Monolignol ferulate transferase introduces chemically labile linkages into the lignin backbone.

Redesigning lignin, the aromatic polymer fortifying plant cell walls, to be more amenable to chemical depolymerization can lower the energy required for industrial processing. We have engineered poplar trees to introduce ester linkages into the lignin polymer backbone by augmenting the monomer pool with monolignol ferulate conjugates. Herein, we describe the isolation of a transferase gene capable of forming these conjugates and its xylem-specific introduction into poplar. Enzyme kinetics, in planta expression, lignin structural analysis, and improved cell wall digestibility after mild alkaline pretreatment demonstrate that these trees produce the monolignol ferulate conjugates, export them to the wall, and use them during lignification. Tailoring plants to use such conjugates during cell wall biosynthesis is a promising way to produce plants that are designed for deconstruction.

Characterization of a family of Arabidopsis genes related to xyloglucan fucosyltransferase1.

To understand primary cell wall assembly in Arabidopsis, we have focused on identifying and characterizing enzymes involved in xyloglucan biosynthesis. Nine genes (AtFUT2-10) were identified that share between 47% and 62% amino acid similarity with the xyloglucan-specific fucosyltransferase AtFUT1. Reverse transcriptase-PCR analysis indicates that all these genes are expressed. Bioinformatic analysis predicts that these family members are fucosyltransferases, and we first hypothesized that some may also be involved in xyloglucan biosynthesis. AtFUT3, AtFUT4, and AtFUT5 were expressed in tobacco (Nicotiana tabacum L. cv BY2) suspension culture cells, and the resulting proteins did not transfer fucose (Fuc) from GDP-Fuc to tamarind xyloglucan. AtFUT3, AtFUT4, and AtFUT5 were overexpressed in Arabidopsis plants. Leaves of plants overexpressing AtFUT4 or AtFUT5 contained more Fuc than wild-type plants. Stems of plants overexpressing AtFUT4 or AtFUT5 contained more xylose, less arabinose, and less galactose than wild-type plants. We suggest that the AtFUT family is likely to include fucosyltransferases important for the synthesis of wall carbohydrates. A targeted analysis of isolated cell wall matrix components from plants altered in expression of these proteins will help determine their specificity and biological function.

Identification and analysis of Arabidopsis expressed sequence tags characteristic of non-coding RNAs.

Sequencing of the Arabidopsis genome has led to the identification of thousands of new putative genes based on the predicted proteins they encode. Genes encoding tRNAs, ribosomal RNAs, and small nucleolar RNAs have also been annotated; however, a potentially important class of genes has largely escaped previous annotation efforts. These genes correspond to RNAs that lack significant open reading frames and encode RNA as their final product. Accumulating evidence indicates that such "non-coding RNAs" (ncRNAs) can play critical roles in a wide range of cellular processes, including chromosomal silencing, transcriptional regulation, developmental control, and responses to stress. Approximately 15 putative Arabidopsis ncRNAs have been reported in the literature or have been annotated. Although several have homologs in other plant species, all appear to be plant specific, with the exception of signal recognition particle RNA. Conversely, none of the ncRNAs reported from yeast or animal systems have homologs in Arabidopsis or other plants. To identify additional genes that are likely to encode ncRNAs, we used computational tools to filter protein-coding genes from genes corresponding to 20,000 expressed sequence tag clones. Using this strategy, we identified 19 clones with characteristics of ncRNAs, nine putative peptide-coding RNAs with open reading frames smaller than 100 amino acids, and 11 that could not be differentiated between the two categories. Again, none of these clones had homologs outside the plant kingdom, suggesting that most Arabidopsis ncRNAs are likely plant specific. These data indicate that ncRNAs represent a significant and underdeveloped aspect of Arabidopsis genomics that deserves further study.

Real-time analysis of methanol in air and water by membrane introduction mass spectrometry.

We present results for the near-real-time, on-line detection of methanol in both air and water using membrane introduction mass spectrometry (MIMS). In these experiments, we compare the sensitivity of a poly(dimethylsiloxane) (PDMS) membrane and an allyl alcohol (AA) membrane to the detection of methanol. In MIMS, the membrane serves as the interface between the sample and the vacuum of the mass spectrometer. Membrane-diffused water was used as the reagent ion (H3O+) for chemical ionization of methanol in an ion trap mass spectrometer. Linear calibration curves have been obtained for methanol using both PDMS and AA membranes. For PDMS, detection limits of methanol are 14 ppmv and 5 ppm in air and water, respectively. For AA, detection limits are 3.3 ppmv and 2 ppm in air and water, respectively. We demonstrate that the sensitivity of the analysis can be altered by the chemistry of the membrane. When the AA membrane is used, the sensitivity of MIMS is enhanced over that of PDMS by a factor of 8.5 for methanol in air and by a factor of 23.4 for methanol in water.

Golgi enzymes that synthesize plant cell wall polysaccharides: finding and evaluating candidates in the genomic era.

Although the synthesis of cell wall polysaccharides is a critical process during plant cell growth and differentiation, many of the wall biosynthetic genes have not yet been identified. This review focuses on the synthesis of noncellulosic matrix polysaccharides formed in the Golgi apparatus. Our consideration is limited to two types of plant cell wall biosynthetic enzymes: glycan synthases and glycosyltransferases. Classical means of identifying these enzymes and the genes that encode them rely on biochemical purification of enzyme activity to obtain amino acid sequence data that is then used to identify the corresponding gene. This type of approach is difficult, especially when acceptor substrates for activity assays are unavailable, as is the case for many enzymes. However, bioinformatics and functional genomics provide powerful alternative means of identifying and evaluating candidate genes. Database searches using various strategies and expression profiling can identify candidate genes. The involvement of these genes in wall biosynthesis can be evaluated using genetic, reverse genetic, biochemical, and heterologous expression methods. Recent advances using these methods are considered in this review.

The unique catalytic subunit of sperm cAMP-dependent protein kinase is the product of an alternative Calpha mRNA expressed specifically in spermatogenic cells.

cAMP-dependent protein kinase has a central role in the control of mammalian sperm capacitation and motility. Previous protein biochemical studies indicated that the only cAMP-dependent protein kinase catalytic subunit (C) in ovine sperm is an unusual isoform, termed C(s), whose amino terminus differs from those of published C isoforms of other species. Isolation and sequencing of cDNA clones encoding ovine C(s) and Calpha1 (the predominant somatic isoform) now reveal that C(s) is the product of an alternative transcript of the Calpha gene. C(s) cDNA clones from murine and human testes also were isolated and sequenced, indicating that C(s) is of ancient origin and widespread in mammals. In the mouse, C(s) transcripts were detected only in testis and not in any other tissue examined, including ciliated tissues and ovaries. Finally, immunohistochemistry of the testis shows that C(s) first appears in pachytene spermatocytes. This is the first demonstration of a cell type-specific expression for any C isoform. The conservation of C(s) throughout mammalian evolution suggests that the unique structure of C(s) is important in the subunit's localization or function within the sperm.

Biochemical characterization and molecular cloning of an alpha-1,2-fucosyltransferase that catalyzes the last step of cell wall xyloglucan biosynthesis in pea.

Pea microsomes contain an alpha-fucosyltransferase that incorporates fucose from GDP-fucose into xyloglucan, adding it preferentially to the 2-O-position of the galactosyl residue closest to the reducing end of the repeating subunit. This enzyme was solubilized with detergent and purified by affinity chromatography on GDP-hexanolamine-agarose followed by gel filtration. By utilizing peptide sequences obtained from the purified enzyme, a cDNA clone was isolated that encodes a 565-amino acid protein with a predicted molecular mass of 64 kDa and shows 62.3% identity to its Arabidopsis homolog. The purified transferase migrates at approximately 63 kDa by SDS-polyacrylamide gel electrophoresis but elutes from the gel filtration column as an active protein of higher molecular weight ( approximately 250 kDa), indicating that the active form is an oligomer. The enzyme is specific for xyloglucan and is inhibited by xyloglucan oligosaccharides and by the by-product GDP. The enzyme has a neutral pH optimum and does not require divalent ions. Kinetic analysis indicates that GDP-fucose and xyloglucan associate with the enzyme in a random order. N-Ethylmaleimide, a cysteine-specific modifying reagent, had little effect on activity, although several other amino acid-modifying reagents strongly inhibited activity.

Serum leptin during recovery following maximal incremental and prolonged exercise.

This study investigated the delayed circulating leptin response to maximal and prolonged treadmill exercise. Six healthy untrained males performed three sessions after an overnight fast: control, maximal exercise, and prolonged exercise at 50% of maximal oxygen consumption. Blood samples were collected prior to exercise, at the end of exercise, and at 60, 120, 180, and 240 min following exercise and control sessions. Blood samples were analyzed for serum leptin, insulin, glucose, free fatty acids, and glycerol. Hemoglobin and hematocrit were measured to correct for plasma volume changes. Resting energy expenditure (REE) and body fat (BF) were also assessed. Immediately at the end of maximal and prolonged exercise, and during the 4 hours of recovery, serum leptin levels did not change significantly compared to their respective baseline values. At 240 min of recovery serum leptin decreased 7% and 9% (p>0.05) from the baseline in the maximal and prolonged sessions, respectively. In the control experiment serum leptin decreased 27% from the baseline at 240 min of the recovery (p < 0.05). No significant differences were found in leptin values between the control and exercise sessions. Control serum leptin was positively correlated (p < 0.05) to BF (r = 0.88) and glucose (r=0.96), and negatively correlated to REE (r= -0.81). In conclusion, maximal or prolonged exercise do not appear to have an influence on circulating serum leptin in the delayed (4 hr) post exercise recovery period.

Comparison of ropivacaine 0.2% and lidocaine 0.5% for intravenous regional anesthesia in volunteers.

UNLABELLED: A longer acting local anesthetic such as ropivacaine may offer advantages over lidocaine for IV regional anesthesia (IVRA). The objective of this investigation was to determine whether the use of ropivacaine improves the quality and duration of IVRA. In a randomized, double cross-over design, 10 volunteers received lidocaine 0.5% or ropivacaine 0.2% for IVRA of the upper extremity on two separate days with a standard double-cuff technique. Sensation to pinprick, response to tetanic stimuli, and tourniquet pain were assessed on a 0-10 verbal numeric score scale at 5-min intervals throughout the period of tourniquet inflation. Motor function was evaluated by grip strength. After release of the second (distal) cuff, pinprick sensation, motor strength, and systemic side effects were evaluated at 3, 10, and 30 min. No significant differences were observed for onset times of anesthesia and times to proximal (38 +/- 3 and 36 +/- 3 min) or distal (34 +/- 13 and 36 +/- 13 min) tourniquet release after the administration of ropivacaine and lidocaine, respectively. However, postdeflation hypoalgesia and motor blockade were prolonged with ropivacaine, and postdeflation light-headedness, tinnitus, and drowsiness were more prominent with lidocaine. We conclude that ropivacaine may be an alternative to lidocaine for IVRA. It may result in prolonged analgesia and fewer side effects after tourniquet release. IMPLICATIONS: In this study, volunteers received lidocaine 0.5% or ropivacaine 0.2% for IV regional anesthesia on two study days. Ropivacaine and lidocaine provided similar surgical conditions. However, after release of the distal tourniquet, prolonged sensory blockade and fewer central nervous system side effects were observed with ropivacaine.

Toxicity and dose-response studies of 1,25-(OH)2-16-ene-23-yne vitamin D3 in transgenic mice.

The vitamin D3 analogue 1,25-(OH)2-16-ene-23-yne vitamin D3 (16,23-D3) in doses with low systemic toxicity has been demonstrated to inhibit retinoblastoma growth in transgenic mice. This study examines the dose-dependent response for inhibition of tumor growth in transgenic mice with retinoblastoma and evaluates the in vivo toxicity of 16,23-D3 in nontransgenic mice. Transgenic 8-10-week-old mice with retinoblastoma (n = 119) were randomly assigned to groups receiving 1.0, 0.75, 0.5, 0.35, 0.2, or 0.05 microg of 16,23-D3 and a vehicle alone (control) group i.p. five times a week for 5 weeks. An additional control group received no injection. Eyes were enucleated one week after the end of treatment, and tumor areas were measured. To determine the toxic dose, transgene-negative littermates received 0.5, 1.0, 1.5, 2.5, 3.5, 4.5, or 5.0 microg of 16,23-D3, and control groups received vehicle alone, 5 days a week for 5 weeks. Serum calcium levels were measured, and necropsies were performed on animals from each group. In the dose-response study, tumor growth inhibition was greatest in the group receiving 0.35 microg (55% inhibition; P = 0.0056) and was also significant in the group receiving 0.5 microg (42% inhibition; P = 0.036). The systemic toxic effects due to hypercalcemia occurred at doses of > or =1.0 microg. 16,23-D3 inhibits tumor growth at doses > or =0.35 microg and shows toxic effects at doses > or =1.0 microg related to hypercalcemia in mice fed an unrestricted diet. No toxicity was observed with lower doses.

A dynein light chain is essential for the retrograde particle movement of intraflagellar transport (IFT).

Several enzymes, including cytoplasmic and flagellar outer arm dynein, share an Mr 8,000 light chain termed LC8. The function of this chain is unknown, but it is highly conserved between a wide variety of organisms. We have identified deletion alleles of the gene (fla14) encoding this protein in Chlamydomonas reinhardtii. These mutants have short, immotile flagella with deficiencies in radial spokes, in the inner and outer arms, and in the beak-like projections in the B tubule of the outer doublet microtubules. Most dramatically, the space between the doublet microtubules and the flagellar membrane contains an unusually high number of rafts, the particles translocated by intraflagellar transport (IFT) (Kozminski, K.G., P.L. Beech, and J.L. Rosenbaum. 1995. J. Cell Biol. 131:1517-1527). IFT is a rapid bidirectional movement of rafts under the flagellar membrane along axonemal microtubules. Anterograde IFT is dependent on a kinesin whereas the motor for retrograde IFT is unknown. Anterograde IFT is normal in the LC8 mutants but retrograde IFT is absent; this undoubtedly accounts for the accumulation of rafts in the flagellum. This is the first mutation shown to specifically affect retrograde IFT; the fact that LC8 loss affects retrograde IFT strongly suggests that cytoplasmic dynein is the motor that drives this process. Concomitant with the accumulation of rafts, LC8 mutants accumulate proteins that are components of the 15-16S IFT complexes (Cole, D.G., D.R. Deiner, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008), confirming that these complexes are subunits of the rafts. Polystyrene microbeads are still translocated on the surface of the flagella of LC8 mutants, indicating that the motor for flagellar surface motility is different than the motor for retrograde IFT.

The Chlamydomonas reinhardtii ODA3 gene encodes a protein of the outer dynein arm docking complex.

We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the uter ynein rm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737- 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83. 4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme.

Functional interaction between Chlamydomonas outer arm dynein subunits: the gamma subunit suppresses the ATPase activity of the alpha beta dimer.

The alpha beta dimer and the gamma subunit of the Chlamydomonas outer arm dynein were solubilized by treating isolated axonemes with 0.6 M KCI, and purified by sucrose density gradient centrifugation. The axonemes were from an ida1 mutant to eliminate contamination of outer arm subunits by inner arm dynein 11, and the axonemes were pre-extracted with 0.6 M CH3COOK to remove non-dynein protein that might otherwise contaminate outer arm dynein fractions in the sucrose gradient. In addition, purer fractions of outer arm dynein subunits were obtained by modifying the centrifugation conditions to take advantage of the propensity of the dynein to dissociate under high hydrostatic pressure in the presence of Mg2+. When sucrose gradient fractions containing the gamma subunit were added to a fraction containing the purified alpha beta dimer under conditions expected to promote reassociation of the subunits to form a trimeric outer arm dynein complex [Takada et al., 1992: J. Biochem, 111:758-762], the total ATPase activity of the mixture was suppressed to a level lower than that of the original alpha beta dimer fraction. The inhibition paralleled the distribution of gamma subunit in the sucrose gradient, was saturable, and was maximum at an approximately equimolar ratio of the gamma subunit to the alpha beta dimer. These results indicate that when the gamma subunit interacts with the alpha beta dimer, the latter's ATPase activity is modulated downward. Previous results showed that interaction of the alpha subunit with the beta subunit suppressed the beta subunit's ATPase activity [Pfister and Witman, 1984: J. Biol. Chem. 259:12072-12080]. Thus, the total ATPase activity of the outer arm dynein is dependent upon communication between all three subunits within the arm.

Melanocytes and iris color. Light microscopic findings.

OBJECTIVE: To systematically evaluate morphologic differences in iris stroma that contribute to clinically perceptible differences in iris color, using immunohistochemical identification of stromal melanocytes and fluorescence microscopy. METHODS: Paraffin-embedded sections from 51 human irides were stained with S100a and fluorescein isothiocyanate. Cells were counted and scored as melanocytes or other. Melanocyte number, proportion, and density were determined for light-colored (blue), medium-colored (hazel) and dark-colored (brown) irides and compared. RESULTS: No statistically significant difference was observed for mean total cellularity or mean melanocyte number among the three color groups. Mean total stromal cell count was 1177 +/- 259 (mean +/- SEM), and mean melanocyte number was 778 +/- 196 per 5-micrometer section. In human irides, 65.9% of the iris stroma is composed of melanocytes. Melanocyte density (number of cells per square millimeter) is not related to iris color. CONCLUSION: The number of melanocytes, the proportion of melanocytes, and iris stromal cellularity are not major contributors to iris color.

A Comparison of Cryofocusing Injectors for Gas Sampling and Analysis in Fast GC.

The performance of two cryofocusing injectors for fast gas chromatography has been studied. The first system traps analytes onto bare metal tubes and rapidly vaporizes them upon ballistically heating the tube using a capacitance discharge. The second is a microloop injector in which analytes are cryotrapped onto short lengths of narrow-bore fused silica tubing with various coatings. The ballistically heated injector is capable of sampling and injecting compounds from air faster than the microloop system, because the metal tube can be heated and cooled more rapidly. Both systems are capable of cryotrapping compounds as volatile as butane at -90 °C, and the microloop system can trap ethane when a section of a porous layer open-tubular (PLOT) column is used as the sample loop. In addition, the microloop injector can be used without cryointegration to analyze compounds regardless of their volatility, as long as they are present in the samples at detectable concentrations. Because the ballistically heated injector is flushed prior to injection, it can introduce only compounds that are adsorbed onto its metal trap. Comparison of chromatograms obtained using the two injectors show similar chromatographic resolution. Both traps are susceptible to freezing during the cryotrapping step, but the use of an inline Nafion dryer allows air saturated with water vapor to be sampled using both systems for 3 min without plugging the trap. Thermal decomposition during the injection step can occur for labile species in the ballistically heated trap, but even the highly unstable compound ethyl diazoacetate may be injected without breakdown in the microloop system.

The 78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein.

A previous study (King et al., 1991. J. Biol. Chem. 266:8401-8407) showed that the 78,000-M(r) intermediate chain (IC78) from the Chlamydomonas outer arm dynein is in direct contact with alpha-tubulin in situ, suggesting that this protein may be involved in binding the dynein to the doublet microtubules. Molecular genetic analysis of this chain recently demonstrated that it is a WD repeat protein essential for outer arm assembly (Wilkerson et al., 1995.J. Cell Biol. 129:169-178). We have now transcribed and translated IC78 in vitro, and demonstrate that this molecule binds axonemes and microtubules, whereas a homologous protein (the 69,000-M(r) intermediate chain [IC69] of Chlamydomonas outer arm dynein) does not. Thus, IC78 is a bona fide microtubule-binding protein. Taken together with the previous results, these findings indicate that IC78 is likely to provide at least some of the adhesive force that holds the dynein to the doublet microtubule, and support the general hypothesis that the dynein intermediate chains are involved in targeting different dyneins to the specific cell organelles with which they associate. Analysis of the binding activities of various IC78 deletion constructs translated in vitro identified discrete regions of IC78 that affected the binding to microtubules; two of these regions are specifically missing in IC69. Previous studies also showed that IC78 is in direct contact with IC69; the current work indicates that the region of IC78 that mediates this interaction is coincident with two of IC78's WD repeats. This supports the hypothesis that these repeats are involved in protein-protein interactions within the dynein complex.

Interspecies conservation of outer arm dynein intermediate chain sequences defines two intermediate chain subclasses.

Immunological analysis showed that antibodies against the intermediate chains (ICs) IC2 and IC3 of sea urchin outer arm dynein specifically cross-reacted with intermediate chains IC78 and IC69, respectively, of Chlamydomonas outer arm dynein. In contrast, no specific cross-reactivity with any Chlamydomonas outer arm polypeptide was observed using antibody against IC1 of sea urchin outer arm dynein. To learn more about the relationships between the different ICs, overlapping cDNAs encoding all of IC2 and IC3 of sea urchin were isolated and sequenced. Comparison of these sequences with those previously obtained for the Chlamydomonas ICs revealed that, although all four chains are homologous, sea urchin IC2 is much more closely related to Chlamydomonas IC78 (45.8% identity), and sea urchin IC3 is much more closely related to Chlamydomonas IC69 (48.5% identity), than either sea urchin chain is related to the other (23.5% identity). For homologous pairs, the similarities extend throughout the full lengths of the chains. Regions of similarity between all four ICs and the IC (IC74) of cytoplasmic dynein, located in the C-terminal halves of the chains, are due primarily to conservation of the WD repeats present in all of these ICs. This is the first demonstration that structural differences between individual ICs within an outer arm dynein have been highly conserved in the dyneins of distantly related species. The results provide a basis for the subclassification of these chains.

The 78,000 M(r) intermediate chain of Chlamydomonas outer arm dynein isa WD-repeat protein required for arm assembly.

We have isolated and sequenced a full-length cDNA clone encoding the 78,000 Mr intermediate chain (IC78) of the Chlamydomonas outer arm dynein. This protein previously was shown to be located at the base of the solubilized dynein particle and to interact with alpha tubulin in situ, suggesting that it may be involved in binding the outer arm to the doublet microtubule. The sequence predicts a polypeptide of 683 amino acids having a mass of 76.5 kD. Sequence comparison indicates that IC78 is homologous to the 69,000 M(r) intermediate chain (IC69) of Chlamydomonas outer arm dynein and to the 74,000 M(r) intermediate chain (IC74) of cytoplasmic dynein. The similarity between the chains is greatest in their COOH-terminal halves; the NH(2)-terminal halves are highly divergent. The COOH-terminal half of IC78 contains six short imperfect repeats, termed WD repeats, that are thought to be involved in protein-protein interactions. Although not previously reported, these repeated elements also are present in IC69 and IC74. Using the IC78 cDNA as a probe, we screened a group of slow-swimming insertional mutants and identified one which has a large insertion in the IC78 gene and seven in which the IC78 gene is completely deleted. Electron microscopy of three of these IC78 mutants revealed that each is missing the outer arm, indicating that IC78 is essential for arm assembly or attachment to the outer doublet. Restriction fragment length polymorphism mapping places the IC78 gene on the left arm of chromosome XII/XIII, at or near the mutation oda9, which also causes loss of the outer arm. Mutants with defects in the IC78 gene do not complement the oda9 mutation in stable diploids, strongly suggesting that ODA9 is the structural gene for IC78.

Potential use of buccal smears for rapid diagnosis of autosomal trisomy or chromosomal sex in newborn infants using DNA probes.

Buccal smears from 3 women and 1 man were probed with alpha satellite DNA probes for chromosomes 8, 18, X, and Y. Buccal smears were also collected from an adolescent phenotypic female with uterine agenesis, as well as from newborn infants with suspected trisomy 18 and trisomy 21. The clinical cases were confirmed with conventional cytogenetic studies of peripheral lymphocytes. Overall probe efficiency at detecting expected chromosome number in interphase cells was found to be 71% +/- 6.8%. Higher than expected n-1 signal numbers may be due to karyopyknotic intermediate epithelial cells present in all collected samples. Overall probe efficiency was found to be consistent using alpha satellite and cosmid probes, both of which accurately reflected the modal copy number of the target chromosomes. False trisomy was less than 1%. This study suggests DNA probes can be used in buccal smears for rapid diagnosis of trisomies and chromosomal sex in newborns, but because of high rates of false hypoploid signals, probed buccal smear specimens may not be accurate at diagnosing mosaicism.

Estrogen induced peroxidase secretion from the endometrial epithelium: a possible function for the luminal enzyme.

Estrogen induced peroxidase (EIP) activity revealed as a diaminobenzidine-H2O2 product in electron micrographs is apparent within cisternae of the RER and in large dilated apical vesicles, of which only a few have been seen to open into the uterine lumen. EIP activity is infrequently present in the trans cisternae of the rather diminutive Golgi complex in uterine epithelial cells from 12-72 h after treatment with estrogen. EIP activity is, however, prominent on the surface of microvilli of epithelial cells and as deposits in the lumen. Desalted uterine fluid (72 h) isolated by rotofor (Biorad) and analysed on isoelectric focusing gels that were stained with the diaminobenzidine-H2O2 reaction, reveal the existence of at least 6 peroxidase isoelectric variants with isoelectric points between pI 3.5 and pI 5.0. In similar preparations doubly-stained by diaminobenzidine-H2O2 and silver, peroxidase positive bands are enhanced, along with other isoelectric variants in the acidic pI range. In SDS-PAGE preparations, five prominent proteins ranging from 29-115 kD are present in 72 h uterine fluid. Luminal EIP coats the surface microvilli of the reproductive tract cells, and of viable spermatozoa incubated in uterine fluid. Peroxidase coated bacteria appear to be in the process of decay, and enzyme activity is present on the surface of most spermatozoa. It is not yet determined if EIP has bactericidal, spermicidal or capacitation functions.