Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.     

Molecular cloning of two paralytogenic, temperature-sensitive mutants, ts1 and ts7, and the parental wild-type Moloney murine leukemia virus.

ts1 and ts7, the paralytogenic, temperature-sensitive mutants of Moloney murine leukemia virus (MoMuLV), together with their wild-type parent, MoMuLV-TB, were molecularly cloned. ts1-19, ts7-22, and wt-25, the infectious viruses obtained on transfection to NIH/3T3 cells of the lambda Charon 21A recombinants of ts1, ts7, and wt, were found to have retained the characteristics of their non-molecularly cloned parents. In contrast to the wt virus, ts1-19 and ts7-22 are temperature-sensitive, inefficient in the intracellular processing of Pr80env at the restrictive temperature, and able to induce paralysis in CFW/D mice. Like the non-molecularly cloned ts7, the ts7-22 virion was also shown to be heat labile. The heat lability of the ts7 virion distinguishes it from ts1. Endonuclease restriction mapping with 11 endonucleases demonstrated that the base composition of MoMuLV-TB differs from that of the standard MoMuLV, but no difference was detected between the molecularly cloned ts1 and ts7 genomes. However, ts1 and ts7 differ from MoMuLV in the loss or acquisition of four different restriction sites, whereas they differ from MoMuLV-TB in the loss or acquisition of three different restriction sites.     

Association between morningness-eveningness and behavioral/emotional problems among adolescents

Adolescent eveningness is associated with age, parental monitoring, daytime sleepiness, sleep problems, moodiness, and the use of coffee. This study investigated the association between adolescent morningness-eveningness and psychopathology, substance use, and suicidality in 1332 students ages 12 to 13. Each student-participant completed the Chinese version of the Child Morningness/Eveningness Scale (CMES), the Pubertal Development Scale, and a questionnaire about their sleep schedule, trouble sleeping, habitual substance use, and suicidality. Their mothers completed the Child Behavioral Checklist and Chinese Health Questionnaire. The morning (n = 412), intermediate (n = 740), and evening (n = 180) groups were operationally defined by the CMES t scores. The mixed model was used for data analysis. The evening group had shorter weekday sleep time, longer weekend sleep time, more daytime napping, and greater sleep compensation on weekends and was more likely than the other 2 groups to have behavioral/emotional problems, suicidality, and habitual substance use. Internalizing and externalizing problems partially explained the association between eveningness, substance use, and suicidality. The findings suggest that eveningness may be an indicator for adolescents with behavioral/emotional problems and risky behaviors and suggest an investigation for possible intervention.     

The positive impact of cytological specimens for EGFR mutation testing in non‐small cell lung cancer: a single South East Asian laboratory’s analysis of 670 cases

B. Pang, D. Matthias, C.W. Ong, A.N. Dhewar, S. Gupta, G.L. Lim, M.‐E. Nga, J.E. Seet, A. Qasim, T.‐M. Chin, R. Soo, R. Soong and M. Salto‐Tellez The positive impact of cytological specimens for EGFR mutation testing in non‐small cell lung cancer: a single South East Asian laboratory’s analysis of 670 cases Objectives: To compare the rejection rates of non‐small cell lung cancer (NSCLC) samples obtained by differing sampling methods for testing by Sanger sequencing for epidermal growth factor receptor (EGFR) mutations. To assess the association between unsatisfactory outcomes and the quantity of DNA extracted from cytological versus histological samples. Methods: Six hundred and seventy NSCLC samples referred to our centre from 2008 to 2010 were reviewed as a consequence of sample rejection, presence of EGFR mutations, cytological versus histological sampling methods, DNA quantity and the unsatisfactory genotyping rate. Results: Eighty samples were rejected for testing in similar proportions of histological and cytological samples (11.9% versus 10.9%) usually (n = 75) because the amount of cellular material was judged insufficient in small biopsies or cytology samples. The remaining 590 samples on which EGFR testing was attempted yielded 51 (8.6%) unsatisfactory test outcomes caused by failure of the polymerase chain reaction (PCR) (n = 47 cases), uninterpretable Sanger chromatograms (n = 3 cases) and insufficient DNA extracted for PCR (n = 1 case). The difference in rates of unsatisfactory outcomes between cytological samples (seven of 147 samples or 4.7%) versus tissue samples (44 of 443 samples or 9.9%) was clinically relevant but not statistically significant (Mann–Whitney test; P < 0.081). There was no association between the concentration of DNA extracted and the likelihood of an unsatisfactory analysis; which was similar in all types of sections (large and small) while 0% of 37 cytology slides were unsatisfactory. Conclusions: Utilizing cytology samples for EGFR testing avoids unnecessary patient re‐biopsing and yields a clinically superior satisfactory rate to the overall satisfactory rate of tissue biopsies of NSCLC. The quality rather than quantity of DNA extracted may be a more important determinant of a satisfactory result.     

Exome Sequencing Identifies GNB4 Mutations as a Cause of Dominant Intermediate Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of inherited neuropathies. Mutations in approximately 45 genes have been identified as being associated with CMT. Nevertheless, the genetic etiologies of at least 30% of CMTs have yet to be elucidated. Using a genome-wide linkage study, we previously mapped a dominant intermediate CMT to chromosomal region 3q28–q29. Subsequent exome sequencing of two affected first cousins revealed heterozygous mutation c.158G>A (p.Gly53Asp) in GNB4, encoding guanine-nucleotide-binding protein subunit beta-4 (Gβ₄), to cosegregate with the CMT phenotype in the family. Further analysis of GNB4 in an additional 88 unrelated CMT individuals uncovered another de novo mutation, c.265A>G (p.Lys89Glu), in this gene in one individual. Immunohistochemistry studies revealed that Gβ₄ was abundant in the axons and Schwann cells of peripheral nerves and that expression of Gβ₄ was significantly reduced in the sural nerve of the two individuals carrying the c.158G>A (p.Gly53Asp) mutation. In vitro studies demonstrated that both the p.Gly53Asp and p.Lys89Glu altered proteins impaired bradykinin-induced G-protein-coupled-receptor (GPCR) signaling, which was facilitated by the wild-type Gβ₄. This study identifies GNB4 mutations as a cause of CMT and highlights the importance of Gβ₄-related GPCR signaling in peripheral-nerve function in humans.     

Lateral diffusion of PEG-Lipid in magnetically aligned bicelles measured using stimulated echo pulsed field gradient 1H NMR

Lateral diffusion measurements of PEG-lipid incorporated into magnetically aligned bicelles are demonstrated using stimulated echo (STE) pulsed field gradient (PFG) proton (1H) nuclear magnetic resonance (NMR) spectroscopy. Bicelles were composed of dimyristoyl phosphatidylcholine (DMPC) plus dihexanoyl phosphatidylcholine (DHPC) (q = DMPC/DHPC molar ratio = 4.5) plus 1 mol % (relative to DMPC) dimyristoyl phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000) at 25 wt % lipid. 1H NMR STE spectra of perpendicular aligned bicelles contained only resonances assigned to residual HDO and to overlapping contributions from a DMPE-PEG 2000 ethoxy headgroup plus DHPC choline methyl protons. Decay of the latter's STE intensity in the STE PFG 1H NMR experiment (g(z) = 244 G cm(-1)) yielded a DMPE-PEG 2000 (1 mol %, 35 degrees C) lateral diffusion coefficient D = 1.35 x 10(-11) m2 s(-1). Hence, below the "mushroom-to-brush" transition, DMPE-PEG 2000 lateral diffusion is dictated by its DMPE hydrophobic anchor. D was independent of the diffusion time, indicating unrestricted lateral diffusion over root mean-square diffusion distances of microns, supporting the "perforated lamellae" model of bicelle structure under these conditions. Overall, the results demonstrate the feasibility of lateral diffusion measurements in magnetically aligned bicelles using the STE PFG NMR technique.     

Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N(2)A cells (EC(50) in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca(2+) in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress.     

Molecular analysis of survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes of spinal muscular atrophy patients and their parents

We have assayed deletions of two candidate genes for spinal muscular atrophy (SMA), the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes, in 101 patients from 86 Chinese SMA families. Deletions of exons 7 and 8 of the telomeric SMN gene were detected in 100%, 78.6%, 96.6%, and 16.7%, in type I, II, III, and adult-onset SMA patients, respectively. Deletion of exon 7 only was found in eight type II and one type III patient. One type II patient did not have a deletion of either exon 7 or 8. The prevalence of deletions of exons 5 and 6 of the NAIP gene were 22.5% and 2.4% in type I and II SMA patients, respectively. We also examined four polymorphisms of SMN genes and found that there were only two, SMN-2 and ^CBCD541-2, in Chinese subjects. In our study, analysis of the ratio of the telomeric to centromeric portion (T/C ratio) of the SMN gene after enzyme digestion was performed to differentiate carriers, normals, and SMA patients. We found the T/C ratio of exon 7 of the SMN gene differed significantly among the three groups, and may be used for carrier analysis. An asymptomatic individual with homozygous deletion of exons 7 and 8 of the SMN gene showed no difference in microsatellite markers in the SMA-related 5q11.2–5q13.3. In conclusion, SMN deletion in clinically presumed child-onset SMA should be considered as confirmation of the diagnosis. However, adult-onset SMA, a heterogeneous disease with phenotypical similarities to child-onset SMA, may be caused by SMN or other gene(s). Received: 13 November 1996 / Accepted: 13 May 1997