Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+-activated K+ channel deficiency.

Malfunctions of potassium channels are increasingly implicated as causes of neurological disorders. However, the functional roles of the large-conductance voltage- and Ca(2+)-activated K(+) channel (BK channel), a unique calcium, and voltage-activated potassium channel type have remained elusive. Here we report that mice lacking BK channels (BK(-/-)) show cerebellar dysfunction in the form of abnormal conditioned eye-blink reflex, abnormal locomotion and pronounced deficiency in motor coordination, which are likely consequences of cerebellar learning deficiency. At the cellular level, the BK(-/-) mice showed a dramatic reduction in spontaneous activity of the BK(-/-) cerebellar Purkinje neurons, which generate the sole output of the cerebellar cortex and, in addition, enhanced short-term depression at the only output synapses of the cerebellar cortex, in the deep cerebellar nuclei. The impairing cellular effects caused by the lack of postsynaptic BK channels were found to be due to depolarization-induced inactivation of the action potential mechanism. These results identify previously unknown roles of potassium channels in mammalian cerebellar function and motor control. In addition, they provide a previously undescribed animal model of cerebellar ataxia.

A molecular switch for specific stimulation of the BKCa channel by cGMP and cAMP kinase.

The cGMP and the cAMP pathways control smooth muscle tone by regulation of BK(Ca) (BK) channel activity. BK channels show considerable diversity and plasticity in their regulation by cyclic nucleotide-dependent protein kinases. The underlying molecular mechanisms are unclear but may involve expression of splice variants of the BK channel alpha subunit. Three isoforms, BK(A), BK(B), and BK(C), which were cloned from tracheal smooth muscle, differed only in their C terminus. When expressed in HEK293 cells, cGMP kinase (cGK) but not cAMP kinase (cAK) stimulated the activity of BK(A) and BK(B) by shifting the voltage dependence of the channel to more negative potentials. In contrast, BK(C) was exclusively stimulated by cAK. BK(C) lacks a C-terminal tandem phosphorylation motif for protein kinase C (PKC) with Ser(1151) and Ser(1154). Mutation of this motif in BK(A) switched channel regulation from cGK to cAK. Furthermore, inhibition of PKC in excised patches from cells expressing BK(A) abolished the stimulatory effect of cGK but allowed channel stimulation by cAK. cAK and cGK phosphorylated the channel at different sites. Thus, phosphorylation/dephosphorylation by PKC determines whether the BK channel is stimulated by cGK or cAK. The molecular mechanisms may be relevant for smooth muscle relaxation by cAMP and cGMP.

Identification and characterization of a tri-partite hydrophobin from Claviceps fusiformis. A novel type of class II hydrophobin.

A new type of hydrophobin is encoded by an abundant mRNA of Claviceps fusiformis. The predicted amino-acid sequence of the protein, dubbed CFTH1, shows a putative signal sequence for secretion, followed by three class II hydrophobin domains each preceded by glycine/asparagine rich regions. SDS/PAGE analysis of 60% ethanol extractions of C. fusiformis mycelia from shaken cultures showed CFTH1 at the 50-55-kDa position. N-terminal sequencing of both untreated mature CFTH1 and of a fragment obtained by trypsin digestion revealed that CFTH1 is not processed between the hydrophobin domains. Mass spectroscopy showed a mass of about 36 500 Da, which is about 1500 Da higher than the mass predicted from the constituent amino acids, indicating post-translational modification but not glycosylation. Purified CFTH1 self-assembled at hydrophilic/hydrophobic interfaces and, after assembly at a water/air interface, it was found to be highly surface active. Antibodies raised against CFTH1 localized the protein in a mucilageous coat surrounding submerged vegetative hyphae in liquid shaken culture and, as a discrete layer of about 10 nm thickness at the surface of aerial hyphae of standing cultures, suggesting a role in the formation of aerial hyphae.

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea.

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3'-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxidoreductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway.

Identification of genes induced in alkaloid-producing cultures of Claviceps sp.

In order to identify genes which are expressed during alkaloid synthesis in an axenic culture of Claviceps sp. (strain ATCC 26245), a cDNA library from a producing culture was differentially screened with cDNA from producing (cDNA+) and non-producing (cDNA-) cultures, respectively. Altogether, ten cDNA clones were obtained, the alkaloid-synthesis-correlated expression of which was confirmed by Northern analyses. Evaluation of their nucleotide and derived amino-acid sequences identified one gene unequivocally, coding for dimethylallyltryptophan-synthase (DMAT-S), the initial enzyme of the specific alkaloid pathway. For two other genes significant homologies to known fungal genes were detected: one clone showed homology to the Neurospora crassa ccg1 gene, coding for a clock-regulated putative general stress protein; seven cDNA clones, derived from the same gene, which is highly expressed under these conditions, contained typical hydrophobin domains and long stretches of asparagine/glycine repeats (like QID3 from Trichoderma harzianum), thus probably representing a cell-wall constituent. These data show that this is not only a successful approach to clone genes specific for the alkaloid-pathway of C. purpurea, but also of genes which might be involved in the differentiation of sclerotial hyphae, the prerequisite for alkaloid synthesis.

The isoprenoid pathway: cloning and characterization of fungal FPPS genes.

Farnesylpyrophosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis. Several classes of essential metabolites, including sterols, quinones, carotenoids and gibberellins, are terpenoids with high biological activity. The structural gene for FPP synthase was isolated from two ascomycete fungi, Neurospora crassa and Gibberella fujikuroi. A comparative analysis of the nucleotide sequences of both FPPS genes revealed the presence of introns at the same positions at the 5' end of the coding regions. Furthermore, the most conserved region of the gene was isolated from two other plant pathogenic fungi, Sphaceloma manihoticola and Claviceps purpurea, by PCR. Sequence analysis showed a high degree of similarity between the deduced proteins of all known FPP synthase genes. In contrast to animals, all analyzed fungi contain a single copy of the gene, although FPP is the precursor for essential sterol and quinone biosynthesis and secondary metabolites, such as gibberellins, as well. Transcription analysis in different light regimes has shown that the FPPS genes in G. fujikuroi and N. crassa are not regulated by light induction.

The Claviceps purpurea glyceraldehyde-3-phosphate dehydrogenase gene: cloning, characterization, and use for the improvement of a dominant selection system.

The GPD 1 gene of Claviceps purpurea coding for glyceraldehyde-3-phosphate dehydrogenase was cloned and sequenced, including 1,800 bp of its 5' upstream region. This gene shows an identical structure to the gpd gene of Podospora anserina and Cryphonectria parasitica (one intron at an identical position) with high homology at both the DNA and amino-acid levels. Two fragments of the promoter spanning from the ATG to -500 bp and to -1,400 bp were fused to the phleomycin-resistance gene. Both constructs transformed C. purpurea at a high rate. The enhanced expression of the long vector construct indicates the presence of additional elements between -500 bp and -1,400 bp upstream of the initiation codon.

Prosthetic replacement of the shoulder for the treatment of defects in the rotator cuff and the surface of the glenohumeral joint.

We operatively treated, between 1978 and 1987, twenty-one shoulders in nineteen patients, fifty-four to eighty-four years old, who had disabling pain attributable to a massive tear of the rotator cuff, accompanied by loss of the surface of the glenohumeral joint. These patients were not candidates for total shoulder replacement because of the massive deficiency in the cuff and the fixed upward displacement of the humeral head. A prerequisite for hemiarthroplasty was a functionally intact coracoacromial arch to provide superior secondary stability for the prosthesis. One important aspect of the operative technique was the selection of a sufficiently small prosthesis so that excessive tightness of the posterior aspect of the capsule could be avoided. Eighteen shoulders in sixteen patients were available for follow-up, which ranged from twenty-five to 122 months. Pain decreased from marked or disabling in fourteen shoulders preoperatively to none or slight in ten and to pain only after unusual activity in four. Active forward elevation improved from an average of 66 degrees preoperatively to an average of 109 degrees postoperatively. One patient, who had had an excellent result, fell and sustained an acromial fracture, so the functional result changed to poor. Three patients had persistent, substantial pain in the shoulder that led to a revision. Neither infection nor prosthetic loosening developed in any shoulder.

Surgical management of complex irreparable rotator cuff deficiency.

The authors surgically treated 23 shoulders in 23 patients with disabling pain associated with irreparable tears of the musculotendinous cuff. In a total of 12 shoulders with preserved passive motion, normal deltoid function, loss of glenohumeral joint surfaces, and sculpturing of the coracoacromial arch, a standard or oversized Neer II humeral prosthesis without glenoid replacement was selected. A total of 11 shoulders that failed to meet these prerequisites or demanded heavy use after operation underwent arthrodesis. Twenty-two patients (12 from the hemiarthroplasty group and 10 from the arthrodesis group) were available for evaluation at an average follow-up period of 37.5 months. Comfort level and overall function were improved in both groups. Active forward elevation improved an average of 44 degrees in the hemiarthroplasty group and an average of 15 degrees in the arthrodesis group. The success of hemiarthroplasty and the problems of glenoid loosening in the presence of cuff deficiency with upward head displacement have led to the conclusion that humeral hemiarthroplasty is the preferred method for managing complex irreparable tears of the rotator cuff in which the articular surface is destroyed, yet the deltoid muscle is functional. Shoulder arthrodesis is reserved for those patients who have both irreparable tears of the rotator cuff and irreparable deficiencies of the deltoid muscle, or the younger patient with demands for substantial strength at low angles of flexion.

Regeneration of the rat sciatic nerve after different conditioning lesions: effects of the conditioning interval.

Regeneration of the rat sciatic nerve was studied after a crush lesion (test lesion) on nerves previously subjected to a conditioning lesion. The test lesion was made approximately 30 mm proximal to the conditioning lesion. The period between the conditioning lesion and the test lesion was varied. Regeneration was measured by the pinch test. The conditioning procedure increased the rate of axonal elongation and decreased the initial delay. Conditioning intervals between 14 hours and 4 days were sufficient to increase the regeneration distance significantly, but only until day 4. If the conditioning interval was prolonged to 7 or 14 days, the conditioning effect persisted until day 6. A conditioning effect also was produced by transection of the sciatic nerve and by local compression produced with a silicone tube. The results of this study demonstrate that the type of injury and the conditioning intervals are important determinants in producing the conditioning effect in the rat.

Fractures and fracture-dislocations of the tarsometatarsal joint.

We are reporting the results in a consecutive series of forty adults in whom, between 1978 and 1984, forty-one tarsometatarsal fracture-dislocations were treated with open reduction followed by temporary internal fixation with AO screws. Ninety per cent of the patients had an intra-articular or a periarticular fracture. An anatomical or nearly anatomical reduction was achieved in all but a few patients, and there was no loss of fixation or displacement. For thirty-four patients (thirty-five injuries), the length of follow-up averaged 3.4 years, and a good or excellent functional result was obtained in all but two of the thirty in whom an anatomical reduction had been achieved. Of the six patients who had a fair or a poor result, five had an associated grade-II or grade-III open injury. The development of post-traumatic arthritis was directly related to damage to the articular surfaces or to inadequate reduction, or to both.

Dislocations and fracture dislocations of the tarsometatarsal joints.

Dislocations and fracture-dislocations of the tarsometatarsal joints are potentially disabling injuries that present challenging therapeutic problems. Early recognition is imperative and is based on a familiarity with the important anatomic features of this joint, mechanism of injury, and subtle radiographic changes that often accompany these lesions. Following injury, a precise anatomic reduction of the tarsometatarsal joint is critical if long-term disability is to be avoided. There appears to be a direct correlation between achieving an accurate reduction and a satisfactory clinical result. In our experience, surgical reduction offers the most effective and reliable means of achieving this goal. We have presented an approach for the management of these lesions, which we believe offers advantages over previously described techniques. Our experience has shown that accurate anatomic operative reduction and rigid internal fixation provide an increased assurance of a pain-free, durable, and functional foot in the great majority of cases.