Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pi for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the gamma-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](1,3,4,6)P4,[3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from PtdIns via PtdIns4P, PtdIns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)P5 is Ins(3,4,5,6)P4. Furthermore, if the gamma- (and/or beta-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the gamma- (and/or beta-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(1,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3.

The Escherichia coli dam gene and upstream sequences were cloned from the Kohara phage 4D4. Five promoters were found to contribute to dam gene transcription. P1 and P2 (the major promoter) were situated approximately 3.5 kb upstream of the structural gene, P3 was within the aroB gene, P4 was within the urf74.3 gene, and P5 was in the urf74.3-dam intergenic region. The nucleotide sequence of 2280 bp of DNA containing P1 and P2 was determined and shown to have the potential to encode a protein of approximately 16 kDa between P1, P2 and the aroB gene. This 16 kDa open reading frame has been identified as aroK, the gene for shikimic acid kinase I. Thus the dam gene is part of an operon containing aroK, aroB, urf74.3, and dam. The transcriptional start points of the promoters were determined. A comparison of their nucleotide sequences suggested that P1-P4 were all recognized by the sigma 70 subunit of the RNA polymerase.

Neonatal rats were exposed to ethanol throughout gestation, or during the early postnatal period (postnatal days 4-10 (P4-10)), and enzyme-linked immunoabsorbent assays were subsequently conducted in order to assess nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) protein content in hippocampus, septum, cortex/striatum and cerebellum. These determinations revealed that following prenatal ethanol treatment, there were significant ethanol-induced increases in NGF in P1 cortex/striatum, but no changes in any of the three neurotrophic factors (NTFs) in the other brain regions. Cortex/striatal NGF protein returned to control levels by P10. Following early postnatal exposure, BDNF was elevated in hippocampus and cortex/striatum (assessed on P10), and NGF was also enhanced in cortex/striatum at this age. Hippocampal and cortex/striatal BDNF returned to control levels by P21, but cortex/striatal NGF levels remained enhanced at this age. This NTF did not differ in ethanol and control animals by P60, however. The possible significance of elevated levels of NTFs as a function of ethanol exposure is discussed, and it is speculated that while such alterations could play a protective role, increases in these substances during critical developmental periods could also prove to be deleterious, and could even contribute to certain of the neuropathologies which have been observed following developmental ethanol exposure.

Autonomous parvoviruses are tightly dependent on host cell factors for various steps of their life cycle. In particular, DNA replication and gene expression of the prototype strain of the minute virus of mice (MVMp) are closely linked to the onset of host cell DNA replication, pointing to the involvement of an S-phase-specific cellular factor(s) in parvovirus multiplication. The viral nonstructural protein NS-1 is absolutely required for parvovirus DNA replication and is able to transcriptionally regulate parvoviral and heterologous promoters. We previously showed that the promoter P4, which directs the transcription unit encoding the NS proteins, is activated at the onset of S phase. This activation is dependent on an E2F motif in the proximal region of promoter P4. An infectious MVM DNA clone was mutated in the E2F motif of P4. The wild type and the E2F mutant derivative were tested for their ability to produce progeny viruses after transfection of permissive cells. In the context of the whole MVMp genome, the E2F mutation abolished P4 induction in S phase and inactivated the infectious molecular clone, which failed to become amplified and generate progeny particles. The virus could be rescued when NS proteins were supplied in trans, showing that P4 hyperactivity in S is needed to reach a level of NS-1 expression that is sufficient to drive the viral replication cycle. These data show that E2F-mediated P4 activation at the early S phase is a limiting factor for parvovirus production. The primary barrier to parvovirus gene expression in G1 is thought to be promoter formation rather than activation, due to the poor conversion of the parental single-strand genome to a duplex form. The S dependence of P4 activation may therefore be a sign of the virus adaptation to life in the S-phase host cell. If the conversion block in G1 were to be leaky, the S induction of promoter P4 could be envisioned as a safeguard against the production of toxic NS proteins until cells reach the S phase and provide the full machinery for parvovirus replication.

Although the phages P2 and P4 build their capsids from the same precursor, the product of the P2 N gene, the two capsids differ in size: P2 builds a 60 nm, T = 7 capsid from 420 subunits, whereas P4 makes a 45 nm, T = 4 capsid from 240 subunits. This difference leads to substantial changes in shell geometry and subunit interactions. Previous results have demonstrated that the P4 sid gene is responsible for the assembly of P4-sized shells. We have used cryo-electron microscopy and image reconstruction to determine the structure of a putative assembly intermediate of P4 capsids, produced in vivo from cloned genes. We demonstrate that Sid forms a P4-specific scaffold with icosahedral symmetry on the outside of the procapsid-like particles. The Sid molecules (60 or 120 copies) form lofty arches that interact with the gpN hexamers on the icosahedral 2-fold axes, and connect as trimers over the 3-fold axes, forming a continuous dodecahedrally shaped outer cage. The gpN shell inside the Sid cage is approximately 40 nm wide, consistent with the previously suggested maturational expansion. The main difference with respect to the mature P4 capsids is found in the hexamers, which appear strongly elongated and more protruding than in the mature shell. These and previous results are discussed in the light of a model for regulation of capsid size.

The spatial patterns of IGF2 and H19 gene expression are strikingly similar during parts of human embryonic/fetal and early postnatal development. Notable exceptions were found with the ciliary anlage of the embryonic retina and the choroid plexus/leptomeninges, where transcripts from the IGF2 but not the H19 locus could be detected. Moreover, in contrast to the other tissue samples examined, the choroid plexus/leptomeninges expressed both parental IGF2 alleles. Whilst RNase protection analysis revealed a weak activity of the P1 promoter in the choroid plexus/leptomeninges, the P2, P3 and P4 promoters were all active wherever IGF2 was expressed. We discuss these observations with respect to a hypothesized coordinated control of the reciprocally imprinted and closely linked IGF2 and H19 loci.

The serum transferrins are monomeric proteins with a molecular mass of around 80 kDa and are responsible for the transport of iron in vertebrates. The three-dimensional structures of diferric porcine and rabbit serum transferrin have been refined against X-ray diffraction data extending to 2.15 and 2.60 A, respectively. Data for both proteins were collected using synchrotron radiation at temperatures of 277 K. The porcine protein crystallizes in the space group C2, with unit-cell parameters a = 223.8, b = 44.9, c = 78.9 A, beta = 105.4 degrees with one molecule in the asymmetric unit. The structure was solved by molecular-replacement methods using rabbit serum transferrin as the search model. The structure was refined using REFMAC, with a final residual of 13.8% (R(free) = 18.2% for a 5% data sample) for all data to 2.15 A. The final model comprises 5254 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, one N-acetyl glucosamine moiety and 494 water molecules. The rabbit protein crystallizes in space group P4(3)2(1)2, with unit-cell parameters a = 127.2, c = 144.9 A and one molecule per asymmetric unit. The structure was solved using the method of multiple isomorphous replacement and refined using REFMAC to give a final residual of 18.6% (R(free) = 22.2% for a 5% data sample) for all data to 2.60 A. The final model comprises 5216 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, a Cl(-) anion and 206 solvent molecules; there is no clear indication of the carbohydrate moiety attached to Asn490 (rabbit serum numbering). Both molecules adopt a bilobal structure typical for members of the transferrin family. Each of the structurally homologous lobes contains two dissimilar domains with a single iron-binding site buried within the interdomain cleft. The porcine serum protein lacks an interdomain disulfide bridge close to the connecting peptide between the lobes, but this seems to have little effect on the overall orientation of the lobes. The N-lobes of both proteins possess lysine residues, one from each of the two domains, that lie in close proximity to one another to form the so-called dilysine trigger. The more acid-labile release of iron from serum transferrins than from lactoferrins is discussed.

In cattle, elevated concentrations of circulating progesterone (P4) in the immediate postconception period are associated with advanced conceptus development, while low P4 is implicated as a causative factor in low pregnancy rates observed in dairy cows. This study aimed to: 1) describe the transcriptional changes that occur in the bovine endometrium during the estrous cycle, 2) determine how elevated P4 affects these changes, 3) identify if low P4 alters the expression of these genes, and 4) assess the impact that low P4 has on conceptus development. Relatively few differences occurred in endometrial gene expression during the early luteal phase of the estrous cycle (Day 5 vs. 7), but comparison of endometria from more distant stages of the luteal phase (Day 7 vs. 13) revealed large transcriptional changes, which were significantly altered by exogenous supplementation of P4. Induction of low circulating P4 altered the normal temporal changes in gene expression, and these changes were coordinate with a delay in the down-regulation of the PGR from the LE and GE. Altered endometrial gene expression induced by low P4 was associated with a reduced capacity of the uterus to support conceptus development after embryo transfer on Day 7. In conclusion, the present study provides clear evidence that the temporal changes in the transcriptome of the endometrium of cyclic heifers are sensitive to circulating P4 concentrations in the first few days after estrus. Under low P4 conditions, a suboptimal uterine environment with reduced ability to support conceptus elongation is observed.

While both 17β-estradiol (E2) and progesterone (P4) are neuroprotective in several experimental paradigms, P4 also counteracts E2 neuroprotective effects. We recently reported that a 4-h treatment of cultured hippocampal slices with P4 following a prolonged (20 h) treatment with E2 eliminated estrogenic neuroprotection against NMDA toxicity and induction of brain-derived neurotrophic factor (BDNF) expression. In the present study, we evaluated the effects of the same treatment on levels of estrogen receptors, ERα and ERβ, and BDNF using a similar paradigm. E2 treatment resulted in elevated ERβ mRNA and protein levels, did not modify ERα mRNA, but increased ERα protein levels, and increased BDNF mRNA levels. P4 reversed E2-elicited increases in ERβ mRNA and protein levels, in ERα protein levels, and in BDNF mRNA levels. Experiments with an ERβ-specific antagonist, PHTPP, and specific agonists of ERα and ERβ, propylpyrazoletriol and diarylpropionitrile, respectively, indicated that E2-mediated neuroprotection against NMDA toxicity was, at least in part, mediated via ERβ receptor. In support of this conclusion, E2 did not protect against NMDA toxicity in cultured hippocampal slices from ERβ-/- mice. Thus, E2-mediated neuroprotection against NMDA toxicity may be because of estrogenic induction of BDNF via its ERβ receptor, and P4-mediated inhibition of E2 neuroprotective effects treatment to P4-induced down-regulation of ERβ and BDNF.

Zones containing actively dividing cells (proliferation zones: PZs), in the brain of adult three-spined sticklebacks, were identified by autoradiographic detection of (3)H-thymidine and immunocytochemical detection of the thymidine analogue 5'-bromodeoxyuridine (BrdU), singly or in combination, and by immunocytochemical detection of proliferating cell nuclear antigen (PCNA) by monoclonal antibodies. The PZs are associated with boundaries between adult brain regions, as well as with defined morphofunctional subdivisions. PZs are located at the border between the telencephalon and diencephalon, and at the border between the mesencephalon and the rhombencephalon. In the midbrain, the PZ follows the dorsomedial, caudal, and ventrolateral aspects of each tectal hemisphere, extending over the caudal aspect of the torus semicircularis to the nucleus lateralis valvulae. In the hindbrain, the major PZ apparently represents the persisting embryonic secondary matrix layer of the developing cerebellum. In the forebrain, the PZs are associated with the ventricular zones of the olfactory bulbs and ventral telencephalic area ("subpallium"), dorsal telencephalic area ("pallium"), preoptic region, ventral thalamus, dorsal thalamus, epithalamus, pretectum, posterior tuberculum, and the hypothalamus. The diencephalic PZs are parcellated according to a neuromeric organisation (a synencephalic, a posterior, and an anterior parencephalic neuromere: p1, p2, and p3). The PZs of the secondary prosencephalon (telencephalon and hypothalamus) thus would belong to neuromeres p4-6, but do not form an immediately recognised serial pattern. The prosencephalic PZs correlate well with parts of embryonic migration areas as defined by Bergquist and Källén ([1954] J. Comp. Neurol. 100:627-659), morphogenetic fields from which postmitotic neurones migrate to their final destination.

HLA-DQA1*0102/DQB1*0602 (DQ0602) is observed at a decreased frequency in insulin-dependent diabetes mellitus in different ethnic groups, suggesting a protective role for DQ0602. Analysis of overlapping peptides from human insulin found that insulin B(1-15) bound well to DQ0602 and exhibited a high degree of allelic specificity. Truncation analysis of insulin B(1-15) identified insulin B(5-15) as the minimal peptide for DQ0602 binding. Insulin B(5-15) bound to DQ0602 with an apparent KD of 0.7 to 1.0 microM and peptide binding reached equilibrium at 96 h. Single arginine substitutions at each position of the insulin B(5-15) peptide identified amino acids 6, 8, 9, 11, and 14 (relative positions P1, P3, P4, P6, and P9) as important for binding. Extensive substitutions for each of these amino acids revealed that amino acids 11 and 14 (P6 and P9) exhibited the highest specificity. Amino acid 11 (P6) preferred large aliphatic amino acids, while amino acid 14 (P9) preferred smaller aliphatic and hydroxyl amino acids. Binding of an overlapping series of peptides from a randomly chosen protein, the herpes simplex virus-2 tegument protein UL49, correlated completely with the presence or absence of the DQ0602 peptide binding motif. Peptides 11 amino acids long were selected from GAD65, IA-2, and proinsulin, that contained the DQ0602 peptide binding motif. Of these, 79% (19 of 24) were able to bind DQ0602. This study identifies a peptide binding motif for DQ0602 and peptides from insulin-dependent diabetes mellitus autoantigens that bind DQ0602 in vitro.

The neuroendocrine granule-associated protein 7B2, unlike many other neuroendocrine precursor proteins stored in secretory granules, carries in its primary structure the Arg-Xaa-Arg/Lys-Arg processing site usually found in constitutively secreted precursor proteins and recognized by the ubiquitously expressed convertase, furin. pro7B2 (30 kDa), when expressed in endocrine (AtT-20, PC12, and GH4C1) or non-endocrine (Ltk-) cell lines using recombinant vaccinia viruses, was converted to a 23-kDa form. Mutation of the P4 Arg to Gly completely prevented this conversion. When excess pro7B2 was coexpressed with the pro-protein convertases PC1, PC2, or furin, only furin could induce complete processing. In addition, coexpression of pro7B2 in LoVo cells, which are devoid of endogenous furin activity, with each one of the three convertases, showed that only furin was able to induce processing of this precursor. pro7B2 processing in AtT-20 was completely abolished when protein transport into Golgi compartments was blocked by cell incubation at either 15 or 37 degrees C in the presence of monensin or brefeldin A. Furthermore, pulse-chase experiments in the presence of Na2[35S]SO4 showed that pro7B2 is Tyr-sulfated in the trans-Golgi network before it is processed. These results demonstrate that pro7B2 is first processed by a furin-like enzyme within the trans-Golgi network into a 23-kDa form that is then sequestered into secretory granules.

The involvement of inositol lipid metabolism in agonist-mediated Ca2+ signaling by Ins 1,4,5-P3 has become firmly established. Recent advances have led to a better understanding of the proteins associated with signal transduction in the plasma membrane. A number of specific receptors (G proteins, phospholipases and inositol lipid kinases) have now been purified and characterized. An Ins 1,4,5-P3 receptor has also been purified which is presumably involved in mediating Ca2+ efflux from intracellular stores. The morphological site of the hormone-sensitive Ca2+ pool has been tentatively identified as discrete, specialized intracellular structures (calciosomes), but further studies are required to demonstrate that these contain Ins 1,4,5-P3-gated Ca2+ channels and their possible functional relationship to the plasma membrane. Receptor occupancy by Ca2+ mobilizing agonists also stimulates Ca2+ entry into the cell, but the mechanism for activation of voltage insensitive Ca2+ channels and the possible involvement of Ins 1,4,5-P3, Ins 1,3,4,5-P4 and/or G proteins in this process has not been established. The Ca2+ signaling pathway is subject to multisite feedback regulation by Ca2+ itself and by a diacylglycerol-mediated activation of protein kinase C. Potential sites for Ca2+ interaction are displacement of Ins 1,4,5-P3 from its receptor by a Ca2+-dependent mechanism, promotion of Ins 1,3,4,5-P4 formation by the Ca2+/calmodulin-regulated Ins 1,4,5-P3 3-kinase, and efflux of Ca2+ from the cell or sequestration into intracellular Ca2+ stores by Ca2+/calmodulin-regulated Ca2+-ATPases. Protein kinase C activation potentially affects the rate of generation of Ins 1,4,5-P3 by negative feedback to the receptor-G protein-phospholipase C transduction system and possibly also the rate of Ins 1,4,5-P3 degradation by activation of an inositol polyphosphate 5-phosphomonoesterase. It may also attenuate the Ca2+ transient directly by increasing the activity of Ca2+-ATPases associated with the plasma membrane and the endoplasmic reticulum. Cell-to-cell heterogeneity in the relative control strengths of these different mechanisms may explain the differences in the Ca2+ signal in different tissues and even in different cells within a population. The ability of Ca2+ and protein kinase C to provide negative feedback at various points in the signal transduction pathway suggests that a complex mechanism involving multiple feedback loops is likely to regulate the generation of Ca2+ oscillations seen in some cells.(ABSTRACT TRUNCATED AT 400 WORDS)

alpha Protein of satellite phage P4 of Escherichia coli is multifunctional in P4 replication with three activities. First, the protein (subunit M(r) = 84,900) complexes specifically the P4 origin and the cis replication region required for replication. alpha Protein interacts with all six type I repeats (TGTTCACC) present in the origin. Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate. All common NTPs except UTP and dTTP can serve as cofactors. Strand separation of partial duplexes containing tailed ends that resemble a replication fork is preferred, although a preformed fork is not absolutely required for the enzyme to invade and unwind duplex DNA. alpha Protein catalyzes unwinding in the 3'-5' direction with respect to the strand it has bound. Finally, the primase activity already demonstrated for alpha protein is due to synthesis of RNA primers. In vitro, alpha protein generates di- to pentaribonucleotides on single-stranded phage fd DNA. The predominant product is the dimer pppApG, on which most of the longer oligoribonucleotides are based. Using DNA oligonucleotides of defined sequence as templates, synthesis of pppApG was also detectable. To date, among prokaryotic and eukaryotic replication systems, gp alpha is the only protein known that combines three activities on one single polypeptide chain.

Cultures of established rat fibroblasts transformed by the avian erythroblastosis virus were more susceptible to the cytopathic effect of the autonomous parvovirus minute virus of mice, prototype strain (MVMp), than were their untransformed homologs. This effect could be ascribed to the presence of a greater fraction of cells that were sensitive to the killing action of MVMp in transformed cultures than in their normal parents. Yet, transformed and normal lines were similarly efficient in virus uptake, DNA amplification, and capsid protein synthesis. In contrast, transformants accumulated 2.5- to 3-fold greater amounts of all three major MVM mRNA species and nonstructural protein than did their normal progenitors. Thus, in this system transformation-associated sensitization of cells to MVMp appears to correlate primarily with an increase in their capacity for the expression of the viral transcription unit which encodes nonstructural proteins and is controlled by the P4 promoter. Consistently, a reporter gene was expressed at a higher level by transformed versus normal cultures, when placed under the control of the MVM P4 promoter. As infectious MVMp was produced in larger amounts by transformed cultures, a late step of the parvoviral cycle, such as synthesis, encapsidation of progeny DNA, or both, was also stimulated in the transformed cells.

Netrin-1 is a bifunctional guidance cue that directs migrating neurons and axons based on specific receptors expressed on the cell surface. Attraction occurs through the receptor Deleted in Colorectal Cancer (DCC) and repulsion occurs through a receptor complex of DCC and UNC5H, the vertebrate homolog to Caenorhabditis elegans UNC-5, but how the specific surface expression of these receptors is achieved remains unknown. Here, we demonstrate that surface expression of UNC5H1 is regulated in neurons by protein interacting with C kinase-1 (PICK1) and protein kinase C (PKC), and show that one mechanism by which cells control their response to netrin-1 is by changing the surface availability of receptors. We identified PICK1 as a binding partner for UNC5H1 using the yeast two-hybrid system and found that the extreme three C-terminal amino acids of UNC5H1 interact with the PSD-95/Dlg/ZO-1 (PDZ) domain of PICK1. Coexpression of UNC5H1 and PICK1 in heterologous cells results in the recruitment of PICK1 to UNC5H1 clusters. Endogenous UNC5H1 and PICK1 coimmunoprecipitate from extracts of cultured hippocampal neurons and P4 cortices, and immunohistochemistry shows that UNC5H1, PICK1, and PKC are all present in growth cones. PKC activation induces the formation of UNC5H1/PICK1/PKC complexes and leads to the specific removal of UNC5H1, but not DCC, from the surface of neurons and growth cones via a PICK1/PKC-dependent mechanism. Lastly, we demonstrate that activating PKC, which decreases surface expression of UNC5H1, inhibits netrin-1-dependent collapse of hippocampal growth cones. Together, our results suggest that by regulating the surface expression of UNC5Hs, an axon can modulate its repellent response to netrin-1.

We have previously developed a new method for the development and maintenance of uveal melanoma (UM) xenografts in immunodeficient mice. Here, we compare the genetic profiles of the primary tumors to their corresponding xenografts that have been passaged over time. The study included sixteen primary UMs and corresponding xenografts at very early (P1), early (P4), and late (P9) in vivo passages. The tumors were analyzed for mutation status of GNAQ, GNA11, GNAS, GNA15, BAP1, and BRAF, chromosomal copy number alterations using Affymetrix GeneChip(®) Genome-Wide Human SNP6.0 arrays, gene expression profiles using GeneChip(®) Human Exon 1.0 ST arrays, BAP1 mRNA and protein expression, and MAPK pathway status using Reverse Phase Protein Arrays (RPPA). The UM xenografts accurately recapitulated the genetic features of primary human UMs and they exhibited genetic stability over the course of their in vivo maintenance. Our technique for establishing and maintaining primary UMs as xenograft tumors in immunodeficient mice exhibit a high degree of genetic conservation between the primary tumors and the xenograft tumors over multiple passages in vivo. These models therefore constitute valuable preclinical tool for drug screening in UM.

The present study examined several aspects of epidermal growth factor receptor (EGF-R) in the mouse uterus during the peri-implantation period and after ovarian hormone treatment of adult ovariectomized mice. The cell-specific distribution, regulation of expression, and binding kinetics were assessed by immunohistochemistry, Northern blot analysis, and ligand binding assays, respectively. Affinity cross-linking studies ascertained the size of the EGF-R, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase activity and receptor autophosphorylation. In the intact uterus and separated cell types, EGF-R was detected in the stroma, deciduum, and myometrium, but not in the luminal or glandular epithelium. Uterine EGF-R mRNA transcript profiles showed some differences between pregnant and ovariectomized mice regardless of steroid hormone treatments. Two major [6.5- and 2.7-kilobase (kb)] and two less abundant (9.6- and 5.0-kb) transcripts were detected in pregnant uterine poly(A)+ RNA. Three additional transcripts (< 2.0 kb) were detected in decidual poly(A)+ RNA, and a larger transcript (8.0 kb) was detected in uterine poly(A)+ RNA isolated from ovariectomized mice. Scatchard analysis of EGF binding also revealed apparent differences in binding kinetics between pregnant and ovariectomized mice, although EGF was cross-linked to a 170-kilodalton protein under these conditions. Two classes (Kd, approximately 0.2 and approximately 2.0 nM) of binding sites were noted in pregnant mice, whereas a single class (Kd, approximately 1.0 nM) was found in ovariectomized mice. 17 beta-Estradiol (E2) caused a rapid transient upregulation of uterine EGF-R mRNA levels and increased the number of EGF-binding sites in ovariectomized mice, as did an injection of progesterone (P4). However, the bioactivity of EGF-R could not be detected in uteri of ovariectomized mice treated with oil or P4. E2 treatment was found to be essential for EGF-R bioactivity. Taken together, the results suggest that in the adult mouse uterus, EGF-R status is influenced by factors other than P4 and E2, the epithelium is not the direct target for the actions of EGF-related growth factors as thought previously, the mitogenic effects of these growth factors on epithelial cells in vivo are perhaps mediated by other uterine cell-types expressing EGF-R, and, lastly, these growth factors are not likely to be functional in the uterus in the absence of estrogen. The present observations are supportive of the concept of paracrine or juxtacrine interactions between EGF-related growth factor ligands of luminal epithelial origin and blastocyst EGF-R in the process of implantation.

OBJECTIVE

To investigate the importance of progesterone (P4) metabolism by the 5alpha-reductase type I enzyme in mitigating P4 antiseizure effects.

METHODS

Ovariectomized, female homozygous and heterozygous 5alpha-reductase type I knockout mice (n = 23) and their wild-type siblings (n = 31) were administered P4 (1.0 mg), and their pentylenetetrazol (PTZ)-induced ictal behaviors were compared with those of vehicle-administered mice (n = 49).

RESULTS

Mice deficient in the 5alpha-reductase type I enzyme administered P4, or vehicle-administered control mice, had significantly shorter latencies and increased incidence of PTZ-induced hindlimb extension and death than did wild-type mice administered P4.

CONCLUSIONS

These data suggest that P4's metabolism by the 5alpha-reductase type I enzyme may mitigate some of P4's antiseizure effects in the PTZ-induced seizure model.

The helper component-proteinase (HC-Pro) encoded by potyviruses functions to cleave the viral polyprotein by an autoproteolytic mechanism at the HC-Pro C-terminus. This protein belongs to a group of viral cysteine-type proteinases and has been shown previously to catalyze proteolysis between a Gly-Gly dipeptide. The amino acid sequence requirements surrounding the HC-Pro C-terminal cleavage site of the tobacco etch virus polyprotein have been investigated using site-directed mutagenesis and in vitro expression systems. A total of 51 polyprotein derivatives, each differing by the substitution of a single amino acid between the P5 and P2' positions, were tested for autoproteolytic activity. Substitutions of Tyr (P4), Val (P2), Gly (P1), and Gly (P1') were found to eliminate or nearly eliminate proteolysis. Substitutions of Thr (P5), Asn (P3), and Met (P2'), on the other hand, were permissive for proteolysis, although the apparent processing rates of some polyproteins containing these alterations were reduced. These results suggest that auto-recognition by HC-Pro involves the interaction of the enzymatic binding site with four amino acids surrounding the cleavage site. Comparison of the homologous sequences of five potyviral polyproteins revealed that the residues essential for processing are strictly conserved, whereas the nonessential residues are divergent. The relationship between HC-Pro and other viral and cellular cysteine-type proteinases is discussed.

In 23 follicular fluids (FF) each yielding an oocyte known to result in a clinical pregnancy after in vitro fertilization, the following substances were measured: free and total concentrations of estradiol (E2), progesterone (P4), testosterone (T), androstenedione (A), immunoreactive inhibin, insulin-like growth factor-binding protein-1, alpha 1-antitrypsin, and placenta protein-14. In addition, FF volume and follicular diameter at the time of oocyte recovery were recorded. The characteristics of these pregnancy-associated follicles were compared with those of FF obtained from women who failed to conceive after embryo transfer. The FF were collected from 14 women who became pregnant and 14 women who did not conceive. The ultrashort GnRH agonist protocol was used for ovarian stimulation. Pregnancy was associated with follicles showing a significantly higher E2/T ratio than follicles in which the oocyte failed to implant or did not cleave in vitro (P < 0.01). In addition, FF volume and follicular diameter were significantly higher in pregnancy-associated follicles than in follicles in which the oocyte failed to implant or did not cleave in vitro (P < 0.05). The E2/A ratio was higher in pregnancy-associated follicles than in follicles in which the oocyte failed to implant, but the difference did not reach significance (0.05 < P < 0.1). No difference was found when pregnancy-associated follicles and follicles not associated with pregnancy were compared with respect to the levels of free and total E2, P4, T, A, immunoreactive inhibin, insulin-like growth factor-binding protein-1, alpha 1-antitrypsin, placental protein-14, and the E2/P4 ratio. This study demonstrates a correlation between the pregnancy potential of oocytes and the ratio of E2 to androgens in FF. It confirms and extends the correlation between the E2/androgen ratio and follicular health and maturity. A low E2/androgen ratio seems to express early follicular atresia, which affects the viability of the enclosed oocyte negatively and limits the chances of a resulting preembryo to implant and establish itself as a pregnancy.

The sequence specificities of human fibroblast and neutrophil collagenases have been investigated by measuring the rate of hydrolysis of 60 synthetic oligopeptides covering the P4 through P'5 subsites of the substrate. The choice of peptides was patterned after both known cleavage sites in noncollagenous proteins and potential cleavage sites (those containing Gly-Ile-Ala, Gly-Leu-Ala, or Gly-Ile-Leu sequences) found in types I, II, III, and IV collagens. The initial rate of hydrolysis of the P1-P'1 bond of each peptide has been measured under first-order conditions ([SO] much less than KM), and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P4 through P'4 all influence the hydrolysis rates for both collagenases. However, the effects of substitutions at each site are distinctive and are consistent with the view that human fibroblast and neutrophil collagenases are homologous but nonidentical enzymes. For peptides with unblocked NH2 and COOH termini, occupancy of subsites P3 through P'3 is necessary for rapid hydrolysis. Compared with the alpha 1(I) cleavage sequence, none of the substitutions investigated at subsites P3, P2, and P'4 produces markedly improved substrates. In contrast, many substitutions at subsites P1, P'1, and P'2 improve specificity. The preferences of both collagenases for alanine in subsite P1 and tryptophan or phenylalanine in subsite P'2, is noteworthy. Human neutrophil collagenase accommodates aromatic residues in subsite P'1 much better than human fibroblast collagenase. The subsite preferences observed for human fibroblast collagenase in these studies agree well with the residues found at cleavage sites in noncollagenous substrates. However, the sequence specificities of these collagenases cannot explain the failure of these enzymes to hydrolyze many potentially cleavable but apparently protected sites in intact collagens. This represents additional support for the notion that the local structure of collagen is important in determining the location of collagenase cleavage sites.

In this study, we determined the boundaries of a 99-kb deletable element of Shigella flexneri 2a strain YSH6000. The element, designated the multiple-antibiotic resistance deletable element (MRDE), had recently been found to contain a 66-kb pathogenicity island (PAI)-like element (designated the SRL PAI) which carries the Shigella resistance locus (SRL), encoding resistance determinants to streptomycin, ampicillin, chloramphenicol, and tetracycline. The YSH6000 MRDE was found to be flanked by two identical IS91 elements present at the S. flexneri homologs of the Escherichia coli genes putA and mdoA on NotI fragment D. Sequence data from two YSH6000-derived MRDE deletants, YSH6000T and S2430, revealed that deletion of the MRDE occurred between the two flanking IS91 elements, resulting in a single IS91 element spanning the two original IS91 loci. Selection for the loss of tetracycline resistance confirmed that the MRDE deletion occurred reproducibly from the same chromosomal site and also showed that the SRL PAI and the SRL itself were capable of independent deletion from the chromosome, thus revealing a unique set of nested deletions. The excision frequency of the SRL PAI was estimated to be 10(-5) per cell in the wild type, and mutation of a P4-like integrase gene (int) at the left end of the SRL PAI revealed that int mediates precise deletion of the PAI.

Previous studies have demonstrated a female disadvantage in airway diseases, such as asthma and bronchiectasis. The basis for this sex disparity is unknown. We hypothesized that the female sex hormone, progesterone (P4), inhibits functions of the normal airway mucociliary apparatus. P4 receptor (PR) expression was evaluated in human lung and cultured primary human airway epithelial cells isolated from male and female lung transplant donors. PR expression was restricted to the proximal region of the cilia of airway epithelia, and was similar in men and women. Expression of isoform PR-B was more abundant than PR-A in cells from both sexes. Airway epithelial cell exposure to P4 decreased cilia beat frequency (CBF) by 42.3% (±7.2). Inhibition of CBF was prevented by coadministration of P4 with the active form of estrogen, 17β-estradiol, or the PR antagonist, mifepristone. P4 inhibition was time and dose dependent, with a significant decrease by 8 hours and maximal effect at 24 hours, accompanied by translocation of PR from the cilia to the nucleus. Inhibition of cilia beat was also prevented by treatment of cells with actinomycin D, suggesting that CBF inhibition is a transcriptionally mediated event. Together, these findings indicate that sex hormones influence the function of a key component of the mucociliary apparatus. These mechanisms may contribute to the sex disparity present in airway diseases and provide therapeutic targets for the treatment of these debilitating airway diseases.