Chromatofocusing profile of purified human alpha-fetoprotein and albumin differs from those of crude samples: effect of protein concentration of the elution of the sample.

Chromatofocusing was utilized to characterize charge microheterogeneity of purified human alpha-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with pIs 4.57 (52%), 4.27 (43%), and less than 4.00 (5%), respectively. In contrast, 10 micrograms of purified AFP or 250,000 cpm of 125I-AFP eluted entirely as isoform AFP-II. 125I-AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With greater than or equal to 5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of 125I-HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with greater than or equal to 5 mg unlabeled HSA. When carrier proteins of varying pI values were chromatofocused along with purified AFP, it was observed that only those proteins with pIs in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared.     

Human alpha-fetoprotein purified from amniotic fluid enhances growth factor-mediated cell proliferation in vitro.

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha-fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.     

Chromosomal localization of uroplakin genes of cattle and mice

The asymmetric unit membrane (AUM) of the apical surface of mammalian urinary bladder epithelium contains several major integral membrane proteins, including uroplakins IA and IB (both 27 kDa), II (15 kDa), and III (47 kDa). These proteins are synthesized only in terminally differentiated bladder epithelial cells. They are encoded by separate genes and, except for uroplakins IA and IB, appear to be unrelated in their amino acid sequences. The genes encoding these uroplakins were mapped to chromosomes of cattle through their segregation in a panel of bovine x rodent somatic cell hybrids. Genes for uroplakins IA, IB, and II were mapped to bovine (BTA) Chromosomes (Chrs) 18 (UPK1A), 1 (UPK1B), and 15 (UPK2), respectively. Two bovine genomic DNA sequences reactive with a uroplakin III cDNA probe were identified and mapped to BTA 6 (UPK3A) and 5 (UPK3B). We have also mapped genes for uroplakins 1A and II in mice, to the proximal regions of mouse Chr 7 (Upk1a) and 9 (Upk2), respectively, by analyzing the inheritance of restriction fragment length variants in recombinant inbred mouse strains. These assignments are consistent with linkage relationships known to be conserved between cattle and mice. The mouse genes for uroplakins IB and III were not mapped because the mouse genomic DNA fragments reactive with each probe were invariant among the inbred strains tested. Although the stoichiometry of AUM proteins is nearly constant, the fact that the uroplakin genes are unlinked indicates that their expression must be independently regulated. Our results also suggest likely positions for two human uroplakin genes and should facilitate further analysis of their possible involvement in disease.     

Alpha-mannosidases involved in N-glycan processing show cell specificity and distinct subcompartmentalization within the Golgi apparatus of cells in the testis and epididymis.

The Golgi apparatus is enriched in specific enzymes involved in the maturation of carbohydrates of glycoproteins. Among them, alpha-mannosidases IA, IB and II are type II transmembrane Golgi-resident enzymes that remove mannose residues at different stages of N-glycan maturation. alpha-Mannosidases IA and IB trim Man9GlcNAc2 to Man5GlcNAc2, while alpha-mannosidase II acts after GlcNAc transferase I to remove two mannose residues from GlcNAcMan5GlcNAc2 to form GlcNAcMan3GlcNAc2 prior to extension into complex N-glycans by Golgi glycosyltransferases. The objective of this study is to examine the expression as well as the subcellular localization of these Golgi enzymes in the various cells of the male rat reproductive system. Our results show distinct cell-and region-specific expression of the three mannosidases examined. In the testis, only alpha-mannosidase IA and II were detectable in the Golgi apparatus of Sertoli and Leydig cells, and while alpha-mannosidase IB was present in the Golgi apparatus of all germ cells, only the Golgi apparatus of steps 1-7 spermatids was reactive for alpha-mannosidase IA. In the epididymis, principal cells were unreactive for alpha-mannosidase II, but they expressed alpha-mannosidase IB in the initial segment and caput regions, and alpha-mannosidase IA in the corpus and cauda regions. Clear cells expressed alpha-mannosidase II in all epididymal regions, and alpha-mannosidase IB only in the caput and corpus regions. Ultrastructurally, alpha-mannosidase IB was localized mainly over cis saccules, alpha-mannosidase IA was distributed mainly over trans saccules, and alpha-mannosidase II was localized mainly over medial saccules of the Golgi stack. Thus, the cell-specific expression and distinct Golgi subcompartmental localization suggest that these three alpha-mannosidases play different roles during N-glycan maturation.     

Expressions of two types of soluble cAMP-dependent protein kinases of the rabbit myometrium in different functional states

Two types of soluble cAMP-dependent protein kinase (I and II) were isolated from rabbit myometrium cytosol at functional rest and characterized. In pregnancy, protein kinase is represented by type II alone. Upon delivery, one isoform of the enzyme was detected, which was eluted from a DEAE-cellulose column with 0.15-0.22 M. NaCl. During the postnatal period, the elution profile of the enzyme is made up of two protein bands, one fraction being eluted with 0.15-0.22 M NaCl (93% of total enzyme content), and the other one being represented by a minor component eluted with 0.07-0.09 M. NaCl (7%). In terms off isoenzyme activity, main kinetic properties, ability to autophosphorylate and Kass for cAMP, the protein kinase isolated during delivery and the major protein kinase fraction obtained in the postnatal period can be related to protein kinases type II. Quantitative and qualitative expression of two types of soluble cAMP-dependent protein kinase from rabbit myometrium isolated at different functional states may be due to differences in their biological activity.     

Purification and characterization of a third isoform of myosin I from Acanthamoeba castellanii

A third isoform of myosin I has been isolated from Acanthamoeba and designated myosin IC. Peptide maps and immunoassays indicate that myosin IC is not a modified form of myosin IA, IB, or II. However, myosin IC has most of the distinctive properties of a myosin I. It is a globular protein of native Mr approximately 162,000, apparently composed of a single 130-kDa heavy chain and a pair of 14-kDa light chains. It is soluble in MgATP at low ionic strength, conditions favoring filament assembly by myosin II. Myosin IC has high Ca2+- and (K+,EDTA)-ATPase activities. Its low Mg2+-ATPase activity is stimulated to a maximum rate of 20 s-1 by the addition of F-actin if its heavy chain has been phosphorylated by myosin I heavy chain kinase. The dependence of the Mg2+-ATPase activity of myosin IC on F-actin concentration is triphasic; and, at fixed concentrations of F-action, this activity increases cooperatively as the concentration of myosin IC is increased. These unusual kinetics were first demonstrated for myosins IA and IB and shown to be due to the presence of two actin-binding sites on each heavy chain which enable those myosins I to cross-link actin filaments. Myosin IC is also capable of cross-linking F-actin, which, together with the kinetics of its actin-activated Mg2+-ATPase activity, suggests that it, like myosins IA and IB, possesses two independent actin-binding domains.     

The purification and characterization of mannosidase IA from rat liver Golgi membranes

Rat liver Golgi membranes contain two alpha 1,2-specific mannosidases (IA and IB) (Tulsiani, D. R. P., Hubbard, S. C., Robbins, P. W., and Touster, O. (1982) J. Biol. Chem. 257, 3660-3668). Mannosidase IA has now been purified to apparent homogeneity by detergent extraction and (NH4)2SO4 precipitation, followed by Sephacryl S-300, ion-exchange, and hydroxylapatite chromatography. The enzyme was homogeneous by nondenaturing polyacrylamide gel electrophoresis with different gel concentrations, and Ferguson plot analysis indicated an Mr of 230,000 for the native enzyme. Although electrophoresis under denaturing conditions generally gave a subunit Mr of 57,000, electrophoresis of less than 1 microgram of protein yielded a faint doublet of Mr 57,000 and 58,000. Thus, the enzyme appears to be a tetramer with four very similar subunits. The enzyme bound to concanavalin A-Sepharose 4B only when it was kept in contact with the lectin for 16 h. Endoglycosidase H treatment resulted in loss of its binding to the lectin, without leading to a detectable change in the size of the enzyme subunit. On electrophoretic gels, the enzyme gave a faint positive stain with periodic acid-Schiff's base. The enzyme contained about 0.9% hexose by direct analysis. It did not bind to affinity resins specific for neuraminic acid, galactose, or N-acetylglucosamine. All these studies suggest that the enzyme is a glycoprotein containing only one or two clusters of high mannose oligosaccharide. Mannosidase IA is active toward oligosaccharides containing alpha 1,2-linked mannosyl residues. [3H]Man9GlcNAc, [3H] Man8GlcNAc, [3H]Man7GlcNAc, and [3H]Man6GlcNAc are good substrates. Man9GlcNAc, the best substrate, yields Man8, Man7, and Man6 derivatives with structures suggesting that the sequence of release of mannose residues is rather specific. Immunoprecipitation studies using polyclonal antibody (IgG) prepared against homogeneous mannosidase IA cross-reacted with mannosidase IB, a result suggesting that these two enzymes share antigenic determinants. However, no cross-reactivity was observed with rat liver cytosolic and lysosomal alpha-D-mannosidases or with Golgi mannosidase II.     

Differential localization of Acanthamoeba myosin I isoforms

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.     

Collagen-induced arthritis in rats. Examination of the epitope specificities of circulating and cartilage-bound antibodies produced by outbred and inbred rats using cyanogen bromide-derived peptides purified from heterologous and homologous type II collagens.

To determine the number and location of antibody binding epitopes on type II collagen, outbred and inbred rats were immunized with chick, bovine, human, and rat type II collagen (CII, BII, HII, and RII); all sera were assayed for reaction with a panel of CB peptides purified and renatured from the immunizing collagen and from RII. Antibody reaction patterns (profiles) varied among individual outbred rats but were essentially constant over time and changed little after boosting. The strongest antibody reactions were to CB11, CB9-7, and CB12 followed by CB8, CB10, and CB6. Antibody profiles varied depending on the species of collagen used for immunization and the strain of rat immunized. Except for CB10, where antibodies were largely specific for heterologous collagens, antibodies reactive with all other CB peptides cross-reacted strongly with renatured rat CB peptides. Sera from inbred BB rats immunized with BII, CII, or HII reacted best with CB11, unlike antisera to RII that reacted strongly with CB9-7. Inbred LEW, COP, WKY, F344, and BUF rats immunized with BII reacted strongest with CB9-7 and variably with CB11 and CB12. BBxLEW F1 hybrid rats reacted almost equally with CB11 and CB9-7 producing an antibody profile intermediate to those elicited in the parent strains. Finally, antibodies reactive with rat CB11, CB9-7, and CB12 could be eluted from normal rat cartilage incubated in anti-BII serum; antibody eluate profiles generally paralleled the profile produced by the sera applied to cartilage. Taken together, these findings indicate that multiple antibody-reactive epitopes on type II collagen may be instrumental in the initiation of collagen-induced arthritis in rats, particularly shared or cross-reactive epitopes located within CB11, CB9-7, CB12, and CB8.     

Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.     

Structural and biophysical coupling of heparin and activin binding to follistatin isoform functions

Follistatin (FS) regulates transforming growth factor-beta superfamily ligands and is necessary for normal embryonic and ovarian follicle development. Follistatin is expressed as two splice variants (FS288 and FS315). Previous studies indicated differences in heparin binding between FS288 and FS315, potentially influencing the physiological functions and locations of these isoforms. We have determined the structure of the FS315-activin A complex and quantitatively compared heparin binding by the two isoforms. The FS315 complex structure shows that both isoforms inhibit activin similarly, but FS315 exhibits movements within follistatin domain 3 (FSD3) apparently linked to binding of the C-terminal extension. Surprisingly, the binding affinities of FS288 and FS315 for heparin are similar at lower ionic strengths with FS315 binding decreasing more sharply as a function of salt concentration. When bound to activin, FS315 binds heparin similarly to the FS288 isoform, consistent with the structure of the complex, in which the acidic residues of the C-terminal extension cannot interact with the heparin-binding site. Activin-induced binding of heparin is unique to the FS315 isoform and may stimulate clearance of FS315 complexes.     

Effects of maturation and co-culture treatments on the developmental capacity of early bovine embryos.

A total of 901 cumulus-oocyte complexes (COCs) were collected from bovine ovaries obtained at a local abattoir. COCs randomly assigned to Treatment I (n = 451), were cultured in TCM-199 + 10% fetal bovine serum (FBS) and hormones, while oocytes in Treatment II (n = 450) were cultured in TCM-199 + 20% estrous cow serum (ECS). Assessment of maturation revealed that 91.3% (42/46) of oocytes in Treatment I had reached metaphase II of meiosis, which was greater (P less than 0.05) than the 73.3% (33/45) in Treatment II. Following in vitro fertilization, 203 oocytes from Treatment I were co-cultured on bovine granulosa cells (Treatment IA) while the remaining 202 oocytes were co-cultured on bovine oviductal cells (Treatment IB). Similarly, 203 oocytes from Treatment II were co-cultured on granulosa cells (Treatment IIA) or oviductal cells (Treatment IIB, n = 202). Co-culture was maintained for 8 days. The proportion of cleaved zygotes was higher (P less than 0.05) in Treatment IB (86.6%) compared to Treatments IA (78.8%), IIA (58.1%), and IIB (64.8%). The proportion of cleaved zygotes that progressed beyond the 16-cell stage was also greater (P less than 0.001) in Treatment IB (71.4%) compared to Treatments IA (50.0%), IIA (35.4%) and IIB (55.8%). Treatment IB also produced the highest proportion of blastocysts (P less than 0.0001) (41.1%) versus 24.6% (IA), 11.3% (IIA) and 18.3% (IIB). The proportion of day 6 morulae that progressed to form day 8 blastocysts was similar for both co-culture treatments (IA, 70.1%; IB 70.2%; IIA, 51.5%; IIB 50.8%) and varied only between in vitro maturation groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-function studies on Acanthamoeba myosins IA, IB, and II

Myosins IA and IB are globular proteins with only a single, short (for myosins) heavy chain (140,000 and 125,000 daltons for IA and IB, respectively) and are unable to form bipolar filaments. The amino acid sequence of IB heavy chain shows 55% similarity to muscle myosins in the N-terminal 670 residues, which contain the active sites, and a unique 500-residue C-terminus highly enriched in proline, glycine, and alanine. The C-terminal region contains a second actin-binding site which allows myosins IA and IB to cross-link actin filaments and support contractile activity. Myosins IA and IB are regulated solely by phosphorylation of one serine on the heavy chain positioned between the catalytic site and the actin-binding site that activates ATPase. Myosin II is a more conventional myosin in composition (two heavy chains and two pairs of light chains), heavy chain sequence (globular head 45% identical to muscle myosins and a coiled-coil helical tail), and structure (bipolar filaments). The tail of myosin II is much shorter than that of other conventional myosins, and it contains a 25 amino acid sequence in which helical structure is predicted to be weak or absent. The position of this sequence corresponds to the position of a bend in the monomer. Myosin II heavy chains also have a 29-residue nonhelical tailpiece which contains three regulatory, phosphorylatable serines. Phosphorylation at the tip of the tail regulates ATPase activity in the globular head apparently through an effect on filament structure.     

Characterization of multiple forms of histone phosphatase in rat liver.

By using chromatography on DEAE-cellulose, aminohexyl-Sepharose 4B and Sephadex G-200, rat liver extract was shown to contain at least three fractions, IA, IB and II, of histone phosphatase. Fractions IA and II are probably the same enzymes as the previously described glycogen synthase phosphatase and phosphorylase phosphatase, respectively, but IB exhibits noticeable activities only with phosphohistone as substrate. Approximate molecular weights of 69 000, 300 000 and 160 000 were determined by gel filtration on Sephadex G-200 for IA, IB and II, respectively.     

Estrogen-binding properties of rat serum alpha 1-fetoprotein and its isoforms. Investigation of the apparent non-integrality of sites on the unfractionated protein.

Rat fetal serum alpha 1-fetoprotein (AFP), a heterogeneous glycoprotein, binds estrogens with high affinity but at a fractional number of sites even after treatment with charcoal (n = 0.6), which may mean 60% of the protein has 1 site and the remainder none. To investigate the origin of this fractional number of sites the "native" protein (purified by negative affinity chromatography) was further purified (step 1) and fractionated (step 2) into its two main charge variants (electrophoretically "slow" and "fast") by a two-step fast-protein liquid chromatography method. The binding parameters for estrone and estradiol-17 beta of the "native" and "repurified" proteins and of each charge variant were determined by equilibrium microdialysis. The molar extinction coefficient at 278 nm of each sample was also determined. (1) The "repurified" AFP and each charge variant had a number of binding sites for estrogens close to unity. This increase in the number of sites could neither be explained by the loss of a non-binding isoform (corresponding to 40% of the protein) during chromatography, nor by the existence of complex negative modulatory interactions between isoforms. (2) The affinities for estrogens of the "repurified" protein and the two charge variants were slightly decreased compared to that of "native" AFP, except that the "fast" form had the "native" protein's high affinity for estrone--but not for estradiol-17 beta. (3) The molar extinction coefficients at 278 nm of the "repurified" AFP and the isoforms were much lower than that of the "native" protein. These results suggest that the presence of (an) inhibitor(s) of estrogen binding on the "native" protein which is/are removed by the ion-exchange fast protein liquid chromatography (FPLC) column. A ligand absorbing at 278 nm, which may or may not be the inhibitor, is also removed. The isoform heterogeneity with respect to estrone binding is discussed.     

Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii

The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70- 100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.     

Activation of rat liver and rabbit skeletal muscle glycogen synthases by rat liver cytosolic protein phosphatases

To gain more insight into the nature of the substrate specificity of protein phosphatases, four forms of glycogen synthase D were used as substrates for previously characterized protein phosphatases, IA, IB, and II, from rat liver cytosol. The phosphatase activity was measured as the conversion of glycogen synthase D to synthase I. While glycogen synthase isolated from rat liver as the D-form was activated mainly by phosphatase IA, rabbit skeletal muscle glycogen synthase previously phosphorylated in vitro by cyclic AMP-dependent protein kinase or phosphorylase kinase was activated efficiently by phosphatases IA, IB, and II. Glycogen synthase isolated from rabbit skeletal muscle as the D-form, however, was a poor substrate for all three phosphatases. These results suggest that the phosphorylation state as well as the primary structure of synthase D markedly affects the rate of its activation by individual protein phosphatases. A protein phosphatase released from rat liver particulate glycogen, on the other hand, activated all forms of synthase D used here readily and at about the same rate.     

The role of frozen section in surgical staging of low risk endometrial cancer

Background

The role of frozen section (FS) in intraoperative decision making for surgical staging of endometrial cancer is controversial. Objective of this study is to assess the agreement rate between the FS and paraffin section (PS); and the potential impact of the role of FS in the intra-operative decision making for the complete surgical staging in low risk endometrial cancer.

Methods

This is a retrospective analysis of patients diagnosed with intra-operative FS stage I, grade I or II endometrial cancer from 1995–2004. FS results were compared with final pathology results with regard to tumor grade, depth of myometrial invasion, cervical involvement, lymphovascular invasion, and lymph node involvement. Agreement statistic with kappa was calculated using SPSS statistical software. Categorical variables were tested using chi-square test with p value of ≤0.05 being statistically significant.

Results

Of the 457 patients with endometrial cancer, 146 were evaluated by intra-operative FS and met inclusion criteria. FS results were in disagreement with permanent section in 35% for the grade (kappa 0.58, p = 0.003), 28% for depth of myometrial invasion (kappa 0.61, p<0.0001), 13% for cervical involvement (kappa 0.78, p = 0.002), and 32% for lymphovascular invasion (kappa 0.6, p = 0.01). Permanent pathology upstaged 31.9% & 23.2% of FS stage IA, & IB specimen respectively. Lymph node dissection was done in 56.8%. Lymph node metastasis was identified in 8.4%. Use of intraoperative FS would have resulted in suboptimal surgical treatment in 13% stage IA and 6.6% of stage IB patients respectively by foregoing lymphadenectomy.

Conclusion

A significant number of patients with low risk endometrial cancer by FS were upstaged and upgraded on final pathology. Before placing absolute reliance on intraoperative FS to undertake complete surgical staging, the inherent limitation of the same in predicting final stage and grade highlighted by our data need to be carefully considered.     

Isolation and characterization of subunits from the predominant form of Dolichos biflorus lectin.

The subunits of the two molecular forms (A and B) of the Dolichos biflorus lectin were isolated by ion-exchange chromatography on DEAE-cellulose in 8.0 M urea. Subunits IA and IIA which comprise the predominant molecular form A of the lectin were found to have molecular weights of 27,700 and 27,300, respectively, as determined by sedimentation equilibrium studies in 8.0 M urea. These subunits have similar amino acid compositions and each have alanine at their amino-terminal ends. Comparison of the IA and IIA subunits by immunodiffusion against antisera to the seed extract as well as to subunits IA and IIA showed no antigenic differences between the two subunits. Carboxyl terminal analyses of subunits IA and IIA with carboxypeptidase A produced an essentially simultaneous release of both leucine and valine residues from subunit IA; no detectable amino acids were released from subunit IIA under identical conditions. The data suggest that the molecular form A of the lectin (molecular weight 113,000, Carter and Etzler, 1975) consists of four subunits with a possible stoichiometry of IA2IIA2. Other possible arrangements of the subunits are discussed.     

Peroxisomal acetoacetyl-CoA thiolase of an n-alkane-utilizing yeast, Candida tropicalis.

Two genes encoding acetoacetyl-CoA thiolase (thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the lambda EMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41,898 Da for thiolase IA and 41,930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl-CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other thiolases reported hitherto.