Human immune response to monoclonal antibody-enzyme conjugates in ADEPT pilot clinical trial.

The human immune response to monoclonal antibody-enzyme conjugates has been studied in patients included in the pilot clinical trial of ADEPT. Each patient received murine monoclonal anti-CEA antibody fragments (A5B7-F(ab')2, conjugated to bacterial enzyme, carboxypeptidase G2 (CPG2) followed by a galactosylated monoclonal anti-CPG2 antibody (SB43), 36-48 h after the conjugate. Some patients were also given a dose of 131I-labeled conjugate (4-8 mg, 7-15 mCi) for blood clearance and gamma camera image studies. All patients studied developed human antimouse antibodies (HAMA) and anti-CPG2 antibodies within 10 d after a single course of treatment with the conjugate. In most cases, IgM response was detected at 7 d after the conjugate followed by the IgG response 14 d later. In one patient, HAMA and anti-CPG2 antibodies of the IgG type could still be detected at 10 mo after treatment. Anti-CPG2 antibodies in serum of one patient were found to inhibit CPG2 activity in vitro. Generation of neutralizing antibodies limits the use of repeat cycles of ADEPT in patients. Use of immunosuppressive agents may allow a useful time window for several ADEPT cycle treatments by delaying the appearance of HAMA and anti-CPG2 antibodies. Patients given cyclosporin A before and during ADEPT are currently being studied for HAMA and anti-CPG2 response.     

Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine.

It is well known that ammonium ion excretion is increased during metabolic acidosis in mammals. The purpose of this study was to determine whether we could isolate from human urine during metabolic acidosis a factor that would stimulate NH+4 and/or H+ excretion in toad urinary bladder. Extracts of urine from six human subjects collected during NH4Cl-induced acidosis were prepared. These extracts were tested for their effect on NH+4 excretion in hemibladders mounted between plastic chambers. The extracts significantly increased NH+4 excretion in the toad urinary bladder. We found no effect on H+ excretion by these extracts. This ammoniuretic activity was not present in the urine when the same individuals were in metabolic alkalosis. We conclude that during metabolic acidosis a humoral factor is present which stimulates the excretion of NH+4. The factor could act as a permease in the bladder cell or as a stimulator of an NH+4 transport system.     

On the sealing of gas-filled glass ampoules

Studies in our laboratories involved the placing of argon-filled hand-sealed glass ampoules (tip-, balloon-, or draw-sealed) containing biological materials dried by sublimation of ice in vacuo in water baths at elevated temperatures. Although these ampoules tested negative for “leakers,” many ampoules upon removal from the water bath contained beads of moisture. To determine if the water within the ampoules entered through openings in the closed ends, we used a laser imaging apparatus to examine the sealed ends. We carried out studies also on machine-sealed ampoules.Two modes of laser imaging were used: dark-field imaging and interference imaging. In tip-sealed ampoules, uniform, long channels were found in the gatherings of glass at the ends of the ampoules; the lengths of the channels were approximately ten times their diameters. In balloon-sealed ampoules, pore-like openings were found; the lengths of the pores were approximately four times their diameters. In draw-sealed ampoules, channels of small diameters were observed; the lengths of the open pathways were approximately 30 times their diameters. Based on the sizes of the images obtained with laser imaging, the magnifications used for photographic reproductions and the original measurements of the sealed ampoules we estimated the openings in tip-sealed and balloon-sealed ampoules to range from 5 to 8 μm and the channels in draw-sealed ampoules to be less than 3 μm in diameter, The diameters of the helix-like openings in machine-sealed ampoules were less than 5 μm.To prevent the migration of molecules into and out of ampoules, we sought for a barrier that could be interposed between the external environments of the ampoules. Many liquid formulations of natural and man-made elastomers were tested; neoprene dissolved in toluol was found best.     

Morphological and chromosomal descriptions of new species in the Anopheles subpictus complex

Abstract. Anopheles subpictus Grassi is shown to comprise four reproductively distinct species, designated A, B, C and D, occurring sympatrically in villages of Pondicherry, southeast India.
Adult females were reared individually from wild larvae and examined for their morphological and chromosomal characters. Paracentric fixed inversions on the X-chromosome serve to distinguish the species cytogenetically, with no inversion heterozygotes (i.e. no interspecific hybrids) among totals of 717 species A (X+^a,+^b), 1863 species B (Xa, b), 869 species C (Xa,+^b) and 1365 species D (X +^a,b) identified.
Morphologically, diagnostic characters for each of the four species are seen in the egg float ridge number, larval mesothoracic seta 4, pupal seta 7-1 and the palpi of female adults. Species A. C and D immatures inhabit freshwater, whereas the malaria vector species B breeds in saltwater and was found only in coastal villages.     

Expression of injected HPRT minigene DNA in mouse embryos and its inhibition by antisense DNA

We have used a highly sensitive biochemical microassay to monitor the expression of a cloned minigene for hypoxanthine phosphoribosyl transferase (HPRT, EC.2.4.2.8) in preimplantation mouse embryos. The mouse HPRT promoter and the mouse metallothionein promoter (MT-I) function equally well in embryos at the 2-cell stage whereas the viral SV40 promoter does not allow HPRT expression. Induced HPRT activity from the MT-I HPRT minigene construct occurs in cleavage embryos cultured in the presence of cadmium. In contrast, negation of enzyme expression from the injected minigene DNA is mediated by simultaneous injection of HPRT antisense DNA.     

A Serine Carboxypeptidase-Like Acyltransferase Is Required for Synthesis of Antimicrobial Compounds and Disease Resistance in Oats

Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthesis of glucose polyesters that may contribute to insect resistance in wild tomato (Solanum pennellii) and enzymes required for the synthesis of sinapate esters associated with UV protection in Arabidopsis thaliana. In our earlier genetic analysis, we identified the Saponin-deficient 7 (Sad7) locus as being required for the synthesis of antimicrobial triterpene glycosides (avenacins) and for broad-spectrum disease resistance in diploid oat (Avena strigosa). Here, we report on the cloning of Sad7 and show that this gene encodes a functional SCPL acyltransferase, SCPL1, that is able to catalyze the synthesis of both N-methyl anthraniloyl- and benzoyl-derivatized forms of avenacin. Sad7 forms part of an operon-like gene cluster for avenacin synthesis. Oat SCPL1 (SAD7) is the founder member of a subfamily of monocot-specific SCPL proteins that includes predicted proteins from rice (Oryza sativa) and other grasses with potential roles in secondary metabolism and plant defense.     

Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine

It is well known that ammonium ion excretion is increased during metabolic acidosis in mammals. The purpose of this study was to determine whether we could isolate from human urine during metabolic acidosis a factor that would stimulate NH₄⁺ and/or H⁺ excretion in toad urinary bladder. Extracts of urine from six human subjects collected during NH₄Cl-induced acidosis were prepared. These extracts were tested for their effect on NH₄⁺ excretion in hemibladders mounted between plastic chambers. The extracts significantly increased NH₄⁺ excretion in the toad urinary bladder. We found no effect on H⁺ excretion by these extracts. This ammoniuretic activity was not present in the urine when the same individuals were in metabolic alkalosis. We conclude that during metabolic acidosis a humoral factor is present which stimulates the excretion of NH₄⁺. The factor could act as a permease in the bladder cell or as a stimulator of an NH₄⁺ transport system.