Geographical differences in cancer treatment and survival for patients with oesophageal and gastro-oesophageal junctional cancers.

BACKGROUND: Only around one-quarter of patients with cancer of the oesophagus and the gastro-oesophageal junction (GOJ) undergo surgical resection. This population-based study investigated the rates of treatment with curative intent and resection, and their association with survival. METHODS: Patients diagnosed with oesophageal and GOJ cancer between 2006 and 2015 in Sweden were identified from the National Register for Oesophageal and Gastric Cancer (NREV). The NREV was cross-linked with several national registries to obtain information on additional exposures. The annual proportion of patients undergoing treatment with curative intent and surgical resection in each county was calculated, and the counties divided into groups with low, intermediate and high rates. Treatment with curative intent was defined as definitive chemoradiation therapy or surgery, with or without neoadjuvant oncological treatment. Overall survival was analysed using a multilevel model based on county of residence at the time of diagnosis. RESULTS: Some 5959 patients were included, of whom 1503 (25·2 per cent) underwent surgery. Median overall survival after diagnosis was 7·7, 8·8 and 11·1 months respectively in counties with low, intermediate and high rates of treatment with curative intent. Corresponding survival times for the surgical resection groups were 7·4, 9·3 and 11·0 months. In the multivariable analysis, a higher rate of treatment with curative intent (time ratio 1·17, 95 per cent c.i. 1·05 to 1·30; P < 0·001) and a higher resection rate (time ratio 1·24, 1·12 to 1·37; P < 0·001) were associated with improved survival after adjustment for relevant confounders. CONCLUSION: Patients diagnosed in counties with higher rates of treatment with curative intent and higher rates of surgery had better survival.

ANTECEDENTES: En los pacientes con cáncer en el esófago y de la unión gastroesofágica (gastroesophageal junction, GOJ), solamente en una cuarta parte se practica una resección quirúrgica. Este estudio de base poblacional analizó las tasas de tratamiento con intención curativa y de resección y su asociación con la supervivencia. MÉTODOS: A partir del Registro Nacional Sueco de Cáncer de Esófago y Estómago (National Register for Oesophageal and Gastric Cancer, NREV), se identificaron los pacientes diagnosticados de cáncer de esófago y de la GOJ entre 2006-2015. El NREV se cruzó con otros registros nacionales para obtener información adicional. Se calculó la proporción anual de pacientes tratados con intención curativa o mediante resección quirúrgica en cada una de las áreas territoriales de los condados y se categorizaron en baja, intermedia y alta. El tratamiento con intención curativa se definió como la quimiorradioterapia definitiva (definitive chemoradiation therapy, dCRT) o la cirugía, con o sin tratamiento oncológico neoadyuvante. Se analizó la supervivencia global con un modelo multinivel basado en el condado de residencia en el momento del diagnóstico. RESULTADOS: Se incluyeron 5.959 pacientes, de los que 1.503 (25,2%) fueron tratados quirúrgicamente. La mediana de supervivencia global después del tratamiento con intención curativa fue de 7,7, 8,8 y 11,1 meses para los condados de volumen bajo, intermedio y alto. Para el grupo de cirugía fue de 7,4, 9,3 y 11,0 meses, respectivamente. En el análisis multivariable, una mayor tasa de tratamiento con intención curativa y una mayor tasa de resección se asociaron con una mejor supervivencia (tiempo ganado 1,17; i.c. del 95% 1,05-1,30, P < 0,001 y tiempo ganado 1,24; i.c. del 95% 1,12-1,37, P < 0,001) después del ajuste para los factores principales de confusión. CONCLUSIÓN: Los pacientes diagnosticados en condados con tasas altas de tratamiento con intención curativa y de cirugía tuvieron una mejor supervivencia.

Characterization and topical delivery of phenylethyl resorcinol.

OBJECTIVE: Phenylethyl resorcinol (PR) has been used widely in the personal care industry as a novel skin lightening ingredient. Surprisingly, there is only limited information describing the physicochemical properties of this active. Therefore, the primary objective of this study was to perform a comprehensive characterization of PR. A secondary objective was to investigate the delivery of this molecule to mammalian skin. METHODS: Phenylethyl resorcinol was characterized using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and nuclear magnetic resonance (NMR). A new high-performance liquid chromatographic (HPLC) method for analysis of PR was developed and validated. The log P (octanol water partition coefficient), value, solubility and short-term stability of PR in a series of vehicles were also determined using HPLC. The evaporation of the selected vehicles was examined using dynamic vapour sorption (DVS). The permeation profiles of PR were investigated under finite dose conditions in porcine and human skin. RESULTS: The melting point of PR was determined to be 79.13 °C and the measured log P (octanol water partition coefficient) at 21 °C was 3.35 ± 0.03. The linearity of the HPLC analytical method was confirmed with an r2 value of 0.99. Accuracy of the method was evaluated by average recovery rates at three tested concentrations, and the values ranged from 99 to 106%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.57 µg mL-1 , respectively. The solubility of PR in PG, DMI, glycerol was within the range of 367 to 877 mg mL-1 . The stability of PR in tested solvents was also confirmed by the 72 h stability studies. From the DVS studies, 70-125% of applied formulations were recovered at 24 h. The permeation through porcine skin at 24 h ranged from 4 to 13 µg cm-2 , while the corresponding amounts of PR delivered through human skin were 2 to 10 µg cm-2 . CONCLUSION: The physicochemical properties of PR confirm it is suitable for dermal delivery. In this study, propylene glycol was the most promising vehicle for PR delivery to human skin. Future work will expand the range of vehicles studied and explore the percutaneous absorption from more complex formulations.

OBJECTIF: Le phényléthyl résorcinol (PR) est largement utilisé dans le secteur des soins personnels comme ingrédient éclaircissant pour la peau. Pour autant, on ne dispose que d'informations limitées concernant les propriétés physicochimiques de ce principe actif. C'est pourquoi cette étude avait pour objectif principal de réaliser une caractérisation exhaustive du PR. Son objectif secondaire était d'étudier l'administration de cette molécule à la peau de mammifères. MÉTHODES: Le phényléthyl résorcinol a été caractérisé par calorimétrie différentielle à balayage (CDB), analyse thermogravimétrique (ATG) et par résonance magnétique nucléaire (RMN). Pour analyser le PR, une nouvelle méthode de chromatographie liquide à haute performance (CLHP) a été développée et validée. On s'est servi de la CLHP pour déterminer les propriétés suivantes du PR : log P (coefficient de partage octanol/eau), valeur, solubilité et stabilité à court terme du PR dans plusieurs véhicules. L'évaporation des véhicules sélectionnés a été examinée par sorption de vapeur dynamique (DVS). Les profils de perméabilité du PR ont été étudiés dans des conditions de dose finie dans des peaux porcine et humaine. RÉSULTATS: On a pu déterminer que le point de fusion du PR était de 79,13 °C et le log P (coefficient de partage octanol/eau) à 21 °C était de 3,35 ± 0.03. La linéarité de la méthode analytique de la CLHP a été confirmée avec une valeur r2 de 0,99. L'exactitude de la méthode a été évaluée par les taux moyens de récupération à trois concentrations testées, avec des valeurs résultantes comprises entre 99 et 106 %. La limite de détection (LD) et la limite de quantification (LQ) ont été déterminées à 0,19 et 0,57 µg/ml_ 1, respectivement. La solubilité du PR dans le PG, le DMI et le glycérol reste dans une plage comprise entre 367 et 877 mg/ml _ 1. La stabilité du PR dans les solvants testés a également pu être confirmée par les études de stabilité à 72 h. Parmi les formulations appliquées lors des études de DVS, 70 à 125 % de celles-ci ont été récupérées à 24 h. La pénétration par la peau porcine à 24 h était comprise entre 4 et 13 µg/cm_ 2, tandis que les quantités de PR correspondantes délivrées à travers la peau humaine étaient de 2 à 10 µg/cm_ 2. CONCLUSION: Les propriétés physicochimiques du PR confirment qu'il est adapté à l'administration dermique. Dans le cadre de cette étude, le propylène glycol est apparu comme le véhicule le plus prometteur pour l'administration de PR dans la peau humaine. De futurs travaux étudieront davantage de véhicules et examineront l'absorption percutanée lors de l'emploi de formulations plus complexes.

3D-printed Franz type diffusion cells.

OBJECTIVE: Franz cells are routinely used to measure in vitro skin permeation of actives and must be inert to the permeant under study. The aim of the present work was to develop and manufacture transparent Franz-type diffusion cells using 3D printing. Printouts were then tested using a range of model active compounds. The study also aims to identify the critical 3D-printing parameters necessary for the process, including object design, choice of printing resin, printout curing and post-curing settings and introduction of model coatings. METHODS: Transparent Franz cells were constructed using an online computer aided design program and reproduced with different stereolithography 3D printers. The two acrylate-based resins used for the fabrication process were a commercially available product and a polymer synthesised in-house. Comparative studies between glass and 3D-printed Franz cells were conducted with selected model actives: terbinafine hydrochloride (TBF), niacinamide (NIA), diclofenac free acid (DFA) and n-methyl paraben (MPB). In preliminary studies, MPB showed the lowest recovery when exposed to the receptor compartment of 3D printed cells. Consequently, in vitro permeation studies were carried out using only MPB with polydimethylsiloxane (PDMS) membrane. RESULTS: A decrease in the amounts of selected compounds was observed for transparent 3D-printed Franz cells compared to glass cells. MPB showed the lowest recovery (53.8 ± 13.1%) when compared with NIA (74.9 ± 4.0%), TBF (81.5 ± 12.0%) and DFA (90.2 ± 12.9%) after 72 h. Permeation studies conducted using 3D-printed transparent cells with PDMS membrane also showed a decrease in MPB recovery of 51.4 ± 3.7% for the commercial resin and 94.4 ± 3.5% for the polymer synthesised in-house, when compared to glass cells. Although hydrophobic coatings were subsequently applied to the 3D-printed cells, the same reduction in MPB concentration was observed in the receptor solution. CONCLUSION: Transparent Franz cells were successfully prepared using 3D printing and were observed to be robust and leak-proof. There are few resins currently available for preparation of transparent materials and incompatibilities between the actives investigated and the 3D-printed cells were evident. Hydrophobic coatings applied as barriers to the printed materials did not prevent these interactions.

Enhancement of the Schottky barrier height of Au/ZnO nanocrystal by zinc vacancies using a hydrothermal seed layer.

Various seed layers were prepared on a Si substrate using the sol-gel (SG) or hydrothermal (HT) method and then ZnO nanocrystal was grown on the seed layer by an HT process. Au/ZnO nanocrystal Schottky diodes (SDs) were fabricated to study the effects of various seed layers on the electrical properties of Au/ZnO SDs. The observations showed that large numbers of Zn vacancies were present near the interface of Au/ZnO with an HT seed layer. The Zn vacancy plays an acceptor-like role, which raises the barrier height of the Au/ZnO SDs to 0.79 eV with a rectifying ratio of more than 8000. Hence, a non-surface-treated Au/ZnO SD was achieved as compared to those of other reported oxygen-plasma treated surfaces. In contrast, oxygen vacancies appear near the interface of Au/ZnO with an SG seed layer. The O vacancy plays a donor-like role, which reduces the barrier height of Au/ZnO, leading to an Ohmic behavior in the I-V characteristics. Zn out-diffusion is found during Au evaporation by of x-ray photoelectron spectroscopy measurements.

Mutations in the tyrosine phosphatase CD45 gene in a child with severe combined immunodeficiency disease.

The hematopoietic-specific transmembrane protein tyrosine phosphatase CD45 functions to regulate Src kinases required for T- and B-cell antigen receptor signal transduction. So far, there have been no reports to our knowledge of a human deficiency in a tyrosine-specific phosphatase. Here, we identified a male patient with a deficiency in CD45 due to a large deletion at one allele and a point mutation at the other. The point mutation resulted in the alteration of intervening sequence 13 donor splice site. The patient presented at 2 months of age with severe combined immunodeficiency disease. The population of peripheral blood T lymphocytes was greatly diminished and unresponsive to mitogen stimulation. Despite normal B-lymphocyte numbers, serum immunoglobulin levels decreased with age. Thus, CD45 deficiency in humans results in T- and B-lymphocyte dysfunction.

Impact of surgical resection rate on survival in gastric cancer: nationwide study.

BACKGROUND: There are marked geographical variations in the proportion of patients undergoing resection for gastric cancer. This study investigated the impact of resection rate on survival. METHODS: All patients with potentially curable gastric cancer between 2006 and 2017 were identified from the Swedish National Register of Oesophageal and Gastric Cancer. The annual resection rate was calculated for each county per year. Resection rates in all counties for all years were grouped into tertiles and classified as low, intermediate or high. Survival was analysed using the Cox proportional hazards model. RESULTS: A total of 3465 patients were diagnosed with potentially curable gastric cancer, and 1934 (55.8 per cent) were resected. Resection rates in the low (1261 patients), intermediate (1141) and high (1063) tertiles were 0-50.0, 50.1-62.5 and 62.6-100 per cent respectively. The multivariable Cox analysis revealed better survival for patients diagnosed in counties during years with an intermediate versus low resection rate (hazard ratio (HR) 0.81, 95 per cent c.i. 0.74 to 0.90; P < 0.001) and high versus low resection rate (HR 0.80, 0.73 to 0.88; P < 0.001). CONCLUSION: This national register study showed large regional variation in resection rates for gastric cancer. A higher resection rate appeared to be beneficial with regard to overall survival for the entire population.

Application of reutilization technology to waste from liquid crystal display (LCD) industry.

This investigation studies the recycling utility of two major waste products from the liquid crystal display (LCD) industry, panel glass and calcium fluoride sludge, which remain after the treatment of waste water. Waste panel glass was mixed with calcium fluoride sludge in various ratios and then subject to conditioning and melting treatment in order to yield glass-ceramics. Heavy metal leaching tests indicated that reductive conditions lowered the heavy metal concentrations in the leachate to an order of magnitude below that in the waste glass and sludge. A 5:5 (wt%) mixture of glass and sludge melted at 1200 degrees C for 60 min achieves a specific gravity, water absorption, unit mass, porosity ratio, and soundness that meet the American Society for Testing and Materials (ASTM) standard for fine aggregates. Therefore, waste panel glass can indeed be efficiently recycled into a useful construction material.

Nationwide study of the impact of D2 lymphadenectomy on survival after gastric cancer surgery.

BACKGROUND: Gastrectomy including D2 lymphadenectomy is regarded as the standard curative treatment for advanced gastric cancer in Asia. This procedure has also been adopted gradually in the West, despite lack of support from RCTs. This study sought to investigate any advantage for long-term survival following D2 lymphadenectomy in routine gastric cancer surgery in a Western nationwide population-based cohort. METHODS: All patients who had a gastrectomy for cancer in Sweden in 2006-2017 were included in the study. Prospectively determined data items were retrieved from the National Register of Oesophageal and Gastric Cancer. Extent of lymphadenectomy was categorized as D1+/D2 or the less extensive D0/D1 according to the Japanese Gastric Cancer Association classification. Overall survival was analysed and, in addition, a variety of possible confounders were introduced into the Cox proportional hazards regression model. RESULTS: A total of 1677 patients underwent gastrectomy, of whom 471 (28·1 per cent) were classified as having a D1+/D2 and 1206 (71·9 per cent) a D0/D1 procedure. D1+/D2 lymphadenectomy was not associated with higher 30- or 90-day postoperative mortality. Median overall survival for D1+/D2 lymphadenectomy was 41·5 months with a 5-year survival rate of 43·7 per cent, compared with 38·5 months and 38·5 per cent respectively for D0/D1 (P = 0·116). After adjustment for confounders, in multivariable analysis survival was significantly higher after D1+/D2 than following D0/D1 lymphadenectomy (hazard ratio 0·81, 95 per cent c.i. 0·68 to 0·95; P = 0·012). CONCLUSION: This national registry study showed that long-term survival after gastric cancer surgery was improved after gastrectomy involving D1+/D2 lymphadenectomy compared with D0/D1 dissection.

ANTECEDENTES: En Asia, la gastrectomía con linfadenectomía D2 asociada se considera el tratamiento curativo estándar para el cáncer gástrico avanzado. Este procedimiento se ha adoptado gradualmente también en el mundo occidental a pesar de la falta de apoyo de los ensayos clínicos aleatorizados. En este estudio hemos tratado de investigar cualquier ventaja sobre la supervivencia a largo plazo tras la linfadenectomía D2 de rutina en una cohorte de base poblacional de cirugía del cáncer gástrico en un país occidental. MÉTODOS: Se incluyeron todos los pacientes que fueron sometidos a gastrectomía por cáncer en Suecia desde 2006-2017. Se recuperaron datos registrados prospectivamente del Registro Nacional de Cáncer de Esófago y Estómago. La extensión de la linfadenectomía se categorizó en D1+/D2 o cuando fue menos amplia en D0/D1 de acuerdo con la clasificación de la Japanese Gastric Cancer Association. Se analizó la supervivencia global y, además, se introdujeron diversos factores de confusión en un modelo de regresión de riesgos proporcional de Cox. RESULTADOS: Un total de 1.677 pacientes fueron sometidos a gastrectomía, de los cuales 471 (28%) fueron clasificados como D1+/D2 y 1.206 (72%) como D0/D1. La linfadenectomía D1+/D2 no se asoció con una mayor mortalidad postoperatoria a los 30 y 90 días. La mediana de la supervivencia global para la linfadenectomía D1+/D2 fue de 41,5 meses con una tasa de supervivencia a los 5 años de 44% comparado con 38,5 meses y 39%, respectivamente, para D0/D1 (P = 0,116). Después de ajustar por los factores de confusión en el análisis multivariable, la supervivencia fue significativamente más alta en la linfadenectomía D1+/D2 comparada con D0/D1 (cociente de riesgos instantáneos, hazard ratio, HR 0,81 (i.c. del 95% 0,68-0,95), P = 0,012)). CONCLUSIÓN: Este estudio del registro nacional mostró que la supervivencia a largo plazo tras cirugía del cáncer gástrico mejoró después de una gastrectomía que incluya linfadenectomía D1+/D2 en comparación con la disección D0/D1.

Prognosis and clinicopathologic features in patients with gastric stump cancer after curative surgery.

Background: Gastric stump ("remnant") cancer is the development of a malignancy related to previous gastric surgery. Prognosis in gastric stump cancer, compared with that in primary gastric cancer, is still controversial. Methods: From January 1988 to December 2012 at a single medical centre in Taiwan, 105 patients with gastric stump cancer, including 85 with previous peptic ulcer disease and 20 with previous gastric cancer, were analyzed for clinicopathologic characteristics and overall survival (os). Results: The 5-year os rates for patients with gastric stump cancer and with primary gastric cancer were 51.2% and 54.5% respectively (p = 0.035). Analysis of clinicopathologic characteristics indicated that, compared with patients having primary gastric cancer, those with gastric stump cancer had more lymph node metastasis (p < 0.001) and had been diagnosed at a more advanced stage (p = 0.047). Multivariate analysis with os as an endpoint showed that age [p = 0.015; hazard ratio (hr): 2.300; 95% confidence interval (ci): 1.173 to 4.509], tumour size (p = 0.037; hr: 1.700; 95% ci: 1.031 to 2.801), stromal reaction (p = 0.021; hr: 1.802; 95% ci: 1.094 to 2.969), and pathologic N category (p = 0.001; hr: 1.449; 95% ci: 1.161 to 1.807) were independent predictors in gastric stump cancer. The os rates for patients with gastric stump cancer who previously had gastric cancer or peptic ulcer disease were 72.9% and 50.0% respectively (p = 0.019). The Borrmann classification was more superficial (p = 0.005), lymph node metastases were fewer (p = 0.004), and staging was less advanced (p = 0.025) in patients with gastric stump cancer who previously had gastric cancer than in their counterparts who previously had peptic ulcer disease. Conclusions: Survival is poorer in patients with gastric stump cancer who previously had peptic ulcer disease than in those who previously had primary gastric cancer. Patients with gastric stump cancer who previously had gastric cancer and could receive curative gastrectomy tended to have a better prognosis because of a more superficial Borrmann classification. Regular follow-up in patients who have undergone gastric surgery is recommended for the early detection of gastric stump cancer.

Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia.

The morphology of the transition zone between the terminal plate of the basal body and the 9 + 2 region of the somatic (non-oral) cilium has been examined in Paramecium tetraurelia. Freeze-fracture and thin-section techniques disclosed both membrane specializations and various internal structural linkages. Freeze-fracture material revealed sets of particles interrupting the unit membrane. The more distal of these form plaquelike arrays while the proximal set of particles forms the ciliary "necklace." The plaque regions correspond to anionic sites on the outer membrane surface as revealed by binding of polycationic ferritin. Both the plaque particles and the necklace particles appear to be in contact with outer doublet microtubules via a complex of connecting structures. In the interior of the transition zone an axosomal plate supports an axosome surrounded by a ring of lightly packed material. Only one of the two central tubules of the axoneme reaches and penetrates the axosome. Below the axosomal plate four rings, each approx. 20 nm wide, connect adjacent outer doublets. An intermediate plate lies proximal to these rings, and a terminal plate marks the proximal boundary of this zone. Nine transitional fibers extend from the region of the terminal plate to the plasmalemma. The observations described above have been used to construct a three-dimensional model of the transition region of "wild-type" Paramecium somatic cilia. It is anticipated that this model will be useful in future studies concerning possible function of transition-zone specializations, since Paramecium may be examined in both normal and reversed ciliary beating modes, and since mutants incapable of reverse beating are available.

Rotation and twist of the central-pair microtubules in the cilia of Paramecium.

The orientation and configuration of the central-pair microtubules in cilia were studied by serial thin-section analysis of "instantaneously fixed" paramecia. Cilia were frozen in various positions in metachronal waves by such a fixation. The spatial sequence of these positions across the wave represents the temporal sequence of the positions during the active beat cycle of a cilium. Systematic shifts of central-pair orientation across the wave indicate that the central pair rotates 360 degrees counterclockwise (viewed from outside) with each ciliary beat cycle (C. K. Omoto, 1979, Thesis, University of Wisconsin, Madison; C. K. Omoto and C. Kung, 1979, Nature [Lond.] 279:532-534). This is true even for paramecia with different directions of effective stroke as in forward- or backward-swimming cells. The systematic shifts of central-pair orientation cannot be seen in Ni++-paralyzed cells or sluggish mutants which do not have metachronal waves. Both serial thin-section and thick-section high-voltage electron microscopy show that whenever a twist in the central pair is seen, it is always left-handed. This twist is consistent with the hypothesis that the central pair continuously rotates counterclockwise with the rotation originating at the base of the cilium. That the rotation of the central pair is most likely with respect to the peripheral tubules as well as the cell surface is discussed. These results are incorporated into a model in which the central-pair complex is a component in the regulation of the mechanism needed for three-dimensional ciliary movement.

Manganese effectively supports yeast cell-cycle progression in place of calcium.

Metal ion requirements for the proliferation of Saccharomyces cerevisiae were investigated. We used bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relatively acid tolerant chelator, to reduce the free metal ion concentrations in culture media. Chelatable metal ions were added back individually and in combination. In addition to a requirement for approximately 10 pM external free Zn2+ we found an interchangeable requirement for either 66 nM free Ca2+ or only 130 pM free Mn2+. Cells depleted of Mn2+ and Ca2+ arrested as viable cells with 2 N nuclei and tended to have very small minibuds. In the absence of added Mn2+, robust growth required approximately 60 microM total internal Ca2+. In the presence of added Mn2+, robust growth continued even when internal Ca2+ was < 3% this level. Chelator-free experiments showed that MnCl2 strongly and CaCl2 weakly restored high-temperature growth of cdc1ts strains which similarly arrest as viable cells with 2 N nuclear contents and small buds. Its much greater effectiveness compared with Ca2+ suggests that Mn2+ is likely to be a physiologic mediator of bud and nuclear development in yeast. This stands in marked contrast to a claim that Ca2+ is uniquely required for cell-cycle progression in yeast. We discuss the possibility that Mn2+ may function as an intracellular signal transducer and how this possibility relates to previous claims of Ca2+'s roles in yeast metabolism.

Microinjection of cytoplasm as a test of complementation in Paramecium.

Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard-to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species.

Intra- and interspecific complementation of membrane-inexcitable mutants of Paramecium.

Membrane excitation was the basis for backward swimming of Paramecium facing stimulus. According to standard genetic tests, inexcitable mutants fell into three complementation groups for both Paramecium tetraurelia (pwA, pwB, and pwC) and Paramecium caudatum (cnrA, cnrB, and cnrC). Cytoplasm from a wild type transferred to a mutant through microinjection restored the excitability. Transfusions between genetically defined complementation groups of the same species effected curing, whereas transfusions between different mutants (alleles) of the same group or between sister cells of the same mutant clone did not. Cytoplasmic transfers of all combinations among the six groups of mutants of the two species showed that any cytoplasm, except those from the same group, was able to cure. Since the pawns and the caudatum nonreversals complement one another through transfusion, they appeared to belong to six different complementation groups. The extent of curing, the amount of transfer needed to cure, and the time course of curing were characteristic of the group that received the transfusion. Variations in these parameters further suggested that the six groups represented six different genes. Because the donor cytoplasms from either species were equally effective quantitatively in curing a given mutant, the curing factors were not species specific. These factors are discussed.

Antibodies to the ciliary membrane of Paramecium tetraurelia alter membrane excitability.

Immobilization of Paramecium followed the binding of antibodies to the major proteins of the ciliary membrane (the immobilization antigens, i-antigens, approximately 250,000 mol wt). Immunoelectron microscopy showed this binding to be serotype-specific and to occur over the entire cell surface. Antibody binding also reduced the current through the Ca-channel of the excitable ciliary membrane as monitored using a voltage-clamp. The residual Ca-current appeared normal in its voltage sensitivity and kinetics. As a secondary consequence of antibody binding, the Ca-induced K-current was also reduced. The resting membrane characteristics and other activatable currents, however, were not significantly altered by the antibody treatment. Since monovalent fragments of the antibodies also reduced the current but did not immobilize the cell, the electrophysiological effects were not the secondary consequences of immobilization. Antibodies against the second most abundant family of proteins (42,000-45,000 mol wt) had similar electrophysiological effects as revealed by experiments in which the Paramecia and the serum were heterologous with respect to the i-antigen but homologous with respect to the 42,000-45,000-mol-wt proteins. Protease treatment, shown to remove the surface antigen, also caused a reduction of the Ca-inward current. The loss of the inward Ca-current does not seem to be due to a drop in the driving force for Ca++ entry since increasing the external Ca++ or reducing the internal Ca++ (through EGTA injection) did not restore the current. Here we discuss the possibilities that (a) the major proteins define the functional environment of the Ca-channel and that (b) the Ca-channel is more susceptible to certain general changes in the membrane.

Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes.

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.

Are ions involved in the gating of calcium channels?

The rates of activation and deactivation of the currents carried by calcium, strontium, or barium ions through the voltage-sensitive calcium channel of Paramecium are different. The differences cannot be attributed to complications due to internal ion concentration, calcium channel inactivation, potassium current activation, surface charge effects, or incomplete space clamping. The findings indicate participation of the divalent cations in the voltage-driven calcium channel gating process.

Temperature-sensitive pawns: conditional behavioral mutants of Paramecium aurelia.

"Pawns" are mutants of Paramecium aurelia in which the process of calcium activation during membrane excitation is genetically impaired, with a corresponding loss of avoiding reactions. Mutants are selected that behave normally when grown at 23 degrees C but as pawns at 35 degrees C. The normal excitation can now be disrupted and restored in the same strain at will.

Calcium-induced ciliary reversal in the extracted models of "Pawn", a behavioral mutant of Paramecium.

"Pawn," a genic mutant of Paramecium aurelia, cannot swim backward as the wild type can upon proper stimulation. In contrast, after membrane disruption by Triton X-100, the adenosine triphosphate-magnesium reactivated models of Pawns swim backward in the presence of calcium as wild-type models do. Thus, the mutant phenotype is due to an impairment in the membrane and not in the calcium-sensitive motile system.

A mechanosensitive ion channel in the yeast plasma membrane.

Mechanosensitive ion channels use mechanical energy to gate the dissipation of electrochemical gradients across cell membranes. This function is fundamental to physiological processes such as hearing and touch. In electrophysiological studies of ion channels in the plasma membrane of the yeast Saccharomyces cerevisiae, channels were observed that were activated by, and adapted to, stretching of the membrane. Adaptation of channel activity to mechanical stimuli was voltage-dependent. Because these mechanosensitive channels pass both cations and anions, they may play a role in turgor regulation in this walled organism.