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Sam68 (Src-associated in mitosis) is an SH3 (Src-homology 3), SH2 (Src-homology 2), and RNA binding protein which associates with and is tyrosine phosphorylated by wild-type and activated forms of c-Src in a mitosis-specific manner. We now show that Sam68 immunoprecipitated from either HeLa S3 or NIH3T3 cells is phosphorylated on threonine residues exclusively during mitosis as well as on serine residues during both interphase and mitosis. Recombinant Sam68, expressed as a glutathione S-transferase (GST) fusion protein, was phosphorylated on threonine and serine residues after incubation with mitotic lysates several-fold more extensively than after incubation with unsynchronized lysates. Cdc2 was identified as the kinase responsible for the mitotic threonine phosphorylation by (1) immunodepletion of the mitotic Sam68 kinase from cell lysates with anti-Cdc2 antibodies, (2) inhibition of Sam68 phosphorylation in vitro and in vivo by the cyclin-dependent kinase inhibitor olomoucine and (3) phosphorylation of Sam68 by purified Cdc2. These data demonstrate that Sam68 is a direct target of Cdc2 and may therefore mediate some of its biological effects during mitosis.

The direct biochemical analysis of protein tyrosine kinases from yeast has been difficult due to their very low activity in crude cell lysates. Here we present a procedure for the enrichment and partial purification of protein tyrosine kinases from Saccharomyces cerevisiae based on single-step substrate affinity chromatography using a synthetic random co-polymer of glutamic acid and tyrosine. Fractionation of cell lysates on a poly-glutamic acid:tyrosine (4:1)-Sepharose affinity column resulted in a 4000-fold increase in tyrosine kinase activity. Active fractions contain at least six potential protein kinases as judged by in situ phosphorylation assay and Western blot analysis using anti-phosphotyrosine. We propose that this protocol may also be useful for the initial identification and purification of tyrosine kinases from other organisms exhibiting low levels of this enzymatic activity in cell lysates.

An unconstrained cadaver ankle model was designed to reevaluate the effect of ankle mortise widening and syndesmotic fixation on the load-bearing characteristics of the tibiotalar joint. Tibiotalar contact area, centroid shift, and mean contact pressure were quantified using a pressure-sensitive film technique. Six fresh-frozen below-knee amputation specimens were axially loaded with 500 N in three positions: neutral, 10 degrees of dorsiflexion, and 20 degrees of plantarflexion. The tibiotalar contact area and centroid position for each specimen in its intact state were first determined and then compared with values obtained after syndesmotic fixation, mortise widening of 2 and 4 mm, and deep deltoid ligament transection. Syndesmotic fixation significantly decreased joint contact area but did not consistently affect centroid position. However, unlike earlier studies, which used more constrained ankle fracture models, mortise widening with or without deltoid rupture was not found to significantly affect contact area, centroid position, or joint contact pressure. When statically loaded, the talus moved to its position of maximal congruence in the mortise, rather than displacing laterally along with the lateral malleolus.

Grandmultiparity (GMP) has long been considered an obstetric complication for both mother and fetus, although recent studies indicate that, with proper perinatal care, women with high-parity rates are no longer at high risk. The current study examines the outcome of delivery in 1700 women in their fifth or more delivery, as compared with two control groups: 622 primiparas and 735 multiparas (two to three previous deliveries). Excellent prenatal care was available free of charge to all parturients. Our objectives were to evaluate the management of GMP in contemporary obstetrics and to assess whether grand multiparas are still high-risk patients. The age of the grandmultiparas was significantly higher compared with with the control groups, which may explain the higher incidence among them of antenatal medical disorders, such as diabetes mellitus and hypertensive disease. No significant differences were found among the three groups for preterm or post-term births, small-for-gestational-age infants, polyhydramnios, oligohydramnios, perinatal death, fetal distress, multiple births, placenta previa, abruptio placentae or cord prolapse. Macrosomia was markedly higher in the grandmultiparas and multiparas than in nulliparas. Thus, our results indicate that good perinatal care can ensure better results in grandmultiparas, and that grandmultiparity no longer needs to be considered a high-risk obstetric category in our population.

This article describes a patient with a 10-year history of persistent ankle pain. Differential diagnosis included osteoid osteoma and anterior ankle impingement. This patient subsequently underwent arthroscopic excision of a lesion on the talar neck following a complete radiographic work-up, which was nondiagnostic. The diagnosis of osteoid osteoma was finalized upon pathologic study of the arthroscopic shavings. The use of a motorized instrument for excision did not preclude pathologic evaluation of the specimen. Therefore, in an accessible location on the talar neck, arthroscopic excision of an osteoid osteoma can be performed.

In this paper we report on a community based sample of heroin addicts not in addiction treatment who responded to the offer of experimental treatment (buprenorphine) for heroin addition. Comparison to a sample of methadone maintenance clients from the same geographic area reveals that on average the community sample began heroin use later, attained a higher level of education, and were most likely to have had a significant cohabitation relationship. The methadone maintenance sample, on the other hand, reported significantly greater regular use of drugs of abuse other than heroin. Each group may represent a different subgroup drawn from the heterogeneous heroin-addicted population. Implications for improved recruitment into treatment and enhanced treatment design are drawn from these findings.

Deltoid ligament forces were studied after observing deltoid ligament insufficiency in several post-triple arthrodesis patients. Six fresh-frozen, below-knee amputation specimens were axially loaded. The results demonstrate that a properly positioned triple arthrodesis produced deltoid ligament forces that were similar to those seen with an intact tibialis posterior tendon. A triple arthrodesis in combination with a lateral displacement calcaneal osteotomy produced deltoid ligament forces that were 76% greater than those seen with the intact tibialis posterior tendon (P < .05). A triple arthrodesis in combination with a medial displacement calcaneal osteotomy produced deltoid ligament forces that were 56% less than those seen with the lateral displacement calcaneal osteotomy (P < .01). Patients with longstanding ruptures of the tibialis posterior tendon and associated peritalar subluxation/dislocation may have less than optimal clinical results after triple arthrodesis, unless the hindfoot can be properly reduced, due to persistent elevated forces in the deltoid ligament and resulting ligament laxity. This study suggests that a medial displacement calcaneal osteotomy in combination with a triple arthrodesis may be a viable treatment when the hindfoot cannot be positioned properly.

The aim of this study was to evaluate whether prolonged pregnancy was associated with dangers to the fetus, and to assess the incidence of cesarean section as well as fetal distress. The outcome for mother and baby in both groups of patients with post-term pregnancies was generally good. Furthermore, the improvement of prognosis for postdate pregnancy is due to the availability of electronic fetal monitoring and ultrasound, making possible the identification of fetal distress, as well as the use of prostaglandin to ripen a stubborn cervix, and induce labor successfully in most cases. Consequently, we feel that fetal jeopardy with continued intrauterine existence, as well as the excellent results of induction of labor, justify the active approach. The routine induction of labor in postdate pregnancy may reduce perinatal morbidity, as indicated in previous reports [2], and confirmed in this study.

Using the model described by Michelsson, 24 mature New Zealand white rabbits underwent mechanical induction of heterotopic ossification in the quadriceps of the right hind limb. The rabbits were equally divided into four groups: indomethacin-treated, piroxicam-treated, and their respective controls. The effect of drug therapy on the production of heterotopic bone was assessed by analyzing plain radiographs of the femur using the grading system described by Scott. The results demonstrated that, while indomethacin is effective in decreasing the formation of heterotopic bone, piroxicam--when used in the dosage previously demonstrated to have no deleterious effect on healing bone--is not. The analysis of piroxicam blood levels demonstrated that the experimental piroxicam dose is inadequate to produce effective plasma blood levels in the rabbit. Therefore, any potential benefit from using piroxicam, as opposed to other nonsteroidal antiinflammatory drugs regarding fracture healing and bone remodeling, cannot be applied for the prophylactic treatment of heterotopic bone formation.

Treatment of cells with platelet-derived growth factor (PDGF) was previously shown to increase the release of unesterified fatty acids from phospholipids. In view of these observations we examined the effect of various fatty acids on PDGF receptor activity. Unsaturated (oleic, linoleic, linolenic, and arachidonic) but not saturated (stearic and arachidic) fatty acids significantly inhibited the tyrosine kinase activity of the PDGF receptor in intact cells and membrane preparations. Half-maximal inhibition (IC50) was observed between 60 and 100 microM and maximal inhibition between 170 and 200 microM. Lysophospholipids and phospholipids had no effect on receptor activity. Activation of endogenous phospholipase A2 by mellitin in vivo or hydrolysis of phosphatidylcholine by purified phospholipase A2 in vitro inhibited PDGF receptor autophosphorylation similar to that of purified fatty acids. Dimerization of PDGF receptors in vivo was significantly reduced by concentrations of arachidonic acid inhibitory for receptor kinase activity while the binding of PDGF was only marginally affected. These data suggest a possible feedback mechanism resulting in the reduction of PDGF receptor activity by unsaturated fatty acids generated downstream within the PDGF-dependent signal transduction pathway and this effect may be as a direct result of decreased receptor dimerization and/or kinase activity.

Attention deficits were studied among 67 consecutive admissions to an adult tertiary care in-patient substance abuse unit. Fifty-four patients completed the testing; of these 15 were excluded due to confounding variables. The majority of the remaining 39 subjects had evidence of current and/or childhood attention dysfunction. There was no correlation with current performance and childhood recollection of dysfunction even when the effect of gender, race and alcohol abuse were taken into account. There are intriguing educational and therapeutic implications of the similarities between cognitive impairments of newly sober substance abusers and adults with persistence of attention deficit-hyperactivity disorder (ADHD).

Eighty-five heroin addicts who were unwilling to receive methadone maintenance or enter therapeutic communities were assessed, single-blind, for the lowest sublingual dose of buprenorphine that blocked heroin craving (8.0 mg max). All doses were administered daily under observation. After maintenance for 4 to 12 weeks, abstinent subjects (confirmed by urine drug screens) entered a double-blind discontinuation trial and were randomly assigned to receive dose reductions (10% twice weekly for 5 weeks to zero dose, then placebo for 2 weeks) or a stable dose for 7 weeks. Subjects were terminated from discontinuation if heroin was used or they had increased craving/symptoms. Subjects completed the trial if they did not use heroin and had no increase in craving/symptoms. A wide dose range (1.5-8.0 mg/day) was effective in reducing heroin craving and use. Of 73 subjects who received buprenorphine for 4 to 52 weeks, 40 had no prior treatment, despite high levels (mean $/day heroin = 70.5 +/- 94.7) and many years (mean years = 10.7 +/- 8.6) of dependence. Subjects who received dose reductions developed abstinence symptoms, low energy most commonly, associated with drug-seeking behavior. Discontinuation trial outcome (n = 51) shows a highly significant difference between 29 subjects who received dose reductions (28 terminated, 1 completed) and 22 subjects who received no dose reductions (3 terminated; 19 completed) (chi-square = 36.08; p less than .00001). The findings suggest that buprenorphine could be an important medication for reducing demand for heroin by many heroin addicts who remain outside the present health-care system.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure of rat-1 fibroblasts to cholera toxin increased aerobic lactate production 3- to 8-fold with maximal stimulation observed between 1 and 2 h at a concentration of 1-2 micrograms/ml. Concomitant with this change was a 10- to 40-fold elevation in the intracellular concentration of cAMP. The cell permeable cAMP analogue, N6,2'-O-dibutyryl cAMP and the cyclic nucleotide phosphodiesterase inhibitor RO-20-1724 also increased lactate production and intracellular cAMP levels, although less effectively. Cholera toxin and dibutyryl cAMP induced a 2- to 3-fold elevation of intracellular fructose 2,6-bisphosphate and 2- to 3-fold increases in both 3-O-methylglucose and inorganic phosphate transport. A survey of five additional cell lines revealed striking variabilities in their individual responses to cholera toxin and dibutyryl cAMP. All were observed to be considerably less sensitive to either agent than rat-1 cells. These data suggest that a cooperative effect involving multiple parameters may be responsible for the observed increases in aerobic lactate production in response to cAMP and that these parameters may vary significantly among cell lines.

Fructose 2,6-bisphosphate (F-2,6-P2) stimulated glycolysis in cell-free extracts of both normal and ras-transfected rat-1 fibroblasts. The extract of the transformed cell glycolyzed more rapidly in both the absence and the presence of F-2,6-P2 than the extract of the parent fibroblast. Addition of mitochondrial ATPase (F1) or inorganic phosphate (Pi) further stimulated lactate production in both cell lines. F-2,6-P2 stimulated the 6-phosphofructo-1-kinase (PFK-1) activity in extracts of normal and transfected cells. The activity in extracts of transformed cells tested with a fructose 6-phosphate regenerating system was considerably higher than in the extract of normal cells. Stimulation of PFK-1 activity by cAMP of both cell lines was not as pronounced as that by F-2,6-P2. In the absence of F-2,6-P2 the PFK-1 activity was strongly inhibited in the transformed cell by ATP concentrations higher than 1 mM, whereas in the normal cell only a marginal inhibition was noted even at 2 or 3 mM ATP. F-2,6-P2 reversed the inhibition of PFK-1 by ATP. Nicotinamide adenine dinucleotide (NAD) at 100 microM (in the presence of 2 mM ATP and 1 microM F-2,6-P2) stimulated PFK-1 activity only in the transformed cell, whereas nicotinamide adenine dinucleotide phosphate (NADP) inhibited PFK-1 activity (in the presence or absence of 1 microM F-2,6-P2) in extracts of both cell lines. No previous observations of stimulation or inhibition by NAD or NADP on PFK-1 activity appear to have been reported. A threefold increase in the intracellular concentration of F-2,6-P2 was observed after transfection of rat-1 fibroblast by the ras oncogene. We conclude from these data that the PFK-1 activity of ras-transfected rat-1 fibroblasts shows a greater response to certain stimulating and inhibitory regulating factors than that of the parent cell.

1. A 26-32 month follow-up of 16 heroin-dependent subjects who entered a pilot trial of treatment with buprenorphine (a mixed agonist/antagonist) suggests that positive response to treatment may identify a subgroup of untreated addicts whose levels of psychosocial functioning are intermediate between those for whom methadone (a pure agonist) or naltrexone (a pure antagonist) would be indicated. 2. Buprenorphine's pharmacologic profile provides a missing link in available modalities for opiate dependence treatment, making it acceptable for many addicts who will not accept methadone maintenance treatment, join a residential therapeutic community, or be successful on naltrexone treatment. 3. Eight of the 16 ss were abstinent from heroin while receiving 0.6-3.9 mg/day buprenorphine and counseling. Responders (mean age 34 yrs) had been heroin dependent for a mean of 9.5 years (range 6-17 yrs), all were self-supporting, 4 lived with a non-addicted spouse, 5 had no prior treatment for addiction and 3 had prior naltrexone treatment, but had discontinued it and relapsed. Non-responders (mean age 30 yrs) had been heroin dependent for a mean of 7.4 yrs (range 2-19 yrs), 7 had no regular employment, all were single and 7 had no prior treatment for addiction. 4. Levels of psychosocial functioning (work, home, leisure) and global assessments of functioning were significantly higher for buprenorphine responders than non-responders (p less than .001 and p less than .01 respectively). 5. A new formulation of buprenorphine needs to be developed for addiction treatment, ideally consisting of 0.5 mg and 2.0 mg sublingual tablets.

We developed a polymerase chain reaction DNA amplification system using two distinct consensus oligonucleotide primer sets for the improved detection and typing of a broad spectrum of human genital papillomavirus (HPV) sequences, including those of novel viruses. The system incorporates one primer set designed to amplify a highly conserved L1 domain and a second primer set designed to amplify a domain within the E6 gene. We used this system to analyze 48 fixed, paraffin-embedded tissue sections (41 specimens from 33 cervical carcinomas, four normal cervical tissues, and several control tissues) for the presence of HPV DNA. HPV sequences were detected in all carcinoma samples and none of the control samples. Hybridization analyses showed that the results obtained with the two amplification schemes concurred completely. This approach allowed rapid confirmation of typing results and may improve the likelihood of detecting a wide variety of HPV sequences, including those of novel HPVs.

Various schedules exist for inducting heroin addicts newly admitted to treatment into methadyl acetate maintenance and crossover schedules for the transition from methadone maintenance to methadyl acetate maintenance. A sample of 255 street addicts was randomized to three induction schedules: methadyl acetate three times a week (tiw) with placebos on alternate days; methadyl acetate tiw supplemented with decreasing doses of methadone on alternate days; methadyl acetate six days a week with diminishing doses on three days. Treatment was double-blind for four weeks and single-blind for six. All schedules were considered feasible, but supplementation with methadyl acetate or methadone had no advantage. A sample of 310 patients receiving methadone maintenance was randomized to comparable supplementation groups, except that the group receiving supplementary methadyl acetate received it along with the regular dose. This schedule was not successful. Supplementing with methadone had no advantage.

In 1964 a 42-year-old woman was hospitalized with clinical and laboratory signs of posttransfusion hepatitis five weeks after administration of six whole blood transfusions. During the following 17 years anicteric chronic liver disease was repeatedly documented by elevations of serum aspartate aminotransferase (SGOT) and alkaline phosphatase enzymes. In 1981 hepatomegaly, progressive jaundice, and a serum alphafetoprotein level of 516,000 ng/ml were observed. Percutaneous liver biopsy showed a primary hepatocellular carcinoma (PHC). Serologic examinations failed to reveal markers for hepatitis B virus including HBsAg, anti-HBs, and anti-HBc by radioimmunoassay; antibody to hepatitis A virus was also absent. This sequence of events demonstrates a presumptive association of PHC and the agent(s) of non-A, non-B viral hepatitis.

The effects of ionic strength, pH, and temperature on three separate aspects of prothrombin-phospholipid membrane binding were studied. The three parameters include a calcium-dependent protein transition, a calcium-membrane interation, and, finally, the binding of calcium-saturated protein to a calcium-saturated phospholipid membrane. The results are consistent with calcium binding to carbonyl groups in the protein and to phosphate in the phospholipids. These interactions show the expected pH profiles and sensitivity to ionic strength. Temperature effects indicate a small negative enthalpy change for each process. The binding of calcium-saturated protein to calcium-saturated membrane shows very little variation between pH 6 and pH 9, is accompanied by no detected enthalpy change, and is relatively insensitive to ionic strength. These latter results indicate that ionic calcium bridging between the protein and membrane is not important. A chelation model for prothrombin-membrane binding is proposed where the two interacting species have no net charge; ligands on the protein complete the coordination sphere of membrane-bound calcium and vice versa.

The authors studied the responses of 28 adult male volunteers who were openly changed from methadone to l-alpha-acetylmethadol (LAAM) maintenance. They found that patients who had been receiving middle-range doses (50-70 mg) of methadone required a significantly lower mean increase in LAAM than patients who had been receiving either high or low methadone doses and that the patients who accepted LAAM differed significantly from those who did not in MMPI 2-point-code ratings and mean social adjustment scale scores. These findings may provide prognostic indicators for response to LAAM, a possible alternative to methadone.