Pyrimidine and s-triazine as structural motifs for ordered adsorption on Si(100): a first principles study.

The chemisorption of pyrimidine and s-triazine, two aromatic molecules with N atoms in the aromatic ring, is studied by first principles calculations to examine not only the chemisorption energy, but also the reaction barriers and the cooperative effects. Among the reaction paths at low coverage, the formation of a cross-bridge structure with two N-Si dative bonds is almost barrierless and should be dominant at low temperature. At higher coverage and low temperature, these cross-row structures should produce an ordered zig-zag pattern, even though it is not the energetically most stable arrangement. Upon heating, the zig-zag pattern could be transformed into lines along dimer rows. These two molecules also provide structural motifs that could facilitate the formation of ordered adsorption structures on Si(100). By symmetry, the complexity of tight-bridge structures could be greatly reduced, while the X-C-X motif, with X being an electronegative atom, could provide the building blocks for cross-row bridges.

Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats.

Genetic and environmental factors are important in the pathogenesis of clinical and experimental chronic intestinal inflammation. We investigated the influence of normal luminal bacteria and several groups of selected bacterial strains on spontaneous gastrointestinal and systemic inflammation in HLA-B27 transgenic rats. Rats maintained germfree for 3-9 mo were compared with littermates conventionalized with specific pathogen-free bacteria. Subsequently, germfree transgenic rats were colonized with groups of five to eight bacteria that were either facultative or strictly anaerobic. Transgenic germfree rats had no gastroduodenitis, colitis, or arthritis, but developed epididymitis and dermatitis to the same degree as conventionalized rats. Colonic proinflammatory cytokine expression was increased in transgenic conventionalized rats but was undetectable in germfree and nontransgenic rats. Colitis progressively increased over the first 4 wk of bacterial exposure, then plateaued. Only transgenic rats colonized with defined bacterial cocktails which contained Bacteroides spp. had colitis and gastritis. Normal luminal bacteria predictably and uniformly induce chronic colonic, gastric and systemic inflammation in B27 transgenic F344 rats, but all bacterial species do not have equal activities.

Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary beta2-adrenergic receptor gene delivery.

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

Adenomas induced by polycyclic aromatic hydrocarbons in strain A/J mouse lung correlate with time-integrated DNA adduct levels.

The induction of DNA adducts and adenomas in the lungs of strain A/J mice has been investigated following the single i.p. administration of each of the following polycyclic aromatic hydrocarbons (PAH): pyrene, dibenz[a,h]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, 5-methylchrysene, and cyclopenta[c,d]pyrene. DNA adducts were measured by 32P-postlabeling at times between 1 and 21 days following injection, while adenomas were counted at 240 days after treatment. Pyrene did not induce either DNA adducts or lung adenomas at any of the doses examined. Each of the remaining PAH induced both adenomas and DNA adducts in a dose-dependent manner, with dibenz[a,h]anthracene > 5-methylchrysene > cyclopenta[c,d]pyrene > benzo[a]pyrene > benzo[b]fluoranthene. DNA adducts reached maximal levels between 3 and 9 days after injection, followed by a gradual decrease. The time-integrated DNA adduct level (TIDAL) was calculated by numerically integrating the areas under the adduct persistence curves extrapolated to 240 days for each PAH at each dose level. This value represents the effective total molecular dose of PAH that was delivered to the lung DNA over the entire course of tumorigenesis. A strong correlation of lung adenoma induction with the TIDAL values was observed for each PAH. The slopes of the tumors versus TIDAL value relationships were essentially identical for 5-methylchrysene, cyclopenta[cd]pyrene, benzo[a]pyrene, and benzo[b]fluoranthene. The slope of this relationship for dibenz[a,h]anthracene was markedly greater. The essentially identical induction of adenomas as a function of TIDAL values for these PAH suggests that the formation and persistence of DNA adducts determines their carcinogenic potency.

Cardiac gene delivery with cardiopulmonary bypass.

BACKGROUND: Cardiac gene therapy offers the possibility of enhancing myocardial performance in the compromised heart. However, current gene delivery techniques have limited myocardial transgene expression and pose the risk of extracardiac expression. Isolation of the coronary circulation during cardiac surgery may allow for more efficient and cardiac-selective gene delivery in a clinically relevant model. Methods and Results-- Neonatal piglets (3 kg) underwent a median sternotomy and cardiopulmonary bypass, followed by aortic cross-clamping with 30 minutes of cardioplegic arrest. Adenoviral vectors containing transgenes for either beta-galactosidase (adeno-beta-gal, n=11) or the human beta(2)-adrenergic receptor (adeno-beta(2)-AR, n=15) were administered through the cardioplegia cannula immediately after arrest and were allowed to dwell in the coronary circulation during the cross-clamp period. After 1 week, the animals were killed, and their heart, lungs, and liver were excised and examined for gene expression. Analysis of beta-galactosidase staining revealed transmural myocardial gene expression among animals receiving adeno-beta-gal. No marker gene expression was detected in liver or lung tissue. beta-AR density in the left ventricle after adeno-beta(2)-AR delivery was 396+/-85% of levels in control animals (P<0.01). Animals receiving adeno-beta(2)-AR and control animals demonstrated similar beta-AR density in both the liver (114+/-8% versus 100+/-9%, P=NS) and lung (114+/-7% versus 100+/-9%, P=NS). There was no evidence of cardiac inflammation. CONCLUSIONS: By using cardiopulmonary bypass and cardioplegic arrest, intracoronary delivery of adenoviral vectors resulted in efficient myocardial uptake and expression. Undetectable transgene expression in liver or lung tissue suggests cardiac-selective expression.

Persistent Strongyloides stercoralis in a blind loop of the bowel. Successful treatment with mebendazole.

Three courses of thiabendazole therapy, including one course given directly into a blind loop of the bowel, failed to eradicate Strongyloides stercoralis from a 55-year-old man who had undergone a Roux-en-Y operation. The patient responded to 1.5 g/day of mebendazole for 14 days, and the infection did not recur. Our case illustrates the difficulty of eliminating S stercoralis from a blind loop of the bowel and indicates that mebendazole therapy used in adequate doses is effective treatment for strongyloidiasis. The lack of toxicity of mebendazole makes it a desirable drug for Strongyloides infestation resistant to thiabendazole therapy.

Studies on the epidemiology of antibiotic-associated Clostridium difficile colitis.

Vancomycin protects hamsters from the development of Clostridium difficile colitis after treatment with clindamycin, and vancomycin is useful in treatment of humans with the disease. Relapses have occurred in both hamsters and humans when vancomycin is discontinued. Vancomycin appears to enhance susceptibility to colonization with C. difficile by eliminating competing intestinal organisms. The nature of these organisms is not known, but various tools are now available to aid in identifying them. Cancer chemotherapeutic agents should be added to the list of factors such as surgery and antibiotics that may predispose to emergence of C. difficile. The number of organisms required for colonization of antibiotic-treated hamsters is low and cross-infection seems to play a role in the disease in hamster colonies. The organism can be detected on surfaces in rooms of patients with the disease, and on the hands of personnel caring for them. Outbreaks of the disease have been recognized. Our results suggest isolation precautions should be used to prevent spread of the organism from patients with the disease to others being treated with antibiotics.

Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification.

Ribosomal RNA sequences are useful for establishing phylogenetic relationships, for oligonucleotide probes and for characterization of uncultured organisms. We describe rapid ribosomal DNA sequencing using PCR with transcript sequencing. Nucleic acid specificity at three steps (amplification, transcription and sequencing) eliminated the need for nucleic acid extraction or purification. Sequence was obtained from a crude lysate from a single colony of bacteria. The basic sequencing method should be adaptable to provide rapid sequence information in a wide variety of applications.

UV absorption complicates PCR decontamination.

UV irradiation is widely used to inactivate contaminating DNA in PCR. Highly UV-absorbent deoxyribonucleoside triphosphates in PCR mixtures reduce the efficiency of UV decontamination. Optimal decontamination may be achieved by irradiating the PCR mixture without the deoxyribonucleoside triphosphates.

Alpha1-adrenergic receptors in human spinal cord: specific localized expression of mRNA encoding alpha1-adrenergic receptor subtypes at four distinct levels.

alpha1-Adrenergic receptors (alpha1ARs) are important in lower urinary tract syndromes such as benign prostatic hypertrophy and bladder irritability. Spinal cord alpha1ARs have been postulated to play a role in modulating these diseases, yet alpha1AR subtype (alpha1a, alpha1b, alpha1d) neuronal localization in human spinal cord has not been described. We therefore tested the hypothesis that alpha1AR subtype distribution varies according to specific spinal cord tract and level. In situ hybridization was performed to identify cell bodies containing alpha1AR subtype mRNA at four levels of human spinal cord (cervical enlargement, thoracic, lumbar, sacral). alpha1AR mRNA is present in ventral gray matter only (ventral>dorsal; sacral>lumbar=thoracic>cervical). Signaling cell bodies were detected in anterior horn motor neurons at all levels; dorsal nucleus of Clarke and intermediolateral columns in cervical enlargement, thoracic and lumbar spinal cord regions; and parasympathetic nucleus in sacral spinal cord. Although all three alpha1AR subtypes are present throughout human spinal cord, alpha1d mRNA predominates overall. If confirmed at a protein level, these findings may contribute to the development of new therapeutic strategies in the treatment of several human diseases.

alpha 2-Adrenergic receptors in human spinal cord: specific localized expression of mRNA encoding alpha 2-adrenergic receptor subtypes at four distinct levels.

alpha 2-Adrenergic receptor (AR) subtype mRNA (alpha 2a, alpha 2b, alpha 2c) neuronal localization in human spinal cord has not been described. We therefore performed in situ hybridization to identify cell bodies at four levels of human spinal cord (cervical, thoracic, lumbar, sacral) containing alpha 2AR subtype specific mRNA. alpha 2AR mRNA is present in gray matter only (ventral > dorsal; sacral > cervical > thoracic = lumbar). In addition to alpha 2AR mRNA in cell bodies in thoracic and lumbar intermediolateral (sympathetic) and sacral intermediate (parasympathetic) cell columns (lamina VII), all levels in dorsal horn laminae I, II, V, and ventral horn lamina IX, we demonstrate alpha 2AR mRNA in dorsal horn laminae III and IV, and dorsal nucleus of Clarke, where alpha 2ARs have not been described. Previously unreported heterogeneity in alpha 2AR subtype distribution (alpha 2a and alpha 2bAR mRNA present, alpha 2cAR mRNA virtually absent) is found at all sites of alpha 2AR mRNA expression in human spinal cord, including locations known to mediate effects of alpha 2AR agonist drugs on nociception, autonomic function and motor tone. Cervical spinal cord demonstrates a predominance of alpha 2a mRNA signal, while thoracic, lumbar, and sacral spinal cord demonstrate an increasing predominance of alpha 2bAR mRNA. If confirmed at a protein level, these findings have profound implications for therapeutic strategies in managing human pain.

Adenovirus-mediated genetic manipulation of the myocardial beta-adrenergic signaling system in transplanted hearts.

OBJECTIVES: Ex vivo perfusion of the cardiac allograft during organ procurement is an ideal environment for adenoviral vectors with transgenes that target improving graft contractility. One such target is the beta-adrenergic receptor-signaling system, in which alterations in transgenic mice have elucidated novel means to improve the function of the heart in vivo. The purpose of the current study was to determine the functional consequences of beta-adrenergic receptor manipulation in a rabbit model of cardiac allograft transplantation. METHODS: New Zealand White rabbits weighing 3 kg served as recipients to 1-kg outbred donors. Donor hearts were arrested and harvested, and 1 of 3 adenoviral constructs was administered into the aortic root perfusing the graft. Transgenes delivered encoded either the human beta(2)-adrenergic receptor, a peptide inhibitor of beta-adrenergic receptor densensitization, or the marker transgene beta-galactosidase. RESULTS: Five days after cervical heterotopic transplantation, left ventricular performance was measured on a Langendorff apparatus. A moderate pattern of rejection was seen in all grafts. Biventricular myocyte expression of beta-galactosidase was observed, and beta(2)-adrenergic receptor density was elevated 10-fold in grafts that received adeno-beta(2)-adrenergic receptor. Left ventricular systolic and diastolic performance was significantly increased in grafts transfected with either adeno-beta(2)-adrenergic receptor or adeno-beta-adrenergic receptor densensitization compared with control grafts that received adeno-beta-galactosidase. CONCLUSIONS: Ex vivo adenovirus-mediated gene transfer is feasible in a rabbit allograft model and, more important, genetic manipulation of beta-adrenergic receptor signaling either by increasing beta(2)-adrenergic receptor density or blocking endogenous receptor desensitization improves graft function acutely in this allograft model.

Evaluation of danazol as an oral contraceptive.

The effect of Danazol as an oral contraceptive in doses of 50, 100, and 200 mg daily for 6 months was studied in 3 groups of 10 women. Both 50 and 100 mg Danazol daily were well tolerated, but one pregnancy occurred among the women receiving 50 mg daily, and 2 pregnancies occurred in women receiving 100 mg daily. There were no pregnancies in women taking 200 mg Danazol daily; however, the side effects were frequent and 5 of the 10 patients withdrew from the study prior to 6 months of therapy. Six patients in this study were followed intensively with blood hormone analysis, vaginal cytology, and pathologic evaluation, and these findings are detailed.

The effect of danazol in the treatment of chronic cystic mastitis.

Danazol in a dose of 400 mg daily was administrated to 40 patients with chronic cystic mastitis and resulted in a marded improvement in both objective and subjective symptoms in 87.5% of the patients studied. Three patients showed partial relief of symptoms and 1 patient showed no improvement after 1 month of treatment at which time she withdrew from the study. One patient had a worsening of her condition and was withdrawn from the study after 3.5 months of therapy. Nineteen of the 40 patients in the study had a secondary diagnosis of pelvic endometriosis confirmed histologically, and all these patients showed a marked improvement of symptoms during the Danazol treatment. Patients developed amenorrhea after 3 to 4 months of Danazol therapy, and symptoms such as dysmenorrhea, premenstrual pelvic pain, and tension abated at the same time. A mild, but well-tolerated weight gain was the major side effect of Danazol administration. No significant changes in the levels of plasma E1, E2, FSH, LH, or progesterone could be demonstrated in specimens drawn from 11 patients before and during the course of Danazol administration.

Management of failed prostaglandin abortions.

Midtrimester abortion was induced in 529 patients by administration of the naturally occurring prostaglandins E2 and F2alpha as well as the 15-methyl analogs, 15-ME-PGE2 and 15-ME-PGF2alpha. Ten patients failed to abort with prostaglandin therapy, even in association with intravenous oxytocin, a failure rate of 1.9%. Two failures were related to uterine malformation; 1 patient had the pregnancy in a blind uterine horn, and the second patient was pregnant in one horn of a uterus didelphys. Five of the 10 patients who failed to abort during prostaglandin administration were subsequently found to have uterine distortion due to myomata uteri. When abortion induced by prostaglandin fails to occur within the expected time for the agent and technic employed, the presence of uterine malformation or abnormality should be considered. Evaluation with ultrasonography is indicated along with a repeat test to confirm the pregnancy. If the sonogram is suggestive of uterin malformation, a hysterosalpinogram should be obtained to determine if there is communication between the cervix and the gestational sac. If no communication is present, an intravenous pyelogram should be performed in view of the 90% correlation of urogenital abnormalities, and an exploratory laparotomy should be performed. When a communication exists between the cervix and the gestational sac, the 24 hours of uterine activity induced by the prostaglandin will have resulted in cervical changes so that the cervix can easily be dilated to either a 14 or 16 Hegar dilator and the conceptus can be removed in parts with minimal bleeding.

A new abortion technique: intravaginal and intramuscular prostaglandin.

The 15-methyl analog of prostaglandin F2 alpha (15-ME-PGF2 alpha), administered in a 3-mg dose via a single vaginal suppository and supplemented at 24 hours by intramuscular injection(s) of 250 micrograms, successfully induced abortion in 80 of 81 patients in the midtrimester of pregnancy. The mean abortion time was 19.6 hours. Two thirds of the patients aborted after treatment with the suppository alone in a mean time of 14.6 hours; the remaining 27 patients required intramuscular injections of 15-ME-PGF2 alpha to effect expulsion of the products of conception. Twenty-six of these 27 patients subsequently aborted in a mean total abortion time of 29.6 hours. Fifty-eight patients aborted within 24 hours of the initial prostaglandin administration, and 78 aborted by 36 hours. Parity and length of gestation did not significantly affect abortion time in this series, although the mean abortion time for parous patients and patients with gestations earlier than 17 weeks tended to be somewhat shorter than that of nulliparous patients and those with more advanced gestations. The placenta was spontaneously expelled in the majority of patients. Abortion was incomplete in 3 patients and required curettage. Uterine activity, as measured via an intraamniotic catheter in 6 patients, developed very gradually with the suppository, peaking at 3 hours after insertion, and was characterized by regular contractions with low intrauterine baseline tonus. The gastrointestinal side effects that occurred in 59% of patients who received the suppository were also most frequently observed at 3 hours after administration. In contrast the gastrointestinal disturbances elicited by intramuscular injections of the analog immediately followed the administration.

The abortifacient effectiveness and plasma prostaglandin concentrations with 15(S)-15-methyl prostaglandin F2alpha methyl ester-containing vaginal silastic devices.

The abortifacient effectiveness of three intravaginal Silastic devices impregnated with 15(S)-15-methyl prostaglandin F2alpha (15(S)-Me-PGF2alpha) methyl ester in concentrations of 0.25%, 0.5%, and 1.0% was investigated. Each concentration was tested in 10 patients with gestations ranging from 8 to 19 weeks. Abortion was successfully induced by prostaglandin alone in 6 patients with the 0.25% device, in 9 patients with the 0.5% device, and all 10 patients treated with the 1.0% device. Additionally, three patients treated with the 0.25% device and one patient treated with the 0.5% device aborted with concomitant, continuous, intravenous oxytocin therapy. The mean abortion time with the 0.25% device was 16.43 hours; with the 0.5% device, 16.49 hours; and with the 1.0% device, 10.18 hours. The peripheral plasma levels of 15(S)-Me-PGF2alpha methyl ester were the most variable with the 0.5% device. The plasma levels of 15(S)-Me-PGF2alpha methyl ester of patients receiving the 0.25% device were similar to the levels of patients receiving the 1.0% device. The intravaginal Silastic device impregnated with 15(S)-Me-PGF2alpha methyl ester appears to be an effective abortifacient, but further study is indicated to determine the most efficient device with the fewest side effects.

The effects of intramuscular injections of 15(S)-15-methyl prostaglandin F2alpha in failed abortions.

Intramuscular injections of 15(S)-15-methyl prostaglandin F2alpha (15-Me-PGF2alpha) induced abortion in 38 patients who had failed to abort with other techniques, such as intra-amniotic instillation of saline or PGF2alpha and intravaginal insertion of prostaglandin-impragnated Silastic devices. The intramuscular injections of 15-Me-PGF2alpha were initiated when the original abortion techniques, even when augmented by intravenous oxytocin, failed to produce expulsion of the fetus. The dose schedule was 250 microgram or 500 microgram every 2 to 4 hours, and the concomitant intravenous oxytocin was continued at a rate of 167 mU/minute. Of the 38 patients, 26 aborted with two or fewer injections of 15-Me-PGF2alpha, and 30 patients required only 1 mg of the drug to expel the fetus successfully. The mean time from the first injection of 15-Me-PGF2alpha to the expulsion of the fetus was 5.25 hours; one-half of the patients aborted in less than 4 hours. The placenta was expelled spontaneously in 15 patients, removed manually from the vagina in 18, and removed by sponge forceps in 3. Two abortions were incomplete and surgical intervention was required. Twenty-eight patients (74%) experienced gastrointestinal disturbances, chiefly vomiting and diarrhea. Intramuscular administration of 15-Me-PGF2alpha eliminates the need for repeated amniocentesis, and the dose may be adjusted to meet the precise requirements of the clinical situation.

In situ hybridization: identification of rare mRNAs in human tissues.

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Early pregnancy interruption with two 15-ME-PGF2a suppositories.

Vaginal administration of 15-ME-PGF2a suppositories successfully terminated pregnancy, as determined by a negative pregnancy test at 14 days following insertion, in 34 of 39 patients with gestations of 34 to 51 days of amenorrhea. Fifteen of 20 patients who received a single long-acting 3-mg suppository had negative pregnancy tests at follow-up, while all 19 patients in whom the 3-mg suppository was preceded by a rapidly acting 1-mg suppository successfully aborted. This two-suppository regimen resulted in a more rapid onset of uterine activity and vaginal bleeding, as well as a slight decrease in gastrointestinal side effects. Peripheral plasma levels of 15-ME-PGF2a as detected by radioimmunoassay were also more consistent with two suppositories. It appeared that the rapidly acting 1-mg suppository provided a "prostaglandin impact" which significantly enhanced the abortifacient effectiveness of the technique. Nine patients on the 2-suppository regimen had the procedure performed on an outpatient basis. Expanded studies are indicated to determine the practicality of this technique as a valid pharmacologic alternative to surgical interruption of very early pregnancy.