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Biomedical subjects

K L Hoffman

Publications and source records attributed to K L Hoffman.

At least 19 recordsLinked to original sources
PubMed2006

Progesterone receptor activation signals behavioral transitions across the reproductive cycle of the female rabbit.

The female rabbit is an exceptional experimental model to define mechanisms by which progesterone (P) controls the expression of reproductive behaviors. In the rabbit, the rise in P levels during pregnancy inhibits estrous scent marking ("chinning"), stimulates the excavation of a nest burrow ("digging"), and primes behaviors later used for nest construction. The pre-parturient fall of P triggers the construction of a straw nest ("straw carrying") that is lined with hair that she pulls from her own body ("hair pulling"). These behaviors can be replicated in ovariectomized (ovx) females given a schedule of estradiol (E) and P that mimics hormone levels during pregnancy (E from days 0 to 4, E + P from days 5 to 17, E from days 18 to 27). We administered PR antagonists RU486 or CDB(VA)2914 to ovx female rabbits during either the initial (days 5-11) or late (days 12-17) phases of P treatment, to determine the role of PR activation in coordinating the expression of these behaviors. Both antiprogestins attenuated the P-mediated decline in chinning and increase in digging when administered during days 5-11. When given across days 12-17, both antiprogestins triggered an early decline in digging, the onset of nest building in some Ss, and the reinstatement of chinning. These results point to a central role of PR activation for establishing and maintaining the behavioral phenotype of pregnancy, and for the behavioral transition from pregnancy to estrus.

Analysis of Variance↗
PubMed2005

Sparse, environmentally selective expression of Arc RNA in the upper blade of the rodent fascia dentata by brief spatial experience.

After a spatial behavioral experience, hippocampal CA1 pyramidal cells express the activity-regulated, immediate early gene Arc in an environment-specific manner, and in similar proportions ( 40%) to cells exhibiting electrophysiologically recorded place fields under similar conditions. Theoretical accounts of the function of the fascia dentata suggest that it plays a role in pattern separation during encoding. The hypothesis that the dentate gyrus (DG) uses a sparse, and thus more orthogonal, coding scheme has been supported by the observation that, while granule cells do exhibit place fields, most are silent in a given environment. To quantify the degree of sparsity of DG coding and its corresponding ability to generate distinct environmental representations, behaviorally induced Arc expression was assessed using in situ hybridization coupled with confocal microscopy. The proportion of Arc(+) cells in the "upper blade" of the fascia dentata (i.e., the portion that abuts CA1) increased in an environment-specific fashion, approximately 4-fold above cage-control activity, after behavioral exploration. Surprisingly, cells in the lower blade of the fascia dentata, which are capable of expressing Arc following electrical stimulation, exhibited virtually no behaviorally-induced Arc expression. This difference was confirmed using "line scan" analyses, which also revealed no patterns or gradients of activity along the upper blade of the DG. The expression of Arc in the upper blade was quantitatively similar after exploring familiar or novel environments. When animals explored two different environments, separated by 20 min, a new group of cells responded to the second environment, whereas two separated experiences in the same environment did not activate a new set of granular cells. Thus, granule cells generate distinct codes for different environments. These findings suggest differential contribution of upper and lower blade neurons to plastic networks and confirm the hypothesis that the DG uses sparse coding that may facilitate orthogonalization of information.

Animals↗
PubMed2002

Cell culture of mechanoreceptor neurons innervating proleg sensory hairs in Manduca sexta larvae, and co-culture with target motoneurons.

The tip of each proleg in Manduca sexta larvae bears a dense array of mechanosensory hairs termed planta hairs (PHs), each innervated by a single sensory neuron (termed a PH-SN) located in the underlying epidermis. In the CNS, axon terminals of PH-SNs make direct, excitatory, nicotinic cholinergic synapses with proleg retractor motoneurons including the accessory planta retractor (APR). These synapses mediate a proleg withdrawal reflex, exhibit multiple forms of activity-dependent plasticity and weaken during the prepupal peak of ecdysteroids. In the present study we developed methods to dissociate PH-SNs from the epidermis and culture them alone or with APRs. The PH-SNs were fluorescently labeled in situ by introducing dye through the cut hair shaft or by retrograde axonal staining. Alternatively, unlabeled PH-SNs were utilized. The epidermis beneath the planta hair array was separated from the cuticle, enzymatically treated and mechanically dissociated into single cells. PH-SNs were cultured on glass coverslips coated with concanavalin A and laminin, in modified Leibovitz's IL-15 medium. Supplementation with medium conditioned by an insect cell line produced the best results. Dissociated PH-SNs had somatic diameters of ~10 micro m and typically bore a stout dendrite consisting of the inner and, occasionally, the outer dendritic segment. An axonal stump was sometimes retained. Viable PH-SNs typically extended new processes and often survived for 2-4 weeks. When co-cultured, PH-SNs and APRs exhibited robust growth and made close anatomical appositions. This culture system provides convenient experimental access to PH-SNs and may potentially permit sensorimotor synapses to be investigated in vitro.

Animals↗
PubMed2002

Coordinated reactivation of distributed memory traces in primate neocortex.

Conversion of new memories into a lasting form may involve the gradual refinement and linking together of neural representations stored widely throughout neocortex. This consolidation process may require coordinated reactivation of distributed components of memory traces while the cortex is "offline," i.e., not engaged in processing external stimuli. Simultaneous neural ensemble recordings from four sites in the macaque neocortex revealed such coordinated reactivation. In motor, somatosensory, and parietal cortex (but not prefrontal cortex), the behaviorally induced correlation structure and temporal patterning of neural ensembles within and between regions were preserved, confirming a major tenet of the trace-reactivation theory of memory consolidation.

Action Potentials↗
PubMed2001

Dentate gyrus and ca1 ensemble activity during spatial reference frame shifts in the presence and absence of visual input.

In rats shuttling between a variably placed landmark of origin and a fixed goal, place fields of hippocampal CA1 cells encode location in two spatial reference frames. On the initial part of the outbound journey, place fields encode location with respect to the origin while on the final segment, place fields are aligned with the goal (Gothard et al., 1996b). An abrupt switch of reference frame can be induced experimentally by shortening the distance between the origin and the goal. Two linked hypotheses concerning this effect were addressed: (1) that the persistent, landmark-referenced firing results from some internal dynamic process (e.g., path integration or "momentum") and is not a result of maintained sensory input from the landmark of origin; and (2) that this hypothetical process is generated by connections either within CA3 or between CA3 and CA1, in which case the effect might be absent from the dentate gyrus. Neuronal ensemble recordings were made simultaneously from CA1 and the dentate gyrus as rats shuttled on a linear track between a variably located box and a goal, under light or dark conditions. The box-referenced firing persisted significantly longer in the dark in both hippocampal subfields, suggesting a competitive interaction between an internal dynamic process and external sensory cues. The similarity between reference frame transitions in the dentate gyrus and the CA1 region suggests that this process probably occurs before CA3, possibly in the entorhinal cortex or subiculum.

Action Potentials↗
PubMed2001

Role of caspases and mitochondria in the steroid-induced programmed cell death of a motoneuron during metamorphosis.

Accessory planta retractor (APR) motoneurons of the hawk moth, Manduca sexta, undergo a segment-specific pattern of programmed cell death (PCD) 24 to 48 h after pupal ecdysis (PE). Cell culture experiments show that the PCD of APRs in abdominal segment 6 [APR(6)s] is a cell-autonomous response to the steroid hormone 20-hydroxyecdysone (20E) and involves mitochondrial demise and cell shrinkage. Twenty-four hours before PE, at stage W3-noon, APR(6)s require further 20E exposure and protein synthesis (as tested with cycloheximide) to undergo PCD, and death can be blocked by a broad-spectrum caspase inhibitor. By PE, death is 20E- and protein synthesis-independent and the caspase inhibitor blocks cell shrinkage but not loss of mitochondrial function. Thus, the commitment to mitochondrial demise precedes the commitment to execution events. The phenotype of necrotic cell death induced by a mitochondrial electron transfer inhibitor differs unambiguously from 20E-induced PCD. By inducing PCD pharmacologically, the readiness of APR(6)s to execute PCD was found to increase during the final larval instar. These data suggest that the 20E-induced PCD of APR(6)s includes a premitochondrial phase which includes 20E-induced synthetic events and apical caspase activity, a mitochondrial phase which culminates in loss of mitochondrial function, and a postmitochondrial phase during which effector caspases are activated and APR(6) is destroyed.

Animals↗
PubMed2001

Antimicrobial effects of corn zein films impregnated with nisin, lauric acid, and EDTA.

Bacterial growth during food transport and storage is a problem that may be addressed with packaging materials that release antimicrobials during food contact. In a series of five experiments, EDTA, lauric acid (LA), nisin, and combinations of the three antimicrobial agents were incorporated into a corn zein film and exposed to broth cultures of Listeria monocytogenes and Salmonella Enteritidis for 48 h (sampled at 2, 4, 8, 12, 24, and 48 h). Four experiments used starting cultures of 10(8) CFU/ml in separate experiments tested against each bacterium; the fifth experiment examined the inhibitory effect of selected antimicrobial agents on Salmonella Enteritidis with an initial inoculum of 10(4) CFU/ml. L. monocytogenes cell numbers decreased by greater than 4 logs after 48 h of exposure to films containing LA and nisin alone. No cells were detected for L. monocytogenes (8-log reduction) after 24-h exposure to any film combination that included LA. Of all film agent combinations tested, none had greater than a 1-log reduction of Salmonella Enteritidis when a 10(8)-CFU/ml broth culture was used. When a 10(4) CFU/ml of Salmonella Enteritidis initial inoculum was used, the films with EDTA and LA and EDTA, LA, and nisin were bacteriostatic. However, there was a 5-log increase in cells exposed to control within 24 h. The results demonstrate bacteriocidal and bacteriostatic activity of films containing antimicrobial agents.

Anti-Bacterial Agents↗
PubMed2000

Mixed-mechanism ionization to enhance sensitivity in atmospheric pressure ionization LC/MS.

A novel dual-mechanism ionization technique for LC/MS/MS has been observed, characterized and applied to the quantitation of a tertiary amine-containing drug compound in dog plasma. This mixed-mechanism ionization approach can improve the sensitivity of the pneumatically assisted electrospray experiment. Under conditions of higher than normal chromatographic flow and lower electrospray voltage, approximately a 4-fold increase in sensitivity was realized. A detection limit of 16 pg (45 fmol) on-column, and inter-day imprecision and inaccuracy of < 11 and < 15%, respectively, were obtained. A trade-off in concentration sensitivity in favor of ease of sample preparation was made to increase sample throughput. Although results strongly suggest that mixed-mechanism ionization is in operation, and that pneumatically assisted electrospray is a partial contributor to the overall ionization process, the exact nature of the second mechanism of ionization is unclear at this time.

Animals↗
PubMed2000

Semi-automated 96-well liquid-liquid extraction for quantitation of drugs in biological fluids.

A semi-automated liquid-liquid extraction (LLE) technique for biological fluid sample preparation was introduced for the quantitation of four drugs in rat plasma. All liquid transferring during the sample preparation was automated using a Tomtec Quadra 96 Model 320 liquid handling robot, which processed up to 96 samples in parallel. The samples were either in 96-deep-well plate or tube-rack format. One plate of samples can be prepared in approximately 1.5 h, and the 96-well plate is directly compatible with the autosampler of an LC/MS system. Selection of organic solvents and recoveries are discussed. Also, precision, relative error, linearity and quantitation of the semi automated LLE method are estimated for four example drugs using LC/MS/MS with a multiple reaction monitoring (MRM) approach. The applicability of this method and future directions are evaluated.

Algorithms↗
PubMed1999

Quantitation of a novel metalloporphyrin drug in plasma by atomic absorption spectroscopy.

A bioanalytical method to quantify cobalt mesoporphyrin (CoMP), a novel therapeutic agent, in plasma has been developed and validated. The approach involves atomic absorption spectroscopy to determine total cobalt in a sample and a back-calculation of the amount of compound present. Endogenous plasma cobalt concentrations were small ( <0.2 ng/ml(-1) Co in rat plasma) in comparison to the quantitation limit (4.5 ng/ml(-1) Co). The inter-day imprecision of the method was 10.0% relative standard deviation (RSD) and the inter-day bias was +/- 8.0% relative error (RE) over a standard curve range of 4.5- 45.0 ng/ml(-1) Co. Because it quantifies total cobalt, the method cannot differentiate between parent drug and metabolites, but negligible metabolism allows reliable estimates of the actual parent drug concentration. A correlation study between the atomic absorption method and 14C-radiometry demonstrated excellent agreement (r = 0.9868, slope = 1.041 +/- 0.028, intercept = 223.7 +/- 190.0) and further substantiated the accuracy of the methods. Methodology was successfully applied to a pharmacokinetic study of CoMP in rat, with pharmacokinetic parameter estimation. The elimination half-lives, after intra-muscular and subcutaneous administration, were 7.7 and 8.8 days, respectively.

Animals↗
PubMed1999

Development of a chiral HPLC method to evaluate in vivo enantiomeric inversion of an unstable, polar radiosensitizer in plasma.

A chiral HPLC method to quantify in vivo enantiomeric inversion of prodrug CI-1010 (IR) or its drug IIR (PD 146923), a radiosensitizer, upon X-irradiation of dosed rats was developed. These polar enantiomers were separated only by using normal-phase chiral HPLC. A Chiralpak AS column provided the best separation. Isolation of analytes from plasma employed solid-phase extraction (SPE), and required conditions that were compatible with normal-phase HPLC. Options for SPE were restricted by the chemically reactive nature of both prodrug and drug, which produced analyte losses as high as 100%. Acceptable recoveries using SPE required evaluation of conditions for analyte chemical stability. The validated method gave a lower-limit of quantitation (LLOQ) of 200 ng/ml for each enantiomer extracted from 0.15 ml of plasma. The LLOQ of the inverted enantiomer could be detected in the presence of 10,000 ng/ml of the dosed enantiomer. Precision (RSD) ranged from 14.2 to 4.4%, and from 24.2 to 5.1% for IIS and IIR, respectively. Accuracy (RE) was +/- 13.1 and +/- 13.2%, respectively. Recoveries ranged from 44.3 to 71.4%, and from 40.7 to 67.9%, for IIS and IIR, respectively.

Animals↗
PubMed1998

Programmed cell death of an identified motoneuron in vitro: temporal requirements for steroid exposure and protein synthesis.

Ecdysteroid hormones trigger the programmed cell death (PCD) of a segmental subset of accessory planta retractor (APR) motoneurons at pupation in the moth, Manduca sexta. APRs from abdominal segment four [APR (4)s] survive through the pupal stage, whereas homologous APR(6)s die 24-48 h after pupal ecdysis (PE) (the shedding of the larval cuticle), in response to the prepupal peak of ecdysteroids. Following retrograde labeling with the vital fluorescent dye, DiI, the morphology of APR(4)s and APR(6)s in vivo was examined at PE and 24-48 h later. During this period, APR(4) somata remained large and ovoid while APR(6)s somata became shrunken and rounded. Similar phenotypes were observed when DiI-labeled APRs were cultured at PE and examined 24 h to 1 week later. During initial shrinkage and rounding of APR(6)s, the plasma membrane remained intact but DNA condensation occurred and mitochondrial activity was lost. The requirements for ecdysteroids and new protein synthesis for APR(6) death were tested by culturing cells with ecdysteroids and cycloheximide (CHX). When cultured at PE, the death of APR(6)s was independent of further exposure to ecdysteroids and could not be blocked by CHX. In contrast, APR(6)s cultured 24 h earlier required additional exposure to ecdysteroids to die and their death was inhibited by CHX. Thus, the final 24 h of larval life represents an important transition period in the commitment of APR(6)s to undergo PCD, and is of interest for pursuing underlying mechanisms of steroid-induced PCD.

Animals↗
PubMed1997

Tandem-in-time mass spectrometry as a quantitative bioanalytical tool.

Tandem-in-time mass spectrometry, as implemented on an ion-trap detector (ITD), is the process whereby precursor ions are created, stored in a radio frequency (rf) trapping field, and then sequentially fragmented to form product ions by application of additional rf waveforms. As with any form of tandem mass spectrometry (MS/MS), tandem-in-time MS is highly selective, by virtue of both mass discrimination and specific gas-phase chemistry. Beyond this, however, tandem-in-time MS offers ion throughput efficiency and cost advantages over either quadrupole or sector instruments. This paper will describe the use of capillary gas chromatography combined with tandem-in-time mass spectrometry to quantify a novel therapeutic agent extracted from human plasma. For an example compound, a quantitation limit of 25 pg/mL (S/N approximately 10, 15 fmol on-column) was attained out of plasma. The interday imprecision was < or = 12.2% over a dynamic range extending to 10 ng/mL. Due to favorable ionization conditions for the test analytes, electron ionization resulted in formation of M+ ions, with very little fragmentation, allowing for maximum assay sensitivity. Although method characterization and validation demonstrated adequate instrumental performance, some lack of ruggedness was encountered during routine application.

Cholinesterase Inhibitors↗
PubMed1997

An HPLC assay utilizing solid-phase extraction for CI-1010, an alkylating radiosensitizer, in rat plasma.

CI-1010, a 2-nitroimidazole, is a chiral prodrug for the active moiety PD 146923 and is under development as an alkylating radiosensitizer to be used as an adjuvant to radiotherapy. Because CI-1010 has an estimated half-life < or = 2 min under physiological conditions its metabolites/degradation products PD 146415, an inactive moiety, and PD 146923 were assayed to support rat toxicology studies. The method involves the processing of plasma samples through phenyl solid-phase extraction cartridges followed by chromatography on CN columns with UV detection at 325 nm. The assay appears linear over the range 0.050-100 micrograms ml-1 for both PD 146415 and PD 146923. Interrun accuracy and precision estimates for PD 146415 and PD 146923 were within +/- 6.50 and < or = 3.27%, respectively, and +/- 12.8 and < or = 4.06%, respectively, for quality controls containing nominal concentrations of 0.400, 4.00 and 40.0 micrograms ml-1. The absolute recovery of CI-1010, PD 146415 and internal standard, PD 126675, were approximately 40, 96 and 95%, respectively. The recovery of PD 146923 appeared concentration dependent and ranged from 68 to 92%. PD 146415 and PD 146923 were both stable in rat plasma at 4 degrees C and -77 degrees C for at least 7 h and 154 days, respectively. CI-1010 was not stable in rat plasma at 4 degrees C. CI-1010, PD 146415 and PD 126675 were stable for at least 63 days in 10 mM phosphate buffer at pH 3.0 and 4 degrees C. Under identical conditions PD 146923 was stable for only 8 days. The applicability of this method to determine concentrations of PD 146415 and PD 146923 in rat plasma is reported in this paper.

Alkylating Agents↗
PubMed1995

Canine oligodendrocytes undergo morphological changes in response to basic fibroblast growth factor (bFGF) in vitro.

The mature oligodendrocyte, though able to divide under certain circumstances, has been regarded as incapable of changing into a phenotypically plastic cell type. To further explore this question, we developed an in vitro system in which a virtually pure population of early postnatal canine oligodendrocytes were cultured in a serum free, defined medium. We tested the oligodendrocytes' morphological and mitotic responses to concentration of basic Fibroblast Growth Factor (bFGF) ranging from 5 ng to 100 ng/ml. We found that bFGF effected both the morphology and mitotic potential of these cells. In addition, oligodendrocytes exposed to bFGF respond to 10% fetal bovine serum (FBS) by undergoing morphological changes that are quite different than naive oligodendrocytes exposed to 10% FBS, suggesting that bFGF causes some fundamental change in plasticity.

Animals↗
PubMed1995

Trace quantitation of the novel cholinesterase inhibitor in human plasma by capillary gas chromatography/ion trap mass spectrometry.

This paper demonstrates the utility of an ion trap mass spectrometer as a detector for trace quantitative determinations of pharmaceuticals in human plasma by capillary gas chromatography/mass spectrometry. A novel acetylcholinesterase inhibitor (CI-1002) was selected as an illustrative example for the technique. When coupled with a selective solid-phase extraction, this approach was capable of quantifying as little as 34 pg (0.50 ng ml-1, RSD = 12.7%) of compound on the column, and the inter-run precision was typically 3-4% RSD over a 0.5-25 ng ml-1 linear range. The advantages and requirements of the technique, in addition to the prospects for improvements in the detection limit, are discussed.

Calibration↗