Elongation of primed DNA templates by eukaryotic DNA polymerases.

The combined action of DNA polymerase alpha and DNA polymerase beta leads to the synthesis of full-length linear DNA strands with phi X174 DNA templates containing an RNA primer. The reaction can be carried out in two stages. In the first stage, DNA polymerase alpha catalyzes the synthesis of a chain that averaged 230 deoxynucleotides long and was covalently linked to the RNA primer. In the second stage, DNA polymerase beta elongates the DNA strand covalently attached to the RNA primer to full length. With DNA primers, DNA polymerase alpha catalyzes only limited deoxynucleotide addition whereas DNA polymerase beta alone elongates DNA primed templates to full length. DNA polymerase beta can also stimulate the synthesis of adenovirus DNA in vitro in the presence of a cytosol extract from adenovirus-infected cells. In all of these systems, dNMP incorporation catalyzed by DNA polymerase beta was sensitive to N-ethylmaleimide; however, this polymerase activity was resistant to N-ethylmaleimide with poly(rA) x (dT) as the primer template.     

Esophageal dysfunction and Raynaud's phenomenon in patients with scleroderma

The relationship of Raynaud's phenomenon (RP) to the degree of esophageal motility dysfunction was evaluated in 12 patients with scleroderma. Motility abnormalities of the smooth muscle esophagus were quantitated using a motility index (MI). MI of the scleroderma patients differed significantly from controls. No relationship was noted between the extent of motility abnormality and the duration or severity of the RP. A nearly uniform finding was the absence of coordinated esophageal peristalsis, occurring regardless of the duration or severity of the RP. The study demonstrates that it is not possible to predict the degree of esophageal motility dysfunction in scleroderma based on RP alone. The disease may have two different and independent pathogenetic components. One component is closely linked to RP and involves impairment of neuromuscular electrical transmission. The other component (independent of RP) involves progressive loss of muscle strength in the distal esophagus and in the lower esophageal sphincter.     

Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures

The minichromosome maintenance (Mcm) proteins 2-7 are required for both the initiation and elongation steps of chromosomal DNA replication. Previous studies have shown that the Mcm complex consisting of the Mcm 4, 6, and 7 proteins contains 3' to 5' DNA helicase activity with limited processivity (displacing duplex DNA regions up to 30 nt). In this report, we show that the presence of both 5' and 3' single-stranded tails in DNA helicase substrates is essential for the processive helicase activity of the Mcm complex. The presence of both 5' and 3' tails facilitated the formation of double heterohexameric complexes of Mcm4/6/7 on substrate DNA, which appeared to be essential for the processive helicase activity. The double heterohexameric complex of Mcm4/6/7, in the presence of a single-strand DNA binding protein, is capable of unwinding duplex DNA region of about 600 bp in length. These results support the hypothesis that the Mcm4/6/7 complex can function as a replication helicase.     

Reduction of opioid use and improvement in chronic pain in opioid-experienced patients after topical analgesic treatment: an exploratory analysis

Objective: There is a need to identify safe and effective opioid-sparing multimodal alternative treatment strategies and approaches, including topical analgesics, for opioid-experienced chronic pain patients to mitigate the risk of addiction, misuse, and abuse of opioids.

Methods: This subset analysis from a prospective, observational study evaluated changes in opioid use, other concurrent medication use, and pain severity and interference in opioid-experienced patients (OEP) treated with topical analgesics for chronic pain with measures obtained at baseline and 3- and 6- month follow-up.

Results: The 3-month opioid-experienced patient (3-month OEP) group included 121 patients who completed baseline and 3-month follow-up assessments; 27 opioid-experienced patients completed baseline and 6-month follow-up assessments (6-month OEP). Demographic characteristics, and mean pain severity and interference scores were similar between groups at baseline. After treatment with topical analgesics, 49% of patients in the 3-month and 56% of patients in the 6-month group reported they had completely discontinued use of opioids. In addition, 31% of patients at the 3-month assessment and 30% at the 6-month assessment reported that they were no longer taking any pain medication. Other concurrent medications decreased by 65% after 3 months, and 74% after 6 months. There were statistically significant decreases from baseline in pain severity and interference scores within the 3- (CI:0.7–1.4, 1.4–2.2) and 6-month (CI:0.7–2.4 (severity); CI:1.2–3.5 (interference)) OEP groups.

Conclusions: Opioid use and other concurrent medications decreased among opioid-experienced chronic pain patients after 3- and 6- months of treatment with topical analgesics. Pain severity and interference scores also decreased. The topical analgesics were reported to be effective and safe for the treatment of chronic pain, with randomized controlled trials needed to confirm these findings.     

Spinal manipulation for low-back pain.

PURPOSE: To review the use, complications, and efficacy of spinal manipulation as a treatment for low-back pain. DATA IDENTIFICATION: Articles were identified through a MEDLINE search, review of articles' bibliographies, and advice from expert orthopedists and chiropractors. STUDY SELECTION: All studies reporting use and complications of spinal manipulation and all controlled trials of the efficacy of spinal manipulation were analyzed. Fifty-eight articles, including 25 controlled trials, were retrieved. DATA ANALYSIS: Data on the use and complications of spinal manipulation were summarized. Controlled trials of efficacy were critically appraised for study quality. Data from nine studies were combined using the confidence profile method of meta-analysis to estimate the effect of spinal manipulation on patients' pain and functional outcomes. RESULTS OF DATA SYNTHESIS: Chiropractors provide most of the manipulative therapy used in the United States for patients with low-back pain. Serious complications of lumbar manipulation, including paraplegia and death, have been reported. Although the occurrence rate of these complications is unknown, it is probably low. For patients with uncomplicated, acute low-back pain, the difference in probability of recovery at 3 weeks favoring treatment with spinal manipulation is 0.17 (for example, increase in recovery from 50% to 67%; 95% probability limits of estimate, 0.07 to 0.28). For patients with low-back pain and sciatic nerve irritation, the difference in probabilities of recovery at 4 weeks is 0.098 (probability limits, -0.016 to 0.209). CONCLUSIONS: Spinal manipulation is of short-term benefit in some patients, particularly those with uncomplicated, acute low-back pain. Data are insufficient concerning the efficacy of spinal manipulation for chronic low-back pain.     

Interleukin-1 is both morphogenic and cytotoxic to cultured rat ovarian cells: obligatory role for heterologous, contact-independent cell-cell interaction.

An increasing body of information now suggests that intraovarian interleukin-1 (IL-1) may play an intermediary role in the ovulatory process. Given that follicular rupture inevitably requires marked tissue remodeling and possibly cell death, we set out to examine the morphogenic potential of IL-1 under in vitro circumstances. Treatment of freshly plated whole ovarian dispersates from immature rats with any one of several batches of IL-1 (10 ng/ml) for up to 96 h produced marked time-dependent morphological alterations, including cellular retraction, rounding, clumping, aggregation, blebbing, swelling, and, ultimately, irreversible detachment. Evidence of (asynchronous) cell death consisted of reduced total cell number, diminished cellular protein content, enhanced cellular release of lactic dehydrogenase, failure to exclude trypan blue, and attenuated reduction of the tetrazolium dye 3-[4,5-dimethylthiazol-2-y]2,5-diphenyltetrazolium bromide to spectrophotometrically detectable formazan. Comparable results were obtained when using established day 4 cultures, arguing against a possible critical action of IL-1 at the time of plating. Dose-response curves revealed IL-1 beta (EC50, 0.2-0.4 ng/ml) to be substantially more potent than IL-1 alpha (EC50, 2.7-2.8 ng/ml). Importantly, the concurrent provision of an IL-1 beta-directed polyclonal antibody yielded complete immunoneutralization of the IL-1 beta effect, arguing against the possible involvement of a non-IL-1 contaminant. An unrelated polyclonal antiserum raised against insulin-like growth factor-I was without effect. IL-1 action proved relatively specific, in that tumor necrosis factor-alpha (10 ng/ml), a putative cytotoxic principle, as well as IL-1-inducible ILs (IL-2 and -6; 100 U/ml) were without effect. Although minimally effective at the level of the isolated granulosa or theca-interstitial cell, IL-1 proved highly potent in heterologous (but not homologous), contact-dependent and independent cocultures of these somatic cell types, strongly suggesting obligatory cell-cell cooperation. These observations further indicate that IL-1 action is indirect and may require the induction of an intermediary soluble principle to serve as the final effector. Taken together, these findings indicate that relatively low concentrations of IL-1 (beta > alpha), possible of somatic ovarian cell or resident ovarian macrophage origin, are capable of exerting specific dose- and time-dependent (immunoneutralizable) morphogenic as well as cytotoxic effects at the level of ovarian cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bone mineral density varies as a function of the rate of joint space narrowing in the hip

OBJECTIVE: To determine whether patients with a rapid rate of joint space narrowing (JSN) in the hip have higher initial bone mineral density (BMD) in the proximal femur and/or lumbar spine than corresponding patients with a slow rate of JSN. METHODS: Twenty-eight patients undergoing unilateral total hip replacement (THR) for osteoarthritis (OA), but whose contralateral hips were asymptomatic and had minimal or no radiographic OA, were evaluated. The contralateral proximal femur (i.e., non-operated hip) and lumbar spine were scanned by dual energy x-ray absorptiometry at baseline (prior to THR) and at 2 years. The rate of JSN was determined by serial longitudinal quantification of the joint spaces over the 2 year period following THR from conventional radiographs, and the patients were divided into a group with a slow rate of JSN (< or = 0.2 mm/yr, n = 20) and a group with a rapid rate of JSN (> 0.2 mm/yr, n = 8). RESULTS: The baseline BMD z and t scores at the femoral neck, Ward's triangle, and lumbar spine of the patients with subsequent rapid rates of JSN were significantly higher than those of patients with slower rates (p < 0.05). There was no difference between the rapid and slow narrowers at the greater trochanter (p > 0.2). Age, sex, weight, height, body mass index, Kellgren- Lawrence scores, and initial joint space width were not significantly different between the 2 groups. CONCLUSION: Patients with a rapid rate of JSN of the asymptomatic hip after unilateral THR are characterized by elevated local and remote BMD. The local elevation in BMD implies that increased local bone density may contribute to or serve as a marker for increased risk of development of OA (assuming that JSN can be used as a predictive marker). The presence of elevated BMD in the spine suggests that there are systemic as well as local aspects of OA pathogenesis, at least in patients with one THR and rapid JSN in the contralateral hip.     

Substrate for endothelial prostacyclin production in the presence of platelets exposed to collagen is derived from the platelets rather than the endothelium

Interactions between vascular endothelial cells and blood platelets have been investigated using a model microcirculation consisting of microcarrier beads colonized with human umbilical vein endothelial cells (HUVECs) and perfused with washed platelet suspensions. To simulate the effects of endothelial desquamation and exposure of subendothelium, fibrillar collagen in suspension was coinjected with the platelets. In this model, neither the passage of platelets alone nor collagen alone stimulated prostacyclin (PGI2) production by the HUVECs. Platelets activated by coinjection with collagen released thromboxane A2 (TXA2), and this was associated with the simultaneous production of PGI2 by the HUVECs. By means of double-isotope experiments with [3H]arachidonic acid (AA) incorporated into platelets and [14C]-AA into HUVECs, it was shown that all the PGI2 generated was derived from platelet AA and/or endoperoxides. This interpretation was strengthened by the finding that PGI2 production was not prevented by treatment of HUVECs with indomethacin followed by perfusion with collagen-stimulated platelets. AA metabolites in double-isotope label experiments were further characterized by reverse-phase chromatography, and it was shown that both cyclooxygenase and lipoxygenase products of the HUVECs were derived from platelet membrane lipid. Thrombin regularly produced transient PGI2 release, but showed rapid tachyphylaxis. Platelet-derived compounds including ADP, ATP, and platelet-activating factor (PAF) did not produce PGI2 release by HUVECs in this system. Thus, the transfer of AA and metabolites from collagen- stimulated platelets is likely to be the mechanism for PGI2 production in the context of minor degrees of endothelial desquamation.