Life or death decisions in the corpus luteum.

The corpus luteum (CL) is an ephemeral endocrine organ. During its lifespan, it undergoes a period of extremely rapid growth that involves hypertrophy, proliferation and differentiation of the steroidogenic cells, as well as extensive angiogenesis. The growth phase is followed by a period in which remodelling of the tissue ceases, but it engages in unparalleled production of steroids, resulting in extraordinarily high metabolic activity within the tissue. It is during this stage that a critical juncture occurs. In the non-fertile cycle, uterine release of prostaglandin (PG)F(2α) initiates a cascade of events that result in rapid loss of steroidogenesis and destruction of the luteal tissue. Alternatively, if a viable embryo is present, signals are produced that result in rescue of the CL. This review article summarizes the major concepts related to the fate of the CL, with particular focus on recent insights into the mechanisms associated with the ability of PGF(2α) to bring about complete luteolysis. It has become clear that the achievement of luteolysis depends on repeated exposure to PGF(2α) and involves coordinated actions of heterogeneous cell types within the CL. Together, these components of the process bring about not only the loss in progesterone production, but also the rapid demise of the structure itself.

Truncated BRCA2 is cytoplasmic: implications for cancer-linked mutations.

BRCA2 mutations predispose carriers mainly to breast cancer. The vast majority of BRCA2 mutations are predicted to result in a truncated protein product. The smallest known cancer-associated deletion removes from the C terminus only 224 of the 3,418 residues constituting BRCA2, suggesting that these terminal amino acids are crucial for BRCA2 function. A series of green fluorescent protein (GFP)-tagged BRCA2 deletion mutants revealed that nuclear localization depends on two nuclear localization signals that reside within the final 156 residues of BRCA2. Consistent with this observation, an endogenous truncated BRCA2 mutant (6174delT) was found to be cytoplasmic. Together, these studies provide a simple explanation for why the vast majority of BRCA2 mutants are nonfunctional: they do not translocate into the nucleus.

Functional characterization of a purified, recombinant NF-kappaB/IkappaB complex.

The NF-kappaB signal transduction pathway involves the interaction of several NF-kappaB and IkappaB family members that are activated by a diverse range of extracellular signals and that stimulate many different cellular responses. The biochemical regulation of this cascade can be studied by establishing a cell-free system using purified proteins. As a first step toward establishing an in vitro model incorporating multiple combinations of NF-kappaB and IkappaB proteins, we produced purified human p65 (RelA) and human IkappaBalpha proteins and tested their functionality. Full-length RelA and IkappaBalpha proteins were overproduced by coinfection of TN5-JE cells with two recombinant baculoviruses. RelA and IkappaBalpha formed a stable complex that could be purified to >95% homogeneity. Protein-protein interactions, protein-DNA binding, and protein phosphorylation were similar to the native proteins.

Identification of oral mitis group streptococci by arbitrarily primed polymerase chain reaction.

"Mitis group" streptococci are commensal but may play some role in dental caries, septicemia or endocarditis. Rapid genotypic identification would aid studies of dental plaque ecology, or diagnostic use. AP-PCR with 58 unpaired arbitrary primers was used to characterize 7 Streptococcus gordonii, 11 Streptococcus sanguis, 2 Streptococcus crista, 5 Streptococcus parasanguis, 18 Streptococcus oralis, and 36 Streptococcus mitis (22 biovar 1 and 14 biovar 2). S. parasanguis 16S rRNA variable region primer RR2 produced species-specific bands with all S. gordonii and S. sanguis. Human V beta 1 T-cell receptor primer 434 yielded concordant genotypic identification of all phenotypically defined S. crista and S. parasanguis, 83% of S. oralis, and 74% of S. mitis biovar 1. Amplicon patterns for S. mitis biovar 2 were heterogeneous. Findings suggest that primers RR2 and 434 in succession will allow rapid identification of genotypic groups corresponding closely to mitis group species established by phenotype.

Freudian symbols on sexual and creative aspects of stories.

Sexual content and creativity of stories and story titles was investigated. 96 college students responded to visual presentation of instances of theoretical Freudian symbols. Analyses subjected responses to a 2 (sex) x 2 (symbol) x 2 (mode) x 6 (subscales) analysis of variance with repeated measures on subscales and to multivariate analysis of variance procedures with four dependent measures. These showed men wrote masculine stories and women wrote feminine stories. Certain subscales were more sensitive to sexual content than others. Pairwise comparisons between the subscales among instances of symbols emerged as significant. In addition, subjects exposed to Male symbols wrote stories containing greater latent sexual content than subjects exposed to Female symbols. Creativity of story titles was evident only on a univariate analysis of variance.

Structure of the human NF-kappaB p52 homodimer-DNA complex at 2.1 A resolution.

The crystal structure of human NF-kappaB p52 in its specific complex with the natural kappaB DNA binding site MHC H-2 has been solved at 2.1 A resolution. Whereas the overall structure resembles that of the NF-kappaB p50-DNA complex, pronounced differences are observed within the 'insert region'. This sequence segment differs in length between different Rel proteins. Compared with NF-kappaB p50, the compact alpha-helical insert region element is rotated away from the core of the N-terminal domain, opening up a mainly polar cleft. The insert region presents potential interaction surfaces to other proteins. The high resolution of the structure reveals many water molecules which mediate interactions in the protein-DNA interface. Additional complexity in Rel protein-DNA interaction comes from an extended interfacial water cavity that connects residues at the edge of the dimer interface to the central DNA bases. The observed water network might acount for differences in binding specificity between NF-kappaB p52 and NF-kappaB p50 homodimers.

Saliva protein binding to streptococcal layers placed at different oral sites in 48 persons.

Preliminary studies of 10 subjects suggested that saliva protein binding to oral bacteria might vary among oral sites. This study investigated saliva protein binding to layers of oral streptococci in an expanded sample of 48 subjects. Those persons were at opposite extremes for unstimulated whole saliva amylase, sIgA, lactoferrin, and lysozyme in an initial screening of 128 individuals. Layers of Streptococcus gordonii Blackburn or Streptococcus oralis 10557 on enamel chips were placed on buccal left and right upper premolars and molars (UL, UR), labial upper central incisors (UC), and lingual lower central incisors (LL). After a 10-minute exposure to saliva, bacterial extracts were assayed for bound amylase, sIgA, lactoferrin, and lysozyme. Those proteins also were quantified in unstimulated whole saliva collected after chip exposure. Both strains bound significantly more amylase at UL and UR, and significantly less at UC. Blackburn bound more amylase than 10557 at all sites. Significantly less sIgA was bound at UC; strain differences for sIgA were inconsistent across sites. Significantly more lactoferrin and lysozyme were bound at LL. There were no strain differences for lactoferrin; 10557 bound significantly more lysozyme at UL and UR. Subjects at opposite extremes for saliva protein concentrations differed for bound amylase and lactoferrin; those differences were smaller than site and strain differences. Bound protein levels were correlated across sites and strains. Correlations between whole saliva and bound proteins were moderate and were most consistent at LL. These findings suggest that saliva protein effects on oral ecology may vary among oral sites.

CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter.

Transcription factors play a key role in the development and differentiation of specific lineages from multipotential progenitors. Identification of these regulators and determining the mechanism of how they activate their target genes are important for understanding normal development of monocytes and macrophages and the pathogenesis of a common form of adult acute leukemia, in which the differentiation of monocytic cells is blocked. Our previous work has shown that the monocyte-specific expression of the macrophage colony-stimulating factor (M-CSF) receptor is regulated by three transcription factors interacting with critical regions of the M-CSF receptor promoter, including PU.1 and AML1.PU.1 is essential for myeloid cell development, while the AML1 gene is involved in several common leukemia-related chromosome translocations, although its role in hematopoiesis has not been fully identified. Along with AML1, a third factor, Mono A, interacts with a small region of the promoter which can function as a monocyte-specific enhancer when multimerized and linked to a heterologous basal promoter. Here, we demonstrate by electrophoretic mobility shift assays with monocytic nuclear extracts, COS-7 cell-transfected factors, and specific antibodies that the monocyte-enriched factor Mono A is CCAAT enhancer-binding protein (C/EBP). C/EBP has been shown previously to be an important transcription factor involved in hepatocyte and adipocyte differentiation; in hematopoietic cells, C/EBP is specifically expressed in myeloid cells. In vitro binding analysis reveals a physical interaction between C/EBP and AML1. Further transfection studies show that C/EBP and AML1 in concert with the AML1 heterodimer partner CBF beta synergistically activate M-CSF receptor by more then 60 fold. These results demonstrate that C/EBP and AML1 are important factors for regulating a critical hematopoietic growth factor receptor, the M-CSF receptor, suggesting a mechanism of how the AML1 fusion protein could contribute to acute myeloid leukemia. Furthermore, they demonstrate physical and functional interactions between AML1 and C/EBP transcription factor family members.

A DNA-bending protein interacts with an essential upstream regulatory element of the human embryonic beta-like globin gene.

The mammalian beta-like globin gene family has served as an important model system for analysis of tissue- and developmental state-specific gene regulation. Although the activities of a number of regulatory proteins have been implicated in the erythroid cell-specific transcription of globin genes, the mechanisms that restrict their expression to discrete stages of development are less well understood. We have previously identified a novel regulatory element (PRE II) upstream from the human embryonic beta-like globin gene (epsilon) that synergizes with other sequences to confer tissue- and stage-specific expression on a minimal epsilon-globin gene promoter in cultured embryonic erythroid cells. Binding of an erythroid nuclear protein (PRE II-binding factor [PRE-IIBF]) to the PRE II control element is required for promoter activation. Here we report on some of the biochemical properties of PREIIBF, including the characterization of its specificity and affinity for DNA. The embryonic and adult forms of PREIIBF recognize their cognate sequences with identical specificities, supporting our earlier conclusion that they are very similar proteins. PREIIBF binds DNA as a single polypeptide with an Mr of approximately 80,000 to 85,000 and introduces a bend into the target DNA molecule. These results suggest a mechanism by which PREIIBF may contribute to the regulation of the embryonic beta-like globin gene within the context of a complex locus.

The prediction of saliva swallowing frequency in humans from estimates of salivary flow rate and the volume of saliva swallowed.

Saliva swallowing frequency is an important factor in models of oral clearance. It varies widely among individuals, and the basis for that variation has not been established. This study evaluated the use of unstimulated flow rate and the volume of saliva swallowed as predictors of swallowing frequency in 128 first-year dental students. A microphone was placed over the larynx, and swallowing activity was recorded for 30 min between 3-6:00 p.m. The average interval between swallows was determined, and individuals retained saliva in the mouth for a period equal to that time. Retained saliva was spat out, and volume was determined gravimetrically. Four replicate tubes were collected. Flow rate was determined as sample volume over average swallow time. A subset of 10 individuals was measured on two further occasions 2-3 months apart. An independent estimate of flow rate was taken on the second occasion. Repeat-measures analysis of variance and intraclass correlations were used to estimate the reproducibility of replicate volumes, and of measurements taken on different occasions. Associations between swallowing interval, saliva volume, and flow rate were evaluated by multiple regression. Replicate volumes were highly reproducible, as were measurements of volumes, swallow times, and flow rates on different occasions. Saliva volume and flow rate jointly accounted for 99% of variance in swallowing intervals. Swallowing intervals were shortest for individuals who combined high flow rates with small saliva volumes; current models suggest that their oral clearance might be most efficient. Swallowing intervals were longest for individuals with low flow rates and large volumes; their oral clearance might be the least efficient.

Saliva protein binding to layers of oral streptococci in vitro and in vivo.

This paper reports a system for measuring saliva protein binding to oral streptococci. Enamel chips with layers of Streptococcus gordonii Blackburn or Streptococcus oralis 10557 were incubated in vitro with whole saliva from eight persons. Blackburn bound significantly more amylase than 10557; no strain differences were seen for lysozyme or lactoferrin. There were significant correlations between saliva and bound amylase and lactoferrin. Blackburn and 10557 chips were then placed in ten subjects. Sites included the buccal left and right upper premolars and molars (UL, UR), labial upper central incisors (UC), and lingual lower central incisors (LL). That study was repeated three months later; chips with Streptococcus sanguis 13379 were also placed then. Blackburn bound significantly more amylase than the other strains. Blackburn and 10557 both bound the most amylase at UL and UR, and the least amylase at UC. However, strain 13379 bound less amylase at UL. That strain also bound significantly less sIgA at UL. All three strains bound the least sIgA at UC. Lysozyme and lactoferrin binding showed few differences among sites or strains. Bound protein concentrations were significantly correlated across sites and strains within subjects, but not correlated with whole saliva. Strain differences may reflect species differences in amylase binding, or differences in species-specific sIgA titers. Site differences may indicate local variation in protein availability. Differences between chip correlations with whole saliva in vitro and in vivo suggest that the salivary film may be modified as it flows over tooth surfaces.

Use of restriction fragment polymorphism analysis of rRNA genes to assign species to unknown clinical isolates of oral viridans streptococci.

This study evaluated restriction fragment length polymorphisms of rRNA genes (ribotyping) for genotypic identification of 53 oral isolates classified as "Streptococcus sanguis" by colony morphology. Isolates were from 8-h buccal plaque on lower first permanent molars of 20 subjects. DNA was digested with AatII and hybridized with digoxygenin-labeled cDNA of Escherichia coli 16S and 23S rRNA. Strains were ribotyped again with AlwNI or PvuII on the basis of the presence or absence of a 2,290-bp AatII band. Band patterns were compared with reference ribotypes for Streptococcus gordonii, Streptococcus sanguis, Streptococcus crista, Streptococcus oralis, Streptococcus mitis, and Streptococcus parasanguis strains. Forty-eight isolates could be assigned to a species (22 S. sanguis, 14 S. oralis, 12 S. gordonii). Multiple species were seen in 14 subjects; multiple strains of the same species occurred in 11 subjects. Our findings suggest that ribotyping can be used for genotypic identification of S. sanguis, S. oralis, and S. gordonii isolates.

Species identification of oral viridans streptococci by restriction fragment polymorphism analysis of rRNA genes.

Oral streptococci formerly classified as Streptococcus sanguis have been divided into six genetic groups. Methods to identify those species by genotype are needed. This study compared restriction fragment polymorphisms of rRNA genes (ribotypes) for seven S. gordonii, three S. sanguis, four S. oralis, three S. mitis, one S. crista, and seven S. parasanguis strains classified in previous DNA hybridization studies, as well as one clinical isolate. DNA was digested with HindIII, PvuII, HindIII and PvuII combined, EcoRI, BamHI, AatII, AlwNI, and DraII. DNA fragments were hybridized with a digoxigenin-labeled cDNA probe obtained by reverse transcription of Escherichia coli 16S and 23S rRNA. S. oralis, S. mitis, and S. parasanguis all showed an isolated 2,290-bp band in AatII ribotypes that was absent from S. gordonii, S. sanguis, and S. crista. The last three groups showed species-specific bands with AatII and also with PvuII. S. oralis could be distinguished from S. mitis and S. parasanguis in AlwNI and DraII ribotypes. S. mitis and S. parasanguis could not be distinguished, since they shared multiple bands in PvuII, AlwNI, and EcoRI patterns. The clinical isolate in the panel was very similar to S. sanguis by all enzymes used. Our findings suggest that ribotyping may be useful for genotypic identification of oral viridans streptococci. Initial digests of clinical isolates might be made with AatII, followed by PvuII or AlwNI. Isolates then could be identified by comparing ribotype patterns with those of reference strains. This approach could facilitate clinical studies of these newly defined species.

Purification of the major histocompatibility complex class I transcription factor H2TF1. The full-length product of the nfkb2 gene.

H2TF1 is a ubiquitous major histocompatibility complex (MHC) class I-specific transcription factor, which binds to the palindromic kappa B enhancer site upstream of MHC class I genes. Here we report that H2TF1 consists of a polypeptide with relative molecular mass 110,000, that corresponds to the predicted 100-kDa product (NF-kappa B2 p100) encoded by the candidate proto-oncogene nfkb2 (lyt-10). H2TF1 was purified by a novel affinity chromatography method and identified as the NF-kappa B2 p100 polypeptide by peptide sequencing as well as by reactivity with a specific antiserum. Purified H2TF1 binds the MHC kappa B site with high affinity (KD = 3 x 10(-11) M), in contrast with previous reports that NF-kappa B2 p100 did not bind DNA.

Human T-cell leukemia virus type I (HTLV-I) and blood transfusion.

Human T-cell leukemia (or T-lymphotropic) virus type I (HTLV-I) is a human exogenous infectious retrovirus of the family Retroviridae. This virus has been associated with adult T-cell leukemia and endemic myelopathies (tropical spastic paraparesis and HTLV-I associated myelopathy). HTLV-I is transmitted by sexual contact, from mother to child, by intravenous drug abuse, and by blood transfusion. The estimated lifetime risk of developing disease in antibody-positive patients is 1 in 80, and a latency period as long as 20 years can intervene. No case of transfusion-transmitted disease has been reported to date. Currently, no testing of blood donors for HTLV-I is required in the United States, and no such test has been approved by the Food and Drug Administration. Because data on the natural history of this virus may take years to accumulate, it is probably wise to begin excluding anti-HTLV-I-positive units from the blood supply in the United States as soon as a licensed test is available.

Fatal acute dissection of the right hepatic artery after appendectomy.

A 53-year-old normotensive man underwent an appendectomy for suppurative appendicitis with peritonitis and Escherichia coli bacteremia. On the third postoperative day, he became severely dyspneic, developed bright red blood flow from his abdominal drains, collapsed, and died. At autopsy, a ruptured intramedial dissection of the right hepatic artery was found. Hepatic artery dissections are rare, but may be associated with abdominal operation, peritonitis, hypertension, or preexisting arterial disease.