Biologic Response to Peripheral and Central Administration of Recombinant Human Leptin in Dogs

LEBEL, CARL, AMY BOURDEAU, DAVID LAU, AND PAMELA HUNT. Biologic response to peripheral and central administration of recombinant human leptin in dogs. Obes Res. Objective: Because leptin is believed to act within the central nervous system, the objective of this study was to test that presumption by comparing the biologic responses to recombinant human leptin (rHuLeptin) when delivered either subcutaneously or intrathecally in a large animal species, the beagle dog. Methods and Procedures: Adult beagle dogs were used for all studies (n = 3 to 14). Treatment with rHuLeptin was either as daily subcutaneous or intermittent intrathecal injections. Results: Subcutaneously administered rHuLeptin was absorbed with peak concentrations appearing at 2 to 4 hours. After intrathecal administration, cerebral spinal fluid concentrations declined in a bi-phasic manner with a terminal half-life of ?6 to 8 hours. When lean beagles were given leptin subcutaneously, at 0. 05 to 5 gkglday for up to 6 months, reductions in body weight (up to 30%) and food intake (up to 75%) were observed. Body fat loss was observed in both lean and obese dogs, and confirmed by dual energy X-ray absorptiometry and histology of adipose tissue. When rHuleptin was delivered intrathecally at 4 to 1000 μg/dose for up to 3 months, the primary effects observed were reductions in body weight and food intake. In general all findings reported in the intrathecal studies were consistent with those noted in the subcutaneous studies; however, the required intrathecal dose was substantially lower than that for subcutaneous delivery. Discussion: These studies demonstrate that both subcutaneous and intrathecal treatment of rHuLeptin was associated with effects on body weight, food intake, and body fat in dogs. These results support the concept that the central nervous system is the probable primary site of action for leptin and suggest that rHuLeptin has similar physiologic activities that influence body weight, body fat, and metabolism in large animals to those reported previously in rodents.     

Narrow hybrid zone between two subspecies of big sagebrush (ARTEMISIA TRIDENTATA: Asteraceae). IX. Elemental uptake and niche separation

The concentrations of selected elements and their biological absorption coefficients were determined for leaves from plants in native stands and reciprocal transplant gardens to determine whether niche differentiation occurs among the parental taxa and their hybrids in the big sagebrush hybrid zone in Utah. The bounded hybrid superiority model predicts such niche differentiation, while the ecologically neutral dynamic equilibrium model predicts complete niche overlap, at least in the vicinity of the hybrid zone. The concentrations of elements in the leaves of site-indigenous sagebrush and the biological absorption coefficients differed significantly between the subspecies and between either parental taxon and hybrids. Within reciprocal transplant gardens, both the elemental concentrations and the biological absorption coefficients differed among the gardens and taxa. Significant genotype-by-environment interactions were observed for several essential elements. Niche differentiation was evident as correspondence analyses ordinated the parental taxa and hybrids into separate groups even when raised in the same garden. These findings support the ecologically based bounded hybrid superiority model and suggest that the big sagebrush parental taxa and their hybrids have adapted to their respective unique habitats.     

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms

Strain SB^T is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SB^T produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SB^T in coculture with Desulfovibrio strain G11. Strain SB^T grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 ± 1 g cell dry mass per mol of crotonate. Strain SB^T did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SB^T was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SB^T in the δ-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SB^T was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SB^T and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus. Received: 2 June 1998 / Accepted: 16 November 1998     

A group of schwannomas with interstitial deletions on 22q located outside the NF2 locus shows no detectable mutations in the NF2 gene

Schwannomas are tumors arising mainly at cranial and spinal nerves. Bilateral vestibular schwannoma is the hallmark of neurofibromatosis type 2 (NF2). The NF2 gene has been cloned and comprehensive analysis of its mutations in schwannomas shows that up to 60% of tumors carry inactivating mutations. Thus, the genetic mechanism behind the development of more than 40% of schwannomas without NF2 mutations is unknown. We have therefore studied tumor tissue from 50 human schwannomas by allelotyping and have found chromosome 22 deletions in over 80% of the cases. We detected 14 cases (27%) that revealed partial deletions of one copy of chromosome 22, i.e., terminal and/or interstitial deletions. We sequenced the NF2 gene in seven of these tumors and detected only one case with mutations. The deletion mapping of chromosome 22 in tumors with partial deletions indicates that several regions, in addition to the NF2 locus, harbor genes involved in schwannoma tumorigenesis. Our findings suggest that heterogeneity in the mechanisms leading to the development of schwannomas probably exists. These findings are in agreement with the recent analysis of schwannomas from familial and sporadic cases of schwannomatosis and point to a possible role of an additional gene, which, in cooperation with the NF2 tumor suppressor, causes schwannomas. Received: 12 November 1998 / Accepted: 1 March 1999     

Statistical considerations for in vitro research: II — Data to presentation

Summary The paper is the second of two papers about statistical considerations that researchers should make while doing in vitro plant biology research. The first paper focused on aspects from developing a plan to do research through the collection of data. This paper continues with information about editing data, handling outliers, analyzing quantitative and qualitative data, comparing treatment means, preparing graphs and tables, and presenting results.     

The human immunodeficiency virus type 1 nef gene can to a large extent replace simian immunodeficiency virus nef in vivo.

The nef gene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1) nef alleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of the nef alleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency. nef long terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6 nef allele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1 nef can, to a large extent, functionally replace SIVmac nef in vivo.     

Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the β3–β4 loop to favor a low-affinity drug-binding state

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.Keywords: PBP2, Neisseria gonorrhoeae, beta-lactam, conformational dynamics, antibiotic resistance

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea, with nearly 80 million cases worldwide each year (1). Without antibiotic treatment, infections persist as a chronic disease and can cause serious sequelae, including pelvic inflammatory disease, infertility, arthritis, and disseminated infections (2). For many years, N. gonorrhoeae was treated with a single dose of penicillin, and more recently, ceftriaxone. In 2012, the emergence of several high-level ceftriaxone-resistant strains led the Centers for Disease Control and Prevention to change its recommended treatment for gonorrhea from monotherapy to dual therapy with ceftriaxone and azithromycin (3, 4, 5). However, treatment failures have been reported for both agents, and in 2018, a strain with high-level resistance to both ceftriaxone and azithromycin was identified (6, 7). Concern about azithromycin resistance led the Centers for Disease Control and Prevention recently to drop the recommendation of dual therapy in favor of an increased dose (500 mg) of ceftriaxone alone (8). Both penicillin and ceftriaxone inhibit cell wall biosynthesis in N. gonorrhoeae by targeting penicillin-binding protein 2 (PBP2).PBP2 is an essential peptidoglycan transpeptidase (TPase) that crosslinks the peptide chains from adjacent peptidoglycan strands during cell-wall synthesis (9). β-lactam antibiotics, including the extended-spectrum cephalosporin (ESC) ceftriaxone, are analogs of the d-Ala-d-Ala C terminus of the peptidoglycan substrate and as such target PBP2 by binding to and reacting with the active-site serine nucleophile (Ser310 in N. gonorrhoeae PBP2) to form a covalently acylated complex (10, 11). The acylation reaction (Equation 1) proceeds first through formation of a noncovalent complex with the β-lactam (defined by the equilibrium constant, Ks), which is then attacked by the serine nucleophile to form a covalent acyl-enzyme complex (k₂). For PBPs, hydrolysis of the acyl-enzyme (k₃) is very slow compared with its formation, and the enzyme is essentially irreversibly inactivated. The acylation of PBPs by β-lactam antibiotics is therefore defined by a second-order rate constant, k₂/Ks (M⁻¹ s⁻¹), which reflects both the noncovalent binding affinity (Ks) and the first-order acylation rate (k₂):E+S↔KsE⋅S→k2E−S→k3E+P(1)The emergence of resistance to penicillin and ceftriaxone in N. gonorrhoeae occurs primarily via the acquisition of mutant alleles of the penA gene encoding PBP2 (12). These alleles are referred to as mosaic because they arise through multiple homologous recombination events with DNA released by commensal Neisseria species. PBP2 from the high-level ceftriaxone-resistant strain, H041, contains 61 mutations compared with PBP2 from the antibiotic-susceptible strain, FA19 (13, 14). Determining how these mutations lower the k₂/Ks of ceftriaxone for PBP2 by over 10,000-fold while still preserving essential TPase activity is fundamental for understanding the evolution of antibiotic resistance.Toward this goal, we have identified a subset of these mutations that, when incorporated into the penA gene from FA19, confer ∼80% of the increase in minimum inhibitory concentration for ceftriaxone relative to that of the penA gene from H041 (penA41) (15, 16). We recently reported the structures of apo and ceftriaxone-acylated PBP2 at high resolution and have detailed conformational changes in β3 and the β3–β4 loop involved in antibiotic binding and acylation (17). Intriguingly, although present in the active site region, most of the mutations conferring resistance are not in direct contact with ceftriaxone in the crystal structure of acylated PBP2 (17, 18). We have proposed that these mutations alter the binding and acylation kinetics of PBP2 with ceftriaxone by restricting protein dynamics (18).To understand further the structural and biochemical mechanisms by which these mutations lower the acylation rates of β-lactam antibiotics, we utilized a combination of solution ¹⁹F NMR, X-ray crystallography, and biochemical approaches to investigate PBP2. We report that the β3–β4 loop in the TPase domain of WT PBP2, which is known to adopt markedly different conformations in the apo versus acylated crystal structures (17), samples two major conformational states in solution. Substitutions of WT PBP2 residues with mutations in H041 that confer ceftriaxone resistance alter the conformational landscape of PBP2 by destabilizing the high-affinity state containing the inward conformation of the β3–β4 loop and stabilizing a low-affinity conformation containing an extended β3–β4 loop conformation, thereby restricting access to the inward conformation required for high-affinity drug binding. Our combined solution NMR and crystallographic analyses of PBP2 and its preacylation drug complexes further support the notion that mutations in PBP2 from ceftriaxone-resistant strains of N. gonorrhoeae confer antibiotic resistance by hindering conformational changes required to form a productive drug-binding state (18).