Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

Urea metabolism in the cyanobacterium Anabaena cycadeae: regulation of urea uptake and urease by ammonia

A total of 160 Escherichia coli positive for F165 fimbrial antigen and isolated from diarrheic and septicemic animals, were examined for the presence of the pap, afa, and sfa/foc operons or related nucleotide sequences using colony hybridization. Most isolates shared DNA sequences with the pap operon sequences alone or in association with afa or sfa. Thus, our results indicate that F165-positive E. coli from diseased animals share DNA sequences with operons coding for adhesins important in human extra-intestinal disease and that multiple adhesin systems are often found in single isolates. However, 20% of the F165-positive isolates did not show any homology with the probes representing the three adhesin systems, suggesting that one of the operons responsible for F165 production could be different from the pap, sfa/foc, and afa operons.     

Assay of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.     

Nitrosyl cytochrome c oxidase. Formation and properties of mixed valence enzyme

We report the first resonance Raman scattering studies of NO-bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+, a2+(3) NO) and the mixed valence enzyme (a3+, a2+(3) NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide-bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe2+ state. In the mixed valence NO-bound samples, a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O-bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme. The carbonyl line of the cytochrome a3 heme formyl group in the fully reduced NO-bound enzyme appears at approximately equal to 1666 cm-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm-1 to approximately equal to 1667 cm-1. Thus, modes in cytochrome a2+(3) NO are sensitive to the redox state of the cytochrome a and/or CuA centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a2+(3) (NO) and the cytochrome a-CuA pair, and is linked to the cytochrome a3 (NO) by the coupling between CuB and the NO-bound cytochrome a3 heme.     

Differing amounts of genetic polymorphism in testes and male accessory glands ofDrosophila melanogaster andDrosophila simulans

We surveyed genetic polymorphism by two-dimensional gel electrophoresis of male reproductive tract proteins in 20 isofemale lines each ofDrosophila melanogaster andDrosophila simulans. After classifying 244 such proteins ofDrosophila melanogaster and 271 ofDrosophila simulans by their distribution between testes and accessory glands within the reproductive tract, significant correlations were found between genetic polymorphism and tissue distribution. In both species, gland-specific proteins were significantly more polymorphic than testis-specific proteins, as well as those found in both testes and glands. Simultaneously, inDrosophila simulans, proteins found in roughly equivalent relative abundance in both testes and glands were significantly less variable than gland-specific and testis-specific proteins, as well as those with a quantitative difference in relative abundance between testes and glands. These correlations may reflect general differences in variability between extracellular and intracellular proteins and between proteins with broad as opposed to tissue-specific distributions.We thank the Natural Sciences and Engineering Research Council of Canada for financial support (Grant A0235 to R.S.S.).     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Degradation of apolipoprotein B-100 in human chylomicrons

The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.