Conjugation of the ubiquitin-like protein NEDD8 to cullin-2 is linked to von Hippel-Lindau tumor suppressor function.

The von Hippel-Lindau tumor suppressor protein pVHL assembles with cullin-2 (hCUL-2) and elongin B/C forming a protein complex, CBCVHL, that resembles SKP1-CDC53-F-box protein ubiquitin ligases. Here, we show that hCUL-2 is modified by the conserved ubiquitin-like protein NEDD8 and that NEDD8-hCUL-2 conjugates are part of CBCVHL complexes in vivo. Remarkably, the formation of these conjugates is stimulated by the pVHL tumor suppressor. A tumorigenic pVHL variant, however, is essentially deficient in this activity. Thus, ligation of NEDD8 to hCUL-2 is linked to pVHL activity and may be important for pVHL tumor suppressor function.

A novel protein modification pathway related to the ubiquitin system.

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.

The ubiquitin-like proteins SMT3 and SUMO-1 are conjugated by the UBC9 E2 enzyme.

The ubiquitin-like protein SMT3 from Saccharomyces cerevisiae and SUMO-1, its mammalian homolog, can be covalently attached to other proteins posttranslationally. Conjugation of ubiquitin requires the activities of ubiquitin-activating (E1) and -conjugating (E2) enzymes and proceeds via thioester-linked enzyme-ubiquitin intermediates. Herein we show that UBC9, one of the 13 different E2 enzymes from yeast, is required for SMT3 conjugation in vivo. Moreover, recombinant yeast and mammalian UBC9 enzymes were found to form thioester complexes with SMT3 and SUMO-1, respectively. This suggests that UBC9 functions as an E2 in a SMT3/SUMO-1 conjugation pathway analogous to ubiquitin-conjugating enzymes. The role of yeast UBC9 in cell cycle progression may thus be mediated through its SMT3 conjugation activity.

Changes of presaccadic cortical activity when performing horizontal, visually guided saccades.

When a visually guided saccade task is running, the presaccadic potential obtained in the initial period of the task differs from those obtained later, while the subject's oculomotor performance remains unaffected. These time-related changes of cortical activity consist both of an overall decreasing electrical activity as well as a selective one over certain cortical areas. The generalised reduced activity already described in earlier studies is considered as an unspecified effect such as fatigue or decreased motivation. On the contrary, the pronounced selective changes of cortical activity obtained over cortical areas such as the centro-parietal and frontal cortices, should be related with more specific, that is, visuomotor function. We assume that at the beginning of the task of the performance of the saccade needs the activation of several cortical areas but later on the same oculomotor plan runs sufficiently under subcortical control.

Characterization of ubiquitin genes and -transcripts and demonstration of a ubiquitin-conjugating system in Entamoeba histolytica.

We have investigated the genomic organization of Entamoeba histolytica ubiquitin and looked for the occurrence of a ubiquitin-conjugating system in this organism. Southern blots indicated the presence of > or = 5 ubiquitin-coding regions. One of these, EhUBI1, was cloned and sequenced and found to correspond to a monoubiquitin gene; as shown by a polymerase chain reaction, E. histolytica lacked polyubiquitin genes altogether. Blots of poly(A)+ RNA from exponentially-growing trophozoite cultures exhibited five ubiquitin transcripts, the most prominent and smallest of which corresponded to EhUBI1 mRNA. Expression of the ubiquitin genes was not influenced by heat shock. Although the predicted amino acid sequence of the ubiquitin from E. histolytica differs significantly (in 7-9 amino acid residues) from that of yeast and animals, expression of the coding sequence of EhUBI1 suppressed the heat-sensitive phenotype of a polyubiquitin gene-deficient yeast mutant. In correlation, trophozoite extract catalyzed an ATP-dependent conjugation of radioiodinated bovine ubiquitin to trophozoite proteins. The latter data indicate that E. histolytica contains a functional ubiquitin-conjugating system.

Cortical potentials with antisaccades.

The term antisaccade refers to saccades that are performed towards the side opposite to that of target appearance. The performance of antisaccades is considered to be determined by intact frontal inhibitory areas as patients with frontal, and especially prefrontal, lesions show a striking impairment in suppressing an unwanted protarget saccade. We recorded cortical slow potentials from subjects performing saccades and antisaccades in a task, antitask and no move conditions in order to investigate possible topographic differences between these two types of eye movement. Our main findings concern both movement related as well as sensory related potentials. With regard to the saccadic potentials, performance of an antisaccade is preceded by a much more pronounced activity during the last 100 ms prior to the eye movement onset over central-anterior leads with a slight ipsilateral lateralization. As for the sensory potentials, the target related with antisaccade performance is followed by smaller, but nonstatistically significant, exogenous responses while at 300-350 ms after target appearance, the activity associated with the antisaccade's target is clearly larger over central midline leads. Although we could not precisely relate the electrical activity obtained with well circumscribed cortical function, the results support the view that the anterior and slightly ipsilateral cortical activation which precedes the performance of an antisaccade could reflect the frontal mechanisms of suppression of the unwanted saccade.

The increased reaction time of antisaccades. What makes the difference?

The aim of this study was the detection of the parameters involved in an already defined phenomenon, namely that the reaction time of antisaccades is greater than that of pro-target saccades. Thus, we performed four different experimental paradigms: (i) The target location was unpredictable (to the left or to the right) but the type of saccade (pro-or anti-target) was predictable. The corresponding mean values of the reaction times were 231 +/- 40 ms for anti- and 179 +/- 50 ms for pro-target saccades. (ii) Both the target position and the type of saccade were unpredictable (437 +/- 91 and 412 +/- 85 for anti-and pro-target saccades, respectively). (iii) The target location remained predictable while the type of saccade was unpredictable (397 +/- 104 and 385 +/- 90 ms) and (iv) Both the target position and the type of saccade were predictable (185 +/- 67 and 180 +/- 61). The statistical analysis (ANOVA and post hoc comparisons) revealed significant differences only in the first two experiments. Our results suggest that the antisaccades present increased latency, compared to that of pro-target saccades, only under certain experimental conditions and especially when the target location is unpredictable. We presume that the antisaccade's latency prolongation is due not to the frontal lobe inhibition but to the double interference of the parietal lobe, which has to re-reconstruct the target location in space.

External ophthalmoplegia with ragged-red fibres and acetylcholine receptor antibodies.

The cases of two elderly women with external ophthalmoplegia, generalized muscle weakness and serum anti-acetylcholine receptor antibodies, are presented. The electophysiological studies showed a myopathic pattern but no indications of myasthenia after repetitive stimulation. The edrophonium test was negative and there was no response to anticholinesterase medication. In addition, elevated serum lactic acid levels and ragged-red muscle fibres in the muscle biopsy, were observed in both patients. These findings are discussed in relation to the fact that anti-acetylcholine receptor antibodies are diagnostic of myasthenia gravis, whereas ragged-red fibres and elevated lactic acid are correlated with mitochondrial myopathies.

Cortical potentials preceding centrifugal and centripetal self-paced horizontal saccades.

Cortical potentials preceding self-paced centrifugal and centripetal saccades were recorded in 15 subjects from F3, Fz, F4, C3, Cz, C4, P3, Pz and P4 versus linked mastoid electrodes. A negative potential starting about 1.0 sec prior to saccade onset, reaching a peak amplitude of 6.8 microV on average, preceded centrifugal saccades. In contrast the negativity preceded centripetal saccades by only 500 msec, and its peak amplitude was smaller (4.6 microV). We conclude that these differences reflect the fact that less 'effort' is needed with centripetal as compared to centrifugal saccades.

Hashish smoke interfers with Sidman avoidance in mice.

Hashish smoke has been proved to be active in the Sidman avoidance. Its activity is similar to that of hallucinogens.

Cannabis interferes with nest-building behavior in mice.

Nest-building, a behavioral model shown to be disrupted by hallucinogens, has never been used to answer questions concerning the psychotomimetic effects of delta9-THC. Several fractions of cannabis and tobacco pyrolysis products were tested consecutively in the same procedure. The following drugs were injected i.p. under a saline-drug-saline schedule: d-amphetamine (6 mg/kg), pentobarbital (25 mg/kg), delta9-THC (10 mg/kg, 5 mg/kg, 2.5 mg/kg), the cannabis fractions designated Is (water soluble products), IIs (nonsoluble, nonvolatile products, IIIs (it comprises what is inhaled by a common hashish smoker), and analogous fractions of tobacco pyrolysis products designated IIIB (what is inhaled by a common tobacco smoker), IIB and IB. The effects of delta9-THC (10 mg/kg), IIs, and IIIs were quite similar as far as the disruption of the normal behavioral pattern is concerned. d-Amphetamine, delta9-THC (5 mg/kg), and IIB disrupted the normal behavioral pattern as well. The similarity of the effects of IIs and IIIs was unexpected in view of the different contents of cannabinoids in these fractions. Also unexpected was the similarity of the effects of delta9-THC (10 mg/kg) and IIIs (40 mg/kg containing 7% delta9-THC) as well as the activity of fraction IIIB.