Increased liver blood flow (LBF) resulting from elevated feed intake in lactating dairy cows may increase steroid metabolism. Continuous infusion of bromosulphthalein (BSP; specifically metabolized in liver) was used to measure LBF. Similarly, progesterone (P4) and estradiol-17beta (E2) were administered by continuous infusion. Circulating concentrations at steady state were used to calculate the metabolic clearance rate (MCR) of BSP, P4, and E2. Experiment 1: Variation in LBF was determined in thee nonlactating and four lactating cows over 3 d at 3 to 5 h after feeding. Coefficients of variation ranged from 14 to 31% among cows within day and from 4 to 8% within cows across days. Experiment 2: Six nonlactating cows were used in a 3 x 3 Latin-square design with three feed regimens: no feed, 0.5 maintenance diet (M), and 1.5 M. Experiment 3: Eight lactating cows were used in a 4 x 4 Latin-square design with four feed regimens: no feed, 0.5 M, 1.5 M, and 2.2 M. In experiments 2 and 3, LBF and MCR of P4 increased immediately after feed consumption and increases persisted longer at higher intakes. The LBF reached a maximum at 2 h after feeding and MCR of P4 reached maximum at 3 h after feeding with a positive correlation (r = 0.92) between LBF and MCR for P4. Experiment 4: A crossover design was used to determine MCR of E2 in unfed or full-fed lactating dairy cows. The MCR of E2 increased immediately after feeding and stayed elevated throughout the 4.5-h infusion period. Thus, LBF and steroid metabolism were acutely elevated by feed consumption in lactating and nonlactating cows. Higher rates of LBF and steroid metabolism in lactating than in nonlactating cows may indicate chronic effects of higher feed intakes as well.

Uterine expression of lactoferrin (LF) during the preimplantation period and its regulation by the ovarian steroids estradiol (E2) and/or progesterone (P4) in ovariectomized adult mice were examined. Immunoblot detection of LF in uterine cell lysates demonstrated the presence of this protein from days 1-8 of pregnancy [day 1 (D1) = day of vaginal plug]. Immunoprecipitation of 35S pulse-labeled uterine proteins showed that the relative rate of LF synthesis was high on D1, but below the level of detection by D4. Immunolocalization of LF in uterine sections showed intense luminal and glandular epithelial staining on D1 and D2, and progressively decreased staining through D4. Immunoreactive protein was also detected in cells, primarily concentrated in the stroma. The relative number of these cells was greatest on D1 and decreased progressively to a low number by D4. These cells were morphologically similar to neutrophils, which are known to contain LF protein, but little or no LF mRNA. Northern blotting showed that uterine LF mRNA levels were very high on D1 and D2 of pregnancy and decreased to low, but detectable, levels by D4. In situ hybridization to uterine sections showed that LF mRNA was highly abundant only in glandular and luminal epithelial cells, and followed the same pattern as immunolocalization on D1-D4 in epithelial cells. These results document two sources of LF in the preimplantation mouse uterus: neutrophils and epithelial cells. The synthesis of LF in the uterus reflects the abundance of epithelial LF mRNA, which is high on the first 2 days of pregnancy. Neutrophils that contain LF are also abundant in the uterine stroma during this time. E2 and/or P4 regulation of uterine LF was examined. LF mRNA was rapidly induced by E2 in ovariectomized adult mice, and this mRNA was localized exclusively to epithelial cells. P4 had little effect on uterine LF mRNA levels, but antagonized the prolonged induction of this gene by E2. E2 induced the accumulation of immunoreactive LF in uterine epithelial cells and the appearance of numerous immunopositive neutrophils distributed throughout the uterine stroma. P4 also antagonized these effects. Thus, E2 regulates LF gene expression in uterine epithelial cells and causes the recruitment of neutrophils into the uterus. These results suggest that LF may play an important role in early pregnancy and that uterine LF gene expression is regulated by a balance between estrogen and P4.

Crystals of an acidic pi class glutathione S-transferase from human placenta have been obtained by the hanging drop method using ammonium sulphate as a precipitant. The crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 60.1 A, c = 244.0 A. They contain a dimer in the asymmetric unit and diffract to a resolution of 2.7 A.

Kinetic analysis of the hydrolysis of the peptide H-Val-Ser-Gln-Asn-Tyr*Pro-Ile-Val-Gln-NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV-1 and HIV-2 proteinases. Deletion of the terminal Val and Gln had only moderate effect on the substrate hydrolysis, while the deletion of the P4. Ser as well as P'3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having high frequency of occurrence in beta turns resulted in good substrates, while large amino acids were unfavorable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV-2 substrate binding pocket.

The P4 helix is an essential element of ribonuclease P (RNase P) that is believed to bind catalytically important metals. Here, we applied a combination of NMR residual dipolar couplings (RDCs) and a recently introduced domain-elongation strategy for measuring "motionally decoupled" relaxation data to characterize the structural dynamics of the P4 helix from Bacillus subtilis RNase P. In the absence of divalent ions, the two P4 helical domains undergo small amplitude (approximately 13 degrees) collective motions about an average interhelical angle of 10 degrees. The highly conserved U7 bulge and helical residue C8, which are proposed to be important for substrate recognition and metal binding, are locally mobile at pico- to nanosecond timescales and together form the pivot point for the collective domain motions. Chemical shift mapping reveals significant association of Mg2+ ions at the P4 major groove near the flexible pivot point at residues (A5, G22, G23) previously identified to bind catalytically important metals. The Mg2+ ions do not, however, significantly alter the structure or dynamics of P4. Analysis of results in the context of available X-ray structures of the RNA component of RNase P and structural models that include the pre-tRNA substrate suggest that the internal motions observed in P4 likely facilitate adaptive changes in conformation that take place during folding and substrate recognition, possibly aided by interactions with Mg2+ ions. Our results add to a growing view supporting the existence of functionally important internal motions in RNA occurring at nanosecond timescales.

We investigated whether combinatorial post-injury treatment with progesterone (P4) and vitamin D hormone (VDH) would reduce ischemic injury more effectively than P4 alone in an oxygen glucose deprivation (OGD) model in primary cortical neurons and in a transient middle cerebral artery occlusion (tMCAO) model in rats. In the OGD model, P4 and VDH each showed neuroprotection individually, but combination of the "best" doses did not show substantial efficacy; instead, the lower dose of VDH in combination with P4 was the most effective. In the tMCAO model, P4 and VDH were given alone or in combination at different times post-occlusion for 7 days. In vivo data confirmed the in vitro findings and showed better infarct reduction at day 7 and functional outcomes (at 3, 5 and 7 days post-occlusion) after combinatorial treatment than when either agent was given alone. VDH, but not P4, upregulated heme oxygenase-1, suggesting a pathway for the neuroprotective effects of VDH differing from that of P4. The combination of P4 and VDH activated brain-derived neurotrophic factor and its specific receptor, tyrosine kinase receptor-B. Under specific conditions VDH potentiates P4's neuroprotective efficacy and should be considered as a potential partner of P4 in a low-cost, safe and effective combinatorial treatment for stroke.

OBJECTIVE

To quantify optical coherence tomography (OCT) images of the central retina in patients with blue-cone monochromatism (BCM) and achromatopsia (ACH) compared with healthy control individuals.

METHODS

The study included 15 patients with ACH, 6 with BCM, and 20 control subjects. Diagnosis of BCM and ACH was established by visual acuity testing, morphologic examination, color vision testing, and Ganzfeld ERG recording. OCT images were acquired with the Stratus OCT 3 (Carl Zeiss Meditec AG, Oberkochen, Germany). Foveal OCT images were analyzed by calculating longitudinal reflectivity profiles (LRPs) from scan lines. Profiles were analyzed quantitatively to determine foveal thickness and distances between reflectivity layers.

RESULTS

Patients with ACH and BCM had a mean visual acuity of 20/200 and 20/60, respectively. Color vision testing results were characteristic of the diseases. The LRPs of control subjects yielded four peaks (P1-P4), presumably representing the RPE (P1), the ovoid region of the photoreceptors (P2), the external limiting membrane (ELM) (P3), and the internal limiting membrane (P4). In patients with ACH, P2 was absent, but foveal thickness (P1-P4) did not differ significantly from that in the control subjects (187 +/- 20 vs. 192 +/- 14 microm, respectively). The distance from P1 to P3 did not differ significantly (78 +/- 10 vs. 82 +/- 5 microm) between ACH and controls subjects. In patients with BCM, P3 was lacking, and P2 advanced toward P1 compared with the control subjects (32 +/- 6 vs. 48 +/- 4 microm). Foveal thickness (153 +/- 16 microm) was significantly reduced compared with that in control subjects and patients with ACH.

CONCLUSIONS

Quantitative OCT image analysis reveals distinct patterns for controls subjects and patients with ACH and BCM, respectively. Quantitative analysis of OCT imaging can be useful in differentiating retinal diseases affecting photoreceptors. Foveal thickness is similar in both normal subjects and patients with ACH but is decreased in patients with BCM.

Peri-implantation conceptus (embryo/fetus and associated extraembryonic membranes) growth and development are primarily regulated by secretions from the uterus. This study investigated the effects of progesterone on preimplantation conceptus development and endometrial galectin 15 (LGALS15). Ewes received daily injections of either corn oil (CO) vehicle or 25 mg progesterone (P4) from 36 h postmating to hysterectomy. Treatment with P4 increased blastocyst diameter by 220% on Day 9 and advanced time of elongation of blastocysts to a filamentous conceptus on Day 12. Effects of P4 treatment on blastocyst development were blocked by administration of RU486, a progesterone receptor antagonist. Consistent with early elongation of blastocysts, interferon tau (IFNT) protein was about 50-fold greater in uterine flushes from Day 12 in ewes receiving P4 compared with those receiving CO. Expression of cathepsin L (CTSL) and radical S-adenosyl methionine domain containing 2 (RSAD2), both IFNT-stimulated genes, was increased in endometria of Day 12 P4-treated ewes. LGALS15 mRNA, expressed only in the endometrial luminal epithelium and superficial glands, was detected between Days 9 and 12 and was more abundant in ewes receiving P4 than in those receiving CO on both Days 9 and 12. RU486 treatment ablated P4 induction of LGALS15 mRNA in the endometrial epithelia. LGALS15 protein in uterine flushings was not different on Day 9 but tended to be greater in P4-treated ewes than in those receiving CO on Day 12. The advanced development of blastocysts in P4-treated ewes is hypothesized to involve early induction of specific genes in the endometrial epithelia, such as LGALS15, and undoubtedly components of uterine histotroph.

The host cell protein cyclophilin A (CypA) binds to CA of human immunodeficiency virus type 1 (HIV-1) and promotes HIV-1 infection of target cells. Disruption of the CypA-CA interaction, either by mutation of the CA residue at G89 or P90 or with the immunosuppressive drug cyclosporine (CsA), reduces HIV-1 infection. Two CA mutants, A92E and G94D, previously were identified by selection for growth of wild-type HIV-1 in cultures of CD4(+) HeLa cell cultures containing CsA. Interestingly, infection of some cell lines by these mutants is enhanced in the presence of CsA, while in other cell lines these mutants are minimally affected by the drug. Little is known about this cell-dependent phenotype of the A92E and G94D mutants, except that it is not dependent on expression of the host factor TRIM5alpha. Here, we show that infection by the A92E and G94D mutants is restricted at an early post-entry stage of the HIV-1 life cycle. Analysis of heterokaryons between CsA-dependent HeLa-P4 cells and CsA-independent 293T cells indicated that the CsA-dependent infection by A92E and G94D mutants is due to a dominant cellular restriction. We also show that addition of CsA to target cells inhibits infection by wild-type HIV-1 prior to reverse transcription. Collectively, these results support the existence of a cell-specific human cellular factor capable of restricting HIV-1 at an early post-entry step by a CypA-dependent mechanism.

During early pregnancy in ruminants, progesterone (P4) from the corpus luteum and interferon tau (IFNT) from the conceptus act on the endometrium to regulate genes important for uterine receptivity and conceptus growth. The use of the uterine gland knockout (UGKO) ewe has demonstrated the critical role of epithelial secretions in regulation of conceptus survival and growth. A custom ovine cDNA array was used to identify alterations in gene expression of endometria from Day 14 cyclic, pregnant, and UGKO ewes (study 1) and from cyclic ewes treated with P4 or P4 with ZK 136,317 antiprogestin and control proteins or IFNT (study 2). In study 1, expression of 47 genes was more than 2-fold different between Day 14 pregnant and cyclic endometria, whereas 23 genes was different between Day 14 cyclic and UGKO endometria. In study 2, 70 genes were different due to P4 alone, 74 genes were affected by IFNT in a P4-dependent manner, and 180 genes were regulated by IFNT in a P4-independent manner. In each study, an approximately equal number of genes were found to be activated or repressed in each group. Endometrial genes increased by pregnancy and P4 and/or IFNT include B2M, CTSL, CXCL10, G1P3, GRP, IFI27, IFIT1, IFITM3, LGALS15, MX1, POSTN, RSAD2, and STAT5A. Transcripts decreased by pregnancy and P4 and/or IFNT include COL3A1, LUM, PTMA, PUM1, RPL9, SPARC, and VIM. Identification and analysis of these hormonally responsive genes will help define endometrial pathways critical for uterine support of peri-implantation conceptus survival, growth, and implantation.

OBJECTIVE

The aim was to determine whether progesterone (P4) or 17-alpha-hydroxyprogesterone caproate (17P) directly inhibit human uterine contractility in vitro and thereby clarify their mechanisms of action.

METHODS

Myometrial tissues were suspended in organ chambers and exposed for 2 to 20 hours to varying concentrations of P4 or 17P or solvent. Contractile activity was registered, stored, and analyzed. Dose response curves were then generated for P4 or 17P at various times.

RESULTS

P4 significantly inhibited spontaneous contractility dose dependently. The inhibition was not blocked by RU486 but was reversible after washing. Surprisingly, 17P dose dependently stimulated contractility. HPLC and GC-MS methods were used to determine the detectable concentrations of progestins in the baths.

CONCLUSIONS

P4, at concentrations equivalent to those present in the placenta and uterus, inhibit spontaneous myometrial contractility in vitro by nongenomic mechanisms.

The neuroendocrinology of menopause is reviewed from a comparative perspective, with emphasis on laboratory rodent models. These changes are compared by the 2011 STRAW criteria (Stages of Reproductive Aging Workshop). Ovarian cell loss begins prenatally in all mammals studied, with exponential depletion of primary follicles and oocytes in association with loss of fecundity by midlife. Rodents and humans also share progressively increasing irregularity in ovulatory cycles and increasing fetal aneuploidy as oocyte depletion become imminent. Hypothalamic impairments of the estrogen-induced surge of pituitary gonadotrophins (luteinizing hormone, LH; follicle stimulating hormone, FSH) are prominent in middle-aged rodents, but sporadic in peri-menopausal women. In aging rodents, hypothalamic impairments of the LH surge have been experimentally associated with prolonged phases of sustained estradiol (E2) and very low progesterone (P4) ('unopposed estradiol'). Although peri-menopausal women also show hyper-estrogenic cycles, there is no indication for irreversible hypothalamic desensitization by E2. Ongoing cognitive assessments in clinical trials of estrogen therapy with and without P4 or other progestins may further inform about possible persisting effects of unopposed estrogens.This article is part of a Special Issue entitled 'Menopause'.

The inhibitory transmitters glycine and GABA undergo a developmental shift from depolarizing to hyperpolarizing action (D/H-shift). To analyse this shift in functionally related nuclei of the rat superior olivary complex (SOC), we employed voltage-sensitive dye recordings in auditory brainstem slices. Complementarily, we analysed single neurons in gramicidin perforated-patch recordings. Our results show a differential timing of the D/H-shift in the four SOC nuclei analysed. In the medial superior olive (MSO), the shift occurred at postnatal day (P) 5-9. In the superior paraolivary nucleus (SPN), it occurred between embryonic day (E) 18 and P1. No D/H-shift was observed in the medial nucleus of the trapezoid body (MNTB) until P10. This is in line with the finding that most of the patched MNTB neurons displayed glycine-induced depolarizations between P0-9. While no regional differences regarding the D/H-shift were found within the MSO, SPN, and MNTB, we observed such differences in the lateral superior olive (LSO). All LSO regions showed a D/H-shift at P4-5. However, in the high-frequency regions, hyperpolarizations were large already at P6, yet amplitudes of this size were not present until P8 in the low-frequency regions, suggesting a delayed development in the latter regions. Our physiological results demonstrate that D/H-shifts in SOC nuclei are staggered in time and occur over a period of almost two weeks. Membrane-associated immunoreactivity of the Cl- outward transporter KCC2 was found in every SOC nucleus already at times when glycine was still depolarizing. This implies that the mere presence of KCC2 does not correlate with functional Cl- outward transport.

Three experimental protocols were devised to induce endometrial maturation in 12 women with ovarian failure. Each was planned to serve a dual purpose: to resolve a particular clinical situation related to synchronization between ovum donor and recipient and to answer a specific question about endometrial physiology. A fourth protocol of sequential estrace (2-6 mg/day) and progesterone (P4; 25-50 mg/day, im) simulating the 28-day natural cycle, served as a control protocol (18 cycles). A short follicular phase protocol consisted of only 6 days of estrogen (E) administration before addition of P4 (13 cycles). In the long follicular phase protocol (5 cycles), estrace was given for 3-5 weeks, and P4 administration was accordingly postponed. In 6 accelerated secretory transformation cycles, 150 mg/day P4 were administered, im, from day 15 onward. The adequacy of the induced endometrial cycles was evaluated by hormonal, morphological, and histochemical criteria relevant to endometrial normalcy and receptivity. Serum estradiol levels and the areas under the estradiol curves for the long and short follicular phase protocols differed significantly from those during the control cycles (P less than 0.005). Areas under the estradiol curves in the accelerated secretory transformation protocol yielded significantly higher P4 values than those in all other protocols (P less than 0.05). All biopsies in the 3 experimental protocols compared favorably with those of the control protocol. Glycocalyx intensity (periodic acid-Schiff) and the amount of galactose residues in the glycocalyx (Ricinus communis-I agglutinin) were greatest during the periimplantation interval. We conclude that a very short exposure of the human endometrium to E or, conversely, prolonged E stimulation will allow normal endometrial maturation with the addition of P4. Supraphysiological doses of P4 in the accelerated secretory transformation protocol significantly enhanced endometrial maturational processes.

Secretion of fully folded extracellular proteins across the outer membrane of Gram-negative bacteria is mainly assisted by the ATP-dependent type II secretion system (T2SS). Depending on species, 12-15 proteins are usually required for the function of T2SS by forming a trans-envelope multiprotein secretion complex. Here we report crystal structures of an essential component of the Xanthomonas campestris T2SS, the 21-kDa N-terminal domain of cytosolic secretion ATPase XpsE (XpsEN), in two conformational states. By mediating interaction between XpsE and the cytoplasmic membrane protein XpsL, XpsEN anchors XpsE to the membrane-associated secretion complex to allow the coupling between ATP utilization and exoprotein secretion. The structure of XpsEN observed in crystal form P4(3)2(1)2 is composed of a 90-residue alpha/beta sandwich core domain capped by a 62-residue N-terminal helical region. The core domain exhibits structural similarity with the NifU-like domain, suggesting that XpsE(N) may be involved in the regulation of XpsE ATPase activity. Surprisingly, although a similar core domain structure was observed in crystal form I4(1)22, the N-terminal 36 residues of the helical region undergo a large structural rearrangement. Deletion analysis indicates that these residues are required for exoprotein secretion by mediating the XpsE/XpsL interaction. Site-directed mutagenesis study further suggests the more compact conformation observed in the P4(3)2(1)2 crystal likely represents the XpsL binding-competent state. Based on these findings, we speculate that XpsE might function in T2SS by cycling between two conformational states. As a closely related protein to XpsE, secretion ATPase PilB may function similarly in the type IV pilus assembly.

Nucleotide sequence of one-third of the genome of coliphage P4 has been obtained and mutations virl, epsilon am104, cI405, sidl, and delta 35 identified. The epsilon gene likely encodes a 10 kd protein with epsilon am104 being located at the beginning of the gene. cI405, a proposed repressor gene mutation, is located in a sequence capable of coding for a 15 kd protein. A new class of P4 mutations, ash, is located in the neighborhood of cI405. Two TATA-like sequences are mapped 5' to this cI (ash) sequence. Virl is possibly a promoter-up mutation and is located near or within the replication origin, which is about 400 bp long and AT rich. A sidl mutation is amber that shortens the sid protein by 9 amino acids. The delta gene may encode a 17 kd protein and appears to be coupled with the sid gene translationally. In the 5' side of the sid gene a sequence of CACAAT is the best TATA-like sequence. Sequences of two possible genes that are previously unrecognized and part of the alpha and psu genes are also identified.

Coaxial stacking of helical elements is a determinant of three-dimensional structure in RNA. In the catalytic center of the Tetrahymena group I intron, helices P4 and P6 are part of a tertiary structural domain that folds independently of the remainder of the intron. When P4 and P6 were fused with a phosphodiester linkage, the resulting RNA retained the detailed tertiary interactions characteristic of the native P4-P6 domain and even required lower magnesium ion concentrations for folding. These results indicate that P4 and P6 are coaxial in the P4-P6 domain and, therefore, in the native ribozyme. Helix fusion could provide a general method for identifying pairs of coaxially stacked helices in biological RNA molecules.

Suppression of streptomycin dependence in Escherichia coli strain K-114, a spectinomycin-sensitive strain, is correlated with modification of 30S ribosomal protein P4, the component modified in spectinomycin-resistant mutants. The mutant is unusual in that reversion from dependence has previously been correlated only with modification in 30S protein P4a. Introduction into K-114 of another mutation conferring spectinomycin resistance results in a further alteration in protein P4.

Drosophila Nedd2-like caspase (DRONC), an initiator caspase in Drosophila melanogaster and ortholog of human caspase-9, is cleaved during its activation in vitro and in vivo. We show that, in contrast to conclusions from previous studies, cleavage is neither necessary nor sufficient for DRONC activation. Instead, our data suggest that DRONC is activated by dimerization, a mechanism used by its counterparts in humans. Subsequent cleavage at Glu352 stabilizes the active dimer. Since cleavage is at a Glu residue, it has been proposed that DRONC is a dual Asp- and Glu-specific caspase. We used positional-scanning peptide libraries to define the P1-P4 peptide sequence preferences of DRONC, and show that it is indeed equally active on optimized tetrapeptides containing either Asp or Glu in P1. Furthermore, mutagenesis reveals that Asp and Glu residues are equally tolerated at the primary autoprocessing site of DRONC itself. However, when its specificity is tested on a natural substrate, the Drosophila executioner caspase DRICE, a clear preference for Asp emerges. The formerly proposed Glu preference is thus incorrect. DRONC does not differentiate between Asp and Glu in poor substrates, but prefers Asp when tested on a good substrate.

We have fused the promoter (PF) for the P2 late FETUD operon to the gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a plasmid vector. Synthesis of CAT in Escherichia coli strains carrying this plasmid requires the product of the P2 ogr gene or the satellite phage P4 transactivation gene, delta. Our results demonstrate that these phage-encoded transcriptional regulatory proteins are necessary and sufficient for activation of P2 late transcription in this reporter plasmid. Positive regulation of cloned PF is severely impaired in a host strain carrying the rpoA109 mutation. Expression from the cloned promoter thus approximates those features of P2 late transcription that have been shown to occur during normal P2 infection. To define sequences required for promoter function, sequential upstream deletions of PF were generated using BAL 31 nuclease, and the mutant promoters were assayed for cat expression. A sequence between nucleotides -69 and -64 from the transcription start point was found to be essential for promoter activity. This coincides with a region of homology conserved among all four P2 late gene promoters and the two P4 late promoters, and includes an element of dyad symmetry.

The mechanism by which RNA molecules assemble into unique three-dimensional conformations is important for understanding their function, regulation and interactions with substrates. The Tetrahymena group I ribozyme is an excellent model system for understanding RNA folding mechanisms, because the catalytic activity of the native RNA is easily measured. Folding of the Tetrahymena ribozyme is dominated by intermediates in which the stable P4-P6 domain is correctly formed, but the P3-P9 domain is partially misfolded. The propensity of the RNA to misfold depends on the relative stability of native and non-native interactions. Circular permutation of the Tetrahymena ribozyme shows that the distance in the primary sequence between native interactions also influences the folding pathway.

Mastitis, an inflammatory response of the mammary tissue to invading pathogenic bacteria, is a common disease in breast-feeding women and dairy animals. Escherichia coli is a leading cause of mastitis in dairy animals. During the course of the disease the host mounts a strong inflammatory response, but specific bacterial virulence factors have not yet been identified. Here we report the use of a murine mastitis model to investigate the innate inflammatory reaction of the mammary gland. We show that lipopolysaccharide (LPS) infusion induces mastitis in wild-type mice (C3H/HeN), but not in mice expressing mutated Toll-like receptor 4 (TLR4) (C3H/HeJ). The wild-type phenotype was restored by adoptive transfer of TLR4-expressing macrophages into the alveolar milk space of C3H/HeJ mice. In contrast to the LPS treatment, infection with E. coli P4 (ECP4) resulted in inflammation even in the absence of LPS/TLR4 signalling, indicating that additional factors play a role in the pathogenesis of the intact bacteria. Furthermore, in the absence of functional TLR4 the infecting ECP4 invade the epithelial cells with high efficiency, forming intracellular microcolonies. However, adoptive transfer with TLR4-expressing macrophages drastically reduced the epithelial invasion. Taken together, these results indicate that ECP4 has an invasive potential, which is restricted by alveolar macrophages in response to the LPS/TLR4 signalling.

In the present study we used human breast cancer cell lines to assess the influence of ceramide and glucosylceramide (GC) on expression of MDR1, the multidrug resistance gene that codes for P-glycoprotein (P-gp), because GC has been shown to be a substrate for P-gp. Acute exposure (72 h) to C8-ceramide (5 microg/ml culture medium), a cell-permeable ceramide, increased MDR1 mRNA levels by 3- and 5-fold in T47D and in MDA-MB-435 cells, respectively. Acute exposure of MCF-7 and MDA-MB-231 cells to C8-GC (10 microg/ml culture medium), a cell-permeable analog of GC, increased MDR1 expression by 2- and 4- fold, respectively. Chronic exposure of MDA-MB-231 cells to C8-ceramide for extended periods enhanced MDR1 mRNA levels 45- and 390-fold at passages 12 and 22, respectively, and also elicited expression of P-gp. High-passage C8-ceramide-grown MDA-MB-231 (MDA-MB-231/C8cer) cells were more resistant to doxorubicin and paclitaxel. Incubation with [1-(14)C]C6-ceramide showed that cells converted short-chain ceramide into GC, lactosylceramide, and sphingomyelin. When challenged with 5 mug/ml [1-(14)C]C6-ceramide, MDA-MB-231, MDA-MB-435, MCF-7, and T47D cells took up 31, 17, 21, and 13%, respectively, and converted 82, 58, 62, and 58% of that to short-chain GC. Exposing cells to the GCS inhibitor, ethylenedioxy-P4, a substituted analog of 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, prevented ceramide's enhancement of MDR1 expression. These experiments show that high levels of ceramide and GC enhance expression of the multidrug resistance phenotype in cancer cells. Therefore, ceramide's role as a messenger of cytotoxic response might be linked to the multidrug resistance pathway.