FLR-4, a novel serine/threonine protein kinase, regulates defecation rhythm in Caenorhabditis elegans.

The defecation behavior of the nematode Caenorhabditis elegans is controlled by a 45-s ultradian rhythm. An essential component of the clock that regulates the rhythm is the inositol trisphosphate receptor in the intestine, but other components remain to be discovered. Here, we show that the flr-4 gene, whose mutants exhibit very short defecation cycle periods, encodes a novel serine/threonine protein kinase with a carboxyl terminal hydrophobic region. The expression of functional flr-4::GFP was detected in the intestine, part of pharyngeal muscles and a pair of neurons, but expression of flr-4 in the intestine was sufficient for the wild-type phenotype. Furthermore, laser killing of the flr-4-expressing neurons did not change the defecation phenotypes of wild-type and flr-4 mutant animals. Temperature-shift experiments with a temperature-sensitive flr-4 mutant suggested that FLR-4 acts in a cell-functional rather than developmental aspect in the regulation of defecation rhythms. The function of FLR-4 was impaired by missense mutations in the kinase domain and near the hydrophobic region, where the latter allele seemed to be a weak antimorph. Thus, a novel protein kinase with a unique structural feature acts in the intestine to increase the length of defecation cycle periods.

Hyperactivation of the G12-mediated signaling pathway in Caenorhabditis elegans induces a developmental growth arrest via protein kinase C.

The G(12) type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells. However, little is known about the molecular nature of the components that act in the G(12)-signaling pathway in an organism. We characterized a C. elegans Galpha subunit gene, gpa-12, which is a homolog of mammalian G(12)/G(13)alpha, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G(12)QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G(12) participates, we screened for suppressors of the G(12)QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCtheta/delta. TPA-1 mediates the action of the tumor promoter PMA. Expression of G(12)QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G(12) signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G(12) proteins.

Morphology of the cricopharyngeal muscle in Zenker and control specimens.

The cricopharyngeal muscle (CPM) is essential for normal deglutition. Pharyngeal dysphagia commonly results from impaired or uncoordinated CPM dilation. Dysfunction of the CPM has also been implicated in the genesis of Zenker's (pharyngoesophageal) diverticulum. Despite the CPM's significance, little is understood about its morphology. We studied CPM biopsy specimens from 20 patients with Zenker's diverticulum and from 5 fresh cadaver patients with detailed histologic techniques to include fiber size and shape and adenosine triphosphatase, reduced nicotinamide adenine dinucleotide, trichrome, succinate dehydrogenase, cytochrome C oxidase, periodic acid-Schiff reaction, oil red O, acid phosphatase, Congo red, crystal violet, and monoadenylate deaminase stains. The normal CPM has unique morphological characteristics, with some myofibers having staining properties that are a hybrid between striated muscle and muscle spindle. The variable orientation of the muscle fibers is also different from that of most other striated musculature. Of the 20 Zenker CPM specimens, 4 specimens did not reveal any significant differences from controls (2 of which had insufficient amounts of tissue for complete analysis). In the remaining 16 specimens, several abnormalities existed, including excessive size variation (16/16), grouping of atrophic fibers (9/16), target or targetoid formations (4/16), cores (2/16), and ragged red fibers (2/16). The final pathological pattern of the 16 specimens was neurogenic in 7, myopathic in 4, and mixed (with neurogenic predominance) in the remaining 5. Two specimens contained significant lymphocytic inflammatory infiltrates. We conclude that the unique neuromuscular function of the CPM in deglutition is likely due to its fiber orientation and the hybrid nature of some of the myofibers. Morphological disturbances of the CPM impair its dilation and may account for the development of Zenker's diverticulum. This disturbance is most often due to progressive denervation of the CPM.

Neuromuscular compartments and fiber-type regionalization in the human inferior pharyngeal constrictor muscle.

The inferior pharyngeal constrictor (IPC) muscle functions during swallowing, respiration, and vocalization. The most-caudal portion of the IPC is believed to be part of the functional upper esophageal sphincter (UES). We hypothesized that the caudal fibers of the human IPC may have enzyme-histochemical characteristics similar to those of the cricopharyngeus muscle, a major component of the UES. In this study, human IPC muscles obtained from autopsy were studied using Sihler's stain to examine innervation patterns, and using myofibrillar ATPase, NADH tetrazolium reductase (NADH-TR), and succinic dehydrogenase (SDH) techniques to investigate the distribution and oxidative capacity of the slow- (type I) and fast- (type II) twitch fibers in the muscle. The results showed that the human IPC consists of at least two neuromuscular compartments (NMCs): rostral and caudal. Each of the NMCs was innervated by a separate nerve branch derived from the pharyngeal branch of the vagus nerve. The rostral NMC is faster (39% type I, 61% type II) than the caudal NMC (70% type I, 30% type II). In addition, two histochemically-delineated fiber layers were identified in the human IPC: a slow inner layer (SIL) with predominantly type I fibers (66%), and a fast outer layer (FOL) with predominantly type II fibers (62%) (P < 0.01). However, the dimensions of both fiber layers and proportions of the muscle fiber types varied with the NMCs. Specifically, the ratio of the thickness of the SIL to FOL was approximately 2:1 for the caudal NMC and approximately 1:2 for the rostral NMC, respectively. In the SIL the type I fibers accounted for 84% for the caudal NMC and 69% and 44% for the lower and upper portions of the rostral NMC. In contrast, the type II fibers in the FOL accounted for 46% for the caudal NMC and 67% and 74% for the lower and upper portions of the rostral NMC, respectively (P < 0.01). The caudal NMC of the IPC shared histochemical characteristics with the cricopharyngeus muscle, in that it contained predominantly slow oxidative fibers. Overall, the caudal NMC and the SIL in the IPC had high NADH-TR and SDH activities. However, different patterns of oxidative enzyme activity were identified in both type I and type II fibers. This study provided histochemical evidence for the concept that the caudal NMC within the IPC contributes to the functional UES. In addition, the two histochemically-defined fiber layers in the IPC may be a specialized adaptation in humans to enable different upper-airway functions during respiration, swallowing, and speech.

Developmental and tissue-specific expression of 2-methyl branched-chain enoyl CoA reductase isoforms in the parasitic nematode, Ascaris suum.

The 2-methyl branched-chain enoyl CoA reductase (ECR) plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode, Ascaris suum. Two-dimensional gel electrophoresis of the purified ECR yielded multiple spots, with two distinct but overlapping N-terminal sequences. These multiple isoforms were not the result of population effects, as the pattern observed on 2-D gels of the purified ECR was identical to those on immunoblots of muscle homogenates isolated from individual worms. A full-length cDNA coding for the major ECR isoform (ECRI) has been cloned and sequenced and compared with that of the minor isoform (ECRII) which has been described previously (Duran et al. J Biol Chem 1993;268:22391-22396). ECRI contained the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, a 5' untranslated region (UTR) of 13 nucleotides, an open reading frame (ORF) of 1257 nucleotides, a 3'-UTR of 110 nucleotides that included the polyadenylation signal AATAAA downstream of the termination codon and a short poly(A) tail. The ORF predicted a 16 amino acid leader sequence not found in the native protein and a mature protein of 403 amino acids with a molecular weight of 43 698 and a predicted pI of 6.2. ECRI and ECRII were 73% identical at the predicted amino acid level and their mRNAs exhibited significant structural similarity even though they were products of separate genes. Comparison of ECRI and ECRII with the sequences of acyl CoA dehydrogenases from a variety of different sources revealed a high degree of interspecies sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene. This result is surprising since the ascarid enzymes function as reductases, not as dehydrogenases. Both ECRs were tissue-specific and developmentally regulated and were found in transitional third-stage larvae (L3) and adult muscle, but not in early, aerobic larval stages or adult testis, ovary, or intestine. The ratio of ECRII to ECRI was greater in L3 than in adult muscle. Interestingly, both ECRs also appeared to be expressed in pharyngeal muscle, suggesting that branched-chain fatty acid synthesis may not be confined exclusively to body wall muscle.

Pharyngeal dysphagia caused by isolated myogen dystrophy of musculus cricopharyngeus.

Five patients suffering from idiopathic cricopharyngeal dysfunction (without Zenker's diverticulum) were treated surgically. Together with cricopharyngeomyotomy biopsies were taken at the level of the cricopharyngeus. Histological, enzyme hystochemical and electronmicroscopic examinations were performed on all patients. In two cases the histology revealed myogen dystrophy (presence of necrosis, myophagocytosis, abnormal fiber structure, basophilic fibers, fibrosis, mild cellular reaction and predominancy of fiber type I). Since the complete patient evaluation (clinical features, electromyography, serum creatinin phosphokinase level, etc.) could rule out any general, muscle disorders, the cause of the idiopathic pharyngeal dysfunction must have been in these two cases an isolated myogen dystrophy of the cricopharyngeus.

A histological and histochemical study of the cricopharyngeus muscle in man.

The human cricopharyngeus muscle was investigated by dissection and by histological, histochemical and morphometric methods. Muscle fibres in the cricopharyngeus were found to be similar in appearance to those of the lateral part of the quadriceps femoris, although they were generally much smaller and more variable in size. The endomysial connective tissue was markedly increased in the cricopharyngeus and muscle spindles were not found. Certain features normally considered to be pathological were also noted in the cricopharyngeus muscles. The fibre type population consisted mainly of histochemically 'slow-twitch' richly oxidative fibres. This finding is consistent with the proposed function of this muscle in its sphincteric role in deglutition, vomiting, eructation and in the control of aerophagia.

A histological and histochemical study of the cricopharyngeus muscle in the guinea-pig.

Histological, histochemical and morphometric methods were used to investigate the cricopharyngeus muscle in the guinea-pig and to compare it with the extensor digitorum longus and soleus muscles. The cricopharyngeus comprised uniformly small diameter fibres otherwise similar in appearance to those found in skeletal limb muscles. Several fibre type profiles were distinguished within the cricopharyngeus, all of which had homogeneously high oxidative activity, whilst the majority were histochemically fast (Type II). Muscle spindles were not observed in the cricopharyngeus muscles. Compared to the surrounding musculature the cricopharyngeus has a higher oxidative activity and may thus be suitably adapted for the maintenance of tonic contraction, forming a part of the upper oesophageal sphincter.

[Myonic makeup of the muscles innervated by the vagus nerve]. / Mionnyi sostav myshts, innerviruemykh bluzhdaiushchim nervom.

Histochemical investigation on succinic dehydrogenase activity and morphometric studies have demonstrated certain differences in the dog sublingual group of muscles. The thyreohyoid and sternohyoid muscles innervated by spinal nerves possess three types of myons differing in succinic dehydrogenase activity and in the area of transversal section. The cricothyreoid muscle and the superior pharyngeal constrictor obtaining their motor innervation from the vagus nerve are composed of unitypical muscular fibres with nearly the same areas of transversal section and high enzymic activity. The differences noted should be explained by different sources of motor innervation.