Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans. A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.


Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus- and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Acholeplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, Lactobacillus, Bacillus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.


[Interferon-inducing activity of hydroxybenzylamine derivative]. Interferon-inducing activity of the interferon inductor savrats, an oxybenzylamine derivative of was studied. It was shown on experimental animals that along with its low toxicity, savrats had a high interferon inducing capacity. There were early and late peaks of interferon production (4-8 and 48-96 hours later, respectively) depending on the route of the inductor administration. It was noted that for optimization of the schemes for using interferon inductors, careful choosing of the drug pairs participating in the induction and employment of various routes for administration of the same drug are required.


Immunohistochemical study of nerve fibres with substance P- or calcitonin gene-related peptide-like immunoreactivity in the junctional epithelium of developing rats. The beginning of innervation in the junctional epithelium of maxillary first molars was examined in gingival tissues from 19 to 32-day-old rats. Substance P- or calcitonin gene-related peptide (CGRP)-like immunoreactivity was demonstrated by the avidin-biotin peroxidase complex method. In 19-day-old rats, nerve fibres with substance P- or CGRP-like immunoreactivity were seen in the connective tissue and oral epithelium, but not in the reduced enamel epithelium, which would be transformed into the junctional epithelium. In 21-day-old rats, the fibres with substance P- or CGRP-like immunoreactivity formed a plexus in the oral sulcular epithelium and thin varicose fibres were seen for the first time entering the adjacent reduced enamel epithelium. These fibres also penetrated the middle portion of the reduced enamel epithelium, but did not reach the cuboidal reduced ameloblasts. More nerve fibres had CGRP-like immunoreactivity than substance P-like immunoreactivity. In 23-day-old rats, many fibres with both immunoreactivities were seen in the basal layers of the junctional epithelium, but only a few were seen in its superficial layers. In 28-32-day-old rats, numerous fibres with both immunoreactivities were distributed in the whole junctional epithelium and showed a similar pattern of innervation. For all immunoreactive fibres, the density in the middle portion in the junctional epithelium was the highest. The nerve plexus was formed in the basal layers and some fibres with a varicose appearance were found in the superficial layers.


Pancreatic juice cytology for monitoring pancreatic grafts in the early postoperative period. Thirty-one pancreas transplant recipients were monitored by pancreatic juice cytology in the early postoperative period. An increase in the total amount of cells and, in particular, signs of immunoactivation with the appearance of two or more blast-transformed cells per specimen were taken as evidence of acute rejection. According to these criteria a total of 38 rejection episodes were diagnosed. The first positive cytology appeared after 9 days (mean) and lasted for 2 days (mean). Immunocytochemical analysis of the juice showed increased amounts of CD3+ cells during rejection. When rejection occurred during prophylaxis with antithymocyte globulin, neutrophils were preponderant in the pancreatic juice while during OKT-3 prophylaxis a high percentage of monocytes was a characteristic finding. Antirejection treatment was started when the cytology became positive and all rejection episodes except one were reversed. A decrease in the pancreatic juice amylase activity occurred in 66% of the rejection episodes, but in only 5 of the 38 episodes was the decrease highly significant. No correlation was found between graft rejection and volume excretion of pancreatic juice. There were no persistent or characteristic changes in serum amylase or peripheral white blood cell count at the time of rejection. Graft pancreatitis was diagnosed cytologically in 7 patients, in 5 of whom the grafts were eventually lost.


Relationship between gonadotrophin subunit gene expression, gonadotrophin-releasing hormone receptor content and pituitary and plasma gonadotrophin concentrations during the rebound release of FSH after treatment of ewes with bovine follicular fluid during the luteal phase of the cycle. The modulation of FSH secretion at the beginning and middle of the follicular phase of the cycle represents the key event in the growth and selection of the preovulatory follicle. However, the mechanisms that operate within the pituitary gland to control the increased release of FSH and its subsequent inhibition in vivo remain unclear. Treatment of ewes with bovine follicular fluid (bFF) during the luteal phase has been previously shown to suppress the plasma concentrations of FSH and, following cessation of treatment on day 11, a rebound release of FSH occurs on days 12 and 13. When luteal regression is induced on day 12, this hypersecretion of FSH results in an increase in follicle growth and ovulation rate. To investigate the mechanisms involved in the control of FSH secretion, ewes were treated with twice daily s.c. injections of 5 ml bFF on days 3-11 of the oestrous cycle and luteal regression was induced on day 12 with prostaglandin (PG). The treated ewes and their controls were then killed on day 11 (luteal), or 16 or 32 h after PG and their pituitaries removed and halved. One half was analysed for gonadotrophin and gonadotrophin-releasing hormone (GnRH) receptor content. Total pituitary RNA was extracted from the other half and subjected to Northern analysis using probes for FSH-beta, LH-beta and common alpha subunit. Frequent blood samples were taken and assayed for gonadotrophins. FSH secretion was significantly (P less than 0.01) reduced during bFF treatment throughout the luteal phase and then significantly (P less than 0.01) increased after cessation of treatment, with maximum secretion being reached 18-22h after PG, and then declining towards control values by 32h after PG. A similar pattern of LH secretion was seen after bFF treatment. Pituitary FSH content was significantly (P less than 0.05) reduced by bFF treatment at all stages of the cycle. No difference in the pituitary LH content was seen. The increase in GnRH receptor content after PG in the controls was delayed in the treated animals. Analysis of pituitary mRNA levels revealed that bFF treatment significantly (P less than 0.01) reduced FSH-beta mRNA levels in the luteal phase. Increased levels of FSH-beta, LH-beta and alpha subunit mRNA were seen 16h after PG in the bFF-treated animals, at the time when FSH and LH secretion from the pituitary was near maximum.(ABSTRACT TRUNCATED AT 400 WORDS)


LH receptor RNA and protein levels after hormonal treatment of porcine granulosa cells in primary culture. Granulosa cells were prepared from small follicles (less than 3 mm) from the ovaries of 5-month-old gilts. They were cultured in plastic dishes coated with a synthetic adhesion peptide in a chemically defined medium supplemented with 2% serum substitute. After 3 days of culture, the cells reached confluence and expression of the LH receptor could be stimulated in a hormonally defined medium. LH receptor RNAs were estimated by autoradiography using Northern blots and dot blots of total cell RNA. LH receptor RNAs were hybridized with a homologous 32P-labelled random-primed DNA probe. The LH receptor was measured using 125I-labelled human chorionic gonadotrophin (hCG) as tracer. Northern blots of LH receptor RNAs revealed a predominant signal of 4.4 kb and two less-intense hybridization bands of 7.5 and 1.9 kb. The 4.4 kb band was used for quantification of LH receptor RNAs because it was the most intense and may be attributed to the full-length messenger RNA. In these conditions, after 72 h stimulation, FSH (0.6 nM), insulin (5 micrograms/ml), oestradiol (30 nM) and deoxycorticosterone (0.3 nM) yielded high LH receptor RNA levels (eight times unstimulated cell level), while dibutyryl cyclic AMP (1 mM), cortisol (5.4 nM), thyroxine (100 nM) and epidermal growth factor (16 pM) gave low LH receptor RNA levels (one to five times). However, the respective amounts of the receptor RNA did not give yield to the same proportion of LH receptor for every factor, indicating some post-transcriptional regulations. The kinetic study of the production of the LH receptor obtained in a defined medium supplemented with FSH, oestradiol and insulin showed that the receptor appeared after 48 h of stimulation and reached a maximum of about 7000 receptors per cell at 72 h. The three hybridization bands on Northern blots evolved in parallel and appeared as early as 24 h. They were at maximal level from 24 to 48 h of stimulation. When the granulosa cells were pulse-treated for 2 h with cycloheximide (10 micrograms/ml), they exhibited a transient rise in LH receptor RNA content which was followed by a delayed receptor increase especially at 72 h of stimulation. Taken together, these results indicate that the LH receptor in primary culture of granulosa cells seems to be regulated by different physiological factors both at the transcriptional and the translational levels.


Differential expression of the insulin-like growth factor binding proteins in spontaneously diabetic rats. Diabetes-induced growth retardation in the rodent is associated with both reduced circulating insulin-like growth factor-I (IGF-I) and enhanced levels of inhibitors of somatomedin activity. IGF-binding proteins (IGFBPs) are present in the circulation and tissue fluids and are believed to modulate the actions of IGF-I. Since elevated concentrations of the IGFBPs may contribute to the enhanced somatomedin-inhibitor activity observed in serum from diabetic animals, we have examined the amounts of hepatic IGFBP-1, -2, -3 and -4 mRNA in the spontaneously diabetic BioBreeding/Worcester rat. The study used two types of diabetic animal: mildly diabetic animals, which received suboptimal insulin treatment (0.5-1 U/day) and diabetic animals, which received intensive insulin treatment (3-6 U/day). A significant increase in the amount of IGFBP-1 and IGFBP-2 mRNA was seen 1 month and 3 months after the onset of diabetes. Intensive insulin treatment for 3 weeks normalized the amount of IGFBP-1 mRNA in diabetic rats and resulted in a decrease in IGFBP-2 mRNA. In contrast to the increase in IGFBP-1 and IGFBP-2 mRNA, a significant decrease in IGFBP-3 mRNA was seen in diabetic rats (54.6% of control, P less than 0.0005 and 64.6% of control, P less than 0.005 for 1 and 3 months respectively) and intensive insulin treatment for 3 weeks did not restore the IGFBP-3 mRNA level in diabetic rats. No significant difference in IGFBP-4 mRNA levels was seen in diabetic compared with non-diabetic rats. When serum was analysed by ligand blotting the major finding was a reduction in the 39-42 kDa binding protein. No increase in 29-30 kDa IGFBP in the serum was detected in the diabetic rats. From these studies we conclude that the major change in IGFBPs in mildly hyperglycaemic spontaneously diabetic rats is a decrease in IGFBP-3. The changes in hepatic IGFBP-1 and -2 mRNA do not appear to be of sufficient magnitude to result in an increase in serum concentrations of these binding proteins.


The characterization and expression of ovine insulin-like growth factor-binding protein-2. We have isolated an ovine insulin-like growth factor-binding protein-2 (IGFBP-2) cDNA from an adult sheep cDNA library, to determine the structure of ovine IGFBP-2 and to examine the pattern of IGFBP-2 gene expression in adult sheep tissues. This cDNA had 81, 96 and 87% identity with the rat, bovine and human sequences respectively. The deduced amino acid sequence of the ovine IGFBP-2 showed 86, 95 and 85% homology with the rat, bovine and human peptide sequences respectively. The ovine IGFBP-2 cDNA encoded a precursor protein of 317 amino acids which comprised a 33 residue hydrophobic leader sequence and a 284 residue, 30.9 kDa, mature peptide. The 18 cysteine residues, which are a characteristic feature of IGFBPs, were conserved. Also, an Arg-Gly-Asp (RGD) sequence near the C terminus was present. A single transcript of approximately 1.5 kb was expressed in abundance in selected tissues of an adult sheep, namely liver, kidney, adrenal, pituitary and choroid plexus. Southern blot analysis of ovine genomic DNA with the cDNA probe demonstrated that IGFBP-2 is encoded by a single gene. These findings indicate that the ovine IGFBP-2 protein is similar to that in other species and that, in the adult, the mRNA is expressed only in selected tissues.


Macromolecular prodrugs. XX. Factors influencing model dextranase-mediated depolymerization of dextran derivatives in vitro. Endo-dextranase-mediated depolymerization of dextran and dextran derivatives under various experimental conditions in vitro was determined. By a simultaneous determination of Mn and MW of dextrans treated with the enzyme in aqueous buffer, an initial increase of the polydispersity of the polysaccharide sample was observed, indicating that dextranase cleaved the dextran molecules into chains which differed significantly in length. A pH optimum of 5 for the enzyme action was found. However, in the pH range 5-8, which prevails in the colon, the initial depolymerization rates differed by a factor of less than 2. Dextranase treatment of a dextran sample resulted in a constant increase of the concentration of terminal reducing glucose residues per time unit suggesting, that the initial depolymerization reaction followed zero-order kinetics. For degrees of substitution below 12 the efficacy of dextranase fragmentation of dextran conjugates decreased almost linearly with increasing DS. The chemical nature of the attached drug did not significantly affect the depolymerization rates. Maximally depolymerized dextran derivatives were obtained by the combined action of dextranase and various alpha-glucosidases. Treatment of such solutions with: a) model esterases b) 80% plasma and c) 20% liver homogenate did not give rise to an acceleration of the initial drug regeneration, as compared to identical experiments carried out in pure buffer solution (pH 7.4 and 37 degrees C).


Studies on human porin. VII. The channel properties of the human B-lymphocyte membrane-derived "Porin 31HL" are similar to those of mitochondrial porins. Porin 31HL was isolated and purified from total membrane preparations of a human B-lymphocyte cell line. The protein showed a single band of apparent molecular mass 31 kDa on SDS-PAGE. Reconstitution of the protein into artificial lipid bilayer membranes defines its function as a channel-forming protein. The distribution of single-channel conductances had two maxima of 2.4 and 4.3 nS in 1M KCl. The channel formed by Porin 31HL of human B-lymphocytes was found to be voltage-dependent and switched to ion-permeable substates at membrane voltage larger than 20mV. In the open state the pore exhibited the characteristics of a general diffusion pore because the mobility sequence of the ions inside the pore was similar to that in the bulk aqueous phase. The effective diameter was estimated to be about 1.7 nm. The properties of the low conductance state of the channel were studied in detail. In this state the pore favored the passage of cations, in contrast to the open state which favored anions slightly. Monoclonal antibodies against the N-terminal end of Porin 31HL blocked its reconstitution but had otherwise no influence on the channel properties. This result suggested that the amphipathic alpha-helical structure at the N-terminal end is probably not involved in channel gating. The channel-forming properties of Porin 31HL were compared to those of porins isolated from mitochondrial outer membranes and to those of the "maxi chloride channel" observed in the cytoplasmic membrane of several eukaryotic cells.


Induction and detection of anti-peptide antibody specificity is critically affected by the mode of hapten presentation. C-Terminal cholecystokinin (CCK)-peptides of increasing chain lengths were all linked at their N-termini to the single surface-exposed cysteine residue 107 of yeast iso-1-cytochrome c by the maleimide/thiol reaction. The resulting CCK/cytochrome 1:1 conjugates with the haptenic peptides in the identical protein environment were used to immunize outbred guinea pigs in order to assess the critical size of CCK peptides required for the expression of a CCK-specific epitope and the induction of antibodies not crossreacting with the homologous gastrin sequence. By using standard ELISA techniques with polystyrene-adsorbed antigen to evaluate the specificity of the antisera, none of the conjugates were found to induce anti-CCK antisera not crossreacting with gastrin. However, when the biotinyl-CCK-antigen was immobilized by polystyrene-adsorbed avidin, i.e. via a procedure which assures maximum accessibility of the bound antigen, we were able to demonstrate that with CCK-12 and particularly CCK-13, linked through their N-termini to the carrier, the critical length for the expression and recognition of a CCK-specific epitope was reached. The related polyclonal antisera did not crossreact with the homologous gastrin in the modified ELISA.


Quaternary structure of apolipoprotein E in solution: fluorimetric, chromatographic and immunochemical studies. Self-association and stability in solution of apolipoprotein E (apoE), isolated from human plasma very low density lipoproteins, were studied in the nanomolar concentration range. Equilibrium denaturation of fluorescein-labelled apoE (induced by guanidine hydrochloride) was studied by measurement of fluorescence anisotropy, total fluorescence emission intensity, the shift in wavelength of maximal fluorescence emission, and gel-chromatographic behaviour. The protein denaturation was reversible, displayed biphasic behaviour, and was dependent on the apoE concentration. As measured by fluorescence anisotropy, the kinetics of apoE denaturation in the presence of 6M denaturant were heterogeneous, and the contribution of the long-lived component increased with the apoprotein concentration. The results are in agreement with the following scheme: Oligomer (in aged preparations) in equilibrium with tetramer in equilibrium with native or partially denatured monomer in equilibrium with fully denatured monomer. It is suggested that self-association of individual apoE molecules in solution is due to their lipid-binding domains, and leads to additional stabilization of apoprotein structure. Monoclonal antibody 3D12F11 of the IgG1 subclass bound with high affinity to apoE (Kd = 3.5 +/- 0.5 nM), and had no effect on apoprotein binding to heparin-Sepharose or the apoprotein-induced destabilization of liposomes formed from dipalmitoyl-phosphatidylcholine. This indicates that the epitope to the antibody is localized outside the heparin- and lipid-binding sites of the apoprotein molecule.


Flow cytometric analysis of protease activities in vital cells. The analysis of lysosomal proteases in cell lysates is complicated by pH-dependent and oxidative changes of their activity and complex formation with cytosolic inhibitors. Therefore, new flow cytometric methods were developed for the intracellular measurement of protease activities in viable cells. Intracellular cleavage of substrates such as Z-Arg-Arg-4-trifluoromethylcoumarinyl-7-amide to green fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) in viable neutrophils and monocytes was only detected following phagocytosis of Escherichia coli. A measurement of the cysteine or serine proteinase activities in resting human leukocytes was, however, not possible with AFC derivatives as the overlapping blue fluorescence of the substrates reduces sensitivity. Nonfluorescent bis-substituted peptide derivatives of rhodamine 110 (R110), which are intracellularly cleaved to green fluorescent mono-substituted R110 and free R110 proved to be more sensitive substrates. The activity of the lysosomal cysteine proteinases of human monocytes or rat macrophages, i.e. cathepsin B and L, was specifically measured with (Z-Arg-Arg)2-R110, (Z-Phe-Arg)2-R110, or (Z-Ala-Arg-Arg)2-R110. Fluorescence generation was completely inhibited by Z-Phe-Ala-diazomethane or E-64. The serine proteinases of human neutrophils were analyzed with Elastase-substrates such as (Z-Ala-Ala)2-R110 or (Z-Ala-Ala-Ala)2-R110. Specificity was shown by inhibition with diisopropylfluorophosphate.


Tumor necrosis factor regulation of endothelial cell extracellular proteolysis: the role of urokinase plasminogen activator. Morphological and functional changes in the endothelial cell phenotype which may be central to proinflammatory processes can be elicited by tumor necrosis factor alpha (TNF). Recent observations have indicated that TNF can promote the expression, synthesis and secretion of urokinase plasminogen activator (uPA) in low passage human umbilical vein endothelial cells which normally synthesize little uPA. To further address this issue, we evaluated the ability of TNF to regulate: 1) PA and plasminogen activator inhibitor (PAI-1) mRNA expression and 2) endothelial cell surface associated PA and PAI-1. TNF (100 U/ml) treatment of endothelial cultures induced steady state levels of uPA and PAI-1 mRNA following a 18 hr treatment both 6-fold and 2-fold, respectively utilizing northern analysis. In accord with Northern analyses, TNF stimulated a time and dose dependent increase in cell surface associated uPA antigen as determined by a cell based ELISA assay and immunofluorescence in conjunction with flow cytometry. Treatment of endothelial cell cultures with 100 U/ml of TNF resulted in a 3-fold increase in cell surface uPA antigen levels which peaked at 8 hr. In contrast, no changes in tissue-PA (tPA) and PAI-1 cell surface antigen expression were evident under analogous conditions over a 24 hr period. The TNF mediated increase in both uPA mRNA and cell surface uPA expression correlated with the increased ability of endothelial cells to invade matrix and organize into tube-like structures when cultured on Matrigel.(ABSTRACT TRUNCATED AT 250 WORDS)


Induction of 2'-5' oligoadenylate synthetase during interferon treatment of chronic myelogenous leukemia. Activity of the interferon-induced enzyme 2'-5' oligoadenylate synthetase (2-5 OAS) was measured in peripheral blood mononuclear cells (PBMCs) and serum of patients with chronic phase Ph'-positive chronic myelogenous leukemia (CML) treated with interferon-alpha (IFN-alpha) (4 x 10(6) IU/m2) alone or in combination with 50 micrograms IFN-gamma. At the beginning of IFN therapy, marked elevation of 2-5 OAS titers was detected in PBMCs (pretreatment 0.03-1.62, median 0.2; during treatment 0.8-13.14, median 4.31; 22 patients studied) and in serum (pretreatment 21-156 pmol/dl, median 62; during treatment 532-1740 pmol/dl, median 800; eight patients studied). However, 2-5 OAS titers were not related to clinical outcome or IFN therapy and also during IFN resistance elevated 2-5 OAS activity in PBMCs (median 3.45; range 1.05-13.14; 11 patients studied) were detected. These data argue against direct involvement of the 2-5 OAS system in the therapeutic effect of IFN in CML. However, 2-5 OAS titers in PBMCs or serum appear to be a reliable control of biologically active IFN therapy.


Modifications of the guanidine hydrochloride procedure for the extraction of RNA: isolation from a variety of tissues and adherent/nonadherent cell types. In a previous report dealing with the guanidine hydrochloride protocol for the extraction of RNA from mouse peritoneal macrophages, we identified a major source of RNA-degrading activity and showed that its removal early in the extraction procedure resulted in a more dependable method for the recovery of high-quality RNA. This report extends these findings and demonstrates the general applicability of the technique to a variety of fresh or frozen adherent cell types, cell suspensions and tissues, further highlighting stages at which degradation is most likely to occur and how to avoid a variety of pitfalls associated with the extraction procedure.


Western blots using stained protein gels. A general method is described for the electrophoretic transfer of proteins from stained gels to membranes and subsequent Western detection of specific proteins on the stained membranes. Proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gels are stained using either of two different methods followed by electrophoretic transfer to nitrocellulose or Immobilon-P membranes. The transferred proteins remain stained during immunodetection, providing a set of background markers for protein location and size determination.


RNA abundance measured by a lysate RNase protection assay. We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.


The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium. The monoclonal antibody (mAb) 80L5C4 is a potent inhibitor of the cell adhesion molecule gp80 of Dictyostelium discoideum. To map the exact location of the epitope recognized by mAb 80L5C4, overlapping hexapeptides were synthesized on plastic pins and the binding p6 mAb 80L5C4 to these peptides was monitored by enzyme-linked immunosorbent assay. The 80L5C4 epitope is mapped to a single hexapeptide sequence GYKLNV, which shares five amino acid residues with the octapeptide sequence YKLNVNDS involved in gp80 homophilic binding. Analogue studies indicate that the hydrophobic residues within this sequence are crucial for antigen recognition.


Comparison of embryonic developmental competence of mouse oocytes grown with and without serum. The first objective of this study was to determine whether oocyte growth in serum-free medium affects the solubility of the zona pellucida to alpha-chymotrypsin digestion, which is an index of zona pellucida "hardening" and reflects the potential penetrability of the zona pellucida by sperm. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum-free medium. The zonae pellucidae of oocytes grown in serum-free medium were four times as hard as freshly isolated germinal vesicle (GV)-stage oocytes grown in vivo or oocytes grown in vitro in FBS-containing medium. The hardening of the zonae pellucidae of oocytes grown in serum-free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum-free vs. FBS-containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum-free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two-cell stage after insemination compared with only 6% of the ova grown in serum-free medium. Addition of fetuin to the serum-free medium used for oocyte growth increased the frequency of cleavage to the two-cell stage. Of the embryos derived from oocytes that grew in FBS-containing medium, 70% completed the two-cell stage to blastocyst transition.(ABSTRACT TRUNCATED AT 250 WORDS)


Human zona pellucida during in vitro fertilization: an ultrastructural study using saponin, ruthenium red, and osmium-thiocarbohydrazide. The human zona pellucida (ZP) and its changes during in vitro fertilization in oocytes at different maturational stages and polypronuclear ova at one- to four-cells stages were studied by transmission electron microscopy (TEM) and correlative scanning electron microscopy (SEM). To define the microstructure of the ZP, its amorphous masking material was removed using a detergent (saponin), and its structural glycoproteins were stabilized with a cationic dye, ruthenium red, followed by osmium-thiocarbohydrazide treatment. These methods allowed in all samples the clear visualization of variously arranged networks of filaments composing the outer and inner surfaces of the ZP. These filaments were straight or curved, 0.1-0.4 microns in length and 10-14 nm thick as seen via TEM or 22-28 nm thick as seen via SEM (the difference in thickness was due to the presence of the metal coating for SEM). The filament arrangement was remarkably different between the inner and outer surfaces of the ZP and among the various stages studied. The filaments of the outer surface of the ZP were basically arranged in "large" and "tight" meshed networks. Mature oocytes and fertilized (polypronuclear) ova had a regular alternating pattern of wide and tight meshed networks of filaments. On the other hand, immature and atretic oocytes displayed almost exclusively a tight meshed network of filaments. The inner surface filaments of the ZP of unfertilized oocytes at any stage were arranged in repetitive structures characterized by numerous short and straight filaments anastomosing with each other and sometimes forming at the intersections small, rounded structures. After fertilization, the inner surface of the ZP displayed numerous areas where filaments fused together. Collectively, these data clearly reveal that oocyte maturation and fertilization in humans are accompanied by changes of ZP filaments arrangement, which may be relevant in the processes of binding, penetration, and selection of spermatozoa.


Identification of a small epitope in domain Ib of Pseudomonas aeruginosa exotoxin A that elicits enzyme-neutralizing antibodies. A peptide corresponding to amino acids 392-404 of the amino acid sequence of Pseudomonas aeruginosa exotoxin A (the last 13 amino acids of domain Ib) was synthesized and coupled to thyroglobulin. The conjugate induced an antiserum in rabbits with high antibody titer against native toxin as measured by ELISA, and this antiserum was highly efficient in inhibiting the ADP-ribosyltransferase activity of exotoxin A. These data corroborate the potential importance of amino acids 400-404 in the enzymatic mechanism of exotoxin A.


Uridine and cytidine nucleotide synthesis in renal hypertrophy: biochemical differences in response to the growth stimulus of diabetes and unilateral nephrectomy. The effects of unilateral nephrectomy (UN) and streptozotocin (STZ) diabetes on the activities of enzymes involved in uridine and cytidine synthesis in early renal growth (3-14 days after stimulus to growth) have been compared. Measurements were also made of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) and of glucose 6-phosphate (G6P), UDP-glucose, and glycogen, in relation to phosphoribosyl pyrophosphate, ribonucleotide, and complex carbohydrate formation. There were striking differences in the activities of CTP synthetase, G6PDH, and 6PGDH in the two conditions, with a three-fold increase in all three enzymes at 3 and 5 days and a two-fold increase above basal values at 14 days of STZ diabetes. The UN group showed no significant change in CTP synthetase at any stage and the activity of G6PDH and 6PGDH only kept pace with renal growth. Changes in routes of uridine synthesis were less marked, with a more rapid rise in carbamoyl-phosphate synthetase (glutamine) and a lesser response of dihydroorotate dehydrogenase in the UN relative to the STZ-diabetic groups. The enzymes of complex II and of uracil phosphoribosyltransferase showed essentially similar patterns during renal hypertrophy in UN and STZ diabetes. The parallel increase in CTP synthetase, G6PDH, and 6PGDH in the kidney in diabetes, also known to increase in growth situations in hepatomas and in renal tumors, is discussed in relation to hormone signals involved in renal growth. The importance of the concentration of CTP, and thus of CTP synthetase, in the CTP-cytidyltransferase reaction, an enzyme with a high Km for CTP, makes the present observation of the striking increase in CTP synthetase in STZ diabetes of particular interest in relation to phosphatidylcholine formation and hormone signal transduction.


Functional activity of an HIV-1 neutralizing IgG human monoclonal antibody: ADCC and complement-mediated lysis. The IgG1 kappa, human monoclonal antibody (HMAb), F105, was studied for functional activity in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). F105 reacts with a discontinuous epitope on the CD4 binding site of the HIV-1 envelope glycoprotein, gp120, expressed on the surfaces of infected cells and neutralizes diverse viral strains at antibody concentrations readily achievable in humans. Neither F105 nor serum (diluted 1:50) from HIV seropositive donors mediate CDC against an SF2-infected cell line with rabbit or human sera as a source of complement. F105 and HIV-1 sera mediate ADCC against the SF2 strain. Normal human serum reduced spontaneous lysis of SF2 by peripheral blood monocytes (PBM). Although mixing of F105 with normal human serum reduced the lysis observed (36 +/- 8 vs. 42 +/- 8%), this still was significantly greater than lysis in media (30 +/- 5%) or normal human serum (23 +/- 6%) (p less than .05). A murine antibody to CD16 significantly reduced spontaneous lysis observed with media (30 +/- 5 vs. 18 +/- 3%) while normal mouse serum had no effect (31 +/- 7%). ADCC mediated by F105 is completely abrogated by the anti-CD16 antibody (42 +/- 8 vs. 22 +/- 4%), while only a fraction of ADCC mediated by HIV sera is inhibited by anti-CD16 (60 +/- 9 vs. 46 +/- 6%), suggesting that several populations of effector cells function in ADCC mediated by the polyclonal sera. Thus, F105, as opposed to polyclonal sera, mediates ADCC through a CD16+ PBM population.


Class II (I-Ad) restricted T-cell recognition of the V3 loop region of HIV-1 gp120. A CD4+ and gp120-specific T-cell line, and subclone, were established from BALB/c mice following immunization with recombinant gp120, using an adjuvant formulation (alum) acceptable for use in the human. The recognition specificity was determined against a panel of synthetic peptides corresponding to the primary sequence of the HXB2 strain of human immunodeficiency virus (HIV). An epitope was identified, corresponding to amino acid residues 322-341, and a further series of truncated peptides established the minimum determinant to be 327-341. Possible reasons for the immunodominance of the V3 loop, and adjacent regions, for both antibody and T-cell recognition are discussed.


Recombinant glycoprotein 120 of human immunodeficiency virus is a potent interferon inducer. Cells infected with human immunodeficiency virus (HIV) induce antiviral activity in peripheral blood mononuclear cells (PBMC) from healthy donors. This activity is neutralized by anti-interferon-alpha antibody and partially destroyed at pH 2. Previous studies with enriched cell populations and monoclonal antibodies suggest that B lymphocytes are the main IFN-producing cells, and that both CD4 and HLA class II antigens are essential for IFN induction. Since the initial event of HIV infection of CD4+ cells is the interaction of the virus coat glycoprotein gp120 with CD4 molecule, we investigated whether gp120 is responsible for IFN induction. Using PBMC and recombinant gp120 obtained from a baculovirus expression system, dose-dependent induction of antiviral activity was observed with titers approaching 10(3) IU/ml. This induction was blocked in the presence of antibody to gp120. The antiviral activity was characterized as IFN-alpha by neutralization with IFN alpha-specific antibody. Preincubation of PBMC with anti-CD4 or the presence of soluble CD4 during incubation inhibited IFN induction, indicating that interaction of gp120 with cell-associated CD4 is responsible for this induction. Neither lymphoproliferation nor interleukin-2 (IL-2) production was observed during IFN induction. However, class G immunoglobulin secretion was enhanced by gp120, indicating that B cells are direct or indirect targets of gp120 stimulation in this experimental system. Since gp120 is shed from HIV-infected cells and occurs in the serum of acquired immunodeficiency syndrome (AIDS) patients, our data suggest that this glycoprotein is responsible for the induction of endogenous IFN and the polyclonal activation of B cells both of which are observed in AIDS patients.


Pyridoxal-5'-phosphate inhibits the polymerase activity of a recombinant RNAase H-deficient mutant of HIV-1 reverse transcriptase. We have investigated the ability of pyridoxal-5'-phosphate to inhibit a recombinant deletion mutant of human immunodeficiency virus type 1(HIV-1) reverse transcriptase (RT) which is missing the last 23 amino acids of the C-terminus. This mutant reverse transcriptase is characterized by normal polymerase activity as compared with full-length enzyme; however, it has no RNase H activity. Inhibition studies with pyridoxal-5'-phosphate showed several differences as compared with inhibition of full-length enzyme: (1) Inhibition of mutant reverse transcriptase was independent of divalent cation, (2) Either substrate alone could protect mutant reverse transcriptase from inactivation by pyridoxal-5'-phosphate, and (3) stoichiometry of pyridoxal-5'-phosphate binding to mutant reverse transcriptase was 2 mol/mol under the same conditions in which 1 mol/mol bound to full-length enzyme. Furthermore, in the presence of either substrate alone, the stoichiometry of pyridoxal-5'-phosphate binding to the mutant was reduced to 1 mol/mol. These results indicate that the second binding site for pyridoxal-5'-phosphate seen in the mutant reverse transcriptase is at or near the primer-template binding site of the enzyme. They also suggest that the RNase H domain of HIV RT plays a functional role in substrate binding at the polymerase domain.


Inhibition of the reverse transcriptase activity and replication of human immunodeficiency virus type 1 by AS 101 in vitro. In a search for compounds active against human immunodeficiency virus type 1 (HIV-1), it was found that the novel low-molecular weight immunoenhancer ammonium trichloro(dioxyethylene-O,O'-) tellurate (AS101) suppresses production of HIV-1 in vitro. Treatment of HIV-1-infected peripheral blood mononuclear cells (PBMC) with increasing concentrations of AS101 resulted in substantial inhibition of virus production as measured by both reverse transcriptase (RT) activity and antigen presence in supernatants of treated cells. AS101 had no effect on PBMC viability, growth, or morphology up to a concentration of 15 microM for 14 days. To elucidate a possible mechanism for the inhibition of AS101, we have analyzed the effect of the drug on the catalytic functions associated with HIV RT, namely the RDDP, DDDP, and RNase H activities. RDDP and DDDP activities were impaired by the drug with calculated IC50 value of about 4 microM. On the other hand, the RNase H activity was less sensitive to AS101, with an apparent IC50 value of about 30 microM. The anti-HIV-1 activity of AS101 as reflected by inhibition of the different catalytic functions associated with viral RT, in the absence of drug-related toxicity to lymphocytes, together with its immunomodulating activity strongly argues in favor of its evaluation, as a therapeutic agent for patients with HIV infection.


Dehydroepiandrosterone (DHEA) and synthetic DHEA analogs are modest inhibitors of HIV-1 IIIB replication. Down-regulation of Epstein-Barr virus (EBV) induced transformation of human lymphocytes in vitro by dehydroepiandrosterone (DHEA), a naturally occurring human steroid secreted by the adrenal gland has been demonstrated. This article reports on the effects of DHEA and its novel synthetic analogs 16 alpha-fluoro-5-androsten-17-one (8354) and 3 beta-hydroxy-16 alpha-fluoro-5 alpha-androstan-17-one (OH8356) on human immunodeficiency virus (HIV-1) replication. Treatment with DHEA, 8354, or OH8356 resulted in a modest down-regulation of HIV-1 replication in phytohemagglutinin-stimulated peripheral blood lymphocytes as measured by syncytia formation, release of p24 antigen, and accumulation of reverse transcriptase activity. DHEA and 8354 also reduced syncytia formation in HIV-1-infected SupT1 lymphoblasts. DHEA and synthetic analogs of DHEA, which have been shown previously to have antiproliferative effects, now are shown to reduce HIV-1 replication. DHEA or synthetic analogs of DHEA could provide an alternative and/or adjuvant for HIV-1 infection.


Studies on a type D retrovirus isolated from an AIDS patient lymphoma. The development of AIDS-related lymphomas (ARL) has been on the rise in recent years. During an analysis of ARL from AIDS patients, one individual developed atypical syncytial variants of high-grade Burkitt's-type B-cell lymphomas, which prompted further study. However, the search for a HIV-1 retrovirus, which we hypothesized was infecting these cells, led to the subsequent discovery of a type D retrovirus in two early-passage lymphoma cell lines derived from this patient. Nucleotide and amino acid sequence analysis, as well as immunologic reactivity, indicated that the virus was closely related to Mason-Pfizer monkey virus (MPMV) or simian retrovirus type 1 (SRV-1). MPMV and SRV-1 are immunosuppressive type D retroviruses that cause an AIDS-like syndrome in rhesus macaques. Amplification of DNA from the patient's diagnostic bone marrow biopsy specimen by the polymerase chain reaction generated MPMV-specific fragments indicative of infection by a retrovirus similar to MPMV. Additionally, the patient's serum contained antibodies that recognized type D retroviral env proteins (gp20 and gp70) and gag proteins (p27 and p14) as assayed by immunoblot and radioimmunoprecipitation techniques. Although there have been reports of human cell lines infected with type D retroviruses and of type D reactive human sera, this is the first report of a type D retrovirus infection in a human confirmed by virus isolation, serum immunoreactivity, and viral DNA identification in tumor tissue.


Clinical and laboratory evaluation of the myeloprotective effect of medroxyprogesterone acetate in head and neck cancer. The action of high-dose medroxyprogesterone acetate (MPA) was studied by analysing the behaviour of colony-forming-unit granulocyte-macrophage (CFU-GM) during chemotherapy. 21 non-pretreated men with locally advanced carcinoma of the head and neck were randomised into two arms: A (11 patients) received three alternating cycles of cisplatin, 5-fluorouracil (CF)/cisplatin, methotrexate, bleomycin, vincristine and then CF every 4 weeks and B (10 patients) were treated with the same schedule plus 1000 mg per day of MPA. MPA was administered 14 days before the start of chemotherapy (day 0) and continued daily up to the 90th day. Bone marrow was harvested in arm A on days 0, +14 and +90, and in B, also on day -14. There was diverse CFU-GM behaviour in the two arms on the 14th day. These data support the hypothesis that the myeloprotective effect of MPA is due to induction of a mitotic rest in the stem cells, which protects them from drug action.


Fulminant hepatic failure in non-Hodgkin lymphoma patients treated with chemotherapy. Chemotherapy is the mainstay of therapy for patients with non-Hodgkin lymphoma. Among side-effects associated with the use of chemotherapy, immunosuppression is one which can be potentially fatal. In hepatitis B carriers, immunosuppression permits widespread infection of the hepatocytes and its subsequent withdrawal causes an "immunological rebound" leading to massive necrosis of hepatocytes. 4 patients who died of fulminant hepatitis following chemotherapy are reported. These were patients with positive hepatitis B serology. Caution is advised when treating non-Hodgkin lymphoma in patients from hepatitis B endemic regions.


Immunoreactive "calcitonin-like" material in heroin addicts: varying reactivity with different antibodies. High levels of immunoreactive calcitonin (iCT) in the blood of heroin addicts were previously reported. As it is well known that multiple forms of calcitonin exist in the blood and in tissues, the purpose of the present study was to investigate the immunological nature of the CT-like immunoreactive material found in the blood of these subjects. We investigated 25 addicts, who had been using heroin for more than one year and were hospitalized for a 2 week detoxication program. Blood samples were drawn at the start of the program (when the subjects were still on heroin) and after 5 and 12 days of abstinence from heroin. Twenty-five healthy subjects served as controls. We used 2 commercial RIA kits, calibrated against the same reference material (WHO 70-234), but employing different antisera. One antiserum substantially confirmed the previous findings of increased levels of calcitonin during heroin use, but the other seemed to exclude any change in the hormone concentrations. This suggests that the "calcitonin like" material found in heroin addicts contains some epitopes similar to those found in the calcitonin standard which are detected by the first antiserum. However it lacks other epitopes which are also present in calcitonin standard and which are recognized by the second antiserum. Therefore, this substance seems to be different from the standard human calcitonin 1-32. A possible involvement of a calcitonin analogue (precursor or metabolite) in the biochemical changes occurring during chronic opiate use is suggested.


Rat prostate cancer cell line-specific production and apparent secretion of heparin-binding growth factors. Cultured C3 and T5 AXC/SSh rat prostate cancer cells and their conditioned media contained 1000-fold more heparin-binding mitogens than did normal AXC/SSh rat prostates. Immunological analyses confirmed that rat ventral prostate contained only a 17.5 kilodalton (kDa) basic fibroblast growth factor (bFGF)-like mitogen. In contrast, combined immunological and metabolic radiolabeling analyses showed that C3 cells contained 23/24 and 14 kDa bFGF-like polypeptides, whereas the principal bFGF-like polypeptides of C3 cell conditioned medium were proteins having masses of 17.5 and 14 kDa. Identical analyses showed that T5 cells contained 17.5 and 14 kDa bFGF-like polypeptides, whereas the principal bFGF-like polypeptides of T5 cell conditioned medium were proteins having masses of 17.5, 16, and 14 kDa. We found that bFGF-like proteins of mass less than 17.5 kDa, which were present in conditioned medium, were not derived by proteolysis of higher molecular weight bFGF-like mitogens after these were processed into conditioned medium. Northern analyses showed that normal prostate and prostate cancer cells contained acidic fibroblast growth factor transcripts of 6.5 and 3.4 kilobases (kb) and bFGF transcripts of 6.0 and 2.5 kb. Prostate cancer cells also contained a 12-kb bFGF transcript that was not present in normal prostate. Southern analysis of restriction endonuclease-digested normal prostate or prostate cancer cell genomic DNA showed that the 12-kb bFGF transcript was not the product of a rearranged bFGF gene. Our data show that rat prostate cancer cells contain bFGF-like polypeptides of mass 14 to 23/24 kDa and suggest that these cells secrete bFGF-like polypeptides.


The RNP motif protein family.

The RNP motif protein family. RNA-protein interactions play critical roles in a variety of important processes in the cell, such as mRNA splicing and translation. Many RNA binding proteins contain an approximately 80-aminoacid conserved sequence, the RNP motif, that has been shown in several cases to constitute the binding domain. As a class, RNP motif proteins bind many different RNAs with varying degrees of sequence specificity. A major area of current interest is the elucidation of the structure-function relationships in these proteins: the structure of the motif and the residues that are important for sequence-specific binding, the roles of the individual motifs in proteins with multiple RNP motifs, and the functions of other domains found in RNP motif proteins. Recent experiments have provided some insight into these issues and have paved the way for a more detailed understanding of this protein family.


FLP-mediated intermolecular recombination in the cytoplasm of Drosophila embryos. We show that when a heat-shock-driven gene that encodes the yeast FLP recombinase is injected into preblastoderm Drosophila embryos, it promotes intermolecular recombination between two coinjected plasmids that bear the specific recombination target sequence, FRT. Minimal, 34-bp FRT sites in the two plasmids are sufficient for their cointegration. The reaction is efficient enough to produce detectable recombinants when one of the plasmids is present in as little as 1000 molecules per embryo. This is comparable to the concentration of unique chromosomal sites, raising the possibility that integration of injected plasmid DNA into FRT-bearing fly chromosomes may also be achievable. Since integrants might be stabilized against the reverse excision reaction if the recombinase could be provided in a sharp pulse, it is encouraging that efficient plasmid cointegration is also achieved when in vitro synthesized FLP RNA rather than DNA is injected into the embryos.


Growth inhibition of human colorectal carcinoma cell lines by tumor necrosis factor-alpha correlates with reduced activity of pp60c-src. One molecular alteration that has been observed in the majority of colorectal carcinomas is the activation of pp60c-src kinase. To address the role of pp60c-src in growth control of colon carcinoma cell lines, the effects of the biologic response modifier, tumor necrosis factor (TNF)-alpha were studied on the established HT29 colorectal carcinoma cell line and two clonal variants derived from the parental line. In one clone, HT29-A34, approximately 55% growth inhibition was observed following a 120-h incubation period with 10(3) U/ml TNF. In this TNF-sensitive cell line, pp60c-src immune complex kinase activity was reduced 3.9-fold accompanied by only a slight reduction in pp60c-src protein levels. Growth inhibition and decreased pp60c-src kinase activity correlated in a dose-dependent manner. The HT29-A14 TNF-resistant clone was not growth-inhibited by TNF, and no changes in pp60c-src were observed following treatment. Growth inhibition also correlated with reduced pp60c-src kinase specific activity in a number of other established colon carcinoma cell lines that were TNF-sensitive. In TNF-resistant colon carcinoma cell lines, pp60c-src kinase activity and levels remained unchanged. When changes in specific activity of pp60c-src were observed in sensitive cells, they occurred after decreased [3H]-thymidine uptake was observed. Therefore, these changes in pp60c-src activity are not the earliest event in TNF-induced growth inhibition. Nevertheless, our results suggest that modulation of pp60c-src kinase activity may be important in growth control of colorectal carcinoma cell lines.


Treatment of hairy cell leukemia with granulocyte colony-stimulating factor and recombinant consensus interferon or recombinant interferon-alpha-2b. Patients with hairy cell leukemia and neutropenia (absolute neutrophil count less than 1.5 x 10(9)/L) were treated with recombinant granulocyte colony-stimulating factor (G-CSF) at doses of 3.6 and 7.2 micrograms/kg by daily subcutaneous injection, until normalization of neutrophil counts occurred. Patients then received either recombinant interferon-alpha-2b (r-IFN-alpha-2b) or a unique IFN, recombinant consensus IFN (rIFN-con-1), each given at doses of 10 micrograms/m2 subcutaneously three times a week, coupled with continued daily G-CSF therapy, for 3 months. After 3 months the G-CSF was discontinued; patients continued to take IFN for 1 year. All 10 patients responded to G-CSF with normalization of neutrophil counts within 2 weeks; the increase in neutrophil counts was greater in previously splenectomized patients. Four patients were treated with r-IFN-alpha-2b, and six were treated with rIFN-con-1. No patients developed recurrent neutropenia with the initiation of IFN therapy. Nine patients are evaluable for response to IFN. Five of six patients demonstrated hematologic improvement with rIFN-con-1, with two patients obtaining complete responses. All three patients receiving r-IFN-alpha-2b demonstrated hematologic improvement; one complete response was observed. Toxicities of both IFNs included influenza-like symptoms. We conclude that G-CSF can abrogate the myelosuppressive effects of IFN, and may be a useful adjunct to this therapy in neutropenic patients. We conclude that rIFN-con-1, the product of a synthetic gene, has activity in the treatment of hairy cell leukemia, and merits clinical investigation in other settings.


Human prostatic aldehyde dehydrogenase of healthy controls and diseased prostates. Aldehyde dehydrogenase (ALDH, EC 1.2.1.3) of the human prostate was the subject of investigation in this study. The possible physiological role of aldehyde dehydrogenase in the human prostate might be to detoxify aldehydes arising from the oxidation of the polyamines via monoamine or diamine oxidases. The specific activity of the enzyme with 1 mM propionaldehyde as substrate and 0.5 mM NAD at pH 7.4 in the control normal prostates and prostates afflicted with the disease, benign prostatic hyperplasia (BPH), was 26.06 +/- 2.96 and 5.17 +/- 0.48 nmol/g prostate per min, respectively. When 100 microM gamma-aminobutyraldehyde was used as a substrate, the specific activity in the normal controls and prostates with benign prostatic hyperplasia was 19.80 +/- 1.33 and 2.95 +/- 2.46 nmol/g prostate per min, respectively. Upon isoelectric focusing of the extracts of the control prostates when the gels were developed for aldehyde dehydrogenase activity, there were three aldehyde dehydrogenase activity bands visible, pI 4.9 (mitochondrial), 5.4 (cytosolic) and about 6.0-6.5, on the IEF gels developed with gamma-aminobutyraldehyde as a substrate. With the extracts of prostates with benign prostatic hyperplasia the pI 4.9 band was significantly reduced, the pI 5.4 band enhanced and the approx. pI 6.0 band was not detectable on the IEF gels with propionaldehyde as a substrate. There was no detectable aldehyde dehydrogenase activity in the extract of the prostate with cancer on IEF gels nor in the activity assays with propionaldehyde or gamma-aminobutyraldehyde as substrates.


Ionic selectivity of volume-sensitive currents in human epithelial cells. The ion selectivity of swelling-activated Cl- currents has been investigated in three different human epithelial cell lines, two derived from the airway epithelium (9HTEo- and CFNPE9o-) and one from a colon carcinoma (T84). The relative permeability of volume-sensitive currents with respect to Cl- is: I- (1.19) greater than NO3- (1.07) approximately Br-(1.05) greater than Cl-(1.0) greater than F-(0.5) approximately HCO3-(0.48) greater than isethionate(0.28) greater than aspartate (0.14) approximately gluconate(0.13) approximately SO4(2-)(0.12). This type of ion selectivity is similar to that described for depolarization-activated outwardly rectifying Cl- channels found in epithelial cells.


Isolation, mass spectrometric characterization, and protein phosphatase inhibition properties of cyclic peptide analogues of gramicidin-S from Bacillus brevis (Nagano strain). Six substitutional variants of gramicidin-S, present in a commercial extract of Bacillus brevis, have been characterized using a combination of liquid chromatographic/mass spectrometric analysis of the crude extract, plus accurate mass measurements, tandem mass spectrometric investigations, and amino acid analyses, of isolated high-performance liquid chromatographic fractions. Two of these variants were shown previously (Nozaki and Muramatsu, J. Antibiotics 37, 689 (1984)) to involve replacement of one or both Val residues in gramicidin-S by aminobutyric acid, and these findings are confirmed here. One of the four unknown variants corresponds to substitution of one Val residue by Leu, while the other three all involve substitution of one of the Orn residues in gramicidin-S by Lys, or by citrulline (gamma-carbamoyl ornithine), or by gamma-formyl ornithine. The biological activities of the purified fractions were assessed with respect to their in vitro inhibition of protein phosphatases PP1 and PP2A. For both protein phosphatases inhibition levels fell in the micromolar range, about four orders of magnitude higher than IC50 values for other small cyclic peptides such as microcystins and nodularins.


Evaluation of commercially available antibodies to cytokeratin intermediate filaments and laminin in normal cat pinna. The pattern of distribution of cytokeratin (CK) intermediate filaments can be used to characterize subsets of epithelial tissues. The purpose of the study was to examine the CK expression of feline pinna skin. Six normal feline pinnae were routinely processed in formalin. An immunohistochemical method was used to stain the pinnae with 8 commercially available anti-human CK antibodies (Abs) (PKK1, CAM 5.2, UCD 10/11, 35BH11, 34BE12, AE1/AE3, MAK 6, A575) and an anti-human laminin Ab. All the CK Abs selectively localized to epithelium except 35BH11, which did not react with any part of the pinna. Some epithelial subsets were identified by their unique staining pattern with CK Abs. Basal cells but not suprabasal cells of the epidermis stained with PKK1; basal but not lumenal cells of apocrine glands stained with 34BE12. Apocrine glands stained with all CK Abs except 35BH11. All epithelial structures were stained with A575. Basal lamina of epithelial and mesenchymal tissues was clearly identified by the anti-laminin Ab. The results indicate that in cat pinna some commercially available anti-human CK Abs selectively stain subsets of epithelium and adnexa. PKK1, 34BE12, and A575 were the CK Abs with the most consistent staining patterns, the other Abs stained more variably from pinna to pinna. The pattern of epithelial and adnexal staining was similar but not identical to that reported for humans.


Fixation of aldehydic dextrans onto human deoxyhemoglobin. A procedure commonly used to transform native adult human hemoglobin (Hb) into a physiological oxygen carrier consists of a pyridoxylation of the protein to lower its oxygen affinity, followed by its polymerization in the presence of glutaraldehyde, with or without further reduction, to increase its circulating half-life. This series of reactions yields derivatives presenting a great molecular heterogeneity that have to be fractionated for use in vivo. Hemoglobin derivatives with low oxygen affinity and a narrow distribution of molecular weights were obtained by linking a dextran polyaldehydic derivative to deoxyhemoglobin at pH 8. From oxygen-binding measurements carried out in the presence of inositolhexaphosphate, a strong effector of hemoglobin, it appeared that the allosteric site of hemoglobin was blocked, probably by crosslinking bonds, which stabilizes its deoxy structure. On the other hand, when the reaction was performed in the presence of inositolhexaphosphate, the resulting conjugates exhibited an oxygen affinity identical to that of unmodified hemoglobin. After treatment with NaBH4, the polymer-hemoglobin derivatives were stable and possessed a reversible oxygen-carrying capacity similar to that of blood. The conjugates prepared from oxyhemoglobin all possessed a lower P50 than native hemoglobin whatever the reaction conditions.


Tumour necrosis factor induction of ELAM-1 and ICAM-1 on human umbilical vein endothelial cells--analysis of tumour necrosis factor-receptor interactions. Induction of the adhesion molecules ELAM-1 and ICAM-1 on endothelial cells is a key pro-inflammatory effect of tumour necrosis factor (TNF). Earlier work in non-human systems has suggested that unlike other cell types, endothelial cells interact with the N-terminus of the TNF molecule, thereby implying novel TNF receptors on endothelial cells. This is also supported by 125I-TNF cross-linking studies on bovine endothelial cells. The present study aimed to see whether TNF induction of ELAM-1 and ICAM-1 on human umbilical vein endothelial cells (HUVECs) involved novel TNF-receptor interactions. Three approaches were employed. First, antibodies directed at different sites on the TNF molecule were tested for inhibition of TNF-induction of ELAM-1 and ICAM-1 on HUVECs. Inhibition was seen only with antibodies reacting with epitopes outside the N-terminal region. Second, an N-terminal TNF peptide (residues 1-26) failed to induce ELAM-1 and ICAM-1 on HUVECs or antagonise TNF induction of these molecules. Third, HUVEC/125I-TNF cross-linking revealed a major complex characteristic of the known 55 kDa TNF receptor: this was confirmed with receptor-specific monoclonal antibodies. It is concluded that (a) the same part of the TNF molecule interacts with TNF-receptors on HUVECs and other cell types and (b) TNF induction of ELAM-1 and ICAM-1 on HUVECs is mediated via the well-characterized 55 kDa TNF receptor.


Preparation and properties of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C-dextran conjugate. Monoclonal antibody (mAb) A7, produced against human colon cancer, was conjugated with a polymeric prodrug of mitomycin C (MMC), the MMC-dextran conjugate with an anionic charge (MMCDan) and a molecular weight of 70,000. The amino groups were introduced into the MMCDan by reacting ethylenediamine with the carboxyl group in the spacer arm of the dextran bridge by a carbodiimide-catalyzed reaction. The coupling to mAb A7 was performed using SPDP. A 15 M excess of ethylenediamine produced an optimal MMCDan with amino groups, which resulted in a homogenous conjugate (A7-MMCD) with minimal formation of high-molecular-weight aggregates in about a 30% yield of both IgG and MMC. The molar binding ratio of IgG:dextran:MMC in A7-MMCD was estimated to be 1:1.2:40. A7-MMCD, having MMC prodrug properties, released active MMC with a half-life of 29.1 h and had an almost neutral electric charge under physiological conditions. A competitive binding assay using 125I-labeled A7 revealed that the A7-MMCD almost fully retained its antibody-binding activity. The cytotoxicity of A7-MMCD was assayed by determining the degree of inhibition of [3H]-thymidine in corporation in two different ways using the human colon cancer cell line SW1116. A 48-h continuous exposure test revealed that the pharmacological activity of MMC in A7-MMCD was completely preserved. In addition, A7-MMCD exhibited about a 14-fold greater cytotoxicity than MMCDan when the IC50 values determined using a 2-h pretreatment exposure system were compared. These results suggest that A7-MMCD could be useful in immunotargeting chemotherapy for colorectal cancer.


Beta/A4 deposits and their relationship to senile plaques in Alzheimer's disease. The density and spatial pattern of immunostained beta/A4 deposits and mature senile plaques (SP) stained by the Glees method were compared in Alzheimer's diseased brain. Thirty-seven percent of the variance in Glees SP density in a tissue could be explained by beta/A4. Both lesions were clustered with the beta/A4 clusters often larger than the Glees SP clusters. Beta/A4 and Glees SP cluster size were not correlated in a tissue. The size of Glees SP clusters was positively correlated with SP density but no correlation could be detected for beta/A4. Hence, the density and spatial pattern of beta/A4 deposits in most tissues did not predict the development of Glees SP.


Galanin- and CGRP-like immunoreactivity coexist in rat spinal motoneurons. We can report, that by the use of indirect immunofluorescence techniques, that rat spinal motoneurons contain galanin-like immunoreactivity (LI). Furthermore, that galanin and calcitonin gene-related peptide (CGRP)-LI coexist in a number of spinal motoneurons. Co-localization could be demonstrated as thoracic, lumbar as well as sacral spinal cord levels in individual neurons by studying adjacent sections each stained by one of the two antisera, as well as by double labelling experiments. Small neurons, within the gamma-motoneuron and interneuron size-range, were also positive for galanin-LI.


Molecular amplifiers: synthesis and functionalization of a poly(aminopropyl)dextran bearing a uniquely reactive terminus for univalent attachment to biomolecules. The synthesis and characterization of the versatile dextran-based molecular amplifier 6 is described. Dextran (Mr = 40,200) was selectively monofunctionalized in high yield at its reducing terminus via reductive amination with 2-(4-nitrophenyl)ethylamine to give 1. The nitro group in 1 serves as a masked amino group which is eventually converted into a reactive isothiocyanato group used for monovalent attachment of the completed assembly to a target molecule. Cyanoethylation of 1 gave the terminally nitrophenylated poly(cyanoethyl)dextran 5 which was selectively reduced to the corresponding poly(aminopropyl) derivative 6 with BH3.THF, a reagent which preserved the end nitro group. Conjugation of amplifier 6 with the isothiocyanate-derivatized Gd(III) chelate 7 gave conjugate 9 containing about 22 mol of chelate/mol of amplifier. The T1 relaxivity per Gd(III) ion of 9 in H2O was 15.0 mM-1 s-1, about 3-fold higher than that of free Gd(III)DTPA in H2O. The nitro group of 9 was then selectively reduced to the corresponding amine 10, which was converted into isothiocyanate 11. The reactivity of the single isothiocyanate group in 11 was demonstrated by coupling to 5-aminoeosin, giving conjugate 12. Amplifier 6 was also conjugated with the acid-labile N-cis-aconityl derivative 8 of the potent anticancer agent daunomycin. The nitro group of the resulting conjugate 13 was then reduced and the resulting amine 14 was converted into mono isothiocyanate 15. Compound 15 reacted with a water-insoluble amine-containing solid support to give 16. Free daunomycin was released from 16 by exposure to citrate-phosphate buffer at pH 4.0.


Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurones. Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.


Structure of the mouse 5-HT1C, 5-HT2 and stomach fundus serotonin receptor genes. By analysis of the mouse 5-HT1C receptor gene we found that its coding region contains three introns. We next isolated cDNA and genomic clones for the closely related 5-HT2 receptor using a probe derived from the 5-HT1C receptor sequence. This probe also hybridized to an additional gene, called SRL (Serotonin Receptor Like). We have evidence demonstrating that it encodes the stomach fundus 5-HT receptor. Two introns are present within the coding regions of the mouse 5-HT2 receptor gene and the SRL gene at positions which correspond to those of introns in the 5-HT1C receptor gene. This intron distribution is unique and distinguishes these receptors from other members of the family of receptors coupled to G-proteins.


Alpha-fetoprotein expression in hepatocellular carcinoma: a clinical study. Alpha-fetoprotein (AFP) levels were studied in 51 consecutive patients with hepatocellular carcinoma that presented to the Surgical Hepatobiliary Unit at Westmead Hospital over 12 years. Twenty-three were hepatitis surface antigen (HBsAg) positive and 13 of those patients were Asian. Thirteen patients drank more than 60 g of alcohol each day. A significantly raised level of AFP was defined as more than 20 ng/mL, and 31 of the 51 patients had AFP levels exceeding this at some stage during surveillance. Twenty-five demonstrated levels above 200 ng/mL. Univariate statistical methods suggested that men were more likely to express raised AFP than women, Asians more likely than other races, patients with chronic active hepatitis more likely than those without and those with chronic hepatitis B infection more likely than those who were HBsAg negative. Those who drank more than 60 g alcohol each day were less likely to demonstrate a raised serum AFP than those who drank less. Multivariate logistic regression demonstrated that HBsAg carriage was the only statistically significant independent determinant of a raised AFP. Age 65 years or more was associated with a chance of a raised AFP.


Isolation and characterization of two wound-regulated tomato extensin genes. Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library by in situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro4-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro4-Tyr3-Lys-Ser-Pro4-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro4 repeat units, a central domain with repeats of the consensus sequence Ser-Pro2-5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro4-Ser-Pro-Ser-Pro4-Thr-Tyr1-3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.


Defective transport as a mechanism of acquired resistance to methotrexate in patients with acute lymphocytic leukemia. Although the mechanisms of resistance to methotrexate (MTX) are known in experimental tumors made resistant to this drug, little information is available regarding acquired resistance to MTX in patients. A competitive displacement assay using the fluorescent lysine analogue of MTX, N-(4-amino-4-deoxy-N10-methylpteroyl)-N epsilon-(4'-fluorescein-thiocarbamyl)-L-lysine (PT430), was developed as a sensitive method of detection of transport resistance to MTX in cell lines, as well as in blast cells from patients with leukemia. Rapid uptake of PT430 at high concentrations (20 mumol/L) in leukemic blasts resulted in achievement of steady-state levels within 2 hours. Subsequent incubation with the folate antagonists, MTX and trimetrexate (TMTX), which differ in the mode of carrier transport, produced characteristic patterns of PT430 displacement. Flow cytometric analysis of the mean fluorescence intensity in the human CCRF-CEM T-cell lymphoblastic leukemia cell line and its MTX-resistant subline clearly identified the presence of transport deficiency in the resistant subline. Analysis of blasts from 17 patients with leukemia, nine with no prior chemotherapy and eight previously treated with chemotherapy, found evidence of MTX transport resistance in two of the four patients who were treated with MTX and considered to be clinically resistant to the drug. The finding that blast cells of some patients with leukemia considered clinically resistant to MTX is due to decreased MTX transport has important implications for clinical use of this drug and for new drug development.


Diethyldithiocarbamate induction of cytokine release in human long-term bone marrow cultures. Diethyldithiocarbamate (DDTC) is a biochemical modulating agent that protects murine bone marrow progenitor cells from the cytotoxicity of a variety of cancer chemotherapeutic agents. However, the mechanism of this protection is not well understood. Long-term human bone marrow cultures (LTBMC) were established and at day 17 treated with 30 mumol/L DDTC for 1 hour, after which DDTC was removed and replaced with complete medium. Conditioned medium was then collected 6, 12, 24, and 48 hours later and analyzed for the presence of cytokines. A time-dependent increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) (12-fold), granulocyte-CSF (G-CSF) (66-fold), interleukin (IL)-6, (three-fold), IL-1 beta (161-fold), and tumor necrosis factor (TNF)-alpha (25-fold) was observed. The maximum increase for the factors other than TNF-alpha was at 24 to 48 hours posttreatment. However, TNF-alpha peaked as early as 6 hours post-DDTC. When conditioned medium from these cultures was tested in a granulocyte-macrophage progenitor cell (GM-CFC) assay, an increase in colony formation was observed that correlated with the increased levels of cytokines in the medium. The specificity of this effect was confirmed by the fact that the closely related congener bis(hydroxyethyl)dithiocarbamate was devoid of colony-stimulating activity. The addition of antibodies for TNF-alpha and/or IL-1 alpha following DDTC treatment did not inhibit the release of GM-CSF, G-CSF, or IL-6 from the LTBMC. These results suggest that DDTC accelerates bone marrow recovery following myelotoxic drug treatment via increased production of cytokines that are known to be essential for hematopoiesis.


Bone marrow stromal fibroblasts secrete interleukin-6 and granulocyte-macrophage colony-stimulating factor in the absence of inflammatory stimulation: demonstration by serum-free bioassay, enzyme-linked immunosorbent assay, and reverse transcriptase polymerase chain reaction. Bone marrow (BM) stromal fibroblasts produce hematopoietic growth factors (HGFs) in response to inflammatory mediators such as tumor necrosis factor-alpha or interleukin-1 alpha (IL-1 alpha). In the absence of such inflammatory stimuli, production of HGFs by BM stromal cells has been problematic and controversial. In vivo, however, basal hematopoiesis maintains blood counts within a normal homeostatic range even in the absence of inflammation, and HGFs are required for progenitor cell differentiation in vitro. To better ascertain the contribution of BM stromal fibroblasts to basal hematopoiesis, we therefore studied HGF production in quiescent BM stromal fibroblasts by three sensitive assays: serum-free bioassay, enzyme-linked immunosorbent assay, and reverse transcriptase polymerase chain reaction. Stromal fibroblasts were cultured in the presence or absence of normal human serum to determine if serum factor(s) present in the noninflammatory (basal) state induce secretion of HGFs. Human serum was found to induce or enhance transcription and secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and enhance secretion of constitutively expressed IL-6. In contrast, no secretion of either granulocyte-CSF (G-CSF) or IL-3 was found. These data indicate that factors in normal human serum are active in enhancing GM-CSF and IL-6 production by stromal fibroblasts and suggest that these growth factors contribute to the maintainance of normal, basal hematopoiesis in vivo.


Effects of human stem cell factor (c-kit ligand) on proliferation of myeloid leukemia cells: heterogeneity in response and synergy with other hematopoietic growth factors. A novel hematopoietic growth factor, the stem cell factor (SCF), for primitive hematopoietic progenitor cells has recently been purified and its gene has been cloned. In this study we tested the mitogenic activity of recombinant human SCF on myeloid leukemia cells as well as the expression of its receptor. We have investigated the proliferation of 31 myeloid leukemia cell lines as well as fresh myeloid leukemic blasts from 17 patients in a 72-hour 3H-thymidine uptake assay in the presence of various concentrations of recombinant human (rh) SCF alone or in combination with saturating concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, interleukin-3 (IL-3), or erythropoietin (EPO). Only five of 31 lines, but fresh leukemic blasts from 12 of 17 patients with acute myeloid leukemia (AML), significantly responded to SCF. The responding cell lines were of the acute promyelocytic, chronic myeloid, megakaryoblastic, and erythroleukemia origin, the responding blast preparations of all French-American-British subtypes. Synergistic activities of SCF were found with G-CSF, GM-CSF, EPO, and IL-3. To determine the SCF binding sites on leukemic cells, we used 125I-radiolabeled SCF in Scatchard analysis and cross-linking studies. The leukemic cell lines responding to SCF expressed from 2,300 up to 29,000 binding sites per cell. The SCF receptor expression was downregulated in vitro by the presence of its ligand. Cross-linking studies demonstrated a 150-Kd SCF receptor on the surface of all responding myeloid leukemias. This study suggests that SCF may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor for other cytokines. Furthermore, using polymerase chain reaction analysis of total RNA from the myeloid leukemia lines, we found expression of SCF-mRNA in 17 of 30 lines, suggesting autocrine mechanisms in the growth of a subgroup of leukemic cells by coexpression of SCF and its receptor.


Kit ligand improves in vitro erythropoiesis in myelodysplastic syndrome. Erythropoiesis in response to erythropoietin (Epo) in myelodysplastic syndrome (MDS) in vitro and in vivo is severely impaired. We investigated the stimulative effect of c-kit ligand (KL) on the erythroid colony-forming abilities of bone marrow cells from 17 patients with MDS. The effects of normal donor-derived marrow were examined in comparison. Suppression of erythroid colony formation in MDS in response to Epo could not be restored by the addition of interleukin-3 (IL-3) to culture. In cultures dishes supplemented with KL, erythroid colony formation was dramatically enhanced, regarding both colony number and size. Colony-forming abilities by MDS progenitors were improved following costimulation with KL, particularly in refractory anemia (RA) and refractory anemia with ring sideroblasts (RARS); however, little enhancement was apparent following KL stimulation of marrow from patients with refractory anemia with excess of blasts (RAEB), refractory anemia with excess of blasts in transformation (RAEB-t), and chronic myelomonocytic leukemia (CMML). These results suggest that KL responsiveness of patients with low-risk MDS may still be intact, and that with progression to high-risk MDS, erythroid progenitors lose proliferative reactivity to both KL and Epo stimulation. KL may have a therapeutic role in restoring erythropoiesis in a subset of patients with MDS.


Interleukin-11 stimulates multiple phases of erythropoiesis in vitro. Interleukin-11 (IL-11), a pleiotropic cytokine originally isolated from a primate bone marrow stromal cell line, has been shown to stimulate T-cell-dependent B-cell maturation, megakaryopoiesis, and various stages of myeloid differentiation, but to inhibit adipogenesis. Because stromal cells are essential for the maintenance of early hematopoietic progenitor cells in long-term culture, we investigated the effects of IL-11 on multipotent and erythroid precursors from murine bone marrow in vitro in suspension and semisolid cultures. Our results show that in the presence of IL-3 or c-kit ligand (KL), IL-11 has profound stimulatory effects on primitive multilineage hematopoietic progenitors, pre-CFC(multi), as well as on precursors representing various stages of erythroid differentiation observable in vitro, including CFC(multi), BFU-E, and CFU-E. In addition, the combination of KL with IL-11 also stimulated highly proliferative erythroid progenitors that yield remarkable macroscopic erythroblast colonies in culture. These results indicate that IL-11 is likely to play a pivotal role in early hematopoiesis and at multiple stages of erythropoiesis.


Functional expression of c-kit by acute myelogenous leukemia blasts is enhanced by tumor necrosis factor-alpha through posttranscriptional mRNA stabilization by a labile protein. The c-kit proto-oncogene encodes a transmembrane glycoprotein identical to the receptor for the recently cloned stem cell factor (SCF). The present study examines constitutive synthesis of transcripts in primary acute myelogenous leukemia (AML) blasts and the effects of recombinant human tumor necrosis factor (TNF)-alpha on c-kit mRNA expression in these cells. The c-kit transcripts were detectable at low levels in 10 of 10 different AML samples investigated. TNF treatment of AML cells was associated with enhanced c-kit mRNA expression in all specimens. Nuclear run-on transcription assays indicated that the c-kit gene was transcriptionally active in all leukemias examined and the rate of transcription was unaffected by exposure to TNF, suggesting posttranscriptional control mechanisms of c-kit mRNA accumulation. In the absence of TNF, the half-life of c-kit transcripts was 2 to 3 hours, while in TNF-treated AML cells, c-kit half-life was found to be 5 to 9 hours. Inhibition of protein synthesis reduced TNF-induced c-kit mRNA expression by Northern blot analysis, but did not affect the rate of c-kit gene transcription. In the presence of inhibition of protein synthesis, the half-life of c-kit transcripts in TNF-induced leukemia cells decreased to 2 to 4 hours. These findings indicate that levels of c-kit mRNA are controlled by a labile protein that is involved in TNF-mediated stabilization of c-kit transcripts. The effects of TNF-alpha also extended to the protein level in that TNF-alpha treatment of primary AMLs was associated with enhanced surface expression of the SCF receptor by some of these cells. While exogenous SCF induced clonogenic growth of all primary AML samples investigated, TNF-alpha failed to stimulate leukemic cells to proliferate. However, the combination of SCF and TNF-alpha resulted in synergistic growth stimulation in seven of nine different AML specimens investigated. The finding of transmodulation of the SCF receptor through posttranscriptional modifications might further contribute to our understanding of the synergistic interplay of TNF-alpha and SCF.


Human fetal liver-derived CD7+CD2lowCD3-CD56- clones that express CD3 gamma, delta, and epsilon and proliferate in response to interleukin-2 (IL-2), IL-3, IL-4, or IL-7: implications for the relationship between T and natural killer cells. Cells from fetal liver or fetal and adult bone marrow that are membrane (m)CD3 negative and have not rearranged TCR genes but express CD3 proteins in their cytoplasm are considered to be committed T-cell progenitors. Recent findings question whether CD3 is T-cell specific because fetal natural killer (NK) cells have been shown to express cCD3 delta and epsilon proteins. To further examine the relationship between T and NK cells, we generated mCD3-cCD3+ clones from fetal liver. Two stable clones, FL412 and FL508, isolated from different donors, were selected on the basis of absence of the NK cell marker CD56. These clones shared a common phenotype of CD7+CD2lowCD3-CD4-CD8-CD5-CD6-CD11b+CD16-CD56 -. Like fetal NK clones, these clones expressed cytoplasmic CD3 delta and epsilon transcripts and proteins. However, the clones exhibited no or very low levels of cytotoxic activity against K562, JY, or Daudi, which were lysed efficiently by fetal NK clones. TCR beta, gamma, and delta genes in these clones were in germline configuration. Furthermore, both FL412 and FL508 responded not only to interleukin-2 (IL-2), IL-4, or IL-7, but also to IL-3 with proliferation. These results suggest that FL412 and FL508 retained some characteristics of a putative T or NK precursor in the fetal liver and may be useful for analyses of this poorly defined cell type.


Interleukin-4 inhibits both paracrine and autocrine tumor necrosis factor-alpha-induced proliferation of B chronic lymphocytic leukemia cells. The proliferative response of purified malignant B cells from 26 patients with chronic lymphocytic leukemia (CLL) was investigated in vitro. In the majority of these patients, a proliferative response could be induced by the combination of tumor necrosis factor (TNF)-alpha and PMA. The concentration of PMA was found to be critical and showed a sharp optimum. In most cases maximal proliferation was obtained with as little as 0.1 ng/mL PMA. In all cases tested, TNF-alpha-induced proliferation could be inhibited completely by the addition of low doses of interleukin-4 (IL-4). Maximal inhibition was already found with 400 pg/mL IL-4. Inhibition by IL-4 was not caused by a downmodulation of TNF receptors. Apart from TNF-alpha, IL-2 was also in synergy with PMA able to induce proliferation in B-CLL cells of some patients. This IL-2-induced proliferation could be inhibited both by IL-4 and by neutralizing anti-TNF-alpha antibodies. This shows that TNF-alpha also can act as an autocrine growth factor. These data indicate that TNF-alpha is an important growth factor for neoplastic B-CLL cells and that IL-4 provides a tool to interfere with this TNF-alpha response.


Accumulation of high levels of methotrexate polyglutamates in lymphoblasts from children with hyperdiploid (greater than 50 chromosomes) B-lineage acute lymphoblastic leukemia: a Pediatric Oncology Group study. Hyperdiploidy (greater than 50 chromosomes, or a DNA index greater than 1.16) confers a favorable prognosis in B-lineage acute lymphoblastic leukemia of childhood. Children with B-lineage acute lymphoblastic leukemia whose lymphoblasts at diagnosis accumulate high levels of methotrexate (MTX) and MTX polyglutamates (MTXPGs) in vitro experience a better event-free survival than those whose lymphoblasts do not (Blood 76:44, 1990). Lymphoblasts from 13 children with hyperdiploidy (greater than 50 chromosomes) accumulated high levels of MTX-PGs (1,095 and 571 to 2,346 pmol/10(9) cells [median and 25% to 75% intraquartile range]). These levels were higher than those in B-lineage lymphoblasts from 19 children with other aneuploidy (326 and 159 to 775 pmol/10(9) cells) and 15 children with diploidy (393 and 204 to 571 pmol/10(9) cells) (P = .0015). Chromosomal trisomies in hyperdiploid cases were highly nonrandom. Chromosome 9 was not one of the chromosomes involved in trisomies, even though this chromosome contains the gene for folate polyglutamate synthetase, which is the enzyme required for MTXPG synthesis. The correlation between MTXPG level and percentage of S-phase cells was weak, suggesting that increased levels of MTXPGs could not be attributed to elevated proportions of cells in active DNA synthesis. The ability of hyperdiploid lymphoblasts to accumulate high levels of MTXPGs may increase their sensitivity to MTX cytotoxicity, accounting in part for the improved outlook for hyperdiploid patients treated with regimens that emphasize MTX as a primary component of continuation therapy.


Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy. The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth.


Recombinant human granulocyte-colony-stimulating factor in the treatment of patients with chronic benign granulocytopenia and congenital agranulocytosis (Kostmann's syndrome). Seven patients with chronic benign granulocytopenia and nine patients with congenital agranulocytosis, received consecutive seven-day courses of recombinant human granulocyte-colony stimulating factor at a starting dose of 50 micrograms/m2/day, subcutaneously. If there was no response the doses were increased to 300 micrograms/m2. All patients with chronic benign granulocytopenia responded rapidly at the minimum dose within 1-3 days after administration. By contrast, only three of the nine patients with congenital agranulocytosis responded within 1-7 days at this dose. Four patients with congenital agranulocytosis showed a response between days 7-19 at a dose of granulocyte-colony-stimulating factor 100-200 micrograms/m2 but in the remaining two cases no response was obtained. The administration of granulocyte-colony-stimulating factor was shown to be safe and effective also in reducing infectious episodes in these patients. Previously it was reported that granulocyte-colony-stimulating factor 10-30 micrograms/kg/day was effective for patients with congenital agranulocytosis. These results indicate that patients with congenital agranulocytosis may require much higher doses of recombinant human granulocyte-colony-stimulating factor than patients with chronic benign granulocytopenia and that the response to ordinary doses of recombinant human granulocyte-colony-stimulating factor may be useful in differentiating between chronic benign granulocytopenia and congenital agranulocytosis.


Tenascin: a potential modulator of cell-extracellular matrix interactions during vertebrate embryogenesis. Tenascin is a large oligomeric extracellular matrix (ECM) glycoprotein whose expression is highly restricted during vertebrate development. It has a characteristic hexameric quaternary structure with six arms linked to a central globular domain. Each arm contains a single polypeptide with the central globular domain formed by the covalent association of the N-terminal ends of the six polypeptides. Tenascin first appears during development, associated with the neural crest cell migration pathways of mammalian, avian and amphibian embryos. During later development, it is observed at sites of cartilage, bone and tendon formation. Tenascin expression also occurs in defined areas in the developing nervous system and in condensing mesenchyme, in response to epithelio-mesenchymal interactions. The function of tenascin in these different morphogenetic processes is not yet clearly understood. Tenascin can promote neurite outgrowth in vitro and can inhibit cell interactions with fibronectin. Results based on antibody mapping and molecular cloning indicate that these properties involve two distinct cell binding sites. Together with its highly regulated expression in the embryo, these properties suggest that tenascin plays a key role in the control of cell migration and differentiation during development.


Cloning and sequencing of a 24-kDa Trypanosoma cruzi specific antigen released in association with membrane vesicles and defined by a monoclonal antibody. In the present study we have used the Tcr7 monoclonal antibody (mAb) previously characterized as directed against Trypanosoma cruzi 24-25-kDa specific antigens, both are immunogenic in man and during experimental T cruzi infections. We have demonstrated that mAb Tcr7 was able to recognize two in vitro translation products of molecular weights of 24 and 25 kDa. This suggested the holoproteic nature of these two related antigens bearing at least a common epitope and allowed us to use Tcr7 for an immunoscreening of a lambda ZAPII T cruzi cDNA library. Indeed, we have obtained several positive clones and completely sequenced the largest one which encoded theoretically for a protein of 23.7 kDa. The sequence analysis revealed a nearly perfect homology between this clone and one already described by other investigators and was shown to express a major flagellar protein of T cruzi able to bind calcium. Using different overlapping peptides derived from the sequence of the cDNA clone, we have localized the immunoreactivity of mAb Tcr7 mainly on several primary sequences present in the N-terminal part of the sequence, suggesting that the mAb could recognize a discontinuous epitope. Moreover, the immunoelectron microscopy allowed us to show that the antigen(s) carrying the epitope reacting with mAb Tcr7 is (are) released in association with membrane vesicles which protruded from the parasite surface and the flagellar pocket. This new mechanism of antigen shedding is likely to be independent of phospholipase C-mediated release of GPI-anchored molecules.