Sex pheromones in snakes.

The majority of pheromones identified to date are insect pheromones, which are volatile in nature. Identification of nonvolatile pheromones have been relatively rare, especially in vertebrates. Male and female garter snakes use pheromones to mediate sexual behavior. The female sex attractiveness pheromone of the Canadian red-sided garter snake, Thamnophis sirtalis parietalis, consists of a novel series of nonvolatile saturated and monounsaturated long-chain methyl ketones, whereas the male sex recognition pheromone contains squalene. These compounds were isolated, identified, and partially synthesized, and field tests show them to be biologically active.

Inhaled nitric oxide augments nitric oxide transport on sickle cell hemoglobin without affecting oxygen affinity.

Nitric oxide (NO) inhalation has been reported to increase the oxygen affinity of sickle cell erythrocytes. Also, proposed allosteric mechanisms for hemoglobin, based on S-nitrosation of beta-chain cysteine 93, raise the possibility of altering the pathophysiology of sickle cell disease by inhibiting polymerization or by increasing NO delivery to the tissue. We studied the effects of a 2-hour treatment, using varying concentrations of inhaled NO. Oxygen affinity, as measured by P(50), did not respond to inhaled NO, either in controls or in individuals with sickle cell disease. At baseline, the arterial and venous levels of nitrosylated hemoglobin were not significantly different, but NO inhalation led to a dose-dependent increase in mean nitrosylated hemoglobin, and at the highest dosage, a significant arterial-venous difference emerged. The levels of nitrosylated hemoglobin are too low to affect overall hemoglobin oxygen affinity, but augmented NO transport to the microvasculature seems a promising strategy for improving microvascular perfusion.

Phospholipase D-catalyzed hydrolysis of phosphatidylcholine occurs with P-O bond cleavage.

Mammalian phospholipase D has been implicated in signal-transduction mechanisms, most recently in association with stimuli that enhance phosphatidylcholine (PC) turnover. It was previously unknown whether hydrolysis of PC by phospholipase D proceeds via P-O or C-O bond cleavage. Commercially available phospholipase D isolated from Streptomyces chromofuscus was used to hydrolyse distearoyl phosphatidylcholine (PC) in a detergent-containing buffer consisting of 90% 18O-water. The product of hydrolysis, phosphatidic acid (PA), was purified by thin-layer chromatography and analyzed using californium-252 plasma desorption mass spectrometry. An increase of two mass units was observed, compared to a distearoyl PA control, consistent with a reaction mechanism involving cleavage of the P-O bond.

Bis(monoacylglycero)phosphate from PC12 cells, a phospholipid that can comigrate with phosphatidic acid: molecular species analysis by fast atom bombardment mass spectrometry.

Phospholipids from pheochromocytoma (PC12) cells were purified by one-dimensional thin-layer chromatography (TLC). Material corresponding in RF to phosphatidic acid (PA) was analyzed by fast atom bombardment mass spectrometry (FAB). The molecular ions of the major constituents corresponded in mass to phosphatidylglycerols (PG), which, however, have a lower RF value. Analysis of the mass spectra demonstrated that this material consists of bis(monoacylglycero)phosphates (BMP, lysobisphosphatidic acid), a structural isomers of PG. Linked scans of individual molecular ions indicate that BMP from PC12 cells is esterified almost exclusively with monounsaturated (16:1 and 18:1) and polyunsaturated (20:4 and 22:6) fatty acids. One of the two major molecular species contains two monounsaturated (18:1/18:1), while the other contains both a monounsaturated (18:1) and a polyunsaturated (22:6) fatty acid ester. FAB in combination with TLC is ideally suited for analysis of molecular species of phospholipids.

Intermolecular aggregations are responsible for the slow kinetics observed in the folding of cytochrome c at neutral pH.

Folding of equine cytochrome c at a low protein concentration (26 microM) eliminated a slow kinetic phase (time constant three seconds) that was observed in the previous hydrogen exchange pulse-labeling experiments at pH 6.2 and 10 degrees C. It was demonstrated that this slow folding phase was caused by intermolecular aggregations. Because heterogeneous kinetics is a very general feature in the folding of proteins characterized by pulsed hydrogen exchange coupled with two-dimensional NMR, our experimental results suggest aggregations might also be responsible for the complex folding kinetics of other proteins. This is possible since these experiments were performed at relatively high protein concentrations.

Solid-state NMR data support a helix-loop-helix structural model for the N-terminal half of HIV-1 Rev in fibrillar form.

Rev is a 116 residue basic protein encoded by the genome of human immunodeficiency virus type 1 (HIV-1) that binds to multiple sites in the Rev response element (RRE) of viral mRNA transcripts in nuclei of host cells, leading to transport of incompletely spliced and unspliced viral mRNA to the cytoplasm of host cells in the latter phases of the HIV-1 life cycle. Rev is absolutely required for viral replication. Because Rev aggregates and fibrillizes in solution at concentrations required for crystal growth or liquid state NMR measurements, high-resolution structural characterization of full-length Rev has not been possible. Previously, circular dichroism studies have shown that approximately 50 % of the Rev sequence adopts helical secondary structure, predicted to correspond to a helix-loop-helix structural motif in the N-terminal half of the protein. We describe the application of solid-state NMR techniques to Rev fibrils as a means of obtaining site-specific, atomic-level structural constraints without requiring a high degree of solubility or crystallinity. Solid-state NMR measurements, using the double-quantum chemical shift anisotropy and constant-time double-quantum-filtered dipolar recoupling techniques, provide constraints on the phi and psi backbone dihedral angles at sites in which consecutive backbone carbonyl groups are labeled with (13)C. Quantitative analysis of the solid-state NMR data, by comparison with numerical simulations, indicates helical phi and psi angles at residues Leu13 and Val16 in the predicted helix 1 segment, and at residues Arg39, Arg 42, Arg43, and Arg44 in the predicted helix 2 segment. These data represent the first site-specific structural constraints from NMR spectroscopy on full-length Rev, and support the helix-loop-helix structural model for its N-terminal half.

The detection of intact double-stranded DNA by MALDI.

DNA fragments have been analyzed by matrix-assisted laser desorption ionization (MALDI) and electrospray mass spectrometry. In many cases, only the single-stranded oligonucleotides have been detected. Recently, spectra of intact double-stranded DNA have been obtained in both electrospray and massive cluster impact ionization. We show here the first MALDI spectra of intact double-stranded DNA (EcoR1 adaptor 12/16) that is clearly not due to nonspecific dimer formation. 6-Aza-2-thiothymine was used as the matrix in the presence of ammonium citrate. Via the same procedure but with other matrices commonly employed for oligonucleotide analysis, the intact DNA duplex was not detected. No sign of the homodimer of either of the single strands is observed. Although the spectrum also shows peaks attributable to each of the single strands, these are demonstrated to arise from the DNA solution and not the sample preparation or desorption process.

Isolation of an anti-HIV diprenylated bibenzyl from Glycyrrhiza lepidota.

The organic soluble extract from the leaves of Glycyrrhiza lepidota showed moderate activity in the US National Cancer Institute in vitro anti-HIV-1 bioassay. Chromatographic separation of this extract resulted in the identification of a new diprenylated bibenzyl as the compound responsible for the observed anti-viral activity. Extensive spectroscopic experiments provide the complete 1H NMR and 13C NMR spectral assignments to support the proposed structure. Known compounds glepidotin B and glepidotin A were also isolated from the extract and shown to be inactive in the anti-viral assay.

Computer storage of toxicology methods and postmortem drug determinations.

Two data bases have been developed by toxicologists from the Department of Scientific and Industrial Research in New Zealand. The data bases are designed to store and retrieve postmortem drug and poison levels (TOXFILE) and methods used in drug and poison determinations (TOXMETH). TOXFILE contains a list of all the analytical results determined in toxicological cases received by the three laboratories. This method of storing the data has been found superior to the previously used card systems. The file also contains a reference to the analytical method which is very important for the interpretation of the results. TOXMETH contains a list of the analytical methods which have been developed and are in use in one or more of the three Chemistry Division toxicology laboratories. Methods can be added, modified and superseded as approaches change. A Chemistry Division Report (Pannell, L.K. et al., Chemistry Division Report No. CD: 2195, 1983) has been published containing all the programs and instructions. Copies of this are available from the authors on request. Computer magnetic tapes of the programs, the report and the data are also available. The names of the deceased on the TOXFILE data file are encrypted to provide increased security of the information. The TOXFILE data file which will be supplied on magnetic tape to overseas laboratories will have the name file on all records changed to 'CD' to provide complete confidentiality.

The analysis of verapamil in postmortem specimens by HPLC and GC.

High levels of verapamil (1) were found in the blood, stomach contents, and liver and kidney specimens in a fatal overdose case. The drug was extracted from both acid and enzyme digested tissue with ether, chloroform, and 1-chlorobutane and the digests analysed by both high-performance liquid chromatography and gas chromatography. Verapamil was extracted directly from the blood and stomach contents. The extraction efficiencies of each method are discussed. The metabolite, norverapamil (II), was observed in liver and kidney extractions and identified by gas chromatography/mass spectrometry.

A modified method for the analysis of carbon monoxide in postmortem blood.

A simple spectrophotometric method is given for the determination of carbon monoxide in postmortem bloods. Duplicate determinations take about 15 minutes and are shown to be unaffected by the presence of methemoglobin. The method is therefore particularly suitable for use in a forensic laboratory. The results obtained are compared to those from the method of Commins and Lawther.

Determination of labetalol in a post mortem case using HPLC.

A case report involving low levels of labetalol is presented. Samples of post mortem stomach contents, liver, kidney, urine, and blood were flocculated with aluminium hydroxide before the labetalol was extracted by using Sep-pakTM C18 cartridges. Quantification of the extracts was performed by HPLC with an ultraviolet detector. The significant of relatively low drug levels found in a patient suffering from a respiratory disease is discussed.

A report on the analysis of orphenadrine in post mortem specimens.

A discussion of the methods reported for the analysis of orphenadrine is given. The drug levels in biological specimens and the methods reported vary widely. The stability and extractability of orphenadrine is investigated and a method suggested, which uses subtilisin digestion of tissue followed by extraction with 1-chlorobutane. Body fluids are extracted directly by 1-chlorobutane after pH adjustment. Analysis is performed by gas chromatography with a nitrogen selective detector and no interferences are observed from bland specimens. The following levels are results from a case of a 19-year-old male who was found dead 2.5 hours after last being seen alive. This laboratory analysed the case using diphenhydramine as an internal standard: blood 18.1 micrograms/mL, liver 242 micrograms/g, urine 7.0 micrograms/mL, stomach contents 1452 mg.

A high performance liquid chromatographic method for the analysis of illicit heroin.

A method using high performance liquid chromatography with an ammonium acetate-buffered acetonitrile/water mobile phase has been developed for the analysis for heroin. The method gives good resolution of the opiates found in illicit heroin.

Increased vascularity and spontaneous metastasis of breast cancer by hedgehog signaling mediated upregulation of cyr61.

The Hedgehog (Hh) pathway is well known for its involvement in angiogenesis and vasculogenesis during ontogeny. The ligand, Sonic Hh (SHH), has an important role in vascular formation during development. However, SHH expression is upregulated on tumor cells and can impact the tumor microenvironment. We have investigated the effects of autocrine as well as paracrine Hh signaling on tumor cells as well as on endothelial cells, respectively. Upon constitutive expression of SHH, breast cancer cells showed aggressive behavior and rapid xenograft growth characterized by highly angiogenic tumors that were spontaneously metastatic. In these cells, SHH caused activation of the Hh transcription factor, GLI1, leading to upregulated expression of the potent pro-angiogenic secreted molecule, CYR61 (cysteine-rich angiogenic inducer 61). Silencing of CYR61 from these SHH-expressing Hh activated cells blunted the malignant behavior of the tumor cells and resulted in reduced tumor vasculature and limited hematogenous metastases. Thus, CYR61 is a critical downstream contributor to the Hh influenced pro-angiogenic tumor microenvironment. We also observed concomitant upregulation of SHH and CYR61 transcripts in tumors from patients with advanced breast cancer, further ratifying the clinical relevance of our findings. In summary, we have defined a novel, VEGF-independent, clinically relevant, pro-angiogenic factor, CYR61, that is a transcriptional target of Hh-GLI signaling.

DNAJB6 chaperones PP2A mediated dephosphorylation of GSK3ß to downregulate ß-catenin transcription target, osteopontin.

Elevated levels of the oncoprotein, osteopontin (OPN), are associated with poor outcome of several types of cancers including melanoma. We have previously reported an important involvement of DNAJB6, a member of heat-shock protein 40 (HSP40) family, in negatively impacting tumor growth. The current study was prompted by our observations reported here which revealed a reciprocal relationship between DNAJB6 and OPN in melanoma specimens. The 'J domain' is the most conserved domain of HSP40 family of proteins. Hence, we assessed the functional role of the J domain in activities of DNAJB6. We report that the J domain of DNAJB6 is involved in mediating OPN suppression. Deletion of the J domain renders DNAJB6 incapable of impeding malignancy and suppressing OPN. Our mechanistic investigations reveal that DNAJB6 binds HSPA8 (heat-shock cognate protein, HSC70) and causes dephosphorylation of glycogen synthase kinase 3ß (GSK3ß) at Ser 9 by recruiting protein phosphatase, PP2A. This dephosphorylation activates GSK3ß, leading to degradation of ß-catenin and subsequent loss of TCF/LEF (T cell factor1/lymphoid enhancer factor1) activity. Deletion of the J domain abrogates assembly of this multiprotein complex and renders GSK3ß inactive, thus, stabilizing ß-catenin, a transcription co-activator for OPN expression. Our in-vitro and in-vivo functional analyses show that silencing OPN expression in the background of deletion of the J domain renders the resultant tumor cells less malignant despite the presence of stabilized ß-catenin. Thus, we have uncovered a new mechanism for regulation of GSK3ß activity leading to inhibition of Wnt/ß-catenin signaling.

Polychlorinated acetamides from the cyanobacterium Microcoleus lyngbyaceus.

Several new compounds were isolated from the organic extract of the cyanobacterium Microcoleus lyngbyaceus, and their structures were determined by spectroscopic means. Polychlorinated acetamidoalkynes and alkanes were the major metabolites. 6-Acetamido-1,1,1-trichloroundecane, a positional isomer of the naturally occurring 5-acetamido-1,1,1-trichloroundecane, was synthesized in six steps from delta-decanolactone.

Analysis of the disulfide linkage pattern in circulin A and B, HIV-inhibitory macrocyclic peptides.

Circulin A and B are members of a family of macrocyclic peptides, originally isolated from the tropical tree Chassalia parvifolia, that have been shown to display anti-HIV activity. Complete structural elucidation of these highly constrained peptides was difficult due to their cyclic amide backbone and the presence of six disulfide-linked cysteines. In the present study, the disulfide pairing motif of circulin A and circulin B was determined. Since the circulins were resistant to enzymatic proteolysis, cysteine residue pairings were identified by analysis of the complex mixture of cleavage products that resulted from partial acid hydrolysis of the native peptides. Combined utilization of HPLC, fast atom bombardment mass spectrometry and peptide recognition software ("F-MASS" and "F-LINK" programs) were employed to identify the cleavage products. Thus, we were able to unambiguously identify the disulfide linkage pattern in circulin A and circulin B as Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6, where the numbers on the cystine residues refer to their respective order in the peptides.

In vivo mechanism-based inactivation of S-adenosylmethionine decarboxylases from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.

S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits. The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation. A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product. Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078-4081].

Structures of five components of the actinomycin Z complex from Streptomyces fradiae, two of which contain 4-chlorothreonine.

Structure elucidation of five components of the actinomycin Z complex (Z(1)-Z(5)) isolated from Streptomyces fradiae is described. The components were separated by Si gel column chromatography and TLC/PLC and analyzed by ESIMS, FABMS, LC-MS of derivatized hydrolysates, and 2D NMR techniques. This permitted determination of the complete structures of actinomycins Z(1)-Z(5). In Z(3) and Z(5,) site 1 of the beta-depsipeptide is occupied by the rare 4-chloro-L-threonine, an amino acid not previously found in an actinomycin. The structural variants of the actinomycin Z complex have the molecular architecture typical of other actinomycins but possess greater structural diversity resulting from the presence of several highly unusual amino acids. Actinomycins Z(3) and Z(5,) but not Z(1), were more potent than actinomycin D in cytotoxicity assays against three tumor cell lines.