An enclosed-chamber labeling system for the safe <Superscript>14</Superscript>C-enrichment of phytochemicals in plant cell suspension cultures

Summary Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans, but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body. While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is problematic because many compounds of interest can be synthesized only in planta at present. In order to generale ¹⁴C-labeled phytochemicals of high radioactive enrichment, we developed an enclosed-chamber labeling system in which cell suspension cultures can be safely and efficiently grown when supplied with ¹⁴C-enriched precursors. The system is designed to hold culture flasks within a clear, polyacrylic compartment that is affixed to the top of a rotary shaker. The flow-through gas exchange nature of the system allows for O₂ replenishment and complete capture of respired ¹⁴CO₂ throughout the entire period of cell culture. Air is circulated internally with the aid of a small fan, and chamber air temperature is monitored continuously with an internal temperature probe and data logger. Production runs of 12–14 d with Vaccinium pahalae (ohelo berry) and Vitis vinifera (grape) suspension cultures, using [¹⁴C]sucrose as the carbon source, demonstrated a 20–23% efficiency of ¹⁴C incorporation into the flavonoid-rich fractions. Further studies with ohelo cell cultures showed that flavonoids were produced with either sucrose or glucose as the carbohydrate source, although flavonoid productivity (measured as anthocyanins) was higher with sucrose. This comprehensive chamber system should have broad applicability with numerous cell types and can be used to generate a wide array of labeled phytochemicals.     

Photoperiod modulates the effects of norepinephrine on lymphocyte proliferation in Siberian hamsters

Siberian hamsters (Phodopus sungorus) rely on photoperiod to coordinate seasonally appropriate changes in physiology, including immune function. Immunity is regulated, in part, by the sympathetic nervous system (SNS), although the precise role of the SNS in regulating photoperiodic changes in immunity remains unspecified. The goal of the present study was to examine the contributions of norepinephrine (NE), the predominant neurotransmitter of the SNS, to photoperiodic changes in lymphocyte proliferation. In experiment 1, animals were maintained in long [16:8-h light-dark cycle (16:8 LD)] or short days (8:16 LD) for 10 wk, and splenic NE content was determined. In experiment 2, in vitro splenocyte proliferation in response to mitogenic stimulation (concanavalin A) was assessed in spleen cell suspensions taken from long- or short-day hamsters in which varying concentrations of NE were added to the cultures. In experiment 3, splenocyte proliferation was examined in the presence of NE and selective alpha- and beta-noradrenergic receptor antagonists (phenoxybenzamine and propranolol, respectively) in vitro. Short-day animals had increased splenic NE content compared with long-day animals. Long-day animals had higher proliferation compared with short-day animals independent of NE. NE (1 microM) further suppressed splenocyte proliferation in short but not long days. Last, NE-induced suppression of proliferation in short-day hamsters was blocked by propranolol but not phenoxybenzamine. The present results suggest that NE plays a role in photoperiodic changes in lymphocyte proliferation. Additionally, the data suggest that the effects of NE on proliferation are specific to activation of beta-adrenergic receptors located on splenic tissue. Collectively, these results provide further support that photoperiodic changes in immunity are influenced by changes in SNS activity.     

Intraventricular melanin-concentrating hormone stimulates water intake independent of food intake

The lateral hypothalamus (LH) has a critical role in the control of feeding and drinking. Melanin-concentrating hormone (MCH) is an orexigenic peptidergic neurotransmitter produced primarily in the LH, and agouti-related protein (AgRP) is an orexigenic peptidergic neurotransmitter produced exclusively in the arcuate (ARC), an area that innervates the LH. We assessed drinking and eating after third ventricular (i3vt) administration of MCH and AgRP. MCH (2.5, 5, and 10 micro g i3vt) significantly increased food as well as water intake over 4 h when administered during either the light or the dark portion of the day-night cycle. When MCH (5 micro g) was administered to rats with access to water but no food, they drank significantly more water than when given the vehicle. AgRP (7 micro g i3vt), on the other hand, increased water intake but only in proportion to food intake during the dark and the light, and water intake was not increased after i3vt AgRP in the absence of food. Hence, in contrast to AgRP, MCH elicits increased water intake independent of food intake. These results are consistent with historical data linking activity of the LH with water as well as food intake.     

Extracellular ATP signaling in the rabbit lung: erythrocytes as determinants of vascular resistance

Previously, it was reported that red blood cells (RBCs) are required to demonstrate participation of nitric oxide (NO) in the regulation of rabbit pulmonary vascular resistance (PVR). RBCs do not synthesize NO; hence, we postulated that ATP, present in millimolar amounts in RBCs, was the mediator, which evoked NO synthesis in the vascular endothelium. First, we found that deformation of RBCs, as occurs on passage across the pulmonary circulation with increasing flow rate, evoked increments in ATP release. Here, ATP (300 nM), administered to isolated, salt solution-perfused (PSS) rabbit lungs, decreased total and upstream (arterial) PVR, a response inhibited by NG-nitro-L-arginine methyl ester (L-NAME, 100 microM). In lungs perfused with PSS containing RBCs, L-NAME increased total and upstream PVR. In lungs perfused with PSS containing glibenclamide-treated RBCs, which inhibits ATP release, L-NAME was without effect. Apyrase grade VII (8 U/ml), which degrades ATP to AMP, was without effect on PVR in PSS-perfused lungs. These results are consistent with the hypothesis that ATP, released from RBCs as they traverse the pulmonary circulation, evokes endogenous NO synthesis.     

Genetic consequences of white-tailed deer (Odocoileus virginianus) restoration in Mississippi

White-tailed deer (Odocoileus virginianus) were nearly extirpated from the southeastern USA during the late 19th and early 20th centuries. Recovery programmes, including protection of remnant native stocks and transplants from other parts of the species' range, were initiated in the early 1900's. The recovery programmes were highly successful and deer are presently numerous and continuously distributed throughout the southeastern USA. However, the impact of the recovery programmes on the present genetic structure of white-tailed deer remains to be thoroughly investigated. We used 17 microsatellite DNA loci to assess genetic differentiation and diversity for 543 white-tailed deer representing 16 populations in Mississippi and three extra-state reference populations. There was significant genetic differentiation among all populations and the majority of genetic variation (> or = 93%) was contained within populations. Patterns of genetic structure, genetic similarity and isolation by distance within Mississippi were not concordant with geographical proximity of populations or subspecies delineations. We detected evidence of past genetic bottlenecks in nine of the 19 populations examined. However, despite experiencing genetic bottlenecks or founder events, allelic diversity and heterozygosity were uniformly high in all populations. These exceeded reported values for other cervid species that experienced similar population declines within the past century. The recovery programme was successful in that deer were restored to their former range while maintaining high and uniform genetic variability. Our results seem to confirm the importance of rapid population expansion and habitat continuity in retaining genetic variation in restored populations. However, the use of diverse transplant stocks and the varied demographic histories of populations resulted in fine-scale genetic structuring.     

Reductions in total body fat decrease humoral immunity

Mounting an immune response requires substantial energy, and it is well known that marked reductions in energy availability (e.g. starvation) can suppress immune function, thus increasing disease susceptibility and compromising survival. We tested the hypothesis that moderate reductions in energy availability impair humoral immunity. Specifically, we examined the effects of partial lipectomy (LIPx) on humoral immunity in two seasonally breeding rodent species, prairie voles (Microtus ochrogaster) and Siberian hamsters (Phodopus sungorus). Animals received bilateral surgical removal of epididymal white adipose tissue (EWATx), inguinal white adipose tissue (IWATx) or sham surgeries and were injected with the antigen keyhole limpet haemocyanin (KLH) either four or 12 weeks after surgery. In prairie voles, serum anti-KLH immunoglobulin G (IgG) did not differ significantly at four weeks. At 12 weeks, serum IgG was significantly reduced in IWATx, but not EWATx animals, compared with sham-operated animals. In Siberian hamsters, both IWATx and EWATx animals reduced serum IgG at four weeks. At 12 weeks, EWATx hamsters displayed a significant compensatory increase in IWAT pad mass compared with sham-operated hamsters, and serum IgG no longer differed from sham-operated animals. There was no significant increase in EWAT in IWATx hamsters compared with sham animals and IgG remained significantly reduced in IWATx hamsters. These results suggest that reductions in energy availability can impair humoral immunity.     

Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

Efficacy of transgenic cotton containing a cry1Ac gene from Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) in northern China

NuCOTN 33B, a Bt transgenic variety of upland cotton (Gossypium hirsutum L.) expressing the insecticidal protein Cry1Ac from Bacillus thuringiensis Berliner sp. kurstaki, was evaluated for resistance to Helicoverpa armigera (Hübner) during 1998-2000 in northern China. The results indicated that there was no significant difference in egg densities between NuCOTN 33B and three nontransgenic varieties (DP5415, Zhongmian12, and Shiyuan321) during the season, although the survival of larvae on NuCOTN 33B seemed significantly reduced. High larval densities observed on non-Bt cotton appeared in great contrast to the low larval populations observed on NuCOTN 33B plants during the seasons. In an environment without insecticide sprays, the annual ginned cotton yields in NuCOTN 33B plots, ranging from 1391.17 to 1511.35 kg/ha, were significantly higher than those in non-Bt cotton (340.34-359.58 kg/ha). These high levels of field efficacy for NuCOTN 33B against H. armigera in northern China may pave the way for reduced pesticide applications and an expansion of alternative pest-control strategies.     

Partial reconstitution of human interstrand cross-link repair in vitro: characterization of the roles of RPA and PCNA

The repair of DNA interstrand cross-links (ICLs) remains largely ill-defined in higher eukaryotic cells. Previously, we have developed assays that can be used to monitor the early stages of processing of ICLs in vitro. Here, we have used P11 phosphocellulose chromatography to fractionate HeLa nuclear extracts and have subsequently reconstituted these assays with the resulting fractions. RPA and PCNA were found in a single fraction, and were the only factors in this fraction required for the reconstitution of these assays. The roles of RPA and PCNA in the formation of incisions at ICLs and in the subsequent DNA synthesis step were assessed. RPA was found to be essential for both stages of ICL processing indicating that it is required for lesion recognition and/or for the subsequent endonucleolytic processing. PCNA is required for the DNA synthesis stage and although it is not critical for the incision stage of the reaction it does enhance this step presumably by a stimulation of lesion recognition by MutSbeta. These findings define novel roles for RPA and PCNA in the processing of ICLs in mammalian cells.