Diverged subpopulations in tropical Urochloa (Brachiaria) forage species indicate a role for facultative apomixis and varying ploidy in their population structure and evolution.

BACKGROUND: Urochloa (syn. Brachiaria) is a genus of tropical grasses sown as forage feedstock, particularly in marginal soils. Here we aimed to clarify the genetic diversity and population structure in Urochloa species to understand better how population evolution relates to ploidy level and occurrence of apomictic reproduction. METHODS: We explored the genetic diversity of 111 accessions from the five Urochloa species used to develop commercial cultivars. These accessions were conserved from wild materials collected at their centre of origin in Africa, and they tentatively represent the complete Urochloa gene pool used in breeding programmes. We used RNA-sequencing to generate 1.1 million single nucleotide polymorphism loci. We employed genetic admixture, principal component and phylogenetic analyses to define subpopulations. RESULTS: We observed three highly differentiated subpopulations in U. brizantha, which were unrelated to ploidy: one intermixed with U. decumbens, and two diverged from the former and the other species in the complex. We also observed two subpopulations in U. humidicola, unrelated to ploidy; one subpopulation had fewer accessions but included the only characterized sexual accession in the species. Our results also supported a division of U. decumbens between diploids and polyploids, and no subpopulations within U. ruziziensis and U. maxima. CONCLUSIONS: Polyploid U. decumbens are more closely related to polyploid U. brizantha than to diploid U. decumbens, which supports the divergence of both polyploid groups from a common tetraploid ancestor and provides evidence for the hybridization barrier of ploidy. The three differentiated subpopulations of apomictic polyploid U. brizantha accessions constitute diverged ecotypes, which can probably be utilized in hybrid breeding. Subpopulations were not observed in non-apomictic U. ruziziensis. Sexual Urochloa polyploids were not found (U. brizantha, U. decumbens) or were limited to small subpopulations (U. humidicola). The subpopulation structure observed in the Urochloa sexual-apomictic multiploidy complexes supports geographical parthenogenesis, where the polyploid genotypes exploit the evolutionary advantage of apomixis, i.e. uniparental reproduction and clonality, to occupy extensive geographical areas.

The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor.

For over 25 years, the cytokine known as macrophage migration inhibitory factor (MIF) has been considered to be a product of activated T lymphocytes. We recently identified the murine homolog of human MIF as a protein secreted by the pituitary in response to endotoxin administration. In the course of these studies, we also detected MIF in acute sera obtained from endotoxin-treated, T cell-deficient (nude), and hypophysectomized mice, suggesting that still more cell types produce MIF. Here, we report that cells of the monocyte/macrophage lineage are an important source of MIF in vitro and in vivo. We observed high levels of both preformed MIF protein and MIF mRNA in resting, nonstimulated cells. In the murine macrophage cell line RAW 264.7, MIF secretion was induced by as little as 10 pg/ml of lipopolysaccharide (LPS), peaked at 1 ng/ml, and was undetectable at LPS concentrations > 1 microgram/ml. A similar stimulation profile was observed in LPS-treated peritoneal macrophages; however, higher LPS concentrations were necessary to induce peak MIF production unless cells had been preincubated with interferon gamma (IFN-gamma). In RAW 264.7 macrophages, MIF secretion also was induced by tumor necrosis factor alpha (TNF-alpha) and IFN-gamma, but not by interleukins 1 beta or 6. Of note, MIF-stimulated macrophages were observed to secrete bioactive TNF-alpha. Although previously overlooked, the macrophage is both an important source and an important target of MIF in vivo. The activation of both central (pituitary) and peripheral (macrophage) sources of MIF production by inflammatory stimuli provides further evidence for the critical role of this cytokine in the systemic response to tissue invasion.

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward.

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with beta-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.

Complications following tarsal arthrodesis using bone plate fixation in dogs.

OBJECTIVES: To report the complications encountered following tarsal arthrodesis surgery with bone plate fixation and describe the previously unreported complication of plantar necrosis. METHODS: Medical records of 40 dogs that had been treated by tarsal arthrodesis with bone plate fixation were reviewed to determine the major and minor complications and the associated risk factors. RESULTS: The major complication rate was 32.5 per cent and the minor complication rate was 42.5 per cent. Pantarsal arthrodeses had a higher major complication rate than partial tarsal arthrodeses. Plantar necrosis was the most common major complication and occurred in 15 per cent of cases. Plantar necrosis occurred more frequently when a bone plate was applied to the medial aspect of the hock, and only occurred in cases where tarsometatarsal joint arthrodesis was performed. CLINICAL SIGNIFICANCE: Plantar necrosis is a catastrophic complication that may be associated with injury to the dorsal pedal artery or perforating metatarsal artery. Application of a bone plate to the medial aspect of the hock should be performed with care during tarsal arthrodesis, particularly where the tarsometatarsal joint is debrided of cartilage. Strict attention to surgical technique and proper postoperative coaptation is critical to reduce the potential for complications with tarsal arthrodesis.

Oral squamous carcinoma cells promote macrophage polarization in an MIF-dependent manner.

BACKGROUND: Tumor-associated macrophages (TAMs) are important determinants of intratumoral immune evasion, neoangiogenesis, extracellular matrix remodeling and dysregulated tumor cell proliferation. Our prior studies revealed that macrophage-derived, but not tumor cell-derived, macrophage migration inhibitory factor (MIF), is an important determinant of TAM alternative activation and M2 polarization. AIM: Because MIF is historically thought to initiate signaling via a receptor-dependent, outside-in mode of action, we wished to investigate the specific contributions of tumor-derived vs. macrophage-derived MIF to M2 marker expression during macrophage polarization. DESIGN: Murine oral squamous cell-carcinoma cells (SCCVII) were co-cultured with either the RAW 264.7 mouse macrophage cell line or mouse primary bone marrow-derived macrophages in the context of MIF genetic loss/inhibition individually or in combination each cell type. METHODS: Twelve well Transwell plates were used to co-culture SCCVII cells and RAW 264.7, MIF+/+ or MIF-/- macrophages treated with/without the small molecule MIF inhibitor, 4-iodo-6-phenylpyrimidine and incubated in the presence or absence of interleukin (IL-4) for 48 h. Macrophages were analyzed by quantitative real-time polymerase chain reaction and/or immunoblotting for relative macrophage polarization marker expression. RESULTS: IL-4 treatment synergizes with SCCVII co-culture in inducing the expression of macrophage M2 markers and loss or inhibition of macrophage-derived MIF significantly reduces both IL-4 alone and IL-4/SCCVII co-culture-induced macrophage M2 marker expression. CONCLUSION: These studies identify an important and dominant requirement for macrophage MIF in maximal Th2-cytokine and oral squamous carcinoma cell-induced macrophage polarization and M2 marker expression.

Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle.

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G1/early S phase and again at the G2 phase in wild-type cells. Interestingly, only the deficit of the G1/early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G2 phase may be required for exit from a DNA damage-induced G2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.

Adaptation of photosynthesis in marama bean Tylosema esculentum (Burchell A. Schreiber) to a high temperature, high radiation, drought-prone environment.

Marama bean, Tylosema esculentum, is a tuberous legume native to the Kalahari region of Southern Africa where it grows under high temperatures (typical daily max 37 degrees C during growing season) and radiation (frequently in excess of 2000 micromol m(-2) s(-1)) in sandy soils with low rainfall. These conditions might be expected to select for increased water-use efficiency of photosynthesis. However, marama was found to give similar leaf photosynthetic rates to other C3 plants for a given internal leaf CO2 concentration and Rubisco content. Under conditions of increasing drought, no increase in water-use efficiency of photosynthesis was observed, but stomata closed early and preceded any change in leaf water potential. The possibility of subtle adaptations of photosynthetic characteristics to its natural environment were investigated at the level of Rubisco kinetics. The specificity factor of marama Rubisco was slightly lower than that of wheat, but the apparent Km for CO2 in air (Km') was about 20% lower than that of wheat. This is consistent with better adaptation for efficient photosynthesis at high temperatures in marama compared to wheat, although the net benefit is predicted to be very small (<0.5% at 35 degrees C). The sequence of marama rbcL gene shows 27 deduced amino acid residue differences from that for wheat, and the possibility that one or more of these cause the difference in Rubisco Km' is discussed.

The neural pathway involved in "efferent inhibition" of chemoreceptors in the cat carotid body.

This study was done to determine whether a pathway of efferent axons in the carotid sinus nerve is necessary for the phenomenon of "efferent inhibition" (inhibition induced in carotid body chemoreceptors by electrical stimulation of the carotid sinus nerve). Our approach was to eliminate efferent axons in the carotid sinus nerve of cats without destroying the sensory axons. This was achieved by cutting the ipsilateral glossopharyngeal and vagus nerves central to their sensory ganglia and/or by removing the nodose and superior cervical ganglia. In neurophysiological studies we found that the response of chemoreceptors in cats 10 days after surgery was the same as that in controls. chemoreceptor activity was decreased by electrical stimulation of the carotid sinus nerve and was increased by hypoxia and cyanide. In operated cats as in control animals, "efferent inhibition" was abolished by haloperidol and dihydroergotamine, drugs that block the inhibitory action of dopamine. Electron microscopic studies disclosed that the number of nerve endings in glomus cell/sheath cell complexes was not measurably different in control and experimental carotid bodies. By contrast, 10 days after the carotid sinus nerve was cut the number of nerve endings next to such ells was reduced by more than 99%. cutting the nerve roots and excising the ganglia eliminated most nerve endings on blood vessels: The number of noradrenergic-type nerve endings was reduced 99% and other types of nerve endings (presumptive cholinergic and peptidergic types) were reduced by more than 90%. Our experiments indicate that "efferent inhibition" is not abolished by operations that destroy inputs to blood vessels and to carotid boy glomus cells from (1) the nodose ganglion, (2) superior cervical ganglion, or from (3) neurons in the brain stem whose axons run in the glossopharyngeal or vagus nerves. We conclude that " efferent inhibition" may be caused by antidromic stimulation of sensory axons.

The architecture of nerves and ganglia of the ferret trachea as revealed by acetylcholinesterase histochemistry.

The goal of this study was to determine the architecture of the nerves and ganglia of the ferret trachea. Tracheas from four newborn ferrets and three adult ferrets were stained histochemically for acetylcholinesterase activity and analyzed in their entirety as whole mounts. The architecture consisted of one or two longitudinal nerve trunks overlying the posterior surface of the trachealis muscle, a dense plexus of nerves superficial to the trachealis muscle that interconnected these longitudinal nerve trunks, and, on the anterior surface, a plexus superficial to the submucosal glands and located between the cartilaginous rings. In addition, deep neural plexuses were associated with the trachealis muscle and with the submucosal glands. Ganglion cell bodies along the longitudinal nerve trunks were large (mean diameter +/- S.E. = 34.3 +/- 0.3 microns), were usually attached to the nerve trunk by a stalk, and were loosely clustered in groups of as many 38 cell bodies. By contrast, those cell bodies of the superficial muscle and gland plexuses were significantly smaller (mean diameter +/- S.E. = 24.2 +/- 0.3 microns), were never attached by a stalk, and were tightly clustered in ganglia of one to four cell bodies. We conclude that nerves and ganglia of the ferret trachea constitute one or two longitudinal nerve trunks containing ganglia with large cell bodies, two superficial nerve plexuses containing ganglia with small cell bodies overlying the smooth muscle and submucosal glands, respectively, and two deep nerve plexuses providing the terminal innervation to the muscle and glands.

Serum interleukin-6 levels in the steady state of sickle cell disease.

In patients with childhood sickle cell disease (SCD) serum interleukin-6 (IL-6) levels were measured during the steady (healthy) state of disease. The corresponding measurements were made in comparable healthy normal controls. Serum IL-6 levels were assessed via ELISA in 27 SCD patients and 19 controls. Results revealed significantly higher circulating levels of IL-6 in the SCD patients (60 +/- 7 pg/ml) compared with the healthy controls (12 +/- 5 pg/ml). IL-6 is a multifunctional cytokine that plays a central role in host defense mechanisms. The impact of high circulating levels of IL-6 may be deleterious to humoral and cell-mediated immune functions in SCD, with resultant heightened risk for morbidity.

Short-chain fatty acids and encephalopathy of Reye's syndrome.

Plasma levels of six short-chain fatty acids (SCFA) were measured in 23 Reye's syndrome patients. In sequential measurements, only propionic acid correlated closely with neurologic severity. Although admission SCFA levels were slightly elevated, there were no significant differences between patients grouped by severity of encephalopathy. Admission SCFA did not predict neurologic outcome; also, they correlated poorly with admission blood ammonia, amino acid nitrogen, and lactate.

The metabolic course of Reye's syndrome: distinction between survivors and nonsurvivors.

We compared the levels of hormones and metabolites in the plasma of 37 survivors of Reye's syndrome with the levels in 8 fatal cases, at four time periods within 72 hours of admission. The most prominent differences were found for norepinephrine (NE), which was significantly elevated in fatal cases compared with survivors at all periods. Lactate and dopamine were elevated in the earlier periods. Epinephrine and alpha-amino acid nitrogen were also elevated in fatal cases, but the differences usually were not significant. NE elevation may reflect an increased sympathoadrenal medullary output associated with brain edema, compounded by impaired hepatic clearance of monoamines. Skeletal muscle ischemia from NE-induced vasoconstriction may explain the association between lactic acidemia and the severity of encephalopathy.

Hepatic and encephalopathic components of Reye's syndrome: factor analysis of admission data from 209 patients.

Factor analysis of admission data from 209 Reye's syndrome patients yielded three factors. Factor 1 was associated with encephalopathy, blood ammonia, creatinine kinase (CK), uric acid and, to a lesser extent, bilirubin. This factor was linked to the encephalopathy and hypermetabolic changes in muscle, possibly prostaglandin-mediated proteolysis. Factor 2 was associated with serum alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT), and was identified as a hepatic lesion component. These factors correspond to two etiologic components of Reye's syndrome. Salicylate was only weakly associated with neuropathic and hypercatabolic indicators and not at all associated with the hepatic damage indicators.

Inhibition of S-nitrosation of reduced glutathione in aerobic solutions of nitric oxide by phosphate and other inorganic anions.

Reduced glutathione is nitrosated in aerobic solutions of nitric oxide under physiological conditions; however, the extent of S-nitrosation was found to be dependent on the inorganic anions present. Of nine anions tested, the bifunctional anions, arsenate, phosphate, and pyrophosphate (40 mM), inhibited the S-nitrosation reaction from 20 to 40%, whereas SO4(2-), H3BO3, SCN-, NO3-, Cl-, and acetate inhibited this reaction < or = 15%. A mechanism of inhibition is presented that involves the catalytic hydrolysis of N2O3 by the bifunctional anions; however, using [18O]phosphate as inhibitor, only 10% of the theoretically produced N2O3 was found to be hydrolyzed to nitrite via this mechanism as calculated from the loss of 18O from phosphate. We conclude that this mechanism accounts for only a minor part of the increased inhibition of S-nitrosation by these bifunctional anions.

In vitro lymphocyte blastogenic responses and cytokine production in sickle cell disease patients with acute pneumonia.

BACKGROUND: Pulmonary infections continue to be a major cause of morbidity and mortality in patients with sickle cell disease (SCD). METHODS: In this study cell-mediated immunity in vitro was evaluated in 62 SCD patients (62 steady state and 16 with acute pneumonia) and compared with 44 normal controls (30 healthy and 14 with acute pneumonia). Lymphocyte blastogenic responses to phytohemagglutinin, tetanus toxoid and Candida albicans antigen were assessed in all subjects. In addition production of tumor necrosis factor, alpha- and gamma-interferon (IFN) were assayed. RESULTS: The results revealed comparable blastogenic responses to all three stimuli in all subjects except SCD patients with pneumonia. This group showed poor responses to all stimuli. The mean counts per minute were decreased 65 to 90% when compared with the other patients. Cytokine production of IFN-alpha and TNF was equivalent in all subjects. Conversely IFN-gamma production in both SCD groups, steady state (35 +/- 6 U/ml) and SCD with pneumonia (14 +/- 6 U/ml), was significantly decreased when compared with those in normal healthy controls (65 +/- 14 U/ml) and with pneumonia (48 +/- 17 U/ml). On analysis of individual titers 15 of 62 (24%) steady state and 10 of 16 (63%) SCD patients with pneumonia were deficient in IFN-gamma production in vitro. CONCLUSIONS: Acute pulmonary infections seem to have a profound effect on cell-mediated immunity in SCD. IFN-gamma deficiency, along with quantitative and qualitative T cell abnormalities, may represent significant factors to explain the frequent and severe infections seen in SCD.

Splanchnic and total body oxygen consumption differences in septic and injured patients.

The splanchnic and total body oxygen exchange and flow dynamics for injured patients (n = 7) and patients with sepsis and stable vital signs (n = 12) were studied. All patients were judged to be in the hyperdynamic phase of the stress response. In both patient groups 27% to 28% of the cardiac index was directed to the splanchnic circulation. However, in sepsis the splanchnic region consumed a significantly larger fraction (p less than 0.05) of the total body oxygen (43.8%) compared with that consumed in injury (30.2%). After injury, the regional splanchnic flow and oxygen consumption appeared to be well matched whereas in sepsis, a disproportionately higher oxygen consumption is found, which must be supplied by increasing blood oxygen extraction. This regional hypermetabolism of the splanchnic area probably results from the increased metabolic demand imposed by the various synthetic processes of this region. In addition, it is proposed that excessive discrepancy between splanchnic flow and oxygen demand may precipitate regional ischemia.

Hepatic metabolic response to injury and sepsis.

BACKGROUND: Experimental reports have indicated that hepatic oxidative and synthetic metabolism may become depressed in sepsis. Because the mechanism of infection-related liver dysfunction has not been established, further study of these functional alterations could contribute to the therapeutic management of septic organ failure syndromes. However, recently controversy has arisen over the existence of these derangements that must be reconciled before further progress in this field can be made. METHODS: Splanchnic balance studies for the measurement of glucose output and oxygen consumption were used to assess hepatic function in fasted normal volunteers (n = 18), injured patients (n = 10), and patients with sepsis (n = 18). The liver's contribution to splanchnic metabolism was estimated from a comparison of splanchnic oxygen utilization in response to increases in the liver-specific process of glucogenesis. In addition, in vivo liver albumin production was determined by using the [14C] carbonate technique. RESULTS: Glucose output after injury and sepsis was increased by 12.8% and 76.6%, respectively, compared with controls. On the basis of substrate balance studies, gluconeogenesis was estimated to account for 46%, 87%, and 93%, respectively, of splanchnic glucose output in each of the three groups. In patients with sepsis glucose output was also noted to be linearly related to regional oxygen consumption, indicating that these processes were coupled and increases in the respiratory activity of the splanchnic cellular mass could be accounted for by increases in new glucose output and gluconeogenic substrate clearance. The mean albumin synthetic rate increased during injury and sepsis by 22% and 29%, respectively, compared with normal volunteers. CONCLUSIONS: These studies cast doubt on the commonly held notion that tissue respiratory dysfunction may occur during sepsis. On the contrary, hepatic function is accelerated during hyperdynamic sepsis, and evidence indicating oxidative or synthetic functional depression is lacking.

Hepatic blood flow and splanchnic oxygen consumption measurements in clinical sepsis.

In an effort to characterize the hemodynamic response of the liver to sepsis, hepatic blood flow (HBF) was measured in 10 normal volunteers and compared with that of 9 patients with sepsis. Flow was determined according to two different indicators and three methods of analysis including indocyanine green dye clearance (HBFICG), galactose clearance (GC), and galactose clearance with splanchnic galactose gradient measurement (HBFGG). For normal subjects, these three analytic methods provided essentially identical results (HBFICG = 0.74 +/- 0.18, GC = 0.72 +/- 0.14, and HBFGG = 0.76 +/- 0.16 L/min-m2). With hepatic venous sampling, HBF in patients with sepsis was significantly higher than normal levels (HBFICG = 1.28 +/- 0.50 and HBFGG = 1.17 +/- 0.52 L/min-m2) (p less than 0.025), but HBF by the GC technique (0.89 +/- 0.41 L/min-m2), which uses peripheral venous sampling, was not significantly increased because of reduced splanchnic galactose extraction, which appears to be characteristic of sepsis. Thus HBF estimates based on peripheral venous sampling must be interpreted with caution in view of the reduced extraction fraction in sepsis. HBF in clinical sepsis tends to increase in response to this inflammatory stress.

Nicotine-induced respiratory effects of cigarette smoke in dogs.

We report that nicotine is responsible for both a blood-borne stimulation of the respiratory center and a direct effect on intrathoracic airway tone in dogs. We introduced cigarette smoke into the lungs of donor dogs and injected arterial blood obtained from them into the circulation of recipient dogs to show that a blood-borne material increased breathing and airway smooth muscle tone. Smoke from cigarettes containing 2.64 mg of nicotine was effective; that from cigarettes containing 0.42 mg of nicotine was not. Nicotine, in doses comparable to the amounts absorbed from smoke, also increased breathing and tracheal smooth muscle tension when injected into the vertebral circulation of recipient dogs. Finally, blockade of nicotine receptors in the central nervous system and in the airway parasympathetic ganglia inhibited the effects of inhaled cigarette smoke and intravenous nicotine on the respiratory center and on bronchomotor tone. We conclude that nicotine absorbed from cigarette smoke is the main cause of cigarette smoke-induced bronchoconstriction. It caused central respiratory stimulation, resulting in increased breathing and airway smooth muscle tension, and had a direct effect on airway parasympathetic ganglia as well.

Inspiratory rhythm in airway smooth muscle tone.

In anesthetized paralyzed open-chested cats ventilated with low tidal volumes at high frequency, we recorded phrenic nerve activity, transpulmonary pressure (TPP), and either the tension in an upper tracheal segment or the impulse activity in a pulmonary branch of the vagus nerve. The TPP and upper tracheal segment tension fluctuated with respiration, with peak pressure and tension paralleling phrenic nerve activity. Increased end-tidal CO2 or stimulation of the carotid chemoreceptors with sodium cyanide increased both TPP and tracheal segment tension during the increased activity of the phrenic nerve. Lowering end-tidal CO2 or hyperinflating the lungs to achieve neural apnea (lack of phrenic activity) caused a decrease in TPP and tracheal segment tension and abolished the inspiratory fluctuations. During neural apnea produced by lowering end-tidal CO2, lung inflation caused no further decrease in tracheal segment tension and TPP. Likewise, stimulation of the cervical sympathetics, which caused a reduction in TPP and tracheal segment tension during normal breathing, caused no further reduction in these parameters when the stimulation occurred during neural apnea. During neural apnea the tracheal segment tension and TPP were the same as those following the transection of the vagi or the administration of atropine (0.5 mg/kg). Numerous fibers in the pulmonary branch of the vagus nerve fired in synchrony with the phrenic nerve. Only these fibers had activity which paralleled changes in TPP and tracheal tension. We propose that the major excitatory input to airway smooth muscle arises from cholinergic nerves that fire during inspiration, which have preganglionic cell bodies in the ventral respiratory group in the region of the nucleus ambiguus and are driven by the same pattern generators that drive the phrenic and inspiratory intercostal motoneurons.