Uses of 3D planning in addition to creating a good treatment: Ongoing studies at MGH/HCL

Summary Ongoing studies and 3D treatment planning and its clinical impact will be described.Invited paper given on the fourth workshop on Heavy Charged Particles in Biology and Medicine GSI, Darmstadt, FRG, September 23–25, 1991     

ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.     

Continuous non-invasive blood pressure monitoring in patients with sleep disorders.

Sleep related breathing disorders are of high prevalence and are often associated with essential hypertension. It is therefore necessary to study blood pressure continuously in all patients with sleep related breathing disorders and arterial hypertension as well as in all patients with essential hypertension and suspected sleep apnoea. To investigate the usefulness of a non-invasive continuous volume-clamp method during sleep we used this technique in parallel with 130 sleep recordings and performed a validation study of the Finapres instrument on a subgroup where continuous invasive blood pressure recordings were available. Absolute pressure values of Finapres are valid when the position and the movement of the sensor were carefully observed and only appropriate segments of the recordings were taken for further evaluation. The high beat to beat resolution of the systolic and diastolic pressure is the main advantage of this non-invasive technique because it reflects rapid blood pressure variations as they occur in sleep related breathing disorders. This could be investigated only invasively until now.     

DNA analysis of malignant effusions. Comparison with cytologic diagnosis and carcinoembryonic antigen content.

Twenty-three consecutive malignant effusions from 19 patients submitted for cytologic examination were analyzed for carcinoembryonic antigen (CEA) content and for DNA analysis by flow cytometry. The study was undertaken to determine if the addition of DNA analysis would improve the sensitivity of cytologic diagnosis and CEA assay. CEA examination was performed on Papanicolaou-stained smears and hematoxylin-and-eosin-stained cell blocks. Final diagnoses were correlated with histologic examination (four patients), clinical and radiologic studies, and follow-up. The malignant effusions in 19 patients were secondary to carcinoma of the breast (5), lung (5), ovary (1), endometrium (1), mucinous carcinoma of the colon (1), unknown primary (1), extraovarian papillary carcinoma (1), mesothelioma (2) and large cell lymphoma (2). The sensitivity of cytologic diagnosis was 100% and specificity 100%. DNA aneuploidy, defined as the presence of two separate peaks in the histogram, was present in 7 of 23 fluids (sensitivity, 30%). Four fluids had insufficient cells for analysis, and one histogram showed debris (following chemotherapy). DNA aneuploidy was detected in effusions secondary to carcinoma of the breast (4), lung (1) and lymphoma (2). Using 5 ng/mL as the cutoff, the sensitivity of CEA was 68%. DNA analysis of cells in malignant effusions is less sensitive than cytologic diagnosis, and CEA assay and is not recommended for routine use in the diagnosis of malignant effusions.     

Biologic markers in ethylene oxide-exposed workers and controls

Ethylene oxide (EtO) is an alkylating agent and a model direct-acting mutagen and carcinogen. This study has evaluated a panel of biologic markers including EtO-hemoglobin adducts (EtO-Hb), sister-chromatid exchanges (SCEs), micronuclei, chromosomal aberrations (CAs), DNA single-strand breaks (SSB) and an index of DNA repair (ratio of UDS to NA-AAF-DNA binding) in the peripheral blood cells of 34 workers at a sterilization unit of a large university hospital and 23 controls working in the university library. Comprehensive environmental histories were obtained on each subject including detailed occupational and smoking histories. Industrial hygiene data obtained prior to the study and personal monitoring during the 8 years preceding the study showed that workers were subject to low-level exposure near or below the current Occupational Safety and Health Administration (OSHA) standard of 1 ppm (TWA). Personal monitoring data obtained during 2 weeks prior to blood sampling were uniformly less than 0.3 ppm (TWA). After adjusting for smoking, EtO workplace exposure was significantly (p less than 0.001) associated with EtO-Hb (a carcinogen-protein adduct) and 2 measures of SCEs [the average number of SCEs/cell (SCE50) and the number of high frequency cells (SCEHFC)]. There was an apparent suppression of DNA repair capacity in EtO-exposed individuals as measured by the DNA repair index; i.e., the ratio of unscheduled DNA synthesis (UDS) and NA-AAF-DNA binding (p less than 0.01). No association of DNA repair index with smoking was found. Another important finding of this study is the highly significant correlation between EtO-Hb adduct levels and SCEHFC (p less than 0.01) and SCEs (p less than 0.02) which provides evidence of a direct link between a marker of biologically effective dose and markers of genotoxic response. In contrast, micronuclei, CAs and SSBs were not significantly elevated in the workers. The activity of the u-isoenzyme of glutathione-S-transferase (GT) was measured as a possible genetic marker of susceptibility and a modulator of biomarker formation. However, possibly because of confounding by age, no significant relationships were found between GT and any of the exposure-related markers by ANOVA or among other independent variables by regression. This study demonstrates significant effects of low-level EtO exposure, independent of smoking history, near or below 1 ppm on multiple biomarkers and suggests that the current OSHA standard may not be adequately protective. Previously described effects of smoking on EtO-Hb adducts, SCEs and SCEHFC were also seen in this study.(ABSTRACT TRUNCATED AT 400 WORDS)     

Investigation of the neutral filter elution technique I. Effects of pore density and pore diameter on elution rate at pH 9.6

To understand better the biophysical mechanism of neutral filter elution (pH 9.6), we eluted genomes of known size and shape: coliphage T4c (Mr 1.15 x 10(8), E. coli (Mr 2.7 x 10(9)), and Chinese hamster lung fibroblasts (V79, Mr 2-4 x 10(10)). DNA eluted through 15% sucrose atop the filter in a biphasic pattern. The elution rate of the initial component correlated (r greater than 0.97) exponentially with 1/Mr for monodisperse samples of DNA eluted through pore sizes 0.1-3.0 microns. Using this relationship between elution rate and Mr, we estimated Mn of polydisperse, X-irradiated (253 Gy) samples of DNA from E. coli or V79 cells to be 3.15 +/- 1.46 and 1.42 +/- 0.33, respectively, compared to expected values of 2.93 and 3.52 (10(8) Da). The best predictor of elution rate for DNA from T4c and intact and X-irradiated V79 cells was pore density, and pore diameter for DNA from X-irradiated E. coli. The rate of elution of DNA from unirradiated E. coli was unrelated to pore density or diameter. While the mechanism of neutral filter elution remains unknown, its use for linear DNAs with Mn ca. 10(8) Da appears to be valid quantitatively.     

Cellulose biosynthesis and function in bacteria.

The current model of cellulose biogenesis in plants, as well as bacteria, holds that the membranous cellulose synthase complex polymerizes glucose moieties from UDP-Glc into beta-1,4-glucan chains which give rise to rigid crystalline fibrils upon extrusion at the outer surface of the cell. The distinct arrangement and degree of association of the polymerizing enzyme units presumably govern extracellular chain assembly in addition to the pattern and width of cellulose fibril deposition. Most evident for Acetobacter xylinum, polymerization and assembly appear to be tightly coupled. To date, only bacteria have been effectively studied at the biochemical and genetic levels. In A. xylinum, the cellulose synthase, composed of at least two structurally similar but functionally distinct subunits, is subject to a multicomponent regulatory system. Regulation is based on the novel nucleotide cyclic diguanylic acid, a positive allosteric effector, and the regulatory enzymes maintaining its intracellular turnover: diguanylate cyclase and Ca2(+)-sensitive bis-(3',5')-cyclic diguanylic acid (c-di-GMP) phosphodiesterase. Four genes have been isolated from A. xylinum which constitute the operon for cellulose synthesis. The second gene encodes the catalytic subunit of cellulose synthase; the functions of the other three gene products are still unknown. Exclusively an extracellular product, bacterial cellulose appears to fulfill diverse biological roles within the natural habitat, conferring mechanical, chemical, and physiological protection in A. xylinum and Sarcina ventriculi or facilitating cell adhesion during symbiotic or infectious interactions in Rhizobium and Agrobacterium species. A. xylinum is proving to be most amenable for industrial purposes, allowing the unique features of bacterial cellulose to be exploited for novel product applications.     

Cryptic dioecy in flowering plants

In some dioecious plant species, mates and/or females have large and presumably costly opposite-sex structures that are sterile. This is termed 'cryptic dioecy'. Several new cases of cryptic dioecy have recently been studied. They may give information about the minimal requirements for the evolution of separate sexes from hermaphroditism, because the most important differences contributing to the initial advantage of the breeding system have not been obscured by further developments. Reviewed in this light, cryptic dioecy can provide evidence on the role of reallocation of reproductive resources in the evolution of dioecy.     

Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins.

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.     

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor

Summary Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cell, the plasmalemmal undercoat was labeled. Antiactin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca²⁺-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.