K252a Modulates the Expression of Nerve Growth Factor-Dependent Capsaicin Sensitivity and Substance P Levels in Cultured Adult Rat Dorsal Root Ganglion Neurones

Abstract: K252a, an inhibitor of trk phosphorylation and nerve growth factor signal transduction in PC12 cells, blocked nerve growth factor-induced responses in cultured adult rat dorsal root ganglion sensory neurones. The nerve growth factor-dependent appearance of capsaicin sensitivity and accumulation of the neuropeptide substance P were inhibited when dorsal root ganglion neurones were grown in the presence of low concentrations (100 n M ) of K252a. At higher concentrations (3 µ M ), however, K252a stimulated the development of capsaicin sensitivity and the accumulation of substance P even in the absence of nerve growth factor. By using a wide dose range, therefore, we showed that K252a could either inhibit or mimic nerve growth factor's actions on sensory neurones. These results may explain the apparent paradox in the literature that some groups show a blocking effect of K252a on nerve growth factor-dependent survival of dorsal root ganglion sensory neurones, whereas others report that K252a can substitute for nerve growth factor or other trophic factors and promote neuronal survival.     

Immunodetection of cytoskeletal structures and the Eg5 motor protein on deep-etch replicas of Xenopus egg cortices isolated during the cortical rotation

Summary— We have developed a new method for immunogold detection on deep-etch replicas of isolated Xenopus egg cortices in order to examine the interactions of different cortical elements in three dimensions at high resolution. We have applied this technique to vegetal cortices isolated during the second half of the first cell cycle. The vegetal cortical region at this time is the site of cellular machinery responsible for the ‘cortical rotation’. The entire cortex translocates with respect to the inner cytoplasm, relocating dorsalising determinants to the future dorsal side of the egg. The aligned microtubules in the shear zone between cytoplasm and cortex, implicated in the cortical rotation, were found to be organised as interweaving loose bundles. Interleaved amongst these aligned microtubules were extensive sheets of ER lying in layers parallel to the egg surface. Cytokeratin filaments were found to associate closely with the microtubules over short stretches. Putative actin filaments were present in the shear zone and in the cortex. Eg5, an abundant kinesin-related microtubule motor protein, and candidate for a role in generating cortical rotation movement, showed an almost exclusive localisation to microtubules. Immunofluorescence studies of cortices treated with detergent to disrupt ER or cold to depolymerise microtubules confirmed that Eg5 associates primarily with microtubules. We propose revised models for the mechanism of cortical rotation based on these observations and conclude that Eg5 is unlikely to move ER relative to microtubules during the cortical rotation.     

Oxygen-dependent electron transport and protection from photoinhibition in leaves of tropical tree species

The roles of photorespiration and the Mehlerperoxidase pathway in sustaining electron transport and protection from photoinhibition were studied in outer canopy leaves of two species of tropical trees: the drought-deciduous Pseudobombax septenatum (Jacq.) Dug. and the evergreen Ficus insipida Willd. Ficus had a higher photosynthetic capacity than Pseudobombax and also a greater capacity for light-dependent electron transport under photorespiratory conditions (in the absence of CO₂). As a consequence, in the absence of CO₂, Ficus was able to maintain a largely oxidized electron-transport chain at higher photon flux densities than Pseudobombax. Under the same light conditions, photoinhibition (reduction in F_v/F_m) was always greater in Pseudobombax than Ficus, was increased when leaves were exposed to 2% O₂ in nitrogen compared to 21% O₂ in CO₂-free air, but was not increased by the absence of CO₂. Rates of electron transport due to the Mehler-peroxidase pathway (assessed in 2% O₂ in nitrogen) ranged between 16–40 mol · m^–2·s^–1 in both species. As the dry season approached and Pseudobombax neared leaf senescence there was a decline in the capacity for photorespiratory flux to maintain electron transport in Pseudobombax, but not in Ficus. Ratios of light-dependent electron transport to net CO₂ fixation for Pseudobombax, Ficus and two other species in the field, Luehea seemannii Tr. & Planch, and Didymopanax morototoni (Aubl.) Dec. & Planch., ranged from 6.2 (Ficus) to 16.7 (Pseudobombax). High in-situ rates of photorespiration combined with the decreased capacity of Pseudobombax for photorespiratory flux as the dry season approached indicates a decreased capacity to protect against photooxidative damage. This may contribute to the promotion of leaf senescence in Pseudobombax during the transition from wet to dry season.Abbreviations F_v/F_m ratio of variable to maximum chlorophyll a fluorescence - NPQ nonphotochemical fluorescence quenching - PFD photon flux density - Q_A primary electron acceptor of PSII This research was supported by a grant from the Mellon Foundation. We thank Monica Mejia and Juan Posada for assisting with the fluorescence measurements and Aurelio Virgo for assisting with the field CO₂-exchange measurements.     

A novel human type I hair keratin gene: evidence for two keratin hHa3 isoforms

We present the nucleotide and amino acid sequence for a novel human type I hair keratin, which could be identified through its high sequence homology and strict carboxyterminal length identity as a human ortholog of the murine hair keratin mHa3. Our hHa3 sequence differs, however, from that of a previously described hHa3 hair keratin (published only as an amino acid sequence; [13]) in 24 amino acid positions, 8 of which occur in the middle of the carboxyterminal domain. PCR of genomic DNA from 25 normal human subjects using a primer pair derived from sequence segments located in the 3-region of our hHa3 clone that encode conserved amino acid sequences in both keratins, resulted in the amplification of two distinct products of 0.38 kbp and 1.0 kbp. DNA sequence analysis of the cloned PCR products allowed identification of the 0.38 kb sequence as that originating from Yuet al. [13] and the 1.0 kb sequence as that being derived from our data. The difference in fragment length was due to unique intron 6 sequences, indicating that these two keratin species are encoded by genes of their own. Moreover, extensive Southern blot analyses with DNA from 25 unrelated individuals of different races using a 3-noncoding sequence from our keratin and the intron 6 sequence of the keratin of Yuet al. [13], as hybridization probes showed that both keratin genes are present as single copy sequences occurring ubiquitously and without gross alterations in the human genome. Collectively, these data demonstrate that the human type I hair keratin described in this paper represents an isoform of the previously described hHa3 keratin. We propose that these hHa3 isoforms be named in chronological order of discovery hHa3-I and hHa3-II.     

mtDNA and Y-chromosome polymorphisms in four Native American populations from southern Mexico.

mtDNA sequence variation was examined in 60 Native Americans (Mixtecs from the Alta, Mixtecs from the Baja, Valley Zapotecs, and Highland Mixe) from southern Mexico by PCR amplification and high-resolution restriction endonuclease analysis. Four groups of mtDNA haplotypes (haplogroups A, B, C, and D) characterize Amerind populations, but only three (haplogroups A, B, and C) were observed in these Mexican populations. The comparison of their mtDNA variation with that observed in other populations from Mexico and Central America permits a clear distinction among the different Middle American tribes and raises questions about some of their linguistic affiliations. The males of these population samples were also analyzed for Y-chromosome RFLPs with the probes 49a, 49f, and 12f2. This analysis suggests that certain Y-chromosome haplotypes were brought from Asia during the colonization of the Americas, and a differential gene flow was introduced into Native American populations from European males and females.     

Chemotaxonomic value of anthocyanins in Podalyria and Virgilia (tribe Podalyrieae: Fabaceae)

Anthocyanin pigments responsible for purple and pink flower colours in the tribe Podalyrieae have been identified. The considerable variation in flower colour is not reflected in the chemical variation and the flower pigments are surprisingly conservative. Virgilia flowers always have the acetic acid esters of cyanidin-3-glucoside and peonidin-3-glucoside as major compounds, together with trace amounts of the coumaroyl ester of cyanidin-3-glucoside. Podalyria flowers invariably have the rather more stable coumaroyl ester of cyanidin. The data support a close affinity between the two genera but also show that flower colour is only partially homologous.     

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congential and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA.Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company'sex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activatedex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody.We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 infusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations. It would be prudent that developers of cellular andex vivo gene therapies establish a similar cell processing and QA/QC infrastructure at an early developmental stage to optimize safety and reproducibility and facilitate regulatory review.     

Short-term photosynthesis measurements predict leaf carbon balance in tropical rain-forest canopy plants

Diel (24 h) courses of net CO₂ exchange of leaves were determined in eight species of tropical rainforest plants on Barro Colorado Island, Panama, during 1990 and 1991. The species included three canopy trees, one liana, two epiphytes and one hemiepiphyte. One of the species studied was growing in a rain-forest gap. Daily carbon gain varied considerably across species, leaf age, and season. The analysis of data for all plants from 64 complete day/night cycles revealed a linear relationship between the diurnal carbon gain and the maximum rate of net CO₂ uptake, A_max. Nocturnal net carbon loss was about 10% of diurnal carbon gain and was positively related to A_max. We conclude that short-term measurements of light-saturated photosynthesis, performed at periodic intervals throughout the season, allow the annual leaf carbon balance in these rain-forest plants to be predicted.