p34cdc2 is physically associated with and phosphorylated by a cdc2-specific tyrosine kinase.

The mammalian homologue of the yeast cdc2 gene encodes a 34-kilodalton serine/threonine kinase that is a subunit of M phase-promoting factor. Recent studies have shown that p34cdc2 is also a major tyrosine-phosphorylated protein in HeLa cells and that its phosphotyrosine content is cell cycle regulated and related to its kinase activity. Here, we show that cdc2 is physically associated with and phosphorylated in vitro by a highly specific tyrosine kinase. Tyrosine phosphorylation of cdc2 in vitro occurs at tyrosine 15, the same site that is phosphorylated in vivo. The association between the two kinases takes place in the cytosolic compartment and involves cyclin B-associated cdc2. Evidence is presented that a substantial fraction of cytosolic cdc2 is hypophosphorylated, whereas nuclear cdc2 is hyperphosphorylated. Finally, we show that the tyrosine kinase associated with cdc2 may be a 67-kilodalton protein and is distinct from src, abl, fms, and other previously reported tyrosine kinases.     

Steroid modulation of the GABA/benzodiazepine receptor-linked chloride lonophore

Recent findings suggest that steroids with sedative-hypnotic properties interact specifically with the gamma-aminobutyric acidA/benzodiazepine receptor-chloride ionophore complex (GBRC). They show positive heterotropic cooperativity by allosterically enhancing the binding of GABA agonists and the clinically useful benzodiazepines (BZs) to their respective recognition sites. These steroids have stringent structural requirements for activity at the GBRC, with the essential requirements for high potency being a 3 alpha-hydroxyl group and a 5 alpha-reduced A-ring. Some of these steroids are naturally occurring metabolites of progesterone and deoxycorticosterone and have nanomolar potencies as potentiators of chloride channel conductance. These 3 alpha-hydroxylated, 5 alpha-reduced steroids do not act through any known sites on the GBRC. Thus, the exact site and mechanism of action remain to be determined. Together with the observation that physiological levels of these metabolites are sufficient to influence the function of the GBRC, the evidence clearly suggests a role for these steroids in the normal regulation of brain excitability by potentiating the postsynaptic effects of gamma-aminobutyric acid (GABA). Pharmacological studies of the GBRC-active steroids show that they possess anxiolytic and anticonvulsant activities. The potential therapeutic application of these steroids in the treatment of mood disorders and catamenial exacerbation of seizures associated with the menstrual cycle is discussed. Collectively, the evidence from the studies of these steroids imply that another mechanism by which the endocrine system influences brain function has been identified. Its characterization will provide important insight into how steroids modulate brain excitability under normal and pathophysiological states.     

CP-60,993, a new dianemycin-like ionophore produced byStreptomyces hygroscopicus ATCC 39305: fermentation,isolation and characterization

Summary CP-60,993, 19-epi-dianemycin, is a novel polycyclic ether antibiotic produced byStreptomyces hygroscopicus ATCC 39305. Fermentation recovery, purification and crystallization were achieved using standard procedures. CP-60,993 was characterized as a monocarboxylic acid. Elemental analysis suggested a molecular formula of C₄₇H₇₈O₁₄ for the free acid and C₄₇H₇₇O₁₄ Na for the sodium salt. Crystalline form CP-60,993 sodium salt shows the following properties: m.p. 193205°C, E _{1 cm} ^1% =157 at 232 nm, [] _D ^25°C +11.0 (c 1, methanol). The structure, determined by MS, PMR and CMR, differs from dianemycin only in the stereochemistry at position 19. This was confirmed by X-ray crystallography carried out on the rubidium salt of CP-60,993. It exhibited activity in vitro against Gram-positive and anaerobic bacteria, efficacy againstEimeria coccidia in vivo in poultry, and stimulation in vitro of rumen propionic acid production.     

CIGAR‐seq,a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators

Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m⁶A and m⁵C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high‐throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR‐seq). Using CIGAR‐seq, we discovered NSUN6 as a novel mRNA m⁵C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non‐overlapping subsets of mRNA m⁵C sites and together contributed to almost all the m⁵C modification in mRNA. Finally, using m¹A as an example, we demonstrated that CIGAR‐seq can be easily adapted for identifying regulators of other mRNA modification.     

Spastic Paraplegia Type 7 Is Associated with Multiple Mitochondrial DNA Deletions

Spastic paraplegia 7 is an autosomal recessive disorder caused by mutations in the gene encoding paraplegin, a protein located at the inner mitochondrial membrane and involved in the processing of other mitochondrial proteins. The mechanism whereby paraplegin mutations cause disease is unknown. We studied two female and two male adult patients from two Norwegian families with a combination of progressive external ophthalmoplegia and spastic paraplegia. Sequencing of SPG7 revealed a novel missense mutation, c.2102A>C, p.H 701P, which was homozygous in one family and compound heterozygous in trans with a known pathogenic mutation c.1454_1462del in the other. Muscle was examined from an additional, unrelated adult female patient with a similar phenotype caused by a homozygous c.1047insC mutation in SPG7. Immunohistochemical studies in skeletal muscle showed mosaic deficiency predominantly affecting respiratory complex I, but also complexes III and IV. Molecular studies in single, microdissected fibres showed multiple mitochondrial DNA deletions segregating at high levels (38–97%) in respiratory deficient fibres. Our findings demonstrate for the first time that paraplegin mutations cause accumulation of mitochondrial DNA damage and multiple respiratory chain deficiencies. While paraplegin is not known to be directly associated with the mitochondrial nucleoid, it is known to process other mitochondrial proteins and it is possible therefore that paraplegin mutations lead to mitochondrial DNA deletions by impairing proteins involved in the homeostasis of the mitochondrial genome. These studies increase our understanding of the molecular pathogenesis of SPG7 mutations and suggest that SPG7 testing should be included in the diagnostic workup of autosomal recessive, progressive external ophthalmoplegia, especially if spasticity is present.     

Topographical relationships among the monovalent cation binding sites of bovine plasma-activated protein C and des(1–41)-light-chain-activated bovine plasma protein C and a nitroxide spin label bound to their active site serine residues

The paramagnetic effect of a spin-labeled sulfonyl fluoride, 4-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-fluorosulfonylbenzamide (p-V), when bound to the active site serine residue of the proteases, bovine plasma-activated protein C (APC) and des(1–41)-light-chain-activated protein C (GDAPC), on the longitudinal relaxation rate (T₁) of Tl⁺ bound to these same proteins has been examined by ²⁰⁵Tl⁺-NMR spectroscopy. The substantial shortening by bound p-V of the T₁ for Tl⁺ has been employed to estimate the distances between Tl⁺ and the unpaired electron on each protein surface. Assuming that a single cation-binding site exists on each enzyme, electron-nuclear distances of 3.4–3.9 Å have been calculated for each protein. This suggests that the removal of 41 amino acid residues and, concomitantly, all γ-carboxyglutamic acid, from the amino-terminal of the light chain of APC, does not significantly affect the protein topography in the region of the molecule probed by this technique.