Characteristics of specific125I-ω-conotoxin GVIA binding in rat whole brain

Characteristics of specific¹²⁵I-omega-conotoxin (-CgTX) binding were systematically investigated in crude membranes from rat whole brain. Kd and Bmax Values for the binding were 49.7 pM and 181.5 fmol/mg of protein, respectively. The effects of various types of Ca channel antagonists on the binding were investigated. Dynorphin A (1–13), in particular, specifically inhibited¹²⁵I--CgTX binding, but not that of [³H](+)PN200-110. Spider venom fromPlectreurys tristes did not specifically inhibit specific binding of¹²⁵I--CgTX, because the venom also inhibited the binding of [³H](+)PN200-110 to a similar degree. The amount of specific binding of¹²⁵I--CgTX was less in the cerebellum than that in any other area of whole brain. The cross-linker disuccinimidyl suberate did not label with¹²⁵I--CgTX and its binding sites in rat whole brain, although it did in chick whole brain, which was used as a positive control. These findings suggested that dynorphine A (1–13) was a selective blocker of -CgTX-sensitive Ca channels in crude membranes from rat whole brain and that -CgTX-sensitive Ca channels were mainly present a rat brain except cerebellum.     

Retention of intact concanavalin A and Lens culinaris lectin in transformed lymphoblasts during mitogenic response.

The fate of cell bound mitogens during mitogenic response of mouse cortisone resistant thymocytes (CRT) was studied using ¹²⁵I-concanavalin A (Con A) and ¹²⁵I-Lens culinaris lectin (LcH). A majority of autoradiographic grains derived from ¹²⁵I-lectins bound to a CRT population were distributed in a single broad peak. Pulse labeling of CRT with ¹²⁵I-Con A or ¹²⁵I-LcH in the initial 60 min of incubation followed by 48 hr of culture with unlabeled mitogens revealed that transformed lymphoblasts carried over half of cell-bound ¹²⁵I-mitogens. ¹²⁵I-Con A and ¹²⁵I-LcH found in the lymphoblasts in the above pulse experiments were electrophoretically identical to the native mitogens. A significant loss of cell-bound ¹²⁵I-mitogens was observed only after extensive cell division.     

Improved procedure for the purification of an iodinated protein: Beta nerve growth factor

An improved procedure for the isolation of iodinated Nerve Growth Factor (¹²⁵I-NGF) has been devised. Use of Centricon microconcentrators (Amicon) has allowed the facile and efficient recovery of ultrapure¹²⁵I-NGF in high yields. Centricon microconcentrators are supplied with two molecular weight cutoffs of 10 K and 30 K daltons. NGF is a basic protein with a molecular weight of 26 K daltons. It is therefore possible to filter the¹²⁵I-NGF through the 30K filter (30 K Filtrate) leaving behing any aggregates or reactants greater than 30 K while the¹²⁵I-NGF can be retained and concentrated on the 10 K filter (10 K Retentate). Any free¹²⁵I is easily removed, passing through the 10 K filter and then being discarded. In this way¹²⁵I-NGF can be easily purified.     

Ultrastructural localization of zinc in rat exocrine pancreas by autoradiography

Summary ⁶⁵Zn was infused at a constant rate for 10 days into a rat. Glutaraldehyde fixed, Epon-araldite embedded ultrathin sections of pancreatic tissue were coated with Ilford L4 emulsion and at 211 days exposure were developed. Silver grains were found over the zymogen granules and over the rough endoplasmic reticulum of exocrine cells. Islet tissue was not observed in these studies. The failure of other zinc localization methods to demonstrate zinc in acinar tissue is discussed as are some of the pitfalls of the autoradiographic method and suggestions for future improvement.Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, Madison. Supported in part by USPHS Research Grant AM-05606 from the Nat. Institute of Arthritis and Metabolic Diseases.Supported by an NIH post-doctoral fellowship.     

A Confirmation of 125I-ω-Conotoxin Labeled Sites in a Crude Membrane Fraction from Chick Brain as the α1 Subunit of N-Type Calcium Channels

Omega-conotoxin GVIA (-CTX), as a selective blocker for an N-type Ca²⁺ channel, has been conveniently used in many molecular biochemical and pharmacological experiments. There has been little elucidation of ¹²⁵I--CTX binding sites (mainly the 135-kDa band) in the crude membranes from chick brain, although the characteristics of specific ¹²⁵I--CTX binding and labeling sites in chick brain membranes have been investigated in our previous research. In this work, our goal is to further identify ¹²⁵I--CTX labeling sites in chick brain membranes by using anti-B1Nt antibodies (against the N-terminal segment B1Nt of N- or P-type Ca²⁺ channel ₁-subunits). The ¹²⁵I--CTX–labeled sites in chick brain membranes could be solubilized and immunoprecipitated by using an anti B1Nt antibody. The molecular weight of the immunoprecipitated protein was determined as 135 kDa, which is inconsistent with that of the specific ¹²⁵I--CTX binding protein reported previously. Moreover, the ¹²⁵I--CTX–labeled protein could be purified by the method of preparative SDS-PAGE and recognized by anti-B1Nt antibodies in Western blotting analysis. These results indicated that anti-B1Nt antibodies could truly recognize ¹²⁵I--CTX–labeled sites as the main band of 135 kDa from chick brain membranes, and the -CTX–labeled site (mainly the 135-kDa band) should be N-type Ca²⁺ channel ₁-subunits.     

The talc-resin-TCA test: rapid screening of radioiodinated polypeptide hormones for radioimmunoassay

The talc-resin-TCA test is a rapid, reliable method for predicting the behavior of ¹²⁵I-hormones in radioimmunoassays (RIA). This test is based on the assessment of three different physicochemical properties of iodinated hormones: adsorption to talc, exclusion from ion exchange resin, and precipitation by TCA. These three parameters produce test patterns that are unique for monomeric ¹²⁵I-hormone, aggregated ¹²⁵I-hormone, and ¹²⁵I-salts. Different iodination methods were used to prepare radioiodinated human and rat PRL, GH, TSH, and LH, and rat FSH. Purified monomeric ¹²⁵I-hormones that gave reliable RIA results all showed a characteristic talc-resin-TCA profile of >90% talc adsorption, <25% resin binding, and >90% TCA precipitation. This profile has successfully predicted reliable RIA results for ¹²⁵I-hormones prepared from more than 80 iodinations.     

The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Application to the radioimmunoassay

1. A new method is described for labelling proteins to high specific radioactivities with ¹²⁵I. The protein is treated with a ¹²⁵I-labelled acylating agent, iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which reacts with free amino groups in the protein molecule to attach the ¹²⁵I-labelled groups by amide bonds. 2. Three protein hormones have been labelled by this method, human growth hormone, human thyroid-stimulating hormone and human luteinizing hormone. Specific radioactivities of up to 170, 120 and 55μCi/μg respectively have been obtained for these hormones. 3. The immunoreactivity of these labelled hormones has been investigated by using a radioimmunoassay system specific for each hormone. These preparations have also been compared with and found to be equal or superior to labelled hormones prepared by chemical substitution of ¹²⁵I into tyrosine residues of the proteins by using the chloramine-t-oxidation procedure. 4. With some antisera the immunoreactivity of the antigen was diminished by the introduction of a single I atom into the tyrosyl groups, whereas antigen containing a single ¹²⁵I-labelled 3-(4-hydroxyphenyl)propionamide group showed the same immunoreactivity as the unmodified antigen.     

Characteristics of the Inhibitory Effect of Calmodulin on Specific [125I]Omega-Conotoxin GVIA Binding to Crude Membranes from Chick Brain

The characteristics of the inhibitory effect of calcium ion (Ca²⁺)/calmodulin (CaM) on specific [¹²⁵I]-omega-conotoxin GVIA (¹²⁵I--CTX) binding and on the labeling of ¹²⁵I--CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca²⁺/CaM depended on the concentrations of free Ca²⁺ and CaM. The IC₅₀ values for free Ca²⁺ and CaM were about 2.0 × 10^–8 M and 3.0 g protein/ml, respectively. The inhibitory effect of Ca²⁺/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290–309), but not by the calcineurin inhibitor FK506. Ca²⁺/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca²⁺ channel ₁ subunits) with ¹²⁵I--CTX using a cross-linker. These results suggest that Ca²⁺/CaM affects specific ¹²⁵I--CTX binding sites, probably N-type Ca²⁺ channel ₁ subunits, in crude membranes from chick whole brain.     

Clearance of circulating desialylated thyroglobulins in the rat

Canine and rat thyroglobulins were labeled with ¹²⁵I either in vitro or in vivo, and were utilized for plasma clearance studies performed with rat. The half-life of physiologically radioiodinated asialothyroglobulins was about 6 min, while that of chemically radioiodinated asialothyroglobulin was about 12 min. No marked species difference was observed in this clearance. The label which had disappeared from the blood was recovered mainly in the liver, and this uptake was blocked by the simultaneous injection of desialylated orosomucoid but not by native orosomucoid. Radiolabeled monoiodotyrosine, diiodotyrosine, triiodothyronine and thyroxine were detected in the liver 17 min after intravenous injection of asialothyroglobulin labeled with ¹²⁵I in vivo, suggesting the possible production of thyroid hormones in extrathyroidal tissues.     

Studies on the inhibitory effect of bacitracin on 125I-labelled insulin internalization in the rat hepatocyte

Previous studies have suggested that transglutaminase has a role in the internalization of some polypeptide hormones and is inhibited by the antibiotic, bacitracin. Bacitracin has been used in insulin-receptor studies to inhibit extracellular degradation of ¹²⁵I-labelled insulin. The aim of this study was to investigate bacitracin's effect on ¹²⁵I-labelled insulin-receptor interactions in isolated rat hepatocytes. 1 g/l bacitracin increased cell-associated ¹²⁵I-labelled insulin at 20, 30 and 37°C (P < 0.001, 0.0005 and 0.0005, respectively). At 5 and 15°C (internalization does not occur), bacitracin did not affect cell-associated ¹²⁵I-labelled insulin. The bacitracin effect was concentration dependent, increasing to 2 g/l. Scatchard analysis showed that bacitracin did not alter insulin receptor affinity or number. 1 g/l bacitracin abolished the effect of chloroquine. The increased cell-associated radioactivity with bacitracin was surface-bound in nature. 0.5 g/l bacitracin decreased ¹²⁵I-labelled insulin degradation in hepatocyte suspensions (P < 0.001) and in buffer previously incubated with hepatocytes (P < 0.0005). More ¹²⁵I-labelled insulin remained associated with cells during dissociation studies at 37°C when the buffer contained 1 g/l bacitracin. Label that appeared in the buffer after 60 min was significantly more intact in the presence of bacitracin (P < 0.025). These results suggest that bacitracin retards the internalization of ¹²⁵I-labelled insulin in isolated rat hepatocytes.     

Peculiarities of distribution of 125I-insulin and 125I-insulin-like growth factor-I (IGF-I) at internalization in isolated rat hepatocytes

One of determining conditions of formation in vertebrate phylogenesis of hormonal systems of insulin and IGF-I—peptides common by origin, similar by structure and by biological action is temperature factor. In differentiation of functional roles of two related hormones an important place is ascribed to mechanism of their intracellular action. Study of formation of the two hormonal systems in vertebrate phylogenesis necessitates knowledge of peculiarities of internalization of two related hormones in mammals. On isolated rat hepatocytes at the identical maximal level of internalization of ^I25I-insulin and ¹²⁵I-IGF-I there are revealed marked differences of dynamics of their internalization. Besides, at internalization of ¹²⁵I-insulin or of ¹²⁵I-IGF-I, their peculiar distribution was observed in the cell and on the plasma membrane. At 37°C, only two thirds of ¹²⁵I-insulin relatively bound to receptors on the membrane were immersed into cell, whereas the portion of internalized ¹²⁵I-IGF-I turned out to be higher than the part located on the membrane. At 12°C, a decrease of ¹²⁵I-insulin inside the cell and its increase on the membrane indicated the interdependent label redistribution under these conditions. The form of the ¹²⁵I-IGF-I distribution at low temperature remained unchained. The pattern of established differences of internalization of ¹²⁵I-insulin and ¹²⁵I-IGF-I as well as different sensitivity of each of the processes to low temperature indicated that each of the peptides triggered individual mechanism of endocytosis of receptors. The high sensitivity of internalization of ¹²⁵I-insulin to low temperature and the temperature lability of the process in isolated rat hepatocytes agree with the earlier suggestion about the late formation of the regulatory insulin system in homoiothermal vertebrates.     

Structural Specificity for the Inhibitory Effect of Calmodulin on Specific 125I-Omega-Conotoxin GVIA Binding

To clear the structural specificity of calmodulin (CaM) on the specific ¹²⁵I--CTX binding to crude membranes from whole chick brain, the following experiments were investigated in this study: (i) the attenuating effect of semisynthetic tetrahydroisoquinoline derivatives on the inhibitory effect of Ca²⁺/CaM, (ii) the effects of chimeras of yeast and chicken Ca²⁺/CaM, and (iii) the effects of Ca²⁺-binding proteins (such as troponin c, S 100 a and b, and annexin I, III–V). The inhibitory effect of Ca²⁺/CaM was attenuated by isoquinoline derivatives (PX 28, 34, 216, 224, and CPU57) and a CaM antagonist W-7. PX 34, a typical synthesized isoquinoline derivative, showed the attenuating effect in a dose-dependent manner. The ED₅₀ value for the attenuating effect of PX 34 was about 20 M, which is similar to that of W-7 reported previously. Some chimeric CaMs such as YC 51–53 (which are close to the properties of vertebrate CaM) showed a significant inhibitory effect on the specific ¹²⁵I--CTX binding, but YC 129 and 130 (which retain the properties of yeast CaM), troponin c, S100 a, b, and annexin I, III–V had no effect on the specific ¹²⁵I--CTX binding. These results suggest that the characteristic structure containing the EF-hand structure of CaM itself is needed to cause the inhibitory effect on the specific ¹²⁵I--CTX binding.     

Covalent Cross-Linking of Radiolabeled N-Formylated Hexapeptide to its Specific Receptor on Rat and Human Neutrophils: Evidence for a ligand induced complex formation

Abstract

The structure of the rat and human neutrophil receptor for N-formylated chemotactic peptides was characterized using ¹²⁵I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys hexapeptide as a ligand and an affinity cross-linking technique. ¹²⁵I-hexapeptide bound to purified rat peritoneal neutrophils was time, temperature and pH-dependent. The average receptor number per cell was about 67.000 and díssociation constant (K_d) 0.41 nM. Formyl-MLLP, fMLP, fNLP, were 750%, 15%, 8.6% respectively and Boc-MLP, Boc-NLP, and MLP 0.6% as potent as the hexapeptide in inhibiting the binding of ¹²⁵I-hexapeptide to rat neutrophils. The same correlation was found between these peptides in their potency to induce chemotaxis. This indicates that the rat neutrophil chemotactic receptor is like human receptor also a highly stereoselective and requires a N-formylated ligand for high affinity binding. Affinity cross-linking of ¹²⁵I-hexapeptide to rat and human neutrophil chemotactic receptor with glutaraldehyde revealed on SDS-PAGE a 85-kDa and 62-kDa major complex and a 170-kDa and 120-kDa minor complex, respectively. The 120-kDa complex was absent in human neutrophils if the cells were treated with glutaraldehyde prior to cross-linking of ¹²⁵I-hexapeptide to its receptor. Likewise, the larger complex was absent if neutrophils were exposed to heterelogous ligand (C5a) prior to glutaraldehyde treatment and cross-linking of ¹²⁵I-hexapeptide to its specific receptor. These results demonstrate that the rat neutrophils possess a functional high-affinity receptor for N-formylated chemotactic peptides and that the size of the monomeric receptor is 85-kDa and about 23-kDa larger than that of the human receptor. Upon homologous ligand binding the receptor seems to form a larger complex.     

The removal of cell surface material by enzymes used to dissociate mammary-gland cells

Summary Treatment of mouse mammary epithelial cells (MMEC) with various enzymes used for dispersing and transferring cells results in extensive digestion of materials on the cell surfaces. MMEC biosynthetially labeled with [³H]fucose, [¹⁴C]fucose and [³H]amino acids or with¹²⁵I by the lactoperoxidase method were exposed to either collagenase plus hyaluronidase, followed by pronase, or to trypsin in concentrations and conditions currently used for cell dispersion. Whereas the latter enzyme preparation solubilized 76% of the trichloroacetic acid precipitable radioactive fucose and 96% of the protein-bound¹²⁵I, collagenase plus hyaluronidase treatment released lesser amounts of each label. Subsequent treatment of the cells with pronase removed additional surface-labeled materials, but the total amounts released were still less than when the trypsin preparation alone was employed. Released cell surface materials were analyzed by gel chromatography. Some of the peaks obtained also were examined by polyacrylamide gel electrophoresis. The labeled materials that remained attached to the MMEC after enzymatic treatment were investigated by these two methods as well. We could show that collagenase plus hyaluronidase solubilized three main glycoprotein components from the cell surface. In addition, we could show that the extensive cell surface damage caused by these two enzyme preparations was due to the high proteolytic activity present in these preparations as judged by their ability to hydrolyze rabbit gamma globulin labeled with¹²⁵I. Even though their membranes were extensively damaged by the enzyme treatments, the dispersed cells could be cultured successfully in vitro and could incorporated fucose into their surfaces in a manner similar to that by intact tissue. Through the use of gel-filtration (cochromatography of [¹⁴C]fucose and [³H]fucose cell surface materials), we could demonstrate the identity of cell surface glycoproteins synthesized by cultured cells and by intact tissue. This work was supported by Grant Nos. CA 11736 and CA 19455 from the National Cancer Institute, and Biomedical Research Support Grant No. RR05467 from the National Institutes of Health, DHEW.     

Suppression of Receptors for Prolactin and Estrogen in Rat Liver Due to Treatment with the Growth Hormone Analogue Produced by the Tapeworm Spirometra Mansonoides

Abstract

Somatogenic hormones play an important role in regulation of receptors for prolactin (PRL) and estrogen. Plerocercoids of the tapeworm, S. mansonoides produce a factor which mimics some, but not all of the actions reported for GH. Intact female rats were subjected to a constant infusion of plerocercoid growth factor (PGF) via a subcutaneous infection for two weeks to determine if PGF influences receptors for PRL, GH or estradiol. The rate of weight gain in the PGF-treated rats was accelerated in spite of a marked reduction in serum GH. Partially-purified PGF specifically displaced [¹²⁵I]hGH from rat liver receptors but microsomes prepared from rats treated with PGF specifically bound significantly less [¹²⁵I]hGH than microsomes from control rats. The reduction in [¹²⁵I]hGH binding was not due to occupancy or to a change in affinity but to a suppression in receptor concentration. Scatchard analysis of [³H]estradiol binding in rat liver cytosols shows a 50% reduction in receptor concentration in the PGF-treated group. Specific binding of [³H]estradiol in anterior pituitary was also suppressed by PGF treatment.     

Triiodothyronine bound to red blood cells is not available for transport through the blood-brain barrier

Many steroid and thyroid hormones and some drugs are bound by circulating red cells. Red cell-bound ligand may not be physiologically inert, as recent studies show that red cell-bound drug is available for uptake by brain. To investigate whether triiodothyronine (T₃) is available for uptake by brain in vivo from the circulating red cell pool, the present investigations measure the effects of human erythrocytes on rat brain uptake of [¹²⁵I]T₃ in vivo. The fraction of circulating T₃ available for uptake in vivo in the presence of 0, 2, 5, 10, 22, or 44% red cells was essentially identical to the fraction of [¹²⁵I]T₃ unbound in vitro. Therefore, [¹²⁵I]T₃ bound to red cells obtained from normal volunteers is not available for uptake by brain in vivo.     

p-Azidophenylglyoxal-Epidermal Growth Factor: A Photoactivatable Affinity Crosslinking Reagent

Abstract

A photoaffinity derivative of highly purified ¹²⁵I-labelled epidermal growth factor (¹²⁵I-EGF) has been synthesized. The heterobifunctional crosslinking reagent p-azidophenylglyoxal (PAPG) was bound to arginine residues in ¹²⁵I-EGF. PAPG-¹²⁵I-EGF bound to EGF receptors on rat fibroblasts and human A431 epidermoid carcinoma cells in culture. An apparent decreased affinity of PAPG-¹²⁵I-EGF for the EGF receptor is in accord with at least one arginine being at or near the EGF receptor binding site. The PAPG-¹²⁵I-EGF:EGF receptor complexes on rat cells were internalized to the same extent as control EGF:receptor complexes. A431 cells treated with PAPG-¹²⁵I-EGF were irradiated with ultraviolet light and the labelled proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The 3 major labelled proteins had apparent molecular weights ranging from 75,000 to 200,000. Only the labelling of the 200,000-M_r protein was prevented by the addition of excess unlabelled EGF with the PAPG-¹²⁵I-EGF. This molecular weight is in agreement with the reported size of the EGF receptor plus EGF. A protein with apparent molecular weight of 100,000 was labelled by ¹²⁵I-EGF by an unknown mechanism which was dependent on the dose of UV light and blocked by the addition of excess unlabelled EGF.     

Analysis of human hypoxanthine-guanine phosphoribosyl transferase isozymes by isoelectric focusing in polyacrylamide gel

The method of isoelectric focusing in polyacrylamide gel was used to separate HGPRT isoenzymes in crude hemolysates of human and rat erythrocytes. HGPRT from erythrocytes of a normal human male donor consistently revealed three peaks of activity. Their mean isoelectric points, using pH 5–7 range ampholytes, were, peak I, pI 6.00; peak II, pI 5.8³; and peak III, pI 5.71. Peak III was wide and tailed. It always had a shoulder with a mean pI of 5.62. HGPRT from rat erythrocytes revealed two peaks of activity, corresponding to isoelectric points of 5.90 and 5.80. The method of isoelectric focusing in polyacrylamide gel is presented as a new way of detecting isoenzyme patterns.This study was supported by Grant No. 38-5768 from the Lebanese National Council for Scientific Research and Grant No. 18-5240 from the Medical Research Committee, American University of Beirut.     

Characterisation of a new reporter system allowing high throughput in planta screening for recombination events before and after controlled DNA double strand break induction

DNA double strand breaks (DSBs) are created either by DNA damaging reagents or in a programmed manner, for example during meiosis. Homologous recombination (HR) can be used to repair DSBs, a process vital both for cell survival and for genetic rearrangement during meiosis. In order to easily quantify this mechanism, a new HR reporter gene that is suitable for the detection of rare recombination events in high-throughput screens was developed in Arabidopsis thaliana. This reporter, pPNP, is composed of two mutated Pat genes and has also one restriction site for the meganuclease I-SceI. A functional Pat gene can be reconstituted by an HR event giving plants which are resistant to the herbicide glufosinate. The basal frequency of intra-chromosomal recombination is very low (10^?5) and can be strongly increased by the expression of I-SceI which creates a DSB. Expression of I-SceI under the control of the 35S CaMV promoter dramatically increases HR frequency (10,000 fold); however the measured recombinant events are in majority somatic. In contrast only germinal recombination events were measured when the meganuclease was expressed from a floral-specific promoter. Finally, the reporter was used to test a dexamethasone inducible I-SceI which could produce up to 200× more HR events after induction. This novel inducible I-SceI should be useful in fundamental studies of the mechanism of repair of DSBs and for biotechnological applications.     

Development of two cloned epithelial cell lines from normal adult mouse and rat ventral prostates

Summary Two epithelial cell lines were established, one from adult C3H mouse and one from adult Fischer rat ventral prostate. These cell lines were obtained from explant cultures, using Ham's F12 medium supplemented with HEPES, insulin, testosterone, hydrocortisone, epidermal growth factor, and 7.5% fetal bovine serum. A low concentration of trypsin and EDTA in Ca⁺⁺-and Mg⁺⁺-free phosphate buffer was used for passaging the cells. The rat cell line was established following implantation of prostate tissue in nude mice. These cell lines stained positively for acid phosphatase and were dependent upon epidermal growth factor for growth. Morphological studies, including electron microscopy, revealed a highly characteristic epithelial morphology of both cell lines. These cell lines have hypotetraploid chromosome numbers and are capable of metabolizing benzo(a)pyrene. We propose the application of these cells as models for the study of prostate carcinogenesis. This work was supported in part by Grant CA-21, 746, and by the Electron Microscope Core Facility on Grant CA-14,089, from the National Cancer Institute, National Institutes of Health, Bethesda, MD.