Site-specifically modified 111In labelled antibodies give low liver backgrounds and improved radioimmunoscintigraphy

Site-specific modification of monoclonal antibodies at their oligosaccharide had previously been demonstrated to produce excellent ¹¹¹In imaging in a xenograft model using a Brown Norway (BN) rat lymphoma and a rat anti-BN MHC monoclonal antibody [Lee C. et al. Fed. Proc. Abstr. 43 3014 (1984)]. These results are due, in part, to lack of liver uptake, so we wanted to evaluate the extent of hepatic uptake observed with different monoclonal antibodies in normal mice. Biodistribution data were obtained for four monoclonal antibodies by first modifying each antibody at its carbohydrate with a diethylenetriaminepentaacetic acid (DTPA) derivative. The antibodies were then labelled with ¹¹¹In and injected into normal mice. Images were obtained 24 h post-injection, and at 48 h the mice were dissected and the tissue-to-blood (T:B) ratios determined. T:B ratios were approximately 1 (or less) for every organ evaluated, indicating minimal non-specific uptake into these organs. Data is also presented for the BN-rat system which shows excellent localization into the tumor xenograft and low non-specific organ uptake. These data indicate that modification of antibodies site-specifically at their oligosaccharide results in minimal non-specific uptake into non-target tissues and enhanced localization into the tumor target, and that this may represent a preferred method for production of ¹¹¹In labelled antibodies.     

Therapeutic Efficacy of C-Kit-Targeted Radioimmunotherapy Using 90Y-Labeled Anti-C-Kit Antibodies in a Mouse Model of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the ⁹⁰Y isotope.

Methods

¹¹¹In- or ¹²⁵I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of ⁹⁰Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7.

Results

[¹¹¹In]12A8 and [¹¹¹In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [¹¹¹In]12A8 and [¹¹¹In]67A2 accumulated in tumors, but not in major organs. [¹¹¹In]67A2 uptake by the tumor was 1.7 times higher than for [¹¹¹In]12A8. [⁹⁰Y]12A8, but not [⁹⁰Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [⁹⁰Y]12A8, and 1.85 and 3.7 MBq of [⁹⁰Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [⁹⁰Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [⁹⁰Y]12A8, whereas no dose-dependent increase was observed following [⁹⁰Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well.

Conclusion

Treatment with [⁹⁰Y]12A8 and [⁹⁰Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.     

Radiolabeled products in rat liver and serum after administration of antibody—amide—DTPA—indium-111

Anti-human serum albumin antibody (Ab) was used as a model antibody. Ab was conjugated with DTPA using cyclic DTPA dianhydride reaction and radiolabeled with ¹¹¹In. The labeled Ab was purified by affinity chromatography. Size exclusion HPLC of this product showed 62% of ¹¹¹In bound to monomeric Ab and 38% of the activity bound to antibody oligomers with molecular weights ranging from 300,000 to 450,000. The labeled antibody preparation was injected into the tail vein of rats. The radioactive substances in serum and the supernatant from liver homogenates were analyzed for molecular weight and immunoreactivity. Size exclusion HPLC of the serum samples indicated that the monomeric and dimeric Abs disappeared from the serum at a similar rate over a 48 h period. In addition, a new radioactive substance with an estimated molecular weight of 35,000 appeared in the serum. The immunoreactive fraction of the circulating ¹¹¹In substances decreased slowly, somewhat proportional to the appearance of the metabolite. On the other hand, the immunoreactivity of the ¹¹¹In substances in the supernatant from the liver homogenate decreased rapidly and no appreciable immunoreactivity was observed after 48 h. The labeled antibody was catabolized very rapidly in the liver and the major activity in the supernatant was associated with a small molecular weight metabolite which had a HPLC retention time identical to that of DTPA-¹¹¹In. The second metabolite had an estimated molecular weight of 35,000. No radioactivity was associated with transferrin.     

Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody: comparison to radioiodinated fragments

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

Uptake of three [3H]progestins by target tissues in vivo: Implications for the design of diagnostic imaging agents

We have investigated the tissue distribution of radioactivity for 0.5–4 h follwing the i.v. injection of three tritium-labeled progestins in estrogen-primed, immature rats. Whereas [³H]progesterone shows minimal uterine uptake (<0.7% injected dose per gram; %ID/g), the two higher affinity, synthetic progestins [³H]R 5020 (promegestrone) and [³H]ORG 2058 show highly selective uptake that reaches 4–5% ID/g by 1–3 h. The uterus to non-target tissue activity ratio at 2–4 h is approximately 12–20 for R 5020 and ORG 2058, but less than 2 for progesterone; the uterus to blood activity ratio for R 5020 is also high (approximately 15), but is lower for ORG 2058, possibly due to the accumulation of radiolabeled metabolites in the blood. The uterine uptake is selectively blocked by simultaneous injection of a large dose of unlabeled steroid, indicating that the uptake is mediated by a high affinity, low capacity binding system, presumably the progesterone receptor. Pronounced uptake is also observed by the liver and into fat, but is not receptor-mediated. The highly selective target tissue uptake by the two synthetic steroids, but not by progesterone, indicates that one must have ligands with sufficiently high affinity for the target tissue receptor, as well as low affinity for certain non-receptor binding proteins, in order to obtain adequate contrast between target and non-target tissues in dynamic uptake studies. These guidelines will be important in the development of suitable in vivo imaging agents based on the progesterone receptor.     

Metabolism and renal clearance of 111In-labeled DOTA-conjugated antibody fragments

Radiometal-labeled antibody fragments are promising reagents for radioimmunotherapy due to their high tumor uptake and rapid pharmacokinetics, but their therapeutic potentials are limited by high uptake and retention in the kidney. Identification of metabolic products is a first step in designing rationale approaches to lower kidney uptake. Previous studies in rats have shown that 111In-labeled DTPA-conjugated antibody fragments (via lysine residues) were degraded to an DTPA-epsilon-amino-lysine derivative and retained in the lysosomal compartments of the liver and kidney [Rogers et al. (1995) Cancer Res. 55, 5714s-5720s]. To determine the metabolic profile of another widely used metal-chelate, [111In]DOTA conjugated to lysines in antibody fragments via active ester chemistry, we analyzed kidney homogenates from nude mice injected with an [111In]DOTA-Fab generated enzymatically from the anti-lymphoma intact antibody Rituxan. The major kidney metabolite was identified as [111In]DOTA-epsilon-amino-lysine by comparison to an authentic synthetic standard. This end product was also identified in the urine, along with relatively small amounts of [111In]DOTA-Fab. Since injection of [111In]DOTA-epsilon-amino-lysine into nude mice resulted in rapid clearance into the urine without kidney retention, it is likely that the renal retention observed was due to kidney uptake of [111In]DOTA-Fab, followed by lysosomal degradation to [111In]DOTA-epsilon-amino-lysine, which is only slowly cleared from this compartment. This observation is supported by autoradiographs of the kidney showing rapid localization of radioactivity into the distal regions of the kidney cortex. To extend this analysis to clinical trials, we have also analyzed urine taken from a patient injected with the intact antibody [111In]DOTA-cT84.66. In that example, we found that the major radioactive species was also [111In]DOTA-epsilon-amino-lysine.     

High Efficiency Diffusion Molecular Retention Tumor Targeting

Here we introduce diffusion molecular retention (DMR) tumor targeting, a technique that employs PEG-fluorochrome shielded probes that, after a peritumoral (PT) injection, undergo slow vascular uptake and extensive interstitial diffusion, with tumor retention only through integrin molecular recognition. To demonstrate DMR, RGD (integrin binding) and RAD (control) probes were synthesized bearing DOTA (for ¹¹¹ In³⁺), a NIR fluorochrome, and 5 kDa PEG that endows probes with a protein-like volume of 25 kDa and decreases non-specific interactions. With a GFP-BT-20 breast carcinoma model, tumor targeting by the DMR or IV methods was assessed by surface fluorescence, biodistribution of [¹¹¹In] RGD and [¹¹¹In] RAD probes, and whole animal SPECT. After a PT injection, both probes rapidly diffused through the normal and tumor interstitium, with retention of the RGD probe due to integrin interactions. With PT injection and the [¹¹¹In] RGD probe, SPECT indicated a highly tumor specific uptake at 24 h post injection, with 352%ID/g tumor obtained by DMR (vs 4.14%ID/g by IV). The high efficiency molecular targeting of DMR employed low probe doses (e.g. 25 ng as RGD peptide), which minimizes toxicity risks and facilitates clinical translation. DMR applications include the delivery of fluorochromes for intraoperative tumor margin delineation, the delivery of radioisotopes (e.g. toxic, short range alpha emitters) for radiotherapy, or the delivery of photosensitizers to tumors accessible to light.     

Streptavidin-biotinylated IgG conjugates: a simple procedure for reducing polymer formation

Disulfide links of the IgG2ak anti-ovarian carcinoma antibody, 5G6.4, were site-specifically biotinylated [≈2 biotins/ IgG2a] using a novel crosslinking procedure using the biotin derivatized ETAC (equilibrium transfer alkylation crosslink reagent) 1a. Complexation of ETAC 1a biotinylated 5G6.4 on a column of immobilized protein A at high dilution, followed by passage of [¹²⁵I]streptavidin, washing and pH change leads to elution of a streptavidin-free product with a molecular mass in the 200–300 kDa range. By contrast, direct mixing with [¹²⁵I]streptavidin rapidly gave larger oligomers of ⪢669 and ≈440–669 kDa molecular mass, respectively. The biodistribution of the 200–300 kDa complex showed significantly diminished liver, kidney and spleen uptake as well as higher blood activity than the 440–669 kDa complex. The methodology represent the first application of ETAC chemistry to disulfide-bond directed biotinylation of antibodies and the synthesis of streptavidin antibody conjugates which minimizes their polymerization.     

Comparative studies using 125I- and 111In-labeled monoclonal antibodies

HDP-1 monoclonal antibody was labeled with ¹¹¹In using deferoxamine, diethylenetriaminepentaacetic acid or 1-(para-bromoacetamidobenzyl)-EDTA as chelating agents or with ¹²⁵I. The in vitro binding capacity and stability of the labeled molecules were evaluated using affinity chromatography. The biodistribution and imaging capabilities were compared using an animal model system that does not involve the use of tumors. Similar studies were done using the corresponding labeled F(ab′)₂ and Fab′ fragments. All labeled molecules, except those treated with deferoxamine, were stable in vitro. When tested in vivo, all retained their capacity to localize in the target tissue (lung). The lung %ID/g levels for the ¹¹¹In-labeled molecules were, however, slightly lower than those observed for the corresponding ¹²⁵I-labeled molecules. High uptake was also observed in the liver or kidneys when the ¹¹¹In-labeled molecules were used; no such results were obtained with the ¹²⁵I-labeled molecules. More work appears to be necessary before the use of bifunctional chelates becomes the optimal method for radiolabeling monoclonal antibodies for use in tumor imaging.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Radiosynthesis of [thiocarbonyl-11C]disulfiram and its first PET study in mice

[Thiocarbonyl-¹¹C]disulfiram ([¹¹C]DSF) was synthesized via iodine oxidation of [¹¹C]diethylcarbamodithioic acid ([¹¹C]DETC), which was prepared from [¹¹C]carbon disulfide and diethylamine. The decay-corrected isolated radiochemical yield (RCY) of [¹¹C]DSF was greatly affected by the addition of unlabeled carbon disulfide. In the presence of carbon disulfide, the RCY was increased up to 22% with low molar activity (A_m, 0.27 GBq/μmol). On the other hand, [¹¹C]DSF was obtained in 0.4% RCY with a high A_m value (95 GBq/μmol) in the absence of carbon disulfide. The radiochemical purity of [¹¹C]DSF was always >98%. The first PET study on [¹¹C]DSF was performed in mice. A high uptake of radioactivity was observed in the liver, kidneys, and gallbladder. The uptake level and distribution pattern in mice were not significantly affected by the A_m value of the [¹¹C]DSF sample used. In vivo metabolite analysis showed the rapid decomposition of [¹¹C]DSF in mouse plasma.     

Pharmacokinetics,Tissue Distribution and Metabolism of Intravenously Administered Digalactosyldiacylglycerol and Monogalactosyldiacylglycerol in the Rat

Abstract

A bilayer forming galactolipid, digalactosyldiacylglycerol (DGalDG) has been identified as a tool with suitable physicochemichal properties for pharmaceutical formulation work. One possible application is as a carrier for liposome entrapped drugs for intravenous administration. The fate of intravenously administered galactolipids is not known. In this study liposomal dispersions of galactolipids, containing [³H]fatty acid labelled DGalDG or monogalactosyldiacylglycerol (MGalDG) were injected intravenously in the rat and the disappearance from blood and uptake by tissues were examined. The T1/2 of [³H]DGalDG in plasma was 3 to 5 minutes. Of the tissues examined (liver, spleen, kidneys, lung, heart, stomach, upper and lower small intestine and colon), the liver contained the highest radioactivity per g tissue after both 15 min. and 4 h. Autoradiographic examinations after 15 min, 1 h and 4 h showed that the uptake of radiolabeled DGalDG and MGalDG occurred mainly to the hepatocytes. Less than 6 % of the injected [³H]DGalDG remained in liver and plasma as [³H]DGalDG after 4 h. [³H]MGalDG exhibited a similar pattern of metabolism although the initial disappearance rate was faster than for [³H]DGalDG. The study thus shows that the hepatocytes take up and hydrolyse galactolipids after intravenous administration.     

Distribution of 95Zr and 181Hf in tumor-bearing animals and mechanism for accumulation in tumor and liver

Tumor uptake rates, concentrations in the mitochondrial fraction (containing lysosome) of liver and tumors, avid accumulations in connective tissue (especially inflammatory tissue) and binding substances in these tissues of ⁹⁵Zr and ¹⁸¹Hf were essentially similar to those for ⁶⁷Ga, ¹¹¹In, ¹⁶⁹Yb and ¹⁶⁷Tm. However, the main binding substance of the above elements in group IV in tumor and liver was acid mucopolysaceharide whose molecular weight exceeded 40,000, although the above elements in group III were bound mainly to the acid mucopolysaccharide with a molecular weight of about 10,000.     

Evaluation of Polyethylene Glycol as a Water-soluble Marker: Absorption and Excretion of 14C-labeled Polyethylene Glycol in Rats

The absorbability of polyethylene glycol (PEG), a water-soluble nutritional marker, from the gastrointestinal tract of rat was examined using the [¹⁴C]-labeled compound ([¹⁴C]PEG) having a molecular weight of 4000. Intravenously injected [¹⁴C]PEG was readily excreted and recovered almost completely in the urine and neither hepatic nor renal uptake of the PEG was observed. Intragastrically administered [¹⁴C]PEG was eliminated in the urine with an average recovery of only 0.43 ± 0.13% (Mean ± S.D., n= 10) of the dose over 24 hr. From the gel column chromatographic profile of the radioactivity excreted in the urine after an oral dose, [¹⁴C]PEG was suggested to be absorbed in two forms, as an original form and as a low molecular weight component. The latter component might be the degraded product of PEG in the gastrointestinal tract. From these results it was confirmed that PEG with a molecular weight of 4000 is a satisfactory marker because of its low absorbability.     

Bovine aorta contains at least two related forms of heparan sulphate proteoglycan

• 1.1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density.
• 2.2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan.
• 3.3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane.
• 4.4. On electron microscopy, both high and low charge density proteoglycans were visualized as ‘tadpole-like’ molecules, which showed a tendency to aggregate via their globular heads.
• 5.5. Bovine aortic smooth muscle cells were cultured in the presence of [³⁵S]sulphate and [³H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above.
• 6.6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium.
• 7.8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.

     

Glucose and fructose uptake by <Emphasis Type="Italic">Limulus polyphemus</Emphasis> hepatopancreatic brush border and basolateral membrane vesicles: evidence for Na<Superscript>+</Superscript>-dependent sugar transport activity

[³H]-fructose and [³H]-glucose transport activities were determined in brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) from Limulus polyphemus (horseshoe crab) hepatopancreas. Glucose transport was equilibrative in the absence of sodium and sodium dependent in the presence of sodium in BBMV, suggesting GLUT-like and SGLT-like transport activity. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and sodium dependent in the presence of sodium. Western blot analysis using a rabbit anti-mouse SGLT-1 polyclonal antibody indicated the presence of a cross-reacting horseshoe crab BBMV protein of similar molecular weight to the mammalian SGLT1. Sequence alignment of the mouse SGLT-4 and SGLT1 with a translated, horseshoe crab-expressed sequence tag also indicated significant identity between species. Fructose and glucose uptake in the absence and presence of sodium by hepatopancreas BBMV and BLMV indicated the presence of sodium-dependent transport activity for each sugar that may result from the presence of transporters similar to those described for other species.     

Microsomal redox systems in brown adipose tissue: high lipid peroxidation,low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-¹⁴C]acetate and [2-¹⁴C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b ₅ in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.Abbreviations BAT Brown Adipose Tissue - MDA malondialdehyde     

Ribonucleic acid synthesis and loss of viability in pea seed

Summary RNA synthesis and protein synthesis in embryonic axis tissue of viable pea (Pisum arvense L. var. N.Z. maple) seed commences during the first hour of germination. Protein synthesis in axis tissue of non-viable pea seed is barely detectable during the first 24 h after the start of imbibition. Nonviable axis tissue incorporates significant levels of [³H]uridine into RNA during this period but the level of incorporation does not increase significantly over the first 24 h of imbibition. In axis tissue of non-viable seed during the first hour of imbibition most of the [³H]uridine was incorporated into low molecular weight material migrating in advance of the 4S and 5S RNA species in polyacrylamide gels but some radioactivity was incorporated into a discrete species of RNA having a molecular weight of 2.7×10⁶. After 24 h, non-viable axis tissue incorporates [³H]uridine into ribosomal RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and a heterogeneous RNA species of molecular weight ranging from 2.2×10⁶ to 2.7×10⁶. No 4S or 5S RNA synthesis is detectable after 24 h of imbibition in non-viable axis tissue. Axis tissue of viable pea seed synthesises rRNA, 4S and 5S RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and the rRNA precursor species at both periods of germination studied. Loss of viability in pea seed appears to be accompanied by the appearance of lesions in the processing of rRNA precursor species and a significant loss of RNA synthesising activity.Abbreviations rRNA ribosomal RNA - TCA trichloroacetic acid - SLS sodium lauryl sulphate - PPO 2,5 Diphenyloxazole - POPOP 1,4-Bis-2-(4-methyl-5-penyloxazolyl)-benzene     

Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain.

An endogenous inhibitor(s) of [³H] diazepam binding to synaptosomes has been demonstrated in bovine brain. The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction. Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography. The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [³H] diazepam binding was competitive. A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain. Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks.