Active transport by the cecropia midgut. II. Fine structure of the midgut epithelium.

A morphological basis for transcellular potassium transport in the midgut of the mature fifth instar larvae of Hyalophora cecropia has been established through studies with the light and electron microscopes. The single-layered epithelium consists of two distinct cell types, the columnar cell and the goblet cell. No regenerative cells are present. Both columnar and goblet cells rest on a well developed basement lamina. The basal portion of the columnar cell is incompletely divided into compartments by deep infoldings of the plasma membrane, whereas the apical end consists of numerous cytoplasmic projections, each of which is covered with a fine fuzzy or filamentous material. The cytoplasm of this cell contains large amounts of rough endoplasmic reticulum, microtubules, and mitochondria. In the basal region of the cell the mitochondria are oriented parallel to the long axes of the folded plasma-lemma, but in the intermediate and apical portions they are randomly scattered within the cytoplasmic matrix. Compared to the columnar cell, the goblet cell has relatively little endoplasmic reticulum. On the other hand, the plications of the plasma membrane of the goblet cell greatly exceed those of the columnar cell. One can distinguish at least four characteristic types of folding: (a) basal podocytelike extensions, (b) lateral evaginations, (c) apical microvilli, and (d) specialized cytoplasmic projections which line the goblet chamber. Apically, the projections are large and branch to form villus-like units, whereas in the major portion of the cavity each projection appears to contain an elongate mitochondrion. Junctional complexes of similar kind and position appear between neighboring columnar cells and between adjacent columnar and goblet cells as follows: a zonula adherens is found near the luminal surface and is followed by one or more zonulae occludentes. The morphological data obtained in this study and the physiological information on ion transport through the midgut epithelium have encouraged us to suggest that the goblet cell may be the principal unit of active potassium transport from the hemolymph to the lumen of the midgut. We have postulated that ion accumulation by mitochondria in close association with plicated plasma membranes may play a role in the active movement of potassium across the midgut.

Arginine uptake through a novel cationic amino acid:K+ symporter, System R+, in brush border membrane vesicles from larval Manduca sexta midgut.

A concentrative uptake of arginine into brush border membrane vesicles (BBMV) from the midgut of Manduca sexta larvae was driven by an inwardly directed K+ gradient. The pH-dependence of the initial rate of arginine uptake between pH 7 and 10.5 paralleled the titration curve of the amino acid, suggesting that cationic arginine is the principal ionic form that is transported. In the presence of K+, at pH 7.4, arginine uptake was cis-inhibited and trans-stimulated by arginine and lysine but not by any other naturally occurring amino acids; it was also cis-inhibited by homoarginine and ornithine. Taken together, these data argue that arginine, lysine and their analogues share a cationic amino acid:K+ symporter (cotransporter), which we will designate as System R+. This novel symporter has a substrate spectrum similar to that of the uniporter, System y+, in that it accepts arginine+, lysine+, homoarginine+ and ornithine+ and rejects histidine. However, it differs from y+ in that it is cation-dependent and is almost inactive at pH 5.5.

Cationic lysine uptake by System R+ and zwitterionic lysine uptake by System B in brush border membrane vesicles from larval Manduca sexta midgut.

Lysine uptake was studied at pH 7.4 and 10.0 by rapid filtration methods in brush border membrane vesicles from fifth instar larvae of a model insect, the tobacco hornworm, Manduca sexta (Lepidoptera, sphingidae). At both pH values the uptake was mediated by K+ coupled symport. The uptake rate increased between pH 5.5 and 10, especially so in the alkaline range. The total lysine uptake could be divided into two components based upon lysine's ionic form as a function of pH. Lysine uptake at pH 7.4 was strongly cis-inhibited by arginine but at pH 10 was cis-inhibited and trans-stimulated by many neutral amino acids, e.g. leucine, but not by arginine. Lysine uptake by the arginine-inhibitable component paralleled the titration curve of cationic lysine whereas uptake by the leucine-inhibitable component paralleled that of zwitterionic lysine. Evidently, the brush border membrane contains at least two separate, K(+)-dependent amino acid symporters (co-transporters) that mediate lysine uptake. A cationic amino acid:K+ symporter selects cationic lysine and arginine but not histidine and other amino acids. One or more zwitterionic amino acid:K+ symporters select zwitterionic lysine, possibly arginine, histidine and neutral amino acids. Based upon these substrate repertoires, the zwitterionic symporters are B-type systems whereas the cationic symporter is identical with System R+, which partially resembles System y+. Arginine uptake in vivo is likely to be mediated mainly by System R+ whereas lysine uptake is likely to be mediated by System B-type symporters.

Cloning, sequencing and expression of cDNA encoding an insect V-ATPase subunit E.

This work presents the first invertebrate cDNA sequence encoding subunit E of a V-ATPase. It was cloned by immuno-shot-gun screening of a Manduca sexta (Insecta, Lepidoptera, Sphingidae) posterior larval midgut cDNA library. The amino acid sequence was 64% identical to that of the mammalian E-subunit and 34% to that of yeast. Southern and Northern blots suggested the existence of only one gene encoding the insect subunit E.

Cation-stimulated ATPase activity in purified plasma membranes from tobacco hornworm midgut.

Purified goblet cell apical membranes from Manduca sexta larval midgut exhibit a specific ATPase activity approx. 20-fold higher than that in the 100 000 X g pellet of a midgut homogenate. The already substantial ATPase activity in this plasma membrane segment is doubled in the presence of 20-50 mM KCl. At ATP concentrations ranging from 0.1 to 3.0 mM, the presence of 20 mM KCl leads to a 10-fold increase in the enzyme's affinity for ATP. ATPase activity is greatest at a pH of approx. 8. In addition to ATP, GTP serves as a substrate, but CTP, ADP, AMP and p-nitrophenyl phosphate do not. Either Mg2+ or Mn2+ is required for activity and cannot be replaced by Ca2+ or Zn2+. The ATPase activity of goblet cell apical membranes is inhibited by neither the typical (Na+ + K+)-ATPase inhibitors, ouabain and orthovanadate, nor by the typical mitochondrial F1F0-ATPase inhibitors, azide and oligomycin. Although 1.5 microM DCCD is ineffective, 150 microM DCCD leads to total inhibition of ATPase activity. The ATPase activity of goblet cell apical membranes is stimulated not only by K+, but also, in order of decreasing effectiveness, by Rb+, Li+, Na+ and even Mg2+. Replacement of Cl- by Br-, F- and HCO3- has less influence than variation of the cations. However, replacement of Cl- by NO3- inhibits strongly this ATPase activity. The ATPase activity described above is characteristic of the alkali metal ion pump containing apical membranes of goblet cells and is not enhanced to a similar degree in other purified midgut epithelial cell plasma membrane segments. Its localization, its broad cation specificity and its insensitivity to ouabain all mimic properties of active ion transport by the lepidopteran midgut and suggest this ATPase as a possible key component of the lepidopteran electrogenic alkali metal ion pump.

Cation-dependent leucine, alanine, and phenylalanine uptake at pH 10 in brush-border membrane vesicles from larval Manduca sexta midgut.

Using the rapid filtration technique, cation gradient driven leucine, alanine and phenylalanine uptake by brush-border membrane vesicles (BBMV) from the highly studied model insect, Manduca sexta, is characterized at the physiological pH of 10. The vesicles are sealed and nonspecific binding is small. Almost identical initial time courses of leucine uptake are obtained whether the vesicles are osmotically balanced initially or at equilibrium. The maximum accumulation values are also similar and the equilibrium values are identical with either treatment. Equilibrium is reached by 60 min. Amino acid accumulation is cation gradient dependent and is abolished by 18 microM valinomycin. Uptake of all three amino acids occurs over a broad pH range with maximum rates at approximately pH 10 and lower rates at pH 7.5. The cation selectivity of phenylalanine and alanine uptake changes with pH; the sequence is K+ > Na+ > Cs+ >> Rb+ = Li+ at pH 10.0, whereas K+ = Na+ at pH 8.0; the selectivity of leucine uptake is K+ = Na+ > Cs+ >> Rb+ = Li+ at pH 10. Maximum K+ driven accumulation of all three amino acids decreases with anions in the order: SCN- > NO3- > Cl- = CO(3)2- = So(4)2- = HPO(4)2- > gluconate-.Vmax values are similar for all three amino acids. There are large differences in initial uptake rates (leucine > phenylalanine = alanine), and maximum accumulation values (leucine > phenylalanine > alanine).

Neutral amino acid symport in larval Manduca sexta midgut brush-border membrane vesicles deduced from cation-dependent uptake of leucine, alanine, and phenylalanine.

Uptake of tritiated leucine, alanine, and phenylalanine was measured at the physiological pH of 10 by rapid filtration in brush-border membrane vesicles from the midgut of the larval tobacco hornworm, Manduca sexta. A 20-fold excess of unlabeled leucine, isoleucine, methionine, valine, alanine, lysine, histidine, phenylalanine, and glutamine inhibited uptake of leucine and phenylalanine, and six of these amino acids inhibited uptake of alanine, by more than 50% both in the presence and absence of a potassium ion gradient. These inhibitory amino acids also drove countertransport of leucine, alanine, and phenylalanine with accumulation ratios exceeding 2. These results are consistent with the hypothesis that leucine, alanine, and phenylalanine share a common uptake system - a broad scope B type symporter - which interacts strongly with half of the commonly occurring amino acids, interacts moderately with an additional quarter of them, but does not interact with cysteine, arginine, glutamate, aspartate, or proline.

Primary structure of V-ATPase subunit B from Manduca sexta midgut.

The amino acid sequence of a vacuolar-type ATPase (V-ATPase) subunit B has been deduced from a cDNA clone isolated from a Manduca sexta larval midgut library. The library was screened by hybridization with a labeled cDNA encoding subunit B of Arabidopsis thaliana tonoplast V-ATPase. The M. sexta V-ATPase subunit B consists of 494 amino acids with a calculated M(r) of 54,902. The amino acid sequence deduced for V-ATPase subunit B of M. sexta is between 98% and 76% identical with that of seven other V-ATPase subunits B and greater than 52% identical with three archaebacterial ATPase subunits B.

The multigene family of the tobacco hornworm V-ATPase: novel subunits a, C, D, H, and putative isoforms.

The plasma membrane V-ATPase from Manduca sexta (Lepidoptera, Sphingidae) larval midgut is composed of at least 12 subunits, eight of which have already been identified molecularly [Wieczorek et al., J. Bioenerg. Biomembr. 31 (1999) 67-74]. Here we report primary sequences of subunits C, D, H and a, which previously had not been identified in insects. Expression of recombinant proteins, immunostaining and protein sequencing demonstrated that the corresponding proteins are subunits of the Manduca V-ATPase. Genomic Southern blot analysis indicated the existence of multiple genes encoding subunits G, a, c, d and e. Moreover, multiple transcripts were detected in Northern blots from midgut poly(A) RNA for subunits B, G, c and d. Thus, these polypeptides appear to exist as multiple isoforms that could be expressed either in different tissues or at distinct locations within a cell. By contrast subunits A, C, D, E, F and H appear to be encoded by single transcripts and therefore should be present in any Manduca V-ATPase, independent of its subcellular or cell specific origin.

Stoichiometry of K+/H+ antiport helps to explain extracellular pH 11 in a model epithelium.

The stoichiometry of K+/H+ antiport was measured fluorometrically by the static head method in highly purified vesicles from goblet cell apical membranes of larval lepidopteran midgut. The measured stoichiometry of 1 K+/2 H+ explains how the antiport results in electrophoretic exchange of extracellular H+ for intracellular K+, driven by the voltage component of the proton-motive force of an H+ translocating V-ATPase that is located in the same membrane. In turn, the exchange of K+ for H+ helps to explain how the midgut contents are alkalinized to a pH of 11.

Sense and antisense RNA for the membrane associated 40 kDa subunit M40 of the insect V-ATPase.

For the first time a cDNA encoding the membrane associated subunit M40 of an invertebrate V-ATPase has been isolated and sequenced, based on a cDNA library from larval midgut of the tobacco hornworm, Manduca sexta. Immunoblotting with monospecific antibodies raised against the recombinant M40 polypeptide demonstrated that it is a subunit of the insect plasma membrane V-ATPase. Since M40 subunits had been identified only in endosomal V-ATPases till now, this result indicates that they are constitutive members of all, endomembrane and plasma membrane V-ATPases. A phagemid clone representing a polyadenylated antisense transcript was also isolated and sequenced. Using RT-PCR, endogenous antisense RNA was detected in poly(A) RNA isolated from the larval midgut. Since Southern blots indicated a single gene locus, both the antisense RNA as well as the sense mRNA encoding subunit M40 seem to originate from the same gene.

Molecular architecture of Manduca sexta midgut V1 ATPase visualized by electron microscopy.

The structure of the V1 ATPase from the tobacco hornworm Manduca sexta has been determined from electron micrographs of isolated, negatively stained specimens. The resulting images clearly show a pseudohexagonal arrangement of six equal-sized protein densities, presumably representing the three copies each of subunits A and B, which comprise the headpiece of the enzyme. A seventh density could be observed either centrally or asymmetrically to the hexamer. The maximum diameter of the V1 complex in the hexagonal projection is 13 nm with each of the six peripheral densities being 3-4 nm in diameter.

Cloning and sequencing of cDNA encoding the putative insect plasma membrane V-ATPase subunit A.

For the first time a cDNA encoding subunit A of an invertebrate V-ATPase has been sequenced. The cDNA library was prepared from larval midgut of the tobacco hornworm, Manduca sexta, and screened with monoclonal antibodies to the midgut plasma membrane subunit A. From the cDNA sequence the insect subunit A is predicted to consist of 617 amino acids with a relative molecular mass of 68,162. The predicted primary structure is similar to that of the published eukaryotic subunit A proteins (Bos, Daucus, Saccharomyces and Neurospora); it most closely resembles the bovine amino acid sequences with which it has an 83% sequence identity.

Delayed presentation of cerebral arterial gas embolism following proven intraoperative venous air embolism.

We describe the case of a 33-year-old woman who suffered a delayed-onset arterial gas embolism following a significant venous air embolism during surgery to remove an acoustic neuroma. We report the management of the problem and discuss the mechanisms by which this event might have occurred.

Is routine carotid screening for coronary surgery needed?

An association between carotid and coronary artery disease is well recognized. Routine preoperative duplex carotid screening of all coronary surgery patients is common, but may delay surgery and increase cost. To evaluate such a policy: A retrospective review of the records of 308 consecutive patients undergoing coronary surgery at one hospital was performed. Duplex studies were done on 210. A history of TIA/RIND, CVA, AS-PVD, AAA, neck bruit, or prior carotid surgery was considered suggestive for carotid disease. The history and/or physical exam (HPE) suggested carotid disease in 114; 37 of these (32%) had a positive scan. Of 96 patients without +HPE, three (3%) had a significant stenosis. A prospective study of cardiac surgery patients was done, categorized into "carotid" (n = 33) or "no-carotid" (n = 50) disease by two independent observers, based on +HPE. Positive scans were found in 27 per cent of the "carotid disease" group; No positive scans were found in the "no-carotid disease" group. We conclude that coronary surgery patients with peripheral or cerebral vascular disease or a neck bruit should have preoperative carotid studies. Duplex carotid screening of all cardiac patients is neither medically efficient nor cost-effective.

Genetic, phenotypic and environmental relationships between sow body weight and sow productivity traits.

Yorkshire and Duroc litter records were used to estimate genetic, phenotypic and environmental relationships between sow body weight and sow productivity traits. Two data sets with two subsets each were used to complete this study; 663 and 460 records included litter traits only, while 522 and 359 records also contained sow body weight for Yorkshires and Durocs, respectively. Heritability estimates for number born (NB), number born alive (NBA), total birth weight of live pigs (BWLIT), litter weight at 3 wk (WT3WK), sow weight at parturition (WTDAMPAR) and sow weight at weaning (WTDAMWN) were .24 +/- .14, .21 +/- .14, .42 +/- .16, .19 +/- .14, .72 +/- .21 and .42 +/- .18, respectively, for Yorkshires and .05 +/- .10, .04 +/- .10, .21 +/- .14, .25 +/- .15, .85 +/- .25 and .87 +/- .26, respectively, for the Durocs. Repeatability estimates for NB, NBA, BWLIT, WT3WK, WTDAMPAR and WTDAMWN were .13 +/- .06, .17 +/- .06, .27 +/- .06, .13 +/- .06, .64 +/- .05 and .54 +/- .05, respectively, for Yorkshires and .17 +/- .06, .21 +/- .06, .14 +/- .06, .17 +/- .06, .28 +/- .07 and .39 +/- .07, respectively, for Durocs. Genetic correlations among litter traits were high and positive in the Yorkshire data. Genetic correlations between NBA and WTDAMPAR, NBA and WTDAMWN, WT3WK and WTDAMPAR, and WT3WK and WTDAMWN were .37 +/- .25, .18 +/- .34, .60 +/- .29 and .29 +/- .45, respectively, in the Yorkshire data. Genetic correlations among litter traits in the Duroc analysis had large standard errors but were generally similar to the estimates obtained from the Yorkshire data. The genetic correlation between WTDAMPAR and WTDAMWN was .93 +/- .09 for Yorkshire sows. The primary conclusion from this study is that as selection increases sow productivity traits, there will be a positive correlated response in sow body weight.

Genetic parameters for ewe productivity traits in the Columbia, Suffolk and Targhee breeds.

Estimates of repeatability and heritability were obtained for the following productivity traits of ewes: litter weight at birth (LWB) and weaning (LWW), litter size at birth (LSB), litter size alive at birth (NBA), litter size at weaning (LSW), neonatal survival rate (SRB) and preweaning survival rate (SRW). Phenotypic and genetic correlations were estimated for litter traits. The data set contained 6,394 ewe breeding records from three state stations over 10 yr on 1,731 ewes that were the progeny of 488 sires among three breeds (Columbia, Suffolk and Targhee). Pooled intra-station estimates of repeatability ranged from .11 to .22 for LWB and LWW among the three breeds. For litter size at birth, number born alive and litter size at weaning these estimates varied from .09 to .17 and for the survival traits (SRB and SRW) the variation was from .11 to .20. Intra-station estimates of heritability for the three breeds varied from .12 to .28 for LWB and LWW, and for LSB, NBA and LSW estimates varied from .05 to .35. Heritability estimates for survival traits (SRB and SRW) were low, ranging from .00 to .14. Phenotypic correlations among LWB, LWW, NBA and LSW ranged from .35 to .92 among the breed-station subclasses, with higher correlations occurring where a part-whole relationship existed. The study suggests that selection of ewes with high litter size at birth or at weaning and(or) litter weight at birth or at weaning will genetically improve total litter weight at weaning per ewe lambing.

Divergent selection for postweaning feed conversion in Angus beef cattle: I. Mean comparisons.

Each year from 1979 through 1983, 35 Angus bull calves were selected from a herd at the Eastern Ohio Resource Development Center to be individually fed in a 140-d postweaning performance test. From these 35 individually fed bulls, the three highest and three lowest for feed conversion (feed:gain) were selected and randomly mated to approximately 20 cows each. A different set of high vs low feed conversion sires was used each year. Four replicates (403 progeny) from high vs low sires were evaluated by sire groups for subsequent postweaning and carcass performance. Progeny were slaughtered when estimated by ultrasonic measurement to have 8.9 mm or more of subcutaneous fat at the conclusion of a 140-d postweaning performance test. Progeny with less than 8.9 mm of subcutaneous fat were fed for additional 28-d periods until they reached the required minimum. No differences were found between high and low feed conversion progeny for 140-d feed intake (P less than .30) although high feed conversion progeny gained .09 kg/d more weight (P less than .01) during the 140-d postweaning test. Differences tended to exist between high and low feed conversion progeny for unadjusted (P less than .15) and maintenance-adjusted (P less than .15) feed:gain ratios. Progeny of the high feed conversion group had greater subcutaneous fat (P less than .05) at the end of the 140-d postweaning test and when slaughtered (P less than .05), indicating a genetic difference for composition of BW gain between high- and low-sired progeny. However, no significant differences existed for any other carcass traits evaluated. Bulls had more desirable unadjusted (P less than .001) and maintenance-adjusted (P less than .001) feed:gain ratios than heifers with increased 140-d ADG (P less than .001) and pen feed intakes (P less than .001).

Divergent selection for postweaning feed conversion in Angus beef cattle: II. Genetic and phenotypic correlations and realized heritability estimate.

A single generation divergent selection study, replicated four times (1983, 1984, 1985, and 1986), was conducted to assess genetic differences between progeny of high and low feed conversion sires in Angus beef cattle and to determine correlated response for weight gain (ADG140), feed intake (AVFD140), and BW (OFFTSTWT) in a time- (140-d) and fat-constant (8.9 mm) period. Realized heritability estimates for unadjusted (feed/gain; FEFF140; .26) and adjusted feed conversion (adjusted as recommended by the BIF, 1986; ADJFDEFF; .46) were obtained. The difference in heritability estimates reflects variation accounted for by adjustment for BW differences, and thus maintenance requirements, of individual progeny. Phenotypic and "pseudo" realized genetic correlations of FEFF140 with ADG140, AVFD 140, and OFFTSTWT were -.33 and -.66, .49 and -.26, and .15 and -.41, respectively. Phenotypic and "pseudo" realized genetic correlations of ADJFDEFF with ADG140, AVFD140, OFFTSTWT, and FEFF140 were -.54 and -.59, .30 and -.23, .27 and -.36, and .97 and .49, respectively. Subcutaneous fat (as estimated by ultrasonic measurement; BF140) had phenotypic and "pseudo" realized genetic correlations with FEFF140 of -.33 and .66, respectively, and with ADJFDEFF of -.44 and -.58, respectively.