Safety and activity of vandetanib in combination with everolimus in patients with advanced solid tumors: a phase I study.

BACKGROUND: Preclinical studies suggest that combining vandetanib (VAN), a multi-tyrosine kinase inhibitor of rearranged during transfection (RET) proto-oncogene, vascular endothelial growth factor receptor (VEGFR), and epidermal growth factor receptor (EGFR), with everolimus (EV), a mammalian target of rapamycin (mTOR) inhibitor, may improve antitumor activity. We determined the safety, maximum tolerated dose (MTD), recommended phase II dose (RP2D), and dose-limiting toxicities (DLTs) of VAN + EV in patients with advanced solid cancers and the effect of combination therapy on cancer cell proliferation and intracellular pathways. PATIENTS AND METHODS: Patients with refractory solid tumors were enrolled in a phase I dose-escalation trial testing VAN (100-300 mg orally daily) + EV (2.5-10 mg orally daily). Objective responses were evaluated using RECIST v1.1. RET mutant cancer cell lines were used in cell-based studies. RESULTS: Among 80 patients enrolled, 72 (90%) patients were evaluable: 7 achieved partial response (PR) (10%) and 37 had stable disease (SD) (51%; duration range: 1-27 cycles). Clinical benefit (SD or PR ≥ 6 months) was observed in 26 evaluable patients [36%, 95% confidence intervals (CI) (25% to 49%)]. In 80 patients, median overall survival (OS) was 10.5 months [95% CI (8.5-16.1)] and median progression-free survival (PFS) 4.1 months [95% CI (3.4-7.3)]. Six patients (7.5%) experienced DLTs and 20 (25%) required dose modifications. VAN + EV was safe, with fatigue, rash, diarrhea, and mucositis being the most common toxicities. In cell-based studies, combination therapy was superior to monotherapy at inhibiting cancer cell proliferation and intracellular signaling. CONCLUSIONS: The MTDs and RP2Ds of VAN + EV are 300 mg and 10 mg, respectively. VAN + EV combination is safe and active in refractory solid tumors. Further investigation is warranted in RET pathway aberrant tumors.

Associations of Dual Task Exergaming with Cognitive-Motor Interference in Older Adults with Mild Cognitive Impairment: A Single-Arm Pilot Study.

Purpose: To examine the feasibility and effectiveness of dual task (DT) exergaming to improve volitional balance control in older adults with mild cognitive impairment (MCI). Methods: Ten older adults with MCI were examined at baseline (week-0) and post-training (week-5) on volitional balance control (maximum excursion of center of gravity, MXE [%]) while performing cognitive task (auditory clock test or letter number sequencing task) and on the NIH-motor and cognitive toolboxes. DT exergaming training lasted for 12 sessions which consisted of performing explicit cognitive tasks while playing the Wii-Fit balance games. Results: From pre- to post-training, MXE improved (p<0.05); however, cognitive accuracy (cognitive task) remained the same (p>0.05). Improvement in NIH motor and cognitive toolbox tests was observed post-training (p<0.05). Conclusion: DT exergaming was associated to improvements in balance control under attention-demanding conditions in MCI. Future studies may focus on examining the efficacy of such training.

DNA methylation of Th2 lineage determination genes at birth is associated with allergic outcomes in childhood.

BACKGROUND: There is now increasing evidence that asthma and atopy originate in part in utero, with disease risk being associated with the altered epigenetic regulation of genes. OBJECTIVE AND METHODS: To determine the relationship between variations in DNA methylation at birth and the development of allergic disease, we examined the methylation status of CpG loci within the promoter regions of Th1/2 lineage commitment genes (GATA3, IL-4, IL-4R, STAT4 and TBET) in umbilical cord DNA at birth in a cohort of infants from the Southampton Women's Survey (n = 696) who were later assessed for asthma, atopic eczema and atopy. RESULTS: We found that higher methylation of GATA3 CpGs -2211/-2209 at birth was associated with a reduced risk of asthma at ages 3 (median ratio [median methylation in asthma group/median methylation in non-asthma group] = 0.74, P = .006) and 6-7 (median ratio 0.90, P = .048) years. Furthermore, we demonstrated that the GATA3 CpG loci associated with later risk of asthma lie within a NF-κB binding site and that methylation here blocks transcription factor binding to the GATA3 promoter in the human Jurkat T-cell line. Associations between umbilical cord methylation of CpG loci within IL-4R with atopic eczema at 12 months (median ratio 1.02, P = .028), and TBET with atopy (median ratio 0.98, P = .017) at 6-7 years of age were also observed. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings provide further evidence of a developmental contribution to the risk of later allergic disorders and suggest that involvement of epigenetic mechanisms in childhood asthma is already demonstrable at birth.

Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence.

Dysregulated activation of the CDK4/6 kinases is a hallmark of most mammary-derived carcinomas. ATP-competitive inhibitors against this complex have been recently advanced in the clinic and have shown significant activity, particularly against tumors driven by the estrogen receptor (ER). However, resistance to these compounds has begun to emerge often months to years after their initiation. We investigated potential mechanisms of resistance using cell line models that are highly sensitive to this class of drugs. After prolonged exposure to the selective and potent CDK4/6 inhibitor LY2835219, clones emerged and several were found to harbor amplification of the CDK6 kinase. Amplification of CDK6 resulted in a marked increase in CDK6 expression and reduced response of the CDK4/6 target, phospho-Rb (pRb), to CDK4/6 inhibitors. Knockdown of CDK6 restored drug sensitivity, while enforced overexpression of CDK6 was sufficient to mediate drug resistance. Not only did CDK6 overexpression mediate resistance to CDK4/6 inhibitors but it also led to reduced expression of the ER and progesterone receptor (PR), and diminished responsiveness to ER antagonism. The reduced ER/PR expression after CDK4/6 inhibitor resistance was additionally observed in tumor biopsy specimens from patients treated with these drugs. Alternative mechanisms of resistance to CDK4/6 inhibitors such as loss of pRb and cyclin E1 overexpression also exhibited decreased hormone responsiveness, suggesting that the clinical paradigm of sequential endocrine-based therapy may be ineffective in some settings of acquired CDK4/6 resistance.

Kommerell's diverticulum: A rare aortic arch anomaly.

Anomalies of the aortic arch associated with diverticulum are rare. We present a case of incidentally detected right-sided aortic arch with Kommerell's diverticulum and aberrant left subclavian artery.

Effect of type of cognitive task and walking speed on cognitive-motor interference during dual-task walking.

OBJECTIVE: We aimed to determine the effect of distinctly different cognitive tasks and walking speed on cognitive-motor interference of dual-task walking. METHODS: Fifteen healthy adults performed four cognitive tasks: visuomotor reaction time (VMRT) task, word list generation (WLG) task, serial subtraction (SS) task, and the Stroop (STR) task while sitting and during walking at preferred-speed (dual-task normal walking) and slow-speed (dual-task slow-speed walking). Gait speed was recorded to determine effect on walking. Motor and cognitive costs were measured. RESULTS: Dual-task walking had a significant effect on motor and cognitive parameters. At preferred-speed, the motor cost was lowest for the VMRT task and highest for the STR task. In contrast, the cognitive cost was highest for the VMRT task and lowest for the STR task. Dual-task slow walking resulted in increased motor cost and decreased cognitive cost only for the STR task. CONCLUSIONS: Results show that the motor and cognitive cost of dual-task walking depends heavily on the type and perceived complexity of the cognitive task being performed. Cognitive cost for the STR task was low irrespective of walking speed, suggesting that at preferred-speed individuals prioritize complex cognitive tasks requiring higher attentional and processing resources over walking. While performing VMRT task, individuals preferred to prioritize more complex walking task over VMRT task resulting in lesser motor cost and increased cognitive cost for VMRT task. Furthermore, slow walking can assist in diverting greater attention towards complex cognitive tasks, improving its performance while walking.

Adaptation and generalization to opposing perturbations in walking.

Little is known on how the CNS would select its movement options when a person faces a novel or recurring perturbation of two opposing types (slip or trip) while walking. The purposes of this study were (1) to determine whether young adults' adaptation to repeated slips would interfere with their recovery from a novel trip, and (2) to investigate the generalized strategies after they were exposed to a mixed training with both types of perturbation. Thirty-two young adults were assigned to either the training group, which first underwent repeated-slip training before encountering a novel, unannounced trip while walking, or to the control group, which only experienced the same novel, unannounced trip. The former group would then experience a mix of repeated trips and slips. The results indicated that prior adaptation to slips had only limited interference during the initial phase of trip recovery. In fact, the prior repeated-slip exposure had primed their reaction, which mitigated any error resulting from early interference. As a result, they did not have to take a longer compensatory step for trip recovery than did the controls. After the mixed training, subjects were able to converge effectively the motion state of their center of mass (in its position and velocity space) to a stable and generalized "middle ground" steady-state. Such movement strategies not only further strengthened their robust reactive control of stability, but also reduced the CNS' overall reliance on accurate context prediction and on feedback correction of perturbation-induced movement error.

Generalization of motor adaptation to repeated-slip perturbation across tasks.

Similar adaptations improve both proactive and reactive control of center-of-mass (COM) stability and limb support against gravity during different daily tasks (e.g., sit-to-stand and walking) as a consequence of perturbation training for resisting falls. Yet it is unclear whether--or to what extent--such similarities actually promote inter-task generalization. The purpose of this study was therefore to determine whether young adults could indeed transfer their adaptive control, acquired from sit-to-stand-slip, to improve their likelihood of a recovery from an unannounced novel slip in walking. Subjects underwent either repeated slips during sit-to-stand before experiencing an unannounced, novel slip during walking (training group, n=20), or they received no prior training before the same gait-slip (control group, n=23). The subjects demonstrated training-induced generalization of their improved proactive control of stability in post-training (unperturbed) gait pattern that was more stable against backward balance loss than was that of their own pre-training pattern as well the gait pattern of the subjects in the control group. Upon the unannounced novel gait-slip, the training group showed significantly lower incidence of both falls and balance loss than that shown by the control, resulting from the improvements in the reactive control of limb support and slip velocity, which directly influenced the control of their COM stability. Such transfer could occur when the subjects' central nervous system recalibrates the non-task-specific, generalized representation of stability limits during the initial training to guide both their feed-forward adjustments and their feedback responses. The findings of the inter-task generalization suggests that behavioral changes induced via the perturbation training paradigm have the potential to prevent falls across the spectrum of cyclic and non-cyclic activities.

Independent influence of gait speed and step length on stability and fall risk.

With aging, individuals' gaits become slower and their steps shorter; both are thought to improve stability against balance threats. Recent studies have shown that shorter step lengths, which bring the center of mass (COM) closer to the leading foot, improve stability against slip-related falls. However, a slower gait, hence lower COM velocity, does the opposite. Due to the inherent coupling of step length and speed in spontaneous gait, the extent to which the benefit of shorter steps can offset the slower speed is unknown. The purpose of this study was to investigate, through decoupling, the independent effects of gait speed and step length on gait stability and the likelihood of slip-induced falls. Fifty-seven young adults walked at one of three target gait patterns, two of equal speed and two of equal step length; at a later trial, they encountered an unannounced slip. The results supported our hypotheses that faster gait as well as shorter steps each ameliorates fall risk when a slip is encountered. This appeared to be attributable to the maintenance of stability from slip initiation to liftoff of the recovery foot during the slip. Successful decoupling of gait speed from step length reveals for the first time that, although slow gait in itself leads to instability and falls (a one-standard-deviation decrease in gait speed increases the odds of fall by 4-fold), this effect is offset by the related decrease in step length (the same one-standard-deviation decrease in step length lowers fall risk by 6 times).

Generalization of gait adaptation for fall prevention: from moveable platform to slippery floor.

A person's ability to transfer the acquired improvements in the control of center of mass (COM) state stability to slips induced in everyday conditions can have profound theoretical and practical implications for fall prevention. This study investigated the extent to which such generalization could take place. A training group (n=8) initially experienced 24 right-side slips in blocked-and-random order (from the 1st unannounced, novel slip, S-1 to the last, S-24) resulting from release of a low-friction moveable platform in walking. They then experienced a single unannounced slip while walking on an oil-lubricated vinyl floor surface (V-T). A control group (n=8) received only one unannounced slip on the same slippery floor (V-C). Results demonstrated that the incidence of balance loss and fall on V-T was comparable to that on S-24. In both trials, fall and balance-loss incidence was significantly reduced in comparison with that on S-1 or on V-C, resulting from significant improvements in the COM state stability. The observed generalization indicates that the control of COM stability can be optimally acquired to accommodate alterations in environmental constraints, and it may be broadly coded and easily modifiable within the CNS. Because of such mechanisms, it is possible that the locomotor-balance skills acquired with the aid of low-friction moveable platforms can translate into resisting falls encountered in daily living.

Immediate and latent interlimb transfer of gait stability adaptation following repeated exposure to slips.

The authors trained 21 participants by using blocked-and-mixed exposure to right-side slips and then caused them to slip unexpectedly on the untrained left side. Authors retested participants with a right slip and a left slip at 1 week, 2 weeks, 1 month, and 4 months. The authors found that preslip stability on the first untrained left slip improved and was significantly greater than that on the first right slip, which probably contributed to the reduction in incidence of falls from approximately 30% to approximately 10%. Postslip stability and base of support (BOS) slip velocity were similar to those on the first right slip and much lower than those on the last right slip. Increases in pre- and postslip stabilities and BOS slip velocity during the left slip led to reductions in backward balance loss (BLOB) from approximately 95% on initial left slip to approximately 60% and to approximately 25% on the 1st and 3rd retest sessions, respectively. In contrast, BLOB remained at a constant approximately 40% level on the right slip of the same retest sessions. The results indicate a partial immediate transfer and a possible latent transfer.

Can observational training substitute motor training in preventing backward balance loss after an unexpected slip during walking?

A person's awareness of potential slippery walking conditions induces a cautious gait pattern. The purposes of this study were to determine whether neuromechanical changes associated with such cognitive conditioning are sufficient to alter the outcome of a slip and whether the effects of such conditioning are comparable to those of motor training. Prior to their own first slip exposure, 18 young subjects watched videos and slides demonstrating where and how the slip would occur and how people adapted to repeated-slip exposure (observe). The outcomes of the first slip exposure experienced by another 16 subjects who did not receive any such information were used as controls (naïve). The latter subjects subsequently experienced an additional 23 slips and thus served in a dual-role as the motor training group (motor). Gait stability as measured against backward loss of balance (BLOB) was obtained for pre- and postslip instances. A protective step landing posterior to the slipping-limb identified each BLOB outcome. The observe group had a greater postslip stability and lower slip displacement and velocity than the naïve group. However, such effects were insufficient to prevent balance loss (100% BLOB). The motor group showed significantly better performance on the last training slip (0% BLOB) than did the observe group. The results indicated that updating the cognitive centers of the CNS with awareness and perceptual knowledge through observational training can yield tangible benefits. Nonetheless observation could not replace the task-specific motor training that adaptively updated the internal representations of stability limits for prevention of BLOB.

Retention of adaptive control over varying intervals: prevention of slip- induced backward balance loss during gait.

Stability improvements made in a single acquisition session with merely five slips in walking are sufficient to prevent backward balance loss (BLOB) at the end of session, but not after 12 mo. The purpose of this study was to determine whether the effect of an enhanced single acquisition session would be retainable if tested sooner, at intervals of < or =4 mo. Twenty-four young subjects were exposed to blocks of slip, nonslip, and both types of trials during walking at their preferred speed in the acquisition session. In each of the four follow-up sessions around 1 wk, 2 wk, 1 mo, and 4 mo later, these same subjects experienced only a single slip after eight to 13 unperturbed walking trials in an otherwise identical setup. Gait stability was obtained as the shortest distance between the measured center of mass (COM) state (position and velocity) and the mathematically predicted threshold for BLOB at pre- and postslip, corresponding to the instants of touchdown of the slipping limb and liftoff of the contralateral limb, respectively. During the acquisition session, pre- and postslip stability improved significantly, resulting in a reduction of BLOB from 100% in the first slip (S1) to 0% in the last slip (S24), with improvements converging to a steady state, that enabled all of the subjects to avoid BLOB, regardless of whether a slip occurred. During retest sessions, subjects' preslip stability was not different from that in S24, but was greater than that in S1. Their postslip stability was also greater than that in S1 but less than that in S24, resulting in BLOB at a 40% level. No difference was found in any of these aspects between each follow-up session. These adaptive changes were associated with a range of individual differences, varying from no detectable deterioration in all aspects (n = 8) to a consistent BLOB in all follow-ups (n = 3). Our findings demonstrated the extent of plasticity of the CNS, characterized by rapid acquisition of a stable COM state under unpredictable slip conditions and retention of such improvements for months, resulting in a reduced occurrence of unintended backward falling.

Adaptive control of gait stability in reducing slip-related backward loss of balance.

The properties of adaptation within the locomotor and balance control systems directed towards improving one's recovery strategy for fall prevention are not well understood. The purpose of this study was to examine adaptive control of gait stability to repeated slip exposure leading to a reduction in backward loss of balance (and hence in protective stepping). Fourteen young subjects experienced a block of slips during walking. Pre- and post-slip onset stability for all slip trials was obtained as the shortest distance at touchdown (slipping limb) and lift-off (contralateral limb), respectively, between the measured center of mass (COM) state, that is, position and velocity relative to base of support (BOS) and the mathematically predicted threshold for backward loss of balance. An improvement in pre- and post-slip onset stability correlated with a decrease in the incidence of balance loss from 100% (first slip) to 0% (fifth slip). While improvements in pre-slip stability were affected by a proactive anterior shift in COM position, the significantly greater post-slip onset improvements resulted from reductions in BOS perturbation intensity. Such reactive changes in BOS perturbation intensity resulted from a reduction in the demand on post-slip onset braking impulse, which was nonetheless influenced by the proactive adjustments in posture and gait pattern (e.g., the COM position, step length, flat foot landing and increased knee flexion) prior to slip onset. These findings were indicative of the maturing process of the adaptive control. This was characterized by a shift from a reliance on feedback control for postural correction to being influenced by feedforward control, which improved pre-slip stability and altered perturbation intensity, leading to skateover or walkover (>0.05 m or <0.05 m displacement, respectively) adaptive strategies. Finally, the stability at contralateral limb lift-off was highly predictive of balance loss occurrence and its subsequent rapid reduction, supporting the notion of the internal representations of stability limits that could be modified and updated, as a key component in the adaptive control.

Long-term retention of gait stability improvements.

Evidence of long-term modification of behavior-in particular, gait alterations in response to repeated exposure to slips-within the locomotor-balance control system is limited. The purpose of this study was to examine whether improvements in fall-resisting behavior as reflected by improvements in gait stability could be retained on a long-term basis. Eight healthy young subjects were exposed to a block of repeated slip trials during a single acquisition session consisting of five repeated slip exposures; the same subjects were then re-tested using the same protocol at a minimum of 12 mo later. Pre- and postslip gait stability for all slip trials was measured at touchdown (slipping limb) and liftoff (contralateral limb) based on the center of mass state (i.e., its instantaneous position and velocity) relative to the base of support (BOS) and the predicted thresholds for backward loss of balance. In the acquisition session, subjects were able to increase pre- and postslip stability, which significantly correlated with a decrease in the incidence of balance loss from 100% (1st slip) to 0% (5th slip). All subjects exhibited a similar balance loss on the first slip of the follow-up session. Nonetheless, subjects were able to retain the acquired preslip stability with feedforward control on the first slip but not the postslip stability related to the reactive response. Also, the subjects demonstrated a faster re-acquisition, with only one balance loss on the second slip of the follow-up session, as compared with seven balance losses on the acquisition session. Such rapid improvements were achieved by the significantly greater increase in post- compared with preslip stability; this increase was for the most part, a consequence of reductions in slip intensity (i.e., the peak BOS velocity). We concluded that a single acquisition session could only produce limited long-term retainable effects within the locomotor-balance control system. It appeared, however, that the CNS was still primed to more rapidly update its internal representation of gait stability during re-acquisition.

Influence of gait speed on stability: recovery from anterior slips and compensatory stepping.

Falls precipitated by slipping are a major health concern, with the majority of all slip-related falls occurring during gait. Recent evidence shows that a faster and/or more anteriorly positioned center of mass (COM) is more stable against backward balance loss, and that compensatory stepping is the key to recovering stability upon balance loss. The purposes of this paper were to determine whether walking speed affected gait stability for backward balance loss at slip onset and touchdown of compensatory stepping, and whether compensatory stepping response resembled the regular gait pattern. Forty-seven young subjects were slipped unexpectedly either at a self-selected fast, natural or slow speed. Speed-related differences in stability at slip onset and touchdown of the subsequent compensatory step were analyzed using the COM position-velocity state. The results indicate that gait speed highly correlated with stability against backward balance loss at slip onset. The low COM velocity of the slow group was not sufficiently compensated for by a more anteriorly positioned COM associated with a shorter step length at slip onset. At touchdown of the compensatory step, the speed-related differences in stability diminished, due to the continued advantage of anterior COM positioning from a short compensatory step retained by the slow group, coupled with an increase in COM velocity. Compensatory step length and relative COM position altered as a function of gait speed, indicating the motor program for gait regulation may play a role in modulating the compensatory step.

Growth characteristics of the chimeric Japanese encephalitis virus vaccine candidate, ChimeriVax-JE (YF/JE SA14--14--2), in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes.

The Japanese encephalitis (JE) virus vaccine candidate, ChimeriVax-JE, which consists of a yellow fever (YF) 17D virus backbone containing the prM and E genes from the JE vaccine strain JE SA14--14--2, exhibits restricted replication in non-human primates, producing only a low-level viremia following peripheral inoculation. Although this reduces the likelihood that hematophagous insects could become infected by feeding on a vaccinated host, it is prudent to investigate the replication kinetics of the vaccine virus in mosquito species that are known to vector the viruses from which the chimera is derived. In this study ChimeriVax-JE virus was compared to its parent viruses, as well as to wild-type JE virus, for its ability to replicate in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes. Individual mosquitoes were exposed to the viruses by oral ingestion of a virus-laden blood meal or by intrathoracic (IT) virus inoculation. ChimeriVax-JE virus did not replicate following ingestion by any of the three mosquito species. Additionally, replication was not detected after IT inoculation of ChimeriVax-JE in the primary JE virus vector, Cx. tritaeniorhynchus. ChimeriVax-JE exhibited moderate growth following IT inoculation into Ae. aegypti and Ae. albopictus, reaching titers of 3.6-5.0 log(10) PFU/mosquito. There was no change in the virus genotype associated with replication in mosquitoes. Similar results were observed in mosquitoes of all three species that were IT inoculated or had orally ingested the YF 17D vaccine virus. In contrast, all mosquitoes either IT inoculated with or orally fed wild-type and vaccine JE viruses became infected, reaching maximum titers of 5.4-7.3 log(10) PFU/mosquito. These results indicate that ChimeriVax-JE virus is restricted in its ability to infect and replicate in these mosquito vectors. The low viremia caused by ChimeriVax-JE in primates and poor infectivity for mosquitoes are safeguards against secondary spread of the vaccine virus.

Expression of the Manduca sexta allatotropin gene in cells of the central and enteric nervous systems.

Manduca sexta allatotropin (Mas-AT) was isolated and first characterized as a peptide that stimulated juvenile hormone biosynthesis in adult lepidopteran corpora allata and was subsequently shown to have cardioacceleratory activity in the pharate adult. In this study, we identified the cells in the nervous system of the insect that contain mRNA encoding Mas-AT and immunoreactivity against a polyclonal antiserum to Mas-AT. In larvae, Mas-AT mRNA and immunoreactivity was most abundant in two cells in the frontal ganglion, which project their axons down the recurrent nerve toward the gut, and in cells in the terminal abdominal ganglion. Lower levels of Mas-AT mRNA were detected in the brain and subesophageal ganglion. In the pupal and pharate adult stages, we detected Mas-AT mRNA and immunoreactivity in cells of the abdominal ganglia and in additional cells in the terminal abdominal ganglion. These additional cells in the ventral nerve cord that express Mas-AT during the pupal and pharate adult stages include cells that differentiate during metamorphosis as well as cells that exist in larvae but do not begin to express Mas-AT until these later developmental stages. Some of the cells that exhibit Mas-AT immunoreactivity lack Mas-AT mRNA, suggesting that the antisera used in this and previous studies recognizes other peptides in addition to Mas-AT. This pattern of expression suggests that Mas-AT may mediate multiple physiological functions during the life cycle of the insect, including the larval stage in which no function has yet been described for the peptide.

Molecular characterization and expression analysis of Manduca sexta allatotropin.

Juvenile hormones (JH) are a class of regulatory sesquiterpenoids that control metamorphosis in immature insects and reproduction in adult insects. The regulation of JH synthesis by the corpora allata (CA), a pair of endocrine glands with nervous connections to the brain, is achieved by a complex interplay of stimulatory and inhibitory factors mediated in part by the brain. The neuropeptide, allatotropin (Mas AT), was recently isolated and sequenced from the brain of the tobacco hornworm Manduca sexta. Mas AT is a 13-residue amidated peptide that activates JH synthesis in adult, but not larval, lepidopteran CA. A 23-nucleotide degenerate oligonucleotide was designed based on the peptide sequence and was used to isolate the Mas AT genomic clone. The Mas AT gene is expressed as three mRNAs which differ from one another by alternative splicing. These mRNAs are predicted to encode three distinct prohormones, each containing Mas AT. A restriction fragment from the genomic clone was then used to isolate the cDNA clone. In situ hybridization and immunohistochemistry studies show that Mas AT is expressed in both the central and enteric nervous systems. Cells expressing Mas AT were identified in the brain, frontal ganglion and terminal ganglion.