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Background: Elevated fibrinogen levels have been linked to increased risk for deep venous thrombosis, although it is not clear whether fibrinogen is causal or rather a marker for the presence of other risk factors. A common G/A polymorphism in the gene for the fibrinogen beta-chain (FGB G-455A) is associated with elevated fibrinogen levels. The present study was designed to analyze the role of this genetic marker for deep venous thrombosis., Materials and Methods: We performed a case-control study including 307 patients with documented deep venous thrombosis and 316 control subjects. beta-fibrinogen genotypes were determined by allele-specific polymerase chain reaction., Results: GG, GA and AA genotype frequencies were similar among the patients (53.1%, 41.0, 5.9) and controls (51.6%, 42.1, 6.3; P = 0.92). Fibrinogen levels of the patients (median 3.72 g l-1; range 1.93-11.6) did not differ significantly from those of the controls (3.76; 2.17-9.99). Carriers of the homozygous AA genotype had significantly higher fibrinogen levels than noncarriers (patients: 5.32 vs. 3.59; P = 0.024; controls: 6.29 vs. 3.72; P = 0.048)., Conclusion: Our data suggest that the fibrinogen-elevating FGB G-455A gene polymorphism is not linked to an increased risk for deep venous thrombosis.

Purpose: Hyperhomocysteinemia has been established as an important risk factor for cardiovascular diseases. The aim of the present study was to investigate whether hyperhomocysteinemia and/or homozygosity for the methylenetetrahydrofolate reductase (MTHFR) C677T mutation are associated with an increased risk for retinal artery occlusion (RAO)., Design: Retrospective case-control study., Methods: We studied 105 consecutive patients with retinal artery occlusion and 105 age and sex-matched control subjects. Fasting plasma homocysteine levels were determined by high-performance liquid chromatography, while genotypes of the MTHFR C677T mutation were determined by polymerase chain reaction., Results: Mean plasma homocysteine levels were significantly higher in patients with RAO compared with control subjects (12.2 +/- 4.8 micromol/l vs 10.3 +/- 3.4 micromol/l; P =.003). Hyperhomocysteinemia was defined by the 95th percentile of control plasma homocysteine levels as 15.8 micromol/l. Twenty (19.1%) patients with RAO exceeded this level and were therefore classified as hyperhomocysteinemic compared with 5 (4.8%) control subjects (P =.003). The odds ratio for these patients was calculated at 4.7 (95% confidence interval [CI], 1.5-15.1). Mean plasma folate levels were significantly lower in patients than in the control group (5.6 +/- 2.3 ng/ml vs. 6.3 +/- 2.5 ng/ml; P =.04). The prevalence of the homozygous genotype of methylenetetrahydrofolate reductase C677T mutation did not significantly differ between patients and controls., Conclusions: Our results suggest that hyperhomocysteinemia, but not homozygosity, for the MTHFR C677T mutation is associated with RAO.

Background: Nitric oxide is synthesized by endothelial nitric oxide synthase and plays a key role in adequate endothelial function. An important effect is the reduction of free radicals caused by cigarette smoking, which is the only established risk factor for thromboangiitis obliterans (TAO). In patients with TAO a genetic defect of endothelial nitric oxide synthase may therefore result in deteriorated endothelial function. The aim of our study was to evaluate whether a genetic defect of the common variant of endothelial nitric oxide synthase (Glu(298)-->Asp) can be found in a higher degree in patients with TAO compared to healthy controls., Methods: We enrolled 42 patients with TAO and 149 healthy subjects., Results: Nineteen patients (45.2%) showed homozygosity for Glu(298) versus 76 (51%) in the control group. The heterozygous status for Glu(298)-->Asp was found among 18 patients (42.9%) compared to 61 (40.9%) members of the control group, which was a nearly equal distribution. Homozygosity for the mutant Asp(298) was slightly elevated in the patient group with 11.9 versus 8.1% in the control group. Allele frequency for Asp(298) was 0.333., Conclusions: Our data do not show elevated homozygosity for the common variant Glu(298)-->Asp in patients with TAO compared to healthy controls. The limitation of our evaluation is the small number of patients, which is a general problem when evaluating patients with TAO, as this is not a common disease.

Objective: To determine whether hyperhomocyst(e)inemia and methylenetetrahydrofolate reductase (MTHFR) C677T mutation are associated with branch retinal vein occlusion (BRVO)., Design: Retrospective, case-control study., Participants: The study cohort consisted of 84 consecutive patients with branch retinal vein occlusion and 84 controls, matched for age and gender., Main Outcome Measures: Fasting plasma homocyst(e)ine, folate, and vitamin B(12) levels, MTHFR C677T genotypes., Results: Mean plasma homocyst(e)ine levels were significantly higher in patients than in controls (11.4 +/- 4.3 micromol/l vs. 9.9 +/- 2.8 micromol/l; P = 0.002). An increase of plasma homocyst(e)ine level by 1 micromol/l was associated with an odds ratio of 1.19 (95% confidence interval 1.06-1.34; P = 0.004). Mean plasma folate levels were significantly lower in patients than in the control group (4.5 +/- 2.1 ng/ml vs. 5.6 +/- 2.1 ng/ml; P = 0.007). The prevalence of the homozygous genotype of the MTHFR C677T mutation did not differ significantly between patients and controls., Conclusions: Our results suggest that hyperhomocyst(e)inemia, but not homozygosity for the MTHFR C677T mutation, is associated with BRVO. Increased plasma homocyst(e)ine levels in our study are not the result of an increased prevalence of the homozygous genotype of MTHFR C677T mutation.

Background: Elevated plasma homocyst(e)ine is a major risk factor for venous thrombosis and cardiovascular disease. Homozygosity for the MTHFR C677T mutation and low plasma folate levels increase plasma homocyst(e)ine concentrations. The aim of this retrospective case-control study was to investigate a possible association between hyperhomocyst(e)inemia and central retinal vein occlusion., Methods: Our study included 78 consecutive patients with central retinal vein occlusion and 78 control subjects, matched for age and sex. High-performance liquid chromatography (HPLC) with fluorescence detection was used to determine fasting plasma homocyst(e)ine concentrations. Plasma folate and vitamin B12 levels were determined by immunological assays. Genotyping for the MTHFR C677T mutation was performed by polymerase chain reaction (PCR)., Results: Hyperhomocyst(e)inemia was defined by the 95th percentile of plasma homocyst(e)ine concentrations in the control group (=14.83 micromol/l). Thus 16 patients with central retinal vein occlusion were diagnosed as hyperhomocyst(e)inemic, compared with three control subjects ( P=0.001). The odds ratio of hyperhomocyst(e)inemia for central retinal vein occlusion was 5.29 (95% CI 1.33-21.13). Mean plasma folate levels were significantly lower in patients than in controls (3.94+/-1.94 ng/ml vs. 5.69+/-2.09 ng/ml; P<0.001). Distribution of MTHFR C677T genotypes did not significantly differ between the two groups., Conclusions: Our study suggests that hyperhomocyst(e)inemia, but not the MTHFR C677T mutation, is associated with central retinal vein occlusion.

Background: Factor XIII catalyzes crosslinking of fibrin in the last steps of the coagulation process. A common polymorphism in the gene for factor XIII A subunit (F13A1 V34L) has been associated with a decreased risk for coronary artery disease, cerebrovascular disease, and deep venous thrombosis., Methods: To analyze the role of this polymorphism in peripheral arterial disease (PAD) we performed a case-control study including 873 patients with documented PAD and a total of 523 controls without vascular disease. The F13A1 genotype was determined by an allele-specific polymerase chain reaction., Results: Genotype distribution and allele frequencies were not significantly different between patients (VV: 51.9%; VL: 40.7%; LL: 7.4%) and controls (VV: 54.7%; VL: 39.2%; LL: 6.1%). Mean age at onset of the disease was significantly higher in LL homozygous subjects than in VV homozygous subjects (67.3 versus 64.1 years, p=0.017). Heterozygous subjects had an intermediate age at onset (65.1 years), suggesting a gene-dose effect. The association of the L34 variant with onset of PAD remained significant after adjustment for other risk factors. The effect was stronger in men than in women., Conclusions: We conclude that the F13A1 V34L polymorphism was not associated with the presence of PAD in our study, but may be linked to a later onset of the disease.

The nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system is a major source of superoxide anion (.O2-) production in the human vasculature and may therefore influence lipid peroxidation and severity of atherosclerosis. This study aimed to investigate a hypothetical influence of the p22 phox C242T polymorphism on the generation of malondialdehyde (MDA), extent and clinical onset of coronary artery disease (CAD) in patients. We studied 108 male Caucasians with angiographically documented CAD and 45 controls free of vascular disease under 60 years of age. p22 phox C242T genotypes and MDA levels were determined. Additional information was obtained from each subject on classic risk factors and clinical events of CAD. Genotype distribution in CAD-patients and controls was thymine-thymine (TT): 13.8% (13.3%), cytosine-thymine (CT): 46.3% (53.3%) and cytosine-cytosine (CC): 39.8% (33.3%), respectively. No significant influence was seen of the p22 phox C242T polymorphism on corresponding mean MDA levels in both groups. Furthermore, age at onset of first time angina pectoris (AP) and myocardial infarction (MCI) was not significantly different between genotype groups. It is concluded that the C242T polymorphism of the p22 phox gene is not associated with lipid peroxidation as measured by MDA, and is not a genetic risk marker for CAD Caucasians.

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.

Elevated plasma homocyst(e)ine is currently accepted as a major, independent risk factor for atherosclerosis and venous thrombosis. Even moderate hyperhomocyst(e)inemia is prospectively associated with increased risk of mortality in patients with cardiovascular disease. However, the underlying mechanisms resulting in vascular damage are not clearly defined. The endothelium exerts fundamental control on the vascular tone, coagulation and fibrinolysis. Injury to the endothelium followed by dysfunction is an early key event preceding manifestation of vessel pathology. Acute and chronic exposure of endothelium to homocyst(e)ine induces impairment of endothelial function associated with altered homeostasis and morphologic changes of the vessel wall. Investigations of the role of homocyst(e)ine in the endothelium-dependent function in healthy subjects and cardiovascular patients have recently added important clinical insight with implications for the treatment of cardiovascular disease. Importantly, the damaging effects of hyperhomocyst(e)inemia on endothelial function are, at least in part, reversible in patients with established vascular disease, supporting further the hypothesis that homocyst(e)ine-lowering through vitamin supplementation may have vasoprotective effects.

Background: Platelet glycoprotein (GP) IIb/IIIa, a fibrinogen and von Willebrand factor binding membrane receptor, has an important role in platelet aggregation. A common leucine33-proline polymorphism (PlA1/A2) of the gene encoding the GP IIIa subunit is associated with platelet reactivity and has been proposed as a risk factor for atherothrombotic disease. The aim of this study was to investigate the role of this polymorphism for deep venous thrombosis (DVT)., Methods: We performed a case-control study including 206 patients with documented DVT and a sex- and age-matched group of 310 control subjects. GP IIIa genotypes were determined by restriction fragment analysis of amplimers containing the polymorphic site., Results: A1/A1, A1/A2 and A2/A2 genotypes were found in 67.0, 31.6 and 1.5 percent of patients and 72.3, 25.8 and 1.9 percent of controls (p=0.35), PlA2 allele frequencies were 0.17 in patients and 0.15 in controls (p=0.92). Odds ratio of the PlA2 allele for DVT was 1.21 (95 percent CI 0.85-1.71, p=0.29) and remained insignificant after adjustment for factor V Leiden and prothrombin 20210A genotypes (1.22, 95 percent CI 0.86-1.75, p=0.27)., Conclusions: Our data suggest that the PlA1/A2 polymorphism of GP IIIa is not associated with DVT.

Alphaviruses are positive stranded RNA viruses that replicate to extremely high titers. Sindbis and Semliki Forest viral vectors are widely used tools for high-level production of recombinant proteins. Recent studies have broadened their scope to vaccine production, gene therapy, and analysis of cell function. Here we discuss the development of non-cytopathic and inducible expression vectors which can be applied to bioprocess development strategies. Furthermore, a Sindbis-based expression cloning system has been developed that allows for the rapid identification of genes encoding proteins with a selected functional activity.

Background: Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis. Strong interindividual variations of VEGF plasma levels have been reported previously. Aim of the present study was to search for mutations in the 3' untranslated region (3'-UTR) of the VEGF gene and to analyze their relation to VEGF plasma levels., Methods: The complete 3'-UTR (nucleotide 700-2622) of the VEGF gene was screened for sequence variations by single-strand conformation polymorphism (SSCP) analysis. Frequencies of mutated alleles were determined in 119 healthy subjects; VEGF plasma levels were analyzed in a subgroup of 23 healthy men aged 18-36 years., Results: Three novel mutations (702 C/T, 936 C/T, 1612 G/A) were found, allele frequencies of 702T, 936T and 1612A were of 0.017, 0.160 and 0.471, respectively. VEGF plasma levels were significantly lower in carriers of the 936T allele (9.1 +/- 2.7 pg/ml, mean +/- SEM) than in noncarriers (28.0 +/- 5.5 pg/ml, p = 0.033), whereas the 702 C/T and the 1612 G/A mutations showed no association with VEGF plasma levels. The 936 C/T exchange led to the loss of a potential binding site for transcription factor AP-4, although the functionality of this binding site remains unclear., Conclusion: We have found three common mutations in the VEGF gene; one of them, a 936 C/T exchange, may be an important determinant of VEGF plasma levels., (Copyright 2000 S. Karger AG, Basel)

Bone mineral density (BMD) is modulated by genetic and environmental factors or certain diseases. In several conditions such as low calcium intake, an influence of vitamin D receptor (VDR) polymorphisms on BMD has been suggested. In the present study, we investigated the relationship of Bsm I and Fok I polymorphisms of the VDR gene and BMD in patients with hyperthyroidism, a disease that often results in low BMD. Bsm I and Fok I genotypes were determined in 76 postmenopausal hyperthyroid patients and 62 healthy postmenopausal women as controls. Patients and controls were matched for age, time since menopause, and lifestyle factors and were free of estrogen medication. BMD evaluation included axial dual X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (PQCT). Low BMD was defined as -2.5 STD below the young adult mean value. Biochemical parameters investigated were thyroid hormones, osteocalcin, and 25-(OH)-vitamin D3 as well as routine laboratory data. Low BMD was found in 61% of hyperthyroid patients and in only 23% of euthyroid controls. In the group of hyperthyroid patients with low bone density, the BB genotype (VDR Bsm I polymorphisms) was significantly more frequent (39%) than in controls (13%; p = 0.003) and hyperthyroid patients with normal BMD (6%; p = 0.013). The odds ratio (OR) for low BMD in patients with BB genotype was 5.7 (95% CI, 1.7-19.1; p < 0.005) as compared with the Bb and bb genotypes and 5.5 (95% CI, 2.3-13.2; p < 0.0001) for hyperthyroidism alone. The cumulative risk for low BMD in patients with hyperthyroidism and BB genotype was 31.4 (95% CI, 3.9-256; p < 0.0003). VDR Fok I genotypes showed no significant relationship with BMD or other general or bone-specific parameters. Thus, hyperthyroidism and the genetic background of a BB genotype may promote synergistically the development of low BMD in hyperthyroid patients. Screening for the BB genotype in these patients therefore could help to identify those with particularly high risk for the development of low BMD and allow early treatment.

We present a temperature-regulated, alphavirus replicon-based DNA expression system. The system is regulated by a viral temperature-sensitive RNA-dependent RNA replicase, creating a temperature-dependent RNA amplification loop. Because of this positive feedback, the system exhibits both low background and high inducibility. We observed 700-fold induction in transiently transfected cells, and over 104-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell lines expressing a highly toxic protein upon temperature shift. These data suggest that the present expression system could simplify bioprocess engineering strategies, especially in situations where the cloned protein has detrimental effects on host cell metabolism.

Background: G to A mutations at positions 20210 of the prothrombin gene (F2) and 1691 of the factor V gene (F5) are established risk factors for venous thrombosis. Several factors associated with coagulation and/or fibrinolysis have been associated with arterial occlusive disease, but the role of F2 20210A and F5 1691A for arterial occlusive disease remains unclear., Objective: To investigate if F2 20210A and F5 1691A are associated with peripheral arterial occlusive disease (PAOD)., Methods and Results: We analyzed the prevalence of F2 20210A and F5 1691A alleles in 336 patients with documented PAOD at Fontaine stage II-IV and 300 controls without vascular disease. Allele frequencies in patients and controls were 0.013 and 0.022 for F2 20210A, and 0.042 and 0.045 for F5 1691, respectively, both differences being not statistically significant., Conclusion: Our data suggest that mutations F2 G20210A and F5 G1691A are not associated with PAOD.

Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis. In vivo expression of four different VEGF isoforms, consisting of 121, 165, 189 or 206 amino acids, has been found in the human organism, with all isoforms arising from a single gene by alternative mRNA splicing. We developed an assay for simultaneous quantitation of VEGF isoform expression using competitive polymerase chain reaction (PCR). RNA was isolated from cells, reverse transcribed to cDNA and coamplified with a synthetical competitor DNA using VEGF specific primers. Amplification products were analyzed electrophoretically and concentration of VEGF transcripts calculated. Concentration of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts was quantitated as above, VEGF gene activity is presented as ratio VEGF mRNA to GAPDH mRNA. Using this assay, we were able to detect and quantitate in vivo expression of VEGF121, VEGF165 and VEGF189 by human peripheral blood mononuclear cells (PBMC). These are the first quantitative data of in vivo VEGF expression by PBMC, suggesting a role for them in the maintenance of the vasculature.

Chronic filarial patients exhibit an occult manifestation, Tropical Pulmonary Eosinophilia, (TPE), caused by an exaggerated immune response to shed and circulating filarial antigens, leading to extensive lung damage. We have attempted to examine the disease in vitro using the human epithelial cell line, HEp2. Filarial sheath proteins induce apoptosis in HEp2 cells characterized by chromatin condensation, internucleosomal DNA cleavage, positive staining for TUNEL assay and shows a sub-G1 peak on FACS analysis. In order to understand subcellular events and to analyse the protective role of bcl2, we engineered HEp2 to overexpress Bcl2 protein. HEp2 bcl2 cells do not undergo apoptosis on exposure to filarial sheath protein, indicating that filarial protein-induced apoptosis in epithelial cells proceeds via a pathway, inhibitable by overexpression of bcl 2., (Copyright 1998 Academic Press.)

Site-directed mutagenesis was used to investigate the functions of the traM gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a transfer-derepressed (finO-) variant of plasmid R1. The frequency of conjugative transfer of the traM null mutant derivative of R1-16 was 10(7)-fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this gene in conjugative transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ, traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM gene of plasmid R1 has at least two functions in conjugation: (i) positive control of transfer gene expression; and (ii) a function in a process distinct from gene expression. Since expression of the negatively autoregulated traM gene is itself affected positively by the expression of the transfer operon genes, this gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the genes necessary for horizontal DNA transfer. Based on our studies, we present a novel model for the regulation of the transfer genes of plasmid R1 that might also be applicable to other IncF plasmids.

The classical method of metabolic engineering, identifying a rate-determining step in a pathway and alleviating the bottleneck by enzyme overexpression, has motivated much research but has enjoyed only limited practical success. Intervention of other limiting steps, of counterbalancing regulation, and of unknown coupled pathways often confounds this direct approach. Here the concept of inverse metabolic engineering is codified and its application is illustrated with several examples. Inverse metabolic engineering means the elucidation of a metabolic engineering strategy by: first, identifying, constructing, or calculating a desired phenotype; second, determining the genetic or the particular environmental factors conferring that phenotype; and third, endowing that phenotype on another strain or organism by directed genetic or environmental manipulation. This paradigm has been successfully applied in several contexts, including elimination of growth factor requirements in mammalian cell culture and increasing the energetic efficiency of microaerobic bacterial respiration. (c) 1996 John Wiley & Sons, Inc.

Engineering of the cell cycle can be an effective means for bypassing growth factor requirements of animal cells. Cloned human E2F-1 from Nalm 6 cells was subcloned into pRc/CMV and transfected into Chinese hamster ovary (CHO) cells. Ten stable transfectant clones isolated from cells cultured under neomycin-resistance selection pressure all expressed significantly higher amounts of E2F-1 than control cells as determined by Western analysis. Confocal immunofluorescent microscopy and Southern analysis of several clones also provided evidence for the expression of cloned E2F-1 in these cells. CHO K1:E2F-1 cells are able to proliferate on well-defined serum- and protein-free basal medium and exhibit an S-phase extended by 65% compared to CHO K1 cells mitogenically stimulated by basic fibroblast growth factor (bFGF). Two-dimensional electrophoresis of the intracellular proteins of E2F-1 clones shows an increase in 236 gene products compared to CHO K1 control cells, further verifying a functional regulatory role of cloned E2F-1 in CHO cells. Among these upregulated species is the cell cycle regulatory protein, cyclin A, which has already been shown to be regulated by E2F-1 in human fibroblasts. Overexpression of cloned E2F-1 in CHO cells is a potentially useful new strategy for bypassing serum requirements in mammalian cell culture. Furthermore, such cell cycle control stimulus-protein pattern response data can contribute to a clearer understanding of complex multigene networks involved in mammalian cell cycle regulation.

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. (c) 1995 John Wiley & Sons Inc.

We have cloned and sequenced a cluster of six open reading frames containing gene kdsA from Escherichia coli K-12. The gene encodes 3-deoxy-D-manno-octulosonate 8-phosphate synthetase (KDO-8-phosphate synthetase), which catalyzes formation of 3-deoxy-D-manno-octulosonic acid (KDO), an essential component of enterobacterial lipopolysaccharide. We have also identified two other genes, hemA and prfA, at the beginning of the cluster. Deletion analysis shows that kdsA, the terminal gene of this putative operon, is transcribed from its own promoter located within the cluster rather than from two promoters preceding this group of six open reading frames. Northern (RNA) blot analysis as well as lacZ operon fusion experiments reveal that the expression of gene kdsA occurs maximally in the early log phase and falls to a low level in the late log and stationary phases. Hence, this gene is subjected to growth phase-dependent regulation at the transcriptional level. Similarly, we show that expression of gene kdsB, which codes for the CTP:CMP-3-deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO-synthetase), is also growth regulated. This enzyme catalyzes the activation of KDO via formation of CMP-KDO, which is necessary for the incorporation of KDO into lipid A. We have identified the promoter of gene kdsB, whose expression is growth regulated in the same way as that of kdsA. Despite the fact that transcription of genes kdsA and kdsB is shut off as cells enter stationary phase, KDO-8-phosphate synthetase as well as CMP-KDO-synthetase activities are still present at various levels during stationary-phase growth of an E. coli K-12 culture.

Completing previous studies in patients with sinus bradycardia (Med. Klin. 82 [1987], 647-650) we compared metoprolol with carteolol and pindolol, pindolol with carteolol, no treatment with carteolol (in two groups) in five series of the paired comparisons of Holter-ECG each. With change from metoprolol to carteolol or pindolol (dose ratio 10:1) lowest heart rate on Holter-ECG increased by 28 or 29% without change of exercise heart rate. Direct comparison of pindolol and carteolol revealed a very similar heart rate profile, indicating equipotent beta blockade and ISA. In patients with previous beta blocker induced bradycardia, carteolol did not change a normal resting heart rate off treatment. However, in patients with spontaneous sinus bradycardia carteolol increased lowest heart rate (+14%, due to overriding ISA) and lowered exercise heart rate (-15%, due to overriding beta blockade). A beta blocker induced sinus bradycardia consistently improved with change of treatment to carteolol and pindolol. With caution carteolol and pindolol may also be used despite spontaneous sinus bradycardia.

A computer algorithm for designing sheet lead tissue compensators is described. Corrections are made for scatter within the radiation field as well as the shape of the patient for the mantle fields used in treating Hodgkin's disease. The method was tested experimentally with a phantom and found to be clinically acceptable. The advantages of employing this technique with parallel opposed fields are emphasized.

The surface topography of a patient can be determined by photogrammetry before beginning radiotherapy. The source light of the therapy unit or simulator is used to project a grid pattern onto the patient, and this is then photographed together with control points consisting of miniature light bulbs mounted on a frame suspended from the wedge slot of the therapy machine. When the photograph is projected onto a graphics terminal for data entry into a computer, the three-dimensional topography of the patient's surface can be reconstructed as a two-dimensional matrix of discrete points. A computer algorithm can then design a tissue compensator to fit the individual patient.

Sector real-time ultrasound examinations of the fetal perineal area were done on fetuses ranging in age from 14 weeks to term. An attempt was made to determine fetal sex in 404 consecutive cases. Seventeen cases were lost to follow-up and 20 fetuses under 16 weeks of age were omitted, as only two predictions were made in this group. The fetal sex was predicted in 266 of the remaining 367 fetuses with 93% accuracy. In this series, 194 fetuses were less than 24 weeks of age; within this group, 118 predictions were made with an accuracy rate of 86%.

The mid-plane dose was measured in an anthropomorphic phantom for parallel opposed mantle fields as typically used in the treatment of Hodgkin's Disease. Doses were measured for four cases: no compensator, a compensator designed by a three-dimensional CT based treatment planning algorithm, a compensator designed from a port film, and a compensator designed from surface topography. The results showed all three compensators gave a significant better dose distribution than using no compensator at all. Without a compensator, doses varied from 92 percent to 131 percent, with a standard deviation of 10.9 percent for 65 measured points. The treatment planning algorithm gave the best performance with a standard deviation of 3.2 percent with all points but three within 5 percent out of the 65 points measured, and no points outside of 10 percent. The port film compensator was next best with a standard deviation of 4.4 percent, with 19 points outside of 5 percent, and doses from 88 to 106 percent. The surface topography compensator had a standard deviation of 6.1 percent with 31 points outside of 5% and doses from 89 to 114 percent.

Missing tissue compensators are typically made to correct for the tissue defect separately for each of two parallel opposed radiation fields. Arguments and experimental evidence are provided to show that a more uniform dose distribution is achieved throughout the irradiated volume if the compensator is designed so that each opposed field delivers an equal dose to a surface defining midmass between the opposed body surfaces. An expedient method to approximate this result is to simply divide equally between the two parallel opposed fields the total amount of compensation otherwise required.

Forty-two patients admitted to the Coronary Care Unit with a diagnosis of acute myocardial infarction were studied within 24 hours of the onset of symptoms. In addition to therapy determined by the attending physician, there was a double-blinded administration of either a placebo or methylprednisolone, 30 mg/kg intravenously every 6 hours for four doses. This drug had no beneficial effect on infarct size, dysrhythmias, complications, or left ventricular function 2 weeks after infarction.

The basic concepts of photogrammetry, the science of making geometric measurements from photographs, are reviewed with an emphasis on applications to implant dosimetry. A seed matching algorithm is developed using those concepts.

A computer software package was developed for the planning and execution of brain biopsy and radioactive implant procedures with the BRW Stereotaxic System. With the application of computer graphics and a zero-one integer variable programming algorithm, an implant plan with accompanying isodose distributions and stereotactic coordinates can be easily accomplished at the time of the operation when the computer imaging terminal and a printer/plotter is placed in the operating room.

Previous authors have suggested that high pass spatial filtering of axial transverse and circular tomograms would improve the perceived image. A preliminary study here shows that good signal-to-noise ratio is critical for the success of image processing for the contrasts obtainable in the clinical situation. The successful image processing of a small object on the Philips Polytome is demonstrated, as well as a clinical example. A similar trial with the Toshiba axial transverse tomography machine was not successful due to lower subject contrast with higher kVp techniques. A derivation of the optical transfer function of circular and axial transverse tomograms is given, with emphasis on those features that image processing can improve. To accomplish image processing, two methods employing incoherent light were investigated. Either method may be regarded as an analogue equivalent to the mathematical operations employed by digital computers in the reconstruction of CT images. First, a simple all-purpose image processor is described that makes use of the Herschel effect. Second, a simple film copying technique that simulates a high pass spatial filter is introduced and used to improve tomographic images.

The radiographic diagnosis of rheumatoid arthritis can be suggested long before bone and joint destruction. Soft tissue swelling at the ulnar styloid is classical, but soft tissue swelling also occurs at the PIP and MCP joints. Joint space widening, loss of the lateral fat planes of the wrist, and radial carpal narrowing can all be seen prior to bony change. The earliest bony change is loss of the cortical white line on the radial aspect of the fourth and fifth metacarpal heads.

Beam compensators are optimally designed to give a uniform dose to any plane or midsurface that intersects a single beam, or to give a uniform dose to the volume defined by the intersection of two or more beams. The primary and scatter components are taken into account separately, as well as the patient's shape and internal heterogeneities. The design of the beam compensators is formulated as a linear programming problem and solved with a variation of the Simplex Method. Beam weighting factors are also obtained as part of the solution.