A scanning electron microscope study of Craspedella sp. from the branchial chamber of redclaw crayfish,Cherax quadricarinatus,from Queensland,Australia

Epidermal topography was examined, including papillate ridges, grooves and ciliated sensory papillae of Craspedella sp. from the branchial chamber of redclaw crayfish, Cherax quadricarinatus, from Queensland, Australia. Rhandites were observed to discharge from ducts opening mainly in a small distal region of the ventral epidermis of the three central (of five) tentacles. These regions, devoid of ciliated sensory papillae, serve to adhere the anterior end of the worms during locomotion. Secretions from glands associated with the posterior attachment organ were observed to discharge from pores on the outside region of the ventral surface of the disc.A comparison of various scanning electron microscopy (SEM) fixation techniques showed that (1) hot fixatives at 90 °C provide most information on the largest number of epidermal structures and (2) different fixation regimes highlight different epidermal features.     

Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3.

The herpes simplex virus 1 (HSV-1) infected-cell protein 0 (ICP0) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection. To identify cellular proteins interacting with ICP0, we used a domain of exon II of ICP0 that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen. Our results were as follows. (i) A cDNA in a positive yeast colony was found to encode cyclin D3, a cell cycle regulator of G1 phase. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to cyclin D3 specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate. (iii) To enhance the expression of cyclin D3, the gene was inserted into the viral genome and overexpressed in infected cells. The overexpressed cyclin D3 colocalized with ICP0 in nuclear structures characteristic of ND10 and which earlier have been reported to contain ICP0. (iv) The accumulation of cyclin D3 protein in Vero cells infected with an alpha0 deletion mutant was reduced relative to that of cells infected with wild-type virus or a recombinant virus in which the deleted alpha0 sequences were restored. (v) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express cyclin D3 and cyclin-dependent kinase 4 (CDK4) phosphorylated GST fused to retinoblastoma protein (GST-pRb) but did not phosphorylate the GST-alpha0(20-241) or GST-alpha0(543-768) fusion protein or immunoprecipitated ICP0 proteins. Moreover, the chimeric GST-ICP0(exon II) protein shown to bind cyclin D3 had no effect on the activity of the kinase on GST-pRb when added to mixtures of lysates of Sf9 cells which coexpressed cyclin D3 and CDK4. These results indicate that ICP0 interacts with, colocalizes with, and stabilizes the cyclin D3 cell cycle regulator and does not affect its interaction with the cyclin-dependent kinase.     

Lipid specificity for the reconstitution of well-coupled ATPase proteoliposomes and a new method for lipid isolation from photosynthetic membranes

The lipid specificity for the enzymatic and proton-translocating functions of a reconstituted thermophilic ATPase complex has been investigated. The proteoliposomes were prepared from the ATPase complex of the thermophilic cyanobacterium Synechococcus 6716 and various lipids and lipid mixtures extracted from this organism and from a related mesophilic strain. Some commercial lipids were used as well. An improved method of lipid extraction from chlorophyll-containing membranes is presented. This method is based on acetone extraction and additional chlorophyll separation and results in higher yields, less chlorophyll contamination and a simpler procedure than the conventional methods based on chloroform/methanol extraction. The lipids of Synechococcus 6716 thus extracted were fractionated by thin-layer chromatography. The fatty acyl chain composition of the separated lipids was analyzed by gas chromatography. The coupling quality of the reconstituted ATPase proteoliposomes made of different lipids was tested by a membrane-bound fluorescent probe and uncoupler stimulation of ATP hydrolysis. None of the separated lipids alone was able to produce a well-coupled system. The best results were obtained with the native lipid mixture. The minimum requirement was the combination of a typical bilayer-forming lipid and the non-bilayer (hexagonal II structure)-forming monogalactosyldiacylglycerol. Lipids from the mesophilic Synechococcus 6301 and commercial lipids (also mesophilic) produced poorly coupled vesicles but significant improvement was obtained when thermophilic monogalactosyldiacylglycerol was included. Both the reconstituted and solubilized ATPase complex have a sharp temperature optimum at 50 degrees C. The effect of reconstitution and measurement temperatures on the yield of well-coupled vesicles from different lipid sources was also studied.     

Detection of a yeast polyphosphate fraction localized outside the plasma membrane by the method of phosphorus-31 nuclear magnetic resonance

Non-penetrating cations, like UO2+(2) and Eu3+, are bound to the outside of yeast cells in a reversible fashion. Binding of these ions was attended with a decrease of the 31P NMR polyphosphate signal. Subsequent addition of EDTA to the suspension restored the original spectrum. These experiments confirm the localization of a polyphosphate fraction outside the plasma membrane of yeast.     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of cell swelling on fluid flow across alveolar epithelium

The effects of decreased temperature, metabolic inhibition, and hyposmolality on osmotically and hydrostatically driven water flux across bullfrog alveolar epithelium were studied. Lungs were isolated from double-pithed Rana catesbeiana and prepared as sacs. Either an osmotic (0.1 M raffinose) or hydrostatic (6.3-6.6 Torr) pressure gradient was imposed across the tissue. These gradients resulted in the volume flow of water from the alveolar to pleural bath. Control water flux and hydraulic conductivity were estimated from the rate of weight loss of the lung sac. Subsequently water flux and hydraulic conductivity were determined under one of the following conditions: 1) temperature of bathing solutions lowered to 2 degrees C; 2) 1 mM 2,4-dinitrophenol added to both alveolar and pleural baths, or 3) both baths changed to half-isosmotic Ringer solution. The control hydraulic conductivities for osmotic (Lpo) and hydrostatic (Lph) pressure gradients were 3.65(+/- 0.94) X 10(-12) and 2.14(+/- 0.63) X 10(-10) ml/dyn X s, respectively. Under conditions of metabolic inhibition, hyposmolality, and decreased temperature, Lph decreased by 77, 83, and 92%, and Lpo decreased by 56, 34, and 59%, respectively. These results are most consistent with the hypothesis that the decrements in hydraulic conductivity under our experimental conditions are due to epithelial cell swelling and perhaps to changes in the characteristics of the paracellular pathway.     

The binding of carbon monoxide to cytochrome c oxidase.

The effect of CO on the optical absorbance spectrum of partially reduced cytochrome c oxidase has been studied. The changes at 432 and 590 nm suggest that the cytochrome alpha2/3+ - CO compound is formed preferentially and that concomitantly a second electron is taken up by the enzyme. From the CO-induced changes at 830 nm it is concluded that in the partially reduced enzyme addition of CO causes reoxidation of the copper component of cytochrome c oxidase. Addition of CO to partially reduced enzyme (2 electrons per 4 metal ions) also brings about a decrease in the intensities of electron paramagnetic resonance signals of high-spin heme iron near g = 6 and of the low-spin heme at g = 2.6. Concomitantly both the low-spin heme a signal at g = 3 and the copper signal at g = 2 increase in intensity. These results demonstrate that formation of the reduced diamagnetic cytochrome a3 - CO compound is accompanied by reoxidation of both the copper component detectable by electron paramagnetic resonance and possibly also by cytochrome a.     

A comparison of membrane components of normal and transformed BALB/c cells.

Membrane glycolipids, glycoproteins, and surface proteins of normal and transformed BALB/c cell lines have been compared. Several virally and spontaneously transformed cell lines showed differences in membrane components compared to normal A31 cells. These differences consisted of increased amounts of simpler gangliosides, absence of the large external transformation sensitive (LETS) protein, and the appearance of a major new glycoprotein band of about 105 000 molecular weight. In contrast, the spontaneously transformed cell line that caused the fastest growing tumors in vivo and the most rapid animal death (3T12T) did not have these changes. A31 and 3T12T glycolipid profiles appear similar as did glycoproteins and cell surface proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When Pronase-generated glycopeptides were analyzed by Sephadex G-50 chromatography, and enrichment in faster-eluting species was seen in two killing tumor lines (c5T and 3T12T) compared to A31. Regressing tumor lines (MSC, c5) did not show this change. Isolated membrane glycoproteins yield glycopeptides of different sized after Pronase digestion. In addition, several 3T12T glycoproteins yield glycopeptides that are larger than those from the corresponding glycoproteins of A31 cells. It appears that glycopeptide alterations associated with transformation occur in several membrane glycoproteins.     

Histiocytic sarcoma of the scrotum in a rat

A 225-day-old male fourth generation rat from a developing recombinant inbred line (Lewis x Brown Norway) had a bilaterally symmetrical enlargement of the scrotum. Palpation indicated the presence of a firm lobulated mass extending from the tip of the scrotum to the abdominal wall. Bilateral nodular masses totally occupied the scrotal sacs, surrounded the testicles, and extended along the spermatic cords into the abdominal cavity. Tumor nodules also were present in the intestinal mesentery, omentum, mesenteric lymph nodes, pancreas, and lung. Histologically, the neoplasm presented a spectrum of characteristics varying from that of a granuloma with giant cells to a diffuse proliferation of spindle-shaped mononuclear cells.     

Column chromatography of plant polyphenols on weak anion exchangers.

Mixtures of phenols and cinnamic acid derived plant polyphenols are separated on several WAX cellulose materials and on DEAE Sephadex. Reference mixtures are also split up into the following distinct groups: phenolic carboxylic acids--neutral o-dihydroxy phenolics--neutral non o-dihydroxy phenolics. Quantitative recoveries are obtained, while no isomerizations occur with the pH labile plant phenolics. It is suggested that these materials can be used for preparative scale purification of plant phenolics, or for cleaning up plant extracts prior to HPLC examination.