The kinetics of lanthanide complexation by EDTA and DTPA in lactate media.

The interaction of trivalent lanthanide and actinide cations with polyaminopolycarboxylic acid complexing agents in lactic acid buffer systems is an important feature of the chemistry of the TALSPEAK process for the separation of trivalent actinides from lanthanides. To improve understanding of metal ion coordination chemistry in this process, the results of an investigation of the kinetics of lanthanide complexation by ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) and diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA) in 0.3 M lactic acid/0.3 M ionic strength solution are reported. Progress of the reaction was monitored using the distinctive visible spectral changes attendant to lanthanide complexation by the colorimetric indicator ligand Arsenazo III, which enables the experiment but plays no mechanistic role. Under the conditions of these experiments, the reactions occur in a time regime suitable for study by stopped-flow spectrophotometric techniques. Experiments have been conducted as a function of EDTA/DTPA ligand concentration, total lactic acid concentration, and pH. The equilibrium perturbation reaction proceeds as a first order approach to equilibrium over a wide range of conditions, allowing the simultaneous determination of complex formation and dissociation rate constants. The rate of the complexation reaction has been determined for the entire lanthanide series (except Pm(3+)). The predominant pathway for lanthanide-EDTA and lanthanide-DTPA dissociation is inversely dependent on the total lactate concentration; the complex formation reaction demonstrates a direct dependence on [H(+)]. Unexpectedly, the rate of the complex formation reaction is seen in both ligand systems to be fastest for Gd(3+). Correlation of these results indicates that in 0.3 M lactate solutions the exchange of lanthanide ions between lactate complexes and the polyaminopolycarboxylate govern the process.

Streptococcus pneumoniae causes experimental meningitis following intranasal and otitis media infections via a nonhematogenous route.

Using two different animal models of Streptococcus pneumoniae infection, we have demonstrated that this organism is able to spread to the central nervous system and cause meningitis by bypassing the bloodstream. Following respiratory tract infection induced via intranasal inoculation, bacteria were rapidly found in the bloodstream and brains in the majority of infected mice. A similar pattern of dissemination occurred following otitis media infection via transbullar injection of gerbils. However, a small percentage of animals infected by either route showed no bacteria in the blood and yet did have significant numbers of bacteria in brain tissue. Subsequent experiments using a galU mutant of S. pneumoniae, which is impaired in its ability to disseminate to the bloodstream following infection, showed that this organism is able to spread to the brain and cerebrospinal fluid. These results demonstrate that, unlike many bacterial pathogens that cause meningitis, S. pneumoniae is able to do so independent of bloodstream involvement upon different routes of infection. This may address the difficulty in treating human infections caused by this organism.

Hurricane Floyd. Response of the Pitt County Medical Community.

The financial impact of Hurricane Floyd on the medical community is still being tabulated. Initial estimates indicate a $5.8 million loss to UHS in operating revenue. Additional hospital costs include $568,000 for overtime pay, $310,000 for special equipment including helicopters and general supplies, and $1.2 million in "bonus pay" to the hospital's 6000 employees. The ECU School of Medicine suffered losses estimated at $3.6 million, including lost revenue, overtime, and salaries paid when clinics and services were shut down. Most private offices suffered from lost revenues and occasionally from flood damage to property. Hurricane Floyd was the most costly disaster to hit Eastern North Carolina and the Mid-Atlantic United States. The medical community and its patients were severely challenged from the terrible effects of flooding, but the collaborative efforts of a great number of individuals and a spirit of teamwork came together to provide continued health care to the region. It is very likely that the preventive measures enacted both before and after the storm averted illness and injury, and saved lives. Ingenuity, innovation, and optimal use of available resources allowed Pitt County Memorial Hospital and its surrounding medical community to stay in operation. We have learned a great deal from these experiences that will help us plan for future natural disasters.

Bifurcated endovascular grafting for abdominal aortic aneurysm.

Endovascular grafting represents a minimally invasive approach to prosthetic aortic grafting for abdominal aortic aneurysms (AAA). We reviewed our initial experience with a bifurcated endovascular graft that shares with conventional grafts the characteristics of discrete transaortic fixation and unitary unsupported woven polyester construction. Twenty-eight patients (26 male, 2 female; age: 58-93) with infrarenal aortic aneurysms between 41 and 82 mm in greatest diameter (x = 55.4 mm) underwent bifurcated endovascular grafting (Guidant/EVT, Menlo Park, CA) over an 18-month period. We concluded that bifurcated endovascular grafting with the EVT(R) device provides reliable and reproducible aneurysm exclusion with short hospital recovery and low morbidity.

Regulation of Leydig cells through a steroidogenic acute regulatory protein-independent pathway by a lipophilic factor from macrophages.

The purpose of this investigation was to study the mechanism of action of a macrophage-derived factor that stimulates steroid production by Leydig cells. This factor increased testosterone production within 30 min, and reached a half-maximal response by 6-8 h. At a maximal dose, it stimulated testosterone production 20-fold at 24 h. Its efficacy was consistently higher than that achieved with a maximal dose of human chorionic gonadotropin (hCG). However, Leydig cells treated with a maximal dose of both the macrophage-derived factor and hCG secreted the same amount of testosterone as when given a maximal dose of only the macrophage-derived factor. The macrophage-derived factor did not require new protein synthesis to stimulate testosterone production, nor did it alter the amount of steroidogenic acute regulatory protein (StAR). While the macrophage-derived factor required an active cholesterol side-chain cleavage complex system, it did not alter the capacity of this enzyme complex. Finally, the macrophage-derived factor was unable to stimulate the production of progesterone by isolated mitochondria. In summary, the macrophage-derived factor is a highly active, acute regulator of steroidogenesis that acts through a high capacity StAR-independent pathway.

Plasma thyroid hormone kinetics are altered in iron-deficient rats.

Iron deficiency anemia is associated with lower plasma thyroid hormone concentrations in rodents and, in some studies, in humans. The objective of this project was to determine if plasma triiodothyronine (T3) and thyroxine (T4) kinetics were affected by iron deficiency. Studies were done at a near-thermoneutral temperature (30 degrees C), and a cool environmental temperature (15 degrees C), to determine plasma T3 and T4 kinetics as a function of dietary iron intake and environmental need for the hormones. Weanling male Sprague-Dawley rats were fed either a low Fe diet [iron-deficient group (ID), <5 microg/g Fe] or a control diet [control group (CN), 35 microg/g Fe] at each temperature for 7 wk before the tracer kinetic studies. An additional ID group receiving exogenous thyroid hormone replacement was also used at the cooler temperature. For T4, the disposal rate was >60% lower (89 +/- 6 vs. 256 +/- 53 pmol/h, P < 0.001) in ID rats than in controls at 30 degrees C, and approximately 40% lower (192 +/- 27 vs. 372 +/- 26 pmol/h, P < 0.01) in ID rats at 15 degrees C. Exogenous T4 replacement in a cohort of ID rats at 15 degrees C normalized the T4 concentration and the disposal rate. For T3, the disposal rate was significantly lower in ID rats in a cool environment (92 +/- 11 vs. 129 +/- 11 pmol/h, P < 0.01); thyroxine replacement again normalized the T3 disposal rate (126 +/- 12 pmol/h). Neither liver nor brown fat thyroxine 5'-deiodinase activities were sufficiently different to explain the lower T3 disposal rates in iron deficiency. Thus, plasma thyroid hormone kinetics in iron deficiency anemia are corrected by simply providing more thyroxine. This suggests a central regulatory defect as the primary lesion and not peripheral alterations.

Effects of clodronate-containing liposomes on testicular macrophages and Leydig cells in vitro.

We undertook the present studies to determine if clodronate-containing liposomes have direct effects on Leydig cells. Macrophages and Leydig cells were isolated and maintained separately in culture. Following treatment with clodronate-containing liposomes, macrophages were killed in a dose-response fashion over a range of 5-200 microliters liposomes. By comparison, a 500 microliters dose was required to kill Leydig cells, but this was not dependent upon clodronate since liposomes containing buffer elicited an identical response. The concentration of testosterone in medium from Leydig cells treated with clodronate-containing liposomes was significantly reduced compared with untreated cells. However, we subsequently found that liposomes can adsorb testosterone. Therefore, testosterone production was determined at various times following removal of liposomes from Leydig cells, thereby circumventing this complication. It was found that testosterone production was not altered by liposomes under these conditions. Finally, free clodronate had no effect on testosterone production, even at doses representing the amount present within the 500 microliters dose of liposomes. In summary, clodronate-containing liposomes killed testicular macrophages at a far smaller dose than required to kill Leydig cells. Most importantly, neither liposomes no free clodronate had a direct effect on testosterone production. Thus, clodronate-containing liposomes represent a valuable tool to study Leydig cell-macrophage interactions.

Isolation and characterization of americanin, a specific inhibitor of thrombin, from the salivary glands of the lone star tick Amblyomma americanum (L.).

A thrombin (EC 3.4.21.5) inhibitor (americanin) was isolated from the salivary glands of the lone star tick Amblyomma americanum (L.) using reversed-phase chromatography and anion-exchange chromatography. Americanin did not inhibit any other protease tested, including factor Xa, plasmin, trypsin, chymotrypsin, elastase, papain, pepsin, and carboxypeptidase. The inhibition of thrombin by americanin decreased dramatically with dilution of the reaction mixture including thrombin, its substrate, and americanin. When thrombin assays were performed in the presence of americanin, the reaction curve showed a time-dependent inhibition. Significant inhibition was observed when americanin concentration was approximately equal to that of thrombin, with a Ki of 0.073 nM. The results suggest that americanin is a specific, reversible, competitive, slow, tight-binding inhibitor of thrombin.

Iron and thermoregulation: a review.

Poor temperature regulation in both human and animal models has been associated with iron deficiency anemia. The review examines the evidence for causality in both animals and humans, as well as provides an overview of temperature regulation in homeotherms. A number of investigations conclude that anemia is a central component of the inability of iron-deficient individuals to temperature regulate when they are cold stressed. This is apparently due to effects both on heat production and heat loss rates. The former is related to poor thyroid function in iron deficiency and the latter to the competing demands for tissue oxygenation vs. decreased flood flow to minimize heat losses to the environment. Future research needs are outlined that require integration of endocrinology, physiology, and nutrition disciplines.

Sex pheromone of caddisflyHesperophylax occidentalis (Banks) (Trichoptera: Limnephilidae).

The main component of the sex pheromone of the caddisflyHesperophylax occidentalis (Banks) (Trichoptera: Limnephilidae) was identified as 6-methylnonan-3-one (enantiomeric composition has not yet been determined). Extracts of adult females elicited strong electroantennogram (EAG) responses from adult male antennae, but elicited significantly smaller EAG responses from adult female antennae. Extracts of adult males did not elicit appreciable EAG responses from antennae of either sex. Racemic 6-methylnonan-3-one was synthesized and elicited EAG responses from male antennae as strong as those obtained with female extracts. In field tests with baited sticky traps near lakes and streams, traps baited with synthetic racemic 6-methylnonan-3-one caught significantly more males than control traps. Female adults contained approximately 1 µg of 6-methylnonan-3-one per individual. Related ketones and alcohols of other chain lengths were also tentatively identified, being present in tiny amounts in female extracts. Extraction of different body parts showed that 6-methylnonan-3-one occurs only in a region near the intersegmental membrane between the fourth and fifth abdominal sternites of the female (no discrete glands were observed). Extracts of males did not contain 6-methylnonan-3-one, nor did pupae of either sex.

Hesperophylax occidentalis (Trichoptera: Limnephilidae): Electroantennogram structure-activity study of sex pheromone component 6-methylnonan-3-one.

Electroantennogram (EAG) analyses ofHesperophylax occidentalis male and female antennae were used to generate dose-response curves for synthetic, racemic 6-methylnonan-3-one and to demonstrate the chemical specificity of male antennal olfactory cells. Male antennae responded to 6-methylnonan-3-one, the main female pheromone component. Females also demonstrated a response to 6-methylnonan-3-one, but a smaller one than males. The chemical specificity of male antennal receptors was determined by comparing EAG activity of synthetic, racemic 6-methylnonan-3-one and a series of structural analogs. The three structural parameters considered, including keto position, chain length, and methyl-group position, were demonstrated to contribute significantly to the reception of synthetic, racemic 6-methylnonan-3-one by male antennae. For straight-chain nonanones with keto groups at different positions, nonan-3-one elicited the greatest EAG responses from male antennae. For straight-chain alkan-3-ones with different chain lengths, the EAG responses of male antennae to nonan-3-one and decan-3-one were the same, but greater than those to other chain lengths. For methyl-branched nonan-3-ones, 6-methylnonan-3-one elicited greater EAG responses from antennae than analogs with the methyl group at another position.

Effect of thyroid hormone replacement in iron-deficient rats.

To determine if the previously observed alterations in norepinephrine (NE) metabolism and resting metabolic rate in iron-deficient (ID) rats result from hypothyroidism, exogenous thyroxine (T4) and 3,5,3'-triiodothyronine (T3) were administered to ID rats in doses sufficient to normalize the plasma concentrations of these hormones, whereas other ID and control (CN) rats received placebo treatment. Resting oxygen consumption was approximately 25% higher in ID than CN rats; T4 but not T3 treatment alleviated this elevated oxygen uptake. The NE content of interscapular brown adipose tissue (IBAT), liver, and heart was 70-80% lower in ID than CN rats, and NE turnover in the same tissues was likewise 40-60% lower in ID than CN rats, with no systematic effect of either T3 or T4 treatment. Liver T(4)5'-deiodinase activity was 70% lower in ID than CN rats and increased with T4 but not T3 treatment. These experiments show that iron deficiency alters NE and energy metabolism in a way that is mostly independent of its effects on thyroid hormone metabolism.

Growth in iron-deficient rats.

Poor growth in iron deficiency is commonly observed in animal studies. Previous studies from our laboratory showed that iron-deficient rats are metabolically inefficient and have less body fat than controls and proposed that iron deficiency was related to increased metabolic rates and heat loss. To examine these points more completely, we examined growth and metabolic rate of iron-deficient rats at two environmental temperatures, 25 degrees C and 32 degrees C, and feed efficiency in separate groups of rats during a period of rapid growth. Iron deficiency (hemoglobin [Hb] approximately 60 g/liter) was associated with a systematic elevation of metabolic rate over the 24-hr day with animals at 25 degrees C. This did not occur in animals living in thermoneutrality. Iron deficiency affected growth of animals at 25 degrees C but not at 32 degrees C. Feed efficiency (kcal retained/kcal absorbed) was 25 +/- 4.2 and 31 +/- 4.9 kcal (P < 0.0001), respectively, in iron deficient rats and animals were not anorexic. Use of food-restricted animals allowed the direct calculation that iron deficiency was associated with a 10%-15% increase requirement for growth. We conclude that iron deficiency anemia is associated with a poor feed efficiency and that it is attenuated when nonshivering thermogenesis is minimized by a thermoneutral environment.

Changes in iron status during competitive season in female collegiate swimmers.

We examined changes in iron status in 25 female varsity collegiate swimmers over the course of a competitive season via weekly capillary blood sampling. We also studied the effectiveness of moderate iron supplementation (39 mg/day) in preventing expected changes in iron status via a double-blinded crossover design. On entry into the study, 17 of the swimmers had depleted iron stores (plasma ferritin < 12 micrograms/L) and 5 were anemic (Hb < 120 g/L). After 5 wk of placebo treatment, hemoglobin decreased (> or = 6 g/L) in 44% and increased in 12% of the subjects. After 5 wk of iron supplementation, hemoglobin decreased in 16% and increased in 24% of the subjects. Likewise, after placebo treatment, plasma ferritin decreased (> or = 5 micrograms/L) in 24% and increased in 4% of the subjects, whereas iron supplementation resulted in decreases in 4% and increases in 68% of the subjects. In the absence of iron supplementation, hemoglobin levels decreased despite mean dietary iron intakes of 16.3 mg/day. These results show that moderate iron supplementation is effective in preventing decline in the iron status of female collegiate swimmers during a competitive season, but a higher dose of iron may be necessary to reverse their iron-deficient state.

The impact of varying degrees of iron nutriture on several functional consequences of iron deficiency in rats.

The impact of varying severities of iron-deficiency anemia on fasting blood glucose, plasma triiodothyronine, heart norepinephrine concentrations and resting oxygen consumption were evaluated. Male weanling Sprague-Dawley rats were assigned to one of six dietary groups (4, 6, 11, 16, 23 or 40 mg Fe/kg diet) for 6 wk. Hemoglobin, liver iron and transferrin saturation were significantly lower in the 4 and 6 mg Fe/kg diet groups relative to the other groups and were indicative of anemia, low tissue iron stores and impaired erythropoiesis. Fasting blood glucose and heart norepinephrine concentrations were significantly higher and lower, respectively, in the 4 and 6 mg Fe/kg diet groups than the three highest dietary Fe groups. Although fasting blood glucose was significantly inversely correlated (r = -0.89, P = 0.0001) with hemoglobin concentration; a significant quadratic relationship (R 2 = 0.70, P = 0.0001) existed between hemoglobin and heart norepinephrine concentration. Differences in plasma triiodothyronine concentrations and resting oxygen consumption were not significant among the groups. Thus, base on hemoglobin concentration as an index of the severity of iron deficiency, these findings demonstrate that certain physiological manifestations of iron deficiency occur at even moderate-to-mild degrees of anemia.

Epithelial downgrowth following wound dehiscence after extracapsular cataract extraction and posterior chamber lens implantation: surgical management.

Epithelial downgrowth occurred along a fistulous (nonfiltering) tract containing an incarcerated anterior capsular flap after extracapsular cataract extraction and posterior chamber lens implantation complicated by wound dehiscence. Months later, a YAG posterior capsulotomy was performed before it was realized that posterior capsule opacification was associated with epithelial downgrowth involving the posterior capsule. Surgical management of epithelial downgrowth after extracapsular cataract extraction and posterior chamber lens implantation is discussed, with emphasis on the role of combined cryotherapy, dissection of the retrocorneal membrane, and complete removal of the capsular bag. Histopathologically, we found it difficult to differentiate lens epithelial cells from corneal epithelial downgrowth within the capsular bag, but monoclonal antibody for keratin may help identify corneal epithelial cells.

A microcomputer-based system for recording and analyzing behavioral data regarding the sexual activity of male rodents.

A microcomputer (TRS-80 Model 4P) based system has been developed to aid in the collection and analysis of behavioral data regarding sexual activity in male rodents. Its capabilities include the recording of events, the online calculation of frequency and latency measures, the monitoring of temporal intervals as demanded by experimental protocols, and the prompting of experimenter activities at prescribed times.