Deletion of mouse MsrA results in HBO-induced cataract: MsrA repairs mitochondrial cytochrome c.

PURPOSE: Considerable evidence indicates a role for methionine sulfoxide reductase A (MsrA) in lens cell resistance to oxidative stress through its maintenance of mitochondrial function. Correspondingly, increased protein methionine sulfoxide (PMSO) is associated with lens aging and human cataract formation, suggesting that loss of MsrA activity is associated with this disease. Here we tested the hypothesis that loss of MsrA protein repair is associated with cataract formation. To test this hypothesis we examined the effect of MsrA deletion on lens opacity in mice treated with hyperbaric oxygen, identified lens mitochondrial proteins oxidized upon deletion of MsrA and determined the ability of MsrA to repair the identified proteins. METHODS: Wild-type and MsrA knockout mice were treated or not treated with 100 treatments of hyperbaric oxygen (HBO) over an 8 month period and lenses were examined by in vivo light scattering measurements documented by slit-lamp imaging. Co-immunoprecipitation of MsrA was conducted against five specific protein representatives of the five complexes of the electron transport chain in addition to cytochrome c (cyt c). Cyt c in lens protein from the knockout and wild-type lenses was subjected to cyanogen bromide (CNBr) cleavage to identify oxidized methionines. Methionine-specific CNBr cleavage was used to differentiate oxidized and un-oxidized methionines in cyt c in vitro and the ability of MsrA to restore the activity of oxidized cyt c was evaluated. Mass spectrometry analysis of cyt c was used to confirm oxidation and repair by MsrA in vitro. RESULTS: HBO treatment of MsrA knockout mice led to increased light scattering in the lens relative to wild-type mice. MsrA interacted with four of the five complexes of the mitochondrial electron transport chain as well as with cyt c. Cyt c was found to be aggregated and degraded in the knockout lenses consistent with its oxidation. In vitro analysis of oxidized cyt c revealed the presence of two oxidized methionines (met 65 and met 80) that were repairable by MsrA. Repair of the oxidized methionines in cyt c restored the activity of cytochrome c oxidase and reduced cytochrome c peroxidase activity. CONCLUSIONS: These results establish that MsrA deletion causes increased light scattering in mice exposed to HBO and they identify cyt c as oxidized in the knockout lenses. They also establish that MsrA can restore the in vitro activity of cyt c through its repair of PMSO. These results support the hypothesis that MsrA is important for the maintenance of lens transparency and provide evidence that repair of mitochondrial cyt c by MsrA could play an important role in defense of the lens against cataract formation.

The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1.

BACKGROUND: Signals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway. RESULTS: We identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis. CONCLUSIONS: We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity.

The v-ski oncogene encodes a truncated set of c-ski coding exons with limited sequence and structural relatedness to v-myc.

The nucleotide sequence of a biologically active v-ski gene from a cloned proviral segment shows that ski is a 1,312-base sequence embedded in the p19 region of the avian leukosis virus gag gene. The v-ski sequence contains a single open translational reading frame that encodes a polypeptide with a molecular mass of 49,000 daltons. The predicted amino acid sequence includes nuclear localization motifs that have been identified in other nuclear oncoproteins. It also contains a proline-rich region and a set of cysteine and histidine residues that could constitute a metal-binding domain. Two regions of the amino acid sequences of v-ski and v-myc are related, and the two proteins exhibit similar distributions of hydrophobic and hydrophilic amino acids. Cloned segments of the chicken c-ski proto-oncogene totaling 65 kilobases have been analyzed, and regions related to v-ski have been sequenced. The results indicate that v-ski is derived from at least five coding exons of c-ski, that it is correctly spliced, and that it is missing c-ski coding sequences at both its 5' and 3' ends. The c-ski and avian leukosis virus sequences that overlap the 5' virus/v-ski junction in Sloan-Kettering virus contain an 18-of-20-base sequence match that presumably played a role in the transduction of ski by facilitating virus/c-ski recombination.

Embryonic development of four different subsets of cholinergic neurons in rat cervical spinal cord.

The developmental stage at which a neuron becomes committed to a neurotransmitter phenotype is an important time in its ontogenetic history. The present study examines when choline acetyltransferase (ChAT) is first detected within each of four different subsets of cholinergic neurons previously identified in the cervical enlargement of the spinal cord: namely, motor neurons, partition cells, central canal cluster cells, and dorsal horn neurons. By examining the temporal sequence of embryonic development of these cholinergic neurons, we can infer the relationships between ChAT expression and other important developmental events. ChAT was first detected reliably on embryonic day 13 (E13) by both biochemical and immunocytochemical methods, and it was localized predominantly within motor neurons. A second group of primitive-appearing ChAT-positive cells was detected adjacent to the ventricular zone on E14. These neurons seemed to disperse laterally into the intermediate zone by E15, and, on the basis of their location, were tentatively identified as partition cells. A third group of primitive ChAT-immunoreactive cells was detected on E16, both within and around the ventral half of the ventricular zone. By E17, some members of this "U"-shaped group appeared to have dispersed dorsally and laterally, probably giving rise to dorsal horn neurons as well as dorsal central canal cluster cells. Other members of this group remained near the ventral ventricular zone, most likely differentiating into ventral central canal cluster cells. Combined findings from the present study and a previous investigation of neurogenesis (Phelps et al.: J. Comp. Neurol. 273:459-472, '88), suggest that premitotic precursor cells have not yet acquired the cholinergic phenotype because ChAT is not detectable until after the onset of neuronal generation for each of the respective subsets of cholinergic neurons. However, ChAT is expressed in primitive bipolar neurons located within or adjacent to the germinal epithelium. Transitional stages of embryonic development suggest that these primitive ChAT-positive cells migrate to different locations within the intermediate zone to differentiate into the various subsets of mature cholinergic neurons. Therefore, it seems likely that spinal cholinergic neurons are committed to the cholinergic phenotype at pre- or early migratory stages of their development. Our results also hint that the subsets of cholinergic cells may follow different migration routes. For example, presumptive partition cells may use radial glial processes for guidance, whereas dorsal horn neurons may migrate along nerve fibers of the commissural pathway. Cell-cell interactions along such diverse migratory pathways could play a role in determining the different morphological, and presumably functional, phenotypes expressed by spinal cholinergic neurons.

Saralasin infusion in renovascular hypertension: increased response rate in seated patients.

Most series report a significant percentage of nondepressor (negative) responses to saralasin infusions in patients with surgically curable renovascular hypertension. Usually the infusions have been performed with the supine position. In the present study saralasin infusions were done in 33 acutely sodium-depleted hypertensive patients--17 with renovascular and 16 with the essential variety. Most patients were infused in the seated and supine positions to test the hypothesis that there might be a higher incidence of positive (vasodepressor) responses to the saralasin infusion when patients are in a sitting position. The results did, in fact, show a larger number of positive responses to saralasin in the seated patients with renovascular hypertension than in those in the supine position (88% versus 71%). There was a modest increase in the rate of false-positive responses in the patients with essential hypertension who were infused in the seated position (31% versus 23%). However, inasmuch as the saralasin infusion test is proposed as a screening procedure for renovascular hypertension, some false-positive responses are acceptable.

Gastrointestinal hormone profile in renal insufficiency.

Fasting serum gastrin, cholecystokinin, glucagon, and gastric inhibitory polypeptide concentrations were simultaneously measured in normal subjects and in patients with different degrees of renal failure. Values of gastrin, cholecystokinin, gastric inhibitory polypeptide, and glucagon were significantly higher in all patients with serum creatinine concentrations greater than 3 mg/dl than in controls (P less than 0.01). The degree of renal insufficiency was significantly correlated (P less than 0.05) with serum concentrations of each hormone, but no significant linear correlation existed among the serum concentrations of different gastrointestinal hormones in individuals. Hemodialysis did not significantly alter predialysis serum gastrin, cholecystokinin, or glucagon concentration, but the serum gastric inhibitory polypeptide concentration decreased by 30% (P less than 0.01) after hemodialysis. The disproportionate increases of hormones with antagonistic actions may alter gastrointestinal function in renal insufficiency.

Use of captopril as early therapy for renal scleroderma: a prospective study.

We conducted a prospective study of captopril therapy in patients with scleroderma and combined hypertension and renal insufficiency. In all seven patients studied during a 1-year period, control of blood pressure was achieved, and in six of the seven, renal function stabilized or improved. The total daily dosage of captopril ranged from 32 to 100 mg, divided into doses taken every 6 to 8 hours. Although one patient had a suspected captopril-induced rash for a short time, none of the other patients had any adverse side effects. Renal biopsies were performed in six patients; in three of them, specimens were obtained both at the beginning and at the end of the study. The initial biopsy specimens showed changes that were similar to those described in other reports. Findings on repeat biopsies were unchanged except for evidence of chronicity. In the six patients with controlled blood pressure and improved or stabilized renal function, the improvement was maintained for 1 1/2 to nearly 3 years on this drug therapy. Using specific measurements of skin compliance and vascular blood flow in the upper extremities, we could detect no evidence, however, of concomitant improvement in these other features of the disease. Although the blood pressure was controlled with captopril, one patient had progressive skin induration, one had progressive pulmonary insufficiency, and another had progressive renal failure.

The Mayo Three-Community Hypertension Control Program. I. Design and initial screening results.

The Mayo Three-Community Hypertension Control Program implemented graduated programs for the control of high blood pressure in three rural southeastern Minnesota communities, beginning in 1974. Prevalence of hypertension (when defined as diastolic blood pressure, at initial screening, of 95 mm Hg or more) was similar to that found for comparable groups by age and sex in the United States generally, but an atypically high frequency of known but untreated hypertension was found. Programs of public and professional information, systematic household screening, continuing professional education (two communities), and a new community hypertension clinic (one community) were initiated, and plans were made to evaluate the programs simultaneously by means of total rescreening of persons found to be hypertensive initially. The present report describes in detail the design of the program and the results of initial screening in relation to findings in other populations at the time. Subsequent reports assess the impact of each program on its target community and of a community hypertension clinic within the one setting where this component of a model program was established.

The Mayo Three-Community Hypertension Control Program. III. Outcome in a community-based hypertension clinic.

As part of a broader community program to evaluate approaches to hypertension control, a Community Hypertension Clinic, staffed by two nurse practitioners, was set up in a rural community. Hypertensive persons were identified either by an initial central blood pressure screening or by a subsequent home screening. Slightly more than half of the hypertensive patients at initial screening, or 256 persons, elected to go to the Community Hypertension Clinic for second-stage screening, whereas the remainder elected to see their physicians or to do neither. After secondary screening at the Clinic, 120 patients eventually came under care and were managed by the nurse practitioners. After 2 years of follow-up, 57% of the Clinic patients had office-recorded diastolic blood pressures of less than 90 mm Hg. The Community Hypertension Clinic dropout rate was only 5% after 30 months of operation, for participants whose duration of follow-up ranged from 12 to 27 months (median 16 months), when a repeat home blood pressure screening examination was performed. Comparison of outcomes was thus possible between persons who attended the Community Hypertension Clinic and those who were referred to their physicians' offices. Persons with more severe hypertension most often elected to go to the Clinic, whereas patients with milder degrees of hypertension tended to go to their private physicians for follow-up or failed to make the recommended second-stage screening contact altogether. Greater declines in blood pressure were observed in the Clinic group.

The Mayo Three-Community Hypertension Control Program. II. Outcome of intervention in entire communities.

A pronounced decline in blood pressure levels of hypertensive patients occurred in each of three rural Minnesota communities 1 to 2 years after the inception of community programs to control high blood pressure in these populations. An experimental hypertension clinic was established in one community to integrate a nurse practitioner into a physician-supervised program of long-term patient management. In the community with this innovative, partially subsidized practice arrangement, we observed declines in diastolic pressures of hypertensives. However, comparable degrees of blood pressure reduction occurred in the two other communities, with traditional solo or small group practice arrangements, where intervention was limited to detection and referral alone or was supplemented with continuing education of physicians in the management of hypertension. The evaluation of these three community programs suggests, among other conclusions, that this innovative community model for hypertension control, based on the recommendations of the Inter-Society Commission for Heart Disease Resources, contributed to favorable short-term blood pressure outcomes for the community. The observation of similar overall outcomes as measured by blood pressure reduction in all three communities was unexpected; the clinic's impact appears to have been matched by the effectiveness of screening and referral, alone or with continuing education, in the two other communities.

Renovascular hypertension. Relationship of surgical curability to renin-angiotensin activity.

Fifteen patients whose renovascular hypertension was cured or improved by renal artery reconstructive surgery or nephrectomy (or both) underwent studies of their renin-angiotensin systems preoperatively. These studies included measurements of peripheral venous renin activity in the erect position without diuretic preparation, blood pressure response to blockade of endogenous angiotensin II with a saralasin infusion in the acutely sodium-depleted state, and levels of renal venous renin activity, also after sodium depletion. In 13 of these 15 patients who had benefitted from surgical intervention for relief of renovascular hypertension, at least one index of renin dependency was positive. Two patients had negative results in all of the tests. On the basis of these findings, we concluded that cure or improvement of renovascular hypertension is possible even though these three parameters of renin-angiotensin overactivity are negative.

The management of acute quinidine intoxication.

A 16-year-old patient survived severe intoxication with quinidine. Hypotension, rapidly progressing to oliguria and shock, was resistant to the usual therapeutic interventions but responded favorably to the use of an intra-aortic balloon pump. Some hemodynamic implications are discussed. Pulmonary edema occurred and was treated with positive end-expiratory pressure. Electrocardiographic disturbances in conduction, transient bradycardia and recurrent ventricular arrhythmias characterized the initial 36-hour critical period. Unexplained electrolyte abnormalities occurred and further complicated management.

Generation patterns of immunocytochemically identified cholinergic neurons in rat brainstem.

Combined [3H]thymidine autoradiographic and choline acetyltransferase (ChAT)-immunocytochemical techniques were used to answer questions concerning the generation of specific classes and subclasses of cholinergic neurons in rat brainstem. First, the generation of rostrally and caudally located neurons of the same class (i.e. somatic efferent oculomotor and hypoglossal nuclei, respectively) were compared. Results indicated that, although embryonic day 11 (E11) was the peak birthday for both nuclei, hypoglossal neurons were generated significantly earlier than oculomotor neurons, indicating a caudorostral generation gradient for brainstem somatic motor nuclei. Second, the generation patterns of 3 different subclasses of motor neurons at the same brainstem level were compared; namely those of the somatic efferent hypoglossal nucleus (XII), the general visceral efferent dorsal nucleus of the vagus (X), and the predominantly special visceral efferent nucleus ambiguus. All 3 subclasses of cholinergic cells had the same peak day (E11) and overall period of generation (E11-12). However, statistical analyses indicated a precocious generation of nucleus ambiguus, but no developmental differences between N, XII and N. X. It is suggested that nucleus ambiguus is formed earlier than N. XII and N. X, due to its more ventral location within a ventrodorsal neurogenetic gradient. Third, the generation patterns of different classes of large cholinergic neurons were examined. Specifically, the birthdays of cholinergic non-motor projection neurons of the pedunculopontine-laterodorsal tegmental nuclei (PPT-LDT) were contrasted to those of the cholinergic brainstem motor neurons. The peak birthdays of both rostrally and caudally located motor neurons were two days earlier than those of the PPT-LDT neurons. Thus, large cholinergic cells projecting to peripheral targets are born significantly earlier than those projecting within the CNS, even though the former are located more rostrally on the caudorostral neurogenetic gradient. This represents an apparent exception to the emerging rule that cholinergic neurons obey the general gradients of neurogenesis manifest in the regions of the central nervous system where they reside.

Concentrations of antidiuretic hormone in plasma during human sodium restriction.

Negative sodium balance was produced in 10 human volunteers. Body weight, plasma sodium, osmolality, hematocrit, renin activity (PRA), and antidiuretic hormone (ADH) concentrations were determined before, during, and after sodium restriction. Body weight declined and PRA rose during the period of low sodium intake. Plasma sodium concentration and osmolality did not change. A statistically significant change in ADH was not observed. It is suggested that a decrease in ADH was prevented by a rising titer of renin and contraction of the extracellular space.

Fate of mutagenic activity during conventional treatment of municipal wastewater sludge.

The mutagenic activity of wastewater was followed during conventional activated sludge treatment at a municipal plant. Raw wastewater was initially screened for mutagenic potential, using the AmesSalmonella/mammalian microsomal test and employer tester strains. The combined raw wastewater produced dose-related mutagenic responses in the presence of S9 metabolic activation. Raw wastewater from domestic sources alone was not mutagenic. Mutagenic activity was observed throughout the treatment process. Activated sludge prior to chlorination contained the highest specific mutagenic activity. Chlorination decreased the specific mutagenic activity at pH 11. Mutagenic activity in municipal wastewater containing industrial discharges is not removed by conventional treatment processes and can be enhanced by activated sludge treatment.

Effects of environmental factors on the in vitro potency of azithromycin.

The effects of media, pH, cations, serum, CO2 or anaerobic atmosphere, inoculum size and time of incubation on the in vitro potency of azithromycin were determined. The potency of azithromycin against all genera was particularly sensitive to changes in pH. The MIC for Staphylococcus aureus strains ranged from 50 micrograms/ml at pH 6 to less than or equal to 0.025 micrograms/ml at pH 8; for erythromycin the MIC change was less (1.6 to 0.05 micrograms/ml). Incubation for 18 h in 5% CO2 or an anaerobic atmosphere (10% CO2, 10% H2, 80% N2) lowered the pH by approximately 0.8 units with gram-negative organisms and 0.4 units with gram-positive organisms. This resulted in an MIC eight times greater than the aerobic MIC. In addition, the MIC100 for azithromycin and erythromycin against Bacteroides strains growing in Wilkins-Chalgren broth fell from 3.1 micrograms/ml in the anaerobic atmosphere to 0.2 and 0.4 micrograms/ml, respectively, when using the Oxyrase enzyme system to remove oxygen. With the Oxyrase system, the pH of the medium at the MIC remained at 7.2, while it fell to 6.7 in the anaerobic gas mixture. An increase in potency for both agents was also observed with other anaerobic species when using the Oxyrase system. The addition of serum produced an increase in potency of azithromycin and erythromycin that correlated with an increase in pH during incubation, despite the use of buffered media. Adding cations to Mueller-Hinton broth resulted in increased MICs for gram-negative organisms; the highest increases observed were four-fold for Escherichia coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Tli1, a resistance locus for carcinogen-induced T-lymphoma.

Within 180 days after injection with N-methyl-N-nitrosourea (MNU), 83.5% of AKR/J mice and 37.5% of BALB/cJ mice developed T-lymphoma. The high tumor incidence was a dominant trait, as 93% of MNU-injected F1 mice developed T-lymphoma. A genome screen of 285 MNU-injected F2 mice identified a locus, designated T-lymphoma Induced 1 or Tli1, in a approximately 10-cM interval on central Chr 1 between D1Mit87 and D1Mit423 with significant linkage to the incidence of MNU-induced T-lymphoma (P = 0.0004). Injection of BALB/cJ.AKR/J-Tli1 congenic mice with MNU confirmed the presence of Tli1 on central Chr 1. Mice homozygous for the BALB/cJ allele (Tli1bb) were over-represented in the tumor-free F2 mice, while the inheritance of parental alleles of Tli1 in tumor-bearing mice was close to expected. This suggests that the Tli1b allele is recessive and suppresses MNU-induced T-lymphoma development in BALB/cJ mice and in Tli1bb F2 mice. Furthermore, the kinetics of lymphoma development in BALB/cJ and the Tli1 congenic mice suggests that Tli1b acts to suppress lymphomas developing late after injection with MNU. Two known genes that map in the identified genomic interval on central Chr 1 are candidates for Tli1:IL10, encoding the lymphokine IL10, and Cmkar4, encoding the chemokine receptor CXCR4.

An improved method of toluidine blue counterstaining of neurons in immunocytochemical preparations.

Immunocytochemical methods can identify individual neurons and processes made immunoreactive by virtue of the antigens they contain. However, frequently it is also useful to visualize surrounding nonimmunoreactive cells, but immunocytochemical procedures often interfere with the quality of subsequent counterstaining. This report describes an improved method of counterstaining immunocytochemical specimens with either aged (at least 1 year) or concentrated solutions of toluidine blue. This technique combines well with immunocytochemical preparations of at least two antigens, i.e., choline acetyltransferase and glutamic acid decarboxylase, to delineate nonimmunoreactive somata. Additionally, a method of photographing these color preparations is described that, by the use of an appropriate filter, allows one to illustrate sections essentially with and without blue counterstain in black and white photomicrographs.

Activated B cells express increased levels of costimulatory molecules in young autoimmune NZB and (NZB x NZW)F(1) mice.

Polyclonal B cell activation is a hallmark of autoimmune disease in NZB and (NZB x NZW)F(1) (NZB/W) mice. However, the mechanism by which this activated cell subset facilitates disease development is unknown. We recently showed that resting B cells from these mice demonstrate enhanced expression of costimulatory molecules in response to CD40 crosslinking (Jongstra-Bilen et al., J. Immunol. 159,5810-5820, 1997). This led us to question whether activated B cells expressed costimulatory molecules in vivo. Using flow cytometry we found that NZB and NZB/W mice have an increased proportion of splenic B cells expressing B7.1 and elevated levels of B7.2 and ICAM-1. These B cells isolate within the low-density activated population and possess the phenotypic characteristics of marginal zone B cells. The levels of B7.1 on the activated B cell population are similar to those induced by CD40 stimulation raising the possibility that activated B cells in NZB and NZB/W mice provide costimulatory signals to self-reactive T cells leading to loss of tolerance.