Comparative study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice.

Milk fat globule membranes and mammary tumour virus particles (d=1.17 g/cm3) have been obtained from the milk of a Swiss albino mice strain. Comparative biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.     

Comparitive study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice

Milk fat globule membranes and mammary tumour virus particles (d = 1.17 g/cm³) have been obtained from the milk of a Swiss albino mice strain. Comparitive biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.     

Proteins in the fossil bone of the dinosaur, seismosaurus

Proteins have been successfully extracted from the fossil vertebra of a 150-million-year-old sauropod dinosaur (Seismosaurus) recently excavated from the Morrison Formation of New Mexico. HCl and guanidine·HCl extracts of the fossil bone and its sandstone matrix were concentrated, demineralized, and resolved into a number of different protein fractions by reversed-phase high-performance liquid chromatography (HPLC). One of these fractions had the same retention time as collagen. Amino acid analysis (Pico-Tag method) of these fractions confirmed they were proteins. Comparison of the correlation coefficients of the amino acid analyses with that of collagen standards indicated that none of the fractions contained significant amounts of collagen. Similar HPLC profiles were obtained for the HCl extracts of fossil bone and its sandstone matrix suggesting they contained the same proteins. However, different HPLC profiles were obtained when these HCl extracts were dried and reextracted with guanidine·HCl. These different fractions represent proteins unique to the fossil and were not found in the sandstone matrix. These differences were confirmed by amino acid analysis. Such information on fossil bone proteins might provide useful knowledge concerning the evolution of skeletal molecules and the fossilization process. Similar information on the proteins from the geological matrix might provide useful fingerprints for reconstructing ancient environments and for assessing sedimentary rocks for fossil fuel exploration.     

Laccase production by a monokaryotic strain of Pycnoporus cinnabarinus derived from a dikaryotic strain

Monokaryotic Pycnoporus cinnabarinus strains were obtained from the dikaryotic strain I-938. One of these, designated MK18, consistently produced high laccase activity. In cultures of MK18 and I-938 where ferulic acid was added as laccase inducer, laccase activity was enhanced about 2.5-fold reaching 3400 U/l for the MK18 strain. Laccase was purified to homogeneity and under the selected growth conditions, only one isoform of the enzyme was produced. The N-terminal sequence was similar to the amino terminal sequence of laccase II from Trametes versicolor. The enzyme was stable at 60 C for more than 1 h.     

The separation of human serum high density lipoproteins by hydroxy apatite column chromatography. Evidence for the presence of discrete subfractions.

Human serum high density lipoprotein subfractions 2 and 3, isolated by preparative ultracentrifugation after blocking the enzyme phosphatidylcholine: cholesterol acyl transferase, have been subfractionated further by hydroxyapatite column chromatography. From subfraction 2 we reproducibly obtained 5 and from subfraction 3, 6 fractions differing in chemical composition and apolipoprotein content. The fractions eluting at low salt concentrations were composed primarily of apolipoprotein-A polypeptides while those eluting at high salt concentrations consisted primarily of apolipoprotein-C. From all the 11 subfractions only one contained the "arginine-rich" polypeptide. The apolipoprotein-C-containing fractions were richer in triacylglycerol, phospholipids and free cholesterol as compared to the apolipoprotein-A-containing ones. Although small differences of their partial specific volumes existed, the obtained values indicate that all subfractions belonged to the parent density class. The implications of these results to the current view of lipoprotein metabolism are discussed.     

Subcellular distribution of Plp-40, a lipoprotein in a serotype A strain of Pasteurella multocida

A 40-kDa lipoprotein (Plp-40) is expressed by serotype A strains of Pasteurella multocida in amounts which correlate with the amount of capsular material present. We hypothesized that Plp-40 is exposed at the outer surface of the outer membrane (OM) of the cell and is associated with the serotype A exopolysaccharide material. The objectives of the present study were to confirm the lipoprotein nature of Plp-40 and to determine its subcellular location. Plp-40 maturation was shown to be sensitive to globomycin, thereby confirming it to be a bacterial lipoprotein. Plp-40 was shown to be present in the OM fractions of P. multocida obtained by both sarkosyl extraction and sucrose density gradient centrifugation, as well as in capsule fractions obtained by either hyaluronidase treatment or warm buffer extraction. [(3)H]palmitic acid-labeled Plp-40 could be removed from the surface of whole cells by exposure to proteinase K. Autoradiography of (125)I-labeled cell surface proteins exhibited a 40-kDa band that was prominent in capsulated strains and greatly diminished in a noncapsulated strain. These results support the hypothesis that Plp-40 is a lipid-modified OM protein, which is exposed on the outer cell surface and is likely associated with serotype A extracellular polysaccharide.     

Decolorization of molasses and a dye by a newly isolated strain of the fungus Geotrichum candidum Dec 1

A fungus, Geotrichum candidum Dec 1, newly isolated as a dye-decolorizing microorganism, was used to decolorize molasses and an anthraquinone dye in shaken flasks. A degree of decolorization of molasses of 87% was achieved after 12 days of cultivation, and the maximum rate of decolorization of the dye in the culture broth was obtained in 7 days. The apparent activity of peroxidase in the molasses, which is responsible for dye decolorization, was significantly lower than that of purified peroxidase, due to the inhibition by molasses, but the inhibition was reduced after the fungus was fully grown. As two ultrafiltered fractions of molasses were similarly decolorized by Dec 1, Dec 1 apparently degraded colored substances of a wide range of molecular weights. When Dec 1 was cultivated in a medium in which sucrose in the molasses was hydrolyzed with invertase, the degree of decolorization of molasses, and rate of decolorization of the dye were similar to these obtained above.     

Autocrine Survival Factors of a Cytotoxic CTLL-2 Cell Line

Cell survival in multicellular organisms is controlled by numerous cytokines, growth factors, and autocrine survival factors. Autocrine survival factors remain the least studied. The aim of this work was to study the autocrine factors which control survival of a CTLL-2 cytotoxic cell line: isolation and characterization of biologic activity along with physicochemical features of the active molecules have been performed. The conditioned medium of CTLL-2 cells containing autocrine factors was separated by gel filtration into four fractions: A, B, C, and D (according to the order of their efflux from the column). The biological activity of the fractions was tested by the MTT assay with the low density 5 days culture as a cell survival model. The testing of the ability of the fractions to support the cell survival in culture has shown that fractions A and B were active, whereas fractions C and D were not. The presence of a peptide of the molecular mass of 1157 Da in active fractions A and B has been detected by MALDI-TOF-mass-spectrometry. Considerable amount of lactate in fractions A and B, which flowed out from the column along with the peptide, has been detected with an enzymatic lactic acid assay. The lactate concentration in fraction A was 3.72 ± 0.11 mM and it was 0.83 ± 0.06 mM in fraction B. The obtained data suggest that fractions A and B contain supramolecular complexes of the peptide (M 1157 Da) with different lactate content. The peptide in a free form has not been found in the CTLL-2 cell conditioned medium.     

Gender-specific differences in cannibalism between a laboratory strain and a field strain of a predatory mite

Many phytoseiid species, including Phytoseiulus persimilis, are known to engage in cannibalism when food is scarce and when there is no possibility to disperse. In nature adult females of P. persimilis are known to disperse when prey is locally depleted. Males, in contrast, are expected to stay and wait for potential mates to mature. During this phase, males can obtain food by cannibalizing. Therefore, we hypothesize that male P. persimilis exhibit a higher tendency to cannibalize than females. Because rearing conditions in the laboratory usually prevent dispersal, prolonged culturing may also affect cannibalistic behavior. We hypothesize that this should especially affect cannibalism by females, because they consume far more food. We tested these hypotheses by comparing males and females from two strains, one of which had been in culture for over 20 years, whereas the other was recently collected from the field. It is known that this predator can discriminate between kin and non-kin and prefers cannibalizing the latter, hence to construct lines with high relatedness we created isofemale lines of these two original strains. We subsequently tested to what extent the adult females and males of the original strains and the isofemale lines cannibalized conspecific larvae from the same strain/line in a closed system. Relatedness with the victims did not affect cannibalistic behavior, but males engaged more often in cannibalism than females, and females of the laboratory strain engaged more in cannibalism than those of the field strain, both in agreement with our ideas. We hypothesize that the difference in cannibalism between the two genders will increase when they have the alternative to disperse.     

The subcellular fractionation of embryonic chick tendon and cartilage cells: a re-examination.

A re-examination of the subcellular fractions obtained from matrix-free chick tendon and cartilage cells has been made since the discovery that three out of four of the micrographs of chick tendon microsomal fractions published in an earlier paper from this laboratory were not authentic. The present studies demonstrate that by using the procedures previously reported it is possible to isolate microsomal and submicrosomal fractions from tendon and cartilage cells which exhibit typical morphology when examined by electron microscopy. These observations are consistent with our original biochemical characterization of subcellular fractions, which we know to be valid. Other publications from this laboratory in which these fractionation procedures have been applied to studies of collagen biosynthesis are in no way compromised, and indeed, most of our data have been confirmed by several other laboratories.     

Characterization with crossed immunoelectrophoresis of some antigens differentiating a virulent Candida albicans from its derived, avirulent strain

Antigenic differences between a wild-type virulent Candida albicans 4918 (wt) and its spontaneous avirulent mutant (m-10) were found with crossed immunoelectrophoresis. Yeast cell extracts as well as soluble protein and mannoprotein fractions obtained by affinity chromatography on concanavalin A (Con A) were analyzed. Sera from patients with candidiasis and antisera from rabbits infected with live wt cells and boosted with wt extracts or rabbits immunized with purified wt cell wall preparation were used as counter reactants. Qualitative differences in serum precipitins formed by patients with suspected or culture-proven candidiasis to polysaccharide antigens of wt and m-10 origin were observed. In comparison, except for a spike-formed precipitate detected only with the wt extract, the serum from infected rabbits precipitated the wt and m-10 cell wall polysaccharide antigens about equally. The same type of precipitate was also found with the Con A wt mannoprotein fractions but was again lacking with the m-10 mannoproteins. This precipitate, with extremely slow electromobility in the first dimension, may be related to some special immunodeterminant of the wt mannan molecule. No substantial differences in the precipitation patterns of the Con A wt and m-10 proteins were found when analyzed with patients' sera or rabbit anti-cell sera. However, using these protein fractions with anti-cell wall sera revealed a larger number of precipitates for the wt as opposed to the m-10 strain. The observed antigenic differences between the virulent- and the avirulent-derived strains seem to be mainly associated with cell wall determinants (components) and might be related to the greater adherence and infectivity of the wild strain.     

A cytochemical study on the pancreas of the guinea pig. IV. Chemical and metabolic investigation of the ribonucleoprotein particles

Five ribonucleoprotein (RNP) fractions were isolated from the postmitochondrial supernatant of the pancreas of the guinea pig. Two were obtained from the microsomes which, by deoxycholate (DOC) treatment, were subdivided into a DOC-soluble and a DOC-insoluble fraction. The latter was taken to represent attached RNP particles. Two other fractions obtained from the microsomal supernatant supposedly represent free RNP particles existing as such in the cytoplasm, while a third fraction resisted sedimentation for 20 hours at 105,000 g and is considered to be a soluble nucleoprotein. These fractions exhibited different RNA/protein ratios and also different RNA turnover patterns, as determined after in vivo labelling with adenine-8-C(14). However, little discernible differences could be detected in the nucleotide composition of the RNA moieties of these RNP fractions. Amino acid-"activating" enzymes were found to occur in the fraction containing the soluble nucleoproteins. The discussion focuses on the relationships between these fractions and protein synthesis in the pancreas, using data given in this and a previous paper, and data contained in the literature.     

Stimulating effect of pancreatic DNase on Bacillus subtilis growth as a function of the physiologic properties of the inoculation culture

The stimulating effect of pancreatic DNAse on Bacillus subtilis growth was studied in relation to the content of "slowly growing" cells in the inoculation culture in the phase of decelerated growth. Three cell fractions of B. subtilis were obtained using the stepwise separation of the population in terms of buoyant density in the phase of decelerated growth. In contrast to fractions II and III, fraction I contained cells with decelerated growth, competent, permeable to exogenous DNAase I, and sensitive to the action of this enzyme. The faster growth of bacterial cells in fraction I was shown to be associated with the shorter lag period of these cells having a longer generation time.     

Male accessory gland secretory proteins in a few members of the Drosophila nasuta subgroup

Male accessory gland secretory proteins in seven members of the Drosophila nasuta subgroup have been analyzed by SDS-PAGE. The study revealed remarkable simplicity in the patterns. The protein fractions, which migrate in three groups, could be categorized as major and minor. The number of major fractions varies from a maximum of eight to a minimum of four. Group I consists of high molecular weight fractions, and group III, low molecular weight fractions. Among different members analyzed, the variation with respect to pattern and the number of fractions are confined largely to group III protein fractions, while group I and II fractions are found to be conserved to a greater extent. These proteins are PAS positive and group III fractions are not sensitive to silver staining. Analysis of these tissue specific proteins in the F₁ and F₂ of interspecific crosses and backcross progeny as well as volume analysis revealed that a 26-kD fraction in D. n. nasuta follows an autosomal pattern of inheritance, while a 55-kD and a 25-kD fraction in D. n. albomicans and a 24-kD fraction in D. n. kepulauana follow an X-linked pattern of inheritance.     

Hematochemical parameters in sheep and goats of Sardinian breeds. I) Electrophoretic picture of plasma proteins

It has been studied the electrophoretic picture of plasma proteins of lactating and dry sheep and goats of Sardinian-breed. The values of the single fractions obtained for the four different groups of subjects have been compared with the "t of Student". The statistically significant differences between values of albumin and gammaglobulin fractions obtained comparing sheep and goats can probably derive from genetic factors. In general significant differences in values of several fractions associated with lactation have not been noticed.     

Basis of a humeomics science: chemical fractionation and molecular characterization of humic biosuprastructures

We propose a mild stepwise fractionation of molecular components of a humic acid (HA) suprastructure and their structural identification by advanced analytical methods. This procedure may be the basis of a "Humeomics" approach to characterize natural humic molecules and clarify their relations with ecosystems functions. Sequential fractionation included: (1) organic solvent extraction, (2) transesterification with boron trifluoride in methanol (BF(3)-CH(3)OH), (3) methanolic alkaline hydrolysis (KOH-CH(3)OH), and (4) cleavage of ether and glycosidic bonds with HI. Structural identification of initial and final material, separated organo-soluble and hydrosoluble fractions, and subfractions was conducted by GC-MS, HPSEC-ESI-MS (high-resolution, Orbitrap), and solid- and liquid-state NMR. GC-MS revealed in organosoluble unbound fractions the presence of both saturated and unsaturated, linear and branched, alkanoic, hydroxyalkanoic and alkandioic acids, n-alkanes, and n-alkanols. These components decreased progressively in fractions obtained after weak and strong ester cleavage. Unsubstituted alkanoic acids with variable chain length were ubiquitously detected in all fractions, thereby suggesting their fundamental function in the architecture of humic suprastructures. An important role in differentiating supramolecular associations should also be attributed to substituted alkanoic acids that were detected in variable amounts in different fractions. The content of aromatic acids and steroids was only noticed in the latter fractions. HPSEC-ESI-MS of initial and final solid fractions showed similar compounds, as indicated by GC-MS, whereas the hydrosoluble fraction after transesterification revealed fewer of these compounds but noticeable nitrogen-containing acids. A large amount of "cyclic" acids were identified by MS empirical formula in initial HA, and, to a lesser extent, in the final fractionation residue as well as in the hydrosoluble fraction. The predominant alkyl NMR signals in organosoluble extracts and those of CH-N, CH-O, and O-CH-O groups in hydrosoluble fraction confirmed mass spectrometry results. Homo- and heterocorrelated liquid-state NMR spectra indicated spin systems interactions varying with separated fractions. Solid-state and dipolar-dephasing NMR spectra of final residue showed predominance of sp(2) carbons, 66% of which were quaternary carbons, and a significant increase in conformational rigidity with respect to initial HA. Separated fractions accounted for 60% of initial HA weight, and losses were attributed to hydration water, liberated volatile compounds, and decarboxylation. Quantization of analytes showed that the sum of compound classes in separated fractions was greater than that for the initial HA, thereby showing that stepwise fractionation increased significantly the analytical identification of humic molecules. Our results suggest this "Humeomics" approach as a valid path for mapping humic molecular composition and assess humus origin and formation.     

The effects of humic substances within the Ah horizon of a Calcic Luvisol on morphological changes related to invertase and peroxidase activities in wheat roots

The chemical and biological characteristics of humus within the Ah horizon (Calcic-Luvisol) have been studied. Attention was paid to variation in the NMR spectra of humic fractions and ¹³C values and to how these changes are related to different biological humic fraction activities.The chemical changes in particular involve the decrease of the aromatic component and the increase of the non-aromatic component within the horizon and the different ¹³C value not only within the horizon but also among the humic fractions distinctive of different molecular sizes.An attempt has been made to explain the vertical chemical changes in terms of processes affecting the biological characteristics of the high and low molecular size humic fractions. The main conclusions are that the low molecular size humic fractions, in the upper part of the horizon, are of greater importance with respect to the other humic fractions in influencing the enzyme activities linked to growth metabolism. The biological role of the high molecular size humic fractions characterised by a relevant content of peptidic- and carbohydratic-C is also presented.     

Amylolytic enzymes produced by a color variant strain ofAureobasidium pullulans

A color variant strain ofAureobasidium pullulans (NRRL Y-12974) produced amylase and -glucosidase activities when grown at 28°C for 4 days in liquid culture on a wide variety of carbon sources such as starch, pullulan, glucose, maltose, cyclodextrins, sucrose, xylose, and xylan. An -glucosidase was separated by Q-Sepharose adsorption from the cell-free culture broth and partially purified by hydroxylapatite and octyl-Sepharose chromatography. After ammonium sulfate treatment of the culture supernatant (obtained after Q-Sepharose adsorption), the amylase fraction was separated into three active fractions by hydroxylapatite column chromatography, which were identified as -amylase, glucoamylase A, and glucoamylase B. The glucoamylase A was further purified by octyl-Sepharose column chromatography. The pH optima for the action of -amylase, glucoamylase A, glucoamylase B, and -glucosidase were 5.0, 4.5, 4.0–4.5, and 4.5, respectively. The -amylase and glucoamylase B were fully stable at pH 3.0–6.0, glucoamylase A at pH 4.5–5.5, and -glucosidase at pH 3.5–7.0 for 1 h at 50°C. The optimum temperatures for the action of these enzymes were 55°, 50°–60°, 65°, and 65°C, respectively. The -amylase, glucoamylase A, and glucoamylase B were adsorbed onto raw corn starch and degraded it. Glucoamylase B readily cleaved pullulan. The -glucosidase was not adsorbed onto raw starch and did not degrade it at all. It hydrolyzed both -1,4 and -1,6 linkages in oligosaccharides. All four enzymes did not require any metal ion for activity and were inhibited by cyclodextrins (-and -, 10mm).     

Vaccination against cutaneous leishmaniasis in a murine model. II. Immunologic properties of protective and nonprotective subfractions of soluble promastigote extract

We have previously demonstrated that BALB/c mice can be protected against a fatal infection with Leishmania major by i.p. immunization with a soluble leishmanial antigen (SLA) preparation in conjunction with the adjuvant, Corynebacterium parvum (CP). In this study, SLA was separated into nine distinct fractions by anion exchange liquid chromatography, and the fractions were analyzed for their ability to stimulate T cells obtained from immunized mice, to be recognized by vaccine-induced antibodies, and to induce protective immunity. While all but one of the fractions were recognized by antibodies from SLA + CP immunized mice, only two fractions (fractions 1 and 9) stimulated lymphocytes to produce macrophage-activating factor and elicited significant delayed-type hypersensitivity in vivo. When mice were immunized with the fractions, only fraction 9 stimulated significant immunity (76% protection in seven experiments). Proteins (accounting for 1.3% of the total in SLA) appear to be responsible for the protection elicited with fraction 9, since protease treatment of this fraction destroyed its immunogenicity. Thus, a partially purified protective protein antigen fraction has been obtained and protection with this fraction correlated with cell-mediated immune responses. However, these results also demonstrate that the ability of leishmanial antigens to be recognized by T cells and produce macrophage-activating factor does not in itself predict whether such molecules will induce immunity, suggesting that protective leishmanial antigens may have additional unique properties.