Three-Dimensional Reconstruction of Rabbit-Derived Pneumocystis carinii from Serial-Thin Sections II: Intermediate Precyst

Three-dimensional reconstruction of a binucleate intermediate precyst of Pneumocystis carinii was performed from serial-thin sections using the CATIA (Conception Assistée Tridimensionnelle Inter Active) Dassault system program. The presence of a mitochondrion, complex well-developed endoplasmic structures, and numerous Golgi vesicles was established. A better understanding of the ultrastructure of rabbit-derived P. carinii stages made it possible to formulate hypotheses on the evolution and physiology of the endomembrane system. Thus, the presence of the well-developed endoplasmic saccular structure and more than 230 Golgi vesicles in its vicinity might be implicated in the differentiation of the parasite surface structures and might also be related to nuclear division and individualization of intracystic bodies.     

Isolation and characterization of a Golgi-rich fraction from the adrenal medulla.

1. A Golgi-rich fraction from bovine adrenal medulla was isolated by centrifugation through discontinuous sucrose density gradients. 2. The specific activity of UDPgalactose-N-acetylglucosamine galactosyl transferase was increased in this fraction. Therefore, this enzyme is a useful marker for Golgi in bovine adrenal medulla. 3. Golgi membranes were reasonably free from mitochondria, lysosomes, endoplasmic reticulum and chromaffin granules as shown by the relatively low activities of marker enzymes. 4. The negative staining techniques of electron microscopy revealed the presence of a system of tubules, vesicles and plate-like center regions which are similar to those structures previously described of the Golgi fraction isolated from the liver. 5. The specific activity of 5'-nucleotidase in the Golgi-rich fraction was 3.5 times greater than that in adrenal homogenates. However, the subcellular distribution patterns of galactosyl transferase and 5'-nucleotidase were similar. The possibility that 5'-nucleotidase might be a conspicious component of the Golgi apparatus is discussed.     

The fine structure of differentiating interstitial cells in Hydra

Summary Interstitial cells of hydra are small undifferentiated cells containing an abundance of free ribosomes and few other cytoplasmic organelles. They are capable of differentiating into epitheliomuscular, digestive, glandular, nerve cells, and cnidoblasts. Developing epitheliomuscular and digestive cells acquire bundles of filaments, 50 Å in diameter, which later are incorporated into the muscular processes. Early gland cells develop an elaborate rough-surfaced endoplasmic reticulum and one or more Golgi apparatus. Secretory granules originate in the Golgi region eventually filling the apex of the cell. Neurons are recognized first by the presence of an elaborate Golgi apparatus, absence of a well-developed endoplasmic reticulum, and later the appearance of cytoplasmic processes. The most striking feature of nematocyst formation by cnidoblasts is the presence of a complex distribution system between protein synthesizing rough-surfaced endoplasmic reticulum and the nematocyst. This system consists of connections between cisternae of the endoplasmic reticulum with smooth Golgi vesicles which in turn are connected to minute tubules, 200 Å in diameter. The tubules extend from the Golgi region around the nematocyst finally entering the limiting membrane of the nematocyst. It is suggested that the interstitial cells of hydra represent a model system for the investigation of many aspects of cell differentiation.This work was supported by grants from the National Cancer Institute (TlCA-5055) and from the National Institute of Arthritis and Metabolic Diseases (AM-03688), National Institutes of Health, Department of Health, Education and Welfare.The author is indebted to Dr. Russell J. Barrnett for his guidance and interest throughout this investigation.     

Golgi complex and lysosomes in rabbit derived Pneumocystis carinii.

The ultrastructural morphology of Pneumocystis carinii obtained from nonimmunosuppressed rabbit is described in details. Golgi complex and primary lysosomes of P carinii are described here for the first time. They are easily revealed by the zinc iodide-osmium tetroxide cytochemical reagent. Thiamine pyrophosphatase and beta-glycerophosphatase activities are found in the parasite but cytidine 5' monophosphatase activity is not observed. A weak thiamine pyrophosphatase activity is detected in Golgi vesicles. An endomembranous saccular structure, present from the intracystic body stage to the precystic stage, apparently plays the role of secondary lysosome. A second type of endomembranous saccular structure, only present in the well developed trophozoitic and precystic forms is also described. The presence of carbohydrates in the cell wall of the parasite was demonstrated by periodic acid-thiosemicarbazide-silver proteinate staining and lectin concanavalin A labeling. The development of Golgi vesicles preceded the transition from double-layered to three-layered parasite stages.     

Cell-free transfer of membrane lipids. Evidence for lipid processing

A latent phospholipase A is concentrated in cis elements of rat liver Golgi apparatus, the presumed sites of fusion of the 50-70-nm transition vesicles formed from endoplasmic reticulum. As a result, conversion of transferred phospholipids to their corresponding lysoforms may provide an index of post transfer lipid processing in a corresponding reconstituted membrane transfer system. To label the phosphatidylcholine of transitional endoplasmic reticulum in vitro, [14C]CDP-choline and endogenous cytidyltransferases were used. In the reconstituted transfer system, the radiolabeled phosphatidylcholine was transferred via transition vesicles to Golgi apparatus immobilized on nitrocellulose strips in a time- and temperature-dependent process. Transfer was promoted by ATP and the ATP-dependent transfer was specific for cis Golgi apparatus elements as acceptor. Trans Golgi apparatus elements were ineffective as acceptors. Median Golgi apparatus elements were intermediate. A portion of the transferred phosphatidylcholine was converted subsequently to lysophosphatidylcholine also in a time- and ATP-dependent manner. The phospholipase A activity of the Golgi apparatus was more than 90% latent (active site located on the lumens of the Golgi apparatus membranes). Therefore, the lipid-containing vesicles derived from endoplasmic reticulum must have combined with cis Golgi apparatus membranes as the basis for Golgi apparatus-dependent phospholipase A processing of endoplasmic reticulum-derived phosphatidylcholine. Since the lipids were processed by phospholipase A in approximately the same proportion as occurs in situ, the findings offer evidence both for the specificity of the ATP-dependent component of cell-free lipid transfer from endoplasmic reticulum to Golgi apparatus and its fidelity to lipid transfer observed in vivo.     

Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine,with special reference to the intestinal pacemaker cells

Two types of interstitial cells have been demonstrated in close association in the deep muscular plexus of rat small intestine, by electron microscopy. Cells of the first type are characterized by a fibroblastic ultrastructure, i.e. a well-developed granular endoplasmic reticulum, Golgi apparatus and absence of the basal lamina. They form a few small gap junctions with the circular muscle cells and show close contact with axon terminals containing many synaptic vesicles. They may play a role in conducting electrical signals in the muscle tissue. Cells of the second type are characterized by many large gap junctions that interconnect with each other and with the circular muscle cells. Their cytoplasm is rich in cell organells, including mitochondria, granular endoplasmic reticulum and Golgi apparatus. They show some resemblance to the smooth muscle cells and have an incomplete basal lamina, caveolae and subsurface cisterns. However, they do not contain an organized contractile apparatus, although many intermediate filaments are present in their processes. They also show close contacts with axon terminals containing synaptic vesicles. These gap-junction-rich cells may be regular components of the intestinal tract and may be involved in the pacemaking activity of intestinal movement.     

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.     

Fine cytochemical detection of acid mucopolysaccharides in growing hyphae of Neurospora crassa.

The presence of mucopolysaccharides (MP) in Neurospora crassa hyphae was detected in samples of cultures from 4 h to 5 days. Using a modification of the method of Rothman (1969), acid MP were identified by electron microscopy in vesicles located at the apex, septae, and throughout the cytoplasm of the hyphae. The size, number, and localization of these vesicles was related to the stage of growth of the fungus. The mechanisms of release and transport of these macromolecules are compared to the process of secretion in cells endowed with a better developed endoplasmic reticulum and Golgi system. The specificity of the method was evaluated histochemically. The structures displaying greater electron density corresponded to the Alcian blue positive areas.     

L'appareil de Golgi des Ciliés. Ultrastructure, particulièrement chez Paramecium

Electronmicroscopic study of Coleps, Colpidium, Stylonychia, and especially of Paramecium confirmed the presence of the Golgi complex in these fresh-water ciliates. The complex consisted of numerous dictyosomes scattered throughout the cytoplasm. Each dictyosome included a few flat, partly reticulated saccules lying parallel to a cistern of rough endoplasmic reticulum which was free of ribosomes on the side exposed to the dictyosome. A unique layer of vesicles, characterized by constant size and a thick wall, separated the endoplasmic reticulum from the dictyosomes. The vesicles could be regarded as transition vesicles. Coated vesicles were seen in continuity with some of the flattened saccules. The possible role of the Golgi complex in the physiology of ciliates is discussed.     

Ultrastructure of the pineal organ of the killifish,Fundulus heteroclitus,with special reference to the secretory function

Summary The pineal organ of the killifish, Fundulus heteroclitus, was investigated by electron microscopy under experimental conditions; its general and characteristic features are discussed with respect to the photosensory and secretory function. The strongly convoluted pineal epithelium is usually composed of photoreceptor, ganglion and supporting cells. In addition to the well-differentiated photosensory apparatus, the photoreceptor cell contains presumably immature dense-cored vesicles (140–220 nm in diameter) associated with a well-developed granular endoplasmic reticulum in the perinuclear region and the basal process. These dense-cored vesicles appear rather prominent in fish subjected to darkness. The ganglion cell shows the typical features of a nerve cell; granular endoplasmic reticulum, polysomes, mitochondria and Golgi apparatus are scattered in the electron-lucent cytoplasm around the spherical or oval nucleus. The dendrites of these cells divide into smaller branches and form many sensory synapses with the photoreceptor basal processes. Lipid droplets appear exclusively in the supporting cell, which also contains well-developed granular endoplasmic reticulum and Golgi apparatus. Cytoplasmic protrusions filled with compact dense-cored vesicles (90–220 nm in diameter) are found in dark-adapted fish. The origin of these cytoplasmic protrusions, however, remains unresolved. Thus, the pineal organ of the killifish contains two types of dense-cored vesicles which appear predominantly in darkness. The ultrastructural results suggest that the pineal organ of fish functions not only as a photoreceptor but also as a secretory organ.We thank Dr. Grace Pickford for the fishes.     

Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Golgi apparatus: going round in circles?

Recent studies have questioned the idea that the Golgi complex is a stable organelle with a unique identity through which secretory cargo is transported by vesicles. Instead, it is proposed that Golgi apparatus proteins continuously recycle via the endoplasmic reticulum by vesicle transport, whereas cargo molecules remain in maturing cisternal structures. Rather than forming a rigid matrix, structural Golgi proteins might be highly dynamic and recycle via the cytoplasm. I will discuss the evidence for these claims and consider whether or not they really disprove older ideas on how the Golgi apparatus is structured and performs its function.     

Fertilization in a Crab. III. Cytodifferentiation of vesicles enclosing ring-shaped elements involved in the cortical reaction

During the cortical reaction, Carcinus maenas eggs successively released a fine granular material and a massive amount of ring-shaped elements that subsequently formed most of the fertilization envelope. The ring-shaped elements came from egg cortical vesicles and, owing to their striking morphology, acted as naturally occurring markers in ultrathin sections, which permitted us to understand the pathway of their intracellular transport. In this respect it was established that the ring-shaped elements and their enclosing vesicles originated in the endoplasmic reticulum both in ripe oocytes and early fertilized eggs maintained under in vitro conditions. The intracellular transport pathway of the endoplasmic reticulum-derived vesicles seemed to bypass the Golgi apparatus. Accordingly, the ring-shaped elements appeared to be released by direct exocytosis from the endoplasmic reticulum system. Finally, a tentative scheme of oocytes functioning is suggested for crustacean decapods, based on the remarkable similarities between the structure and ER origin of the ring-shaped elements involved in the cortical reaction and the disc-shaped granules traditionally considered as endogenous yolk precursors. The scheme implies that the oocyte ER system might produce a precursor common to the cortical reaction exudate and to the endogenous yolk, in the form of the ring- or disc-shaped elements.     

Intracellular transport of albumin through the secretory apparatus of rat liver parenchymal cells. An immunocytochemical study

The fine structural localization of albumin in rat liver parenchymal cells was determined by an improved immunocytochemical method and serial sectioning. Albumin in the secretory apparatus of the parenchymal cells was present in segments of the rough endoplasmic reticulum, interrupted with negative segments, in transport vesicles, Golgi saccules, finely anastomosed tubules and vesicles on the trans side of the Golgi complex, and in secretion granules. Horizontally sectioned Golgi saccules contained lipoprotein particles on one side and albumin on the other side. After transport, the vesicles that contained albumin fused with the so-called rigid lamellae on the trans-side of the Golgi complex. Ultrathin serial sections revealed no true structural continuity between the endoplasmic reticulum and the cis-aspect of the Golgi complex. We concluded that secretory proteins are transported from the endoplasmic reticulum to the Golgi complex by transport vesicles that bud from the endoplasmic reticulum and fuse with the Golgi saccules. These vesicles fuse regularly with the Golgi saccules on the cis-side and occasionally with tubular elements on the trans-aspect that may belong to the so-called GERL.     

Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.     

A cytochemical ultrastructural study of the lysosomal system of different species of malaria parasites

We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR3 strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.     

Ultrastructural study of synovial membrane from the antebrachiocarpal joint of calves

The synovial intima from the antebrachiocarpal joint of 4-month-old calves was between 1 and 3 cells in thickness and did not have a basal lamina. Numerous areas of the intimal matrix were in direct contact with the joint lumen. The synovial membrane was comprised mainly of A-type synoviocytes usually located adjacent to the joint lumen. These cells were characterized by numerous filopodia (or lamellipodia), large, empty-appearing vacuoles, numerous lysosomes, large vacuoles containing granular material separated from the vacuolar membrane by a radiolucent band, and coated micropinocytotic vesicles. Smooth micropinocytotic vesicles were seen only rarely in these cells. In contrast, B-type cells had few filopodia, numerous smooth micropinocytotic vesicles, few coated micropinocytotic vesicles, a well-developed Golgi apparatus and rough endoplasmic reticulum, mitochondria that were longer and had a denser matrix than that of A cell mitochondria, and surprisingly, only few maturing or fully formed secretory granules. A distinct intermediate (C or AB) type synoviocyte could not be unequivocally identified. Desmosome-like structures were present between synoviocytes, although it was considered questionable if these were true intercellular junctions. No other junctions were present.     

Cell-Free Transfer of Phosphatidylinositol between Membrane Fractions Isolated from Soybean

Transfer of phosphatidylinositol (PI) between membranes was reconstituted in a cell-free system using membrane fractions isolated from dark-grown soybean (Glycine max [L.] Merr.). Donor membrane vesicles contained [3H]myo-inositol-labeled PI. A fraction enriched in endoplasmic reticulum was a more efficient donor than its parent microsomal membrane fraction. As acceptor, cytoplasmic side-out plasma membrane vesicles were more efficient than cytoplasmic side-in plasma membrane vesicles. Endoplasmic reticulum was also an efficient acceptor, suggesting that transfer occurred to cytoplasmic membrane leaflets. PI transfer was time and temperature dependent but did not require cytosolic proteins, ATP, GTP, cytosol, and acyl-coenzyme A. These results suggest that neither lipid transfer proteins nor transition vesicles, similar to those involved in vesicle trafficking from endoplasmic reticulum to the Golgi apparatus, were involved. In the presence of Mg2+ and ATP, endoplasmic reticulum PI was not metabolized, whereas PI transferred to the plasma membrane was metabolized into phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. To summarize, the cell-free transfer of endoplasmic reticulum-derived PI was distinct from, for example, vesicle transport from endoplasmic reticulum to Golgi apparatus, not only in its regulation but also in its acceptor unspecificity.     

Fine structure of the oxynticopeptic cells in the gastric glands of the ruin lizard, Podarcis sicula campestris De Betta, 1857

The results of an ultrastructural investigation of the gastric glands of the ruin lizard are reported. In this reptile the stomach can be divided into a larger fundus and a smaller pars pilorica. Fundic glands are characterized by three main kinds of cells: mucous, endocrine, and oxynticopeptic; the latter were not observed in the pyloric glands. The morphological features of the oxynticopeptic cells change from the proximal to the distal region of the fundic mucosa. In the proximal region, numerous electron-dense secretory granules, a well-developed granular endoplasmic reticulum, an evident Golgi complex, and a reduced system of smooth-surfaced vesicles and tubules in the apical cytoplasm characterize these cells. In the distal fundic region, oxynticopeptic cells possessed numerous mitochondria and a well-developed smooth-surfaced endoplasmic reticulum, but secretory granules were rare. These data suggest the existence of a gradient in the production of proteolytic enzymes, and perhaps also of hydrochloric acid, along the oral-aboral axis of the stomach. The results are discussed with regard to the evolution of the gastric glands and of the digestive mechanism in vertebrates.     

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

 Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway. Accepted: 26 January 1998