Insertion of viral DNA into host chromosomes is an ancient process essential for propagation in the proviral form. Many present-day bacteriophages insert at specific sites on the host chromosome. Insertion by two coliphage families (lambdoid and P4-like) is compared. For both families, insertion sites frequently lie within tRNA genes. The lambdoid phages insert at anticodon loops, whereas the p4-like phages insert in the TpsiC loops downstream from them. The association of both groups with tRNA genes suggests that the primordial insertion site of both groups may have been within a tRNA gene. The integrase proteins used in phage insertion may have originated at that stage, with subsequent diversification of specificity.

In a cytosolic fraction derived from insulin-secreting RINm5F cells, the rate of conversion of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) was half-maximally stimulated by 0.8 microM Ca2+ (Biden, T. J., and Wollheim, C. B. (1986) J. Biol. Chem. 261, 11931-11934). In the present study we show that after initial purification by anion exchange chromatography, the Ins-1,4,5-P3 kinase activity responsible for that conversion is stimulated by Ca2+-calmodulin, but not by Ca2+ alone. This is almost certainly due to a specific interaction of the enzyme and its activator since kinase activity was retained on a calmodulin-linked Sepharose 6B column in the presence of Ca2+ but eluted upon chelation of the cation. After this two-step purification, Ins-1,4,5-P3 kinase activity was maximally stimulated 5-fold by 10 microM calmodulin in the presence of 10(-5) M Ca2+, and 2 1/2-fold at 10(-6) M Ca2+. Under these conditions the minimum concentrations of calmodulin needed to stimulate activity were in the 10-50 nM range. At 10(-7) M Ca2+, calmodulin (up to 30 microM) was without effect. Stimulated Ins-1,4,5-P3 kinase activity was inhibited in a dose-dependent fashion by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) although the calmodulin antagonist had no effect on the residual activity seen at 10(-7) M Ca2+. These results strongly support our previous suggestion that alterations in cytosolic free Ca2+ concentrations play an important role in regulating the levels of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 during cellular stimulation.

Healthy uterine function depends on the balanced interaction of the ovarian steroids estrogen and progesterone (P4) signaling through their respective receptors. The expression of each receptor is regulated by the other through crucial cross talk between the epithelial and stromal compartments. Ablation of the progesterone receptor (PR) results in complete infertility in mice, and evidence increasingly demonstrates that the PR is a major mediator of epithelial-stromal cross talk and events leading to the disruption of this communication can lead to P4 resistance in the uterus. This resistance, through impaired P4 signaling, can be at the level of the PR itself, coregulators, and downstream effectors. The mechanisms underlying P4 resistance is of critical importance in women's health because this defect is seen in a wide variety of diseases including infertility, endometriosis, endometrial carcinoma, polycystic ovarian syndrome, and leiomyomas. By using mouse models of PR signaling, many of these mechanisms are beginning to be elucidated and aid in the development of effective therapies for treatment of uterine diseases.

The effects of consumption of low doses of ethanol (0.75 mg/kg) on spectral components of the EEG were investigated in 24 21-25-year-old males. A comparison of mean spectral power in 4 frequency bands (4-7 Hz, 7.5-9 Hz, 9-12 Hz, and 12-20 Hz) at 90 min post consumption revealed that ethanol, as opposed to placebo, significantly increased power in the two slower frequency bands, in both posterior and anterior leads. A reduction in the frequency at which power values peaked in the theta (4-7 Hz), fast alpha (9-12 Hz), and beta (12-20 Hz) frequency ranges was also seen following ethanol consumption. The ethanol induced lowering of peak power in the fast alpha range was observed in all 4 leads (F4-C4, P4-O2, F3-C3, and P3-O1), whereas the frequency changes in the theta and beta frequencies were only significant in a frontal lead. Ethanol was also noted to produce a decrease in the stability of the EEG in the fast alpha band as quantified by the coefficient of variation of power. Subjective response to ethanol was observed to vary depending on the subjects' pre-drug EEG pattern. Men with high amounts of fast alpha activity at baseline, in lead P4-O2, reported feeling less 'high' and less overall feelings of intoxication after ethanol than the men with lower amounts of pre-drug fast alpha waves. In addition, subjects were sorted into 'low' and 'moderate' drinkers depending on their quantity and frequency of drinking over the previous 6 month period.(ABSTRACT TRUNCATED AT 250 WORDS)

Human kallikrein-related peptidases (KLKs) are (chymo)-trypsin-like serine proteinases that are expressed in a variety of tissues such as prostate, ovary, breast, testis, brain, and skin. Although their physiological functions have been only partly elucidated, many of the KLKs appear to be useful prognostic cancer markers, showing distinct correlations between their expression levels and different stages of cancer. Recent advances in the purification of 'new type' recombinant KLKs allowed solution of the crystal structures of KLK4, KLK5, KLK6, and KLK7. Along with these data, enzyme kinetic studies and extended substrate specificity profiling have led to an understanding of the non-prime-side substrate preferences of KLK4, 5, 6, and 7. The shape and polarity of the specificity pockets S1-S4 explain well their substrate preferences. KLK4, 5, and 6 exhibit trypsin-like specificity, with a strong preference for Arg at the P1 position of substrates. In contrast, KLK7 displays a unique chymotrypsin-like specificity for Tyr, which is also preferred at P2. All four KLKs show little specificity for P3 residues and have a tendency to accept hydrophobic residues at P4. Interestingly, for KLK4, 5, and 7 extended charged surface regions were observed that most likely serve as exosites for physiological substrates.

Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. Recently, an antibiotic-resistant strain of this serovar was implicated in a large 2011 multistate outbreak resulting from consumption of contaminated ground turkey that involved 136 confirmed cases, with one death. In this study, we assessed the evolutionary diversity of 44 S. Heidelberg isolates using whole-genome sequencing (WGS) generated by the 454 GS FLX (Roche) platform. The isolates, including 30 with nearly indistinguishable (one band difference) Xbal pulsed-field gel electrophoresis patterns (JF6X01.0032, JF6X01.0058), were collected from various sources between 1982 and 2011 and included nine isolates associated with the 2011 outbreak. Additionally, we determined the complete sequence for the chromosome and three plasmids from a clinical isolate associated with the 2011 outbreak using the Pacific Biosciences (PacBio) system. Using single-nucleotide polymorphism (SNP) analyses, we were able to distinguish highly clonal isolates, including strains isolated at different times in the same year. The isolates from the recent 2011 outbreak clustered together with a mean SNP variation of only 17 SNPs. The S. Heidelberg isolates carried a variety of phages, such as prophage P22, P4, lambda-like prophage Gifsy-2, and the P2-like phage which carries the sopE1 gene, virulence genes including 62 pathogenicity, and 13 fimbrial markers and resistance plasmids of the incompatibility (Inc)I1, IncA/C, and IncHI2 groups. Twenty-one strains contained an IncX plasmid carrying a type IV secretion system. On the basis of the recent and historical isolates used in this study, our results demonstrated that, in addition to providing detailed genetic information for the isolates, WGS can identify SNP targets that can be utilized for differentiating highly clonal S. Heidelberg isolates.

Since the appearance of bovine spongiform encephalopathy (BSE) in cattle and its linkage with the human variant of Creutzfeldt-Jakob disease, the possible spread of this agent to sheep flocks has been of concern as a potential new source of contamination. Molecular analysis of the protease cleavage of the abnormal prion protein (PrP), by Western blotting (PrP(res)) or by immunohistochemical methods (PrP(d)), has shown some potential to distinguish BSE and scrapie in sheep. Using a newly developed enzyme-linked immunosorbent assay, we identified 18 infected sheep in which PrP(res) showed an increased sensitivity to proteinase K digestion. When analyzed by Western blotting, two of them showed a low molecular mass of unglycosylated PrP(res) as found in BSE-infected sheep, in contrast to other naturally infected sheep. A decrease of the labeling by P4 monoclonal antibody, which recognizes an epitope close to the protease cleavage site, was also found by Western blotting in the former two samples, but this was less marked than in BSE-infected sheep. These two samples, and all of the other natural scrapie cases studied, were clearly distinguishable from those from sheep inoculated with the BSE agent from either French or British cattle by immunohistochemical analysis of PrP(d) labeling in the brain and lymphoid tissues. Final characterization of the strain involved in these samples will require analysis of the features of the disease following infection of mice, but our data already emphasize the need to use the different available methods to define the molecular properties of abnormal PrP and its possible similarities with the BSE agent.

The 3C protease (3C(pro)) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C(pro), a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a beta-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This beta-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the beta-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C(pro) with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the beta-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C(pro) inhibitors.

To investigate if insulin-like growth factor-1 (IGF-1) provides neuroprotection to oligodendrocyte progenitor cells (OPCs) following cerebral hypoxia-ischemia, a previously developed neonatal rat model of white matter damage was used in this study. Postnatal day 4 (P4) SD rat pups were subjected to bilateral common carotid artery ligation, followed by exposure to 8% oxygen for 10 min. IGF-1 (0.5 microg) or vehicle was injected into the left ventricle after artery ligation and before the hypoxic exposure. Cerebral hypoxia-ischemia caused death of O4+ late OPCs in the P5 rat brain and impaired myelination in the P9 and P21 rat brain. Caspase-3 activation was involved in the death of OPCs. Moreover, cerebral hypoxia-ischemia impaired neurobehavioral performance in juvenile rats. IGF-1 treatment attenuated damages to OPCs and improved neurological functions after cerebral hypoxia-ischemia. It reduced death of O4+ OPCs by 39% on P5 and enhanced myelination on P9 and P21. Bromodeoxyuridine uptake assay showed that cerebral hypoxia-ischemia inhibited proliferation of stem/progenitor cells in the subventricular zone and NG2+ early OPCs in the white matter area. IGF-1 treatment increased cell proliferation in the subventricular zone by 31% 1 day following hypoxic-ischemic insult. Proliferation of early and late OPCs in the IGF-1-treated group was 1.5- and 2.4-fold of that in the vehicle-treated group, respectively. In conclusion, IGF-1 provided potent neuroprotection to OPCs and improved neurological functions following cerebral hypoxia-ischemia in the neonatal rat. The neuroprotection of IGF-1 was associated with its antiapoptotic and mitogenic effects.

The Escherichia coli eda gene, which encodes the Entner-Doudoroff aldolase, is central to the catabolism of several sugar acids. Here, we show that Eda synthesis is induced by growth on gluconate, glucuronate, or methyl-beta-D-glucuronide; phosphate limitation; and carbon starvation. Transcription of eda initiates from three promoters, designated P1, P2, and P4, each of which is responsible for induction under different growth conditions. P1 controls eda induction on gluconate and is regulated by GntR. P2 controls eda induction on glucuronate and galacturonate and is regulated by KdgR. P4 is active under conditions of phosphate starvation and is directly controlled by PhoB. In addition, CsrA activates Eda synthesis, apparently by an indirect mechanism that may be involved in the modest changes in expression level that are associated with carbon starvation. The complex regulation of eda is discussed with respect to its several physiological roles, which apparently accommodate not only sugar acid catabolism but also detoxification of metabolites that could accumulate during starvation-induced stress.

There is a growing body of evidence that the rapid but transient increase in male androgens, particularly testosterone (T), following a single social encounter such as a territorial intrusion occurs in a wide array of vertebrate taxa. Yet, this phenomenon, often called the Challenge Hypothesis, has rarely been investigated in females. Moreover, when studying male challenge effects, researchers have rarely investigated other hormones that can be important to the expression of aggression, such as progesterone (P4) and estradiol (E2). We conducted 10-min aggression trials using the resident-intruder paradigm in cycling female California mice, Peromyscus californicus, a species in which both sexes show territorial behavior. By comparing the hormone levels of test females to control females, we found a decrease in P(4) and the P4/T ratio, but no change in T, E2, corticosterone, E2/P4, or E2/T. Interestingly, these hormone changes were observed even when the resident was not aggressive toward the intruder, suggesting that the stimulus cueing the hormone changes was the mere presence of the intruder and not the amount of aggression displayed by the resident. Generally, T has a positive relationship with aggression, whereas P4 inhibits male and nonmaternal female aggression. Thus, decreasing the P4/T ratio following an encounter may serve to increase future aggression in females. These results suggest that females may use different hormonal mechanisms than do males to mediate aggression in a challenge situation.

BACKGROUND

Treatment failure for esophageal carcinoma is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate invasion- and metastasis-related genes in esophageal carcinoma cells in vitro and in vivo.

METHODS

A metastasis model using a Matrigel invasion clonal selection approach was employed to establish a highly invasive subline EC9706-P4 from the esophageal carcinoma cell (ESCC) line EC9706. The differentially expressed genes of the subline and the parental cells determined by gene microarrays were further analyzed by RT-PCR and Western blotting.

RESULTS

We identified sphingosine kinase 1 (SPHK1) as an invasion and metastasis-related gene of esophageal cancer. SPHK1 was overexpressed in the EC9706-P4 subline with high invasive capacity. Among six ESCC lines tested, KYSE2 and KYSE30 cells showed the highest SPHK1 mRNA and protein expressions as well as the most invasive phenotype. By Western blotting, in 7/12 cases (58%), SPHK1 expression was higher in esophageal carcinomas than in the companion normal tissue. In 23/30 cases (76%), SPHK1 protein expression was upregulated in the tumors compared to matched normal tissue by immunohistochemistry (IHC). Esophageal carcinoma tissue microarray analysis indicated that SPHK1 expression correlated with the depth of tumor invasion (P < 0.0001) and lymph node metastasis (P = 0.016). By Kaplan-Meier analysis, strong SPHK1 expression was significantly associated with clinical failure (P < 0.01), suggesting the involvement of SPHK1 in aggressiveness of human esophageal carcinoma. SPHK1 overexpression significantly increased the invasiveness of EC9706 cells in vitro and also increased EC9706 cell growth and spontaneous metastasis in vivo, promoting significant increases in tumor growth, tumor burden and spontaneous lung metastasis in nude mice. SPHK1 expression significantly correlated with the expression of many EGFR pathway genes associated with invasion of cancer cells. SPHK1 protein expression also significantly correlated with the phosphorylation of EGFR.

CONCLUSIONS

In summary, our data implicate SPHK1 in the metastasis of esophageal cancer. Our study also identified downstream mediators of SPHK1 in esophageal cancer cells that may mediate enhanced malignant behavior, and several of these mediators may be useful as therapeutic targets.

Changes in the ability of substances to diffuse in the intersticial space of the brain are important factors in the pathophysiology of cerebrovascular diseases. Extracellular space (ECS) volume fraction alpha (alpha = ECS volume/ total tissue volume), tortuosity lambda (lambda 2 = free diffusion coefficient/apparent diffusion coefficient), and nonspecific uptake (k')-three diffusion parameters of brain tissue were studied in cortex and subcortical white matter (WM) of the developing rat during anoxia. Changes were compared with the rise in extracellular potassium concentration ([K+]e), extracellular pH (pHe) shifts, and anoxic depolarization (AD). Diffusion parameters were determined from extracellular concentration-time profiles of tetramethylammonium (TMA+) or tetraethylammonium (TEA+), TMA+, TEA+, K+, and pH changes were measured using ion-selective microelectrodes. In the cortex and WM of animals at 4-12 postnatal days (P4-P12), the volume fraction, alpha, is larger than that of animals at>> or = P21. Anoxia evoked by cardiac arrest brought about a typical rise in [K+]e to approximately 60-70 mM, AD of 25-30 mV, decrease in alpha, increase in lambda, and increase in k'. At P4-P6, alpha decreased from approximately 0.43 to 0.05 in cortical layer V and from approximately 0.45 to 0.5 in WM. Tortuosity, lambda, increased in the cortex from 1.50 to 2.12 and in WM from approximately 1.48 to 2.08. At P10-P12 and at P21-P23, when alpha in normoxic rats is lower than at P4-P6 by approximately 25 and 50%, respectively, the final changes in values of alpha and lambda evoked by anoxia were not significantly different from those in P4-P6. However, the younger the animal, the longer the time course of the changes. On P4-P6 final changes in alpha, lambda and k' in cortex and WM were reached after 37 +/- 3 min and 54 +/- 2 min; on P10-P12, after 24 +/- 2 and 27 +/- 3 min; and on P21-P23 at 15 +/- 1 and 17 +/- 3 min, respectively (mean +/- SE, n = 6). The time course of the changes was longer in WM than in gray matter (GM), particularly during the first postnatal week, i.e., in the period during which WM is largely unmyelinated. Changes in diffusion parameters occurred in three phases. The first slow and second fast changes occurred simultaneously with the rise in [K+]e and AD. Peaks in [K+]e and AD were reached simultaneously; the younger the animal, the longer the time course of the changes. The third phase outlasted the rise in [K+]e and AD by 10-15 min and correlated with the acid shift in pHe. Linear regression analysis revealed a positive correlation between the normoxic size of the ECS volume and the time course of the changes. Slower changes in ECS volume fraction and tortuosity in nervous tissue during development can contribute to slower impairment of signal transmission, e.g., due to lower accumulation of ions and neuroactive substances released from cells and their better diffusion from the hypoxic area in uncompacted ECS.

Three crystal forms of the sweet-tasting protein thaumatin from the African berry Thaumatococcus daniellii have been grown. These include two naturally occurring isoforms, A and B, that differ by a single amino acid, and a recombinant form of isoform B expressed in yeast. The crystals are of space groups C2 with a = 117.7, b = 44.9, c = 38.0 A, and beta = 94.0 degrees, P2(1)2(1)2(1) with a = 44.3, b = 63.7 and c = 72.7 A, and a tetragonal form P4(1)2(1)2 with a = b = 58.6 and c = 151.8 A. The structures of all three crystals have been solved by molecular replacement and subsequently refined to R factors of 0.184 for the monoclinic at 2.6 A, 0.165 for the orthorhombic at 1.75 A, and 0.181 for the tetragonal, also at 1.75 A resolution. No solvent was included in the monoclinic crystal while 123 and 105 water molecules were included in the higher resolution orthorhombic and tetragonal structures, respectively. A bound tartrate molecule was also clearly visible in the tetragonal structure. The r.m.s. deviations between molecular structures in the three crystals range from 0.6 to 0.7 A for Calpha atoms, and 1.1 to 1.3 A for all atoms. This is comparable to the r.m.s. deviation between the three structures and the starting model. Nevertheless, several peptide loops show particularly large variations from the initial model.

BACKGROUND

Evidence suggests that a number of microRNA (miRNA) are aberrantly expressed in endometrial disorders with potential posttranscriptional regulation of their specific target genes, including ovarian steroid receptors.

OBJECTIVE

Our objective was to assess the endometrial expression of miR-98 and miR-181a and their respective target genes, progesterone (P4) receptor membrane component 1 (PGRMC1) and P4 receptor (PGR).

METHODS

We evaluated tissue expression and in vitro regulation at an academic university medical center in endometrial biopsies and endometrial tissues from follicular and luteal phases with and without exposure to hormonal therapies and grade I-III endometrial cancer (n = 52).

METHODS

INTERVENTIONS included endometrial biopsies and in vitro transfection.

METHODS

We evaluated expression and function of miR-98 and miR-181a.

RESULTS

Aberrant expression of miR-98 and miR-181a is associated with endometrial transition from normal into cancerous states, which to some extent is influenced by hormonal milieu, and exhibited an inverse relationship with PGMRC1 and PGR expression, respectively. Treatments of Ishikawa cells with 17β-estradiol, P4, or medroxyprogesterone acetate had limited effects on miR-98, miR-181a, and PGRMC1 expression, whereas 17β-estradiol treatment increased PGR expression. In Ishikawa cells, gain of function of miR-98 repressed PGRMC1 and CYP19A1, and miR-181a repressed PGR, DDX3X, and TIMP3 at mRNA and protein levels through direct interactions with their respective 3'-untranslated regions and CCNE1 through miR-181a-induced DDX3X repression, with miR-98 reducing the rate of cell proliferation as compared with controls.

CONCLUSIONS

miR-98 and miR-181a through their regulatory functions on PGRMC1, PGR, CYP19A1, TIMP3, and DDX3X expression may influence a wide range of endometrial cellular activities during normal menstrual cycle and transition into disease states, including endometrial cancer.

A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1'-Ser/Ala/Gly/Thr, P2'-not acidic and P3'-Arg. Our analysis also identifies an important role for a P3'-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3'-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200-400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1-30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times.

Two components of the ERPs elicited by emotional visual stimuli, N300 and P300, were investigated. The emotional charge is explained through two dimensions: arousal (relaxing (R) or activating (A)) and valence (attractive (+) or repulsive (-)). Stimuli were slides of nudes (A+), human remains (A-), landscapes (R), and buildings (neutral (N)). The peculiar structure of the stimuli, along with a distracting task which allowed us to disguise the real aim of the experiment, helped to avoid a sort of 'relevance-for-task effect', mainly related to cognitive processes, which could explain P300 reactions in response to emotional visual stimuli found in several experiments. The ERPs were recorded from 32 subjects at F3, Fz, F4, C3, Cz, C4, P3, Pz and P4. In contrast to previous studies, P300 did not show greater amplitudes in response to emotional stimuli than to N. N300 showed greater amplitudes in response to A+ at parietal sites, the greatest differences being those with respect to A-. No inter-hemispheric differences were found. N300 confirms its usefulness as a variable for studying emotional reactions to visual stimuli.

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

Numerous studies of the proliferative effects of basic fibroblast growth factor (bFGF) in culture, including neonatal and adult hippocampal precursors, suggest that the factor plays a ubiquitous and life-long role in neurogenesis. In contrast, in vivo, bFGF is devoid of effects on neurons in mature hippocampus, raising the possibility that bFGF exhibits developmental stage-specific activity in the complex animal environment. To define neurogenetic effects in the newborn, a single subcutaneous injection of bFGF (20 ng/gm) was administered to postnatal day 1 (P1) rats, and hippocampal DNA content was quantified: bFGF elicited an increase in total DNA throughout adulthood, by 48% at P4, 25% at P22, and 17% at P180, suggesting that bFGF increases hippocampal cell number. To define mechanisms, bromodeoxyuridine (BrdU) was injected at P1 and mitotically labelled cells were assessed at P22: there was a twofold increase in BrdU-positive cells in the dentate granule cell layer (GCL), indicating that bFGF enhanced the generation of neurons, or neuronogenesis, from a cohort of precursors. Moreover, enhanced mitosis and survival led to a 33% increase in absolute GCL neuron number, suggesting that neuron production depends on environmental levels of bFGF. To evaluate this possibility, bFGF-knockout mice were analyzed: hippocampal DNA content was decreased at all ages examined (P3, -42%; P21, -28%; P360, -18%), and total GCL neuron and glial fibrillary acidic protein (GFAP)-positive cell number were decreased by 30%, indicating that bFGF is necessary for normal hippocampal neurogenesis. We conclude that environmental levels of bFGF regulate neonatal hippocampal neurogenesis. As adult hippocampal neuronogenesis was unresponsive to bFGF manipulation in our previous study [Wagner, J.P., Black, I.B. & DiCicco-Bloom, E. (1999) J. Neurosci., 19, 6006], these observations suggest distinct, stage-specific roles of bFGF in the dentate gyrus granule cell lineage.

In cattle, the majority of pregnancy loss can be attributed to early embryonic loss which occurs prior maternal recognition of pregnancy on Day 16 (Day 0 = ovulation). During this time, carefully orchestrated spatio-temporal alterations in the transcriptomic profile of the endometrium are required to drive conceptus elongation, via secretions from the endometrium (termed histotroph) and establish uterine receptivity to implantation. The two main modulators of these processed are progesterone (P4) and the pregnancy recognition signal interferon tau (IFNT). Altered concentrations of P4 in circulation mediate its effects via the endometrium and have been associated with different rates of conceptus elongation in cattle. Transcriptomic analysis of the endometrium has shown that modulation of circulating P4 alters endometrial expression of genes that can contribute to histotroph composition, which is beneficial (when P4 is supplemented) or detrimental (when P4 is reduced) to the developing conceptus. In addition, down-regulation of the progesterone receptor, required to establish uterine receptivity, is altered in the endometrium of heifers with altered P4 concentrations. IFNT, a type 1 interferon, also significantly impacts on the endometrial transcriptome. It induces the expression of a large number of classical interferon stimulated genes as early as Day 15 of pregnancy. In summary, the successful establishment of pregnancy in cattle requires a sequence of key events to ensure appropriate maternally derived secretions, establish uterine receptivity to implantation as well as an adequate endometrial response to IFNT production.

BACKGROUND

Prostase/KLK4 is a member of the human kallikrein (KLK) gene family that is expressed in prostate epithelial cells under the regulation of androgenic hormones. In this study, we sought to characterize the substrate specificity of KLK4 in order to gain insight into potential physiological roles of the enzyme.

METHODS

A chimeric form of KLK4 was constructed in which the pro-region of KLK4 was replaced with the signal and propeptide sequence of trypsinogen (proT-KLK4) to create an activation site susceptible to enterokinase cleavage. proT-KLK4 was expressed in Drosophila S2 cells, purified, and activated with enterokinase to generate mature KLK4. The extended substrate specificity of KLK4 was defined by screening tetrapeptide positional scanning synthetic combinatorial libraries (PS-SCL).

RESULTS

The preferred P1-P4 positions as determined by PS-SCL were: P1-Arg; P2-Gln/Leu/Val; P3-Gln/Ser/Val; P4-Ile/Val. The trypsin-like specificity of KLK4 was further confirmed using synthetic chromogenic peptides. Based upon the optimal cleavage site residues, a database search for potential KLK4 substrates identified several proteins with potential roles mediating normal prostate physiology or neoplastic growth including KLK3/PSA, parathyroid hormone-related peptide (PTHrP), and members of the bone morphogenetic protein (BMP) family. Recombinant KLK4 was able to activate pro-PSA/KLK3 and degrade members of the insulin-like growth factor (IGF) binding protein (IGFBP) family.

CONCLUSIONS

These results identify potential KLK4 substrates that may serve to define the role of this protease in normal prostate physiology, and facilitate studies of the consequences of KLK4 expression in pathological conditions.

Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines.

Brain event-related potentials (ERPs) to probe tones in a dichotic complex tone test were recorded from right-handed depressed patients (n = 44) and normal subjects (n = 19) at homologous sites over left and right hemispheres (F3, F4; C3, C4; P3, P4; O1, O2). There were no differences between groups N1 or P2 amplitude, but patients had smaller P3 amplitude than did normal subjects. Depressed patients failed to show either the left ear advantage or behavior-related hemispheric asymmetry of P3 seen for normal subjects. Depressed patients also showed less difference in hemispheric asymmetry between same and different judgments. These findings indicate that the abnormal behavioral asymmetry for dichotic pitch discrimination in depressed patients reflects a reduction in hemispheric asymmetry and is related to relatively late stages of cognitive processing.