Identification of the primary caspase 3 cleavage site in alpha II-spectrin during apoptosis.

Alpha II-spectrin is one of the major proteins responsible for maintaining the cytoskeletal integrity of the cell. The caspase 3-mediated cleavage of alpha II-spectrin during apoptotic cell death may play an important role in altering membrane stability and the formation of apoptotic bodies. In this study, we identified the primary caspase 3 cleavage site in alpha II-spectrin. We found that the transcriptional inhibitor, actinomycin D, induced caspase 3 activation and that caspase 3 activation is coincident with the cleavage of alpha II-spectrin protein at a primary cleavage site. Deletion analysis and site directed mutagenesis identified the primary cleavage site in alpha II spectrin at amino acid 1185 (DETD). The primary caspase 3 cleavage site in alpha II spectrin is conserved in immature and mature B cells. Our results indicate that alpha II-spectrin is initially cleaved at a caspase 3 consensus site and this primary event likely alters the structural conformation of the protein exposing subsequent cleavage sites and altering cytoskeletal integrity. Identification of the primary cleavage site for caspase 3 may help to elucidate the role of alpha II-spectrin in membrane stability and apoptosis as well as provide new insights into alpha II-spectrin autoantibody formation associated with the autoimmune disease, Sjögren's syndrome.

Calpain proteolysis of alpha II-spectrin in the normal adult human brain.

The proteolysis of alphaII-spectrin by calpain may be physiologically involved with synaptic remodeling, long-term potentiation, and memory formation. Calpain activation may also mediate neuronal apoptosis, responses to hypoxic insult, and excitotoxic injury. Surprisingly little is known of the activity of these calpain-mediated processes in the adult human brain. Using an antibody that specifically recognizes calpain-cleaved alphaII-spectrin, we have mapped the topographic distribution of the major alphaII-spectrin break-down product (alphaII-bdp1) in six adult brains examined post-mortem. All brains were from patients without evident neurological disease. Focally positive alphaII-bdp1 was consistently detected in the neuropil of the cortical gray matter, in occasional pyramidal neurons, and in rare reactive astrocytes in the cerebral cortex and hippocampus. Cerebellar Purkinje cells were more frequently, and more intensely, immunopositive. In all fields, staining was most intense in the soma and dendrites of neurons. There was no correlation of the frequency of positive cells with the postmortem interval or clinical condition. While these findings do not rigorously exclude contributions from postmortem calpain activation, they do suggest that a low-level of calpain processing of alphaII-spectrin is likely to be a constitutive process in the adult human brain.

Dynamic molecular modeling of pathogenic mutations in the spectrin self-association domain.

Disruption of spectrin self-association underlies many inherited hemolytic disorders. Using dynamic modeling and energy minimization, the 3-dimensional structure of the self-association domain has been estimated in human erythrocyte spectrin and the structural consequences of 17 elliptogenic mutations determined. The predicted structure of the normal self-association domain was remarkably similar to the crystal structure of the Drosophila alpha-spectrin 14th repeat unit, despite replacement in the human sequence of over 70% of the amino acids relative to fly spectrin, including 2 prolines in the human sequence that appear in helical regions of the fly structure. The predicted structure placed all hydrophilic residues at the surface and identified 4 salt bridges, 9 hydrophobic interactions, and 4 H-bonds that stabilize the native self-association unit. Remarkably, every pathologic point mutation, including seemingly conservative substitutions such as G for A, A for V, or K for R (single-letter amino acid codes), led to conformational rearrangements in the predicted structure. The degree of structural disruption, as measured by root-mean-square deviation of the predicted backbone structure from the Drosophila structure, correlated strongly with the severity of clinical disease associated with each mutation. This approach thus enables an accurate prediction, from the primary sequence, of the clinical consequences of specific point mutations in spectrin. The 3-dimensional structure of the self-association domain derived here is likely to be accurate. It provides a powerful heuristic model for understanding how point mutations disrupt cytoskeletal function in a variety of hemolytic disorders.

beta III spectrin binds to the Arp1 subunit of dynactin.

Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated betaIII spectrin isoform. We identify two Arp1-binding sites in betaIII spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to betaIII spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and betaIII spectrin co-purify on vesicles isolated from rat brain, and betaIII spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, betaIII spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, betaIII spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for betaIII spectrin. We hypothesize that the interaction between betaIII spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.

Spectrin oligomerization is cooperatively coupled to membrane assembly: a linkage targeted by many hereditary hemolytic anemias?

In the erythrocyte, ankyrin is the major adapter protein linking tetramers of band 3 to the spectrin-actin cytoskeleton. This linkage involves a direct interaction between ankyrin and the 14th-15th repeat unit of beta-spectrin. The spectrin cytoskeleton itself is stabilized by the self-association of spectrin heterodimers into tetramers and larger oligomers, a process mediated by the 17th repeat unit of beta-spectrin and a short NH(2) -terminal sequence in alpha-spectrin. The self-association of spectrin and its ankyrin-mediated membrane binding have generally been considered independent events. We now demonstrate that spectrin self-association, the binding of spectrin to ankyrin, and the binding of ankyrin to the 43-kDa cytoplasmic domain of band 3 (cdb3) are coupled in a positively cooperative way. In solution, [(125)I]-labeled ankyrin was found by ND-PAGE3 to enhance the affinity of spectrin self-association by 10-fold. The reciprocal process was also true, in that spectrin tetramers and oligomers bound ankyrin with enhanced affinity relative to dimer spectrin. Saturation of the beta-spectrin self-association site by an NH(2) -terminal 80-kDa alpha-spectrin peptide enhanced the affinity of spectrin dimer for ankyrin, indicating a direct relationship between ankyrin binding and the occupancy of the beta-spectrin self-association site. cdb3 accentuated these cooperative interactions. Several inherited spectrin mutations that cause hemolytic disease but that do not directly destabilize the self-association or ankyrin-binding sites can be explained by these results. Three classes of mutations appear to disrupt cooperative coupling between self-association and ankyrin binding: (i) mutation of the linker sequences that join helices C and A in repeat units that intervene between the two functional sites, mutations that presumably block repeat-to-repeat transfer of conformational information; (ii) mutations in alpha-spectrin repeats 4 to 6 that disrupt the ability of this region to trans-regulate ankyrin binding by the adjacent beta-spectrin repeats 14-15; and (iii) exon-skipping mutations that shorten alpha-spectrin and force repeats 4 to 6 to fall out-of-register with the ankyrin-binding motif in beta-spectrin. Collectively, these results demonstrate a molecular mechanism whereby a membrane receptor can directly promote cytoskeletal assembly.

Caspase remodeling of the spectrin membrane skeleton during lens development and aging.

Terminal differentiation of lens fiber cells resembles the apoptotic process in that organelles are lost, DNA is fragmented, and changes in membrane morphology occur. However, unlike classically apoptotic cells, which are disintegrated by membrane blebbing and vesiculation, aging lens fiber cells are compressed into the center of the lens, where they undergo cell-cell fusion and the formation of specialized membrane interdigitations. In classically apoptotic cells, caspase cleavage of the cytoskeletal protein alpha-spectrin to approximately 150-kDa fragments is believed to be important for membrane blebbing. We report that caspase(s) cleave alpha-spectrin to approximately 150-kDa fragments and beta-spectrin to approximately 120- and approximately 80-kDa fragments during late embryonic chick lens development. These fragments continue to accumulate with age so that in the oldest fiber cells of the adult lens, most, if not all, of the spectrin is cleaved to discrete fragments. Thus, unlike classical apoptosis, where caspase-cleaved spectrin is short lived, lens fiber cells contain spectrin fragments that appear to be stable for the lifetime of the organism. Moreover, fragmentation of spectrin results in reduced membrane association and thus may lead to permanent remodeling of the membrane skeleton. Partial and specific proteolysis of membrane skeleton components by caspases may be important for age-related membrane changes in the lens.

Dynactin-dependent, dynein-driven vesicle transport in the absence of membrane proteins: a role for spectrin and acidic phospholipids.

We reconstituted dynein-driven, dynactin-dependent vesicle transport using protein-free liposomes and soluble components from squid axoplasm. Dynein and dynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds from cytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requires acidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, we show that spectrin is required for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. We verify this model in the context of liposomes, isolated axonal vesicles, and whole axoplasm. We conclude that spectrin has an essential role in retrograde axonal transport.

alpha -Catenin binds directly to spectrin and facilitates spectrin-membrane assembly in vivo.

The anchorage of spectrin to biological membranes is mediated by protein and phosphoinositol phospholipid interactions. In epithelial cells, a nascent spectrin skeleton assembles in regions of cadherin-mediated cell-cell contact, and conversely, cytoskeletal assembly is required to complete the cell-adhesion process. The molecular interactions guiding these processes remain incompletely understood. We have examined the interaction of spectrin with alpha-catenin, a component of the adhesion complex. Spectrin (alphaIIbetaII) and alpha-catenin coprecipitate from extracts of confluent Madin-Darby canine kidney, HT29, and Clone A cells and from solutions of purified spectrin and alpha-catenin in vitro. By surface plasmon resonance and in vitro binding assays, we find that alpha-catenin binds alphaIIbetaII spectrin with an apparent K(d) of approximately 20-100 nm. By gel-overlay assay, alpha-catenin binds recombinant betaII-spectrin peptides that include the first 313 residues of spectrin but not to peptides that lack this region. Similarly, the binding activity of alpha-catenin is fully accounted for in recombinant peptides encompassing the NH(2)-terminal 228 amino acid region of alpha-catenin. An in vivo role for the interaction of spectrin with alpha-catenin is suggested by the impaired membrane assembly of spectrin and its enhanced detergent solubility in Clone A cells that harbor a defective alpha-catenin. Transfection of these cells with wild-type alpha-catenin reestablishes alpha-catenin at the plasma membrane and coincidentally recruits spectrin to the membrane. We propose that ankyrin-independent interactions of modest affinity between alpha-catenin and the amino-terminal domain of beta-spectrin augment the interaction between alpha-catenin and actin, and together they provide a polyvalent linkage directing the topographic assembly of a nascent spectrin-actin skeleton to membrane regions enriched in E-cadherin.

Spectrin tethers and mesh in the biosynthetic pathway.

The paradox of how the Golgi and other organelles can sort a continuous flux of protein and lipid but maintain temporal and morphological stability remains unresolved. Recent discoveries highlight a role for the cytoskeleton in guiding the structure and dynamics of organelles. Perhaps one of the more striking, albeit less expected, of these discoveries is the recognition that a spectrin skeleton associates with many organelles and contributes to the maintenance of Golgi structure and the efficiency of protein trafficking in the early secretory pathway. Spectrin interacts directly with phosphoinositides and with membrane proteins. The small GTPase ARF, a key player in Golgi dynamics, regulates the assembly of the Golgi spectrin skeleton through its ability to control phosphoinositide levels in Golgi membranes, whereas adapter molecules such as ankyrin link spectrin to other membrane proteins. Direct interactions of spectrin with actin and centractin (ARP1) provide a link to dynein, myosin and presumably other motors involved with intracellular transport. Building on the recognized ability of spectrin to organize macromolecular complexes of membrane and cytosolic proteins into a multifaceted scaffold linked to filamentous structural elements (termed linked mosaics), recent evidence supports a similar role for spectrin in organelle function and the secretory pathway. Two working models accommodate much of the available data: the Golgi mesh hypothesis and the spectrin ankyrin adapter protein tethering system (SAATS) hypothesis.

Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: marker for neuronal apoptosis.

Alpha II-spectrin (alpha-fodrin) is a demonstrated endogenous substrate for caspase-3 in neurons undergoing unscheduled apoptotic death. We have previously identified the caspase cleavage site that yields the distinctive 120 kDa spectrin breakdown product (SBDP120) as (DSLD(1478)*SVEAL). Here, by using a synthetic peptide (NH(2)-SVEALC) mimicking the neo-N-terminal of SBDP120 as antigen, we report the development of chicken antibodies that specifically recognize the SBDP120 generated by in vitro caspase-3 digestion of bovine alpha-spectrin on Western blot. These anti-SBDP120 antibodies recognize SBDP120 generated by two apoptotic challenges (staurosporine, EGTA) to human neuroblastoma SH-SY5Y cells. Yet they neither react with intact alpha-spectrin nor its other fragments on Western blots. These anti-SBDP120 work equally well in detecting SBDP120 generated in rat cerebellar granule neurons undergoing potassium withdrawal-induced apoptosis. In immunocytochemical studies, these antibodies also specifically stained apoptotic SH-SY5Y or CGN's undergoing apoptosis in a caspase- inhibitor-sensitive manner. These anti-SBDP120s might become powerful markers for apoptotic neurons in various neurological or neurodegenerative conditions in vivo.

Identification and characterization of beta V spectrin, a mammalian ortholog of Drosophila beta H spectrin.

Four mammalian beta-spectrin genes are currently recognized, all encode proteins of approximately 240-280,000 M(r) and display 17 triple helical homologous approximately 106-residue repeat units. In Drosophila and Caenorhabditis elegans, a variant beta spectrin with unusual properties has been recognized. Termed beta heavy (beta(H)), this spectrin contains 30 spectrin repeats, has a molecular weight in excess of 400,000, and associates with the apical domain of polarized epithelia. We have cloned and characterized from a human retina cDNA library a mammalian ortholog of Drosophila beta(H) spectrin, and in accord with standard spectrin naming conventions we term this new mammalian spectrin beta 5 (betaV). The gene for human betaV spectrin (HUBSPECV) is on chromosome 15q21. The 11, 722-nucleotide cDNA of betaV spectrin is generated from 68 exons and is predicted to encode a protein with a molecular weight of 416,960. Like its fly counterpart, the derived amino acid sequence of this unusual mammalian spectrin displays 30 spectrin repeats, a modestly conserved actin-binding domain, a conserved membrane association domain 1, a conserved self-association domain, and a pleckstrin homology domain near its COOH terminus. Its putative ankyrin-binding domain is poorly conserved and may be inactive. These structural features suggest that betaV spectrin is likely to form heterodimers and oligomers with alpha spectrin and to interact directly with cellular membranes. Unlike its Drosophila ortholog, betaV spectrin does not contain an SH3 domain but displays in repeat 5 a 45-residue insertion that displays 42% identity to amino acids 85-115 of the E4 protein of type 75 human papilloma virus. Human betaV spectrin is expressed at low levels in many tissues. By indirect immunofluorescence, it is detected prominently in the outer segments of photoreceptor rods and cones and in the basolateral membrane and cytosol of gastric epithelial cells. Unlike its Drosophila ortholog, a distinct apical distribution of betaV spectrin is inapparent in the epithelial cell populations examined, although it is confined to the outer segments of photoreceptor cells. The complete cDNA sequence of human betaV spectrin is available from GenBank(TM) as accession number.

Identification and characterization of human SLP-2, a novel homologue of stomatin (band 7.2b) present in erythrocytes and other tissues.

Human stomatin (band 7.2b) is a 31-kDa erythrocyte membrane protein of unknown function but implicated in the control of ion channel permeability, mechanoreception, and lipid domain organization. Although absent in erythrocytes from patients with hereditary stomatocytosis, stomatin is not linked to this disorder. A second stomatin homologue, termed SLP-1, has been identified in nonerythroid tissues, and other stomatin related proteins are found in Drosophila, Caenorhabditis elegans, and plants. We now report the cloning and characterization of a new and unusual stomatin homologue, human SLP-2 (stomatin-like protein 2). SLP-2 is encoded by an approximately 1.5-kilobase mRNA (GenBank(TM) accession no. AF190167). The gene for human SLP-2, HUSLP2, is present on chromosome 9p13. Its derived amino acid sequence predicts a 38,537-kDa protein that is overall approximately 20% similar to human stomatin. Northern and Western blots for SLP-1 and SLP-2 reveal a wide but incompletely overlapping tissue distribution. Unlike SLP-1, SLP-2 is also present in mature human erythrocytes ( approximately 4,000 +/- 5,600 (+/- 2 S.D.) copies/cell). SLP-2 lacks a characteristic NH(2)-terminal hydrophobic domain found in other stomatin homologues and (unlike stomatin) is fully extractable from erythrocyte membranes by NaOH, pH 11. SLP-2 partitions into both Triton X-100-soluble and -insoluble pools in erythrocyte ghost membranes or when expressed in cultured COS cells and migrates anomalously on SDS-polyacrylamide gel electrophoresis analysis with apparent mobilities of approximately 45,500, 44,600, and 34,300 M(r). The smallest of these protein bands is believed to represent the product of alternative translation initiated at AUGs beginning with nt 217 or 391, although this point has not been rigorously proven. Collectively, these findings identify a novel and unusual member of the stomatin gene superfamily that interacts with the peripheral erythrocyte cytoskeleton and presumably other integral membrane proteins but not directly with the membrane bilayer. We hypothesize that SLP-2 may link stomatin or other integral membrane proteins to the peripheral cytoskeleton and thereby play a role in regulating ion channel conductances or the organization of sphingolipid and cholesterol-rich lipid rafts.

Transforming growth factor beta induces caspase 3-independent cleavage of alphaII-spectrin (alpha-fodrin) coincident with apoptosis.

Transforming growth factor beta (TGF-beta) is a potent growth inhibitor and inducer of cell death in B-lymphocytes and is essential for immune regulation and maintenance of self-tolerance. In this report the mouse immature B cell line, WEHI 231, was used to examine the mechanisms involved in TGF-beta-mediated apoptosis. Induction of apoptosis is detected as early as 8 h after TGF-beta administration. Coincident with the onset of apoptosis, the cytoskeletal actin-binding protein, alphaII-spectrin (alpha-fodrin) is cleaved into 150-, 115-, and 110-kDa fragments. The broad spectrum caspase inhibitor (Boc-D-fmk (BD-fmk)) completely abolished TGF-beta-induced apoptosis and alphaII-spectrin cleavage. Caspase 3, although present in WEH1 231 cells, was not activated by TGF-beta, nor was its substrate, poly(ADP-ribose) polymerase. These results identify alphaII-spectrin as a novel substrate that is cleaved during TGF-beta-induced apoptosis. Our data provide the first evidence of calpain and caspase 3-independent cleavage of alphaII-spectrin during apoptosis and suggests that TGF-beta induces apoptosis and alphaII-spectrin cleavage via a potentially novel caspase. This report also provides the first direct evidence of caspase 3 activation in WEH1 231 cells and indicates that at least two distinct apoptotic pathways exist.

Exclusion of the stomatin, alpha-adducin and beta-adducin loci in a large kindred with dehydrated hereditary stomatocytosis.

Defects in stomatin, alpha-adducin, and beta-adducin have been implicated in erythrocyte disorders of cation permeability. We performed linkage analysis of the genetic loci for these proteins in a large kindred with xerocytosis (dehydrated hereditary stomatocytosis). Using polymerase chain reaction-based genotyping techniques, all three loci are excluded as disease gene candidates.

Brain and muscle express a unique alternative transcript of alphaII spectrin.

Alternative splicing of pre-mRNA transcripts of alpha and beta spectrin has emerged as an important generator of diversity in this gene family, yet the functional consequences and extent of this diversity remains unknown. We have cloned and characterized full-length alphaII spectrin cDNA from human fetal brain (GenBank and ). On the basis of the predicted amino acid sequence, 11 amino acid substitutions, presumably representing polymorphisms, have been identified that distinguish this alphaII spectrin from human lung fibroblast alphaII spectrin. In addition, human fetal brain spectrin displays a novel five amino acid insertion in repeat 15 that arises from alternative mRNA splicing and that distinguishes this spectrin from lung fibroblast alphaII++ spectrin. This discovery, together with two previously identified regions of alternative mRNA splicing in alphaII spectrin suggest that as many as eight different splice forms of the mature protein might exist if all combinations (at inserts 1, 2, and 3) of alternative mRNA splicing are utilized. To assess this possibility, the tissue distribution of alternative exon usage was investigated by semiquantitative PCR with intron-jumping primer sets. Tissues examined were from mouse and included heart, kidney, lung, liver, thymus, spleen, brain, ovary, testis, and skeletal muscle, as well as mouse embryonic tissue. Transcripts both with and without insert 1, representing a 60 bp insertion within alphaII spectrin repeat 10, were identified in all tissues. In contrast, transcripts with insert 2, the novel 15 bp insertion reported here, were only expressed in brain, heart, skeletal muscle, and embryonic tissue. In all tissues examined only transcripts positive for insert 3, an 18 bp insertion in repeat 21, were amplified, even under conditions in which a 30% level of insert 3 negative transcript could be easily detected in artificially prepared control samples. All combinations of insert 1 and insert 2 were identified together in individual transcripts, verifying at least four distinct isoforms of alphaII spectrin. These have been named alphaIISigma1 through alphaIISigma4, in accord with current spectrin naming conventions. Dynamic molecular modeling of the 15th repeat unit incorporating insert 2 predicts that the spliced sequence forms a loop between helices A and B, and suggests that this insert might constitute a novel protein interaction site. The presence of this sequence in alphaIISigma3 and alphaIISigma4 spectrin suggests a specialized and heretofore unanticipated function for the 15th repeat of this molecule.

A widely expressed betaIII spectrin associated with Golgi and cytoplasmic vesicles.

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the beta-spectrin gene family by homology searches of the GenBank databases and by 5' rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called betaIII spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrin-homology domain. betaIII spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human betaIII spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human betaIII spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that betaIII spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, trans-Golgi network (TGN)38, and beta-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with betaIII spectrin. betaIII spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.

Pain control following elective gastrointestinal surgery: is epidural anesthesia warranted?

Fifty-nine patients undergoing elective major gastrointestinal surgery were entered into a prospective, randomized trial between January 1993 and July 1994 comparing the effectiveness, side effects, and hospital costs of postoperative epidural anesthesia (Group 1, n = 29) and intramuscular narcotic injections (Group 2, n = 30). Epidural catheters were inserted by a team that supervised catheter care and infusion rates in the postoperative period. The nonepidural group received intramuscular injections on a regular basis. Patients filled out visual analog scales to measure levels of pain ( 1 = minimal, 10 = maximal) every eight hours. Patient activity, bowel, and urinary function were recorded by the nursing staff. Control of pain (as measured by the daily average visual analog score) was more effective in Group 1 (P < .001) on postoperative days 1-3 (1.3 vs 3.6 on day 1, 0.7 vs 2.6 on day 2, 0.9 vs 3 on day 3). There was no significant difference in mean values between groups 1 and 2 with respect to first ambulation on the hospital ward, onset of liquid diet, intake of solid food, first spontaneous voiding, first bowel movement, length of hospitalization, or charge of hospitalization ($13,439 +/- 7,452 vs $11,821 +/- 6,630). We conclude that epidural anesthesia significantly lessens incisional pain following major elective lower gastrointestinal surgery when compared to analgesic injections alone. However, while not statistically significant, the overall charge was increased by 14% in the epidural group. This finding should be examined in light of the relatively low pain level in patients receiving narcotic injections alone.

The role of ankyrin and spectrin in membrane transport and domain formation.

Recent discoveries reveal a Golgi-centric spectrin-ankyrin skeleton required for Golgi integrity and anterograde protein trafficking. Identification of specific functional domains in spectrin that mediate its association with motor proteins and the Golgi complex has allowed novel insights into the structure and function of the secretory pathway, and into how this process is controlled by ADP-ribosylation factor and phosphoinositides. Alternative models of Golgi spectrin function that have been recently proposed are reviewed.