 3.4.99.45) has been difficult to categorize using only its natural substrates. By exploiting the fact that two substrates competing for the same enzyme inhibit one another, we have found some new substrates of the insulin proteinase from porcine muscle. Two of these substrates, a tryptic fragment of BSA and a fragment of cytochrome c, have been shown to be cleaved at a single site. The albumin fragment, as well as another fragment of cytochrome c., have susceptibilities (Vmax/Km) comparable with that of insulin. In a second aspect of the study, the porcine-muscle enzyme was shown to be related to other members of its superfamily in that it was immunoprecipitated by a monoclonal antibody raised against the insulin-degrading enzyme from human red blood cells and has the same cleavage sites on insulin as has the rat skeletal-muscle insulin proteinase. We note, however, a possible discrepancy between our results and those of another group regarding the subunit size (110 kDa) of the immunoprecipitated material.

Amino Acid Sequence↗
PubMed1994

Chemo-enzymic backbone engineering of proteins. Site-specific incorporation of synthetic peptides that mimic the 64-74 disulfide loop of granulocyte colony-stimulating factor.

We present the concept of chemo-enzymic backbone engineering of proteins. Recombinant DNA techniques are used to produce appropriate proteins that are enzymically fragmented to give the starting materials. These fragments are modified specifically at their chain termini either enzymically (coupling of a hydrazide to the C terminus) or chemically (periodate oxidation of N-terminal serine to a glyoxylyl function). The modified fragments, which need no side protection whatever, are mixed together and religate themselves spontaneously under mild conditions. The hydrazone bond thus formed can be reduced if desired, which stabilizes the linkage and enhances the flexibility of the local conformation. In this way biologically or chemically derived structures can be incorporated into the protein, and the choice of the chemical ones is free of all of the constraints of the genetic code. We believe that this combined approach gives access to constructions that could not be derived by either recombinant or chemical methods alone. We illustrate the particularity of this concept by the engineered modifications of the 64-74 disulfide loop region of human granulocyte colony-stimulating factor. Analogs constructed include one which, in spite of having a nonpeptide link in its backbone, has full biological activity.

Amino Acid Sequence↗
PubMed1994

Trolox attenuates cortical neuronal injury induced by iron, ultraviolet light, glucose deprivation, or AMPA.

The vitamin E analog, trolox, protected cultured cortical neurons against damage induced by exposure to either iron ions or ultraviolet (UV) light, consistent with an ability to inhibit free radical-mediated cytotoxicity. Trolox also reduced neuronal death induced by 24 h exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), but not that induced by N-methyl-D-aspartate (NMDA). When combined with the NMDA receptor antagonist dextrorphan, trolox also reduced the neuronal injury induced by glucose deprivation.

Animals↗
PubMed1994

The semisynthesis of [octadeutero-PheB1-octadeutero-ValB2]-porcine insulin and its characterization by mass spectrometry.

Insulin analogues labelled with stable isotopes (e.g. deuterium, 18O, 15N, etc.) are authentic (the native structure is rigorously maintained), non-radioactive (preferred for injection into man) and can easily be distinguished from endogenous insulin by mass spectrometry by virtue of their molecular masses. Appropriate combinations of amino-protecting groups (methylsulphonylethyloxycarbonyl and t-butoxy carbonyl), Edman degradation and chemical coupling were used to produce [octadeutero-PheB1]-porcine insulin and [octadeutero-PheB1-octadeutero-ValB2]-porcine insulin. The analogues were characterized by electrospray ionization mass spectrometry. Standard mixtures of labelled and unlabelled insulins were successfully studied by mass spectrometry. Isotope dilution mass spectrometry could therefore provide a useful direct measure of insulin under true physiological conditions, without many of the drawbacks of existing methods. In this regard, the analogue with 16 deuteriums was more suitable than the octadeuterated analogue, since the greater mass difference between the labelled and unlabelled forms enabled a lower mass spectrometric resolution to be used, resulting in higher sensitivity.

Animals↗
PubMed1993

Site-specific conjugation of a radioiodinated phenethylamine derivative to a monoclonal antibody results in increased radioactivity localization in tumor.

The preparation of a novel radioiodination reagent, the (aminooxy)acetyl derivative of (p-[125]-iodophenyl)ethylamine, is described. Conventional radioiodination of proteins involves the formation of iodotyrosine residues, but for in vivo applications such as thyroid or stomach immunoscintigraphy, the susceptibility of these residues to tissue dehalogenases constitutes a serious disadvantage. Using our new compound, which has a particularly nonreactive aromatic ring, we confirm and extend studies published by other workers indicating the much greater in vivo stability of iodophenyl compounds compared to the more conventional iodophenolic ones. In addition, the aminooxy group of our reagent gives a stable and specific linkage to aldehyde groups formed by periodate oxidation on the sugar moiety of antibody molecules. In vitro, favorable binding activity and high stability was obtained with a (([125I]iodoaryl)amino)oxy labeled monoclonal antibody directed against carcinoembryonic antigen. In vivo, using paired labeling experiments in nude mice bearing colon carcinoma xenografts, the (([125I]iodoaryl)amino)oxy-MAb (MAb = monoclonal antibody) was compared with the same MAb 131I-labeled by conventional chloramine-T method. Tumor 125I concentration of (arylamino)oxy MAb (measured as percent injected dose per gram) was significantly higher as compared to values obtained with a conventionally labeled 131I antibody. Additionally, thyroid uptake, an indicator of iodine release from the antibody, was up to 25 times lower after injection of 125I-MAb obtained by the new method as compared to the conventionally iodinated 131I-MAb.

Animals↗
PubMed1993

Facile identification by electrospray mass spectrometry of the insulin fragment A14-21-B17-30 produced by insulin proteinase.

We confirm the cleavage at position B16-17 of porcine insulin which occurs during in vitro digestion by insulin proteinase. The fragment A14-21-B17-30 was purified by reversed-phase high performance liquid chromatography and characterized by electrospray ionization mass spectrometry. Fast-atom bombardment mass spectrometry, on the other hand, failed to detect the presence of this fragment.

Animals↗
PubMed1993

In vitro changes in the properties of rabbit colonic muscularis mucosae in colitis.

BACKGROUND: The muscularis mucosae is the muscle layer closet to the site of elevated inflammatory mediator production in inflammatory bowel disease. Thus, it is the first muscle layer subject to their influence. METHODS: Using a rabbit trinitrobenzene sulfonic acid model of colitis, changes in the properties of the muscularis mucosae resulting from the inflammatory process were studied in vitro. RESULTS: Animals developed a mild colitis-like inflammation that was confined to the epithelium, lamina propria, and submucosa. Colitic muscularis mucosae contractile responses to leukotriene D4 and prostaglandins E2 and F2 alpha were significantly attenuated relative to the maximum tissue response to acetylcholine, whereas responses to histamine, substance P, and vasoactive intestinal polypeptide were unchanged. In addition, the stress-generating capacity of the colitic muscularis mucosae was compromised in a stimulus-independent manner and passive tension increased relative to active tension. CONCLUSIONS: The muscularis mucosae undergoes two significant alterations in colitis: (a) a selective desensitization to the effects of arachidonic acid metabolites and (b) an impairment of its excitation-contraction coupling mechanism. A loss of the ability of the muscularis mucosae to cause mucosal movement and alter luminal surface area may be an important early stage in the pathophysiology of inflammatory bowel disease.

Animals↗
PubMed1993

Hyperinsulinemia, insulin resistance, and hyperglycemia: contributing factors in the pathogenesis of hypertension and atherosclerosis.

Subtle abnormalities of carbohydrate metabolism and overt diabetes mellitus are both associated with a substantial increase in the prevalence of hypertension and the accelerated development of atherosclerosis. Hypertension is also a presumed independent risk factor for atherosclerosis, although some of the atherogenic properties of hypertension may be related to the recently recognized subtle metabolic abnormalities commonly found in persons with essential hypertension. The results of epidemiologic studies suggest that the elevated fasting and postprandial insulin levels that often occur in patients with essential hypertension, as well as in patients with type II diabetes mellitus, are an independent risk factor for atherosclerotic cardiovascular disease. Elevated glucose levels in patients with diabetes and hypertension appear to contribute to the acceleration of atherosclerosis, perhaps through toxic effects on the vascular endothelium. Other cardiovascular risk factors that are accentuated in persons with carbohydrate intolerance and hypertension include abnormalities in platelet function, clotting factors, the fibrinolytic system, and dyslipidemia. The goals of both nonpharmacologic and pharmacologic therapy for patients with abnormal carbohydrate metabolism and hypertension are to decrease cardiovascular risk as well as lower blood pressure.

Arteriosclerosis↗
PubMed1993

Pharmacological basis of contractile effects of peptidoleukotrienes on rabbit colonic muscularis mucosae.

The present study was designed to determine the mechanism(s) underlying the excitatory effects of several sulfidopeptide leukotrienes (LTs) on the muscularis mucosae in three regions of the rabbit colon. Proximal colonic muscularis mucosae was refractory to LTs C4, D4, and E4. In addition, it exhibited no responses to prostaglandin (PG) E2 and only a minimal contractile response to PGF2 alpha. Mid and distal colonic muscularis mucosae each responded to LTs C4, D4, and E4 and PGs E2 and F2 alpha with concentration-dependent contractions. In both regions, responses to LTD4 and LTE4 were abolished by indomethacin (10(-6) M) pretreatment. LTC4-induced responses were reduced approximately 50% by this procedure. The residual contraction to LTC4 was resistant to both tetrodotoxin (10(-6) M) and atropine (10(-6) M). In separate experiments, responses to LTC4 were also reduced by approximately 50% if LTC4 conversion to LTD4 and LTE4 was first prevented by L-serine borate (45 mM) in combination with L-cysteine (10 mM). It is concluded that proximal colonic muscularis mucosae lacks the appropriate functional excitatory LT and PG receptors. On mid and distal colonic muscularis mucosae, the actions of LTD4 and LTE4 and, in part, LTC4 are the result of PG production, whereas LTC4 has an additional direct action, possibly mediated through a selective LTC4 receptor.

Animals↗
PubMed1993

Defective modulation of colonic secretomotor neurons in a rabbit model of colitis.

The present in vitro study was conducted to investigate possible alterations in the control of colonic electrolyte transport in an experimental model of colitis. Intrarectal administration of trinitrobenzenesulfonic acid induced a colitis-like inflammation in the rabbit distal colon. Responses to amiloride and residual short-circuit current after this treatment were unchanged, suggesting that the absorptive and secretory mechanisms remained intact. Electrical field stimulation and vasoactive intestinal polypeptide, a candidate secretomotor neurotransmitter, both elicited similar responses in control and colitic tissue. This suggests that communication at the neuroepithelial junction was unimpaired. In untreated tissue, the effects of prostaglandin E2 (PGE2) and of acetylcholine were attenuated by tetrodotoxin, suggesting, therefore, that both play a role in the modulation of secretomotor neurons. In addition, PGE2 had an appreciable direct epithelial effect. Responses to both of these agonists were absent in colitis. The effects of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate were unchanged in colitis, suggesting that altered PGE2 responsiveness may involve changes in epithelial receptor number, affinity, or in their ability to mediate an increase in adenosine 3',5'-cyclic monophosphate levels. It is concluded that this rabbit model of colitis exhibits 1) defects in the modulation of secretomotor neurons by acetylcholine and PGE2 and 2) an attenuated epithelial response to PGE2.

Acetylcholine↗
PubMed1993

Black-white comparisons of 20-year coronary heart disease mortality in the Evans County Heart Study.

The Evans County Heart Study was initiated to compare the epidemiology of coronary heart disease (CHD) of black and white residents of a southeastern United States community. The study was prompted by a clinical observation that CHD appeared to be less frequent in black than white patients, despite a much higher prevalence and greater severity of hypertension in blacks. A total community-based, closed cohort study was initiated with a prevalence survey in 1960-1962. The 20-year follow-up experience of the Evans County cohort confirmed the importance of the major risk factors of serum cholesterol, smoking and blood pressure for middle age and older, black and white men and women for CHD, cardiovascular disease (CVD) and all-cause mortality. Black men were less likely to die of CHD than white men, and this relative protectiveness increased upon adjusting for risk factors and also increased with age. The relative protectiveness of black men for CHD mortality was also reflected in lower black than white CVD and all-cause mortality in men 65 years of age and older.

Adult↗
PubMed1993

Subsite affinities of Aspergillus niger glucoamylase II determined with p-nitrophenylmaltooligosaccharides.

Kinetic parameters were obtained for glucoamylase catalysed hydrolysis of substrates of an alpha-(1,4)-maltooligosaccharide series and of a p-nitro-phenyl-alpha-maltooligosaccharide series. p-Nitrophenyl substrates of chain length 11 and 17 were synthesized in 97% and 95% purity, respectively, to test the significance of binding at remote subsites. The affinities of the subsites > 4 are demonstrated to be insignificant. The subsite binding contributions for D-glucopyranosyl and for p-nitrophenyl residues were calculated.

Aspergillus niger↗
PubMed1992

Pyruvic acid is attached through its central carbon atom to the amino terminus of the recombinant DNA-derived DNA-binding protein Ner of bacteriophage Mu.

Ner protein of bacteriophage Mu, produced by recombinant DNA techniques in Escherichia coli, has been found to possess a molecule of pyruvic acid attached covalently through carbon-2 to the amino-terminal cysteine residue. The intact protein and the amino-terminal chymotryptic peptide were found by mass spectrometry to be 70 mass units heavier than expected. The modified peptide was unstable under mildly acid or mildly basic conditions. Two-dimensional nuclear magnetic resonance spectroscopy of the modified and unmodified forms of the amino-terminal chymotryptic peptide was consistent with the presence of pyruvate linked through carbon-2 to the amino-terminal Cys residue. Treatment of the modified form with 2,4-dinitrophenylhydrazine in acid medium led to the expected hydrazone of pyruvic acid, which was identified by high pressure liquid chromatography. Of the two proteins known to be modified by pyruvate through its central carbon (the other being human adult hemoglobin, in which the modified form represents only a very minor fraction), Ner is the first protein found to be modified quantitatively. Given the instability of the modification, it may be more prevalent than recognized hitherto. Incubation with 2,4-dinitrophenylhydrazine may offer a useful means of detecting the presence of pyruvate linked to proteins in this way.

Amino Acid Sequence↗
PubMed1992

Partial characterization of natural and recombinant human soluble CD23.

The purification to homogeneity of an active soluble 25 kDa fragment of CD23, produced in insect cells using the baculovirus expression system, is described. Peptide mapping and analysis by Edman degradation and mass spectrometry permitted partial characterization of the protein. A total of 165 out of 172 residues, including N-terminal and C-terminal regions, were mapped. The positions of the two disulphide bonds in the IgE-binding region were also determined: residue 110 is joined to residue 124, and residue 42 to residue 133. Natural CD23 25 kDa fragment was also analysed and found to possess the same disulphide bond arrangement. These results extend the previously noted sequence similarity with lectins to elements of secondary structure.

Amino Acid Sequence↗
PubMed1992

Human interleukin-5 expressed in Escherichia coli has N-terminal modifications.

Recombinant human interleukin-5 exists as four major isoforms all possessing N-terminal methionine. Peptide mapping and subsequent analysis by fast-atom-bombardment mass spectrometry (f.a.b.-m.s.) have shown that N-terminal modifications are the cause of the charge heterogeneity. In order of decreasing abundance, these are unmodified methionine, retention of N-terminal formyl group, oxidation of N-terminal methionine to sulphoxide and carbamoylation of the N-terminus. These results were confirmed by analysis of the reduced and alkylated intact protein by electrospray-ionization mass spectrometry. The implications of these findings for the production and characterization of recombinant proteins are briefly discussed.

Chromatography, High Pressure Liquid↗
PubMed1992

Colon carcinoma immunoscintigraphy by monoclonal anti-CEA antibody labeled with gallium-67-aminooxyacetyldeferroxamine.

Previous experimental results in nude mice showing that radiolabeling the monoclonal antibody anti-CEA 35 with 67Ga-aminooxyacetyldeferroxamine could give better tumor localization than radioiodination prompted us to initiate the present clinical study. The 67Ga-labeled antibody anti-CEA 35 (185 MBq, 0.7-1.7 mg) was injected preoperatively into 14 patients for colorectal carcinoma imaging. The same antibody labeled with 125I (3.7 MBq, 0.25 mg) was injected simultaneously to compare the 67Ga and 125I dose recoveries in surgical specimens. Twelve of 14 primary tumors gave a positive 67Ga scintigraph. The mean %ID/g recovered in all tumors 3-9 days after injection was significantly higher for 67Ga (0.019%) than for 125I (0.005%) (p < 0.001, paired t test). The tumor-to-normal tissue ratios were generally higher for 67Ga, with the exception of liver. We conclude that 67Ga-aminooxyacetyldeferroxamine improved immunoscintigraphy outside the liver, particularly in the pelvic region. We also show that deferroxamine infusion accelerates the excretion of 67Ga in eight patients and propose that this could lead to further improvement of immunoscintigraphy.

Aged↗