The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix.

A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenase, named after their metal requirements, containing either a MoFe-, VFe- or FeFe-cofactor, that converts N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase proteins within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.

Vasopressin and renin response to dehydration in aged rats.

Abnormalities in neurohypophyseal function have been postulated to contribute to the alterations in fluid and electrolyte balance observed during aging. In this study, parameters of fluid and electrolyte balance were evaluated during chronic water deprivation in old (30 months) and young (3 months) Fischer 344 rats. The increase in serum vasopressin (VP) and renin concentrations observed in the 3 month animals following chronic water deprivation were absent in the aged rats (p less than 0.05 and p less than 0.02, respectively). This occurred in spite of apparently comparable alterations in fluid volume and osmolality (assessed by changes in body weight, hematocrit and plasma osmolality). Relative to body weight, VP content of the neural lobe was significantly reduced and was more severely depleted by dehydration in aged rats than in young rats. Thus, inadequate neurohypophyseal hormone stores may contribute to the inability of the aged animals to attain elevated serum VP concentrations during chronic stimulation. Several parameters of renal function were examined in the aged rats. Although none of the old rats were in renal failure, they all showed some indication of reduced renal function. In spite of renal abnormalities including reduced concentrating capabilities, the old rats did demonstrate a significant antidiuretic response to dehydration. However, with prolonged fluid deprivation, they were unable to attain serum VP or renin concentrations comparable to that achieved by the young rats.

Recovery of glomerular and tubular function in autotransplanted dog kidneys preserved by hypothermic storage or machine perfusion. Relation of initial function to long-term function.

Sixteen male dogs had split renal function studies prior to unilateral nephrectomy and autotransplant. Kidneys were preserved for 24 hr by either simple hypothermic storage in Collins C2 solution (SHS) or machine-perfused (MP) on a Waters machine (MOX 100) with plasmanate perfusate. Renal function studies were repeated at 1 hr and at 7, 14, and 28 days, and the statistical relationship between initial and 1-month function was determined for a number of parameters. All MP kidneys functioned immediately, whereas 1/3 of SHS kidneys had delayed function. Recovery was more rapid in MP kidneys and was essentially complete by 14 days, at which time MP kidneys had higher rates of creatinine clearance and sodium reabsorption. However, by one month 3/7 MP kidneys (P = 0.15 compared with SHS) had lower creatinine clearance rates than at 2 weeks, and para-aminohippurate (PAH) clearance and fractional sodium reabsorption were significantly decreased. During the same period SHS kidneys either showed continued improvement or maintained stable function. Thus, by one month there were no differences between the groups in clearances of creatinine and PAH, plasma creatinine and blood urea nitrogen concentrations, or fractional reabsorption of sodium, potassium, and water. For SHS kidneys, the 1-hr creatinine clearance and the absolute rate of sodium reabsorption were strong predictors of the eventual function of the kidneys at one month (r = 0.93 and r = .83, P less than 0.05, respectively). No such correlations were found in MP kidneys (r = less than .01, P greater than 0.9 for both variables). The data show that MP results in significantly better function early after transplant, but this advantage does not persist, and that SHS kidneys early function is a good predictor of long-term recovery, but this is not true for MP kidneys.

Early indices of warm ischemic damage in autotransplanted canine kidneys preserved by simple hypothermic storage.

After measurement of normal renal function, dog kidneys (n = 52) were subjected to 3, 15, or 30 min of normothermic warm ischemia (WI). After 24 hr of preservation by simple hypothermic storage (SHS) in a modified Collins solution, autotransplant was done and renal function was again measured beginning at 1 hr. Compared with preharvest values, kidneys with minimal (3 min) WI had significantly decreased clearances of creatinine (Cr) and para-aminohippuric acid (PAH), PAH extraction, absolute and fractional Na reabsorption, Na excretion, and urinary Na concentration; no change in urine flow rate or K excretion; and significantly increased fractional excretion of Na, K, and H2O. Compared with minimal WI, 30-min WI produced further significant decreases in clearances of Cr, PAH, and PAH extraction; and further increases in fractional excretion of Na, K, and H2O. Urine flow was also decreased by about half and urine Na concentration rose significantly. Several parameters were very significantly correlated with the length of WI, but the most reliable index was the fractional reabsorption of Na. When several functional parameters were used together, kidneys with significant (30 min) WI prior to preservation could be identified with a high degree of statistical reliability.

Violence in the health care environment.

The rapid rise in the incidence of violence in our society is an increasing risk to all Americans. As violence increases for our patients and in society around us, it becomes an ever greater problem for all health care personnel. The number of threats and violent acts against health care providers has been steadily increasing in recent years. The most effective method of managing violence is to prevent it. A critical step in prevention is in differentiating between an angry individual and a potentially violent individual based on his or her speech, appearance, and behavior. Heightened security and changes in the medical environment are additional ways of preventing violence. Management of a violent incident includes early recognition, de-escalation techniques, and a collaborative effort with security personnel.

DNA content and tumor response to induction chemotherapy in patients with advanced laryngeal squamous cell carcinoma.

Recent results indicate that the adjusted DNA Index (aDI), a measure of nuclear DNA content, is a significant prognostic factor for patients with advanced laryngeal cancer treated with surgery and radiation therapy. Because DNA aneuploidy is an indirect measure of the proliferative activity of a cell population, a study was conducted to examine differences in tumor response to induction chemotherapy based on aDI values. Pretreatment tumor specimens were obtained from 50 patients with stage III and IV laryngeal squamous cell carcinoma who underwent induction chemotherapy (cisplatin/5-FU). With the use of computerized cytomorphometry, DNA content and nuclear area were measured and associations with tumor site, stage, chemotherapy response, tumor recurrence, and survival were examined. An elevated aDI was more frequent in patients with a chemotherapeutic response (p = 0.08), and mean aDI was higher among the complete responders. There were no complete responders among patients with a low aDI value (< 0.024). Neither aDI nor nuclear area correlated significantly with organ preservation or patient survival. Our results indicate that a complete response is more likely for patients with tumors with an elevated aDI and that pretreatment aDI may be useful in selecting high-risk patients who might benefit from chemotherapy.

Rosai-Dorfman disease (extranodal sinus histiocytosis) in a patient with HIV.

Rosai-Dorfman disease, or sinus histiocytosis with massive lymphadenopathy (SHML), is a rare histiocytosis of unknown etiology that most commonly involves the cervical lymph nodes. Extranodal involvement occurs in 30-40% of cases, most often in the head and neck. Characteristic histologic findings include emperiopolesis (engulfment) of lymphocytes and S-100 protein positivity. Treatment of Rosai-Dorfman disease is unnecessary unless the disorder becomes life- or organ-threatening, since the disease will resolve spontaneously in most patients. We present what, to the best of our knowledge, is the first reported case of Rosai-Dorfman disease limited to the skin in a patient infected with human immunodeficiency virus. SHML is described and diagnostic and therapeutic measures are reviewed.

Effect of cholinergic antagonists on basal and osmotically stimulated vasopressin release in compartmentalized hypothalamo-neurohypophysial explants.

The effects of cholinergic antagonists on vasopressin (VP) release were studied in an organ-cultured, compartmentalized, rat hypothalamo-neurohypophysial system which allows selective application of stimuli to either hypothalamus or pituitary without disrupting axonal connections. Release of vasopressin from the neurohypophysis was measured by radioimmunoassay. Hexamethonium (10(-5) M) and atropine (5 X 10(-5) M) were tested both alone and in combination with hypothalamic osmotic stimulation (+ 15 mosm/kg H2O). In hypothalamus, neither hexamethonium nor atropine had any effect on basal VP release from pituitary. Hexamethonium, but not atropine, prevented the increase in VP release produced by increased osmolality of the hypothalamus side culture medium. In contrast, hexamethonium had no effect when applied to pituitary side, whereas atropine suppressed both basal and osmotically stimulated VP release. Atropine had no effect on basal or KCl-induced VP release in detached neural lobes. Acetylcholine (Ach) (10(-5) M) to pituitary plus simultaneous, hypothalamic stimulation (osmotic or 10(-5) M Ach) did not increase VP release above the hypothalamic stimulus alone. The results support a role for a hypothalamic excitatory nicotinic mechanism in osmoregulation. The presence of a muscarinic mechanism affecting VP release in pituitary was reconfirmed, but the data did not support the hypothesis that Ach stimulates VP release in pituitary by a presynaptic facilitatory mechanism.

The compartmentalized hypothalamo-neurohypophysial system: evidence for a neurohypophysial action of acetylcholine on vasopressin release.

A portion of medial basal hypothalamus containing the supraoptic nuclei with the neurohypophysis attached was organ cultured. Hypothalamus and neurohypophysis were maintained in separate compartments, and the intact infundibular stalk passed through a hole in a fluid-tight barrier which separated the two compartments. After 24, 48 and 72 h in culture, vasopressin (VP) release from the neurohypophysis was measured during a control hour and again during an immediately subsequent test hour. Test hour VP release was expressed as a percentage of control hour release. Test substances were added to either the pituitary or the hypothalamus compartment. Acetylcholine stimulated pituitary VP release both when added to hypothalamus (10(-5) M) and when added directly to neural lobe (10(-6) M and above). Acetylcholine 10(-5) M had no effect when isolated neural lobes (severed from hypothalamus to culture) were similarly tested. Hexamethonium blocked the stimulation of pituitary VP release evoked by addition of acetylcholine to hypothalamus. However, in pituitary, atropine prevented the stimulatory effect of acetylcholine. Atropine had no effect on VP release from severed neural lobes. These data show that high concentrations of acetylcholine can stimulate VP release from pituitary both by a hypothalamic action and also by a direct effect in neural lobe. Further, a nicotinic cholinergic receptor mediates the action of acetylcholine in hypothalamus whereas a muscarinic cholinergic receptor is involved in the direct pituitary response to acetylcholine. Intact axonal connections between hypothalamus and pituitary are required in order for acetylcholine to stimulate VP release in neurohypophysis.

Model for evaluating avian renal hemodynamics and glomerular filtration rate autoregulation.

Multiple vascular connections in normal avian kidneys make it difficult to experimentally manipulate renal blood flow patterns and perfusion pressures. In this study, hemostatic clips were used to obstruct the ureter of one kidney at the level of the ischiadic artery (IA) in anesthetized 3-wk-old chicks (Gallus domesticus). Kidney tissue upstream from the ureteral obstruction degenerated, leaving an intact caudal renal division with one route of arterial inflow branching from the IA. Renal function studies were conducted, using general anesthesia, when the birds reached 12-15 wk of age. A snare placed around the IA was used to unilaterally decrease renal arterial perfusion pressure (RAPP) for the experimental kidney. Under control conditions (snare loose), urine flow rates (UFR), glomerular filtration rates (GFR), clearance of p-aminohippuric acid, and fractional excretion of Na, K, Ca, and PO4 did not differ significantly, per gram of kidney weight, when experimental and intact contralateral kidneys were compared. Gradual tightening of the IA snare reduced RAPP stepwise. UFR decreased significantly from the initial control value when RAPP reached 40 mmHg, and urine flow ceased completely when RAPP reached 30-35 mmHg. In four of five birds, GFR did not decrease significantly between 110 and 60 mmHg but did decrease significantly below 60 mmHg. Urine osmolality was inversely correlated with UFR. Clearance of PAH did not decrease significantly from control values as RAPP ranged from 100 to 37 mmHg, possibly caused by increased renal portal blood flow. Overall, these results provide the first direct demonstration that in domestic fowl GFR is autoregulated at reduced RAPP.

In vitro evidence for an inhibitory effect of atrial natriuretic peptide on vasopressin release.

The effect of Arg-atriopeptin III (ANP) on basal and stimulated (angiotensin II, acetylcholine and KCl depolarization) arginine vasopressin (AVP) release was characterized in the intact hypothalamo-neurohypophysial explant (HNS) and in isolated neurointermediate pituitary lobes (NIL). In initial experiments using 15-min incubation periods, ANP 10(-10) and 10(-9) M slightly inhibited basal AVP release in both NIL and HNS after a delay of at least 15 min. The most effective ANP concentration was 10(-10) M, and the inhibitory effect on AVP release was more marked in HNS (-52 +/- 5% of control compared to -29 +/- 8% for NIL). However, ANP 10(-10) M did not significantly attenuate KCl- or AII (10(-5) M)-stimulated AVP release from HNS after 15 min of exposure. When the incubation periods were increased to 30 min ANP 10(-10) and 10(-9) M significantly decreased AII-stimulated (10(-5) M) AVP release in a dose-dependent manner (p less than 0.05; p less than 0.01, respectively). The same concentrations of ANP did not significantly depress ACH-stimulated (10(-5) M) AVP release (p less than 0.1 for both concentrations). In summary, ANP generally inhibits AVP release in vitro by a slowly activated mechanism which appears to be specific for certain physiological stimuli. Although the site(s) of action cannot be absolutely localized to the ventral hypothalamus and/or the neurohypophysis, an effect in the hypothalamus seems very likely.

A compartmentalized, organ-cultured hypothalamo-neurohypophysial system for the study of vasopressin release.

In the compartmentalized hypothalamo-neurohypophysial system ( CHNS ), a portion of medial basal hypothalamus (HT) containing the supraoptic nuclei (SON) and the neurohypophysis (PP) were organ-cultured in separate compartments. The intact axonal projections from SON to PP passed through a hole in a fluid-tight barrier which separated the two compartments. When properly sealed, the leak rate from one side to the other is less than 1%/24 h and the only connection between the 2 compartments is axonal. This system had relatively stable basal vasopressin (VP) release rates from both HT and PP for up to 72 h in culture. Basal neurohypophysial VP release rate was unchanged during two successive 1-hour periods on any given day. Physiological responsiveness was confirmed by osmotic challenge. When HT osmolality was changed by +/- 15 mosm, VP release from PP was appropriately and significantly increased or decreased. Equivalent changes in PP side osmolality had no effect on VP release. After 72 h in culture, the VP content of neural lobes from CHNS explants was more than double that of lobes which were severed from HT prior to culture. Finally, the presence of numerous VP neurophysin-containing cells in 72-hour cultured explants was demonstrated by immunocytochemistry. This system will be useful to localize sites of action for agents affecting VP release to either HT and/or PP.

Morphological and functional comparisons of normal and hypertrophied kidneys of adult domestic fowl (Gallus gallus).

Similar to mammals, kidneys of domestic fowl undergo compensatory hypertrophy after loss of functional renal mass. Because this species continues to develop new nephrons for up to 12-wk posthatch, renal hyperplasia might play a significant role in compensatory growth. Either transient or permanent loss of approximately 60% of the right kidney was produced in 2- to 3-wk-old roosters by simple ureteral transection or by removing a 1-mm segment of ureter at the level of the ischiadic artery, respectively. In the latter (experimental) group, right anterior and medial divisions atrophied leaving only the posterior division intact. Spontaneous reanastomosis occurred in the former (reconnected) group, and all three divisions were present at death. Control birds were untouched as were the left kidneys of experimental and reconnected birds. At 40-50 wk, renal function was measured separately in right and left kidneys of all groups during five different infusion protocols. Compared with control kidneys, experimental kidneys had a 50-60% weight gain, and their glomerular size distribution profile was shifted to the right (larger glomeruli). Reconnected kidneys were not hypertrophied, and their profile was shifted to the left (smaller glomeruli). Neither group had significant formation of new nephrons. Once variations in kidney weight were taken into account, there were no differences between hypertrophied (experimental) and control kidneys in urine flow rate (UFR), glomerular filtration rate (GFR), paraaminohippuric acid (PAH) clearance, UFR/GFR, urine osmolality, urine/plasma osmolality, osmolal clearance, free water clearance, Na and K load, absolute Na and K excretion, and fractional Na and K excretion except as follows: 1) during infusion of isotonic mannitol-dextrose at 0.1 ml.min-1.kg body wt-1 experimental kidneys had a lower fractional excretion of K than control kidneys, and 2) during brisk osmotic diuresis (isotonic mannitol-dextrose at 0.4 ml.min-1.kg body wt-1) experimental kidneys had higher UFR and free water clearance and lower urine osmolality and urine/plasma osmolality than control kidneys. Reconnected kidneys differed from control kidneys in only 1 of 210 comparisons. Permanent loss of functional renal mass in young birds produces significant compensatory renal hypertrophy that is due to enlargement of existing nephrons rather than formation of new nephrons. Hypertrophied kidneys function like normal kidneys except under conditions of brisk osmotic diuresis.

Localization of central sites of action of angiotensin II on ADH release in vitro.

It is now thought that angiotensin II can stimulate antidiuretic hormone (ADH) release in vivo by a direct action in the central nervous system but it is not known whether the locus of stimulation is the hypothalamus or the neurohypophysis or both. Isolated rat neural lobes incubated for 10 min in buffer containing angiotensin II (200 ng/ml or 2 microgram/ml) did not increase ADH release compared to control values, but addition of KCl (60 mM) to the bath markedly stimulated ADH release. However, intact hypothalamoneurohypophysial systems (containing the supraoptic nuclei) incubated with angiotensin II (200 ng/ml or 2 microgram/ml) did show a pronounced stimulation of ADH release. The data support the hypothesis that angiotensin II, at least in vitro, has a central effect on ADH release which is at the level of the hypothalamus.

Effects of atriopeptin and chicken heart extract in Gallus domesticus.

Effects of Ser-Leu-Arg-Arg-atriopeptin III (ANP) and chicken heart extract (CHE) were compared during unilateral renal portal infusion in anesthetized Single Comb White Leghorn chickens. The purpose was to determine whether renal effects were glomerular and/or tubular. Both CHE and ANP caused substantial decreases in mean arterial pressure but had different renal actions. ANP caused small but significant increases in both absolute and fractional sodium excretion, but these effects were modest compared with those reported in mammals. Although there was a tendency for higher fractional sodium excretion in the portal infused kidney, the difference was not significant (0.1 less than P greater than 0.05). ANP also increased glomerular filtration rate (GFR), urine flow rate (UFR), and osmolal clearance and decreased estimated renal vascular resistance. In contrast, CHE decreased GFR and increased resistance. In contrast, CHE decreased GFR and increased fractional potassium excretion in the infused kidney. After CHE infusion was stopped, GFR and UFR increased, and there was a further transient kaliuresis. No natriuretic effects were ever seen with CHE. Chickens apparently lack potent mammalian-type cardiac natriuretic factor(s) and/or have a limited capacity for natriuresis in response to mammalian ANP. Because hypotension was the most prominent avian response to both CHE and ANP, endogenous vasoactive factor(s) in chicken hearts may function to regulate blood pressure rather than blood volume.

A potent hypotensive factor in chicken left ventricle.

Chicken artria and ventricles both have membrane-bound granules which resemble those containing atriopeptin (ANP) in mammals. However, nothing is known about the contents of the avian granules. A previous study in chickens showed that although extracts of whole chicken heart or synthetic rat ANP both caused profound hypotension, ANP caused both natriuresis and diuresis, while chicken heart extract did not. The present study sought to locate the region(s) of chicken heart containing the hypotensive activity, and to observe the effect on sodium and water excretion and blood pressure in rats. Acid extracts of either atrium, either ventricle, ventricular septum, skeletal muscle, and liver were identically prepared from chickens and rats. Extracts were adjusted to the same protein concentration and injected (0.15 ml/kg) into anesthetized Single Comb White Leghorn roosters. Mean arterial pressure (MAP) and the time for recovery were measured. The most potent extract from chicken hearts was from the left ventricle (-38 +/- 1 mm Hg, 149 +/- 9 sec to recover). All other extracts (including right ventricle) produced only small (10-20 mm Hg), short-lived (20-30 sec) decreases in MAP. In contrast, only rat atrial extracts evoked long-lasting hypotension (greater than 40 mm Hg, recovery time greater than 200 sec). A 30-min infusion of the most potent chicken extract (left ventricle, CLV) into rats produced a small but significant natriuresis and diuresis compared to the vehicle time control (P less than 0.05) and the hypotensive response to bolus injection was about one-third that seen in the chicken. The location of potent spasmolytic activity primarily in chicken left ventricle, the different avian renal responses to chicken heart extract and synthetic rat ANP (5), and the weak diuretic, natriuretic, and hypotensive effects of CLV extract in rats all suggest that the chicken heart substance may be different from mammalian ANP.

Comparison of the oxidation rates of glucose and lactate in relation to support of Na+ reabsorption.

The renal oxidation rates of glucose and lactate in the dog in vivo, in the dog cortical slice and in the isolated perfused rat kidney were compared. Lactate decarboxylation rate, on a carbon-atom basis, was from 2 to 10 fold greater than that of glucose. In the substrate-limited perfused kidney, glucose replaced only 30-40% of the substrates oxidized in vivo, while lactate replaced up to 80% of the substrates oxidized in vivo. Insulin lack does not account for these differences in the rates of lactate and glucose oxidation. Glucose and lactate support GFR and Na+ reabsorption to approximately the same extent in spite of their different rates of oxidation. Thus Na+ reabsorptive rate: CO2 production rate is not a constant and depends on the substrate being oxidized. The virtual absence of glucose oxidation by the dog cortical slice suggests either that: 1) glucose oxidation supports primarily medullary Na+ reabsorption while lactate oxidation supports cortical Na+ reabsorption as well of 2) glucose oxidation is more selectively coupled to Na+ reabsorptive work than is lactate oxidation.