Role of ß3 subunit of the GABA type A receptor in triple negative breast cancer proliferation, migration, and cell cycle progression.

Triple negative breast cancer (TNBC) is known for its heterogeneous nature and aggressive onset. The unresponsiveness to hormone therapies and immunotherapy and the toxicity of chemotherapeutics account for the limited treatment options for TNBC. Ion channels have emerged as possible therapeutic candidates for cancer therapy, but little is known about how ligand gated ion channels, specifically, GABA type A ligand-gated ion channel receptors (GABAAR), affect cancer pathogenesis. Our results show that the GABAA ß3 subunit is expressed at higher levels in TNBC cell lines than non-tumorigenic cells, therefore contributing to the idea that limiting the GABAAR via knockdown of the GABAA ß3 subunit is a potential strategy for decreasing the proliferation and migration of TNBC cells. We employed pharmacological and genetic approaches to investigate the role of the GABAA ß3 subunit in TNBC proliferation, migration, and cell cycle progression. The results suggest that pharmacological antagonism or genetic knockdown of GABAA ß3 subunit decreases TNBC proliferation and migration. In addition, GABAA ß3 subunit knockdown causes cell cycle arrest in TNBC cell lines via decreased cyclin D1 and increased p21 expression. Our findings suggest that membrane bound GABAA receptors containing the ß3 subunit can be further developed as a potential novel target for the treatment of TNBC.

An in vitro model of tumor heterogeneity resolves genetic, epigenetic, and stochastic sources of cell state variability.

Tumor heterogeneity is a primary cause of treatment failure and acquired resistance in cancer patients. Even in cancers driven by a single mutated oncogene, variability in response to targeted therapies is well known. The existence of additional genomic alterations among tumor cells can only partially explain this variability. As such, nongenetic factors are increasingly seen as critical contributors to tumor relapse and acquired resistance in cancer. Here, we show that both genetic and nongenetic factors contribute to targeted drug response variability in an experimental model of tumor heterogeneity. We observe significant variability to epidermal growth factor receptor (EGFR) inhibition among and within multiple versions and clonal sublines of PC9, a commonly used EGFR mutant nonsmall cell lung cancer (NSCLC) cell line. We resolve genetic, epigenetic, and stochastic components of this variability using a theoretical framework in which distinct genetic states give rise to multiple epigenetic "basins of attraction," across which cells can transition driven by stochastic noise. Using mutational impact analysis, single-cell differential gene expression, and correlations among Gene Ontology (GO) terms to connect genomics to transcriptomics, we establish a baseline for genetic differences driving drug response variability among PC9 cell line versions. Applying the same approach to clonal sublines, we conclude that drug response variability in all but one of the sublines is due to epigenetic differences; in the other, it is due to genetic alterations. Finally, using a clonal drug response assay together with stochastic simulations, we attribute subclonal drug response variability within sublines to stochastic cell fate decisions and confirm that one subline likely contains genetic resistance mutations that emerged in the absence of drug treatment.

Induction of Dectin-1 and asthma-associated signal transduction pathways in RAW 264.7 cells by a triple-helical (1, 3)-ß-D glucan, curdlan.

People living in damp buildings are typically exposed to spore and mycelial fragments of the fungi that grow on damp building materials. There is experimental evidence that this exposure to triple-helical (1, 3)-ß-D glucan and low molecular weight toxins may be associated with non-atopic asthma observed in damp and moldy buildings. However, the mechanisms underlying this response are only partially resolved. Using the pure (1, 3)-ß-D glucan, curdlan, and the murine macrophage cell line, RAW 264.7, there were two objectives of this study. The first was to determine whether signal transduction pathways activating asthma-associated cell signaling pathways were stimulated using mouse transduction Pathway Finder(®) arrays and quantitative real-time (QRT) PCR. The second objective was to evaluate the dose and temporal responses associated with transcriptional changes in asthma-associated cytokines, the signal transduction receptor gene Dectin-1, and various transcription factor genes related to the induction of asthma using customized RT-PCR-based arrays. Compared to controls, the 10(-7) M curdlan treatment induced significant changes in gene transcription predominately in the NFkB, TGF-ß, p53, JAK/STAT, P13/AKT, phospholipase C, and stress signaling pathways. The 10(-8) M curdlan treatment mainly induced NFkB and TGF-ß pathways. Compared to controls, curdlan exposures also induced significant dose- and time-dependent changes in the gene translations. We found that that curdlan as a non-allergenic potentiator modulates a network of transduction signaling pathways not only associated with TH-1, TH-2, and TH-3 cell responses including asthma potentiation, but a variety of other cell responses in RAW 264.7 cells. These results help provide mechanistic basis for some of the phenotypic changes associated with asthma that have been observed in in vitro, in vivo, and human studies and open up a hypothesis-building process that could explain the rise of non-atopic asthma associated with fungi.

A common nonsense mutation in EphB2 is associated with prostate cancer risk in African American men with a positive family history.

BACKGROUND: The EphB2 gene was recently implicated as a prostate cancer (PC) tumour suppressor gene, with somatic inactivating mutations occurring in approximately 10% of sporadic tumours. We evaluated the contribution of EphB2 to inherited PC susceptibility in African Americans (AA) by screening the gene for germline polymorphisms. METHODS: Direct sequencing of the coding region of EphB2 was performed on 72 probands from the African American Hereditary Prostate Cancer Study (AAHPC). A case-control association analysis was then carried out using the AAHPC probands and an additional 183 cases of sporadic PC compared with 329 healthy AA male controls. In addition, we performed an ancestry adjusted association study where we adjusted for individual ancestry among all subjects, in order to rule out a spurious association due to population stratification. RESULTS: Ten coding sequence variants were identified, including the K1019X (3055A-->T) nonsense mutation which was present in 15.3% of the AAHPC probands but only 1.7% of 231 European American (EA) control samples. We observed that the 3055A-->T mutation significantly increased risk for prostate cancer over twofold (Fisher's two sided test, p = 0.003). The T allele was significantly more common among AAHPC probands (15.3%) than among healthy AA male controls (5.2%) (odds ratio 3.31; 95% confidence interval 1.5 to 7.4; p = 0.008). The ancestry adjusted analyses confirmed the association. CONCLUSIONS: Our data show that the K1019X mutation in the EphB2 gene differs in frequency between AA and EA, is associated with increased risk for PC in AA men with a positive family history, and may be an important genetic risk factor for prostate cancer in AA.

Uterine tumours are a phenotypic manifestation of the hyperparathyroidism-jaw tumour syndrome.

The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is an autosomal dominant disorder characterized by parathyroid tumours, which are frequently carcinomas, and ossifying jaw fibromas. In addition, some patients may develop renal tumours and cysts. The gene causing HPT-JT, which is referred to as HRPT2 and is located on chromosome 1q31.2, encodes a 531 amino acid protein called PARAFIBROMIN. To date 42 mutations, of which 22 are germline, have been reported and 97% of these are inactivating and consistent with a tumour suppressor role for HRPT2. We have investigated another four HPT-JT families for germline mutations, searched for additional clinical phenotypes, and examined for a genotype-phenotype correlation. Mutations were found in two families. One family had a novel deletional-insertion at codon 669, and the other had a 2 bp insertion at codon 679, which has been reported in four other unrelated patients. These five unrelated patients and their families with the same mutation were not found to develop the same tumours, thereby indicating an absence of a genotype-phenotype correlation. An analysis of 33 HPT-JT kindreds revealed that affected women in 13 HPT-JT families suffered from menorrhagia in their second to fourth decades. This often required hysterectomy, which revealed the presence of uterine tumours. This resulted in a significantly reduced maternal transmission of the disease. Thus, the results of our analysis expand the spectrum of HPT-JT-associated tumours to include uterine tumours, and these may account for the decreased reproductive fitness in females from HPT-JT families.

HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome.

We report here the identification of a gene associated with the hyperparathyroidism-jaw tumor (HPT-JT) syndrome. A single locus associated with HPT-JT (HRPT2) was previously mapped to chromosomal region 1q25-q32. We refined this region to a critical interval of 12 cM by genotyping in 26 affected kindreds. Using a positional candidate approach, we identified thirteen different heterozygous, germline, inactivating mutations in a single gene in fourteen families with HPT-JT. The proposed role of HRPT2 as a tumor suppressor was supported by mutation screening in 48 parathyroid adenomas with cystic features, which identified three somatic inactivating mutations, all located in exon 1. None of these mutations were detected in normal controls, and all were predicted to cause deficient or impaired protein function. HRPT2 is a ubiquitously expressed, evolutionarily conserved gene encoding a predicted protein of 531 amino acids, for which we propose the name parafibromin. Our findings suggest that HRPT2 is a tumor-suppressor gene, the inactivation of which is directly involved in predisposition to HPT-JT and in development of some sporadic parathyroid tumors.

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.

Cloning and characterization of 13 novel transcripts and the human RGS8 gene from the 1q25 region encompassing the hereditary prostate cancer (HPC1) locus.

The aim of this study was to develop a saturated transcript map of the region encompassing the HPC1 locus to identify the susceptibility genes involved in hereditary prostate cancer (OMIM 176807) and hyperparathyroidism-jaw tumor syndrome (OMIM 145001). We previously reported the generation of a 6-Mb BAC/PAC contig of the candidate region and employed various strategies, such as database searching, exon-trapping, direct cDNA hybridization, and sample sequencing of BACs, to identify all potential transcripts. These efforts led to the identification and precise localization on the BAC contig of 59 transcripts representing 22 known genes and 37 potential transcripts represented by ESTs and exon traps. Here we report the detailed characterization of these ESTs into full-length transcript sequences, their expression pattern in various tissues, their genomic organization, and their homology to known genes. We have also identified an Alu insertion polymorphism in the intron of one of the transcripts. Overall, data on 13 novel transcripts and the human RGS8 gene (homologue of the rat RGS8 gene) are presented in this paper. Ten of the 13 novel transcripts are expressed in prostate tissue and represent positional candidates for HPC1.

Evaluation of linkage and association of HPC2/ELAC2 in patients with familial or sporadic prostate cancer.

To investigate the relationship between HPC2/ELAC2 and prostate cancer risk, we performed the following analyses: (1) a linkage study of six markers in and around the HPC2/ELAC2 gene at 17p11 in 159 pedigrees with hereditary prostate cancer (HPC); (2) a mutation-screening analysis of all coding exons of the gene in 93 probands with HPC; (3) family-based and population-based association study of common HPC2/ELAC2 missense variants in 159 probands with HPC, 249 patients with sporadic prostate cancer, and 222 unaffected male control subjects. No evidence for linkage was found in the total sample, nor in any subset of pedigrees based on characteristics that included age at onset, number of affected members, male-to-male disease transmission, or race. Furthermore, only the two previously reported missense changes (Ser217Leu and Ala541Thr) were identified by mutational analysis of all HPC2/ELAC exons in 93 probands with HPC. In association analyses, family-based tests did not reveal excess transmission of the Leu217 and/or Thr541 alleles to affected offspring, and population-based tests failed to reveal any statistically significant difference in the allele frequencies of the two polymorphisms between patients with prostate cancer and control subjects. The results of this study lead us to reject the three alternative hypotheses of (1) a highly penetrant, major prostate cancer-susceptibility gene at 17p11, (2) the allelic variants Leu217 or Thr541 of HPC2/ELAC2 as high-penetrance mutations, and (3) the variants Leu217 or Thr541 as low-penetrance, risk-modifying alleles. However, we did observe a trend of higher Leu217 homozygous carrier rates in patients than in control subjects. Considering the impact of genetic heterogeneity, phenocopies, and incomplete penetrance on the linkage and association studies of prostate cancer and on the power to detect linkage and association in our study sample, our results cannot rule out the possibility of a highly penetrant prostate cancer gene at this locus that only segregates in a small number of pedigrees. Nor can we rule out a prostate cancer-modifier gene that confers a lower-than-reported risk. Additional larger studies are needed to more fully evaluate the role of this gene in prostate cancer risk.

Abnormal morphological and functional organization of the hippocampus in a p35 mutant model of cortical dysplasia associated with spontaneous seizures.

Cortical dysplasia is a major cause of intractable epilepsy in children. However, the precise mechanisms linking cortical malformations to epileptogenesis remain elusive. The neuronal-specific activator of cyclin-dependent kinase 5, p35, has been recognized as a key factor in proper neuronal migration in the neocortex. Deletion of p35 leads to severe neocortical lamination defects associated with sporadic lethality and seizures. Here we demonstrate that p35-deficient mice also exhibit dysplasia/ heterotopia of principal neurons in the hippocampal formation, as well as spontaneous behavioral and electrographic seizures. Morphological analyses using immunocytochemistry, electron microscopy, and intracellular labeling reveal a high degree of abnormality in dentate granule cells, including heterotopic localization of granule cells in the molecular layer and hilus, aberrant dendritic orientation, occurrence of basal dendrites, and abnormal axon origination sites. Dentate granule cells of p35-deficient mice also demonstrate aberrant mossy fiber sprouting. Field potential laminar analysis through the dentate molecular layer reflects the dispersion of granule cells and the structural reorganization of this region. Similar patterns of cortical disorganization have been linked to epileptogenesis in animal models of chronic seizures and in human temporal lobe epilepsy. The p35-deficient mouse may therefore offer an experimental system in which we can dissect out the key morphological features that are causally related to epileptogenesis.

A 6-Mb high-resolution physical and transcription map encompassing the hereditary prostate cancer 1 (HPC1) region.

Several hereditary disease loci have been genetically mapped to the chromosome 1q24-q31 interval, including the hereditary prostate cancer 1 (HPC1) locus. Here, we report the construction of a 20-Mb yeast artificial chromosome contig and a high-resolution 6-Mb sequence-ready bacterial artificial chromosome (BAC)/P1-derived artificial chromosome (PAC) contig of 1q25 by sequence and computational analysis, STS content mapping, and chromosome walking. One hundred thirty-six new STSs, including 10 novel simple sequence repeat polymorphisms that are being used for genetic refinement of multiple disease loci, have been generated from this contig and are shown to map to the 1q25 interval. The integrity of the 6-Mb BAC/PAC contig has been confirmed by restriction fingerprinting, and this contig is being used as a template for human chromosome 1 genome sequencing. A transcription mapping effort has resulted in the precise localization of 18 known genes and 31 ESTs by database searching, exon trapping, direct cDNA hybridization, and sample sequencing of BACs from the 1q25 contig. An additional 11 known genes and ESTs have been placed within the larger 1q24-q31 interval. These transcription units represent candidate genes for multiple hereditary diseases, including HPC1.

Norepinephrine-deficient mice have increased susceptibility to seizure-inducing stimuli.

Several lines of evidence suggest that norepinephrine (NE) can modulate seizure activity. However, the experimental methods used in the past cannot exclude the possible role of other neurotransmitters coreleased with NE from noradrenergic terminals. We have assessed the seizure susceptibility of genetically engineered mice that lack NE. Seizure susceptibility was determined in the dopamine beta-hydroxylase null mutant (Dbh -/-) mouse using four different convulsant stimuli: 2,2,2-trifluroethyl ether (flurothyl), pentylenetetrazol (PTZ), kainic acid, and high-decibel sound. Dbh -/- mice demonstrated enhanced susceptibility (i.e., lower threshold) compared with littermate heterozygous (Dbh +/-) controls to flurothyl, PTZ, kainic acid, and audiogenic seizures and enhanced sensitivity (i.e., seizure severity and mortality) to flurothyl, PTZ, and kainic acid. c-Fos mRNA expression in the cortex, hippocampus (CA1 and CA3), and amygdala was increased in Dbh -/- mice in association with flurothyl-induced seizures. Enhanced seizure susceptibility to flurothyl and increased seizure-induced c-fos mRNA expression were reversed by pretreatment with L-threo-3, 4-dihydroxyphenylserine, which partially restores the NE content in Dbh -/- mice. These genetically engineered mice confirm unambiguously the potent effects of the noradrenergic system in modulating epileptogenicity and illustrate the unique opportunity offered by Dbh -/- mice for elucidating the pathways through which NE can regulate seizure activity.

Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays.

Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.

Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130.

To study intracellular pathways by which the human papillomavirus 16 oncogene E7 participates in carcinogenesis, we expressed an inducible chimera of E7 by fusion to the hormone-binding domain of the estrogen receptor. The chimeric protein (E7ER) transformed rodent fibroblast cell lines and induced DNA synthesis on addition of estradiol. In coimmunoprecipitation experiments, E7ER preferentially bound p130 when compared to p107 and pRb. After estradiol addition, E7ER localization changed to a more intense intranuclear staining. Induction of E7 function was not correlated with binding to p130 or pRb but rather with intranuclear localization and modest induction of binding to p107.

Evolutionary sequence comparisons using high-density oligonucleotide arrays.

We explored the utility of high-density oligonucleotide arrays (DNA chips) for obtaining sequence information from homologous genes in closely related species. Orthologues of the human BRCA1 exon 11, all approximately 3.4 kb in length and ranging from 98.2% to 83.5% nucleotide identity, were subjected to hybridization-based and conventional dideoxysequencing analysis. Retrospective guidelines for identifying high-fidelity hybridization-based sequence calls were formulated based upon dideoxysequencing results. Prospective application of these rules yielded base-calling with at least 98.8% accuracy over orthologous sequence tracts shown to have approximately 99% identity. For higher primate sequences with greater than 97% nucleotide identity, base-calling was made with at least 99.91% accuracy covering a minimum of 97% of the sequence. Using a second-tier confirmatory hybridization chip strategy, shown in several cases to confirm the identity of predicted sequence changes, the complete sequence of the chimpanzee, gorilla and orangutan orthologues should be deducible solely through hybridization-based methodologies. Analysis of less highly conserved orthologues can still identify conserved nucleotide tracts of at least 15 nucleotides and can provide useful information for designing primers. DNA-chip based assays can be a valuable new technology for obtaining high-throughput cost-effective sequence information from related genomes.

Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays.

Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.

Genomic organization of the murine Miller-Dieker/lissencephaly region: conservation of linkage with the human region.

Several human syndromes are associated with haploinsufficiency of chromosomal regions secondary to microdeletions. Isolated lissencephaly sequence (ILS), a human developmental disease characterized by a smooth cerebral surface (classical lissencephaly) and microscopic evidence of incomplete neuronal migration, is often associated with small deletions or translocations at chromosome 17p13.3. Miller-Dieker syndrome (MDS) is associated with larger deletions of 17p13.3 and consists of classical lissencephaly with additional phenotypes including facial abnormalities. We have isolated the murine homologs of three genes located inside and outside the MDS region: Lis1, Mnt/Rox, and 14-3-3 epsilon. These genes are all located on mouse chromosome 11B2, as determined by metaphase FISH, and the relative order and approximate gene distance was determined by interphase FISH analysis. The transcriptional orientation and intergenic distance of Lis1 and Mnt/Rox were ascertained by fragmentation analysis of a mouse yeast artificial chromosome containing both genes. To determine the distance and orientation of 14-3-3 epsilon with respect to Lis1 and Mnt/Rox, we introduced a super-rare cutter site (VDE) that is unique in the mouse genome into 14-3-3 epsilon by gene targeting. Using the introduced VDE site, the orientation of this gene was determined by pulsed field gel electrophoresis and Southern blot analysis. Our results demonstrate that the MDS region is conserved between human and mouse. This conservation of linkage suggests that the mouse can be used to model microdeletions that occur in ILS and MDS.

Loss of the p53 tumor suppressor gene protects neurons from kainate-induced cell death.

The tumor suppressor gene p53 recently has been associated with the induction of cell death in response to some forms of cellular damage. A possible role for p53-related modulation of neuronal viability has been suggested by the finding that p53 expression is increased in damaged neurons in models of ischemia and epilepsy. We evaluated the possibility that p53 expression (in knockout mice) is required for induction of cell damage in a model of seizure activity normally associated with well defined patterns of cell loss. Subcutaneous injection of kainic acid, a potent excitotoxin, induced comparable seizures in both wild-type mice (+/+) and mice deficient in p53 (-/-). Using a silver impregnation technique to examine neurodegeneration in animals killed 7 d after kainate injection, we found that a majority of +/+ mice exhibited extensive cell loss in the hippocampus, involving subregions CA1, CA3, the hilus, and the subiculum. Apoptotic cell death, as identified with an in situ nick end labeling technique to detect DNA fragmentation, was confirmed in CA1- but not CA3-degenerating neurons. In marked contrast, a majority of p53 -/- mice displayed no signs of cell damage; in the remaining p53 -/- mice, damage was mild to moderate and was confined almost entirely to cells in CA3b of the dorsal hippocampus. In +/+ mice, but not in -/- mice, damaged neurons also were observed in the amygdala, piriform cortex, cerebral cortex, caudate-putamen, and thalamus after kainate treatment. The pattern and extent of damage in mice heterozygous for p53 (+/-) were identical to those seen in +/+ mice, suggesting that a single copy of p53 is sufficient to confer neuronal vulnerability. These results demonstrate that p53 influences viability in multiple neuronal subtypes and brain regions after excitotoxic insult.

Prevalence of cervical pathogens in women with and without inflammatory changes on smear testing.

OBJECTIVE: To assess correlation between nonspecific cervicitis, inflammation, or exudate on cervical smears tests and confirmed presence of known cervical pathogens. DESIGN: Investigation of women attending a family practice clinic for smear test by microbiological screening for Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Trichomonas vaginalis, Candida species, group B streptococcus, Gardnerella vaginalis, and Neisseria gonorrhoeae. SETTING: Family practice teaching clinic in a university hospital. PATIENTS: 411 women presenting for a smear test. MAIN OUTCOME MEASURES: Prevalence of genital infections associated with presence or absence of inflammatory changes on cervical smear. RESULTS: Of the 132 women with inflammatory changes on cervical smear, 64 (48%) had positive cultures. Of the 248 without inflammatory changes, 117 (47%) had positive cultures. Subgroup analysis on individual organisms also showed no significant difference between the two groups. CONCLUSION: Reports of inflammatory changes on cervical smear testing are a poor indicator of infection.

Role of Tannins in Defending Plants Against Ruminants: Reduction in Dry Matter Digestion?

Polyphenolic allelochemicals, such as tannins, are widely thought to reduce the digestibility of plants consumed by herbivores by binding to digestive enzymes and dietary proteins. While the apparent digestibility of protein and, therefore, cell solubles is reduced in mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus) consuming tanniferous forages, digestion of the plant cell wall is not reduced beyond that predicted from its content of lignin, cutin, and silica. The lack of a tannin effect on cell wall digestion in deer is in contrast to studies with domestic sheep and numerous in vitro studies. Herbivores adapted to consume tanniferous forages may defend against such allelochemicals by producing salivary proteins that bind tannins in a highly specific manner. These tannin-salivary protein complexes would reduce apparent digestibilities of protein and cell solubles and, if completely effective, would not reduce cell wall digestion. The occurrence of such proteins in ruminants is reported here for the first time. The saliva composition of mule deer (a mixed feeder that commonly consumes browse) and domestic cattle and sheep (predominant grazers) are compared, and the higher potential of the deer saliva to neutralize tannins is related to their feeding habits. Salivary proteins that preferentially bind tannins may minimize fecal nitrogen losses by maximizing the efficiency of tannin-binding per unit of protein and may reduce the absorption of hydrolyzable tannins and the potential for tannin toxicity.