A bound iron porphyrin is redox active in hybrid bacterial reaction centers modified to possess a four-helix bundle domain.

In this paper we report the design of hybrid reaction centers with a novel redox-active cofactor. Reaction centers perform the primary photochemistry of photosynthesis, namely the light-induced transfer of an electron from the bacteriochlorophyll dimer to a series of electron acceptors. Hybrid complexes were created by the fusion of an artificial four-helix bundle to the M-subunit of the reaction center. Despite the large modification, optical spectra show that the purified hybrid reaction centers assemble as active complexes that retain the characteristic cofactor absorption peaks and are capable of light-induced charge separation. The four-helix bundle could bind iron-protoporphyrin in either a reduced and oxidized state. After binding iron-protoporphyrin to the hybrid reaction centers, light excitation results in a new derivative signal with a maximum at 402 nm and minimum at 429 nm. This signal increases in amplitude with longer light durations and persists in the dark. No signal is observed when iron-protoporphyrin is added to reaction centers without the four-helix bundle domain or when a redox-inactive zinc-protoporphyrin is bound. The results are consistent with the signal arising from a new redox reaction, electron transfer from the iron-protoporphyrin to the oxidized bacteriochlorophyll dimer. These outcomes demonstrate the feasibility of binding porphyrins to the hybrid reaction centers to gain new light-driven functions.

Heat stress response in slick vs normal-haired Criollo Limonero heifers in a tropical environment.

To assess the effect of hair type on the heat stress response, 20 Criollo Limonero heifers with slick (n = 11) or normal hair (n = 9) were studied. Under a high temperature-humidity index (THI) environment, heat stress response was assessed through physiological variables that included respiration rate (RR), heart rate (HR), ruminal frequency (RMF), rectal temperature (RT), saliva pH (SPH), and lymphocyte count (LC) in the morning (5:00 AM, 27.4 °C, 64% relative humidity, THI = 77) and afternoon (1:00 PM, 34.5 °C, 70% relative humidity, THI = 88). A case-control study using a split plot design was used. Data were analyzed using ANOVA (PROC MIXED SAS 2010) and a statistical model comprising the fixed effects of hair length, sampling hour, interaction of hair length by sampling hour, and the random effect of animal nested within hair type on physiological variables associated with heat stress response. Sampling hour influenced (P < 0.0001) RR, RT, and (P < 0.003) SPH. Hair length influenced RR (P < 0.01) and RT (P < 0.04) and tended to influence LC (P < 0.07). The interaction of sampling hour by hair influenced RR (P < 0.04), RT (P < 0.0002), and both SPH and LC (P < 0.05). During afternoon hours, slick-haired heifers had lower values for RR (81 ± 4.2 vs 102 ± 4.7 bpm; P < 0.01), RT (39.5 ± 0.1 vs 40.3 ± 0.1 C°; P < 0.002), and LC (60 ± 3.2 vs 72.3 ± 3.6; P < 0.09) than normal-haired heifers. In normal-haired heifers, SPH increased during afternoon compared to morning-hours (8.66 ± 0.1 vs 9.11 ± 0.1; P < 0.04). It was concluded that slick-coated heifers exhibited an enhanced capability to cope with heat stress compared to normal-haired heifers likely due to an enhanced capacity for heat dissipation.

Comparison of preoperative versus postoperative transversus abdominis plane and rectus sheath block in patients undergoing minimally invasive colorectal surgery.

AIM: Ultrasound-guided transversus abdominis plane and rectus sheath block (TAPRSB) decreases pain scores and narcotic use postoperatively after colorectal surgery (CRS). It is unclear if the effectiveness of TAPRSB varies according to whether it is performed preoperatively or postoperatively. Our aim was to investigate this. METHOD: We compared patients who underwent preoperative TAPRSB or postoperative TAPRSB during minimally invasive CRS. Primary end-points were pain scores and oral morphine milligram equivalent (MME) use postoperatively. Secondary end-points included perioperative factors affecting pain scores and postoperative MME. Summary statistics and univariate analysis by nonparametric tests were utilized. The mixed-effect model was applied to model the repeatedly measured pain score. RESULTS: From April 2015 until May 2018 168 patients received TAPRSB before (115) or after (53) minimally invasive CRS. The cohort included 79 (47.0%) women, and had an average age of 59.11 (±12.32) years and mean body mass index of 28.32 (±5.82) kg/m2 . Indication for surgery was cancer in 66 (39.3%), polyp in 43 (25.6%) and diverticulitis in 43 (25.6%). Right colectomy was performed in 61 (36.3%), low anterior resection in 46 (27.4%) and sigmoid colectomy in 40 (23.8%) patients. The demographics of the groups were similar. Postoperative TAPRSB was only associated with lower pain scores at 12 h postoperatively. As secondary outcomes, average pain scores and MME were lower in patients who were older, had right colectomy or intracorporeal anastomosis. CONCLUSIONS: Postoperative TAPRSB resulted in lower pain scores than preoperative TAPRSB 12 h after minimally invasive CRS, but otherwise no differences were seen in pain scores or MME use.

Influence of protein interactions on oxidation/reduction midpoint potentials of cofactors in natural and de novo metalloproteins.

As discussed throughout this special issue, oxidation and reduction reactions play critical roles in the function of many organisms. In photosynthetic organisms, the conversion of light energy drives oxidation and reduction reactions through the transfer of electrons and protons in order to create energy-rich compounds. These reactions occur in proteins such as cytochrome c, a heme-containing water-soluble protein, the bacteriochlorophyll-containing reaction center, and photosystem II where water is oxidized at the manganese cluster. A critical measure describing the ability of cofactors in proteins to participate in such reactions is the oxidation/reduction midpoint potential. In this review, the basic concepts of oxidation/reduction reactions are reviewed with a summary of the experimental approaches used to measure the midpoint potential of metal cofactors. For cofactors in proteins, the midpoint potential not only depends upon the specific chemical characteristics of cofactors but also upon interactions with the surrounding protein, such as the nature of the coordinating ligands and protein environment. These interactions can be tailored to optimize an oxidation/reduction reaction carried out by the protein. As examples, the midpoint potentials of hemes in cytochromes, bacteriochlorophylls in reaction centers, and the manganese cluster of photosystem II are discussed with an emphasis on the influence that protein interactions have on these potentials. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

The three-dimensional structures of bacterial reaction centers.

This review presents a broad overview of the research that enabled the structure determination of the bacterial reaction centers from Blastochloris viridis and Rhodobacter sphaeroides, with a focus on the contributions from Duysens, Clayton, and Feher. Early experiments performed in the laboratory of Duysens and others demonstrated the utility of spectroscopic techniques and the presence of photosynthetic complexes in both oxygenic and anoxygenic photosynthesis. The laboratories of Clayton and Feher led efforts to isolate and characterize the bacterial reaction centers. The availability of well-characterized preparations of pure and stable reaction centers allowed the crystallization and subsequent determination of the structures using X-ray diffraction. The three-dimensional structures of reaction centers revealed an overall arrangement of two symmetrical branches of cofactors surrounded by transmembrane helices from the L and M subunits, which also are related by the same twofold symmetry axis. The structure has served as a framework to address several issues concerning bacterial photosynthesis, including the directionality of electron transfer, the properties of the reaction center-cytochrome c 2 complex, and the coupling of proton and electron transfer. Together, these research efforts laid the foundation for ongoing efforts to address an outstanding question in oxygenic photosynthesis, namely the molecular mechanism of water oxidation.

Electronic structure of the Mn-cofactor of modified bacterial reaction centers measured by electron paramagnetic resonance and electron spin echo envelope modulation spectroscopies.

The electronic structure of a Mn(II) ion bound to highly oxidizing reaction centers of Rhodobacter sphaeroides was studied in a mutant modified to possess a metal binding site at a location comparable to the Mn4Ca cluster of photosystem II. The Mn-binding site of the previously described mutant, M2, contains three carboxylates and one His at the binding site (Thielges et al., Biochemistry 44:389-7394, 2005). The redox-active Mn-cofactor was characterized using electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopies. In the light without bound metal, the Mn-binding mutants showed an EPR spectrum characteristic of the oxidized bacteriochlorophyll dimer and reduced quinone whose intensity was significantly reduced due to the diminished quantum yield of charge separation in the mutant compared to wild type. In the presence of the metal and in the dark, the EPR spectrum measured at the X-band frequency of 9.4 GHz showed a distinctive spin 5/2 Mn(II) signal consisting of 16 lines associated with both allowed and forbidden transitions. Upon illumination, the amplitude of the spectrum is decreased by over 80 % due to oxidation of the metal upon electron transfer to the oxidized bacteriochlorophyll dimer. The EPR spectrum of the Mn-cofactor was also measured at the Q-band frequency of 34 GHz and was better resolved as the signal was composed of the six allowed electronic transitions with only minor contributions from other transitions. A fit of the Q-band EPR spectrum shows that the Mn-cofactor is a high spin Mn(II) species (S = 5/2) that is six-coordinated with an isotropic g-value of 2.0006, a weak zero-field splitting and E/D ratio of approximately 1/3. The ESEEM experiments showed the presence of one (14)N coordinating the Mn-cofactor. The nitrogen atom is assigned to a His by comparing our ESEEM results to those previously reported for Mn(II) ions bound to other proteins and on the basis of the X-ray structure of the M2 mutant that shows the presence of only one His, residue M193, that can coordinate the Mn-cofactor. Together, the data allow the electronic structure and coordination environment of the designed Mn-cofactor in the modified reaction centers to be characterized in detail and compared to those observed in other proteins with Mn-cofactors.

First Report of Bacterial Spot of Tomato Caused by Xanthomonas gardneri in Pennsylvania.

Recent investigation of bacteria isolated from samples submitted to the Plant Disease Diagnostic Laboratory, Pennsylvania Department of Agriculture indicated that in 1995, Xanthomonas gardneri (ex Sutic 1957) (2) caused a leaf spot on tomato plants (Lycopersicon esculentum Mill.). In 1995, we examined 185 tomato and 36 pepper samples (13 field, 2 garden center, 38 greenhouse, 4 residence, 16 field-grown transplant, and 148 greenhouse-grown transplant samples). A processing company representative collected samples showing symptoms of bacterial spot of tomato on a hybrid, whole pack processing tomato, from a 16-ha field in Northumberland County, PA exhibiting almost 50% crop infection. Symptoms consisted of circular- to irregularly shaped, dark brown spots, <5 mm in diameter, and frequently with chlorotic haloes on leaves and stems. The center of a spot may be raised and scabby. Several spots on a single leaflet may coalesce and a portion or the entire leaflet may turn yellow or die. These symptoms were indistinguishable from those of bacterial spot caused by X. euvesicatoria, X. vesicatoria, and X. perforans. Bacterial streaming from lesions was evident under dark-field microscopy. Aerobic, gram-negative, yellow-pigmented, mucoid bacteria were isolated from the leaf spots and purified and stored in nutrient broth with 10% glycerol at -80°C. The 16S rRNA gene from a strain (PDA80951-95) typical of the cultures from these samples was sequenced (GenBank Accession No. GU573763). A BlastN search of GenBank revealed 100% nucleotide identity with the type strain of X. gardneri (XCGA2; No. AF123093). This strain also exhibited repetitive sequence-based (rep)-PCR profiles (4) identical to profiles of X. gardneri type strain XCGA2 DNA and produced a ~425-bp PCR product with BSX primers, a genetic marker indicative of X. gardneri (1). The strain was not amylolytic or pectolytic (2) and failed to utilize maltose, gentiobiose, and melezitose (3). For pathogenicity tests, inoculum was grown in nutrient broth with shaking for 24 h at 28°C. Inoculum was centrifuged, resuspended in sterile tap water, and adjusted to 2.5 × 108 CFU/ml. Lower leaf surfaces of tomato (cvs. Bonnie Best and Walter) and pepper (cvs. California Wonder and Early Niagara) plants were gently rubbed with sterile cheesecloth that was moistened with the inoculum. Strain PDA80951-95 caused leaf spots, with chlorotic haloes and occasional coalescence on both tomato and pepper, within 2 weeks at 15 s of mist per 20 min at 20 to 35°C in a secured greenhouse chamber. X. gardneri was only reisolated from symptomatic plants and its identity was confirmed by rep-PCR and absence of amylolytic and pectolytic activities. Negative controls consisting of X. campestris pv. campestris and sterile tap water did not show symptoms. A known type strain of X. gardneri was not included as a positive control for pathogenicity studies because this species is not known to occur in the United States (2). To our knowledge, this is the first report of bacterial spot on tomato plants caused by X. gardneri in Pennsylvania and the United States. Since the first occurrence in 1995, bacterial spot caused by X. gardneri reoccurred in Pennsylvania tomato fields in 2001 and consecutively from 2003 to 2009. Reference: (1) D. A. Cuppels et al. Plant Dis. 90:451, 2006. (2) J. B. Jones et al. Syst. Appl. Microbiol. 27:755, 2004. (3) A. M. Quezado-Duval et al. Plant Dis. 88:15, 2004. (4) D. J. Versalovic et al. Methods Mol. Cell Biol. 5:25, 1994.

Extracorporeal membrane oxygenation in peripartum cardiomyopathy: A review of the ELSO Registry.

AIMS: Data on the use of extracorporeal membrane oxygenation (ECMO) for cardiogenic shock in peripartum cardiomyopathy (PPCM) is limited. We queried the Extracorporeal Life Support Organization (ELSO) Registry for PPCM patients treated with ECMO in order to characterize demographic and clinical features, complications, survival, and variables associated with mortality. METHODS AND RESULTS: This was a retrospective review of patients voluntarily entered into the ELSO Registry. De-identified data was collected on patients with a diagnosis of PPCM based on ICD-9/ICD-10 coding who received ECMO between 2007 and 2019. Collected data included demographics, ECMO mode, cannulation strategies, pre-ECMO ventilator, biochemical, and hemodynamic parameters, run duration, complications, and survival to wean off ECMO and hospital discharge. Our primary outcome measure was survival to discharge. In the final analysis, 88 veno-arterial (VA) ECMO patients were included. Overall, 72% of patients were weaned off ECMO, including 10% who were weaned to ventricular assist device or heart transplantation, and 64% survived to hospital discharge. Extracorporeal cardiopulmonary resuscitation (ECPR) was performed in 11% of patients with 60% survival. Factors associated with decreased survival included neurologic complications (p = 0.03), specifically central nervous system hemorrhage (p = 0.01). CONCLUSION: Our review is the largest to date of PPCM patients supported with VA ECMO for cardiogenic shock. ECMO and ECPR are valuable forms of short-term mechanical circulatory support with acceptable mortality profiles for PPCM patients who remain refractory to aggressive medical management. Complications should be meticulously avoided, especially neurologic complications.

Actin mutations in dilated cardiomyopathy, a heritable form of heart failure.

To test the hypothesis that actin dysfunction leads to heart failure, patients with hereditary idiopathic dilated cardiomyopathy (IDC) were examined for mutations in the cardiac actin gene (ACTC). Missense mutations in ACTC that cosegregate with IDC were identified in two unrelated families. Both mutations affect universally conserved amino acids in domains of actin that attach to Z bands and intercalated discs. Coupled with previous data showing that dystrophin mutations also cause dilated cardiomyopathy, these results raise the possibility that defective transmission of force in cardiac myocytes is a mechanism underlying heart failure.

Cardiac troponin T mutation in familial cardiomyopathy with variable remodeling and restrictive physiology.

We identified a unique family with autosomal dominant heart disease variably expressed as restrictive cardiomyopathy (RCM), hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM), and sought to identify the molecular defect that triggered divergent remodeling pathways. Polymorphic DNA markers for nine sarcomeric genes for DCM and/or HCM were tested for segregation with disease. Linkage to eight genes was excluded, but a cardiac troponin T (TNNT2) marker cosegregated with the disease phenotype. Sequencing of TNNT2 identified a heterozygous missense mutation resulting in an I79N substitution, inherited by all nine affected family members but by none of the six unaffected relatives. Mutation carriers were diagnosed with RCM (n = 2), non-obstructive HCM (n = 3), DCM (n = 2), mixed cardiomyopathy (n = 1), and mild concentric left ventricular hypertrophy (n = 1). Endomyocardial biopsy in the proband revealed non-specific fibrosis, myocyte hypertrophy, and no myofibrillar disarray. Restrictive Doppler filling patterns, atrial enlargement, and pulmonary hypertension were observed among family members regardless of cardiomyopathy subtype. Mutation of a sarcomeric protein gene can cause RCM, HCM, and DCM within the same family, underscoring the necessity of comprehensive morphological and physiological cardiac assessment in familial cardiomyopathy screening.

Differences in thermoregulatory ability between slick-haired and wild-type lactating Holstein cows in response to acute heat stress.

Animals inheriting the slick hair gene have a short, sleek, and sometimes glossy coat. The objective of the present study was to determine whether slick-haired Holstein cows regulate body temperature more effectively than wild-type Holstein cows when exposed to an acute increase in heat stress. Lactating slick cows (n = 10) and wild-type cows (n = 10) were placed for 10 h in an indoor environment with a solid roof, fans, and evaporative cooling or in an outdoor environment with shade cloth and no fans or evaporative cooling. Cows were exposed to both environments in a single reversal design. Vaginal temperature, respiration rate, surface temperature, and sweating rate were measured at 1200, 1500, 1800, and 2100 h (replicate 1) or 1200 and 1500 h (replicate 2), and blood samples were collected for plasma cortisol concentration. Cows in the outdoor environment had higher vaginal and surface temperatures, respiration rates, and sweating rates than cows in the indoor environment. In both environments, slick-haired cows had lower vaginal temperatures (indoor: 39.0 vs. 39.4 degrees C; outdoor 39.6 vs. 40.2 degrees C; SEM = 0.07) and respiration rate (indoor: 67 vs. 79 breaths/ min; outdoor 97 vs. 107 breaths/min; SEM = 5.5) than wild-type cows and greater sweating rates in unclipped areas of skin (indoor: 57 vs. 43 g x h(-1)/m(2); outdoor 82 vs. 61 g x h(-1)/m(2); SEM = 8). Clipping the hair at the site of sweating measurement eliminated the difference between slick-haired and wild-type cows. Results indicate that slick-haired Holstein cows can regulate body temperature more effectively than wild-type cows during heat stress. One reason slick-haired animals are better able to regulate body temperature is increased sweating rate.

Exercise on-transition uncoupling of ventilatory, gas exchange and cardiac hemodynamic kinetics accompany pulmonary oxygen stores depletion to impact exercise intolerance in human heart failure.

AIM: In contrast to knowledge that heart failure (HF) patients demonstrate peak exercise uncoupling across ventilation, gas exchange and cardiac haemodynamics, whether this dyssynchrony follows that at the exercise on-transition is unclear. This study tested whether exercise on-transition temporal lag for ventilation relative to gas exchange and oxygen pulse (O2 pulse) couples with effects from abnormal pulmonary gaseous oxygen store (O2store ) contributions to V˙O2 to interdependently precipitate persistently elevated ventilatory demand and low oxidative metabolic capacity in HF. METHODS: Beat-to-beat HR and breath-to-breath ventilation and gas exchange were continuously acquired in HF (N = 9, ejection fraction = 30 ± 9%) and matched controls (N = 10) during square-wave ergometry at 60% V˙O2peak (46 ± 14 vs 125 ± 54-W, P < .001). Temporal responses across V˙E , V˙O2 and O2 pulse were assessed for the exercise on-transition using single exponential model Phase II on-kinetic time constants (τ = time to reach 63% steady-state rise). Breath-to-breath gas fractions and respiratory flows were used to determine O2stores . RESULTS: HF vs controls: τ for V˙E (137 ± 93 vs 74 ± 40-seconds, P = .03), V˙O2 (60 ± 40 vs 23 ± 5-seconds, P = .03) and O2 pulse (28 ± 18 vs 23 ± 15-seconds, P = .59). Within HF, τ for V˙E differed from O2 pulse (P < .02), but not V˙O2 . Exercise V˙E rise (workload indexed) differed in HF vs controls (545 ± 139 vs 309 ± 88-mL min-1 W-1 , P < .001). Exercise on-transition O2store depletion in HF exceeded controls, generally persisting to end-exercise. CONCLUSION: These data suggest HF demonstrated exercise on-transition O2store depletion (high O2store contribution to V˙O2 ) coupled with dyssynchronous V˙E , V˙O2 and O2 pulse kinetics-not attributable to prolonged cardiac haemodynamics. Persistent high ventilatory demand and low oxidative metabolic capacity in HF may be precipitated by physiological uncoupling occurring within the exercise on-transition.

Aminoglycoside-induced translational read-through in disease: overcoming nonsense mutations by pharmacogenetic therapy.

A third of inherited diseases result from premature termination codon mutations. Aminoglycosides have emerged as vanguard pharmacogenetic agents in treating human genetic disorders due to their unique ability to suppress gene translation termination induced by nonsense mutations. In preclinical and pilot clinical studies, this therapeutic approach shows promise in phenotype correction by promoting otherwise defective protein synthesis. The challenge ahead is to maximize efficacy while preventing interaction with normal protein production and function.

A common polymorphism in SCN5A is associated with lone atrial fibrillation.

The cardiac sodium channel (SCN5A) is a target for the treatment of arrhythmias. We hypothesized that vulnerability to atrial fibrillation (AF) could be caused by genetic variation in SCN5A. We recruited 157 patients with early-onset AF who lacked traditional risk factors, and 314 matched controls. SCN5A was subject to targeted genotyping of a common loss-of-function H558R polymorphism and comprehensive mutation scanning. Genotype frequencies in the AF cohort vs controls were as follows: HH, 50 vs 63%; HR, 40 vs 33%; and RR, 10 vs 4% (P=0.008). Additional coding sequence mutations were ruled out. The R558 allele was more common in patients than in controls (30 vs 21%, P=0.002), conferring an odds ratios for AF of 1.6 (95% confidence interval 1.2-2.2). The SCN5A R558 allele, present in one-third of the population, thus constitutes a risk factor for lone AF and may increase susceptibility to sodium channel blocker-induced proarrhythmia.

Mapping a cardiomyopathy locus to chromosome 3p22-p25.

Dilated cardiomyopathy (DCM) is a common disorder characterized by cardiac dilation and reduced systolic function. To identify a cardiomyopathy gene, we studied a family with DCM associated with sinus node dysfunction, supraventricular tachyarrhythmias, conduction delay, and stroke. A general linkage approach was used to localize the disease gene in this family. Linkage to D3S2303 was identified with a two-point lod score of 6.09 at a recombination fraction of 0.00. Haplotype analyses mapped this locus to a 30 cM region of chromosome 3p22-p25, excluding candidate genes encoding a G-protein (GNAI2), calcium channel (CACNL1A2), sodium channel (SCN5A), and inositol triphosphate receptor (ITPR1). These data indicate that a gene causing DCM associated with rhythm and conduction abnormalities is located on chromosome 3p, and represent the first step toward disease gene identification.

Vascular permeability factor gene expression in normal and neoplastic human ovaries.

Epithelial ovarian cancer is an aggressive malignancy with a generally poor outcome. To improve survival, novel therapeutic strategies for this disease are needed and require elucidation of the biological events that underlie transformation and tumor growth. Vascular permeability factor (VPF), also known as vascular endothelial growth factor, is a homodimeric glycoprotein that acts on vascular endothelium as a potent permeability-inducing agent and mitogen. The present study demonstrates for the first time the constitutive gene expression of VPF in normal and neoplastic human ovaries. Abundant levels of VPF have been identified by an immunoassay in the ascites of patients with epithelial ovarian cancer (K-T. Yeo et al., Cancer Res., 53: 2912-2918, 1993). We have identified the malignant epithelium as one source of VPF in the ascites. Reverse transcription-polymerase chain reaction has demonstrated the expression of the two secreted isoforms, VPF121 and VPF165, in normal and neoplastic ovaries. Western blotting and an endothelial cell proliferation assay confirmed secretion of a biologically active product. VPF may be an important mediator of ascites formation and tumor metastasis observed in neoplastic conditions of the ovary.

Vascular endothelial growth factor-toxin conjugate specifically inhibits KDR/flk-1-positive endothelial cell proliferation in vitro and angiogenesis in vivo.

Inhibition of tumor neovascularization has profound effects on the growth of solid tumors. An endothelial cell-specific cytotoxic conjugate was prepared by chemically linking recombinant vascular endothelial growth factor (VEGF165) and a truncated diphtheria toxin molecule (DT385). The treatment of subconfluent cultures of human umbilical vein endothelial cells and human microvascular endothelial cells with the VEGF165-DT385 conjugate resulted in a selective, dose-dependent inhibition of growth. Parallel experiments with either the free toxin or a mixture of VEGF and the toxin polypeptide did not affect proliferation (DNA synthesis) of these cells. The selective cytotoxicity correlated with the appropriate receptor expression (KDR/flk-1 positive) on the target cells. VEGF-toxin conjugate inhibited the growth of a murine hemangioma-derived endothelial cell line (Py-4-1), which was positive for flk-1 expression. Under similar conditions, the conjugate did not affect the proliferation of a receptor-negative ovarian cancer cell line in vitro. In an in vivo model of angiogenesis, the VEGF165-DT385 conjugate blocked basic fibroblast growth factor-induced neovascularization of the chick chorioallantoic membrane. These studies demonstrate the successful targeting of a cytotoxic polypeptide to proliferating vascular endothelial cells (normal and tumorigenic) and the potential utility of such conjugates in blocking tumor neovascularization.

Multipoint imprinting analysis indicates a common precursor cell for gonadal and nongonadal pediatric germ cell tumors.

Pediatric germ cell tumors (GCTs) commonly arise at extragonadal sites. It has been proposed that nongonadal GCTs arise from ectopic primordial germ cells that have aberrantly migrated during embryogenesis. During a time between their migration and development to mature gametes, primordial germ cells are characterized by their lack of imprinting, which can be assessed by the evaluation of allelic gene expression and DNA methylation in differentially methylated control regions. To elucidate the cellular origin of nongonadal GCTs, we evaluated the imprinting status of 21 gonadal and 21 nongonadal pediatric GCTs. Allele-specific H19 and IGF-2 expression was assessed with reverse transcription-PCR followed by digestion at polymorphic restriction sites. DNA methylation was evaluated after bisulfite modification, PCR amplification, and restriction digestion at a consistently methylated CpG dinucleotide within the 5' flanking region of the SNRPN gene. These results were compared with genetic gains and losses determined by comparative genomic hybridization. Seven of 15 informative tumors showed biallelic H19 expression, and 8 of 17 informative tumors showed biallelic IGF-2 expression. The frequency of biallelic gene expression was comparable in gonadal and nongonadal GCTs. Sixteen of 19 gonadal GCTs and 17 of 21 nongonadal GCTs showed absence of methylation of SNRPN consistent with loss of imprinting. One testicular GCT and three nongonadal GCTs showed a somatic methylation pattern. Two ovarian teratomas and one mediastinal teratoma showed only methylated SNRPN, consistent with entry into meiosis. Twenty-one of 22 non-GCT control samples showed a somatic methylation pattern. Gonadal and nongonadal germ cell tumors are derived from primordial germ cells that have consistently lost the imprinting of SNRPN and partly lost imprinting of H19 and IGF-2. Because the imprinting pattern of the latter genes differs from that found in testicular GCTs of adult patients, our data suggest that pediatric GCTs arise from a different stage of germ cell development.