Bringing Machine-Learning Enhanced Quantum Chemistry and Microwave Spectroscopy to Conformational Landscape Exploration: the Paradigmatic Case of 4-Fluoro-Threonine.

A combined experimental and theoretical study has been carried out on 4-fluoro-threonine, the only naturally occurring fluorinated amino acid. Fluorination of the methyl group significantly increases the conformational complexity with respect to the parent amino acid threonine. The conformational landscape has been characterized in great detail, with special attention given to the inter-conversion pathways between different conformers. This led to the identification of 13 stable low-energy minima. The equilibrium population of so many conformers produces a very complicated and congested rotational spectrum that could be assigned through a strategy that combines several levels of quantum chemical calculations with the principles of machine learning. Twelve conformers out of 13 could be experimentally characterized. The results obtained from the analysis of the intra-molecular interactions can be exploited to accurately model fluorine-substitution effects in biomolecules.

Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase (PNP) in prostate cancer.

BACKGROUND: Our recent analyses of miRNA expression signatures showed that miR-1 and miR-133a were significantly reduced in several types of cancer. Interestingly, miR-1 and miR-133a are located on the same chromosomal locus in the human genome. We examined the functional significance of miR-1 and miR-133a in prostate cancer (PCa) cells and identified the novel molecular targets regulated by both miR-1 and miR-133a. METHODS AND RESULTS: The expression levels of miR-1 and miR-133a were significantly downregulated in PCa compared with non-PCa tissues. Restoration of miR-1 or miR-133a in PC3 and DU145 cells revealed significant inhibition of proliferation, migration, and invasion. Molecular target identification by genome-wide gene expression analysis and luciferase reporter assay showed that purine nucleoside phosphorylase (PNP) was directly regulated by both miRNAs. Silencing of the PNP gene inhibited proliferation, migration, and invasion in both PC3 and DU145 cells. Immunohistochemistry detected positive staining of PNP in PCa specimens. CONCLUSIONS: Downregulation of miR-1 and miR-133a was a frequent event in PCa and both function as tumour suppressors. The PNP is a novel target gene of both miRNAs and potentially functions as an oncogene. Therefore, identification of novel molecular networks regulated by miRNAs may provide new insights into the underlying causes of PCa oncogenesis.

Pulse dye densitometry using indigo carmine is useful for cardiac output measurement, but not for circulating blood volume measurement.

BACKGROUND AND OBJECTIVE: We evaluated the validity of a newly developed pulse dye densitometer for indigo carmine for measuring cardiac output and circulating blood volume. METHODS: Measurements of cardiac output and circulating blood volume were performed with the indigo carmine densitometer during normovolaemia, hypovolaemia and hypervolaemia in nine mongrel dogs under general anaesthesia. The validity was evaluated by comparison of the values of cardiac output and circulating blood volume obtained by the thermodilution technique and the 51Cr-labelled red blood cell method, respectively. We also examined indigo carmine removal by continuous veno-venous haemofiltration after indigo carmine injection. RESULTS: There was good agreement between dye densitometer- and thermodilution-derived cardiac output (r = 0.885, P < 0.001). The bias and limits of agreement of these values were 0.09 and+/-1.07 L min(-1) (2 SD, n 22), respectively. The dye-densitometer-derived circulating blood volume was greater than that of the 51Cr-labelled red blood cell method, and both values showed weak agreement (r = 0.587, P < 0.027). The sieving coefficient of indigo carmine through continuous veno-venous haemofiltration was 0.34+/-0.06. CONCLUSIONS: These data indicate that indigo carmine densitometry is a reliable method for cardiac output determination, but it overestimates circulating blood volume, probably due to the transition of indigo carmine into the extravascular space in the systemic circulation.

Characterization and baculovirus-directed expression of a myosuppressin encoding cDNA from the true armyworm, Pseudaletia unipuncta.

Insect myosuppressins are a highly conserved sub-family of peptides which are primarily characterized by the ability to suppress contraction of visceral muscles in a variety of insect species. We have isolated a cDNA from the true armyworm, Pseudaletia unipuncta, that encodes a prohormone containing a peptide identical to ManducaFLRFamide. We have shown that this myosuppressin gene appears to be expressed in late larval and adult insects. In Manduca sexta, a number of extended-FLRFamide peptides have previously been purified including ManducaFLRFamide, F7D (DPSFLRFamide), F7G (GNSFLRFamide) and two larger peptides F24 and F39 that contain the shorter ManducaFLRFamide sequence at their C-terminus. Comparison with the true armyworm prepropeptide characterized here identifies F24 and F39 as partially processed products from the same precursor. Expression in the true armyworm was shown by in situ hybridization to occur in over 150 cells throughout the adult brain and nerve cord, and also to occur in both open and closed endocrine type cells of the gut. Overexpression of the P. unipuncta FLRFamide cDNA from a baculovirus vector in cabbage looper caterpillars was used to assess the potential for myosuppressin expression as a means of enhancing virus efficacy. Viral expression of the armyworm prohormone cDNA resulted in raised levels of RFamide-like products in the hemolymph of infected insects, but the products were found to be chemically distinguishable from authentic mature peptide and probably represent partially processed hormone.

Effects of an allatostatin and a myosuppressin on midgut carbohydrate enzyme activity in the cockroach Diploptera punctata.

Neuropeptides of the cockroach allatostatin (AST) family are known for their ability to inhibit the production of juvenile hormone by the corpora allata of cockroaches. Since their discovery, they have also been shown to modulate myotropic activity in a range of insect species as well as to act as neurotransmitters in Crustaceans and possibly in insects. The midgut of cockroaches contains numerous endocrine cells, some of which produce AST whereas others produce the FMRFamide-related peptide, leucomyosuppressin (LMS). We have determined if ASTs and LMS are also able to influence carbohydrate-metabolizing enzyme activity in the midgut of the cockroach, Diploptera punctata. Dippu-AST 7 stimulates activity of both invertase and alpha-amylase in a dose-dependent fashion in the lumen contents of ligatured midguts in vitro, but not in midgut tissue, whereas the AST analog AST(b)phi2, a cyclopropyl-ala, hydrocinnamic acid analog of Dippu-AST 6, has no effect. Leucomyosuppressin also stimulates enzyme activity in lumen contents only, although the EC50 is considerably greater than for Dippu-AST. Dippu-AST is also able to inhibit proctolin-induced contractions of midgut muscle, and this action had already been described for LMS [18]. Thus, in this organ, AST and LMS have at least two distinct physiological effects.

[The anesthetic management of tracheal T-tube exchange using Patil-Syracuse mask].

We exchanged the tracheal T-tube inserted to a 17-year-old female, who wanted to be able to enunciate again, with relapsing polychondritis and difficulty in enunciation which the proximal end of tracheal T-tube above the false vocal cords was causing. The procedure was performed using Patil-Syracuse mask, without tracheal intubation, under-general anesthesia. This method will ensure precise length adjustment and correct placement of the T-tube under fiberoptic bronchoscope.

[Fiberoptic bronchoscopy-aided tracheal intubation using a patil-syracuse mask in a "can ventilate/cannot intubate" situation].

We could perform endotracheal intubation in three patients whose ventilation had been anticipated possible preoperatively but endotracheal intubation impossible, using a fiberoptic bronchoscope while ventilating via Patil-Syracuse mask. This method is an alternative in a "can ventilate/cannot intubate" situation.

The muscular contractions of the midgut of the cockroach, Diploptera punctata: effects of the insect neuropeptides proctolin and leucomyosuppressin.

We have previously shown differential expression of leucomyosuppressin (LMS) mRNA in apparent endocrine cells in the anterior region of midguts of the cockroach Diploptera punctata, using in situ hybridization. In contrast, other FMRFamide-related peptides, as revealed by immunohistochemistry, have been found most abundantly in the posterior region in both apparent endocrine cells and nerve tracts. Here, we partially purified extracts of anterior and posterior cockroach midguts, using HPLC coupled with radioimmunoassay, and found, among multiple FMRFamide-like immunoreactive fractions, one fraction co-eluting with LMS in both regions. The presence of a co-eluting fraction in the posterior region, in the absence of LMS mRNA positive endocrine cells suggests that LMS might therefore be present in nerve tracts running along the length of the midgut. Using a circular muscle contraction assay from different portions of midgut, we determined the effects of LMS, proctolin and a variety of other midgut peptides on contractions of the midgut of Diploptera. Proctolin caused a sustained tonic contraction in the anterior midgut, the amplitude of which was dose-dependent. In contrast, LMS, and its relative SchistoFLRFamide, reduced the amplitude of these contractions. LMS and SchistoFLRFamide also inhibited spontaneous phasic contractions, which were elicited by proctolin application in only a few preparations. Other postulated midgut peptides did not induce or inhibit contractions, nor augment the proctolin-induced contractions. The C-terminal truncated sequences of LMS, HVFLRFamide and VFLRFamide, were sufficient to reduce the amplitude of the proctolin-induced contractions. This work illustrates a possible physiological role for LMS in Diploptera midguts, in the passage of food along the alimentary canal.

In situ hybridization analysis of leucomyosuppressin mRNA expression in the cockroach, Diploptera punctata.

In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system, stomatogastric nervous system, and midgut suggests that LMS may play a central role in Diploptera and may be associated with feeding and digestion.

Elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities in the sera and milk of cows with naturally occurring coliform mastitis.

The presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities was determined in milk and serum of cows with naturally occurring coliform mastitis (CFM). TNF-alpha was detected in the sera from 26 of 32 cows with CFM. TNF-alpha levels were higher in the sera than in the milk. IL-6 was high in the sera of surviving CFM animals, but was low in animals that died and in healthy controls. Furthermore, the mean level of IL-6 was 20-fold higher in the milk than in the sera of mastitic cows. The level of IL-6 in the serum was correlated to that in the milk in individual animals. The presence of IL-6 and TNF-alpha in the sera appears to relate to severe clinical condition of CFM, in the milk whereas they may play a role in generating inflammation of the mammary gland.

Cardiac output and circulating blood volume analysis by pulse dye-densitometry.

OBJECTIVE: Pulse dye-densitometry (PDD) is a newly developed method for monitoring the indocyanine green (ICG) concentration in an artery with which cardiac output (CO) and circulating blood volume (CBV) can be determined. We evaluated its accuracy for clinical use. METHODS: In 7 patients under general anesthesia, ICG-sensitive optical probes (805 and 890 nm) were attached to a finger. Following injection of ICG, the arterial concentration of dye was recorded optically by the non-invasive test instrument and sampled arterial blood ICG concentration was also measured photometrically for comparison. In order to validate the PDD analysis, CO was also measured by both the dye dilution cuvette method and by thermodilution in 8 patients scheduled for coronary artery bypass grafting. In 30 other patients, CBV assessed by PDD was compared with its value estimated from body size. RESULTS: The blood dye concentration correlated well with the values obtained by PDD (r = 0.953, p < 0.01). Mean bias for the test PDD CO was +0.15 +/- 0.72 min l-1 (not significant (n.s.)) compared with the cuvette method while the mean bias of the thermodilution method vs the cuvette method was +0.79 +/- 0.84 min l-1 (p < 0.0001.). The average value of CBV obtained by PDD was 3.81 +/- 1.39 L compared with that estimated value, 3.72 +/- 0.77 L (n.s.). CONCLUSIONS: CO determined by PDD agrees well with cuvette densitometry, and somewhat less well with CO by thermodilution. The new method, by not requiring a pulmonary arterial catheter, is less invasive than either older method, and yields in addition a value of CBV.

Molecular characterization of the inhibitory myotropic peptide leucomyosuppressin.

The myoinhibitory peptide leucomyosuppressin (LMS) (pQDVDHVFLRFamide) has been identified and characterized at the molecular level in the cockroach Diploptera punctata through analysis of the organization of both brain cDNA and genomic DNA. Processing of the precursor predicted from DNA sequence would release a single LMS peptide. The organization of the precursor appears to be conserved in other insects and may reflect a functional organization for this subfamily of extended FLRFamides. The expression of the LMS gene appears in numerous cells of the pars-intercerebralis of the cockroach protocerebellum as well as in numerous endocrine cells of the midgut.

The distribution and partial characterization of FMRFamide-related peptides in the salivary glands of the locust, Locusta migratoria.

The distribution and partial characterization of FMRFamide-related peptides in the salivary glands of the locust, Locusta migratoria, were investigated by means of immunohistochemistry, radioimmunoassay and reversed-phase high performance liquid chromatography. Whole-mount preparations of glands stained positively against anti-FMRFamide antisera, and contained the equivalent of 837 +/- 80 fmol FMRFamide/gland pair, as determined by radioimmunoassay. FMRFamide-like immunoreactivity occurred in the processes of the transverse nerves of both the prothoracic and mesothoracic ganglia, but was not found in the salivary motoneurons 1 or 2 of the suboesophageal ganglion, both of which directly innervate the salivary glands via the salivary nerve 7b; nor was it found within the salivary nerve 7b itself, leading to the salivary glands. It was, however, found as a superficial nerve plexus on the surface of nerve 7 at the suboesophageal ganglion, but did not appear to extend to the salivary glands. The origin of this staining is unclear. High performance liquid chromatography of salivary gland tissue extracts, monitored by radioimmunoassay, revealed 4 peaks of immunoreactive material, 2 of which co-migrated with AFIRFamide and GQERNFLRFamide, previously isolated from the locust ventral nerve cord. These 2 synthetic peptides did not elevate basal levels of the second messengers cyclic AMP or cyclic GMP in the salivary glands.

Characterization of the gene for leucomyosuppressin and its expression in the brain of the cockroach Diploptera punctata.

Using HPLC separation, radioimmunoassay, and subsequent bioassay, we have detected the presence of an active peptide, which co-elutes with the insect myoinhibitory peptide leuco-myosuppressin, in the brain of the cockroach Diploptera punctata. We have isolated a cDNA encoding the precursor for this peptide from cDNA libraries representing D. punctata brain RNA. The cDNA sequence contains an open reading frame that upon translation would result in a prepropolypeptide of 96 amino acids. Proteolytic cleavage of the predicted precursor could result in several peptides, including a 10 amino acid C-terminal peptide that would, upon modification of the NH2 and COOH-terminal amino acids, be identical to the insect FLRFamide, leucomyosuppressin. No other RFamide products are predicted to be processed from the precursor. Southern blot analysis indicates that the gene is present in the D. punctata genome in a single copy. Northern blot analysis shows that the gene is predominantly expressed as a 3.8 kb mRNA in cockroach brain. Study of the expression of the leucomyosuppressin gene in D. punctata brain, using in situ hybridization, indicates that expression occurs primarily in the pars intercerebralis of the protocerebrum, a region showing abundant FMRFamide-like immunoreactive neurosecretory cells. Immunohistochemistry and HPLC coupled to radioimmunoassay indicates that leucomyosuppressin represents a significant proportion of FMRFamide-related peptide production in the brain. However, HPLC analysis also indicates the presence of significant levels of other related peptides, demonstrating the presence of more than one FMRFamide-related gene in this insect.

Poisoning by the red alga 'ogonori' (Gracilaria verrucosa) on the Nojima Coast, Yokohama, Kanagawa Prefecture, Japan.

A food poisoning case due to the ingestion of 'ogonori', an edible red alga, occurred at Yokohama, Kanagawa Prefecture, Japan, in late October 1993, resulting in two victims, including one death (female). No causative agent present in the ogonori was found from a routine bioassay for marine toxins. From the production of increased amounts of prostaglandins (PGs), mainly PGE2, by the alga on stimulation by cutting or soaking in fresh water, and further increase of PGE2 by addition of arachidonic acid, it appeared that an enzyme, probably fatty acid cyclooxygenase, in the ogonori and the body of the victim, was acting on the highly unsaturated fatty acids in the oil of the ingested seafood and in the blood hemorrhaged from the stomach of the victim. This resulted in the production of over 30 mg of PGE2 and small amounts of other PGs in a comparatively short time. With this dosage the victim suffered from nausea, vomiting, and hypotension, and died of hypotensive shock. PGE2 seems to work more selectively on females. This type of poisoning is very unusual, and differs from the more familiar forms of poisoning occurring after ingestion of marine organisms.

Preventive equipment for urinary incontinence: a device employing lower abdominal impendance changes.

The object of this work was to develop a monitoring device for measuring the volume of urine in the bladder to signal, in advance, the need to urinate and thus help prevent urinary incontinence. A high-frequency electric current of 50 KHz transmitter was applied to a pair of terminals placed on the surface of a human body. The constant current was 2mA (p-p). A pair of voltage electrodes was placed in the path of the electric current to pick up high-frequency voltage signals. The best position for current electrodes was found to be on the surface of both femoral joints. Positioning the voltage electrode above the bladder on the lower abdomen was most efficient for detecting the volume of urine. New current electrodes sutured onto underpants were fitted on the surface above the femoral joints, which did not shift in the case of movement by the subjects. The reproducibility and stability were remarkably good with this electrode. The alarm level which foretold both maximum storage volume and maximum desire to void was examined. After that, when the alarm level was set within the range of impedance charge, 60% to 70%, the possibility of prediction of urination exists.

Stress-induced failure of osmoregulation in the parasitic nematode Pseudoterranova decipiens: indirect evidence for hormonal regulation.

When third-stage larvae of Pseudoterranova decipiens maintained at 5 degrees C are placed in either 40% artificial sea water (ASW, iso-osmotic) or 15% ASW (hypo-osmotic) and weighed once at 0 h and again at 24 h, they neither lose nor gain weight, and the osmotic pressure (OP) of their pseudocoelomic fluid (PCF) remains unchanged. In contrast, when worms are weighed six additional times during the 24 h interval, those maintained in isoosmotic conditions lose weight, while those maintained in hypo-osmotic conditions gain weight. Worms which had been exposed to hypo-osmotic conditions and weighed at various times between 0 and 24 h exhibited an increase in weight which was correlated with the number of weighings. Worms exposed to hypo-osmotic conditions and weighed three additional times between 0 and 24 h also gained weight, and the OP of the PCF decreased such that worms experiencing the greatest increase in weight suffered the greatest dilution of the PCF. In worms ligatured at the head or tail or at the head and tail, and then exposed to either 15% or 40% ASW, the effect of multiple weighings is exaggerated in a complex way. The presence of a ligature on the tail in worms immersed in an iso-osmotic medium leads to an increase in weight and to a very marked additional increase in weight in worms immersed in a hypo-osmotic medium. The presence of a head ligature in worms in an iso-osmotic medium leads to a decrease in weight and to a smaller weight gain in a hypo-osmotic medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Osmoregulation in the parasitic nematode Pseudoterranova decipiens.

When subjected to hyper- or hypo-osmotic stress at 5 degrees C for 24 h, third-stage larvae of the parasitic nematode Pseudoterranova decipiens do not exhibit changes in mass or in the osmotic pressure of the pseudocoelomic fluid. Immersion in solutions containing 3H2O demonstrates that exchange with the water in the pseudocoelomic fluid is substantially complete within 24 h. Sacs composed of cylinders of body wall without the intestine and pseudocoelomic fluid do not gain weight when immersed for 24 h in hypotonic medium. Metabolic poisons abolish the ability of whole worms and sacs to maintain their weight when immersed in hypotonic media. These observations support the conclusion that the nematode is capable of at least short-term osmoregulation and that the site of osmoregulation is the body wall. The observations that more fluid is passed from the anus in some hypo-osmotically stressed worms and that worms ligatured at the tail exhibit a small increase in mass when exposed to hypo-osmotic conditions may indicate that the intestine plays a minor and subsidiary role in osmoregulation.

Water compartments and osmoregulation in the parasitic nematode Pseudoterranova decipiens.

A study of the time course of penetration of 3H2O into whole worms suggests that worms immersed in a hypo-osmotic environment (15% artificial sea water) reach full exchange equilibrium more slowly than worms in an iso-osmotic environment (40% artificial sea water). The apparent water content, determined by dry mass, matches that determined by 3H2O exchange when worms are immersed for 24 h in 40% artificial sea water (ASW), but the water content measured by 3H2O exchange is lower when worms are kept in a hypo-osmotic environment for 24 h. These differences disappear after 48 h. No such differences are apparent when sacs, consisting of cylinders of body wall lacking their intestines and pseudocoelomic fluid and closed at both ends by ligatures, are immersed in either 40% or 15% ASW for 24 h. The placing of ligatures at the head, but not at the tail, results in a failure of worms immersed in 40% ASW or 15% ASW containing 3H2O to achieve full exchange equilibrium within 24 h. These results suggest that although worms immersed in an iso-osmotic environment drink, those immersed in a hypo-osmotic environment do not, a conclusion supported by studies involving the addition of [14C]inulin to the medium. The application of ligatures to the head and tail of worms immersed in 40% ASW results in a slower penetration of 3H2O into the pseudocoelomic fluid, whereas similar ligatures do not further retard the penetration in worms exposed to 15% ASW. The results are consistent with a model which sees the pseudocoelomic fluid as consisting of two compartments containing water, one of which exchanges more slowly than the other.