Regression of human breast tumor xenografts in response to (E)-2'-deoxy-2'-(fluoromethylene)cytidine, an inhibitor of ribonucleoside diphosphate reductase.

(E)-2'-Deoxy-2'-(fluoromethylene)cytidine (MDL 101,731) is a mechanism-based inhibitor of ribonucleoside diphosphate reductase (J. Stubbe, personal communication), an enzyme involved in DNA synthesis and therefore a potential target for cancer chemotherapy. In the present report, we show that MDL 101,731 inhibits the proliferation of several human breast cancer cell lines, including the estrogen-dependent cell line, MCF-7, and the estrogen-independent cell lines MDA-MB-231, MDA-MB-468, and MDA-MB-435 in vitro at nanomolar concentrations (50% inhibitory concentration, 15-26 nM). Administration of MDL 101,731 caused marked regression of tumors which formed after s.c. inoculation of all four of the cell lines in athymic (nude) mice. MDA-MB-231 tumors were found to be most sensitive to MDL 101,731 with a 90-100% cure rate at doses of MDL 101,731 between 2 and 20 mg/kg, given as once daily i.p. injections, 5 days/week for as little as 3 weeks. Almost complete cessation of MDA-MB-231 tumor growth was obtained with a dose of 0.5 mg/kg MDL 101,731 following the same dosing regimen. MDA-MB-468, MDA-MB-435, and MCF-7 tumors were not as sensitive as MDA-MB-231, but tumor regression of 50, 65, and 80%, respectively, was obtained after 5-6 weeks of treatment. The effects of MDL 101,731 on spontaneous metastasis of MDA-MB-435 cells from the mammary fat pad to the lung was also examined, and it was found that the number of lung metastases was significantly decreased if mice received MDL 101,731 while the primary tumors were growing and after primary tumors were surgically excised. Additionally, preliminary evidence raises the possibility that MDL 101,731 may induce apoptosis in MDA-MB-231 tumors. Our data suggest that the use of MDL 101,731 for the treatment of breast cancer and possibly other solid tumors should be pursued.

Isolation of the transfer RNA genes of bacteriophage T4 and transfer RNA synthesis in vitro.

Non-glucosylated T4 DNA was restricted with the endonuclease EcoRI and the mixture of DNA fragments separated by gel electrophoresis and transcribed with purified Escherichia coli RNA polymerase. Three purified fragments were shown to act as templates for tRNA synthesis. A smaller fragment, shown to be hybridizable to 32P-labeled T4 tRNA was not transcribable. It was concluded that the promoter for T4 tRNA synthesis had been separated from the structural genes in the smaller fragment by EcoRI and that the distal portion of the tRNA gene cluster lacks internal promoters which display in vitro activity. Preparations of non-glucosylated T4 DNA were never fully restricted with EcoRI and when the larger purified fragments carrying the tRNA were restricted with excess enzyme only a slight cleavage to yield the smaller fragments was obtained. The property of the DNA-limiting complete restriction is not know.

Chromogenic detection of antigen in bacteriophage plaques: a microplaque method applicable to large-scale screening.

We have developed a simple and rapid (24 h) enzyme-linked immuno-detection method to screen for rare antigen-positive phage among large numbers of antigen-negative ones. Horse-radish peroxidase-antibody conjugate, incorporated into the soft agar layer of a plaque assay system, is precipitated locally by antigen produced during plaque formation, and is detected by standard chromogenic methods. The method has been used to screen plaques of bacteriophage beta tox+ for the presence of diphtheria toxin and related cross-reacting material. When phage were plated on very dense bacterial lawns, they formed minute plaques (microplaques). Because of the high local concentration of antigen generated by lysis of the dense lawn, the microplaques gave more intense chromogenic signals than larger plaques formed on less dense Corynebacterium diphtheriae lawns. Thus, antigen-positive microplaques could be easily recognized even in the presence of very large numbers of antigen-negatives. In a reconstruction experiment, small numbers of antigen-positive phage were detected with high efficiency (greater than 75%) against a background of 3.8 X 10(4) antigen-negatives/cm2 of agar surface (equivalent to 2.4 X 10(6) plaques/9 cm petri plate). This screening method should facilitate isolation of phage mutants affecting production of given antigens and may be of particular value in detecting specific genes cloned into phage vectors.

Appendix: a model of plaque formation.

Equations describing plaque formation in soft agar have been based on certain simplifying assumptions, for which data are presented. The derived equations permit one to calculate (i) average plaque size as a function of the initial density of indicator cells (Do), (ii) the number of cells lysed per plaque as a function of Do, and (iii) the cumulative number of cells lysed at various stages of plaque development. The calculated values agree well with those determined experimentally.

Hybrid plasmids containing the araBAD genes of Escherichia coli B/r.

The DNA fragments generated by restriction endonuclease BamI which contain the araCBAD genes from E.coli B/r have been cloned. The DNA fragments containing ara genes were idenified by a compairson of the BamI fragments of lambdah80dara phages containing different ara deletion mutations. The ara genes were cloned into the plasmid pBR317, a derivative of ColE1. The cloned DNA fragments were analyzed by digestion with pairs of restriction endonucleases to determine the molecular weight of the chimeras and to identify the cloned ara DNA fragments. The cloned ara fragments were also identified by genetic complementation and recombination tests.

Identification of cephapirin metabolites and degradants in bovine milk by electrospray ionization--ion trap tandem mass spectrometry.

Liquid chromatography-ion trap tandem mass spectrometry (LC-MS/MS) with electrospray ionization was used to identify cephapirin metabolites and degradants in milk from cows dosed with cephapirin. The milk was extracted according to a previously published procedure. Structures for various components were tentatively identified by their molecular weight, product ion mass spectra, and/or correspondence to standard mass spectra. These components may have occurred as metabolites or as degradants that occurred on storage or during extraction. Compounds identified in the milk included cephapirin, desacetylcephapirin, cephapirin lactone, hydrolyzed cephapirin, and a reduced cephapirin lactone that has not previously been reported. Methylcephapirin was also identified, possibly as a trace contaminant in the formulation. Analysis of incurred milk extracts showed that cephapirin and desacetylcephapirin are the major residues in milk. Desacetylcephapirin residues persisted about as long as the parent drug. The detection limit for both residues by LC-MS/MS was approximately 1 ng/mL in milk. These results have implications for microbiological methods or rapid test kits, if such methods or kits respond to cephapirin metabolites and degradants present in the milk.

Note: Integration of trapped ion mobility spectrometry with mass spectrometry.

The integration of a trapped ion mobility spectrometer (TIMS) with a mass spectrometer (MS) for complementary fast, gas-phase mobility separation prior to mass analysis (TIMS-MS) is described. The ion transmission and mobility separation are discussed as a function of the ion source condition, bath gas velocity, analysis scan speed, RF ion confinement, and downstream ion optical conditions. TIMS mobility resolution depends on the analysis scan speed and the bath gas velocity, with the unique advantage that the IMS separation can be easily tuned from high speed (~25 ms) for rapid analysis to slower scans for higher mobility resolution (R > 80).

Cleavage of Nonglucosylated Bacteriophage T4 deoxyribonucleic acid by Restriction Endonuclease Eco RI.

DNAs lacking the glucosyl modification (Glc-) and additionally lacking the 6-methylaminopurine (N6-methyladenine) modification (Glc-, MeAde-) were prepared from appropriate T4 mutants. These DNAs were cleaved by the purified restriction endonuclease Eco TI from Escherichia coli. Normally modified DNA (Glc+, MeAde+) was not attached. The Eco RII and the hemophilus enzymes Hin dII and Hin dIII do not attack Glc-, MeAde- T DNA, possibly due to the presence of 6-hydroxymethylcytosine. Eco RI produces approximately 40 specific fragments from Glc- DNA ranging in molecular weights from 0.3 to 10.5 X 10-6.

Initiation and transcription of a set of transfer ribonucleic acid genes in vitro.

Using RNA polymerase purified from Escherichia coli, DNA isolated from the bacteriophage T4, and a bacterial supernatant fraction containing the necessary processing enzymes, a set of transfer RNAs can be formed in vitro. To characterize the site or sites of initiation of this tRNA transcription, rifampicin-resistant complexes of RNA polymerase, DNA, and either ATP (UTP and CTP) or GTP (UTP and CTP) were formed, and tRNA was transcribed from these stabilized sites. It is concluded that transcription of the entire set is initiated by ATP. To study the transcription of the tRNAs, the time sequence of the appearance of individual species was determined during synchronous transcription of a preformed RNA polymerase-DNA complex. The appearance of three RNA species is found to be consistent with the sequential transcription of a large polycistronic cluster; the order and distances, inferred from the times of transcription, are as required by the existing gene map. It is concluded that the initiation of tRNA transcription can occur, without accessory factors, with the insertion of ATP at a single or a few closely spaced sites, and that the tRNAs encoded by the bacteriophage T4 are present in a single operon.

Combined immunohistochemical and autoradiographic analyses of antigen/antibody interactions in tumor xenograft models.

A study was conducted to establish optimal conditions which would allow for the simultaneous localization of a carcinoma antigen and its complementary radiolabeled antibody. Immunoperoxidase staining was used to identify the tumor distribution of antigen, while tissue localization of the radiolabeled antibody was identified by autoradiography. The tumor associated glycoprotein-72 (TAG-72) antigen and the high affinity murine monoclonal antibody, CC49 IgG were used as the model antigen/antibody pair. Athymic female mice bearing either CX-1 or LS-174T human colorectal adenocarcinoma xenografts were used as animal/tumor test systems. Experimental mice each received a bolus intravenous injection of the CC49 antibody which was labeled with 125I (specific activity, 0.17 to 0.26 microCi/microgram). Control mice were given a bolus injection of MOPC-21 IgG monoclonal antibody (tumor irrelevant antibody) which was also radiolabeled with 125I (specific activity, 0.24 to 0.35 microCi/microgram). At 24 hours postinjection, all tumors removed, counted for radioactivity, and fixed in formalin. The avidin/biotin immunoperoxidase complex technique was used to identify TAG-72 antigenic sites on slide-mounted tissue sections. Nonradiolabeled CC49 IgG (0.5 micrograms/ml) was used as the specific antigen binding primary antibody in the immunostaining procedures. Nonradiolabeled MOPC-21 IgG (0.5 micrograms/ml) served as the negative control. Immunohistochemically stained tissue sections were coated with photographic emulsion and processed for autoradiographic localization of 125I-CC49 or 125I-MOPC-21. After an optimal exposure time of 6 days, slides were processed and examined under a light microscope. Results of the biolocalization experiment revealed that the % of injected dose/gram of 125I-CC49 in both LS-174T and CX-1 tumors (30.4 +/- 5.2% and 20.6 +/- 5.4%, respectively) were significantly greater (p greater than 0.01) than those for 125I-MOPC-21 (4.9 +/- 0.5% and 5.1 +/- 0.7%, respectively). In both tumor lines from mice injected with 125I-CC49, dense clusters of silver grains were found over those regions which were positive for TAG-72 immunoreactivity. These dual-labeled structures were also found in contact with, or in close proximity to the microvasculature. Tumors from mice which were injected with the control radioconjugate showed a random distribution of silver grains within stromal tissue but no specific localization to TAG-72 positive regions. We conclude that intravenously administered 125I-CC49 IgG localizes specifically to antigen-containing sites in the LS-174T and CX-1 tumor models. The methods described herein should serve as useful tools for the direct study of antigen-antibody interactions in tumor biology.

Meeting the need for public education about dementia.

Research continues to advance the knowledge of pathophysiology and development of effective methods for treating patients with Alzheimer disease and other dementias. Dissemination of information is likely to be slowest among the general population, who may be the first to recognize dementia symptoms but may also be reticent to discuss concerns because of fear, embarrassment, and/or inadequate knowledge. The feasibility of providing public education and access to dementia resources was studied using a toll-free interactive voice response (IVR) telephone system. Public interest in this service and willingness to use this technology were evaluated in a 1-month study conducted in a predominantly rural upper Midwest county (population of 102,565). One hundred ninety-three calls were received during November 1999, with an average length of 9 minutes and 29 seconds. One in six calls lasted 15 minutes or longer. One third of the calls were received outside typical business hours (8:00 AM to 6:00 PM). Concern for a parent or grandparent was the most frequent reason (50.6%) given for the call. Self-concern was indicated by 24.7% of the callers. Callers provided positive feedback. Such IVR technology may provide a cost-effective bridge to the "digital divide" existing among elderly, lower socioeconomic status, and rural populations underrepresented as computer and Internet users.

Preferential use of a H chain V region in antitumor-associated glycoprotein-72 monoclonal antibodies.

The DNA sequence of the mouse H chain V regions from five hybridomas directed against the human tumor Ag tumor-associated glycoprotein-72 (TAG-72) have been determined. This includes a previously determined VH gene sequence from a first-generation anti-TAG-72 mAb, B72.3, and the VH gene sequences from four second-generation anti-TAG-72 mAb, CC49, CC83, CC46, and CC92. A sequence comparison revealed a high degree of shared sequence identity between the five productively rearranged VH genes, suggesting derivation from a common germ line V region gene. In the process of cloning the unrearranged germ line gene, two highly related VH germ line genes were identified and designated VH alpha TAG-1 and VH alpha TAG-2. A comparison of the productively rearranged anti-TAG-72 VH sequences with the two germ line VH genes demonstrated that they were all derived from VH alpha TAG-1. In contrast, the L chain V regions are all derived from separate germ line V region genes. The preferential use of VH alpha TAG-1 in these five mouse hybridomas suggests that VH alpha TAG-1 is a preferred anti-TAG-72 H V chain region germ line gene and that the H chain plays a predominant role in the recognition of this Ag.

Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta.

A 1,942-base-pair DNA segment encoding the structural gene for diphtheria toxin was sequenced, and the primary structure of the toxin was deduced. Restriction enzyme fragments corresponding to nontoxic or hypotoxic peptides of the toxin were isolated from corynebacteriophage beta and cloned into Escherichia coli on plasmid pBR322, and the sequence was determined. The mature toxin molecule deduced from the sequence has 535 amino acid residues and a molecular weight of 58,342. The deduced sequence for the fragment A moiety was the same as that determined at the protein level, except for a single serine residue, which had been mispositioned in the earlier study. Several differences were noted with respect to the partial sequence data available on the fragment B moiety, some or all of which may reflect genetic variations among populations of corynephages carrying the toxin gene. The DNA sequence predicts a 25-residue leader peptide preceding the mature protein, which is presumably involved in secretion of the toxin from lysogenized Corynebacterium diphtheriae. We infer that initiation of translation probably occurs at a GTG codon (codon -25). Cloned restriction fragments containing sequences for the amino-terminal region of toxin, together with 5' flanking regions, were expressed in E. coli. Toxin-related peptides were synthesized and secreted into the periplasmic space. These results provide a basis for applying recombinant DNA methods to the study of diphtheria toxin and for producing novel, genetically altered forms of the toxin suited to the construction of new classes of immunotoxins.

Minocycline in rheumatoid arthritis. A 48-week, double-blind, placebo-controlled trial. MIRA Trial Group.

OBJECTIVE: To assess the safety and efficacy of minocycline in the treatment of rheumatoid arthritis. DESIGN: A double-blind, randomized, multicenter, 48-week trial of oral minocycline (200 mg/d) or placebo. SETTING: 6 clinical centers in the United States. PATIENTS: 219 adults with active rheumatoid arthritis who had previous limited treatment with disease-modifying drugs. MEASUREMENTS: As the primary outcomes, 60 diarthrodial joints were examined for tenderness, and 58 joints were examined for swelling (hips excluded). Grip strength, evaluator's global assessment, morning stiffness, Modified Health Assessment Questionnaire, patient's global assessment, hematocrit, erythrocyte sedimentation rate, platelet count, and IgM rheumatoid factor levels were also assessed; radiographs of both hands and wrists were taken. RESULTS: 109 and 110 patients were randomly assigned to receive minocycline and placebo, respectively. At entry, demographic, clinical, and laboratory measurements were similar in both groups. Most patients had mild to moderate disease activity and some evidence of destructive disease. At the week 48 visit, 79% of the minocycline group and 78% of the placebo group continued to receive the study medication. At 48 weeks, more patients in the minocycline group than in the placebo group showed improvement in joint swelling (54% and 39%) and joint tenderness (56% and 41%) (P < 0.023 for both comparisons). The minocycline group also showed greater improvement in hematocrit, erythrocyte sedimentation rate, platelet count, and IgM rheumatoid factor levels (all P values < 0.001), and more patients receiving minocycline had laboratory values within normal ranges at 48 weeks. For the remaining outcomes, P values ranged from 0.04 to 0.76, all greater than the critical value of 0.005 (Bonferroni adjustment for multiple comparisons). The frequency of reported side effects was similar in both groups, and no serious toxicity occurred. CONCLUSIONS: Minocycline was safe and effective for patients with mild to moderate rheumatoid arthritis. Its mechanisms of action remain to be determined.

Radiographic results from the Minocycline in Rheumatoid Arthritis (MIRA) Trial.

OBJECTIVE: To assess radiographically determined disease progression in patients in the Minocycline in Rheumatoid Arthritis (MIRA) Trial. METHODS: A double blind, randomized, multicenter, 48 week trial of oral minocycline (200 mg/day) or placebo in 6 clinical centers in the United States. Patients include 219 adults with active RA previously receiving limited treatment with disease modifying drugs. Posteroanterior films of the hands from baseline and final visits, blinded for sequence, were read for erosions and joint space narrowing by trained observers. Outcomes included rate of disease progression (change/month) and percentage of patients with progression from baseline, newly involved joints, and newly erosive disease. RESULTS: Using intent-to-treat analyses, progression rates for erosions (0.11 +/- 0.42 minocycline, 0.17 +/- 0.41 placebo; p = 0.47) and joint space narrowing (0.16 +/- 0.55 minocycline and 0.23 +/- 0.71 placebo; p = 0.14) were similar. (Power 43% to detect a 50% difference.) Newly erosive joints occurred more frequently in the placebo group (44 vs 32%; p = 0.08), not a statistically significant difference. CONCLUSION: Radiographic measurement of disease progression using 4 measures failed to show a significant difference between minocycline and placebo treatment, although for all methods there was a trend toward treatment benefit, consistent with reported clinical results. A one year trial duration, high measurement variability, and slow rate of radiographic progression in this cohort may explain the low power to detect a treatment effect. The measurement that denoted "newly involved" joints was most sensitive in detecting change. In future trials longer term assessment (minimum 2 years) of radiographic changes and further comparison of measures of disease progression are warranted.