Gene expression profiling of human placentas from preeclamptic and normotensive pregnancies.

The aim of this study was to investigate patterns of gene expression in placental samples from patients with preeclampsia (PE), persistent bilateral uterine artery notching (without PE), and normal controls. This study included placental tissue from nine women with PE, seven with uncomplicated pregnancies and five with bilateral uterine artery notching in Doppler velocimetry tracings. Human cDNA microarrays with 6500 transcripts/genes were used and the results verified with real-time PCR and in-situ hybridization. Multidimensional scaling method and random permutation technique demonstrated significant differences among the three groups examined. Within the 6.5K arrays, 6198 elements were unique cDNA clones representing 5952 unique UniGenes and 5695 unique LocusLinks. Multidimensional scaling plots showed 5000 genes that met our quality criteria; among these, 366 genes were significantly different in at least one comparison. Differences in three genes of interest were confirmed with real-time PCR and in-situ hybridization; acid phosphatase 5 was shown to be overexpressed in PE samples and calmodulin 2 and v-rel reticuloendotheliosis viral oncogene homolog A (RELA) were downregulated in PE and uterine artery notch placentas. In conclusion downregulation of RELA and calmodulin 2 might represent an attempt by the placenta to compensate for elevations in intracellular calcium, possibly caused by hypoxia and/or apoptosis, in both pregnancies with uterine artery notching and preeclampsia.

Probe generation directly from small numbers of cells for DNA microarray studies.

Recently, we described a technique that allows us to prepare probes for expression profiling from 0.5-1 microgram RNA without template or signal amplification. However, we were unable to use this method to study cells harvested by needle biopsy, cell sorting, or laser capture microdissection. Here we give a new protocol for amplifying RNA with multiple reaction cycles and preparing fluorescent probes from approximately 10 cells. We use random 9-mers with a T3 RNA polymerase recognition sequence on the 5' end for every round of cDNA synthesis except the first. The latter is primed with oligo(dT) with a T7 RNA polymerase recognition sequence on the 5' end. Results were highly reproducible and reliable, and the products generated using our method seemed comparable to those produced using the RiboAmp RNA kit when both were used to do two cycles of amplification. To test our method's utility, we lysed cells directly into reverse transcription buffer containing RNase inhibitor and performed three rounds of RNA amplification. The expression profiles of mouse C2 and NIH 3T3 cells obtained with 11,232-element arrays using amplified RNAs were similar to those seen when probes were prepared from unamplified templates.

Germline mutations in the ribonuclease L gene in families showing linkage with HPC1.

Although prostate cancer is the most common non-cutaneous malignancy diagnosed in men in the United States, little is known about inherited factors that influence its genetic predisposition. Here we report that germline mutations in the gene encoding 2'-5'-oligoadenylate(2-5A)-dependent RNase L (RNASEL) segregate in prostate cancer families that show linkage to the HPC1 (hereditary prostate cancer 1) region at 1q24-25 (ref. 9). We identified RNASEL by a positional cloning/candidate gene method, and show that a nonsense mutation and a mutation in an initiation codon of RNASEL segregate independently in two HPC1-linked families. Inactive RNASEL alleles are present at a low frequency in the general population. RNASEL regulates cell proliferation and apoptosis through the interferon-regulated 2-5A pathway and has been suggested to be a candidate tumor suppressor gene. We found that microdissected tumors with a germline mutation showed loss of heterozygosity and loss of RNase L protein, and that RNASEL activity was reduced in lymphoblasts from heterozyogous individuals compared with family members who were homozygous with respect to the wildtype allele. Thus, germline mutations in RNASEL may be of diagnostic value, and the 2-5A pathway might provide opportunities for developing therapies for those with prostate cancer.

Alteration in Kupffer cell function after mild hemorrhagic shock.

Functional changes in Kupffer cells occur after profound hemorrhagic shock. This study was performed to demonstrate if Kupffer cell changes also occur after mild hemorrhagic shock. Sprague-Dawley rats were bled to a systolic blood pressure of 60 to 70 mmHg and resuscitated with Lactated Ringers solution (twice the shed blood volume) after 30 min. Resuscitation produced immediate recovery of blood pressure and allowed long-term recovery of the animals. Sham animals received anesthesia and monitoring only. Thirty minutes after resuscitation, Kupffer cells were isolated by centrifugal elutriation and cultured for 48 h. In Kupffer cells isolated from shocked animals, phorbol ester-stimulated superoxide production increased 7-fold and lipopolysaccharide- (LPS) stimulated prostaglandin E2 (PGE2) production increased 4-fold. Tumor necrosis factor-alpha (TNFalpha) production, on the other hand, was decreased by 50%. A non-significant trend toward increased phagocytosis was also observed, whereas LPS-stimulated nitric oxide production was unchanged. In conclusion, mild hemorrhagic shock produced increases in superoxide and PGE2 production, and decreases in TNFalpha production by Kupffer cells, changes that may be appropriate to defend against the infectious challenges that often follows trauma and hemorrhage.

Alpha-synuclein immunoreactivity of huntingtin polyglutamine aggregates in striatum and cortex of Huntington's disease patients and transgenic mouse models.

Polyglutamine expansions in proteins are implicated in at least eight inherited neurodegenerative disorders, including Huntington's disease. These mutant proteins can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. Polyglutamine aggregates are ubiquitinated and sequester molecular chaperone proteins and proteasome components. To investigate other protein components of polyglutamine aggregates, cerebral cortex and striata from patients with Huntington's disease and full-length cDNA transgenic mouse models for this disease were examined immunohistochemically for alpha-synuclein reactivity. Our findings demonstrate that alpha-synuclein can be used as a marker for huntingtin polyglutamine aggregates in both human and mice. Moreover in the HD transgenic mice, the intensity of immunoreactivity increases with age. The significance of recruitment of alpha-synuclein into huntingtin aggregates and its translocation away from the synapses remains to be determined. We propose that aberrant interaction of mutant huntingtin with other proteins, including alpha-synuclein, may influence disease progression.

Diagnosis and management of blunt small bowel injury: a survey of the membership of the American Association for the Surgery of Trauma.

BACKGROUND: Blunt small bowel injury (SBI) may be difficult to diagnose accurately. Diagnostic delays are associated with increased morbidity and mortality. METHODS: A cross-sectional survey of members of the American Association for the Surgery of Trauma was conducted. A Likert-type multiple-choice scale was used to evaluate use and usefulness of diagnostic and laboratory tests. Data were analyzed by using univariate and multivariate techniques. RESULTS: A total of 461 of the 702 members (68%) surveyed responded, of which 133 members (29%) were excluded because they did not currently manage adult SBI. Of the remaining 328 respondents, 244 members (74%) reported prior experience as the most important influence on their current practice of diagnosing blunt SBI. None of the standard laboratory tests were reported as useful. Seventy-seven percent of respondents use computed tomographic (CT) scan most or all of the time for diagnosis (p < 0.05 compared with other modalities). Most respondents estimated their annual incidence of SBI at 5% with a >15% frequency of delay in diagnosis. Forty-four percent of the respondents estimated the mortality associated with a delay in diagnosis at < or =5%. Respondents varied significantly in their management of the patient with an unreliable abdominal exam and a CT scan finding of isolated free fluid. In patients with head injuries, 28% observe, 12% repeat the CT scan, 42% perform diagnostic peritoneal lavage, and 16% operate. For intoxicated patients, 51% observe, 11% repeat the CT scan, 26% perform diagnostic peritoneal lavage, and 10% operate. A more aggressive approach with diagnostic and operative intervention was significantly (p < 0.05) advocated by respondents practicing without residents, more than 15 years out from residency, or by those with a perception of higher morbidity and mortality from delays in diagnosis. CONCLUSION: There is significant variation in the diagnostic approach to the patient with suspected SBI. The perceived mortality of delayed diagnosis is much less than reported. Those surgeons with more experience or perception of greater morbidity and mortality from a delayed diagnosis are more aggressive. Further investigation into the diagnosis and treatment of this injury is needed.

Bovine hemoglobin-based oxygen carrier (HBOC-201) for resuscitation of uncontrolled, exsanguinating liver injury in swine. Carolina Resuscitation Research Group.

In the setting of rapidly exsanguinating hemorrhage, resuscitation with intravenous (i.v.) crystalloid solution may not sustain survival before availability of allogenic blood transfusion and surgery. This study tested the hypothesis that bovine hemoglobin-based oxygen carrier, HBOC-201, would improve resuscitation and extend early survival from exsanguinating hemorrhage. This study simulated the prehospital scenario of rapidly exsanguinating hemorrhage with prolonged prehospital time and lack of blood availability. Severe hemorrhagic shock was induced in swine by using multiple liver lacerations. At 9 min after the onset of bleeding, swine were randomized to receive approximately 10 mL/kg/min of i.v. lactated Ringer's solution (n = 10) or HBOC-201 (n = 7) to achieve a mean aortic pressure (MAP) of 60 mmHg. Thereafter, infusion rate was adjusted to maintain MAP at 60 mmHg for up to 2 h. All animals were initially successfully resuscitated. The results showed 2-h survival was 1 of 10 with lactated Ringer's and 7 of 7 with HBOC-201 (P = 0.0004). Nine lactated Ringer's swine had cardiovascular collapse at 36 +/- 10 min. Lactate at 30 min was 18 +/- 3 mmol/L with lactated Ringer's and 12 +/- 2 mmol/L with HBOC-201 (P < 0.05). Hematocrit was <1% in 9 of 10 lactated Ringer's and 6 of 7 HBOC-201 animals. These data indicate that HBOC-201 improved early survival and stabilized hemodynamic and metabolic parameters vs. lactated Ringer's in this swine model of liver injury with uncontrolled, lethal hemorrhage that simulates the prehospital care environment where allogenic blood is unavailable.

Prospective randomized trial of early initiation and hospital discharge on a liquid diet following elective intestinal surgery.

Length of hospital stay after elective intestinal surgery may be related to patient tolerance of a diet. We hypothesized that early initiation and discharge home on a clear liquid diet would decrease the length of hospital stay without increasing morbidity. The aim of this study was to determine if early initiation and discharge on a clear liquid diet decreases the length of hospital stay and is safe. Forty-four patients were randomly assigned to either a standard diet or a clear liquid diet. A standard diet (n = 17) was begun after the passage of flatus or stool, and consisted of clear liquids to a volume of approximately 750 ml, then three solid meals, and discharge thereafter. Patients randomized to a clear liquid diet (n = 27) received 30 ml/hr of clear liquids on postoperative day 2, unlimited clear liquids on postoperative day 3, and were dismissed on a clear liquid diet on postoperative day 4. All patients were followed by a daily telephone call and clinic visit. The primary outcome variable was length of hospital stay. The incidence of postoperative intestinal-related sequelae, complications, and readmission rates did not differ between groups. Postdischarge intestinal symptoms were common in both groups but tended to resolve faster in the patients on a standard diet. The length of hospital stay was decreased in the patients on a clear liquid diet compared to those on a standard diet (6.1 +/- 1.1 days vs. 4.4 +/- 0.2 days; P = 0.09), but total hospital costs did not differ. Early initiation and hospital discharge on a clear liquid diet after elective intestinal surgery decreases the length of hospital stay and is safe.

A genetic epidemiological study of hereditary prostate cancer (HPC) in Finland: frequent HPCX linkage in families with late-onset disease.

Several predisposition loci for hereditary prostate cancer (HPC) have been suggested, including HPC1 at 1q24-q25 (OMIM #601518) and HPCX at Xq27-q28 (OMIM #300147). Genetically homogeneous populations, such as that of Finland, and distinct subsets of families may help to minimize the genetic heterogeneity that complicates the genetic dissection of complex traits. Here, the role of the HPC1, and HPCX loci in a series of Finnish prostate cancer families was studied, especially in subgroups of families defined by age, number of affected cases, and the mode of disease transmission. DNA samples were collected from 57 Finnish HPC families with at least two living prostate cancer patients. Linkage analysis was carried out with 39 microsatellite markers for the HPC1 region and 22 markers for the HPCX region. The maximum two-point LOD score for the HPCX was 2.05 (marker DXS1205, at theta = 0.14), whereas HPC1 LOD scores were all negative. In HOMOG3R analyses, significant evidence of heterogeneity was observed. Subgroup analyses performed to explore the nature of this heterogeneity indicated that families with no male-to-male (NMM) transmission and a late age of diagnosis (>65 years) accounted for most of the HPCX-linked cases. The maximum HPCX LOD score in this subgroup was 3.12 (theta = 0.001). Nonparametric sibling pair analyses gave a peak LOD score of 3.04 (P < 0.000093) for the NMM transmission subgroup. No subgroup showed any positivity for HPC1. This study suggests that the HPCX-linked prostate cancer families represent a distinct subgroup characterized by NMM transmission of disease and late age of diagnosis.

Molecular cloning and functional characterization of a vasotocin receptor subtype that is expressed in the shell gland and brain of the domestic chicken.

In chickens, oviposition is correlated with increased plasma levels of the neurohypophysial hormone vasotocin, and vasotocin stimulates contraction of uterine strips in vitro. A gene encoding a vasotocin receptor subtype that we have designated the VT1 receptor was cloned from the domestic chicken. The open reading frame encodes a 370-amino acid polypeptide that displays seven segments of hydrophobic amino acids, typical of guanine nucleotide-protein-coupled receptors. Other structural features of the VT1 receptor include two potential N-linked glycosylation sites in the extracellular N-terminal region, a conserved aspartic acid in transmembrane domain 2 that is found in nearly all guanine nucleotide-protein-coupled receptors, and two potential protein kinase C phosphorylation sites in the third intracellular loop and C-terminal tail. Expressed VT1 receptors in COS7 cells bind neurohypophysial hormones with the following rank order of potency: vasotocin congruent with vasopressin > oxytocin congruent with mesotocin > isotocin. In addition, the expressed VT1 receptor mediates vasotocin-induced phosphatidylinositol turnover and Ca(2+) mobilization. In the chicken, expression of VT1 receptor gene transcripts is limited to the shell gland (uterus) and the brain. Thus, the VT1 receptor that we have cloned may mediate contractions of the shell gland during oviposition and activate reproductive behaviors known to be stimulated by vasotocin in lower vertebrates.

Evidence for a prostate cancer susceptibility locus on the X chromosome.

Over 200,000 new prostate cancer cases are diagnosed in the United States each year, accounting for more than 35% of all cancer cases affecting men, and resulting in 40,000 deaths annually. Attempts to characterize genes predisposing to prostate cancer have been hampered by a high phenocopy rate, the late age of onset of the disease and, in the absence of distinguishing clinical features, the inability to stratify patients into subgroups relative to suspected genetic locus heterogeneity. We previously performed a genome-wide search for hereditary prostate cancer (HPC) genes, finding evidence of a prostate cancer susceptibility locus on chromosome 1 (termed HPC1; ref. 2). Here we present evidence for the location of a second prostate cancer susceptibility gene, which by heterogeneity estimates accounts for approximately 16% of HPC cases. This HPC locus resides on the X chromosome (Xq27-28), a finding consistent with results of previous population-based studies suggesting an X-linked mode of HPC inheritance. Linkage to Xq27-28 was observed in a combined study population of 360 prostate cancer families collected at four independent sites in North America, Finland and Sweden. A maximum two-point lod score of 4.60 was observed at DXS1113, theta=0.26, in the combined data set. Parametric multipoint and non-parametric analyses provided results consistent with the two-point analysis. Significant evidence for genetic locus heterogeneity was observed, with similar estimates of the proportion of linked families in each separate family collection. Genetic mapping of the locus represents an important initial step in the identification of an X-linked gene implicated in the aetiology of HPC.

Characteristics of women surgeons in the United States.

BACKGROUND: Women surgeons are becoming increasingly prevalent. Despite this, there have been few studies of personal or professional characteristics of US surgeons of either gender. METHODS: Data were taken from the Women Physicians' Health Study, a nationally representative random sample (n = 4,501 respondents) of US women physicians, and data were analyzed in SUDAAN. RESULTS: Surgeons were younger, and more likely to be US born, white, unmarried, and childless than were other women physicians; their personal health behaviors were similar to those of others. They worked significantly more clinical hours and call nights, but were not more likely to report feeling that they worked too much, had too much work stress, or had less control of their work environment. Their career satisfaction was similar to that of other women physicians, and satisfaction with their specialty was greater. They were less avid preventionists than were primary care practitioners, and somewhat less avid than other specialists. CONCLUSIONS: Women surgeons differ in interesting and important ways from other women physicians.

Mapping a gene involved in regulating dietary cholesterol absorption. The sitosterolemia locus is found at chromosome 2p21.

The molecular mechanisms regulating the amount of dietary cholesterol retained in the body as well as the body's ability to selectively exclude other dietary sterols are poorly understood. Studies of the rare autosomal recessively inherited disease sitosterolemia (OMIM 210250) may shed some light on these processes. Patients suffering from this disease appear to hyperabsorb both cholesterol and plant sterols from the intestine. Additionally, there is failure of the liver's ability to preferentially and rapidly excrete these non-cholesterol sterols into bile. Consequently, people who suffer from this disease have very elevated plasma plant sterol levels and develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. Identification of this gene defect may therefore throw light on regulation of net dietary cholesterol absorption and lead to an advancement in the management of this important cardiovascular risk factor. By studying 10 well-characterized families with this disorder, we have localized the genetic defect to chromosome 2p21, between microsatellite markers D2S1788 and D2S1352 (maximum lodscore 4.49, theta = 0.0).

LY320135, a novel cannabinoid CB1 receptor antagonist, unmasks coupling of the CB1 receptor to stimulation of cAMP accumulation.

LY320135 is a selective antagonist for the brain CB1 receptor, having greater than 70-fold higher affinity for the CB1 than the peripheral CB2 receptor. The Ki values for LY320135 at the CB1 and CB2 receptors, transfected and stably expressed in cell lines, were 224 nM and > 10 microM, respectively. Similar Ki values were measured in binding studies performed on cerebellum and spleen membrane preparations endogenously expressing the CB1 (203 nM) and CB2 (> 10 microM) receptors, respectively. LY320135 functionally reversed anandamide-mediated adenylate cyclase inhibition in Chinese hamster ovary (CHO) cells stably expressing the CB1 receptor. Pertussis toxin treatment of CHO cells expressing the CB1 receptor attenuated the anandamide-mediated inhibition of adenylate cyclase and unmasked a stimulatory effect of anandamide on adenylate cyclase. The stimulatory component was blocked with LY320135. This compound also blocked WIN 55212-2-mediated inhibition of N-type calcium channels and activation of inwardly rectifying potassium channels in N18 and AtT-20-CB2 cells, respectively. LY320135 is a promising lead compound for the further development of novel, potent and selective cannabinoid antagonists of novel structure.

Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome-wide search.

Despite its high prevalence, very little is known regarding genetic predisposition to prostate cancer. A genome-wide scan performed in 66 high-risk prostate cancer families has provided evidence of linkage to the long arm of chromosome 1 (1q24-25). Analysis of an additional set of 25 North American and Swedish families with markers in this region resulted in significant evidence of linkage in the combined set of 91 families. The data provide strong evidence of a major prostate cancer susceptibility locus on chromosome 1.

Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping.

Taq DNA polymerase can catalyze non-templated addition of a nucleotide (principally adenosine) to the 3' end of PCR-amplified products. Recently, we showed that this activity, which is primer-specific, presents a potential source of error in genotyping studies based on the use of short tandem repeat (STR) markers. Furthermore, in reviewing our data, we found that non-templated nucleotide addition adjacent to a 3' terminal C is favored and that addition adjacent to a 3' terminal A is not. It was clear, however, that features of the template in addition to the 3' terminal base also affect the fraction of product adenylated. To define consensus sequences that promote or inhibit product adenylation, we transplanted sequences between the 5' ends of the reverse primers of markers that are adenylated and those of markers that are not adenylated. It proved difficult to identify a single sequence capable of protecting the products of all markers from non-templated addition of nucleotide. On the other hand, placing the sequence GTTTCTT on the 5' end of reverse primers resulted in nearly 100% adenylation of the 3' end of the forward strand. This modification or related ones (called "PIG-tailing") should facilitate accurate genotyping and efficient T/A cloning.

Use of peptide combinatorial libraries in drug design: the identification of a potent serotonin reuptake inhibitor derived from a tripeptide cassette library.

BACKGROUND: Medicinal chemistry traditionally requires the identification of biologically active molecules by synthesizing and screening each purified substrate. Further progress in drug discovery then requires definition of the structure-activity relationship of the lead compound. More recently, combinatorial chemistry has emerged as a way to examine structure-activity relationships by screening a large mixture of compounds synthesized in a predictably random manner, without the labor-intensive costs of molecular isolation and purification. We set out to use this approach to examine the structural requirements for peptide binding to serotonin and dopamine transporters. RESULTS: We screened a tripeptide cassette library for serotonin and dopamine reuptake inhibition using cloned transporter assay systems. The method has afforded a number of tripeptide pharmacophores with inhibitory IC50 values ranging from 10 microM to < 1 microM in the dopamine and serotonin reuptake systems. The conformation of one of these tripeptides, N-acetyl-D-Trp-L-Phe-D-Lys-CONH2 (which inhibits serotonin uptake with an IC50 of 10 microM) was compared to that of the serotonin uptake inhibitor s-fluoxetine, and was shown to be more similar in conformation to fluoxetine than was an analogous tripeptide containing L-Lys (IC50 > 50 microM). CONCLUSIONS: We have identified five tripeptides with inhibitory IC50 values of < 10 microM in the serotonin reuptake system. One tripeptide was predicted to have pharmacophore features similar to that of fluoxetine, a selective and potent non-peptide serotonin reuptake inhibitor. Our results suggest that tripeptides derived from combinatorial libraries will help to define the important structural elements of pharmacophores.

Gastric inhibitory polypeptide receptor, a member of the secretin-vasoactive intestinal peptide receptor family, is widely distributed in peripheral organs and the brain.

Gastric inhibitory polypeptide (GIP), or glucose-dependent insulinotropic peptide, is released from endocrine cells in the small intestine after meals. It is involved in several facets of the anabolic response and is thought to be particularly important in stimulating insulin secretion. We have cloned, functionally expressed, and mapped the distribution of the receptor for GIP. It is a member of the secretin-vasoactive intestinal polypeptide family of G-protein-coupled receptors. When expressed in tissue culture cells, it stimulates cAMP production (EC50 0.3 nM) and also increases intracellular calcium accumulation. GIP receptor mRNA is present in the pancreas as well as the gut, adipose tissue, heart, pituitary, and inner layers of the adrenal cortex, whereas it is not found in kidney, spleen, or liver. It is also expressed in several brain regions, including the cerebral cortex, hippocampus, and olfactory bulb. These results suggest that GIP may have previously undescribed actions. GIP receptor localization in the adrenal cortex suggests that it may have effects on glucocorticoid metabolism. Neither GIP nor its effects have been described in the central nervous system, and mRNA for the known peptide ligand for the receptor cannot be detected in the brain by in situ hybridization or polymerase chain reaction. This suggests that a novel peptide may be present in the brain.

Basaloid follicular hamartoma: solitary and multiple types.

BACKGROUND: Multiple skin tumors often show autosomal dominant inheritance; solitary neoplasms are typically nonhereditary. OBJECTIVE: The purpose of this study was to investigate a possible hereditary pattern in patients with multiple and solitary basaloid follicular hamartomas. METHODS: Four new familial cases of multiple basaloid follicular hamartomas and 56 solitary nonhereditary examples were identified and their inheritance pattern recorded. RESULTS: Clinically, basaloid follicular hamartomas were typically 1 to 2 mm, smooth facial papules. Histologically, they were well-circumscribed lesions composed of anastomosing strands of squamoid and basaloid cells in a loose stroma. Horn cysts and pigmentation were common. No significant association with other cutaneous or internal disease was found. CONCLUSION: Basaloid follicular hamartoma, a unique benign follicular tumor, was often diagnosed previously as trichoepithelioma or basal cell carcinoma. It is another cutaneous neoplasm in which patients with multiple lesions show autosomal dominant inheritance, whereas solitary growths with identical clinical, microscopic, and biologic features are nonhereditary.