Identification and characterization of a pregnane steroid recognition site that is functionally coupled to an expressed GABAA receptor

Conclusion Based on the pharmacological and biochemical evidence to date, especially that derived from the recombinantly expressed receptor studies, the suggestion that a novel GBRC-linked steroid recognition site exists becomes a cogent argument. The high affinity of the steroid site for certain naturally occurring metabolites of progesterone and glucocorticoids favors a physiologic role for these steroids in the regulation of brain excitability. Clearly, investigations of such a regulatory role is warranted. If present, it provides an important example of endocrine control of a major inhibitory neurotransmitter in the CNS. Moreover, as we gain a greater understanding of the molecular organization of the GBRC, the putative steroid site provides a novel target for the rational design of therapeutic agents for the treatment of anxiety, epilepsy, and insomnia.Special issue dedicated to Dr. Eugene Roberts.     

Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [³H]diazepam binding was lost in photolabeled membranes, specific [³H]propyl β-carboline-3-carboxylate ([³H]PCC) binding was spared. In this study, the binding of the BZD antagonists [³H]PCC, [³H]Ro15 1788 and [³H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent K_D and small reductions in the B_max of ³H antagonist binding were observed. However, in the same membranes, up to 89% of specific [³H]FLU binding was lost. When [³H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [³H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[³H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.     

Diversity and conservation of blackwater fishes in Peninsular Malaysia,particularly in the North Selangor peat swamp forest

One of the most extreme freshwater habitats in Peninsular Malaysia is the peat swamp forest, with dark-coloured and highly acidic waters. Surprisingly, little is known about blackwater fishes in Peninsular Malaysia. Until 1968, only 26 fish species were known from blackwaters throughout Peninsular Malaysia, of which only one can be regarded as stenotopic. A recent intensive survey of part of the North Selangor peat swamp forest yielded 47 species, of which 14 are probably stenotopic taxa. These include four undescribed species and several new records for western Peninsular Malaysia. These discoveries are significant in that they include the family Chaudhuriidae which until 1985, was not reported from Sundaic Southeast Asia, and the rare genus Encheloclarias which had not been encountered for over 50 years. The rapid rate of destruction of the peat swamp forest owing to development, forestry and agricultural activities must be halted or slowed significantly to enable the proper zoological surveys and studies to be conducted. Conservation plans and environmental impact assessments based on inadequate sampling and knowledge of species present is acutely dangerous. There are no longer substantial undisturbed blackwater peat swamp forests left in most of Peninsular Malaysia. Conservation of the remaining blackwater biotopes is critically important if extinction of many species, here regarded as economically valuable renewable resources, is to be prevented.     

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3–8.3-fold and 19–22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.     

The purified myxoma virus gamma interferon receptor homolog M-T7 interacts with the heparin-binding domains of chemokines.

The myxoma virus T7 protein M-T7 is a functional soluble gamma interferon receptor homolog that has previously been shown to bind gamma interferon and inhibit its antiviral activities in a species-specific manner, but gene knockout analysis has suggested a further role for M-T7 in blocking leukocyte influx into infected lesions. We purified M-T7 to apparent homogeneity and showed that M-T7 is an N-linked glycoprotein that appears to be a stable homotrimer with a molecular mass of approximately 113 kDa in solution. M-T7, in addition to forming inhibitory complexes with rabbit gamma interferon, was also shown to bind to human interleukin-8, a prototypic member of the chemokine superfamily. Moreover, M-T7 was able to interact promiscuously with all members of the CXC, CC, and C chemokine subfamilies tested. Binding of human RANTES to M-T7 can be competed by rabbit gamma interferon and also by cold RANTES competitor with a 50% inhibitory concentration of 900 nM. Although M-T7 retains binding to a number of interleukin-8 N-terminal (ELR) deletion mutants, binding to mutants containing deletions in the C-terminal heparin-binding domain of interleukin-8 is abrogated. Furthermore, heparin effectively competes the interaction of M-T7 with the chemokine RANTES but not with rabbit gamma interferon. We propose that this novel M-T7 interaction with members of the chemokine superfamily may be facilitated through the conserved heparin-binding domains found in a wide spectrum of chemokines and that M-T7 may function by modulating chemokine-glycosaminoglycan interactions in virus-infected tissues.     

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.