Identification and Implications of Trimethyl-n-Alkylbenzenes in Marine Oils from the Deep Tarim Basin

The source of marine oils from the deep Tarim Basin is still in debate due to several alteration processes of source indicators. A series of trimethyl-alkylbenzenes has been detected in marine oils from this old, composite basin, besides the reported aryl isoprenoids with 2,3,6-trimethyl substitution (AIPs). They are characterized by regular gas chromatography elution pattern, which is similar to that of n-alkylbenzenes, and suggest a strong possibility of n-alkyl side chains. C15 trimethyl-n-alkylbenzenes were synthesized by Friedel–Crafts acylation of trimethylbenzene isomers to determine their structures. Based on the chromatography and mass spectra data and the coinjection of synthesized compounds, this series of compounds has been assigned as the 2,4,5-trimethyl-n-alkylbenzenes that coeluted with 2,3,5-trimethyl-n-alkylbenzenes, and other trimethyl-n-alkylbenzene isomers were also detected. This series of trimethyl-n-alkylbenzene (AAs) shows much higher relative abundances in light and waxy oils than in normal and heavy oils, which is opposite to the variation in relative abundances of aryl isoprenoids. The ratios of these trimethyl-n-alkylbenzenes to the aryl isoprenoids (AA/AIP ratio) generally show a good correlation with the maturity indicators for most of studied oils despite of some outliers (mainly condensates). The pyrolysis of asphaltenes has confirmed these trends. These results support an important control of thermal stress on the molecular compositions of marine oils from the deep Tarim Basin, besides other secondary alteration processes (such as oil mixing and migration fractionation, among others). These factors should be given a full consideration for the source determination of deep and ultradeep oils.

Novel cysteine proteinase inTrypanosoma cruzimetacyclogenesis

With the aim to study proteinases released to the culture medium duringTrypanosoma cruzimetacyclogenesis, the presence of cysteine proteinases (CPs) was analysed in culture supernatants obtained throughout the differentiation induced by stimulation of epimastigotes withTriatoma infestanshindgut homogenate. In SDS-gelatin containing gels, an important endopeptidase activity with apparent molecular weight range between 97 and 116 kDa was encountered at pH 6, which was abolished by the specific cysteine proteinase inhibitor E-64 and TLCK, but not by pepstatin, 1,10 phenantroline or PMSF. This novel CP, namedTcCPmet, showed affinity to cystatin-Sepharose, denoting its thiol-proteinase character as well as to ConA-Sepharose, indicating it contains N-linked oligosaccharides. However, it presented a different elution pattern on ConA-Sepharose than cruzipain and, in addition, it was not recognized by anti-cruzipain serum, facts that strongly suggest the different nature of both CPs. Moroever, evidence is presented indicating thatTcCPmet was able to hydrolyse the same chromogenic peptides as cruzipain at optimal alkaline pH values, although with a different order of effectiveness. Our results indicate the presence of a novel CP secreted by metacyclic trypomastigotes and reinforces the important role of these enzymes in metacyclogenesis.

Inactivation of Retroviruses with Preservation of Structural Integrity by Targeting the Hydrophobic Domain of the Viral Envelope

ABSTRACT We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.

Phosphorylation of androgen receptor isoforms

Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser–Ala substitution mutant S650A (Ser-650→Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed.

Fiber-optic spectrophotometry of streaking in pork loins injected with sodium chloride and tripolyphosphate

Enhanced pork produced by needle injection of sodium chloride and tripolyphosphate loses its visual appeal if it has pale streaks on a dark background. Reflectance (R) spectrophotometry was used to test the hypothesis needle injection causes pale streaks by elution of myoglobin. Pale streaks in commercial pork loins (n = 10) had higher R than dark streaks (P < 0.01 from 350 to 590 nm) but differences between pale and dark streaks were almost a linear function of wavelength (r = -0.92, P < 0.0005) with no evidence of myoglobin elution. Using the same apparatus, myoglobin elution was detected in small disks of pork perfused experimentally for 1 h (n = 31 spectra, 2 min apart), comparing water (control, n = 5 disks) with commercial injection solution (n = 5 disks). Myoglobin elution by water increased R (P < 0.005) from 350 to 650 nm with a maximum effect at 440 nm, close to the Soret absorbance band for myoglobin. Perfusion of disks with commercial injection solution produced a complex result with the myoglobin elution pattern (increased R peaking at 440 nm) superimposed on an overall decrease in R, probably from sodium salts dissolving myofibrillar proteins (P < 0.005 from 350 to 360 nm, from 400 to 420 nm, at 440 nm, and from 460 to 540 nm). Thus, there was no support for the initial working hypothesis pale streaks are caused by myoglobin elution because the myoglobin elution pattern (increased R peaking at 440 nm) was absent in commercial pork loins. If perfusing small disks of pork is a valid experimental model of what happens in needle-injected loins, transient pale streaks might be muscle fasciculi not yet reached by injected solution. Key words: Enhanced pork, streaking, needle injection

Demonstration of altered signaling responses in bone marrow extracellular fluid during increased hematopoiesis in rats using a centrifugation method

The composition and characteristics of the bone marrow extracellular fluid supposedly modify the transport of cytokines, drugs, and other signaling molecules involved in the regulation of bone marrow function. Direct access to the bone marrow extracellular fluid surrounding hematopoietic cells is complicated by the virtually noncompliant surrounding bone tissue. We examined the applicability of a centrifugation method to obtain representative samples of bone marrow extracellular fluid from rats and humans. Perforated rat bones or human bone marrow biopsies were wrapped in nylon mesh baskets before being centrifuged at 180–239 g. In the rats, we found an only minor contribution of fluid from other sources than the bone marrow extracellular fluid as indicated by the average ratio of centrifugate-to-plasma activity of the extracellular tracer fluid51Cr-labeled EDTA of 0.85. The colloid osmotic pressure in the centrifugate was consistently lower than that in the corresponding plasma in both species. In rats and humans, high-performance liquid chromatography showed a protein elution pattern from the bone marrow fluid similar to that of plasma, except for a peak eluting in the ∼40-kDa molecular mass range. Western blotting of the cytokines erythropoietin and granulocyte colony-stimulating factor revealed generally higher amounts in the centrifugate than in the plasma. This difference was augmented during increased hematopoietic activity induced by inflammation or bleeding in rats. We conclude that the centrifugation method provides representative samples of bone marrow extracellular fluid and that extracellular signaling responses to altered hematopoiesis are more clearly reflected locally in the bone marrow interstitium than in plasma.

Active-site-mutagenesis study of rat liver betaine-homocysteine S-methyltransferase

A site-directed-mutagenesis study of putative active-site residues in rat liver betaine—homocysteine S-methyltransferase has been carried out. Identification of these amino acids was based on data derived from a structural model of the enzyme. No alterations in the CD spectra or the gel-filtration chromatography elution pattern were observed with the mutants, thus suggesting no modification in the secondary structure content or in the association state of the proteins. All the mutants obtained showed a reduction of the enzyme activity, the most dramatic effect being that of Glu159, followed by Tyr77 and Asp26. Changes in affinity for either of the substrates, homocysteine or betaine, were detected when substitutions were performed of Glu21, Asp26, Phe74 and Cys186. Interestingly, Asp26, postulated to be involved in homocysteine binding, has a strong effect on affinity for betaine. The relevance of these results is discussed in the light of very recent structural data obtained for the human enzyme.

Characterization of Different Reactive Lysines in Bovine Heart Mitochondrial Porin

Incubation of mitochondrial outer membrane porin with citraconic anhydride prior to treatment with fluorescein isothiocyanate (FITC) resulted in the labeling of a set of lysines located at a boundary between the water phase and lipid phase. The elution pattern of porin from the cation exchanger has been considered as indicative for the location of lysines. Electrical measurements after reconstitution of the modified protein in lipid bilayer membranes revealed that certain specific lysine residues are more susceptible to alterations. The innermost positive residues were only slightly influenced, while the outermost lysines exhibited a substantial change in channel properties. These results suggest the presence of critical charged residues in mitochondrial outer membrane porin that may be responsible for both the channels selectivity and its voltage dependence.