Synthesis of novel 3'-C-methylene thymidine and 5-methyluridine/cytidine H-phosphonates and phosphonamidites for new backbone modification of oligonucleotides.

Novel 5'-O-DMT- and MMT-protected 3'-C-methylene-modified thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates 1-7 with O-methyl, fluoro, hydrogen, and O-(2-methoxyethyl) substituents at the 2'-position have been synthesized by a new effective strategy from the corresponding key intermediates 3'-C-iodomethyl nucleosides and intermediate BTSP, prepared in situ through the Arbuzov reaction. The modified reaction conditions for the Arbuzov reaction prevented the loss of DMT- and MMT-protecting groups, and directly provided the desired 5'-O-DMT- and/or MMT-protected 3'-C-methylene-modified H-phosphonates 1-6 although some of them were also prepared through the manipulation of protecting groups after the P-C bond formation. The modified Arbuzov reaction of 3'-C-iodomethyl-5-methylcytidine 53, prepared from its 5-methyluridine derivative 42, with BTSP provided the 5-methylcytidine H-phosphonate 54, which was further transferred to the corresponding 4-N-(N-methylpyrrolidin-2-ylidene)-protected H-phosphonate monomer 7. 5'-O-MMT-protected 3'-C-methylene-modified H-phosphonates 5, 3, and 7 were converted to the corresponding cyanoethyl H-phosphonates 50, 51, and 56 using DCC as a coupling reagent. One-pot three-step reactions of 50, 51, and 56 provided the desired 3'-C-methylene-modified phosphonamidite monomers 8-10. Some of these new 3'-methylene-modified monomers 1-10 have been successfully utilized for the synthesis of 3'-methylene-modified oligonucleotides, which have shown superior antisense properties including nuclease resistance and binding affinity to the target RNA.

Investigation of the proposed interdomain ribose zipper in hairpin ribozyme cleavage using 2'-modified nucleosides.

The hairpin ribozyme achieves catalytic cleavage through interaction of essential nucleotides located in two distinct helical domains that include internal loops. Initial docking of the two domains is ion dependent and appears to be followed by a structural rearrangement that allows the ribozyme to achieve a catalytically active state that can undergo cleavage. The proposed structural rearrangement may also be ion dependent and is now of increased importance due to recent evidence that docking is not rate limiting and that metal ions are unlikely to be involved in the chemical cleavage step. An initial structural model of the docked hairpin ribozyme included a proposal for a ribose zipper motif that involves two pairs of hydroxyl groups at A(10) and G(11) in domain A pairing with C(25) and A(24) in domain B, respectively. We have used a chemical functional group substitution technique to study whether this proposed ribose zipper is likely to be present in the active, conformationally rearranged ribozyme that is fit for cleavage. We have chemically synthesized a series of individually modified hairpin ribozymes containing 2'-analogues of nucleosides, that include 2'-deoxy and 2'-deoxy-2'-fluoro at each of the four nucleoside positions, 2'-amino-2'-deoxy, 2'-deoxy-2'-thio, and 2'-arabino at position C(25), and 2'-oxyamino at position A(10), as well as some double substitutions, and we studied their cleavage rates under both single- and multiple-turnover conditions. We conclude that at least some of the hydrogen-bonding interactions in the ribose zipper motif, either as originally proposed or in a recently suggested structural variation, are unlikely to be present in the active rearranged form of the ribozyme that undergoes cleavage. Instead, we provide strong evidence for a very precise conformational positioning for the residue C(25) in the active hairpin. A precise conformational requirement would be expected for C(25) if it rearranges to form a base-triple with A(9) and the essential residue neighboring the cleavage site G(+1), as recently proposed by another laboratory. Our results provide further support for conformational rearrangement as an important step in hairpin ribozyme cleavage.

Oral nicardipine versus intravenous magnesium sulfate for the treatment of preterm labor.

OBJECTIVE: The aim of this study was to compare the efficacy and safety of oral nicardipine in acute therapy for preterm labor with those of parenteral magnesium sulfate. STUDY DESIGN: Patients between 24 and 34 weeks' gestation with documented preterm labor were randomly assigned to receive oral nicardipine (n = 57) or intravenous magnesium sulfate (n = 65) as initial tocolytic therapy. Patients in the nicardipine group received a 40-mg loading dose and then 20 mg every 2 hours as needed to stop contractions (total 80 mg). Patients in the magnesium sulfate group received a 6-g bolus followed by 2 to 4 g/h to provide uterine quiescence. Patients could be switched to another tocolytic regimen if they continued to have contractions after 6 hours of therapy. The main outcome variables examined were time to uterine quiescence, time gained in utero, recurrence of preterm labor, failure of tocolysis, and pertinent maternal and neonatal outcomes. RESULTS: There were no significant differences in maternal demographic characteristics between the groups. Among patients who responded with uterine quiescence within 6 hours, there was a significant decrease in the time to uterine quiescence in the nicardipine group (P <.01). Patients in the magnesium sulfate group were more likely to have recurrence of preterm labor necessitating further tocolytic attempts (P =.048). The patients in the magnesium sulfate group had more adverse side effects, mainly nausea and vomiting (P =.004). There were no differences in birth weight, estimated gestational age at delivery, or neonatal complications between the 2 groups. CONCLUSIONS: Oral nicardipine is an effective, safe, and well-tolerated tocolytic agent. In this prospective clinical trial patients randomly assigned to receive oral nicardipine had arrest of preterm labor more rapidly than did those randomly assigned to receive parenteral magnesium sulfate. Patients who received magnesium sulfate were more likely to have adverse medication effects and recurrent preterm labor.

Phytoestrogens and the management of menopause.

Phytoestrogens are plant compounds that are structurally or functionally similar to steroidal estrogens produced by the body, such as estradiol. Phytoestrogens are derived from dietary precursors. Methodologically rigorous studies demonstrate the benefit of phytoestrogens in addressing the climacteric syndrome--including vasomotor symptoms--and postmenopausal health risks. Studies suggest that phytoestrogen supplementation offers a potential alternative or complement to conventional HRT for osteoporosis prevention. Whether these effects will translate into reduced fracture rates or enhanced function and well-being remains for further study. The majority of evidence about the impact of plant estrogen consumption on the risk of cancer is epidemiologic. Rates of breast, endometrial and ovarian cancers are low in Asian cultures, where the diet is rich in soy isoflavones.

Pulsed dye laser treatment of warts: an update.

BACKGROUND: Warts are a therapeutic challenge. New studies indicate that pulsed dye laser therapy may be effective, with clearance rates of 72 to 93%. OBJECTIVE: To determine clearance rate in pulsed dye laser treatment of warts and compare our rate to those of other published studies. METHODS: Thirty-three patients with 96 warts received pulsed dye laser treatment for recalcitrant plantar, digital, peri- and subungual, and body warts. RESULTS: Forty-eight percent of patients had complete wart clearance; 45% partially cleared. Sixty-nine percent of those who cleared remained wart-free for an average of 11 months. Mean fluence was 9.4 J/cm2, with an average of 3.4 treatments. Body and palmar warts responded best, digital and peri- and subungual next, and plantar lesions worst. No significant side effects were observed. CONCLUSION: Pulsed dye laser is an effective treatment option for recalcitrant warts with an excellent side effect profile. However, our response rates were not as high as those previously reported, and we feel that further studies would be useful.

Medical therapy for chronic gastrointestinal bleeding of obscure origin.

OBJECTIVE: The aim of this study was to evaluate the effectiveness and safety of combined hormonal therapy in patients with recurring occult gastrointestinal bleeding of obscure origin. METHODS: This was a prospective longitudinal observational study. The setting was an outpatient private practice affiliated with a large university-based hospital. A total of 43 patients, comprising 14 men and 29 women with a mean age of 74 yr (range 48-86 yr), were included. They had a history of recurrent gastrointestinal bleeding of unknown origin for a period of > 1 yr and had required multiple hospitalizations and transfusions. Patients were initially treated with one Enovid 5-mg tablet containing 5 mg norethynodrel and 75 microg of mestranol. Enovid became commercially unavailable and treatment was changed to Ortho-Novum 1/50, containing 1 mg norethindrone and 0.05 milligrams of mestranol, given one tablet b.i.d. Patients were treated and followed for a mean time of 535 days (range 25-1551 days). All patients acted as their own controls and were followed for compliant behavior with periodic hematocrit, serial stool hemoccults, medication counts, and clinical histories regarding transfusion requirements or hospitalization for bleeding or anemia. RESULTS: Of 43 patients who initially entered the study, 38 were treated with combination hormonal therapy. The remaining five patients were treated with estrogen alone. In 25 patients, initial enteroscopy revealed AVMs in the stomach or proximal small bowel and these were cauterized. In the remaining 18 patients no source of bleeding was found. None of the 38 patients who were treated with combination hormonal therapy rebled as long as they continued their prescribed dosage. All five of the patients treated with estrogen alone had rebleeding episodes. There was no statistical difference with respect to AVM cauterization in the rebleeding rate between those patients who underwent cauterization of their AVMs and those who did not. Side effects of combination hormonal therapy occurred in 11 patients and all were considered to be mild. Seven of these 11 patients (64%) elected to continue treatment. CONCLUSION: In this long-term observational study, combination hormonal therapy was shown to stop rebleeding in patients with occult gastrointestinal bleeding of obscure origin.

Clinical utility of amniotic fluid volume assessment.

Abnormal amniotic fluid volume is associated with increased maternal risk and perinatal morbidity and mortality. Until the advent of ultrasonography, the invasive nature of amniotic fluid volume assessment limited its clinical utility. Refinements in quantifying the noninvasive sonographic assessment of oligohydramnios and hydramnios have improved the ability of clinicians to identify at-risk pregnancies. This article reviews the available methods of amniotic fluid volume assessment and outlines a comprehensive approach to sonographic screening and monitoring.

Squamous cell carcinoma of the penis in a circumcised man: a case for dermatology and urology, and review of the literature.

Squamous cell carcinoma of the penis is most frequent in uncircumcised men. Other contributing factors include human papillomavirus infection, phimosis, balanitis, and smoking. We present a patient, circumcised at birth, who showed penile squamous cell carcinoma in situ and was treated with carbon dioxide laser ablation. Squamous cell carcinoma rarely presents in patients circumcised as infants. Factors contributing to chronic inflammation may predispose to this disease. Carbon dioxide and neodymium:YAG lasers are two modalities that are successful in the treatment of in situ and probably early invasive penile carcinoma. Optimal treatment includes coordination with a urologist if urethral disease is present.

Laser treatment of acquired vascular lesions.

Several quasi-continuous wave and pulsed lasers can effectively treat a variety of vascular lesions. The PDL follows the theory of selective photothermolysis, is safe for infants and children, and has a low incidence of side effects. It is successful in treating telangiectasias, spider and cherry angiomas, pyogenic granulomas, venous lakes, and poikiloderma of Civatte, as well as small leg telangiectasias. Quasi-continuous wave lasers such as the APTDL, copper vapor, krypton, and KTP lasers can be used to treat telangiectasias and other vascular lesions as well. Although they carry a higher risk of complications, they may prove more useful in treating larger caliber vessels. Although the PDL often produces superior clinical results than the quasi-continuous wave lasers, some patients may prefer these latter lasers because of the lack of post-operative purpura. Lastly, newer lasers, as well as noncoherent light sources, are being developed for the treatment of leg telangiectasias. Continuing advances in laser technology will enhance results, decrease side effects, improve equipment, and reduce costs, with great benefit to an increasing patient population.

Erbium-YAG and holmium-YAG laser ablation of the lens.

Er-YAG (2.9 microns, 200 microseconds pulsewidth) and Ho-YAG (2.12 microns, 250 microseconds pulsewidth) lasers were used to irradiate bovine crystalline lenses. Mass ablated increased with increasing fluence for both lasers and was greater for the Er-YAG than the Ho-YAG laser at all fluences. The mass loss vs. fluence curve was nonlinear for the Er-YAG and linear for the Ho-YAG laser. Ablation threshold was lower for the Er-YAG. Grossly, the Er-YAG laser produced less charring and expressed fewer, smaller tissue pieces than the Ho-YAG. Scanning electron microscopy indicated that the Er-YAG produced smoother walled craters. The Ho-YAG laser produced more dessicated and disrupted craters. The Er-YAG radiation, delivered by fiberoptic probes, may provide a method of performing minimally invasive cataract surgery.

Characterization of sciellin, a precursor to the cornified envelope of human keratinocytes.

The cornified envelope, located beneath the plasma membrane of terminally differentiated keratinocytes, is formed as protein precursors are cross-linked by a membrane associated transglutaminase. This report characterizes a new precursor to the cornified envelope. A monoclonal antibody derived from mice immunized with cornified envelopes of human cultured keratinocytes stained the periphery of more differentiated cells in epidermis and other stratified squamous epithelia including hair and nails. The epitope was widely conserved among mammals as determined by immunohistochemical and Western analysis. Immunoelectron microscopy localized the epitope to the cell periphery in the upper stratum spinosum and granulosum of epidermis. In the hair follicle, the epitope was present in the internal root sheath and in the infundibulum, the innermost aspect of the external root sheath. The antibody recognized a protein of relative mobility (M(r)) 82,000, pI 7.8. The protein was a transglutaminase substrate as shown by a dansylcadaverine incorporation assay. Purified cornified envelopes absorbed the reactivity of the antibody to the partially purified protein and cleavage of envelopes by cyanogen bromide resulted in release of immunoreactive fragments. The protein was soluble only in denaturing buffers such as 8 M urea or 2% sodium dodecyl-sulfate (SDS). Partial solubility could be achieved in 50 mM TRIS pH 8.3 plus 0.3 M NaCl (high salt buffer); the presence of a reducing agent did not affect solubility. Extraction of cultured keratinocytes in 8 M urea and subsequent dialysis against 50 mM TRIS pH 8.3 buffer resulted in precipitation of the protein with the keratin filaments. Dialysis against high salt buffer prevented precipitation of the protein. The unique solubility properties of this protein suggest that it aggregates with itself and/or with keratin filaments. The possible role of the protein in cornified envelope assembly is discussed. We have named this protein Sciellin (from the old english "sciell" for shell).

Selection of solvent load and first-stage pressure to reduce interference effects in inductively coupled plasma-mass spectrometry.

In inductively coupled plasma-mass spectrometry the first-stage pressure and solvent characteristics can strongly influence spectral and nonspectroscopic interference effects. By manipulating the pressure and solvent load, one can regulate the degree of analyte signal suppression observed in the presence of high concentrations (> 10 mM) of concomitants. Importantly, the same operating conditions that eliminate the matrix effects maintain the analytical utility of the system. However, for some interferent-analyte combinations, the identity of the concomitant anion and subsequent pH of the solution determine whether the interference effects can be eliminated entirely. The first-stage pressure does not appear to significantly affect the oxide-ion and doubly charged ion ratios; the solvent characteristics are the dominant factors that dictate these ratios.

Characteristics and operative management of supination external rotation ankle fractures.

Identification and management of the acutely fractured ankle is discussed by the authors. The Lauge Hansen classification system, and in particular, supination-external rotation injuries, is evaluated. Intraoperative technique, perioperative considerations, and generalized management of these pathologic conditions are reviewed, according to the authors' experiences.

Synthesis and biological properties of some 6H-pyrido[4,3-b]carbazoles.

The effect of methyl substitution on the biological properties of the ellipticines was reexamined. 9-Hydroxy-6H-pyrido[4,3-b]carbazole was synthesized and shown to be devoid of antitumor activity in murine P388 lymphocytic leukemia in mice. 5-(Hydroxymethyl)-11-methyl-6H-pyrido[4,3-b]carbazole (46) and its N-methylcarbamate (48) were synthesized and their effect on macromolecular synthesis in HeLa cells and their antitumor properties were compared with those of ellipticine. In contrast to the alkaloid 1 and the hydroxymethyl derivative 46, which produced partially reversible inhibition of [3H]thymidine incorporation, the carbamate ester irreversibly blocked incorporation of the tritiated pyrimidine. The ester was also a more potent antitumor agent in P388 lymphocytic leukemia than 1 or 46.

Tetracyclic pyridazines as potential psychopharmacological agents.

Since the Z isomer of chlorprothixene (1) is far more active than its E counterpart, it was of interest to develop a stereoselective synthesis for this class of compounds. Insertion of a benzenesulfonamido group at the peri position of a chlorprothixene precursor did affect the stereochemistry of side-chain olefin formation, but after hydrolysis attempted removal of the resulting amine led to a Widman-Stoermer cyclization to afford the corresponding tetracyclic pyridazine-containing compound (4). Since this material displayed encouraging activity in neurotransmitter uptake inhibition studies, compounds in which the sulfur bridge was replaced with an ethano bridge similar to that found in imipramine (8) and with sulfur removed (7) were also prepared. These, together with the corresponding peri amino compounds (3, 5, and 6), were tested as neurotransmitter-uptake The two bridged arylamines 3 and 6 displayed potent and selective inhibition of norepinephrine uptake both when tested in vitro and after in vivo administration. The pyridazine-containing compounds exhibited reasonable activity in vitro, but the activity was lost when they were administered in vivo. None of the compounds displayed significant ability to interfere with spiroperidol binding.

Osteolysis in Trichinella spiralis infected vs noninfected rats.

1. Based upon the present inability to describe the mechanism by which calcification takes place, and the unexplored metabolism of calcium during trichinosis, studies of rat bone involvement were undertaken. 2. Significant (P less than 0.05) differences were found in experimental (Trichinella spiralis infected) rat femurs vs those of noninfected rats. 3. Analysis by atomic absorption spectroscopy, dry femur weight, and tensile stress resistance indicated an overall reduction in calcium content. 4. This calcium reduction is due to active cyst calcification. 5. Results confirming this finding revealed significant (P less than 0.05) differences at days 60-80 postinfection (the beginning of cyst calcification.

The pharmacologic effects of furosemide therapy in the low-birth-weight infant.

The pharmacologic effects of furosemide were studied in six infants (mean gestation 30.7 weeks; mean birth weight 1,490 gm) at ages 10 to 57 days. Furosemide (for clinical indication, standardized at 1 mg/kg) was given intravenously over one minute; data were collected over the ensuing 24 hours. For three hours following furosemide administration, a significant diuresis was observed. Sodium excretion, percent fractional sodium excretion, and potassium excretion were significantly increased and urinary pH significantly decreased for six hours following the administration of furosemide. Creatinine and free water clearances were slightly elevated, although not significantly. Furosemide is an effective diuretic, the onset of pharmacologic action was within one hour, the peak action was sustained for three hours, and the duration of action was six hours. The net fluid, sodium, and potassium losses following a 1 mg/kg single dose were 28 ml, and 3.6 and 0.3 mEq/kg, respectively.