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.     

Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants

Medicago truncatula (barrel medic) is an annual legume of agricultural and biological interest. In this report regeneration from isolated mesophyll protoplasts is described. A specifically developed, highly regenerable seed line is essential for regeneration. Other critical requirements for regeneration are the starting plant material, the use of agarose droplets incubated in a shallow layer of liquid medium, and protoplast density. Plants are grown in controlled environment conditions. Protoplasts are purified using a Percoll-based flotation procedure, then embedded in 100 l agarose droplets containing a basal medium plus 25 M NAA and 4 M BAP (the same medium as in the surrounding shallow liquid layer) to induce protoplast division. A protoplast density of 6–8×10⁵ ml^–1 is required for maximum colony formation. M. truncatula plants previously transformed for kanamycin resistance yielded embryogenic callus and also regenerated plants. Protoplasts from other annual Medicago (M.intertexta and M.scutellata) species readily form calli by the procedure we have described.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

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 nature of cells generating human myeloma colonies in vitro.

Freshly explanted human myeloma cells formed colonies of monoclonal plasma cells in soft agar in the presence of medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. The medium showed peak activity at a dilution of 1:4. 2-mercaptoethanol or monothioglycerol was necessary for colony formation. Other thiols tested were ineffective in promoting colony growth. Colony-forming cells adhered to nylon wool, but not glass beads or plastic dishes. The presence of E-rosetting cells was not required for myeloma colony formation. Antibody prepared against a human myeloma cell line, RPMI 8226, reduced colony formation. These studies demonstrate the usefulness of this bioassay for determining functional properties of the myeloma colony-forming cell.     

Genetic recombination in Micromonospora rosaria by protoplast fusion

Auxotrophic strains of Micromonospora rosaria were isolated by N-methyl-N'-nitro-N'-nitrosoguanidine mutagenesis and used in intraspecific recombination by protoplast fusion. High-frequency fusion of protoplasts of M. rosaria strains was induced by polyethylene glycol (molecular weight, 1,000) (PEG 1,000). The optimum concentration of PEG 1,000 for fusion of M. rosaria was 50% (wt/vol). PEG 4,000 was slightly better than PEG 1,000 at concentrations lower than 50% (wt/vol). The recombinant frequency did not increase after treatment with PEG 1,000 (50% [wt/vol]) for longer than 20 min. Under these conditions, fusion with many auxotrophic strains of M. rosaria resulted in a high frequency of formation of true recombinants (sometimes more than 10%). Additionally, when ros (rosamicin nonproducing) strains were crossed by protoplast fusion; about 5% of the resultant prototrophic recombinants were shown to have the ros+ (rosamicin producing) characteristic restored. Rosamicin production by M. rosaria colonies was clearly distinguished by the broth overlay method. The results of fusion experiments between ros and ros+ strains indicated that either the chromosomal mutation or pleiotrophic effect of some auxotrophic markers is involved.     

Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase three-dimensional structure

Bacillus subtilis has an alkaline phosphatase multigene family. Two members of this gene family, phoAIII and phoAIV, were cloned, taking advantage of in vitro constructed strains containing a plasmid insertion within one or the other of the structural genes. The DNA sequences of the two genes showed approximately 64% identity at the DNA level and 63% identity in the deduced primary amino acid sequences. The phoAIII and phoAIV genes code for predicted proteins of 47,149 and 45,935 Da, respectively. Comparison of the deduced primary amino acid sequence of the mature proteins with other sequenced alkaline phosphatases from Escherichia coli, yeast, and humans shows 25-30% identity. Based on the refined crystal structure of E. coli alkaline phosphatase, it appears that the active site and the core of the structure are retained in both Bacillus alkaline phosphatases. However, both proteins are truncated at the amino terminus compared with other mature alkaline phosphatases, three sizable surface loops of E. coli are deleted, and a minidomain is replaced with a larger domain in the model. Neither Bacillus alkaline phosphatase sequenced contains any cysteine residues, an amino acid implicated in intrachain disulfide bond formation in other alkaline phosphatases.     

Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation.

The biochemical signaling mechanisms involved in transducing the effects of tumor necrosis factor alpha (TNF alpha) and gamma-interferon (gamma-IFN) on leukemia cell differentiation are poorly defined. Recent studies established the existence of a sphingomyelin cycle that operates in response to the action of vitamin D3 on HL-60 cells and that may transduce the effects of vitamin D3 on cell differentiation (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). The effects of TNF alpha and gamma-IFN on sphingomyelin turnover were determined, and the specificity and role of sphingomyelin hydrolysis in HL-60 human promyelocytic leukemia cells with 20% hydrolysis of sphingomyelin at 15 min, 40% hydrolysis at 30-60 min, and return to base line at 2 h. The hydrolyzed sphingomyelin (18 pmol/nmol total phospholipid) was accompanied by the concomitant generation of ceramide (11.2 pmol/nmol total phospholipid). gamma-IFN also caused reversible hydrolysis of sphingomyelin with onset at 1 h and peak effect at 2 h. This sphingomyelin cycle appeared to be specific to the monocytic pathway of HL-60 differentiation, since it was not activated by retinoic acid or dibutyryl cAMP, inducers of granulocytic differentiation, nor with phorbol myristate acetate, an inducer of macrophage-like differentiation. Addition of synthetic ceramide or bacterial sphingomyelinase induced monocytic differentiation of HL-60 cells. Cell-permeable ceramide also caused prompt down-regulation of mRNA for the c-myc protooncogene. The time course of c-myc down-regulation was consistent with the action of ceramide as the mediator of TNF alpha action. These results suggest that sphingomyelin turnover may be an important signaling mechanism transducing the actions of TNF alpha and gamma-IFN with specific function in cell differentiation.     

Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis.

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.     

Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.