Formation of diacetyl by cell-free extracts of Leuconostoc lactis

Abstract Diacetyl formation was linear with time and with protein concentration when a cell-free extract of Leuconostoc lactis NCW1 was added to a buffer system containing pyruvate, thiamine pyrophosphate and MgS₄ (final concentrations 60 mM, 0.11 mM and 0.22 mM, respectively). No diacetyl was detected in the absence of pyruvate or cell-free extract and no increase in diacetyl formation was detected on the addition of acetyl-CoA. When 2-acetolactate (1.6 mM) was the substrate, autodecarboxylation to diacetyl and acetoin occurred under aerobic and anaerobic conditions. When cell-free extract was added, decarboxylation of 2-acetolactate to acetoin and diacetyl increased 4–6-fold, under aerobic and anaerobic conditions. When the cell-free extract was boiled, diacetyl formation from 2-acetolactate was reduced to the level of autodecarboxylation. The results suggest that diacetyl is formed enzymatically in the presence and absence of oxygen, as well as spontaneously, from 2-acetolactate.     

Fourier transform infrared microspectroscopy is a new way to look at plant cell walls

Highly reproducible Fourier transform infrared (FTIR) spectra from both single onion (Allium cepa) cell walls and their constituent polymers were obtained under a variety of sampling conditions. The specificity of the chemical extraction sequence used in the preparation of the material was confirmed: pectins only are extracted by cyclohexanediaminetetraacetic acid and sodium carbonate, whereas xyloglucans are extracted by increasing concentrations of potassium hydroxide. There was very little contamination of the first potassium hydroxide extract with residual pectin. The low abundance of both phenolics and protein was also confirmed. The first sodium carbonate extraction almost completely removes esters remaining in the cell wall. We have demonstrated that FTIR spectroscopy can detect large conformational changes in pectic polymers on removal from the cell wall and on drying. FTIR spectroscopy provides a powerful and rapid assay for wall components and putative cross-links by identifying polymers and functional groups nondestructively in muro. The availability of micro-sampling and data acquisition techniques that permit subtraction of the blanket absorption of water make FTIR spectroscopy particularly suitable for studies of cell wall architecture. The use of polarizers with the microscope accessory permits determination of the orientation of particular functional groups with respect to the direction of cell elongation in carrot suspension cells.     

Detection of cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies raised against halothane metabolite adducts

Antibodies raised against halothane metabolite adducts cross-react with S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine metabolite adducts. Using these antibodies in immunohistochemical experiments, metabolite binding was localized to the damaged areas of the proximal tubule after treatment of male rats with TFEC. Immunoblot analysis of subcellular fractions of rat kidney tissue after in vivo treatment with TFEC revealed a high specificity for binding of metabolites to proteins of the mitochondrial fraction. These proteins may represent target molecules which play a role in cysteine conjugate induced nephrotoxicity.     

Effect of estrogen on the response of thyroid stimulating hormone to substance P in rats

The role of substance P (SP) in the control of thyroid stimulating hormone (TSH) release and the influence of gonadal steroid were investigated. Intravenous administration of SP failed to alter plasma levels of TSH in ovariectomized (OVX) rats, whereas SP induced a significant increase in plasma TSH in estradiol benzoate-primed (Eb-primed) OVX rats (P less than 0.001). Further, intravenously administered SP did not affect the plasma TSH concentration in normal male rats, but significantly increased it in Eb-primed castrated male rats (P less than 0.01). These data suggest possible roles for SP at the level of the hypothalamus and/or the pituitary gland in stimulating TSH secretion under the influence of estrogen.     

Electrical properties of developing rat heart. Effects of dexamethasone

Action potentials recorded from perinatal rat ventricles exhibited a plateau (phase 2), followed by a rapid repolarization characteristics of all mammalian ventricular cells. Within the second postnatal week, a number of distinct changes occurred in the contour of action potentials. An early slow depolarization, at the foot of the action potential, preceded the beginning of phase zero. The early slow depolarization was observed until day 12 and disappeared by day 13. A second slow depolarization occurred during the terminal phase of the rapid upstroke of the action potential, persisted through day 13 and disappeared by day 14. On day 12, what had been a homogeneous contour of action potentials seen during the first week converted into a heterogeneous contour. Occasionally, action potentials similar to those recorded from Purkinje fibres in adult heart were recorded from hearts as young as 12 days. By day 14, signs of a spike (the hallmark of action potentials from adult heart) were apparent in some fibres. Treatment of newborn rats with dexamethasone on the second day after birth prevented the disappearance of the second slow depolarization. In adult and aged rat hearts, dexamethasone treatment induced a slow depolarization and a plateau in the region of overshoot. In view of the time-dependent change of the second slow depolarization it is suggested that this phase of the action potential is influenced by the levels of circulating glucocorticoid in developing heart and by changes in calcium sensitivity observed in this species. Heterogeneity of action potentials observed on day 12 postnatal may precede structural differentiation of myofilaments.     

Effects of the gonadotropin-releasing hormone associated peptides (GAP) on the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in vivo

Six peptide sequences residing between basic amino acid residues in GAP were tested for effects on the release of FSH, LH and PRL in vivo in ovariectomized, estrogen-progesterone-primed (OEP) rats. Synthetic GAP peptides (1–13, 1–23, 15–23, 25–36, 38–53 and 41–53) were injected intravenously (IV) into conscious OEP rats and plasma levels of FSH, LH and PRL were measured by RIA. The activity of GAP peptides in the control of PRL was further examined in ether-stressed male rats which were injected IV with GAP peptides just prior to a 1-min etherization. GAP(1–13) significantly stimulated FSH release at doses of 1, 10 and 100 μg, whereas it stimulated LH release only at the highest dose of 100 μg. GAP(1–23) elevated plasma levels of FSH and LH only at a dose of 100 μg. The other 4 peptides had no effect on the release of gonadotropins. Of these 6 peptides, only GAP(1–13) partially lowered the plasma levels of PRL at the high dose of 100 μg in OEP rats, but it had no effect on the ether-induced PRL surge at doses of 10 and 100 μg. In conclusion, both GAP(1–13) and GAP(1–23) stimulate FSH and LH release in vivo; these 2 peptides are much less potent in stimulating gonadotropin release than is LHRH. GAP(1–13) exerts a preferential FSH-releasing activity, but its PRL-inhibiting activity is minimal.     

Metabolic and hemodynamic responses of lower limb during exercise in patients with COPD

Premature lacticacidosis during exercise in patients with chronic obstructive pulmonarydisease (COPD) may play a role in exercise intolerance. In this study,we evaluated whether the early exercise-induced lactic acidosis inthese individuals can be explained by changes in peripheralO₂ delivery(O₂).Measurements of leg blood flow by thermodilution and of arterial andfemoral venous blood gases, pH, and lactate were obtained during astandard incremental exercise test to capacity in eight patients withsevere COPD and in eight age-matched controls. No significantdifference was found between the two groups in leg blood flow at restor during exercise at the same power outputs. Blood lactateconcentrations and lactate release from the lower limb were greater inCOPD patients at all submaximal exercise levels (allP < 0.05). LegO₂at a given power output was not significantly different between the twogroups, and no significant correlation was found between this parameterand blood lactate concentrations. COPD patients had lower arterial andvenous pH at submaximal exercise, and there was a significant positivecorrelation between venous pH at 40 W and the peakO₂ uptake(r = 0.91, P < 0.0001). The correlation betweenvenous pH and peak O₂ uptakesuggests that early muscle acidosis may be involved in early exercisetermination in COPD patients. The early lactate release from the lowerlimb during exercise could not be accounted for by changes inperipheralO₂. The present results point to skeletal muscle dysfunction as being responsible for the early onset of lactic acidosis inCOPD.

     

Median eminence lesions reveal separate hypothalamic control of pulsatile follicle-stimulating hormone and luteinizing hormone release

The effects of hypothalamic lesions designed to destroy either the anterior median eminence (ME) or the posterior and mid-ME on pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined in castrated male rats. In sham-operated animals, mean plasma FSH concentrations rose to peak at 10 min after the onset of sampling, whereas LH declined to a nadir during this time. In the final sample at 120 min, the mean FSH concentrations peaked as LH decreased to its minimal value. In rats with anterior ME lesions, there was suppression of LH pulses with continuing FSH pulses in 12 of 21 rats. On the other hand, in animals with posterior to mid-ME lesions, 3 out of 21 rats had elimination of FSH pulses, whereas LH pulses were maintained. Fifteen of 42 operated rats had complete ME lesions, and pulses of both hormones were abolished. The remaining 12 rats had partial ME lesions that produced a partial block of the release of both hormones. The results support the concept of separate hypothalamic control of FSH and LH release with the axons of the putative FSH-releasing factor (FSHRF) neuronal system terminating primarily in the mid- to caudal ME, whereas those of the LHRH neuronal system terminate in the anterior and mid-median eminence. We hypothesize that pulses of FSH alone are mediated by release of the FSHRF into the hypophyseal portal vessels, whereas those of LH alone are mediated by LHRH. Pulses of both gonadotropins simultaneously may be mediated by pulses of both releasing hormones simultaneously. Alternatively, relatively large pulses of LHRH alone may account for simultaneous pulses of both gonadotropins since LHRH has intrinsic FSH-releasing activity.     

Localization of hypophysiotropic neurohormones by assay of sections from various brain areas.

The results of studies of the localization of the hypothalamic hypophysiotropic factors based on their direct determination in sections or nuclear punches are described. Luteinizing hormone-releasing hormone was found in high concentrations in the median eminence-arcuate nucleus complex, in lower concentrations in the mediobasal zone of the preoptic area. In addition to these hypothalamic sites, it is present in all four periventricular organs, especially in the organum vasculosum laminae terminalis. Thyrotropin releasing hormone has a widespread distribution. High concentrations are in the median eminence, arcuate nucleus, dorsomedial nucleus, and anterior part of the ventromedial nucleus. Lower concentrations are in several other structures of the hypothalamus, preoptic area and septum, and low but measurable quantities are found in most of the structures of the brain. Somatostatin is also present in most structures of the central nervous system, with highest concentrations in the median eminence, arcuate nucleus, ventromedial nucleus and periventricular nucleus. There are indications that the ventromedial nucleus or its immediate vicinity contains growth hormone releasing factor. Prolactin releasing activity was present in the median eminence and mediobasal parts of the anterior hypothalamus, whereas prolactin inhibitory activity was in the dorsolateral parts of the anterior hypothalamus and/or preoptic area.     

Role of transmitters in mediating hypothalamic control of electrolyte excretion.

Changes in urinary volume and electrolyte excretion were monitored after the injection of cholinergic and monoaminergic drugs into the third cerebral ventricle of conscious male rats made diuretic by an intravenous infusion of 5% dextrose. A natriuretic and kaliuretic response was induced by the intraventricular injection of norephrine (NE) or carbachol, whereas dopamine (DA) had no effect. The beta-receptor stimulator isoproterenol (ISO) induced an antinatriuretic and antikaliuretic effect. Intraventricular injection of the alpha-adrenergic blocker phentolamine abolished the natriuretic response to NE and carbachol and to intraventricular hypertonic saline (HS). By contrast, the beta-adrenergic blocker propranolol induced a natriuresis and kaliuresis when injected alone and an additive effect when its injection was followed by NE or HS. Propranolol potentiated the natriuretic response to carbachol. Cholinergic blockade with atropine diminished the response to NE and blocked the natriuretic response to HS. It is suggested that sodium receptors in the ventricular wall can modify renal sodium excretion via a stimulatory pathway involving cholinergic and alpha-adrenergic receptors and can inhibit sodium excretion via a tonically active beta-receptor pathway.