MAXAMIZE. A DNA sequencing strategy advisor.

The MAXAMIZE advisory system determines from user-provided restriction maps an optimal strategy to do nucleotide sequencing by methods involving end-labeled fragments. The maps may be either simple linear restriction maps of fragments or complex circular maps including restriction sites of a vector. The whole system is interactive and is written in the Genetic English language provided by the GENESIS System, a molecular genetics knowledge representation and manipulation package. In addition, MAXAMIZE provides bookkeeping facilities for sequencing and offers advise on how to verify the newly obtained sequence data.     

Purification and characterization of two β-N-acetylhexosaminidases from the tobacco hornworm,Manduca sexta (L.) (Lepidoptera: Sphingidae)

β-N-Acetylhexosaminidases were detected in 10 insects including species of Lepidoptera, Coleoptera, Hemiptera, and Orthoptera. Two enzymes were purified from the tobacco hornworm, Manduca sexta (L.). EI was detected in larval and pharate pupal molting fluid, integument, and pupal hemolymph while EII was found in larval and pupal hemolymphs. They are acidic hydrolases with similar molecular weights (6.1 × 10⁴), molar extinction coefficients at 280 nm (1.9 × 10⁵ liters mol^?1 cm^?1), and pH optima (pH 6). They differ in the number of polypeptide chains per molecule (EI is a single chain and EII consists of two polypeptide chains), amino acid composition, extent of glycosylation (EII is probably a glycoprotein), isoelectric point (pI_EI = 5.9 and pI_EII ～- 5.1), tissue distribution, and reactivities toward nitrophenylated N-acetylglucosamine (k_cat,I = 328 s^?1 and k_cat,II = 103 s^?1) and N,N′-diacetylchitobiose (k_cat,I = 307 s^?1 and k_cat,II = 3 s^?1). These results suggest that EI is a chitinase and that EII may function as a hexosaminidase in vivo.     

Release of leukotrienes from guinea pig lung stimulated by C5ades Arg anaphylatoxin

The complement anaphylatoxins C5a and C5Ades Arg contract guinea pig peripheral airway preparations and trachea by a mechanism largely independent of histamine release. In trachea the contractions are inhibited by FPL 55712, a relatively specific inhibitor of slow-reacting substance of anaphylaxis (SRS-A). SRS-A is now known to be a mixture of leukotrienes C4, D4, and E4 (LTC4, LTD4, LTE4). These data suggest that C5-derived anaphylatoxins stimulate production and release of leukotrienes in pulmonary tissues. To define these observations more precisely, fragments of guinea pig lung were incubated with porcine C5ades Arg, and the supernatant fluids were analyzed for leukotrienes by using both pharmacologic and chemical methods. In addition to histamine, a smooth muscle contracting activity characteristic of SRS-A was released from C5a-treated lung preparations. The contractile substance was identified as a leukotriene based on: 1) the characteristic contraction of guinea pig ileum, 2) inhibition of the contractile activity by FPL 55712, 3) enhanced release of activity in the presence of indomethacin or L-cysteine, 4) chromatographic behavior of ethanol-extracted active material on Amberlite XAD-7 resin, and 5) cochromatography of the active material on reverse-phase, high performance liquid chromatography with standard LTD4. We therefore concluded the humoral factor C5ades Arg induces a leukotriene release reaction in guinea pig lung tissue. This particular response of pulmonary tissue to anaphylatoxin has not been appreciated previously as an immediate effect of complement activation on the pathophysiology of the lung.     

Sensitization of lymphocytes against pooled allogeneic cells. II. Characterization of effector cells cytotoxic for autologous lymphoblastoid cell lines

Stimulation of human lymphocytes in mixed leukocyte culture (MLC) with x-irradiated pooled allogeneic normal cells (poolx) was previously shown to result in generation of effector cells cytotoxic for autologous Epstein-Barr virus- (EBV) transformed lymphoblastoid cell lines (LCL). This study was undertaken to determine whether lysis of the autologous EBV- transformed LCL cells by pool-stimulated cells is mediated by cytotoxic Tc lymphocytes (Tc) or natural killer- (NK) like cells, both of which are generated in MLC. In the first series of experiments, proliferating cells were eliminated by treatment of pool-stimulated cells with 5 X 10(-5) M 5-bromodeoxyuridine (BUdR) and light. The remaining cells failed to lyse allogeneic normal lymphocytes and autologous LCL cells, whereas cytotoxicity against NK-sensitive K562 leukemia cells was retained. In the second series of experiments, pool-stimulated effector cells were treated with monoclonal anti-human Tc cell antibodies, OKT3 or OKT8, and complement (C). The cells recovered after antibody and C treatment were diminished in their ability to lyse allogeneic normal lymphocytes as well as autologous LCL cells, whereas their cytotoxicity against K562 leukemia cells was unaffected. These combined results provide strong evidence that lysis of autologous LCL cells by lymphocytes stimulated with pooled allogeneic normal cells is mediated by Tc rather than NK-like cells.     

Les Radiolaires d'âge jurassique supérieurà crétacé supérieur dans les Radiolarites du Pinde-Olonos (Presqu'île de Koroni; Péloponnèse méridional,Grèce)

Radiolarians of several radiolarites sections of the Pindos-Olonos zone, southern Peloponnesus, lead to propose direct datations of mesozoic sediments. The acuity of such datations allow to distinguish two periods for depositionof radiolarites s.s. in the tethyan region. The first period would be Upper Jurassic and general in tethyan realm, radiolarites depositing under various latitudes (0–35°N). This would be the result of a strong ocean surface current. The second period would be Upper Cretaceous (Vraconian—Coniacian) and be geographically much more restricted (0–15°N). This could result from a weaker current. The absence of radiolaritic sedimentation is possibly the result of the destruction or insulation of the accurate basins during the obduction of ophiolites on the apulo-african realm. The inventory of Late Cretaceous radiolarian fauna, rarely done on alpine series so far, shows similar result to those obtained in Central American and California.     

Metabolic and temporal studies on pancreatic exocrine cells in culture

Summary An approach is described whereby cells with definitive markers are followed from their source through dissociation and fractionation, then during long-term maintenance in vitro. Such sequential studies should enable investigators to define factors regulating proliferation and function of specific cells since ambiguity concerning identity is readily avoided.Pancreatic cells of guinea pigs were isolated by enzymic dissociation, and exocrine cells were enriched by centrifugation with solutions of serum albumin. Resulting populations consisting of up to 95% exocrine cells were then incubated with gyration to produce aggregates, and these were seeded to standard culture plates for further study. Colonial aggregates of exocrine epithelia develop in culture and can be maintained for 20–30 days. The cells exhibit changes with time that are qualitatively similar to those known to occur during serial cultivation of diploid fibroblastlike cells from human and other species. The uptake of tritiated thymidine decreases with maintenance time. Autoradiographic examination indicates that this is due to a reduction in the number of epithelial cells incorporating the isotope. Cell diameters increase from an average of 21 m at day 0 to 44 m by day 26, and a marked increase in heterogeneity of this parameter is also evident. Cellular DNA and protein accumulate during the same interval. Incorporation of tritiated leucine during 24-h exposures increases until about the 10th day in vitro and remains relatively constant for at least 2 weeks thereafter.The data are consistent with the hypothesis that exocrine pancreatic cells like other diploid cells in culture, progress to terminal differentiation under the culture conditions employed. The role of physical, nutritional, and humoral evironmental factors on this process will be the subject of future reports.Supported in part by National Cancer Institute Contracts NO1-CP-43231 and NO1-CP-65751     

Human placental N-acetyl-beta-D-hexosaminidase isozymes. Activity toward native hyaluronic acid.

The activity of purified human hexosaminidases A and B toward hyaluronic acid (HA) isolated from cultured human skin fibroblasts was investigated. The cleavage of N-acetylglucosaminyl residues to monosaccharide N-acetylglucosamines by hexosaminidase isozymes was determined in the presence and absence of purified human β-glucuronidase. The pH optima of this reaction, with and without β-glucuronidase, were 4.5 for hexosaminidase A and 4.0 for hexosaminidase B. The hydrolysis of HA by both hexosaminidase isozymes proceeds linearily for at least 18 h in the presence of β-glucuronidase. Concentrations of 0.5–5 units of either isozyme showed a linear relationship with rate of hydrolysis. Without β-glucuronidase, hexosaminidase only cleaved the terminal N-acetylglucosamine residue. However, under optimal conditions, with β-glucuronidase, the hydrolytic activity of hexosaminidase B was about 30% as efficient as that of hexosaminidase A. Approximately 70% of the HA could be degraded by 5 units of hexosaminidase A in the presence of 0.5 unit of β-glucuronidase, as opposed to 25% degraded by hexosaminidase B. These results probably reflect intrinsic differences in the activities of the two isozymes. Since the substrate (HA) did not inhibit the hydrolysis of a synthetic substrate (4-methylumbelliferyl-β-glucosaminide) by hexosaminidase B, the linear kinetics of HA hydrolysis implies no product inhibition. These data indicate that native HA can be hydrolyzed by the combined activities of β-glucuronidase with hexosaminidase A or hexoaminidase B.     

Differential scanning calorimetry of dipalmitoyl phosphatidylcholine analogues and of their interaction products with basic polypeptides

The thermotropic behaviour of dipalmitoyl phosphatidylcholine analogues with a varying number (n) of CH₂ groups between the phosphate and the quaternary ammonium has been investigated. The temperature (T_m) and the enthalphy (ΔH) of the phase transition are non-monotonous functions of the number of CH₂ groups. T_m oscillates between 40 and 45°C and ΔH between 7 and 13 kcal/mol for a variation of n between 2 and 11.It is concluded that the hydrocarbon chains in the head groups do not penetrate the hydrocarbon region and do not contribute directly to the melting of the acyl chains. It is suggested that their length may affect the critical ballance between the attractive and the repulsive forces within the bidimensional lattice of the head groups.Copolypeptides of lysine with phenylalanine do not appreciably affect the T_m but have a pronounced effect on ΔH of the lipid phase transition, which depends strongly on the ratio of the two amino acids in the polypeptide. The effect of copolypeptide of any defined composition on ΔH is also a non-monotonous function of the number of CH₂ groups in the phosphatidylcholine head group, but it does not parallel completely the oscilations in the T_m and ΔH of the pure lipids.     

Thymic function in NZB mice. II. Regulatory influence of a circulating thymic factor on antibody production against polyvinylpyrrolidone in NZB mice.

Administration of a circulating thymic factor isolated from normal pig blood prevented the development of the exaggerated production of anti-polyvinyl pyrrolidone (PVP) antibody in young NZB mice. However, treatment was ineffective if initiated after the 4th week of life at a time when endogenous serum thymic factor (TF) normally disappears in these mice. These data suggest that circulating TF is necessary for the survival of short-lived suppressor T cells normally implicated in the regulation of the production of antibodies against PVP, a thymus-independent antigen. In older NZB mice, TF treatment increased paradoxically anti-PVP antibody production, which suggests that "amplifier" T cell activity could also be under TF influence.     

Implications of electrostatic potentials on ribosomal proteins.

Potentiometric studies of ribosomal particles 30S, 50S, and 70S, were designed to investigate possible implications of the electrostatic potentials developed by the 16S and 23S rRNA fractions. Release of protons and proton titrations of these ribosomal fractions were examined as a function of Mg2+ and K+ concentrations. The effects of these cations fit the polyelectrolyte theory remarkably well and are discussed accordingly.