Limitations to intergenerational inheritance: subchronic paternal stress preconception does not influence offspring anxiety.

Independent studies have observed that a paternal history of stress or trauma is associated with his children having a greater likelihood of developing psychopathologies such as anxiety disorders. This father-to-child effect is reproduced in several mouse models of stress, which have been crucial in developing a greater understanding of intergenerational epigenetic inheritance. We previously reported that treatment of C57Bl/6J male breeders with low-dose corticosterone (CORT) for 28 days prior to mating yielded increased anxiety-related behaviours in their male F1 offspring. The present study aimed to determine whether subchronic 7-day CORT treatment of male mice just prior to mating would be sufficient to induce intergenerational modifications of anxiety-related behaviours in offspring. We report that subchronic CORT treatment of male breeders reduced their week-on-week body weight gain and altered NR3C1 and CRH gene expression in the hypothalamus. There were no effects on sperm count and glucocorticoid receptor protein levels within the epididymal tissue of male breeders. Regarding the F1 offspring, screening for anxiety-related behaviours using the elevated-plus maze, light-dark box, and novelty-suppressed feeding test revealed no differences between the offspring of CORT-treated breeders compared to controls. Thus, it is crucial that future studies take into consideration the duration of exposure when assessing the intergenerational impacts of paternal health.

Impaired p53 function leads to centrosome amplification, acquired ERalpha phenotypic heterogeneity and distant metastases in breast cancer MCF-7 xenografts.

In this study, we establish an MCF-7 xenograft model that mimics the progression of human breast carcinomas typified by loss of p53 integrity, development of centrosome amplification, acquired estrogen receptor (ERalpha) heterogeneity, overexpression of Mdm2 and metastatic spread from the primary tumor to distant organs. MCF-7 cells with abrogated p53 function (vMCF-7(Dnp53)) maintained nuclear ERalpha expression and normal centrosome characteristics in vitro. However, following mitogen stimulation, they developed centrosome amplification and a higher frequency of aberrant mitotic spindles. Centrosome amplification was dependent on cdk2/cyclin activity since treatment with the small molecule inhibitor SU9516 suppressed centriole reduplication. In contrast to the parental MCF-7 cells, when introduced into nude mice as xenografts, tumors derived from the vMCF-7(DNp53) cell line developed a strikingly altered phenotype characterized by increased tumor growth, higher tumor histopathology grade, centrosome amplification, loss of nuclear ERalpha expression, increased expression of Mdm-2 oncoprotein and resistance to the antiestrogen tamoxifen. Importantly, while MCF-7 xenografts did not develop distant metastases, primary tumors derived from vMCF-7(DNp53) cells gave rise to lung metastases. Taken together, these observations indicate that abrogation of p53 function and consequent deregulation of the G1/S cell cycle transition leads to centrosome amplification responsible for breast cancer progression.

Characterization of the X-linked murine centrin Cetn2 gene.

A multi-gene family (Cetn1, Cetn2, and Cetn3) encodes the calcium-binding protein, centrin, in the mouse. This work characterizes the Cetn2 gene. Structurally, Cetn2 consists of five exons and four introns, and contains a classical TATA-less promoter. Cetn2 has two alternate transcription start sites, and a single length 3' untranslated region. Fluorescence in situ hybridization demonstrates that Cetn2 is an X-linked gene whose alleles replicate asynchronously during S-phase. Cetn2 encodes a 172 amino acid protein, with a predicted molecular mass of 19,795 Da (pI=4.71), that contains all of the defining characteristics of centrin. Northern blot analysis indicates that Cetn2 is ubiquitously expressed in the tissues of adult mice. RT-PCR shows that Cetn2 and Cetn3, but not Cetn1, are expressed in NIH 3T3 cells. Immunofluorescence microscopy demonstrates that mouse centrin 2 protein localizes to the region immediately surrounding the centrioles in the centrosome of NIH 3T3 cells.

Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein.

The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.

Site-directed mutagenesis and biochemical analysis of the endogenous ligands in the ferrous active site of clavaminate synthase. The His-3 variant of the 2-His-1-carboxylate model.

The facial 2-His-1-carboxylate (Asp/Glu) motif has emerged as the structural paradigm for metal binding in the alpha-ketoglutarate (alpha-KG)-dependent nonheme iron oxygenases. Clavaminate synthase (CS2) is an unusual member of this enzyme family that mediates three different, nonsequential reactions during the biosynthesis of the beta-lactamase inhibitor clavulanic acid. In this study, covalent modification of CS2 by the affinity label N-bromoacetyl-L-arginine near His297, which is within the HRV signature of a His-2 motif, suggested this histidine could play a role in metal coordination. However, site-specific mutagenesis of eight His residues to Gln identified His145 and His280, but not His297, as involved in iron binding. Weak homology of His145 and its flanking sequence and the presence of Glu147 fitting the canonical acidic residue of the His-Xaa-Asp/Glu signature are consistent with His145 being a coordinating ligand (His-1). His280 and its flanking sequence, which give poor alignments to most other members of this enzyme family, are similar among a subset of these enzymes and notably to CarC, an apparent oxygenase involved in carbapenem biosynthesis. The separation of His145 and His280 is more than twice that seen in the current 2-His-1-carboxylate model and may define an alternative iron binding motif, which we propose as His-3. These ligand assignments, based on kinetic measurements of both oxidative cyclization/desaturation and hydroxylation assays, establish that no histidine ligand switching occurs during the catalytic cycle. These results are confirmed in a recent X-ray crystal structure of CS1, a highly similar isozyme of CS2 (81% identical). Tyr299, Tyr300 in CS2 modified by N-bromoacetyl-L-arginine, is hydrogen bonded to Glu146 (Glu147 in CS2) in this structure and well-positioned for reaction with the affinity label.

The MCMI-II and race.

In this study, we investigated MCMI-II profile differences in a sample of 65 Black and 164 White psychiatric inpatients. A multivariate analysis of variance (MANOVA) yielded a significant multivariate effect associated with race, with Black patients scoring significantly higher on the Histrionic, Narcissistic, Paranoid, Drug Dependent and Delusional Disorder scales. A second MANOVA was conducted on these 5 scales with a smaller sample of 46 Black and 46 White patients, who were matched for primary Axis I discharge diagnosis and matched for substance abuse comorbidity. This MANOVA did not yield a significant multivariate effect associated with race, and scale differences were attenuated.

A single monomeric iron center in clavaminate synthase catalyzes three nonsuccessive oxidative transformations.

The trifunctional oxygenase clavaminate synthase 2 (CS2) catalyses a hydroxylation reaction and two coupled oxidative reactions, a cyclization and a desaturation, in a nonsuccessive manner. A series of experiments was performed to elucidate the number of CS2 catalytic site(s) utilized in the three oxidative transformations. The stoichiometry of FeII required by CS2 was determined to be one ion per catalytically active enzyme molecule for the cyclization/desaturation reactions, and an affinity label, modeled after the substrate for the hydroxylation reaction, was synthesized and effectively inactivated CS2. The kinetics of this process showed concentration dependence and substrate protection consistent with active site direction. In addition, when this affinity label was incubated with CS2, the enzyme showed the same first-order rate of activity loss over time in both the hydroxylation activity assay and the cyclization/desaturation activity assay. These results support the view that all of the reactions catalysed by CS2 occur in a single catalytic site containing one FeII.

Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis.

Proclavaminate amidino hydrolase (PAH) catalyzes the reaction of guanidinoproclavaminic acid to proclavaminic acid and urea, a central step in the biosynthesis of the beta-lactamase inhibitor clavulanic acid. The gene encoding this enzyme (pah) was tentatively identified within the clavulanic acid biosynthetic cluster in Streptomyces clavuligerus by translation to a protein of the correct molecular mass (33 kDa) and appreciable sequence homology to agmatine ureohydrolase (M.B.W. Szumanski and S.M. Boyle, J. Bacteriol. 172:538-547, 1990) and several arginases, a correlation similarly recognized by Aidoo et al. (K. A. Aidoo, A. Wong, D. C. Alexander, R. A. R. Rittammer, and S. E. Jensen, Gene 147:41-46, 1994). Overexpression of the putative open reading frame as a 76-kDa fusion to the maltose-binding protein gave a protein having the catalytic activity sought. Cleavage of this protein with factor Xa gave PAH whose N terminus was slightly modified by the addition of four amino acids but exhibited unchanged substrate specificity and kinetic properties. Directly downstream of pah lies the gene encoding clavaminate synthase 2, an enzyme that carries out three distinct oxidative transformations in the in vivo formation of clavulanic acid. After the first of these oxidations, however, no further reaction was found to occur in vitro without the intervention of PAH. We have demonstrated that concurrent use of recombinant clavaminate synthase 2 and PAH results in the successful conversion of deoxyguanidinoproclavaminic acid to clavaminic acid, a four-step transformation. PAH has a divalent metal requirement, pH activity profile, and kinetic properties similar to those of other proteins of the broader arginase class.

Posttraumatic stress disorder and the MCMI-II.

This study investigated the MCMI-II profile characteristics of 39 veterans diagnosed with Posttraumatic Stress Disorder. Characteristics of the mean group profile were similar to prior findings reported in the literature on the MCMI and Posttraumatic Stress Disorder with highest mean elevations found on the Avoidant, Passive-Aggressive, Schizoid, and Antisocial basic personality scales, the Borderline and Schizotypal pathological personality scales, and with elevations on the Anxiety, Dysthymia, Alcohol Dependence, Drug Dependence, and Major Depression clinical syndrome scales. A multivariate analysis of variance comparing the group with Posttraumatic Stress Disorder with a non-PTSD comparison group of 39 on the basic personality, pathological personality, and the clinical syndrome scales of the MCMI-II was not statistically significant. Nonetheless, univariate analyses of variance comparing the two groups on the individual modifier scales and the individual personality and clinical syndrome scales of the MCMI-II using a Bonferroni adjusted probability indicated significant differences on the Desirability and Histrionic scales. Response-style bias as a possible factor in MCMI-II profiles for the group with Posttraumatic Stress Disorder is also discussed.

Expression and purification of two isozymes of clavaminate synthase and initial characterization of the iron binding site. General error analysis in polymerase chain reaction amplification.

Clavaminate synthase is an Fe(2+)-, O2-, and alpha-ketoglutarate-dependent oxygenase that catalyzes three transformations in the biosynthesis of the important beta-lactamase inhibitor clavulanic acid. The genes from Streptomyces clavuligerus encoding two isoenzymes of clavaminate synthase have been over-expressed in Escherichia coli to give soluble proteins whose reactions, kinetic properties, and molecular masses are in excellent agreement with the wild-type isozymes. Preliminary investigation of the active site of clavaminate synthase was undertaken using diethyl pyrocarbonate and N-ethylmaleimide. Each was inhibitory to catalytic activity. Protection from inactivation in the presence of these reagents by Fe2+, O2, and alpha-ketoglutaric acid was thwarted by the rapid self-inactivation of the enzyme in the absence of substrate. However, protection was achieved when Co2+, a potent competitive inhibitor of clavaminate synthase 2 with respect to Fe2+, was substituted. This is consistent with the presence of histidine and cysteine, respectively, at or near the active site and possibly involved in iron binding. In the course of constructing the expression vector, a simply applied general error analysis of the polymerase chain reaction was formulated to calculate the proportion of correctly replicated DNA and guide the design of experiments using this method.

Post-traumatic stress disorder and the MMPI-2.

This study compared the MMPI-2 profiles of 27 veterans diagnosed with post-traumatic stress disorder with a non-PTSD comparison group of 27 veteran patients receiving inpatient treatment for other mental disorders. Three multivariate analyses of variance were conducted comparing the two groups on the 10 traditional clinical scales, the 12 supplemental scales and the 15 new content scales on the MMPI-2. The PTSD group obtained a mean profile with peak elevations on the F validity scale and on clinical Scales 2 (D) and 8 (Sc). The multivariate analysis of variance comparing the PTSD and non-PTSD groups across the 10 traditional clinical scales was not significant. The multivariate analyses of variance comparing the two groups on the 12 supplemental scales and the 15 content scales were significant. Significant univariate supplemental scale differences were found on the Keane PTSD scale (PK) and the Post-Traumatic Stress Disorder (PS) scale with the PTSD group scoring higher on PK and PS. Significant univariate content scale differences were found for the Anger (ANG) scale with the PTSD group scoring higher. A cut-off score of 28 on the PK scale correctly classified 76% of the overall sample, 67% of the PTSD group and 85% of the non-PTSD-comparison group.

MMPI-2 negative treatment indicators scale and irregular discharge.

A sample of 18 psychiatric inpatients who had completed the MMPI-2 and subsequently received an irregular discharge from inpatient treatment were compared on the MMPI-2 Negative Treatment Indicators scale (TRT) with a random sample of 18 inpatients who received a regular discharge from inpatient care. Analysis showed no significant difference between the two groups on the Negative Treatment Indicators scale. The two groups did differ in K scale elevation. The possible need to interpret the Negative Treatment Indicators scale in the context of K scale elevation is discussed.

Prediction of twenty-four-hour energy expenditure in a respiration chamber in smokers and non-smokers.

Fourteen subjects (seven smokers, seven non-smokers) underwent at least one measurement of 24-h energy expenditure (EE) in a respiration chamber, smoking or not smoking as normal. Activity levels (AL) were calculated as multiples of basal metabolic rate (BMR) from records of time spent in specified activity categories and their average published energy costs. Predicted 24-h EE was estimated by multiplying AL by BMR measured on exit from the chamber. Smokers smoked an average of 18.6 cigarettes during the 24-h EE measurement (range 9-29). Measured 24-h EE was higher than predicted in the smokers (7.1%, P < 0.001) but not significantly different from predicted in the non-smokers (2.6% lower). We conclude that smoking increases 24-h EE and that factorial prediction of EE using average published energy costs of activities under-estimates 24-h EE in smokers but not in non-smokers.

A strategy for identifying novel, mechanistically unique inhibitors of topoisomerase I.

While the design of molecules that inhibit or antagonize the functions of specific macromolecules is now well precedented, in many cases the structural information requisite to the design process is lacking. The tools of molecular biology can now furnish the target macromolecules for use in mechanism-based exploration; highly defined assays can be devised based upon the known biochemistry of these macromolecules to permit the discovery of novel inhibitors or antagonists present in chemical collections. Presently, we describe a set of assays directed toward the discovery of novel inhibitors of eukaryotic topoisomerase I, an enzyme critical to maintenance of chromosomal DNA topology and therefore essential for normal replication and transcription. The identification of chebulagic acid as an extraordinarily potent and mechanically novel inhibitor of topoisomerase I illustrates the potential of this approach.

Perineal hidradenitis suppurativa: presentation of two unusual complications and a review.

Two patients with advanced perineal hidradenitis suppurativa, complicated by fecal incontinence and squamous cell carcinoma, are presented. The first patient was a 58-year-old man who had a 30-year history of chronic recurring perianal abscesses and perineal sinuses. At the time of presentation, he had extensive perineal suppurative disease, and scarring and fixation of the anal sphincters with resultant fecal incontinence. He was treated with wide excision and skin graft closure. The second patient was a 27-year-old man with an 11-year history of recurrent gluteal abscesses and perineal sinuses. At the time of presentation, his inflammatory disease was only mildly active, but he had a nonhealing gluteal lesion. The nonhealing lesion was diagnosed as a squamous cell carcinoma and was managed with wide excision and primary closure. The inflammatory disease was excised and grafted. Complications of advanced hidradenitis suppurativa can be debilitating and life threatening. We review the etiology, pathophysiology, complications, and treatment options of hidradenitis suppurativa, including a literature review of the association with malignancy. We propose that the incidence of disabilities and complications may be reduced by early diagnosis and treatment, by emphasis on prevention of recurrence, and by more aggressive surgical intervention for recurrent and extensive disease.

Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site.

Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA and topoisomerase I. The nature of the interaction of CPT with the DNA-topoisomerase I binary complex was studied by the use of two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) and 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown to trap the DNA-topoisomerase I complex irreversibly. Although cleavage of DNA plasmid mediated by topoisomerase I and camptothecin was reduced significantly by treatment with high salt or excess competitor DNA, enzyme-mediated DNA cleavage stabilized by BrCTPMe persisted for at least 4 h after similar treatment. The production of irreversible topoisomerase I-DNA cleavage was time-dependent, suggesting that BrCPTMe first bound noncovalently to the enzyme-DNA complex and, in a second slower step, alkylated the enzyme or DNA in a manner that prevented DNA ligation. The formation of a covalent linkage was supported by experiments that employed [3H]BrCPT, which was shown to label topoisomerase I within the enzyme-DNA complex. [3H]BrCPT labeling of topoisomerase I was enhanced greatly by the presence of DNA; very little labeling of isolated topoisomerase I or isolated DNA occurred. Even in the presence of DNA, [3H]BrCPT labeling of topoisomerase I was inhibited by camptothecin, suggesting that both CPT and BrCPT bound to the same site on the DNA-topoisomerase I binary complex. These studies provide further evidence that a binding site for camptothecin is created as the DNA-topoisomerase I complex is formed and suggest that the A-ring of camptothecin is proximate to an enzyme residue.

Influence of mild cold on 24 h energy expenditure in 'normally' clothed adults.

Ten subjects aged 19-35 years (four men and six women) underwent two measurements of 24 h energy expenditure (EE) in a whole-body respiration calorimeter, one at a temperature of 28 degrees and one at 20 degrees. Choice of clothing was allowed. Dietary intake was standardized and subjects were asked to follow the same pattern of activity during both measurements. Mean 24 h EE was significantly greater at the cooler temperature by 5.0 (SD 5.5)%, with individual differences ranging from 4.6% lower to 12.6% higher. The difference in EE at the two temperatures was similar during the day and the night and occurred even though subjects wore more clothes and used more bedding at 20 degrees. No relationship was observed between response to 20 degrees and body-weight status. In conclusion, the assumption that mild cold is unlikely to affect EE in subjects wearing normal clothing may be incorrect.

Improved imaging of the augmented breast.

The breast containing an augmentation implant presents a challenge to the mammographer and is often considered unsuitable for adequate mammographic evaluation. A modified positioning technique is described. By displacing the implant posteriorly against the chest wall and pulling breast tissue over and in front of the implant, marked improvement in compression and visualization of substantially more breast tissue is achieved. Over 250 patients with augmentation implants have been successfully studied with this modified compression technique. After review of 50 consecutive cases, two experienced mammographers confirmed a significant improvement in image quality, amount of breast tissue visualized, and overall benefit of the modified technique. Modified positioning for women with breast implants substantially improves both image quality and amount of breast tissue imaged.

A mathematical overview of a computer simulation model of maternity histories with illustrative examples.

A mathematical overview of a stochastic computer simulation model of maternity histories is provided. Various components of human reproduction are accommodated in the model through distributions of waiting times among live births. Included in these components are distributions of age at first marriage in a cohort of women, waiting times to pregnancy for fecundable women, and the lengths of infecundable periods following live births. Probabilities that pregnancies end in either a live birth, induced abortion, or some other type of outcome are also included. Elements of renewal theory and semi-Markov processes in discrete time were the basic mathematical concepts used in the construction of the model. A brief description of an interactive software package called MATHIST, which may be used to implement the model on a computer, is also included. Four illustrative computer runs with MATHIST, pertinent to the operation of family planning programmes in Africa, are also described and discussed.