Exchangeability of N termini in the ligand-gated porins of Escherichia coli.

The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin beta-barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty beta-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane beta-barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037-1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization.

Quantum-cascade laser measurements of stratospheric methane and nitrous oxide.

A tunable quantum-cascade (QC) laser has been flown on NASA's ER-2 high-altitude aircraft to produce the first atmospheric gas measurements with this newly invented device, an important milestone in the QC laser's future planetary, industrial, and commercial applications. Using a cryogenically cooled QC laser during a series of 20 aircraft flights beginning in September 1999 and extending through March 2000, we took measurements of methane (CH(4)) and nitrous oxide (N(2)O) gas up to ~20 km in the stratosphere over North America, Scandinavia, and Russia. The QC laser operating near an 8-mum wavelength was produced by the groups of Capasso and Cho of Bell Laboratories, Lucent Technologies, where QC lasers were invented in 1994. Compared with its companion lead salt diode lasers that were also flown on these flights, the single-mode QC laser cooled to 82 K and produced higher output power (10 mW), narrower laser linewidth (17 MHz), increased measurement precision (a factor of 3), and better spectral stability (~0.1 cm(-1) K). The sensitivity of the QC laser channel was estimated to correspond to a minimum-detectable mixing ratio for methane of approximately 2 parts per billion by volume.

Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB.

The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the K(d) of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (K(d) = 30 nM) and ferric enantioenterobactin (K(d) = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (K(d) = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.

Effect of loop deletions on the binding and transport of ferric enterobactin by FepA.

The siderophore ferric enterobactin enters Escherichia coli through the outer membrane (OM) porin FepA, which contains an aqueous transmembrane channel that is normally occluded by other parts of the protein. After binding the siderophore at a site within the surface loops, FepA undergoes conformational changes that promote ligand internalization. We assessed the participation of different loops in ligand recognition and uptake by creating and analysing a series of deletions. We genetically engineered 26 mutations that removed 9-75 amino acids from nine loops and two buried regions of the OM protein. The mutations had various effects on the uptake reaction, which we discerned by comparing the substrate concentrations of half-maximal binding (Kd) and uptake (Km): every loop deletion affected siderophore transport kinetics, decreasing or eliminating binding affinity and transport efficiency. We classified the mutations in three groups on the basis of their slight, strong or complete inhibition of the rate of ferric enterobactin transport across the OM. Finally, characterization of the FepA mutants revealed that prior experiments underestimated the affinity of FepA for ferric enterobactin: the interaction between the protein and the ferric siderophore is so avid (Kd < 0.2 nM) that FepA tolerated the large reductions in affinity that some loop deletions caused without loss of uptake functionality. That is, like other porins, many of the loops of FepA are superficially dispensable: ferric enterobactin transport occurred without them, at levels that allowed bacterial growth.

Airborne laser infrared absorption spectrometer (ALIAS-II) for in situ atmospheric measurements of N2O, CH4, CO, HCl, and NO2 from balloon or remotely piloted aircraft platforms.

The Airborne Laser Infrared Absorption Spectrometer II (ALIAS-II) is a lightweight, high-resolution (0.0003-cm(-1)), scanning, mid-infrared absorption spectrometer based on cooled (80 K) lead-salt tunable diode laser sources. It is designed to make in situ measurements in the lower and middle stratosphere on either a balloon platform or high-altitude remotely piloted aircraft. Chemical species that can be measured precisely include long-lived tracers N(2)O and CH(4), the shorter-lived tracer CO, and chemically active species HCl and NO(2). Advances in electronic instrumentation developed for ALIAS-I, with the experience of more than 250 flights on board NASA's ER-2 aircraft, have been implemented in ALIAS-II. The two-channel spectrometer features an open cradle, multipass absorption cell to ensure minimal contamination from inlet and surfaces. Time resolution of the instrument is

Prototypes of intrafamily homicide and serious assault among insanity acquittees.

Public concern with societal violence is intensified when persons who have been found not guilty by reason of insanity (NGRI) of having committed a homicide or serious assault are returned to the community. Successful management of such acquittees in the community requires a sophisticated understanding of the person and the illness within the larger context of the violent incident, the family, the community, and the culture. In this article, we present an analysis of psychotic violence within a family context. A qualitative study of 64 subjects who were found NGRI of killing or seriously injuring a family member resulted in four prototypes of intrafamilial homicide/assault: Till Death Us Do Part; Overwhelming Burden, Elimination of the Limit Setter; and Family-Focused Delusional Killing. The prototypes are presented as a model for developing management strategies both for future risk assessment and for successful transition of the insanity acquittee into the community.

The use of two different APC resistance assay systems provides optimal sensitivity and specificity for diagnosing genetic APC resistance.

We compared the performance characteristics of a commercial dilute Russell's viper venom (DRVV)-based APC resistance assay (Gradipore PC Impedance Test) to a routinely utilized commercial APTT based assay (Coatest APC Resistance Assay). The DRVV based assay offers improved sensitivity and specificity for the factor V Leiden mutation. However, the routine use of both assays provides optimum reliability for diagnosis of genetic APC resistance. Our results suggest that when both tests are either positive or negative, DNA analysis is unnecessary. Interference by lupus anticoagulants is dramatically minimized by the phospholipid rich DRVV reagent used in the assay and it is insensitive to high factor VIII activity. Additionally, discrepant functional assay results allow identification of patients who may have an acquired APC resistant phenotype.

A simple method for synthesis of B-cell clonospecific probes.

A sensitive PCR-based method was developed to produce B-cell clonogenic probes without the need for sequencing and specific oligonucleotide synthesis. Specificity and sensitivity were assessed and found to be comparable to that achieved using established methods. Possible applications include the detection of MRD, bone marrow involvement with lymphoma, and the contamination of autologous bone marrow or peripheral blood progenitor cell harvests with malignant cells carrying IgH rearrangements.

Effects of insulin-like growth factors I and II and insulin on the immortalized hypothalamic GTI-7 cell line.

Insulin and insulin-like growth factor I (IGF-I) participate in energy metabolism, regulate cellular growth and differentiation, and are thought to act locally in a paracrine manner through specific receptors. Systemic levels of these peptides in humans and primates are directly associated with levels of activity of the reproductive axis. To date, it is unclear whether these peptides participate in reproductive function by acting at the level of the GnRH neuron. In this study we examined the effects of IGF-I, IGF-II and insulin on immortalized GnRH-secreting neurons, the GTI-7 cell line. The GTI-7 cells expressed all three members of the insulin receptor family as determined by analysis of 125I-IGF-I, 125I-IGF-II and 125I-insulin binding sites. Insulin receptors bound insulin, IGF-II and IGF-I with a ratio of potency of 1:5:20. IGF-I and IGF-II receptors bound both IGF-I and IGF-II. The ratio of potency of IGF-I/IGF-II was 1:5 for the IGF-I receptor and 100:1 for the IGF-II receptor. The binding characteristics of the growth factors at 22 degrees C suggested the possibility that these cells may secrete IGF binding proteins. To ensure that changes in GnRH levels in the media were due to secretion and not to changes in cell number, the mitogenic effect of these peptides on GTI cells was evaluated. Both insulin and IGF-I were strong mitogens (48-hour incubation), restoring cell number to that of serum-replete cultures at a dose of 0.1 ng/ml. A 100-fold higher dose of IGF-II was required to produce a similar level of mitogenicity, implicating an action through the IGF-I and/or insulin receptor. Due to these mitogenic effects, the effect of insulin, IGF-I and IGF-II on GnRH secretion was studied after short-term exposure. Insulin and IGF-I did not affect GnRH secretion, but IGF-II had a biphasic effect on GnRH release after 2 h of incubation (a maximum stimulatory effect occurred with a 0.1 ng/ml dose). In order to examine the signal transduction mechanism, the role of cytoplasmic calcium mobilization in IGF-II-induced GnRH secretion was examined in single cells using calcium imaging. The effect of IGF-II on GnRH secretion appeared to operate via a calcium-independent mechanism. The studies document an insulin/IGF system in the GTI-7 neuronal cell line and show that insulin and IGFs can exert direct effects on the immortalized GnRH neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Pseudohypoparathyroidism type Ia and growth hormone deficiency in two siblings.

Pseudohypoparathyroidism type Ia (PHP type Ia) is associated with several endocrinopathies. We describe two siblings with PHP type Ia, hypothyroidism and growth hormone deficiency (GHD). To our knowledge, these patients are the first to have PHP type Ia and GHD. The defect which is most likely responsible for the three endocrinopathies is a deficiency of the stimulatory guanine nucleotide-binding regulatory protein, Gs.

Pregnancy: a clue to normal regulation of B lymphopoiesis.

The large-scale production of lymphocytes in the bone marrow reflects a delicate balance between positive and negative regulatory signals. For instance, interleukin 7 (IL-7) provides a positive signal, and appears to be both essential and limiting in the mouse. However, much less is understood concerning the negative molecular signals that may limit the output of lymphocytes. Here, Paul Kincade and colleagues discuss how a chance observation with pregnant mice revealed that sex steroids can act as negative regulators of B lymphopoiesis, and may do so under normal steady-state conditions.

Design and manufacture of a zero-order sustained-release pellet dosage form through nonuniform drug distribution in a diffusional matrix.

A theoretical basis is presented for the design of dosage forms containing a drug which is initially distributed throughout a noneroding diffusional matrix in a nonuniform manner, for the purpose of achieving zero-order release. Modeling of these dosage forms is based on Higuchi's square root of time diffusional model. Derivations of theoretical drug release profiles for both flat slab and spherical geometry are included. Pellets were prepared utilizing this nonuniform distribution concept using fluid bed suspension layering technology. A gradient pumping system used in conjunction with a specially fabricated miniature fluid bed apparatus was utilized to prepare pellets containing drug nonuniformly dispersed. Actual drug release profiles were obtained for pellets containing drug distributed in the traditional uniform manner and the nonuniform manner described above. Nonuniform distribution of drug in a noneroding diffusional matrix, in theory and practice, is shown to linearize dissolution profiles.

Prediction of vitamin K response using the Echis time and Echis-prothrombin time ratio.

Echis carinatus venom contains proteases capable of activating both normal and descarboxy prothrombin. We showed this venom (Sigma) principally activates prothrombin with almost no factor X activation. Echis time in combination with prothrombin time can predict vitamin K responsiveness since the Echis time is usually normal in the presence of descarboxy prothrombin associated with vitamin K deficiency. 38 patients with abnormal routine prothrombin times (PT) had both coagulant and immunogenic factor II assays along with Echis times done before and after vitamin K. Of 22 patients responding to vitamin K, based on correction of PT, 21 had normal initial Echis times and of 16 not responding, 11 had abnormal Echis times, giving a sensitivity of 95.4% and specificity of 68.8% for vitamin K responsiveness. 90% of patients with a PT/Echis time ratio less than 1.3 and a prolonged Echis time did not correct their PTs with vitamin K therapy. The 5 non-responders with normal Echis times all showed normal initial coagulant and antigenic prothrombin, but 3 had low F V and/or F VII.

Monitoring insanity acquittees: Connecticut's psychiatric security review board.

In 1985 Connecticut established an administrative psychiatric security review board to monitor the postverdict disposition of defendants found not guilty by reason of mental disease or defect. The five-member board may confine an insanity acquittee in a mental hospital, grant temporary leave, order conditional release, and terminate confinement or conditional release. Judicial review is required before a patient is discharged from the board's custody. Between 1985 and 1989, a total of 173 insanity acquittees were under the board's jurisdiction; most were hospitalized in state psychiatric facilities. The authors discuss issues that must be addressed in creating a psychiatric security review board, including the conflict between protecting the public and treating insanity acquittees, clinicians' resistance to treating these patients, and problems posed by patients who do not appear to be mentally ill or who are well known to the public.

Sweet's syndrome, acute leukemia, and t(3;5).

We report herein the case of a 36-year-old woman who was diagnosed as having Sweet's syndrome 13 months prior to developing acute myeloid leukemia (FAB type M2). Her bone marrow karyotype was 46,XX,t(3;5)(q21;q31). Translocation t(3;5) has been reported in seven other cases of acute nonlymphocytic leukemia. None of these cases have been associated with Sweet's syndrome.