Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium.

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni(2+) chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction.

A rapid practical RT-PCR-based approach for the detection of the PML/RAR alpha fusion transcript in acute promyelocytic leukemia.

The t(15;17) and its molecular equivalent, PML/RAR alpha gene fusion, is strongly associated with acute promyelocytic leukemia (APL). Since treatment response to all-trans retinoic acid correlates directly with PML/RAR alpha, expeditious documentation is critical to patient care. We have designed an extremely rapid, practical, polymerase chain reaction (PCR)-based method using a rapid air thermal cycler to detect type A, B, and B-variant fusion patterns of PML/RAR alpha. We examined 15 cases of APL and 13 cases of leukemias other than APL with a nested reverse-transcription PCR assay. Three APL samples were type A, 11 were type B, and 1 was a B variant based on gel band patterns. PCR products exhibited positive probe hybridization signals and had sequences containing type A, B, or B-variant fusion patterns. PCR amplification of PML/RAR alpha was complete in 22 minutes, and the entire test required 4 1/2 hours. This method permits exceptional turnaround time and is an alternative to cytogenetics and slower PCR assays.

Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus.

A locus for familial melanoma, MLM, has been mapped within the same interval on chromosome 9p21 as the gene for a putative cell cycle regulator, p16INK4 (CDKN2) MTS1. This gene is homozygously deleted from many tumour cell lines including melanomas, suggesting that CDKN2 is a good candidate for MLM. We have analysed CDKN2 coding sequences in pedigrees segregating 9p melanoma susceptibility and 38 other melanoma-prone families. In only two families were potential predisposing mutations identified. No evidence was found for heterozygous deletions of CDKN2 in the germline of melanoma-prone individuals. The low frequency of potential predisposing mutations detected suggests that either the majority of mutations fall outside the CDKN2 coding sequence or that CDKN2 is not MLM.

Localization of the VHR phosphatase gene and its analysis as a candidate for BRCA1.

The VH1-related human protein (VHR) gene was localized to human chromosome 17q21 in a region thought to contain the BRCA1 locus, a locus that confers susceptibility to breast and ovarian cancer. VHR encodes a phosphatase with dual specificity for tyrosine and serine residues. Thus it is a plausible candidate for a tumor suppressor gene such as BRCA1. To test this possibility, the VHR coding sequence was screened in individuals with familial breast cancer and in sporadic breast tumor and breast cancer cell lines. No mutations were detected, suggesting that the VHR gene is not BRCA1.

Genetic similarity among ecotypes of Arabidopsis thaliana estimated by analysis of restriction fragment length polymorphisms.

Genetic similarity was estimated among a sample of 28 ecotypes of Arabidopsis thaliana. Twenty-five previously mapped genomic clones were used as probes in Southern hybridizations to detect restriction fragment length polymorphisms (RFLPs). A total of 62 polymorphic restriction fragments were classified as to their presence or absence for each genotype. The genetic similarity between each pair of ecotypes was calculated as the ratio of concordant to total bands scored. The mean genetic similarity among the 28 ecotypes was 0.69 and ranged from 0.32 to near 1.0. No relationship was observed between genetic similarity and geographical origin of the 28 ecotypes. The ecotype most distantly related to the other 27 was Niederzenz, with a mean genetic similarity of 0.55 ± 13. A bootstrap procedure was used to generate 200 random samples of bands of size n (n=8,12,16,..., 55), and the coefficient of variance (CV) was estimated for each sample. The plot of the first two principal components provided a description of the relative genetic similarity among ecotypes. The results provide information useful to investigators interested in sampling the genetic variation among Arabidopsis ecotypes.

The cause of coagulopathy after peritoneovenous shunt for malignant ascites.

Thirty-five patients with malignant ascites who received a peritoneovenous shunt were studied to determine the type and duration of postoperative coagulopathy. Coagulation factors were measured before and on the first and third day after the placement of a Denver peritoneovenous shunt; 1 to 10 L of ascites was removed at operation. Levels of platelets, antithrombin III, plasminogen, antiplasmin, fibrinogen, and factors V and VIII decreased by the first postoperative day but did not change further through the third day. The levels of fibrinolytic split products increased on day 1 but were lower by day 3. The platelet count reduction by the third day correlated with the hematocrit change (-0.031). The prothrombin and activated partial thromboplastin times remained normal postoperatively. The patterns of change were similar for patients with positive (n = 18) and negative (n = 17) ascites cytologic findings, with elevated (n = 24) and normal (n = 11) preoperative fibrinolytic split product levels, and elevated bilirubin value (greater than 25 mumol/L; n = 9), and no jaundice (n = 26). Bleeding did not occur. The data indicated that plasminogen-rather than thromboplastin-activated fibrinolysis occurred and that platelet reduction was largely dilutional. The reactions were not progressive when ascites was removed operatively.

Preoperative coagulation indices in patients with morbid obesity.

Preventive measures against venous thromboembolism are used frequently in surgical patients with morbid obesity because the risk has been claimed to be higher than usual. We addressed this risk in 81 morbidly obese patients by measuring preoperative plasma proteins associated with coagulation and by correlating variations in these indices to excess weight, liver histology, patient sex, tobacco use and serum triglycerides. Plasma levels of antithrombin III and plasminogen were normal in these patients. Plasma fibrinogen concentrations were elevated in 34 percent of patients; the values did not relate to excess weight and they correlated negatively (P less than 0.04) with increasing serum triglycerides and grades of liver fat content. Premenopausal women had higher plasminogen and fibrinogen levels than menopausal women or men, although their mean levels were normal. Smokers had lower plasminogen levels than nonsmokers (P less than 0.01). However, none of the measured levels of preoperative coagulation proteins identified increased risk for venous thrombosis in cohorts of this population, and the coagulation indices were not different from those reported in normal weight patients.

Medicare patients and alternatives to the acute care hospital.

There are many instances where hospitalized elderly patients no longer need intensive hospital care but are not ready to be discharged. One solution to this dilemma is the use of a skilled nursing facility (SNF) or some type of step-down bed, where patients can continue to receive managed care at a less intense and costly level. A major concern with using step-down beds is that while costs may be controlled, quality of care may suffer and patient outcomes may be jeopardized. From an efficiency perspective, the step-down bed can provide an excellent alternative to hospital care for the less acutely-ill patient. For alternative managed care environments to be satisfactorily received by patients and physicians, however, they must produce outcomes equal or better than those in the hospital. Some patients and diagnoses are more appropriate than others for treatment in alternative managed care environments. The judgement of appropriateness must take into account patient and physician satisfaction with the efficiency and effectiveness of the care. This paper describes how the Fallon Community Health Plan, Worcester, Mass., which regularly uses a step-down facility for patient care, assessed outcomes in terms of efficiency and effectiveness. An overview of this alternative managed care facility and its relationship to the HMO is discussed below. Several studies examining outcomes and patient and physician satisfaction are presented. Finally, the ways in which these studies represent a merger of quality assurance (QA) and utilization review (UR) methodologies are discussed.

Abnormalities of the fibrinolytic system as a cause of upper extremity ischemia: a preliminary report.

Abnormalities of the fibrinolytic system can result in unusual or unexplained clotting that occurs spontaneously or after minor trauma. We identified five patients with limb-threatening arterial thrombosis of the upper extremity associated with either a low level of plasminogen or an abnormal immunoreactive plasminogen. All patients had extensive thrombosis of the brachial, radial, and ulnar arteries. Two patients had concomitant thrombus of the subclavian artery, which in one patient was associated with distal embolization to the hand. There was no evidence of atherosclerosis in any patient. Detection of an abnormal plasminogen level was done by immunoelectrophoresis of the patient's serum with antiplasminogen sera. In these patients a separate immunoreactive band located near the anode and distinct from the normal single plasminogen band was detected. Because of extensive thrombosis of the arterial system, exploration of the brachial artery, as well as the origin of all the forearm vessels, was necessary for complete balloon catheter thrombectomy. Prompt diagnosis and treatment are necessary to prevent the catastrophic complication of arm or hand amputation. Patients with an abnormal plasminogen level should receive perioperative heparin therapy and long-term warfarin to prevent recurrent thrombotic episodes.

Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein.

The cDNA clone (pNP24) coding for a protein induced by exogenous NaCl has been isolated from a tomato root cDNA library with the use of an inosine containing synthetic oligomer. The authenticity of the clone has been established by comparing the sequence of the clone to the NH2-terminal sequence of the protein which has been purified to homogeneity by HPLC. The nucleotide sequence of pNP24 reveals a 5' signal sequence, an open reading frame of 718 nucleotides, a 3' AT rich untranslated region containing a probable polyadenylation signal sequence, and a poly A stretch. The mature polypeptide sequence as deduced from the nucleotide sequence reveals a protein with a molecular weight of 24226. This protein has been named NP24. It is slightly basic and has an unusually high number of cysteines (15). Northern blot analyses reveal that the abundance of mRNA for NP24 is at least 100-fold greater in tomato suspension cells in log phase grown in medium with NaCl than in cells grown in the control medium. The mRNA for NP24 is below the level of detection in roots of young control tomato plants until several weeks after germination but it is induced earlier and to higher levels in roots stressed by 0.171 M NaCl. Thus salt stress accelerates the accumulation of message in tomato roots. A comparison of the steady state levels of mRNA for NP24 to the accumulation of NP24 by immuno analyses indicates that the accumulation of this protein is determined by its mRNA level. The protein is not secreted and is localized within the cytoplasm or the soluble fraction of the nucleus, vacuole, or microbodies. NP24 has a high degree of homology (58%) with thaumatin, a protein which has considerable value as an artificial sweetener.

A platelet aggregating factor in thrombotic thrombocytopenic purpura: initial activity, fluctuations, and removal by plasma exchange.

A factor capable of aggregating normal platelets was found in the plasma of six consecutive patients with thrombotic thrombocytopenic purpura (TTP). The activity of the aggregating factor in whole plasma, the cold protein fraction from plasma and the residual supernatant was monitored during each patient's course of therapeutic plasma exchange. Although two patients demonstrated the highest level of aggregating activity at the time of diagnosis, the level fluctuated in five of six patients. Increasing levels of activity were usually accompanied by signs of clinical deterioration. Activity repeatedly within the normal range was not seen until remission of the syndrome. Neutralization of the aggregating activity in vivo through removal of patient plasma and replacement with fresh frozen plasma (plasma exchange) was accomplished less readily and less predictably than by mixing patient plasma and normal plasma in vitro. Use of the aggregating factor level in evaluating the need of plasma exchange is discussed.

A protein induced by NaCl in suspension cultures ofNicotiana tabacum accumulates in whole plant roots.

We have characterized a 26 000 dalton (26 000 D) protein which accumulates inNicotiana tabacum cuspension cells grown in media containing 10-25 g/l NaCl (7, 11, 17). Antibody was prepared against this protein and used to examine protein accumulation in both suspension cells and whole plants. Western blot analysis revealed that the 26 000 D protein also accumulates in suspension cells grown in the absence of NaCl as they approach stationary phase but the accumulation never reaches the level seen in the salt adapted cells. This protein also accumulates after treatment with other agents which lower the water potential, such as PEG and KCl, but no increase is seen after nonosmotic stresses such as heat shock and growth in cadmium chloride. The 26 000 D protein is found not only in whole tobacco plants but also in other members of the Solanaceae that were tested, as well as in alfalfa and green beans. The accumulation of the protein seems to be tissue specific as there is considerably more accumulation in roots than in stems or leaves of greenhouse grown plants. We have been unable to correlate accumlation of the 26 000 D protein with salt in wild tomato species but have demonstrated an increase in the accumulation of this protein with salt stress in hydroponically grown tomato plants. These results lead to speculation as to the role of this protein in responding to lowered water potential in the whole plant.

Severe bleeding diathesis associated with invasive aspergillosis in transplant patients.

A severe bleeding disorder developed in eight renal transplant patients with invasive aspergillosis. The hemorrhagic diathesis was characterized by wound oozing, severe upper and lower gastrointestinal tract hemorrhage, and mucosal bleeding at other sites. This unusual coagulopathy was characterized by a prolonged thrombin time, which was corrected with protamine sulfate, and an abnormal Reptilase time. The bleeding disorder antedated the diagnosis of invasive aspergillosis in all cases. The probability that the coagulopathy was due to proteolytic enzymes elaborated by Aspergillus sp. is discussed.

Thermo-inducible gene expression in Bacillus subtilis using transcriptional regulatory elements from temperate phage phi 105.

A Bacillus subtilis/Escherichia coli shuttle plasmid vector containing a transcriptionally silent chloramphenicol acetyl transferase gene (cat-86) was constructed by ligation of pPL603 (Williams et al., 1981a) and pUC8 (Vieira and Messing, 1982) at their unique EcoRI sites. Using this "promoter probe" vector we have obtained, by direct Cm resistance selection, a collection of cloned Sau3A fragments from the temperate phage phi 105 genome exhibiting promoter activity in B. subtilis. 18 promoter plasmids were subsequently transferred to an acceptor cell containing a functional repressor gene of phage phi 105 inserted into the temperature-sensitive replicon pE194. A repressor-controlled promoter was identified on the basis of its ability to confer thermo-inducible Cm resistance. The promoter is located on a 650-bp Sau3A fragment, mapping within the 3.2-kb EcoRI-F fragment, which also contains the phi 105 repressor gene. By assaying cloned subfragments of EcoRI-F for expression of immunity against phi 105 infection, the repressor gene could be assigned to a 1.1-kb EcoRI-HindIII fragment, which partially overlaps the promoter fragment. Taken together, these results suggest that, like the cI-coded repressor in coliphage lambda, the phi 105 repressor interacts with an operator sequence mapping very close to its own gene.

Abnormal plasminogen: a genetically determined cause of hypercoagulability.

An inherited disorder of the fibrinolytic system has been discovered as a cause of unusual clotting. An abnormal immunoreactive plasminogen was identified in eight patients who presented with unexplained thrombosis. Six patients presented with spontaneous arterial or venous thrombosis, and two patients developed postoperative occlusion of an arterial reconstruction. Five of the six patients with spontaneous thrombosis had recurrent episodes involving both the arterial and venous system at time intervals between the thrombotic episodes varying from 1 month to several years. Detection of an abnormal plasminogen was made by immunoelectrophoresis of the patient's serum with an antiplasminogen sera. In normal patients, plasminogen migrates as a single band toward the anode. In these eight patients a separate immunoreactive band located nearer the anode and distinct from the normal band was detected. Examination of family members of two patients identified a similar abnormal plasminogen with an overall incidence suggestive of an autosomal dominant inheritance pattern. This study suggests the presence of a genetically determined plasminogen variant resulting in a functional deficiency of the plasminogen system causing a reduction of fibrinolytic activity and a latent thrombotic tendency. Recommended treatment is long-term warfarin anticoagulation.