Morphogenetic characteristics of the aggregates formed in epithelial and mesenchymal recombinations of the lungs from intact and urethane-exposed mouse embryos

A study was made of the morphogenesis of organotypic aggregates obtained by epithelial mesenchymal recombinations from the lungs of embryonic mice, intact and treated with urethane. Normal growth and differentiation of organotypic structures were observed in long-term cultures of aggregates obtained by recombinations of the lung epithelium (E) and mesenchyma (M) from intact (i) embryonic mice (EiMi). Hyperplasia and squamous-cell metaplasia (with or without keratinization) of the epithelium were found in aggregates obtained from E and M of the treated mouse embryos (EtMt) and in aggregates obtained by recombinations of lung E and M from intact and treated embryos (EtMi, EiMt). The data obtained suggest that the alterations in epithelial mesenchymal interactions are of great significance for transplacental lung blastomogenesis and that the mesenchymal lung cells play an important part in mediation of the transplacental carcinogenous effects on epithelial target cells via subsequent epithelial mesenchymal tissue interactions.     

Effects of follicle-stimulating hormone and 17beta-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.     

Effects of follicle-stimulating hormone and 17β-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.     

Targeted disruption of fibulin-4 abolishes elastogenesis and causes perinatal lethality in mice

Elastic fibers provide tissues with elasticity which is critical to the function of arteries, lungs, skin, and other dynamic organs. Loss of elasticity is a major contributing factor in aging and diseases. However, the mechanism of elastic fiber development and assembly is poorly understood. Here, we show that lack of fibulin-4, an extracellular matrix molecule, abolishes elastogenesis. fibulin-4-/- mice generated by gene targeting exhibited severe lung and vascular defects including emphysema, artery tortuosity, irregularity, aneurysm, rupture, and resulting hemorrhages. All the homozygous mice died perinatally. The earliest abnormality noted was a uniformly narrowing of the descending aorta in fibulin-4-/- embryos at embryonic day 12.5 (E12.5). Aorta tortuosity and irregularity became noticeable at E15.5. Histological analysis demonstrated that fibulin-4-/- mice do not develop intact elastic fibers but contain irregular elastin aggregates. Electron microscopy revealed that the elastin aggregates are highly unusual in that they contain evenly distributed rod-like filaments, in contrast to the amorphous appearance of normal elastic fibers. Desmosine analysis indicated that elastin cross-links in fibulin-4-/- tissues were largely diminished. However, expression of tropoelastin or lysyl oxidase mRNA was unaffected in fibulin-4-/- mice. In addition, fibulin-4 strongly interacts with tropoelastin and colocalizes with elastic fibers in culture. These results demonstrate that fibulin-4 plays an irreplaceable role in elastogenesis.     

The dose-effect of benz(a)pyrene in organ cultures of embryonic mouse lungs resistant and predisposed to pulmonary blastomogenesis

A direct effect of benz(a)pyrene (BP) was studied on organ cultures from embryonic lungs of C57Bl and A mice. The toxic effect of 3, 6, 12 micrograms/ml BP was observed in alveolar epithelium and mesenchymal cells after 7 days of cultivation. After 14-21 days diffuse and focal hyperplasia of the epithelium was discovered in 35.5, 20.0 and 36.1% of treated cultures (explants) from embryonic lungs of C57Bl mice and in 45.5, 56.3 and 53.2% of treated explants from embryonic lungs of A mice. Squamous epithelial metaplasia was observed in 3.0, 2.2 and 4.2% of treated explants from C57Bl mice and in 2.4, 10.4 and 23.4% of treated explants from A mice. Total papillary adenomatous growth of the epithelium was discovered in 10.0, 18.3 and 36.1% of treated explants from embryonic lungs of A mice only. Thus, a direct effect of BP on organ cultures from embryonic lungs of C57B1 and A mice depended on BP doses and the mouse line.     

Sensitivity to benzo(a)pyrene of epithelial and mesenchymal cells of embryonic lungs of mice susceptible (line A) and resistant (line C57BL) to lung blastomogenesis

Proliferative activity of alveolar (EA) and bronchial (EB) epithelial cells, mesenchymal cells of explants (ME) and mesenchymal cells migrated on the cellulose filter (MF) was studied autoradiographically in the normal cultures and after treatment with benz(a)pyrene (BP). Labeling index (LI) of cells from the treated explants was higher or lower than in normal cultures, or did not differ from normal values. The effect of the treatment depended on BP dose, mouse strain, and the type of cells. Epithelial cells, especially EB, and ME cells from embryonic lungs of A mice were very sensitive to growth-stimulating effect of BP. In treated cultures from C57BL mice LI increased only in EB cells, however, unlike A mice, the effect inversely correlated with BP doses. Proliferative activity of EA, ME and MF cells was lower than in untreated explants from C57BL, the effect of treatment increasing with higher BP doses. The importance of local cell-tissue factors, namely epithelial-mesenchymal interactions, and the role of lung mesenchyma in the realization of genetically determined sensitivity to spontaneous and induced lung blastomogenesis in C57BL and A mice are discussed.     

Increasing the toxic effect of pulmonary carcinogens by destruction of epithelial-mesenchymal interaction in explants of embryonic respiratory tract of mice

The explants from 17-day old embryos of A and C57BL mice were used for long-term organ cultures. The following series of explants were studied: 1) the intact control explants; 2) the explants treated by NMU; 3) the explants treated by BP; 4) the explants isolated from mesenchyma (M); 5) the explants isolated from M and treated by NMU; 6) the explants isolated from M and treated by BP. The survival of explants treated by NMU or BP did not differ from the intact control explants (p less than 0.1). The removal of M decreased the survival only in the explants from the distal RT of A mice (p 0.01). The survival of explants isolated from M and treated by NMU or BP was significantly lower than it was in intact explants (p less than 0.01-0.001). Thus, the destruction of epithelial-mesenchymal interaction induced by removal of M can modulate the toxic effect of pulmonotropic carcinogens.     

Estrogen- and progesterone-dependent secretory changes in the uterus of the sheep.

This study was undertaken to determine the effects of 17 beta-estradiol (E) and progesterone (P) on polypeptide synthesis and release from the uterus of the sheep. Uterine flushings (UF) and endometrium were obtained from ovariectomized untreated animals, ovariectomized animals treated with E (approximately 5-10 pg/ml) for 6 days (6E) and ovariectomized animals primed with E for 6 days then treated with P (approximately 1.5-3 ng/ml), in the continued presence of E, for an additional 6 days (6EP). Endometrium was cultured (24 h) in the presence of 3H-leucine (3H-leu) or 3H-glucosamine (3H-glcN), and newly synthesized and released proteins were detected in culture media by fluorography of 10% SDS gels. The quantity of proteins in UF and radiolabeled proteins in explant culture media did not change between treatment groups (p < 0.05). Qualitative changes in the synthesis and release of proteins were observed depending on the steroid treatment. An M(r) 57,000 protein was present in UF and 3H-leu-labeled culture media obtained from animals treated only with E and an M(r) > 200,000 was present in 3H-leu-labeled culture media of endometrium obtained from 6E and 6EP animals. An M(r) 44,000 protein was present only in UF from 6EP animals but could not be detected in endometrial culture media from animals undergoing this steroid treatment. These data show that the endometrium of the ovariectomized sheep undergoes alterations in secretory protein patterns which depend on the presence of E and P.     

Hoxb-5 control of early airway formation during branching morphogenesis in the developing mouse lung

Hox proteins control structural morphogenesis, pattern formation and cell fate in the developing embryo. To determine if Hoxb-5 participates in patterning of early airway branching during lung morphogenesis, gestational day 11.5 embryonic lung cultures were treated with retinoic acid (RA) to up-regulate and antisense oligonucleotides to down-regulate Hoxb-5 protein expression. RA (10^?6 M) and Hoxb-5 antisense oligonucleotide (20 μM) treatment each significantly decreased branching morphogenesis (P<0.001), but the morphology of branching under these conditions was very different. RA-treated lungs had elongated primary branches but decreased further branching with increased Hoxb-5 immunostaining in subepithelial regions underlying these elongated airways. Western blots confirmed that Hoxb-5 protein was increased by 189±20% (mean±S.E.M., P<0.05) in RA-treated lungs compared to controls. In contrast, lungs treated with Hoxb-5 antisense oligos plus RA had foreshortened primary branches with rudimentary distal clefts resulting in decreased numbers of primary and subsequent branches. Immunohistochemistry confirmed that Hoxb-5 antisense oligos inhibited Hoxb-5 protein expression even in the presence of RA. We conclude that regional and quantitative changes in Hoxb-5 protein expression influence morphogenesis of the first airway divisions from the mainstem bronchi. RA-induced alterations in branching are mediated in part through regulated Hoxb-5 expression.     

Analysis of metopirone-induced hypertrophy of adrenals in fetal rats encephalectomized in utero

Pregnant rats were treated with 30 mg metopirone (M) each day for 2 days and autopsied on the third day in various gestational periods (Days 18-20, 19-21, and 20-22). Control rats were treated with saline alone (S). The adrenals of intact fetuses in M-treated dams were significantly heavier than those of intact fetuses in S-treated dams in every experimental period. In both M- and S-treated dams, the adrenals of encephalectomized (E) fetuses were lighter than those of intact littermates. However, in the experimental period of Days 18-20 and 19-21, the adrenals of E fetuses in M-treated dams were slightly but significantly heavier than those of similar E fetuses in S-treated dams. In contrast, in the experimental period Days 20-22, there was no significant difference in the weight of adrenals of E fetuses of M- and S-treated dams. These changes in fetal adrenal weight were reflected histologically in parallel changes in the size of adrenocortical cells. The observations suggest that the fetal adrenal hypertrophy following maternal treatment with metopirone can occur to some extent independent of the fetal brain, but that close to the end of gestation the hypertrophy occurs completely under the control of the fetal brain.     

Action in vivo and in vitro of 2-tetradecylglycidic acid, 2-tetradecylglycidyl-CoA and 2-tetradecylglycidylcarnitine on hepatic carnitine palmitoyltransferase.

The effects of 2-tetradecylglycidic acid (TDGA), TDGA-CoA and TDGA-carnitine were examined in purified hepatic CPT (carnitine palmitoyltransferase) and in hepatic mitochondria and inverted submitochondrial vesicles derived from Sprague-Dawley rats. Since TDGA has been reported as a specific inhibitor of carnitine palmitoyltransferase-A (CPT-A), the focus was on kinetics and inhibition of CPT-A, and the relationship of this key enzyme to beta-oxidation. After administration of TDGA in vivo to overnight-starved rats, the Vmax. of CPT in intact mitochondria and in inverted vesicles (CPT-B) was depressed by 66%. The S0.5 for palmitoyl-CoA and Km for carnitine were unchanged. The I50 (concn. giving 50% inhibition) for malonyl-CoA was significantly increased from 20 to 141 microM in intact mitochondria, but unchanged (199 versus 268 microM) in inverted vesicles. The addition in vitro of TDGA-CoA (0-1.0 microM) gave I50 values of 0.29 and 0.27 microM (S.E.M. = 0.19) in intact mitochondria from fed and 48 h-starved rats, and 0.81 and 1.57 microM (S.E.M. = 0.29) for inverted vesicles derived from fed and starved rats. Addition in vitro of TDGA-carnitine to mitochondria from starved rats yielded an I50 value of 27.7 mM (S.E.M. = 12.2) for L-[methyl-14C]carnitine release from palmitoyl-L-[methyl-14C]carnitine and 0.64 mM (S.E.M. = 0.07) for palmitoyl-L-[methyl-14C]carnitine formation from L-[methyl-14C]carnitine in intact mitochondria. Inverted vesicles were not measurably sensitive to TDGA-carnitine up to 500 microM for the assay of L-[methyl-14C]carnitine release, but were as sensitive as intact mitochondria when inhibition was determined in the direction of palmitoyl-L-[methyl-14C]carnitine formation (I50 = 0.54 +/- 0.07 microM). When TDGA-CoA was added to intact mitochondria, then incubated for 5 min at room temperature and subsequently washed out, Vmax. of CPT decreased from 5.8 to 3.5 (S.E.M. = 0.6) in intact mitochondria, and from 17.2 to 6.3 (S.E.M. = 4.8) in inverted vesicles. The Km for L-carnitine and the S0.5 for palmitoyl-CoA increased 2-fold with TDGA-CoA pretreatment in both intact mitochondria and inverted vesicles. Detergent solubilization (0.05% Triton X-100) resulted in a complete loss of TDGA-CoA sensitivity (up to 1.0 microM measured). Sonicated mitochondria exhibited an I50 of 0.72 +/- 0.03 microM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.     

Biosynthesis and immunocytochemical localization of an estrogen-dependent glycoprotein and associated morphological alterations in the sheep ampulla oviduct.

Published reports have shown that an M(r) 90,000-92,000 protein is released into the oviductal lumen of the sheep, during estrus at a time corresponding to ovulation and fertilization, where it associates with the embryo. The objectives of this study were (a) to determine whether estradiol-17 beta (E) alone or in combination with progesterone (P) induces the synthesis of the M(r) 90,000-92,000 protein from the ampulla and/or isthmus oviduct; (b) to monitor structural alterations in oviductal epithelial cells associated with the synthesis of this protein; and (c) to generate a polyclonal antiserum to the protein and use the antiserum to verify its cellular location and tissue specificity. Oviductal flushings and explant culture media were obtained from ovariectomized animals treated with E alone or with E plus P. The M(r) 90,000-92,000 protein was present in 3H-leucine- and 3H-glucosamine-labeled culture media of the ampulla (not isthmus) oviduct in animals treated with E alone or with E plus P. The glycoprotein was detected in gels of oviductal flushings obtained from animals treated only with E. A specific polyclonal antiserum to the protein was made and cross-reacted on Western blots of oviductal flushings from E-treated animals and ampulla (not isthmus) oviduct culture media from animals treated with E alone or with E plus P. The secretory apparatus of the epithelial cells of the ampulla oviduct matured and differentiated in response to E. Light and electron microscopic immunocytochemistry localized the M(r) 90,000-92,000 glycoprotein to secretory granules in the nonciliated cells of the ampulla oviduct. Immunoperoxidase reaction product was absent in tissue sections and Western blots of other reproductive and nonreproductive tract tissues obtained from steroid-treated animals. Therefore, the secretory cells of the ampulla oviduct of the sheep synthesize and release an E-induced, oviduct-derived M(r) 90,000-92,000 glycoprotein.     

Embryonic loss in pony mares induced by intrauterine infusion of Candida parapsilosis

Pony mares which were detected pregnant by transrectal ultrasonography received a single intrauterine infusion of either sterile saline (control, n = 12 mares) or 10(6)Candida parapsilosis (treated, n = 12 mares) between Days 11 to 14 postovulation. Subsequent embryonic loss was studied by daily ultrasonography of the mare's uterus, by serum progesterone levels, by endometrial swabs for cytologic and microbiologic examination and by endometrial biopsies that were taken after embryonic loss was detected. Significantly fewer (P<0.01) embryonic losses occurred in control than in treated mares (4 12 vs 12 12 ). The mean interval from intrauterine infusion until embryonic loss was 5.8 +/- 2.8 d for control mares (n = 4) and 2.1 +/- 0.2 d for treated mares (n = 12). Prior to embryonic loss, moderate to marked edema of the endometrial folds in 12 of 12 treated mares and free fluid in the uterine lumen of 5 of 12 treated mares were detected by ultrasonography. After embryonic loss, Candida parapsilosis was cultured from the uteri of 8 of 12 treated mares, and E . coli was cultured from the uteri of 2 of 4 control mares. Postloss endometrial smears had cytologic evidence of inflammation in 10 of 12 treated mares and 3 of 4 control mares. Intrauterine inoculation of C. parapsilosis consistently induced embryonic loss and may provide a basis to further study the relationship between endometritis and embryonic loss in mares.     

Passive serum therapy with polyclonal antibodies against Mycobacterium tuberculosis protects against post-chemotherapy relapse of tuberculosis infection in SCID mice

We investigated the protective role of immune-sera against reactivation of Mycobacterium tuberculosis infection in SCID mice and found that passive immunization with sera obtained from mice treated with detoxified M. tuberculosis extracts (delivered in liposomes in a composition known as RUTI) exerted significant protection. Our SCID mouse model consisted of aerosol infection by M. tuberculosis, followed by 3 to 8weeks of chemotherapy with isoniazid+rifampicin (INH+RIF) (25 and 10mg/kg, respectively). After infection and antibiotic administration, two groups of mice were treated for up to 10weeks with intraperitoneal passive immunization using hyperimmune serum (HS) obtained from mice infected with M. tuberculosis, treated with chemotherapy (INH+RIF) for 8weeks and inoculated with RUTI (HS group) or with normal serum (CT group). Significant differences were found between HS and CT groups in the number of bacilli in the lungs (3.68+/-2.02 vs. 5.72+/-1.41log(10) c.f.u.), extent of pulmonary granulomatomous infiltration (10.33+/-0.67 vs. 31.2+/-1.77%), and percentage of animals without pulmonary abscesses (16.7% vs. 45.5%). These data strongly suggest a protective role of specific antibodies against lung dissemination of M. tuberculosis infection.     

Progesterone in vitro stimulates secretion of a specific protein by ovine epithelial endometrial cells

Cultured ovine epithelial endometrial cells from oestrogen-treated ovariectomized ewes were treated in vitro with combinations of oestradiol-17 beta (E), progesterone (P) and the P-receptor antagonist RU486 (each 10(-6) to 10(-9) M), in the presence of [35S]methionine. Neither DNA content of dishes nor total protein were increased in treatment compared with control dishes. Incorporation of [35S] into secreted protein was lower from cells treated in vitro with P or E + P (10(-9) M) than from those treated with E (10(-9) M, P less than 0.01). Incorporation of [35S] into cellular protein was decreased by P (10(-9) M, P less than 0.025). SDS-PAGE analysis of secreted proteins enabled measurement of levels of a 46K protein which is secreted maximally following E + P administration in vivo. In vitro, P either alone or with E (each 10(-7) M) increased the abundance of the 46K protein in cell secretions by a factor of 1.5 +/- 0.1 (N = 9) or 1.8 +/- 0.3 (N = 10) respectively (P less than 0.01) compared with controls. The administration of E (10(-7) M) or either or both steroids at 10(-9) M, was without effect. RU486 alone (10(-6) to 10(-8) M) was also without effect but in the presence of E + P or P, blocked the increase in the 46K protein, suggesting this effect is mediated via binding of P to its receptor.     

Growth fraction of myelocytes in normal human granulopoiesis

The labelling index (LI) of myelocytes (M) after flash labelling of normal human bone marrow cells with [3H]-thymidine ([3H]TdR) is always lower that the LI obtained for myeloblasts (MB) and for promyelocytes (PM). This fact can be interpreted in two ways: it may mean that the duration of the G1 phase of the cell cycle is longer in M than in MB or PM, or it may mean that the proportion of cells in cycle, i.e., the growth fraction (GF), is lower in the M population than in MB or PM. The evolution of the LI and of the mean number of grains per cell was monitored in [3H]TdR-labelled normal bone marrow during in vitro incubation for 50 hr. The generation time, measured by the halving time of the mean number of grains per cell after flash labelling, was similar for M to that for MB and PM. During continuous labelling, the LI of MB and PM reached 1 and the LI value for M never rose to more than 50% of the values recorded for MB and PM after 30 hr. These findings give support to the second hypothesis, i.e., a lower GF in the M population. Good correlation was found between the LI of M and the proportion of mature polymorphonuclear cells in the bone marrow of normal subjects and of patients with chronic benign neutropenia or hyperleucocytosis. Variations in the M growth fraction could be a medium-term (2-3 days) regulatory factor in granulocyte production.     

Grading follicular lymphoma on fine needle aspiration specimens. Comparison with proliferative index by DNA image analysis and Ki-67 labeling index

OBJECTIVE: To determine whether follicular lymphoma (FL) can be graded on fine needle aspiration (FNA) biopsies by determining the percentage of centroblasts in the neoplastic follicles on the smears. STUDY DESIGN: Eighty-nine cases of histologically confirmed cases of FL, including 31 grade 1, 46 grade 2 and 12 grade 3, were evaluated. Proliferative index (PI) by DNA image analysis (DIA) and Ki-67 labeling index (LI) were obtained on all cases. A minimum of 200 cells were counted per case (range, 200-800 cells) at 40x magnification, and the number of large cells (centroblasts) was expressed as a percentage of the total number of cells counted within the follicles. RESULTS: The percentage of centroblasts in the follicular aggregates was 9.7 +/- 2.9% in grade 1 FLs, 24.7 +/- 5.6% in grade 2 and 48.4 +/- 7.5% in grade 3. These differences were significant (P < .05). DNA image analysis of PI and Ki-67 LI differed significantly between grade 1 FLs and grade 2 and 3 FLs (P < .05), but there were no significant differences between grade 2 and 3 FLs. CONCLUSION: Determining the percentage of centroblasts in the follicular aggregates on FNA specimens is a good method of grading FLs. Using the percentage of centroblasts per follicular structure, FL grades 1, 2 and 3 were adequately distinguished. PI by DIA and Ki-67 LI clearly distinguished FL grade 1 from FL grades 2 and 3; however, it did not clearly distinguish between grades 2 and 3.     

Bile salt hydrolase, the member of Ntn-hydrolase family: differential modes of structural and functional transitions during denaturation

Conformational transitions and functional stability of the bile salt hydrolase (BSH; cholylglycine EC: 3.5.1.24) from Bifidobacterium longum (BlBSH) cloned and expressed in E. coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and CD spectroscopy. Thermal and Gdn-HCl-mediated denaturation of BlBSH is a multistep process of inactivation and unfolding. The inactivation and unfolding of the enzyme was found to be irreversible. Enzyme activity seems sensitive to even minor conformational changes at the active site. Thermal denaturation as such did not result in any insoluble protein aggregates. However, on treating with 0.25 - 1 M Gdn-HCl the enzyme showed increasing aggregation at temperatures of 40 - 55 degrees C indicating more complex structural changes taking place in the presence of chemical denaturants. The enzyme secondary structure was still intact at acidic pH (pH 1 - 3). The perturbation in the tertiary structure at the acidic pH was detected through freshly formed solvent exposed hydrophobic patches on the enzyme. These changes could be due to the formation of an acid-induced molten globule-like state.     

Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.