Bernard F. Erlanger

The preparation and properties of two new chromogenic substrates of trypsin

Abstract The synthesis and properties of l -lysine p -nitroanilide dihydrobromide and benzoyl dl -arginine p -nitroanilide hydrochloride are described. Both compounds are hydrolyzed by trypsin, the latter being a substrate at least as sensitive as benzoyl l -argininamide. Their hydrolysis can be followed conveniently by colorimetric procedures, since one of the products, p -nitroaniline, is yellow. Values of K m and k 3 for their reaction with trypsin were determined, as well as the K 4 of benzoyl d -arginine p -nitroanilide, which was isolated from a tryptic digest of the dl isomer. The pH-activity curves were also determined, that of l -LPA being in a more alkaline region than normally found for trypsin substrates. The possible significance of this shift is discussed. Preliminary studies indicate that benzoyl dl -arginine p -nitroanilide hydrochloride is also hydrolyzed by papain.

Cellular localisation of a water-soluble fullerene derivative

Fullerenes are a new class of compounds with potential uses in biology and medicine and many insights were made in the knowledge of their interaction with various biological systems. However, their interaction with organised living systems as well as the site of their potential action remains unclear. In this work, we have demonstrated that a fullerene derivative could cross the external cellular membrane and it localises preferentially to the mitochondria. We propose that our finding supports the potential use of fullerenes as drug delivery agents as their structure mimics that of clathrin known to mediate endocytosis.

[4] The preparation of antigenic Hapten-Carrier conjugates: A survey

Publisher Summary This chapter discusses the preparation of antigenic hapten-carrier conjugates. Substances of molecular weight less than 1000 are not ordinarily antigenic. However, antibodies can be raised to small molecules, by immunization, with conjugates made up of low molecular weight substances (haptens) covalently linked to proteins or synthetic polypeptides. The protein carriers used in various laboratories include: globulin fractions, the serum albumins of various species, hemocyanin, ovalbumin, thyroglobulin, and fibrinogen. Hapten-protein conjugates of serum albumin are, in general, more soluble than conjugates of γ-globulin or of ovalbumin. In this study, it is attempted to incorporate it into a general survey of the methods of preparing immunogenic haptenprotein conjugates, in which the hapten is a pharmacologically interesting compound. The arrangement is governed by the nature of the reactive functional groups of the hapten. In this way, the information can be applied most easily to new compounds being considered for use as determinant groups. Various procedures described are discussed, by specific examples, presenting practical aspects of the experimental methods.

The action of chymotrypsin on two new chromogenic substrates

Two new chromogenic substrates of chymotrypsin have been synthesized: N -succinyl- L -phenylalanine p -nitroanilide (SPNA) and N -glutaryl- L -phenylalanine p -nitroanilide (GPNA). Both are stable in the absence of enzyme and release p -nitroaniline (yellow) upon hydrolysis. GPNA is the more sensitive substrate being capable of quantifying less than 10 μg per milliliter chymotrypsin. The presence of Ca ++ decreases K m of the enzyme-substrate reaction but has a negligible effect upon k 3 . Na ++ , on the other hand, increases k 3 as well, and provides a suitable means of increasing the sensitivity of GPNA down to microgram levels of enzyme. In combination with an “all or none” assay the rate of hydrolysis of GPNA can be related to the molar concentration of enzyme. The reaction of chymotrypsin with GPNA is not subject to substrate activation over a 200-fold range of substrate concentration, nor is it affected by the presence of ethyl ammonium chloride. In these respects it differs from the reaction of trypsin with susceptible substrates.

Preirradiation paclitaxel in glioblastoma multiforme: efficacy, pharmacology, and drug interactions. New Approaches to Brain Tumor Therapy Central Nervous System Consortium.

PURPOSE The purpose of this study was to determine the response rate of paclitaxel administered at maximal tolerated doses (MTD) in patients with newly diagnosed glioblastoma multiform. PATIENTS AND METHODS All patients in this multicenter study were 45 years or older and had measurable residual tumor on postoperative MRI scans. Up to 3 cycles of paclitaxel were administered as a continuous 96-hour intravenous infusion prior to radiation, provided that the tumor did not enlarge on serial MRIs. The initial 10 patients were treated with the previously recommended phase II dose of 140 mg/m2. Less than anticipated toxicity led to the development of a phase I/II study in 24 patients in which paclitaxel doses were escalated separately in patients receiving (+EIAED) or not receiving (-EIAED), concomitant enzyme-inducing antiepileptic drugs. Paclitaxel plasma steady-state concentrations (Css) were measured during the first cycle of chemotherapy. Response was the primary efficacy endpoint for this study, although survival was also assessed. RESULTS The MTD was 140 mg/m2 in the -EIAED, and 200 mg/m2 in the +EIAED patient groups. The mean Css for the -EIAED patients treated at 140 mg/m2 was 38 nM, whereas the mean Css for +EIAED patients were 17 nm at 140 mg/m2, 27 nM at 175 mg/m2, 46 nM at 200 mg/m2, and 51 nM at 230 mg/m2. One patient, who had a verified partial response, had his diagnosis changed to an anaplastic oligodendroglioma on subsequent central neuropathologic review. None of the 15 assessable glioblastoma patients treated at or above the MTD doses showed a radiographic response to paclitaxel. The median survival of eligible patients on this protocol was 355 days (95% CI, 255 to 485 days), which is similar to the survival of comparable patients treated with conventional therapy. CONCLUSIONS These results suggest that (1) paclitaxel given as a 96-hour infusion at the MTD has minimal activity in patients with untreated glioblastoma, (2) the concomitant administration of EIAEDs alters the pharmacology of paclitaxel, resulting in a lower Css, reduced systemic toxicity, and higher dose requirements, (3) this study design, in which a new agent is given prior to radiation therapy (with serial monitoring of MRI), did not adversely affect survival in this patient population.

Routine large-scale production of monoclonal antibodies in a protein-free culture medium.

Abstract A defined culture medium and cultivation technique are described which allow the long-term production on a large scale of monoclonal antibodies and myeloma proteins without the use of serum, serum proteins, or protein-containing liposomes. The high initial purity of the culture supernatants obtained with protein-free medium facilitates the purification of the monoclonal antibodies, the immunoglobulin (Ig) purity of which is limited only by the genetic homogeneity of the cells. Successful growth of 8 hybridoma and 2 myeloma lines has been achieved. SDS-PAGE analysis revealed that the levels of Ig production in the protein-free medium were comparable to those achieved with serum-supplemented media and with media supplemented with purified serum proteins. Stability of Ig production during continuous cultivation in the protein-free medium over an extended period was studied for a myeloma and a hybridoma line, both of which produced high levels (50–200 μg/ml) of Ig for periods of 11 and 22 weeks, respectively.

Preferential derivation of abnormal human G-group-like chromosomes from chromosome 15

SummaryThe marked binding of antibodies specific for 5-methylcytidine to the short arm of chromosome 15 distinguishes this chromosome from the other human acrocentrics. This method has been used to study over 60 individuals including 12 who did not have Downs syndrome, but who did have an extra G-group sized acrocentric chromosome. In six cases the extra chromosome did not show intensive binding of anti-5-methylcytidine. In the other six cases, the extra chromosome contained a 5-methylcytidine rich band at each end indicating that both ends were derived from chromosome 15 and contained centromeric heterochromatin normally present on the short arm of chromosome 15. The duplication of short arm material in the abnormal chromosomes was confirmed in all cases by quinacrine staining, nucleolar organizer (Ag-AS) staining or C-banding. In three cases, the abnormal chromosome appeared to arise from two different chromosomes 15. Several possible mechanisms for the production of the abnormal chromosome are discussed. The individuals with this abnormal chromosome all showed some degree of mental retardation, but few common physical findings.

Photoregulation of an Enzymic Process by Means of a Light-Sensitive Ligand

A specific inactivator of chymotrypsin, p-azophenyldiphenylcarbamyl chloride, exists as two geometric isomers, cis and trans, which are interconvertible by means of light. The cis-isomer is five times more reactive than the more stable trans-isomer, and is obtained by exposure of the latter to light of 320 nanometer wavelength. The trans-isomer can be regained by exposure of the cis-isomer to light of 420 nanometer wavelength. This interconversion can be made to occur in aqueous solution in the presence of the enzyme under conditions in which the trans-isomer reacts relatively slowly with chymotrypsin. Thus, it is possible to regulate the rate of inactivation of chymotrypsin by using light of the appropriate wavelength. This system is presented as a model for some of the light-sensitive metabolic systems present in living organisms.

The use of antinucleoside antibodies to probe the organization of chromosomes denatured by ultraviolet irradiation

Abstract Ultraviolet irradiation of methanol: acetic acid-fixed human and mouse metaphase chromosomes rendered them capable of binding antibodies specific for purine or pyrimidine bases. Since these antibodies react with single-stranded but not with native DNA, our results indicate that UV irradiation generated single-stranded regions in chromosomal DNA. Using an indirect immuno-fluorescence technique to detect antibody binding, highly characteristic, nonrandom patterns of antibody binding were observed. Antibodies to adenosine (anti-A) and thymidine (anti-T) produced identical patterns of binding which in most respects matched the chromosome banding patterns produced by quinacrine. However, additional foci of intense fluorescence were seen in the paracentromeric regions of constitutive heterochromatin on chromosomes 1, 9 and 16, regions which had been shown by in situ DNA-RNA hybridization to be the locations of AT-rich human satellite DNA. Antibodies to cytidine also bound to the same region of chromosome 9. In mouse chromosome preparations, both anti-A and anti-T produced bright fluorescence of the region containing centromeric heterochromatin, which had been shown to be the location of the AT-rich satellite DNA of this species.

5-Methylcytosine in heterochromatic regions of chromosomes: chimpanzee and gorilla compared to the human

Fixed metaphase chromosomes of gorilla and chimpanzee were UV-irradiated to produce regions of single-stranded DNA and then treated with antibodies specific for the minor DNA base 5-methylcytosme (5 MeC). An indirect immunofluorescence technique was used to visualize sites of antibody binding. In the gorilla six pairs of autosomes contained major fluorescent regions, indicating localized regions of highly methylated DNA. These corresponded, with the exception of chromosome 19, to the major regions of constitutive heterochromatin as seen by C-banding. The Y chromosome also contained a highly fluorescent region which was located just proximal to the intense Q-band region. In the chimpanzee no comparable concentrations of highly methylated DNA were seen. Smaller regions of intense 5 MeC binding were present on perhaps six chimpanzee chromosomes, including the Y. Five of these corresponded to chromosomes which were highly methylated in the gorilla. — There is diversity among the human, gorilla and chimpanzee in both the size and location of concentrations of 5 MeC, supporting the idea that satellite DNA evolves more rapidly than DNA in the remainder of the chromosome.