The Bradyrhizobium Sp. LmicA16 Type VI Secretion System Is Required for Efficient Nodulation of Lupinus Spp.

Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and Lupinus angustifolius. A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild-type (wt) strain and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26-kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidity than the wt strain in free-living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free-living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain.

Functional and expression analysis of the metal-inducible dmeRF system from Rhizobium leguminosarum bv. viciae.

A gene encoding a homolog to the cation diffusion facilitator protein DmeF from Cupriavidus metallidurans has been identified in the genome of Rhizobium leguminosarum UPM791. The R. leguminosarum dmeF gene is located downstream of an open reading frame (designated dmeR) encoding a protein homologous to the nickel- and cobalt-responsive transcriptional regulator RcnR from Escherichia coli. Analysis of gene expression showed that the R. leguminosarum dmeRF genes are organized as a transcriptional unit whose expression is strongly induced by nickel and cobalt ions, likely by alleviating the repressor activity of DmeR on dmeRF transcription. An R. leguminosarum dmeRF mutant strain displayed increased sensitivity to Co(II) and Ni(II), whereas no alterations of its resistance to Cd(II), Cu(II), or Zn(II) were observed. A decrease of symbiotic performance was observed when pea plants inoculated with an R. leguminosarum dmeRF deletion mutant strain were grown in the presence of high concentrations of nickel and cobalt. The same mutant induced significantly lower activity levels of NiFe hydrogenase in microaerobic cultures. These results indicate that the R. leguminosarum DmeRF system is a metal-responsive efflux mechanism acting as a key element for metal homeostasis in R. leguminosarum under free-living and symbiotic conditions. The presence of similar dmeRF gene clusters in other Rhizobiaceae suggests that the dmeRF system is a conserved mechanism for metal tolerance in legume endosymbiotic bacteria.

Inactivation of a serotonin-gated ion channel by a polypeptide toxin from marine snails.

The venom of predatory marine snails is a rich source of natural products that act on specific receptors and ion channels within the mammalian nervous system. A 41-amino acid peptide, final sigma-conotoxin GVIIIA, was purified on the basis of its ability to inactivate the 5-HT3 receptor, an excitatory serotonin-gated ion channel. final sigma-Conotoxin contains a brominated tryptophan residue, which may be important for peptide activity because the endogenous ligand for the 5-HT3 receptor is a hydroxylated derivative of tryptophan. final sigma-Conotoxin inactivates the 5-HT3 receptor through competitive antagonism and is a highly selective inhibitor of this receptor. Serotonin receptors can now be included among the molecular targets of natural polypeptide neurotoxins.

Alanine aminotransferase levels are not significantly elevated in patients with HIV/HBV co-infection and lamivudine resistance.

In hepatitis B virus (HBV) monoinfection, alanine aminotransferase (ALT) levels are linearly correlated with HBV DNA levels and lamivudine resistance. In human immunodeficiency virus (HIV)/HBV co-infection, little is known about the association between ALT, HBV DNA, and lamivudine resistance. We assessed HBV DNA, lamivudine resistance and ALT levels in 45 time points in 11 patients with HIV/HBV co-infection during lamivudine-containing antiretroviral therapy. High HBV DNA levels (>10(6) copies/mL) and lamivudine resistance developed in 45% and 91% of patients, respectively. However, ALT levels were not elevated in the setting of high HBV DNA levels (mean ALT, 48 IU/mL) or lamivudine resistance (mean ALT, 44 IU/mL). HBV viraemia and lamivudine resistance during extended lamivudine-containing antiretroviral therapy are common in HIV/HBV co-infection, occurring in the absence of significant ALT elevations. In HIV/HBV co-infection, measurement of HBV DNA and HBV resistance mutations may identify HBV virological failure before biochemical changes and should be routinely used in the management of HIV/HBV co-infection.

RNA sequencing and analysis of three Lupinus nodulomes provide new insights into specific host-symbiont relationships with compatible and incompatible Bradyrhizobium strains.

Nitrogen fixation in the legume root-nodule symbiosis has a critical importance in natural and agricultural ecosystems and depends on the proper choice of the symbiotic partners. However, the genetic determinism of symbiotic specificity remains unclear. To study this process, we inoculated three Lupinus species (L. albus, L. luteus, L. mariae-josephae), belonging to the under-investigated tribe of Genistoids, with two Bradyrhizobium strains (B. japonicum, B. valentinum) presenting contrasted degrees of symbiotic specificity depending on the host. We produced the first transcriptomes (RNA-Seq) from lupine nodules in a context of symbiotic specificity. For each lupine species, we compared gene expression between functional and non-functional interactions and determined differentially expressed (DE) genes. This revealed that L. luteus and L. mariae-josephae (nodulated by only one of the Bradyrhizobium strains) specific nodulomes were richest in DE genes than L. albus (nodulation with both microsymbionts, but non-functional with B. valentinum) and share a higher number of these genes between them than with L. albus. In addition, a functional analysis of DE genes highlighted the central role of the genetic pathways controlling infection and nodule organogenesis, hormones, secondary, carbon and nitrogen metabolisms, as well as the implication of plant defence in response to compatible or incompatible Bradyrhizobium strains.

Molybdate binding by ModA, the periplasmic component of the Escherichia coli mod molybdate transport system.

ModA, the periplasmic-binding protein of the Escherichia coli mod transport system was overexpressed and purified. Binding of molybdate and tungstate to ModA was found to modify the UV absorption and fluorescence emission spectra of the protein. Titration of these changes showed that ModA binds molybdate and tungstate in a 1:1 molar ratio. ModA showed an intrinsic fluorescence emission spectrum attributable to its three tryptophanyl residues. Molybdate binding caused a conformational change in the protein characterized by: (i) a shift of tryptophanyl groups to a more hydrophobic environment; (ii) a quenching (at pH 5.0) or enhancement (at pH 7.8) of fluorescence; and (iii) a higher availability of tryptophanyl groups to the polar quencher acrylamide. The tight binding of molybdate did not allow an accurate estimation of the binding constants by these indirect methods. An isotopic binding method with 99MoO42- was used for accurate determination of KD (20 nM) and stoichiometry (1:1 molar ratio). ModA bound tungstate with approximately the same affinity, but did not bind sulfate or phosphate. These KDs are 150- to 250-fold lower than those previously reported, and compatible with the high molybdate transport affinity of the mod system. The affinity of ModA for molybdate was also determined in vivo and found to be similar to that determined in vitro.

Identification of a gene for a chemoreceptor of the methyl-accepting type in the symbiotic plasmid of Rhizobium leguminosarum bv. viciae UPM791.

The 4 kb DNA region located immediately upstream of the Rhizobium leguminosarum bv. viciae UPM791 hydrogen structural genes was sequenced and found to encode a chemoreceptor of the methyl-accepting type, the first to be described in a rhizobial symbiotic plasmid. Two additional open reading frames were found. Their protein products showed sequence homology to dehydrogenases and isomerases involved in the metabolism of aromatic compounds. Mutant analysis showed that this region is not required for hydrogenase activity.

Prevalence of impaired glucose tolerance and diabetes in women with polycystic ovary syndrome.

OBJECTIVE: NIDDM occurs commonly among women with polycystic ovary syndrome (PCOS). The prevalence and natural history of its precursor, impaired glucose tolerance (IGT), is less well known. The objective of this study was to characterize the prevalence and incidence of glucose intolerance in a large cohort of women with well-characterized PCOS. RESEARCH DESIGN AND METHODS: A total of 122 women with clinical and hormonal evidence of PCOS were recruited from the Medicine, Endocrinology, Gynecology, and Pediatrics Clinics at the University of Chicago. All women had a standard oral glucose tolerance test (OGTT) with measurement of glucose and insulin levels. A subset of 25 women were subsequently restudied with the aim of characterizing the natural history of glucose tolerance in PCOS. RESULTS: Glucose tolerance was abnormal in 55 (45%) of the 122 women: 43 (35%) had IGT and 12 (10%) had NIDDM at the time of initial study. The women with NIDDM differed from those with normal glucose tolerance in that they had a 2.6-fold higher prevalence of first-degree relatives with NIDDM (83 vs. 31%, P < 0.01 by chi 2) and were significantly more obese (BMI 41.0 +/- 2.4 vs. 33.4 +/- 1.1 kg/m2, P < 0.01). For the entire cohort of 122 women, there was a significant correlation between fasting and 2-h glucose concentrations (r = 0.76, P < 0.0001); among the subset with IGT, the fasting glucose concentration was poorly predictive of the 2-h level (r = 0.25, NS). After a mean follow-up of 2.4 +/- 0.3 years (range 0.5-6.3), 25 women had a second OGTT. The glucose concentration at 2 h during the second glucose tolerance test was significantly higher than the 2-h concentration during the first study (161 +/- 9 vs. 139 +/- 6 mg/dl, P < 0.02). CONCLUSIONS: The prevalence of IGT and NIDDM in women with PCOS is substantially higher than expected when compared with age- and weight-matched populations of women without PCOS. The conversion from IGT to NIDDM is accelerated in PCOS. The fasting glucose concentration does not reliably predict the glucose concentration at 2 h after an oral glucose challenge, particularly among those with IGT, the subgroup at highest risk for subsequent development of NIDDM. We conclude that women with PCOS should periodically have an OGTT and must be closely monitored for deterioration in glucose tolerance.

Low serum free thyroxine index in ambulating elderly is due to a resetting of the threshold of thyrotropin feedback suppression.

Subnormal free T4 index (FT4I) values (less than 80) with inappropriately normal serum TSH concentrations that could not be attributed to illness or drugs were found in 2.5% of ambulating elderly clinic patients. Six such individuals (three men and three women, aged 68.8 +/- 4.8 yr) were selected for their persistent thyroid test abnormalities and were sex and age matched to six subjects (67.7 +/- 4.9 yr) with normal FT4I (greater than 90) and TSH levels. The former also had low serum total T4 (TT4) and rT3 (TrT3) concentrations, but total T3 (TT3) and basal TSH values were normal and did not differ between the groups. Responses of ACTH, LH, FSH, TSH, and PRL to stimulation with CRH, GnRH, and TRH showed no differences between the two groups, indicating that the normal TSH concentration, inappropriate for the low FT4I level, is not due to generalized hypothalamic or pituitary dysfunction. Administration of 3 g iopanoic acid (IOP) daily for 3 days produced significant increases in the TT4 and TrT3 concentrations to the same degree in both groups. Also, in both groups the IOP-induced suppression of T4 to T3 conversion in the pituitary gland provoked similar increases in basal TSH (280 +/- 47% and 288 +/- 33%) and TSH secretion in response to TRH (173 +/- 7% and 156 +/- 13%). These results indicate that the low FT4I is not the consequence of reduced pituitary TSH reserve. In addition, evidence for normal thyroid gland reserve and the secretion of TSH of normal biological activity was obtained by comparing the acute iodothyronine responses to TRH-induced TSH release in both groups. It is concluded that the normal serum TSH concentration, inappropriate for the low FT4I value in some elderly subjects, is due to an apparent resetting of the thyroid hormone feedback regulation threshold of TSH secretion. It may, in turn, be the result of enhanced pituitary conversion of T4 to T3 or increased T4 uptake by the thyrotrophs.

Effects of metformin on insulin secretion, insulin action, and ovarian steroidogenesis in women with polycystic ovary syndrome.

Hyperinsulinemia contributes to the ovarian androgen overproduction and glucose intolerance of polycystic ovary syndrome (PCOS). We sought to determine whether metformin would reduce insulin levels in obese, nondiabetic women with PCOS during a period of weight maintenance and thus attenuate the ovarian steroidogenic response to the GnRH agonist leuprolide. All subjects (n = 14) had an oral glucose tolerance test, a GnRH agonist (leuprolide) test, a frequently sampled iv glucose tolerance test, graded and oscillatory glucose infusions, and a dual energy x-ray absorptiometry scan before and after treatment with metformin (850 mg, orally, three times daily for 12 weeks). With weight maintenance (body mass index: pretreatment, 39.0 +/- 7.7 kg/m2, posttreatment, 39.1 +/- 7.9 kg/m2), oral glucose tolerance, insulin sensitivity (Si; 0.87 +/- 0.82 vs. 0.74 +/- 0.63 x 10(-5) min-1/ pmol.L), and the relationship between Si and first phase insulin secretion (AIRg vs. Si) were not improved by metformin. The insulin secretory response to glucose, administered in both graded and oscillatory fashions, was likewise unaltered in response to metformin. Free testosterone levels remained about 2-fold elevated (pretreatment, 26.6 +/- 12.7 pg/mL; posttreatment, 22.4 +/- 9.8 pg/mL). Both basal and stimulated LH and FSH levels were unaffected by metformin. The mean responses to leuprolide of 17-hydroxyprogesterone (pretreatment, 387 +/- 158 ng/dL; posttreatment, 329 +/- 116 ng/dL) as well as those of the other ovarian secretory products (androstenedione, dehydroepiandrosterone, progesterone, and estradiol) were not attenuated by metformin. We conclude that hyperinsulinemia and androgen excess in obese nondiabetic women with PCOS are not improved by the administration of metformin.

Troglitazone improves defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis in women with polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) are characterized by defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis. We administered the insulin-sensitizing agent troglitazone to 13 obese women with PCOS and impaired glucose tolerance to determine whether attenuation of hyperinsulinemia ameliorates these defects. All subjects had oligomenorrhea, hirsutism, polycystic ovaries, and hyperandrogenemia. Before and after treatment with troglitazone (400 mg daily for 12 weeks), all had 1) a GnRH agonist (leuprolide) test, 2) a 75-g oral glucose tolerance test, 3) a frequently sampled iv glucose tolerance test to determine the insulin sensitivity index and the acute insulin response to glucose, 4) an oscillatory glucose infusion to assess the ability of the beta-cell to entrain to glucose as quantitated by the normalized spectral power for the insulin secretion rate, and 5) measures of fibrinolytic capacity [plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator]. There was no change in body mass index (39.9 +/- 1.4 vs. 40.2 +/- 1.4 kg/m2) or body fat distribution after treatment. Both the fasting (91 +/- 3 vs. 103 +/- 3 mg/dL; P < 0.001) and 2 h (146 +/- 8 vs. 171 +/- 6 mg/dL; P < 0.02) plasma glucose concentrations during the oral glucose tolerance test declined significantly. There was a concordant reduction in glycosylated hemoglobin to 5.7 +/- 0.1 from a pretreatment level of 6.1 +/- 0.1% (P < 0.03). Insulin sensitivity increased from 0.58 +/- 0.14 to 0.95 +/- 0.26 10(-5) min-1/pmol.L (P < 0.01) after treatment as did the disposition index (745 +/- 135 vs. 381 +/- 96; P < 0.05). The ability of the beta-cell to appropriately detect and respond to an oscillatory glucose infusion improved significantly after troglitazone treatment; the normalized spectral power for the insulin secretion rate increased to 5.9 +/- 1.1 from 4.3 +/- 0.8 (P < 0.05). Basal levels of total testosterone (109.3 +/- 15.2 vs. 79.4 +/- 9.8 ng/dL; P < 0.05) and free testosterone (33.3 +/- 4.0 vs. 21.2 +/- 2.6 pg/mL; P < 0.01) declined significantly after troglitazone treatment. Leuprolide-stimulated levels of 17-hydroxyprogesterone, androstenedione, and total testosterone were significantly lower posttreatment compared to pretreatment. The reduction in androgen levels occurred independently of any changes in gonadotropin levels. A decreased functional activity of PAI-1 in blood (from 12.7 +/- 2.8 to 6.3 +/- 1.4 AU/mL P < 0.05) was associated with a decreased concentration of PAI-1 protein (from 64.9 +/- 9.1 to 44.8 +/- 6.1 ng/mL; P < 0.05). No change in the functional activity of tissue plasminogen activator (from 5.3 +/- 0.4 to 5.1 +/- 0.5 IU/mL) was observed despite a decrease in its concentration (from 9.6 +/- 0.9 to 8.2 +/- 0.7 ng/mL; P < 0.05). The marked reduction in PAI-1 could be expected to improve the fibrinolytic response to thrombosis in these subjects. We conclude that administration of troglitazone to women with PCOS and impaired glucose tolerance ameliorates the metabolic and hormonal derangements characteristic of the syndrome. Troglitazone holds potential as a useful primary or adjunctive treatment for women with PCOS.

Retinal ganglion cell death after acute retinal ischemia is an ongoing process whose severity and duration depends on the duration of the insult.

In adult Sprague-Dawley rats we have investigated retinal ganglion cell survival after transient intervals of retinal ischemia of 30, 45, 60, 90 or 120 min duration, induced by ligature of the ophthalmic vessels. Animals were killed 5, 7, 14, 21, 30, 60, 90 or 180 days later and densities of surviving retinal ganglion cells were estimated in retinal whole mounts by counting cells labelled with diAsp. This dye was applied, 3 days prior to death, to the ocular stump of the intraorbitally transected optic nerve. We found that retinal ganglion cell loss after retinal ischemia proceeds for different lengths of time. All the ischemic intervals induced loss of retinal ganglion cells whose severity and duration was related to the length of the ischemic interval. Following 30 or 45 min of ischemia, cell loss lasted 14 days and caused the death of 46 or 50%, respectively, of the population of retinal ganglion cells. Sixty, 90 or 120 min of retinal ischemia were followed by a period of cell loss that lasted up to 90 days and caused the death of 75%, 87% or 99%, respectively, of the population of retinal ganglion cells. We conclude that retinal ganglion cell loss after retinal ischemia is an ongoing process that may last up to 3 months after the injury and that its severity and duration are determined by the ischemic interval.

Neuroprotective effects of alpha(2)-selective adrenergic agonists against ischemia-induced retinal ganglion cell death.

PURPOSE: To investigate in adult rats the effects of two alpha(2)-selective adrenergic agonists (alpha(2)-SAs; AGN 191103 and AGN 190342) on retinal ganglion cell (RGC) survival after transient retinal ischemia. METHODS: RGCs were labeled with a Fluorogold (FG) tracer applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 60 or 90 minutes. In one group, a single dose of saline or one alpha(2)-SA was administered intraperitoneally (IP) or topically 1 hour before ischemia. In another group, a single dose of AGN 190342 was administered IP, 1, 2, 4, 24, or 72 hours after ischemia. Rats were processed 7, 14, or 21 days later. Densities of surviving RGCs were estimated by counting FG-labeled cells in 12 standard retinal areas. RESULTS: Seven days after 60 or 90 minutes of retinal ischemia, death had occurred in 36% or 47%, respectively, of the RGC population, and by 21 days the loss of RGCs amounted to 42% or 62%, respectively. Systemic pretreatment with an alpha(2)-SA resulted in enhanced survival of ischemic-injured RGCs. Topical pretreatment with an alpha(2)-SA prevented up to 100% of the ischemia-induced RGC loss. Pretreatment with an alpha(2)-SA abolished the secondary slow RGC loss that occurred between days 7 and 21 after ischemia. When administered shortly after ischemia (up to 2 hours) AGN 190342 rescued substantial proportions of RGCs destined to die and diminished slow RGC death. CONCLUSIONS: Pretreatment and early posttreatment with an alpha(2)-SA induces marked long-lasting neuroprotective in vivo protection against ischemia-induced cell death in RGCs.

Effects of intramuscular injection of botulinum toxin and doxorubicin on the survival of abducens motoneurons.

PURPOSE: To investigate in vivo the survival of abducens motoneurons (AMNs) at different periods of time after a single intramuscular injection of the neurotoxin botulinum toxin A (BTxA) or doxorubicin (DXR). METHODS: In Sprague-Dawley rats, the AMNs were labeled with fluorogold (FG), which was applied intramuscularly in the lateral rectus muscle. The numbers of labeled neurons were determined in adult control animals; in young animals that had received intramuscular injections of 0.125, 0.250, 1, or 2 U BTxA; and in adult rats that had received 100 microg, 200 microg, or 300 microg DXR, at various survival times. RESULTS: In control animals, the numbers of FG-labeled motoneurons were similar to the numbers found by other investigators with the use of other retrogradely transported tracers; motoneuron numbers diminished with time after FG application. The numbers of FG-labeled neurons in the animals that had been injected with BTxA were similar to those found in control animals. However, there were fewer FG-labeled neurons in the animals injected with DXR. CONCLUSIONS: Fluorogold injected into the lateral rectus muscle can be used to label the AMNs. However, this tracer does not persist within the cytoplasm of the labeled neurons for more than 37 days. The intramuscular injection of 0.125, 0.250, 1, or 2 U BTxA does not induce significant motoneuron death in young rats 30, 60, or 90 days after the injection. Doxorubicin injected intramuscularly causes variable amounts of motoneuron death that is related both to the survival period and to the amount of DXR injected.

Retinal ganglion cell death induced by retinal ischemia. neuroprotective effects of two alpha-2 agonists.

We have investigated in adult Sprague-Dawley rats the neuroprotective effects of two alpha-2-selective agonists [AGN 191,103 (AGN) and brimonidine tartrate (BMD)] on retinal ganglion cell (RGC) survival after transient retinal ischemia. RGCs were labelled with Fluorogold (FG) applied to both superior colliculi. Seven days later, 90 min of retinal ischemia were induced in the left eyes by ligature of the ophthalmic vessels (LOV). In one group of animals, vehicle or AGN (0.01 mg/kg) were administered systemically 1 hr before ischemia. In another group of animals, two 5 microl drops of vehicle, AGN (0.05%) or BMD (0.1%) were administered topically in the left eye 1 hr before ischemia. The animals were processed 7 or 21 days later. RGC survival was estimated by counting FG-labelled cells in 12 standard areas of each retina. In control retinas of systemically pretreated animals, mean densities of labelled RGCs were 2372 +/- 49 cells/mm(2) (mean +/- SEM; n = 6). In experimental retinas of systemically pretreated animals, mean RGC densities had decreased 7 days after ischemia to 53% (n = 6) or 81% (n = 6) of control in the groups treated with vehicle or AGN, respectively. Twenty-one days after ischemia, mean RGC densities had decreased to 38% (n = 6) or 79% (n = 6) of control in the groups treated with vehicle or AGN, respectively. In control retinas of topically pretreated animals, mean densities of labelled RGCs were 2208 +/- 29 cells/mm(2) (n = 6). In experimental retinas of topically pretreated animals, mean RGC densities had decreased 7 days after ischemia to 54% (n = 6), 95% (n = 6) or 96% (n = 6) of control in the groups treated with vehicle, AGN or BMD, respectively. These results indicate that pretreatment with a single systemic or topical dose of AGN or BMD can prevent completely the early rapid phase of RGC loss and abolish the delayed RGC loss observed after 90 min of retinal ischemia induced by ligature of the ophthalmic vessels.

Orthotopic liver transplantation for hepatocellular carcinoma. Factors affecting long-term patient survival.

OBJECTIVE: To determine the influence of several clinicopathologic factors on the 3-year actuarial survival of patients with nonfibrolamellar hepatocellular carcinoma (HCC) following orthotopic liver transplantation (OLT). DESIGN: A case series of 26 consecutive patients with HCC treated with OLT, with a maximum follow-up of 90 months. SETTING: A tertiary care center. PATIENTS: Between March 1988 and December 1993, 521 OLTs were performed in 480 patients, 27 of whom had HCC. One patient was excluded because of donor-transmitted melanoma. Of the remaining 26 patients, there were 18 adults and 8 children, with a mean age of 41 years (range, 0.2-67.4 years). Fourteen patients (54%) had either hepatitis B (n = 6) or hepatitis C (n = 8), while 15 (58%) had coincidental tumor. INTERVENTION: OLT was performed using standard techniques. MAIN OUTCOME MEASURES: The effect of several clinicopathologic factors on 3-year actuarial patient survival. RESULTS: The overall actuarial survival rates for the 26 patients with HCC were 73%, 65.4%, and 65.4%, at 1, 2, and 3 years, respectively. Sixteen patients (62%) were alive at the time of this report, with 14 (54%) free of disease. None of the clinicopathologic factors significantly affected the 3-year patient survival rate. However, the rate of recurrent HCC was significantly higher in nonincidental vs coincidental tumors and in solitary vs multiple tumors. CONCLUSION: Our results suggest that HCC should not contraindicate OLT, as long-term patient survival and cure can be achieved. While patient selection is important, survival in patients with HCC after OLT is not always predictable using the usual clinicopathologic prognostic factors.

Identification of gene products from the Azotobacter vinelandii nifBfdxNnifOQ operon.

The Azotobacter vinelandii nifBfdxNnifOQ operon is required for synthesis of the nitrogenase iron-molybdenum cofactor. To further characterize the roles of its gene products, specific antibodies against NifB and NifO were generated, and the NifB, NifO and NifQ gene products were visualized and identified in nitrogen-fixing A. vinelandii cell extracts by a combination of two-dimensional gel electrophoresis of radiolabelled extracts and immunological detection methods. The three proteins showed apparent pI and M(r) values similar to those expected from sequence data, except for NifO, which showed an apparent M(r) of ca. 23 kDa (vs. 16 kDa expected).

Rhizobium leguminosarum bv. viciae hypA gene is specifically expressed in pea (Pisum sativum) bacteroids and required for hydrogenase activity and processing.

Rhizobium leguminosarum bv. viciae strain UPM791 induces in symbiosis with peas the synthesis of a nickel-containing hydrogenase which recycles the hydrogen evolved by nitrogenase. The genes required for synthesis of this hydrogenase, hupSLCDEFGHIJKhypABFCDEX, are clustered in the symbiotic plasmid. Analysis of a hypA-deficient mutant showed that HypA is essential for symbiotic hydrogenase activity and required for correct processing of the hydrogenase large subunit. Unlike other microoxically induced hyp genes, the hypA gene was only expressed in pea bacteroids from its own promoter. The hypA mRNA 5' end was mapped 109 bp upstream of the translational start codon. This distinct pattern of expression suggests a different role for HypA and the remaining Hyp proteins in hydrogenase synthesis.

The distribution of omega-conotoxin MVIICnle-binding sites in rat brain measured by autoradiography.

An analogue of omega-conotoxin MVIIC, [125I]omega-MVIICnle, has been employed in an autoradiographic assay to define the distribution of binding sites in rat brain of this neuronal calcium channel antagonist. In comparison with N-type channels (labeled by [125I]omega conotoxin GVIA), omega-MVIICnle sites are much denser in cerebellum (molecular layer) than in forebrain. Binding in thalamus is also comparatively high for omega-MVIICnle. Under these conditions, [125I]omega-MVIICnle binding to rat brain sections is not displaceable by the N-channel antagonist, omega-conotoxin GVIA. The calcium channel blocker [125I]omega-conotoxin MVIICnle labels a unique set of binding sites in mammalian brain.

Precursor structure of omega-conotoxin GVIA determined from a cDNA clone.

The Ca2+ channel blocking neurotoxin, omega-conotoxin GVIA, is a 27-amino acid peptide with three disulfide bonds. We have determined the precursor structure of this peptide by analyzing a cDNA clone obtained from a Conus geographus venom duct library. The omega-conotoxin GVIA prepropeptide is 73 amino acids in length comprising a 22 amino acid signal sequence, an intervening region of 23 amino acids, and finally, a 27 amino acid toxin region. A C-terminal glycine residue is later processed to a C-terminal amide moiety. The omega-conotoxin GVIA precursor exhibits regions of strong homology to the previously characterized King-Kong peptide precursor, but shows surprising divergence as well. The possible significance of the precursor organization is discussed.