Localization of pulmonary surfactant protein during mouse lung development

During lung development type II alveolar epithelial cells produce extracellular pulmonary surfactant. Polyclonal antibodies were produced against nonserum proteins associated with human surfactant. The present studies were designed (i) to determine if mouse surfactant proteins were antigenically cross-reactive with polyclonal antibodies directed against human surfactant proteins; and (ii) to determine surfactant protein localization during fetal, neonatal, and adult mouse lung development. Two-dimensional gel electrophoresis studies in conjunction with immunologic techniques provided evidence that mouse and human surfactant proteins shared antigenic determinants. The major monomeric form of mouse surfactant protein in a glycoprotein of approximately Mr 35,000 under reducing conditions. A less abundant form was identified as a Mr 45,000 polypeptide. Immunohistochemical localization showed that type II cells contain surfactant protein at Theiler stage 26. A gradient of immunostaining was localized within alveolar surfaces. The antigen was not detected in heart, blood vessels, or pulmonary interstitial cells. Surfactant protein was detected lining alveolar surfaces in mature adult lung. The distribution of this protein during fetal and neonatal lung morphogenesis suggests that this extracellular constituent of pulmonary surfactant may be extremely useful as a phenotypic marker with which to evaluate normal and abnormal lung development.     

Responses of Sorghum and Pennisetum Species to the N2-Fixing Bacterium Azospirillum brasilense

Three field inoculation experiments, two in Florida and one in New Mexico, were conducted with Azospirillum brasilense Cd. Each of the Florida experiments evaluated two crop species. One species in each of the Florida experiments responded to inoculation with a significant dry matter yield increases of 11 to 24% and nitrogen yield increases of 9 to 39%. No inoculation response was noted in the New Mexico experiment. The responding species were Sorghum bicolor (L.) Moench (sorghum) and the interspecific hybrid between Pennisetum americanum (L.) K. Schum. (pearl millet) and P. purpureum Schumach. (napiergrass). Nonresponding species were pearl millet (Florida) and Sorghum sudanense (Piper) Staph. (New Mexico). Acetylene reduction activity of inoculated plots in Florida was low, showing no increase over the natural uninoculated background rates and, in one case, was negatively correlated with yield. Acetylene reduction activity was not measured in New Mexico. In Florida, A. brasilense populations were found to decline from 5 × 10³ to 5 × 10² bacteria g of soil⁻¹ in about 3 weeks (quadratic regressions). Continued decline to less than 10² by week 5 indicated that the inoculated bacteria did not become established in the soil in high numbers. The A. brasilense population declined at about the same rate in the New Mexico experiment. The erractic inoculation responses in these experiments are similar to those observed in earlier work at the University of Florida. The lack of acetylene reduction activity response to inoculation and the rapid population decline of the inoculated bacteria suggest that N₂ fixation is not the major mechanism causing yield responses after inoculation.     

Microbial models of mammalian metabolism: microbial reduction and oxidation of pentoxifylline.

Fourteen microorganisms, including fungi, yeasts, and bacteria, were screened for their ability to metabolize the xanthine drug pentoxifylline. Thirteen cultures either reduced the drug to the alcohol metabolite or oxidatively cleaved the ketonic side chain to homologous carboxylic acid metabolites. The alcohol metabolite was the predominant or sole metabolite in all organisms, with conversions ranging from 6 to 91%. Preparative-scale production of the alcohol metabolite with Rhodotorula rubra (ATCC 20129) allowed for the isolation of this product with a 40% yield. Two organisms also produced the carboxylic acid metabolites at low levels (2 to 10%). The routes of metabolism in microbial cultures are the same as those reported in mammalian systems.     

Isolation of the fibrinogen-binding region of platelet thrombospondin

Purified platelet thrombospondin binds to immobilized fibrinogen if both Ca++ and Mg++ are present. Digestion of the purified molecule with thermolysin results in a limited number of discrete proteolytic fragments. When such digests are subjected to affinity chromatography on immobilized fibrinogen, only the fragments with Mr of 120,000 and 140,000 are specifically bound and subsequently eluted by the addition of EDTA to the column buffer. Examination by SDS-PAGE under both reducing and nonreducing conditions reveals that the fibrinogen-binding domain is derived from the region of the thrombospondin molecule containing the interchain disulfide bonds. The requirement for Ca++ and Mg++ for optimal binding to fibrinogen is also manifest by the Mr 120,000/140,000 thermolytic fragments.     

Effects of an anion channel blocker, 4, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), on human neutrophil function

We investigated the capacity of DIDS to influence degranulation and superoxide anion (O-2) generation by human neutrophils exposed to soluble, surface-active stimuli. DIDS caused a concentration-related (25-200 microM) inhibition of myeloperoxidase (MPO) release from N-formyl-methionyl-leucyl-phenylalanine (FMLP), 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB4), 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) and phorbol myristate acetate (PMA)-stimulated neutrophils. In contrast, DIDS had no effect on O-2 production by FMLP and PMA activated cells, whereas it enhanced LTB4 and AGEPC-elicited O-2 generation. The respective effects of DIDS on these neutrophil activities were reversed by washing the cells prior to exposure to stimulus. Thus, DIDS represents a useful pharmacologic probe for investigating the role(s) of anions in the mechanisms of neutrophil activation with structurally and chemically dissimilar stimuli.     

Assay of mouse liver uroporphyrinogen decarboxylase by reverse-phase high-performance liquid chromatography

A method for the estimation of hepatic uroporphyrinogen decarboxylase activity employing reverse-phase HPLC is described. Mouse liver homogenate in 0.25 M sucrose was pretreated with a suspension of cellulose phosphate and then centrifuged to remove hemoglobin and debris. The supernatant was used as the enzyme source. Incubations were acidified, oxidized, and centrifuged only before analysis of the porphyrins formed, using a Spherisorb ODS column and a gradient solvent system constructed from methanol/lithium citrate mixtures. Coproporphyrinogen formation by BALB/c mouse liver supernatant was estimated as about 5.0 and 9.1 pmol/min/mg protein from uroporphyrinogens I and III, respectively, at 10 microM substrate concentration and pH 6.8. Decarboxylation of pentacarboxyporphyrinogens (the last step in coproporphyrinogen formation) proved to be easily measured. Coproporphyrinogen formation from pentacarboxyporphyrinogen III abd (20 microM) at pH 6.8 was about 109 pmol/min/mg protein. Pentacarboxyporphyrinogen I was not as good a substrate as III abd but was decarboxylated faster at pH 5.4 than at 6.8, and at the lower pH and at 10 microM concentration of substrate 42 pmol of coproporphyrinogen was formed/min/mg protein. These results compared favorably with those obtained by previously published procedures involving time-consuming extraction and esterification steps.     

Effects of polyamines on the first division cycle of Xenopus laevis eggs

The involvement of polyamines in cytokinesis has been examined in eggs of the amphibian X. laevis. Microinjection of spermine or spermidine into unfertilized eggs induced a shortening of the first division cycle and early formation of cleavage-like constrictions. Eggs were activated by injection and developed furrows about 45 min later, whereas the first division normally occurred around 120 min after activation. In terms of concentration, spermine was slightly more effective than spermidine, but putrescine had no influence on the division cycle.     

Mutant hemoglobin stability depends upon location and nature of single point mutation

The temperature dependence of the rates of heme release from the beta subunits of methemoglobin A and 5 beta mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the alpha 1/beta 2 interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.     

Angiotensin-converting enzyme inhibitory and antihypertensive activities of pivalopril (RHC 3659-(S))

Pivalopril (RHC 3659-(S); (S)-N-cyclopentyl-N-(2-methyl-3-pivaloylthiopropionyl) glycine) is a new compound with a hindered sulfur group that has been compared to captopril for oral angiotensin-converting enzyme (ACE) inhibition in rats and dogs and antihypertensive activity in rats. In separate groups of conscious normotensive rats, pivalopril (0.03-1.0 mg/kg, orally [p.o.]) produced a dose-related antagonism of angiotensin I (AngI)-induced pressor effects. The ED50 for pivalopril and captopril was 0.1 mg/kg. In conscious normotensive dogs, pivalopril (incremental doses of 0.01-1.0 mg/kg, p.o.) produced a dose-related antagonism of AngI pressor effects. The ED50 was 0.17 mg/kg for pivalopril and 0.06 mg/kg for captopril. At equieffective doses the two compounds had similar durations of action. In sodium-deficient, conscious spontaneously hypertensive rats (SHR), pivalopril (1-100 mg/kg, p.o.) produced a dose-related reduction in mean arterial pressure. The potency and duration were similar to those of captopril. In the sodium-replete SHR, 5 days of oral dosing with pivalopril, 100 mg/(kg . day), decreased mean arterial pressure more effectively than captopril, 100 mg/(kg . day). No tolerance developed to the antihypertensive effect of either drug. It is concluded that pivalopril is a potent, orally effective ACE inhibitor and antihypertensive agent.