WITHDRAWN: Profiles in Comparative Endocrinology: Carl B. Schreck Howard Bern Lecturer, Annual Meeting of Society of Integrative and Comparative Endocrinology, Seattle, January 5, 2010.

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Duplication of the POMC gene in the paddlefish (Polyodon spathula): analysis of gamma-MSH, ACTH, and beta-endorphin regions of ray-finned fish POMC.

The proopiomelanocortin (POMC) gene, which encodes the common precursor for MSH-related and beta-endorphin-related end products, appeared early in chordate evolution and features a variety of lineage-specific modifications. Key among these has been the apparent degeneration and subsequent deletion of the gamma-MSH region during the evolution of POMC in the ray-finned fish. A second area of increasing focus has been the role of gene duplication in the evolution of POMC in particular and the opioid/orphanin gene family in general. The cloning and phylogenetic analysis of two POMC cDNAs from the paddlefish (Polyodon spathula) is reported here and biochemical data on their processed end products are presented. Based on conceptual amino acid translations, the paddlefish cDNAs encode all functional domains and, in most cases, the flanking paired-basic amino acid cleavage sites characteristic of gnathostome POMCs (i.e., signal sequence, gamma-MSH-like region, ACTH (alpha-MSH and CLIP), gamma-LPH, beta-MSH, and beta-endorphin). Phylogenetic analysis of the paddlefish POMC sequences in the context of the duplicated POMCs of sturgeon and salmonids suggests that degeneration of the gamma-MSH core sequence and its amino-terminal proteolytic cleavage site was initiated prior to divergence of the sturgeon and paddlefish lineages over 150 mya. Finally, a comparison of the relative rates of evolutionary divergence between paralogously related POMC genes within chondrostean and salmonid lineages provides potential support for the hypothesis that some taxa (e.g., the Chondrosteii) represent relic species as a result of an exceptionally slow rate of evolutionary change.

Long-term predictable nipple projection following reconstruction.

The creation of the nipple-areola complex is often the final step in the surgical treatment of breast cancer patients, and it consequently has important symbolic and aesthetic implications. Patient expectations and the need for symmetry make nipple projection a crucial aesthetic determinant of nipple reconstruction. We hypothesize that long-term nipple projection and shape can be achieved in a predictable fashion using the modified star dermal fat flap technique. Prospectively, 93 nipples were reconstructed by a single surgeon using a modified star dermal fat flap technique in 44 implant and 49 TRAM flap breast reconstructions. Flap dimensions (base diameter and flap length) were designed according to patient desire or to the base diameter and projection of the opposite breast nipple. A standardized, 3-month postoperative care regimen was observed in all patients. Nipple projection was assessed by the same observer at each follow-up examination. The average length of follow-up was 730 days (745 for TRAM reconstructions and 713 for implants). Consistently, an average of 41 percent of the intraoperative projection remained intact in both groups at final evaluation (SD 12 percent). The total flap length was strongly predictive of intraoperative and long-term projection (r = 0.64 and 0.86, p < 0.0001). Flap lengths ranged from 5.5 to 9.0 cm, and in a linear correlation, resulted in intraoperative projection of 1.0 to 2.1 cm, respectively, and long-term projection of 0.4 to 0.83 cm, respectively. Based on the linear relationship, every 1-cm increase in flap length could be expected to result in a 0.16-cm increase in projection. When controlled for flap length and intraoperative projection, there was no difference between TRAM and implant nipple reconstruction in predicting postoperative nipple projection. Intraoperative planning and execution are critical to achieve predictable nipple shape, size, and projection. The dimensions of the star dermal fat flap can be strategically modified to allow the surgeon predictable projection with a consistent 41-percent preservation of intraoperative nipple projection in both TRAM and implant patients at 2 years.

Biochemical analysis of programmed cell death during premeiotic stages of spermatogenesis in vivo and in vitro.

Control points of regulator action during spermatogenesis are not completely known. Using the shark testis model, which facilitates analysis of spermatogenesis stage-by-stage in vivo and in vitro, an early biochemical marker of programmed cell death (PCD) was detected. Nucleosomal oligomers were seen in DNA extracts of testis and isolated spermatocysts (clonal germ cell/Sertoli cell units) at premeiotic (PrM), but not meiotic (M) or postmeiotic (PoM), stages. Cell nuclei isolated from M stages of development were susceptible to cleavage by micrococcal nuclease, suggesting that developmental control of factors other than a nuclease-insensitive chromatin structure may account for stage specificity. Cytological features of apoptosis were seen in germ cells, but not Sertoli cells, of a subset of isolated PrM spermatocysts and appeared to be all-or-none in affected clones. In culture, DNA fragmentation occurred on schedule with or without various additives, but the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased accumulation of DNA breakdown products. Identification of the apoptotic form of PCD as a major, variable component of normal spermatogenesis and the use of PrM spermatocysts as an in vitro test system will allow further definition of mechanisms and developmental and physiological controls.

Effects of curved mirrors in waveguide resonators.

Waveguide gas lasers (CO(2) ones especially) continue to be widely used. We have previously studied simple resonator designs with plane mirrors close to each end of the waveguide. Here we examine theoretical predictions concerning hybrid waveguide/free-space resonators with square-bore guides and curved mirrors. We show how resonator mode losses vary as functions of guide length and width, guide-to-mirror distance, mirror radius of curvature, and mirror tilt. We have tested a 7-W cw rf-excited CO(2) laser with unusually good near-TEM(00) transverse-mode quality; it is one of many promising resonator geometries not covered by earlier published research. The common case 3 reflector, sometimes viewed as guaranteeing near-TEM(00) mode performance, is shown to be alarmingly sensitive to small misalignments for certain guide geometries.

Analysis of the post-translational processing of alpha-MSH in the pituitaries of the chondrostean fishes, Acipenser transmontanus and Polyodon spathula.

Multiple forms of alpha-MSH were isolated from the pituitaries of the white sturgeon, Acipenser transmontanus, and the paddlefish, Polyodon spathula, following fractionation of the pituitary extracts by reversed-phase HPLC. In both species, immunoreactive forms which eluted with the same retention times as synthetic mammalian ACTH(1-13)NH2, monoacetylated alpha-MSH, and diacetylated alpha-MSH were detected. In the pituitary of the sturgeon, N-acetylated forms of alpha-MSH represented 91% of the total alpha-MSH isolated. However, in the pituitary of the paddlefish, N-acetylated forms of alpha-MSH accounted for only 45% of the total alpha-MSH isolated. The presence of N-acetylated forms of alpha-MSH in the pituitaries of representatives of the two extant genera of chondrostean fish coupled with the observation that N-acetylated forms of alpha-MSH are present in the pituitaries of cladistian and neopterygian fishes indicates that the alpha-MSH specific N-acetylation mechanism evolved prior to the radiation of the Actinopterygii. A conspicuous feature of the chondrostean alpha-MSH N-acetylation reaction was the low levels of diacetylated alpha-MSH produced relative to the levels of monoacetylated alpha-MSH. Similar observations have been made for the cladistian fish, Calamoichthys calabaricus, and the cartilaginous fish, Hydrolagus colliei. However, in holostean fishes, lungfishes, and tetrapods, the diacetylated form of alpha-MSH is the major end product produced in the intermediate pituitary. The phylogenetic implications of these observations will be discussed.

Evolution of renal insufficiency in ischemic nephropathy.

This retrospective review of data collected during a recent 42-month period from 58 consecutive patients with ischemic nephropathy submitted to operative management in the authors' center was undertaken to report the rate of decline in their renal function during the period before intervention and to examine the impact of operation on their outcome. Based on serum creatinine values, immediate preoperative estimated glomerular filtration rates (EGFR) ranged from 0 to 46 mL/minute (mean, 23.85 +/- 9.76 mL/minute). Eight patients were dialysis dependent or anuric at the time of operation. Patients with at least three sequential measurements for calculations of EGFR changes during the 6 months before operation (n = 50) and the first 12 months after operation (n = 32) were used to describe the preoperative rate of decline in EGFR and the impact of operation on this decrease in the operative survivors. In addition comparative analyses of data from patients with unilateral versus bilateral lesions and patients classified as having improvement in EGFR versus no improvement after operation were performed. Comparison of the immediate preoperative EGFR with the immediate postoperative EGFR for the entire group showed significant improvement in response to operation. Likewise the rate of deterioration in EGFR for the total group was improved after operation. A similar improvement in the rate of deterioration in EGFR was seen in the subgroup of patients who received an immediate improvement in EGFR in response to operation. These data argue that ischemic nephropathy is a rapidly progressive form of renal insufficiency. Although individual responses to operation were heterogeneous, renal revascularization may provide both immediate improvement in renal function and an improvement in its rate of deterioration during follow-up in patients with ischemic nephropathy.

Cortisol effects on plasma electrolytes and thyroid hormones during smoltification in coho salmon Oncorhynchus kisutch.

Plasma concentrations of Na+, K+, triiodothyronine (T3), and thyroxine (T4) and muscle water content were measured in yearling coho salmon. Oncorhynchus kisutch, after injection of cortisol in April, May, and June in fresh water (FW) and during acclimation to seawater (SW). Cortisol (17-21 micrograms/g), injected intraperitoneally in a melted cocoa butter suspension, suppressed the rise of plasma Na+ during SW acclimation in April but not in May or June. Muscle water content increased during SW acclimation in cortisol-treated fish in April and June. These observations suggest a hypoosmoregulatory function for cortisol during SW acclimation. Cortisol treatment also induced elevations of plasma K+ in FW (April only) and SW (April and May only). Cortisol treatment increased plasma T3 during SW acclimation in June and T4 in FW in May. The results suggest that cortisol may modify osmoregulatory processes and thyroid hormone activity during smoltification and acclimation to SW in yearling coho salmon.

Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual differentiation.

Sex steroids were measured by radioimmunoassay in whole-body extracts of coho salmon, Oncorhynchus kisutch, during early development and sexual differentiation. Profiles were developed for fish from the time of fertilization until 87 days postfertilization (dpf) for six steroids: testosterone (T), 11-ketotestosterone (KT), androstenedione (A), progesterone (P4), 17 alpha-hydroxy-20 beta-dihydroprogesterone (DHP), and 17 beta-estradiol (E2). Ovarian fluid was also examined for steroid content. Steroid profiles of unfertilized eggs essentially paralleled those of ovarian fluid. In one experiment, steroids in developing embryos declined precipitously after fertilization until 30 dpf; at hatching, all steroids increased slightly and then declined during yolk sac absorption. Results from a second experiment basically supported those of the first except that only testosterone increased at the time of hatching. Bimodality was evident in the data on steroid levels for fish collected between 42 and 56 dpf and again after 87 dpf. The hormone levels generally decreased or remained constant after the onset of exogenous feeding. Histological analyses during the first experiment showed the presence of undifferentiated gonads between hatching and 70 dpf, but by 77 dpf ovarian development was evident. In the second experiment, in which fish were more frequently sampled for histological analysis, undifferentiated gonads were present from hatching to 59 dpf. Development of oogonia was observed between 66 and 73 dpf and by 75 dpf ovarian development could be easily discerned. The sex of fish sampled at 101 dpf was determined by examining gonadal morphology, and steroid levels of those fish were determined. A sexual dimorphism was apparent in levels of T, KT, and A, but not of DHP or E2. The dynamics of steroid content of developing coho salmon at hatch, coupled with their bimodal distributions during yolk sac absorption, may suggest a role of sex steroids in the process of sexual differentiation apparent later in development. Changes in whole-body steroid levels at hatch may also be indicative of the onset of sexual differentiation even though no signs of gondal differentiation were histologically discernible at that time.

Antimicrobial resistance among respiratory isolates of Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in the United States.

A national surveillance study was conducted to determine trends in antimicrobial resistance patterns among three common causes of community-acquired respiratory tract infections. Fifteen participating U.S. medical centers submitted clinically significant isolates of Haemophilus influenzae, Moraxella (Branhamella) catarrhalis, and Streptococcus pneumoniae to two central laboratories for testing with a group of 12 antimicrobial agents. The majority of isolates were recovered from adult males greater than 50 years old. Overall, 84.1% of 378 M. catarrhalis and 16.5% of 564 H. influenzae (29.5% of type b strains; 15.0% of non-type b strains) produced beta-lactamase and were thus resistant to penicillin, ampicillin, and amoxicillin. Resistance in H. influenzae to other agents was 2.1% to tetracycline, 0.7% to trimethoprim-sulfamethoxazole, 1.1% to cefaclor, and 0.2% to cefuroxime and amoxicillin-clavulanate, while the M. catarrhalis isolates yielded very low MICs of these latter drugs. As demonstrated in prior studies, erythromycin showed little activity against H. influenzae. Of 487 S. pneumoniae isolates, 1 (0.2%) was penicillin resistant, while 3.8% were relatively resistant to penicillin, 4.5% were resistant to trimethoprim-sulfamethoxazole, 2.3% were resistant to tetracycline, 1.2% were resistant to chloramphenicol, and 0.2% were resistant to erythromycin. Overall, the lowest resistance rates for these common bacterial respiratory pathogens were noted with amoxicillin-clavulanate, cefuroxime, and cefaclor.

Antibacterial activity of the new glycopeptide antibiotic SKF104662.

The inhibitory activity of the new glycopeptide antibiotic SKF104662 was generally equivalent (+/- 1 concentration increment) to the activities of vancomycin, teicoplanin, and daptomycin against selected gram-positive bacteria. However, SKF104662 demonstrated greater activity against Staphylococcus epidermidis and S. haemolyticus than did teicoplanin and was more active than the other drugs against Clostridium difficile isolates. SKF104662 possessed bactericidal activity quite similar to that of vancomycin against selected isolates of Staphylococcus, Streptococcus, and Enterococcus species.

Influence of storage and susceptibility test conditions on stability and activity of LY163892 and four other cephalosporins.

A new carbacephem analog of cefaclor, LY163892, was examined along with four other cephalosporins for chemical stability and for antibacterial activity under a variety of susceptibility test conditions. LY163892 was found to be markedly more stable than cefaclor during storage at 4 and 35 degrees C in human serum, human urine, and various bacteriological media. Under these conditions, LY163892 demonstrated stability equal or superior to the stabilities of cephalexin, cephalothin, and cefuroxime. Broth and agar dilution susceptibility tests did not reveal significant differences in LY163892 activity in a variety of test media, although LY163892 MICs were somewhat higher in Mueller-Hinton chocolate agar or Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) than in other media. The activity of LY163892 against selected pathogens was only slightly influenced by the increased or decreased pH of the test medium, in a manner similar to that of the other cephalosporins tested. Thus, the carbacephem structure of LY163892 appears to represent a more stable cephalosporin than cefaclor, while retaining a similar antimicrobial spectrum.

Disk diffusion interpretive criteria for extended-spectrum cephalosporins with Haemophilus influenzae.

Disk diffusion interpretive zone size criteria for use with Haemophilus test medium are proposed for five extended-spectrum cephalosporins. With cefuroxime, zones greater than or equal to 24 mm in diameter indicated susceptibility, while zones less than or equal to 20 mm implied resistance. With cefotaxime, ceftizoxime, and ceftriaxone, a single susceptible-zone size, greater than or equal to 26 mm, is proposed.

Salt-supplemented medium for testing methicillin-resistant staphylococci with newer beta-lactams.

The addition of 2% NaCl to cation-supplemented Mueller-Hinton broth (CSMHB) was evaluated for microdilution testing of the susceptibility of staphylococci to five cephalosporins, imipenem, amoxicillin-clavulanate, and ticarcillin-clavulanate. With Staphylococcus aureus, NaCl improved the recognition of methicillin (oxacillin) resistance to cefamandole, imipenem, or ticarcillin-clavulanate. Resistance to amoxicillin-clavulanate was readily determined, irrespective of the presence of added salt. The addition of 2% NaCl to CSMHB did not significantly improve detection of resistance to any of the beta-lactams among coagulase-negative staphylococci. Since the addition of NaCl did not have significant adverse effects on tests with coagulase-negative staphylococci, the routine addition of 2% NaCl to oxacillin or methicillin tests with staphylococci may be justifiable on the basis of convenience or standardization. However, addition of NaCl to susceptibility tests of other beta-lactams does not consistently improve recognition of resistance among staphylococci and thus cannot be recommended for routine use.

National collaborative study of the prevalence of antimicrobial resistance among clinical isolates of Haemophilus influenzae.

A total of 2,811 clinical isolates of Haemophilus influenzae were obtained during 1986 from 30 medical centers and one nationwide private independent laboratory in the United States. Among these, 757 (26.9%) were type b strains. The overall rate of beta-lactamase-mediated ampicillin resistance was 20.0%. Type b strains were approximately twice as likely as non-type b strains to produce beta-lactamase (31.7 versus 15.6%). The MICs of 12 antimicrobial agents were determined for all isolates. Ampicillin resistance among strains that lacked beta-lactamase activity was extremely uncommon (0.1%). Percentages of study isolates susceptible to cefamandole, cefaclor, cephalothin, and cephalexin were 98.7, 94.5, 87.3, and 43.3%, respectively. For 14 strains (0.5% of the total), chloramphenicol MICs were greater than or equal to 8.0 micrograms, and thus the strains were considered resistant. All of these resistant strains produced chloramphenicol acetyltransferase. In addition, all 14 strains were resistant to tetracycline; 11 produced beta-lactamase. The percentage of isolates susceptible to tetracycline was 97.7%. In contrast, erythromycin and sulfisoxazole were relatively inactive. The combination of erythromycin-sulfisoxazole (1/64) was more active than erythromycin alone but essentially equivalent in activity to sulfisoxazole alone. Finally, small numbers of clinical isolates of H. influenzae were resistant to trimethoprim-sulfamethoxazole and rifampin.

Susceptibility of multiply resistant Haemophilus influenzae to newer antimicrobial agents.

One hundred and six isolates of Haemophilus influenzae from a national antimicrobial surveillance study demonstrated resistance to two or more of 10 primary antimicrobial agents by mechanisms other than or in addition to beta-lactamase. Of particular note were strains multiply resistant to ampicillin (by beta-lactamase production), chloramphenicol, trimethoprim/sulfamethoxazole, and tetracycline in various combinations. All of the aforementioned strains were shown to be highly susceptible to amoxicillin/clavulanate, the second generation cephalosporins cefuroxime and cefonicid, and the third generation cephalosporins cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, moxalactam, and cefixime. However, 68 strains that demonstrated resistance or marginal susceptibility (MIC greater than or equal to 2 micrograms/ml) to ampicillin by mechanisms other than beta-lactamase, also demonstrated reduced susceptibility to amoxicillin/clavulanate (MICs up to 8 micrograms/ml) and the second generation cephalosporins (MICs up to 32 micrograms/ml). While the latter strains were susceptible to the third generation cephalosporins, MICs were often 10-fold higher than MICs of ampicillin susceptible isolates or of beta-lactamase producing isolates. All of the multiply antimicrobial-resistant strains were highly susceptible (MIC less than or equal to 0.25 micrograms/ml) to the two quinolones ciprofloxacin and pefloxacin.

Interrenal secretion of corticosteroids and plasma cortisol and cortisone concentrations after acute stress and during seawater acclimation in juvenile coho salmon (Oncorhynchus kisutch).

We determined the major corticosteroids secreted by interrenal tissue and those present in plasma of juvenile coho salmon (Oncorhynchus kisutch). Incubation medium of interrenal tissue, unstimulated or stimulated with exogenous ACTH in vitro, and plasma of resting or acutely stressed salmon were extracted and qualitatively analyzed for steroid composition using high-performance liquid chromatography. Concentrations of plasma cortisone and cortisol following an acute handling stress or exposure to seawater were quantitatively measured by radioimmunoassay (RIA). Cortisol was the only corticosteroid detected by HPLC in media after incubation of interrenal tissue in the absence or presence of ACTH in vitro. However, both cortisone and cortisol were detected by HPLC in plasma sampled 1 hr after fish were acutely stressed by handling. Stress and seawater acclimation produced marked elevations in plasma levels of both steroids as determined by RIA and also resulted in long-lasting changes in the plasma cortisone:cortisol ratios. In resting fish, cortisone concentrations were similar or higher than cortisol levels. We concluded that cortisol is the primary steroid secreted by the interrenal tissue of coho salmon, and that plasma cortisone arises primarily from the peripheral conversion of cortisol to cortisone. The relatively high levels of cortisone in resting fish and its increase following stress and seawater acclimation suggest the possibility of a biologically significant role for this hormone.

Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae.

The need for complex growth media has complicated routine susceptibility testing of Haemophilus influenzae because of antagonism of certain antimicrobial agents by the medium or because of difficulties in interpretation of growth endpoints. Haemophilus test medium (HTM) is a simple, transparent medium for broth- or agar-based tests with H. influenzae. HTM incorporates Mueller-Hinton medium with additions of 15 micrograms of hematin per ml, 15 micrograms of NAD per ml, and 5 mg of yeast extract per ml as growth-promoting additives. Agar or broth microdilution MICs of 10 antimicrobial agents for a collection of 179 H. influenzae isolates determined by using HTM compared favorably with MICs determined by the conventional agar or broth dilution methods recommended by the National Committee for Clinical Laboratory Standards. Disk diffusion tests performed with HTM allowed accurate categorization of susceptible and resistant strains and were easier to interpret than tests performed with Mueller-Hinton chocolate agar. A particular advantage of HTM was the reliability of broth- or agar-based test results with trimethoprim-sulfamethoxazole. The results of the study suggest modification of current National Committee for Clinical Laboratory Standards MIC-interpretive criteria for H. influenzae with amoxicillin-clavulanate, chloramphenicol, and trimethoprim-sulfamethoxazole. Error rate-bounded analysis of MICs and disk diffusion zone sizes also suggest modified zone-interpretive criteria for ampicillin, amoxicillin-clavulanate, chloramphenicol, and tetracycline with HTM or conventional media. Interpretive zone sizes are newly proposed for cefaclor and rifampin disk diffusion tests.

In vitro activity of LY146032 (daptomycin) against selected aerobic bacteria.

The in vitro activity of the new lipopeptide antibiotic LY146032 was generally four-fold greater (MIC 90 less than or equal to 0.5 microgram/ml) than that of vancomycin against methicillin-susceptible or methicillin-resistant Staphylococcus aureus and coagulase-negative species of Staphylococcus. Enterococci, Streptococcus bovis, group B and viridans streptococci, and Corynebacterium group J-K isolates were inhibited by less than or equal to 4 micrograms/ml of LY146032, which represented activity equivalent to or greater than that of vancomycin. Unlike vancomycin, LY146032 was bactericidal for Enterococcus faecalis, Enterococcus faecium and Listeria monocytogenes. Due to its bactericidal properties LY146032 appeared to represent an improvement over vancomycin and teicoplanin.