Intercellular junctions in the reticular lamina of the organ of Corti

Junctions between the cells in the reticular lamina of the organ of Corti were examined in thin sections and after freeze-fracturing to find a structural basis for the large ionic differences between the endolymph and perilymph. The apices of the cells in the reticular lamina are joined by a band of tight junctions spaced at 140 A intervals. Beneath this apical band the organization of the tight junctions depends on whether they join a supporting cell and a hair cell, or two supporting cells. At hair cell junctions with supporting cells, there is an extensive labyrinth of tight junctions enclosing lengthy, tortuous passages whose walls are composed of either multiple parallel or single junctions. At appositions between two supportinc cells, maculae or fasciae occludentes lie immediately beneath the apical bands of closely spaced tight junctions, near the top of the zonulae adherentia which are characteristic of appositions between supporting cells. The complexes of tight junctions, or zonulae occludentes, between extralaminar supporting cells differ from those in the reticular lamina. The extralaminar cells are joined by a band of four to seven branching, anastomotic tight junctions. Thus, these junctions are like zonulae occludentes in other tissues. The novel organizations of the tight junctions in the reticular lamina, different from those between the extralaminar supporting cells, suggests a special role for these junctions in the reticular lamina. Two sizes of gap junctions link, and presumably couple, supporting cells in the reticular lamina.     

Organization of cell junctions in the peritoneal mesothelium

Intercellular junctions in the mesothelium of the visceral (mesentery and omentum), and parietal (diaphragm, pre-aortic, and iliac region) peritoneum were examined in rats and mice by using freeze-cleaved preparations. In addition to usual intercellular junctions (cell body junctions), special junctions are found between cell processes and the surface of the neighboring cell (cell process junctions). Cell body junctions are provided with tight junctions and communicating (gap) junctions. The former consist of one to two junctional strands which show a characteristic staggered arrangement, and focal discontinuities. In cell process junctions, the strands form loops or appear as short, free-ending elements; their polymorphism suggests considerable lability, probably in connection with their assembly and disassembly. The existence of free-ending strands indicates that such structures can be used as attachment devices without being concomitantly involved in the formation of occluding zonules. In both types of junctions, the strands can be resolved into bars, approximately 80- 100nm long, frequently provided with terminal enlargements and intercalated particles which occur singly or in small clusters. These particles are morphologically similar to those present in communicating (gap) junctions. The mesothelium is also provided with isolate composite macular junctions. Throughout the mesothelium, the cleavage plane follows the outer contour of junctional strands and particles, suggesting that strand-to-strand interactions in the apposed membranes are weaker than interactions between each strand and underlying cytoplasmic structures. In their general geometry and cleavage characteristics, the mesothelial junctions resemble the junctions found in the venular endothelium.     

Terminal dendritic sprouting and reactive synaptogenesis in the postnatal organ of Corti in culture

Synaptogenesis in the organ of Corti between the primary receptors, the inner hair cells, and the peripheral processes of their afferent spiral ganglion neurons in the mouse lasts for 5 days postnatally (Sobkowicz et al. [1986] J. Neurocytol. 15:693-714). The transplantation of the organ into culture at the fifth postnatal day induces a reactive sprouting of dendritic terminals and an extensive formation of new ribbon synapses within 24 hours. This reactive synaptogenesis differs strikingly from the primary synaptogenesis and has been seen thus far only in the inner hair cells. The synaptically engaged neuronal endings sprout a multitude of filopodia that intussuscept the inner hair cells. The filopodial tips contain a heavy electron-dense matter that appears to attract the synaptic ribbons, which form new synaptic contacts with the growing processes. The intensity of the filopodial growth and synaptogenesis subsides in about 3 days; the filopodia undergo resorption, leaving behind fibrous cytoplasmic plaques mostly stored in the supranuclear part of the hair cells. However, occasional filopodial growth and formation of new synaptic connections continued. The data demonstrate that any disruption or disturbance of the initial synaptic contacts between the inner hair cells and their afferent neurons caused by transplantation results in prompt synaptic reacquisition. Furthermore, we suggest that the transitory phase of terminal sprouting and multiribbon synapse formation manifests a trophic dependence that develops postnatally between the synaptic cells.     

Postnatal maturation of the organ of Corti in gerbils: morphology and physiological responses

The organ of Corti, the sensory epithelium of hearing in mammals, matures postnatally in the gerbil. Quantitative analyses of the postnatal development of the organ of Corti, including supporting cells and the basilar membrane, were carried out. The morphological study confirmed that maturation of the sensory cells proceeds with a base-to-apex gradient, with the outer hair cells appearing to mature before the inner hair cells. Maturation of the supporting cells and the basilar membrane commenced first in the middle turn. Expansion of the second row of Deiters' cells began at 6 days after birth in the middle turn, before enlargement of the pillar cell heads at 8 days postnatally. Pillar cell head enlargement continued until 20 days postnatally in the middle turn. The tunnel of Corti and spaces of Nuel appeared first in the middle turn between 8 and 10 days postnatally. The maturation of the basilar membrane involved the thickening of the central hyaline layer and a reduction in the epithelial cells on the tympanic aspect. This process continued until about 20 days after birth. The cochlear microphonic potential, whole nerve action potential, and stimulus frequency otoacoustic emissions were recorded from 12 days after birth onward and related to changes in organ of Corti morphology. The results show that changes in the accessory structures continue throughout the period of onset and development of cochlear responses between 12 and 20 days after birth, and may therefore influence the micromechanical responses of the organ of Corti to acoustic stimuli during this period.     

Gap junction change in supporting cells of the organ of Corti with ryanodine and caffeine

It has been demonstrated that the gap junctions of the supporting cells of the organ of Corti are controlled by H+ and Ca2+. Inside these cells there is a tubular structure. It is supposed that this network is endoplasmic reticulum. Calcium release from inside the cells, and the effect of calcium on the gap junctions of these cells, were investigated under whole cell clamping application of ryanodine and caffeine. Membrane capacitance and membrane resistance were calculated, with corrections for changes in whole cell parameters. Ryanodine-treated cells (1 microM-10 mM), caffeine-treated cells (5 mM 500 nM) and A23187-treated cells were uncoupled at their gap junctions. Therefore, Ca2+ plays a role in the uncoupling of the gap junctions in supporting cells of the organ of Corti from inside the cells.     

Organization of the visual reticular thalamic nucleus of the rat

The visual sector of the reticular thalamic nucleus has come under some intense scrutiny over recent years, principally because of the key role that the nucleus plays in the processing of visual information. Despite this scrutiny, we know very little of how the connections between the reticular nucleus and the different areas of visual cortex and the different visual dorsal thalamic nuclei are organized. This study examines the patterns of reticular connections with the visual cortex and the dorsal thalamus in the rat, a species where the visual pathways have been well documented. Biotinylated dextran, an anterograde and retrograde tracer, was injected into different visual cortical areas [17; rostral 18a: presumed area AL: (anterolateral); caudal 18a: presumed area LM (lateromedial); rostral 18b: presumed area AM (anteromedial); caudal 18b: presumed area PM (posteromedial)] and into different visual dorsal thalamic nuclei (posterior thalamic, lateral geniculate nuclei), and the patterns of anterograde and retrograde labelling in the reticular nucleus were examined. From the cortical injections, we find that the visual sector of the reticular nucleus is divided into subsectors that each receive an input from a distinct visual cortical area, with little or no overlap. Further, the resulting pattern of cortical terminations in the reticular nucleus reflects largely the patterns of termination in the dorsal thalamus. That is, each cortical area projects to a largely distinct subsector of the reticular nucleus, as it does to a largely distinct dorsal thalamic nucleus. As with each of the visual cortical areas, each of the visual dorsal thalamic (lateral geniculate, lateral posterior, posterior thalamic) nuclei relate to a separate territory of the reticular nucleus, with little or no overlap. Each of these dorsal thalamic territories within the reticular nucleus receives inputs from one or more of the visual cortical areas. For instance, the region to the reticular nucleus that is labelled after an injection into the lateral geniculate nucleus encompasses the reticular regions which receive afferents from cortical areas 17, rostral 18b and caudal 18b. These results suggest that individual cortical areas may influence the activity of different dorsal thalamic nuclei through their reticular connections.     

Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti

Spectrin is a cytoskeletal protein found in the cortex of many cell types. It is known to occur in cochlear outer hair cells (OHCs) with previous immunoelectron microscopical studies showing that it is located in the cuticular plate and the cortical lattice. The latter is a network of filaments associated with the lateral plasma membrane that is thought to play a role in OHC motility. Spectrin has also been found in inner hair cells (IHCs) and supporting cells using immunofluorescent techniques, but its ultrastructural distribution in these cells has not yet been described. This has, therefore, been investigated using a monoclonal antibody to alpha-spectrin in conjunction with pre- and post-embedding immunogold labelling for transmission electron microscopy. Labelling was found in a meshwork of filaments beneath the plasma membranes of both IHCs and supporting cells and, in pillar cells, close to microtubule/microfilament arrays. It was also found in association with the stereocilia of OHCs and IHCs and, as expected, in the cortical lattice and cuticular plate of OHCs. Thus, spectrin is a general component of cytoskeletal structures involved in maintaining the specialised cell shapes in the organ of Corti and may contribute to the mechanical properties of all the cell types examined.     

Regional variation in the lamina propria T cell receptor V beta repertoire in normal human colon

The Ewing's sarcoma cell line RD-ES, which carries the EWS/FLI-1 fusion gene, responded to the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin with growth arrest. Replenishment of mevalonate (MVA) to the arrested cells restored cell growth. However, if tunicamycin (TM), which is an inhibitor of N-linked glycosylation, was present together with MVA the cells remained arrested, indicating that N-linked glycosylation is of importance for growth of Ewing's sarcoma cells. Inhibition of the biosynthesis of EWS/FLI-1 fusion protein by treatment with antisense oligonucleotides also led to growth arrest, suggesting that this protein is of importance for cell growth. We investigated whether MVA synthesis and N-linked glycosylation could be involved in regulation of the expression of the EWS/FLI-1 fusion protein, which in fact contains four potential sites for N-linked glycosylation. We found that inhibition of both HMG-CoA reductase and N-linked glycosylation drastically decreased the expression of the fusion protein, which mainly appears in the cell nuclei. There was no significant difference in the inhibitory effect on the fusion protein between the cytoplasm and the cell nuclei, indicating that the transport of the fusion protein to the cell nucleus is not affected. The fusion protein did not exhibit any gel electrophoretic mobility shift after treatment of the cells with lovastatin or TM, and it did not incorporate [3H]glucosamine. Therefore we can conclude that the fusion protein is not a glycoprotein. The decreased expression of the fusion protein following lovastatin or TM treatment was found to be due to a lowered stability of de novo-synthesized fusion protein. The down-regulation of the fusion protein was correlated to growth arrest. Furthermore, the kinetics between the expression of EWS/FLI-1 fusion protein and the initiation of DNA synthesis in MVA-stimulated cells were similar. Taken together, our data suggest that the regulatory role of N-linked glycosylation in the expression of the EWS/FLI-1 fusion protein is important for growth of Ewing's sarcoma cells. Possible mechanisms underlying TM-induced decrease in EWS/FLI-1 expression may involve the breaking of growth factor receptor pathways.     

Postnatal development of the organ of Corti in cats: a light microscopic morphometric study

This study quantitatively characterizes the development of the major morphological features of the organ of Corti during the first 2 weeks postnatal, the period when the cat auditory system makes the transition from being essentially non-functional to having nearly adult-like responses. Four groups of kittens (n = 3) were studied at one day postnatal (P1), P5, P10, P15, and compared to adults. Measurements were made of the organ of Corti at 3 cochlear locations: 20%, 60% and 85% of basilar membrane length from the base cochlear locations which in the adult correspond to best frequencies of approximately 20 kHz, 2 kHz and 500 Hz, respectively. In addition, measurements of basilar membrane length and opening of the tunnel of Corti were made in 20 cochlear specimens from kittens aged P0-P6. Results indicate that: (i) at P0 the basilar membrane has attained adult length, and the tunnel of Corti is open over approximately the basal one-half of the cochlea; (ii) the initial opening of the tunnel of Corti occurs at a site about 4 mm from the cochlear base (best frequency of approximately 25 kHz in the adult cochlea); (iii) the thickness of the tympanic cell layer decreases markedly at the basal 20-kHz location; (iv) the areas of the tunnel of Corti and space of Nuel and the angulation of the inner hair cells (IHC) relative to the basilar membrane all show marked postnatal increases at both the middle and apical locations; (v) IHC are nearly adult-like in length and shape at birth, whereas the OHC (at 2-kHz and 500-Hz locations) undergo marked postnatal changes; (vi) disappearance of the marginal pillars and maturation of the supporting cells are not yet complete by P15.     

The development of the reticular lamina in the hamster: an examination of transitory features and their functional roles

This study examines the development of the reticular lamina in the Syrian golden hamster postriatally from birth to adulthood at 2 day intervals using the scanning electron microscope. During this period, numerous transitory features emerged whose roles were concerned primarily with the development of the tectorial membrane (TM). The principal findings were as follows. (1) The surface of the developing organ of Corti produced all the fibrous material composing the minor tectorial membrane (mTM) including radial and longitudinal fiber bundles which formed the skeleton of the TM, and spongy, amorphous material which formed its intervening ground substance. (2) Throughout most of the cochlear spiral, radial fiber bundles were seen extending from the microvilli of supporting cells and projecting toward the major tectorial membrane (MTM). In most of the basal turn, but not in the apical turn, these radial bundles were interwoven with longitudinal fiber bundles which emerged from the surface of Hensen's cells. These findings indicate that the architecture of the TM is more complex in the basal turn than in the apex. (3) Increases in the dimensions of the reticular lamina resulted from the emergence of pillar cell headplates and growth in the diameter of hair cells and supporting cells. The emergence of pillar cell headplates was the principal factor contributing to increases in the radial dimension of the reticular lamina. This emergence was most dramatic between 10 and 12 days after birth (DAB) after the mTM completed its growth. Since the mTM appears to be bound medially to the MTM and laterally to the marginal pillars by 10 DAB, it seems likely that the growth of the reticular lamina after 10 DAB causes some stretching of the mTM both radially and longitudinally. (4) Completion of outer hair cell stereocilia growth at 8 DAB was followed by loss of supporting cell attachments of the TM (trabeculae) by 10 DAB, and coincided with the formation of marginal pillars from the third row of supporting cells. It is suggested that the formation of marginal pillars may be required for coupling of the TM to the tips of outer hair cell stereocilia and for induction of radial tension of the mTM. (5) Removal of the marginal pillar attachments occurred following completion of hair cell growth. (6) All structures on the reticular lamina appeared to have adult-like characteristics by 20 DAB.     

The localization of synaptophysin in the organ of Corti of the human as shown by immunoelectron microscopy

Synaptophysin, or p38, a polypeptide of molecular weight 38 kD, is a calcium-binding membrane protein found in synaptic vesicles of neurons and smooth surfaced vesicles of neuroendocrine cells. Six human neonatal and infant temporal bones were fixed in paraformaldehyde and glutaraldehyde, decalcified in EDTA and were than immunoreacted for synaptophysin (ICN Biomedicals) using the avidin-biotin reaction (ABC kit, Vector Labs). The tissue was then prepared for light microscopic surface preparation, radial sections of 5 microns, and serial section electron microscopy. At a light microscopic level, the inner spiral bundle, tunnel spiral bundle, upper tunnel crossing fibers and the base of outer hair cells were stained. At the base of outer hair cells, the immunoreactivity was seen to decrease from the base to the apex and from the first to third outer hair cells. At an electron microscopic level, immunoreactivity at the base of outer hair cells was limited to vesiculated efferent fibers. The degree of immunoreactivity between adjacent efferent fibers varied significantly. Immunoreactive vesiculated endings were also found in the supranuclear region of outer hair cells.     

Efferent nerve fibers associated with the outermost supporting cells of the organ of Corti in the guinea pig

In whold-mount preparations of the organ of Corti nerve fibers are sometimes seen peripheral to the third-row outer hair cells where they appear to wander among the Hensen cells. These fibers were studied in the guinea pig by both light and electron microscopy. In the upper second, third and fourth cochlear turns nerve fibers were seen between and on the peripheral side of the third-row Deiters cells as well as in the floor of the outer tunnel space. They were not found in the intercellular spaces between the Hensen cells. These nerve fibers are continuous with the spiral efferent fibers beneath the outer hair cells. Also their ultrastructural and histochemical characteristics indicate that they are efferent neural elements. This identification was confirmed by the finding that they degenerate following interruption of the olivo-cochlear bundle in the brain stem.     

Quantitative measures reflect degeneration, but not regeneration, in the deafness mouse organ of Corti

The deafness mouse (dn/dn) is a well known model of hereditary deafness uncomplicated by behavioral and motor disturbances. The organ of Corti in this mouse develops a normal complement of sensory and supporting cell structures, yet animals homozygous for this gene never demonstrate any hearing capacity. They are profoundly deaf from birth. Soon after development, the organ of Corti rapidly degenerates, most sensory cells having vanished by 50 days of age. Published observations have suggested that apical regions of the organ of Corti may regenerate some supporting cell structures by 90 days of age. We have quantified changes in organ of Corti structure from 15 to 130 days of age using several different measures. Measures of peak height and total cross-sectional area. as well as a subjective rating scale, all demonstrate consistent degenerative changes during this time period. No evidence for regeneration of supporting or sensory cell structures is noted, although a surprising degree of variability is present in all regions of the organ of Corti which may account for previous claims.     

Does the organ of Corti attempt to differentiate new hair cells after antibiotic intoxication in rat pups?

In the adult mammalian cochlea, post-injury hair cell losses are considered to be irreversible. Recent studies in cochlear explants of embryonic rodents show that the organ of Corti can replace lost hair cells after injury. We have investigated this topic in vivo during the period of cochlear development. Rat pups were treated with a daily subcutaneous injection of 500 mg/kg amikacin for eight consecutive days between postnatal day 9 (PND 9) and PND 16. During this period the organ of Corti is not fully mature, but hair cells are hyper-sensitive to aminoglycoside antibiotics. Scanning and transmission electron microscopy was used to evaluate morphological changes in the organs of Corti during the treatment and at different post-treatment periods, up until PND 90. A massive loss in outer and inner hair cells was observed at least as early as PND 14. A prominent feature in the apical part of cochleas at PND 21 and 35 was the transient presence of small atypical cells in the region of pre-existing outer hair cells. These atypical cells had tufts of microvilli reminiscent of nascent stereociliary bundles. A second striking observation was the replacement of degenerating inner hair cells by pear-shaped supporting cells throughout the cochlea. These cells were covered with long microvilli, and their basal pole was contacted by both afferent and efferent fibers, as in the early stages of inner hair cell maturation. At PND 55 and 90, these features were not clearly observed due to further cytological changes in the organ of Corti. It is possible that an attempt at hair cell neodifferentiation could occur in vivo after an amikacin treatment in the rat during the period of cochlear hyper-sensitivity to antibiotic.     

Dynamic changes of gap junctions and cytoskeleton during in vitro culture of cattle oocyte cumulus complexes

Changes in cell-to-cell contact and distribution of cytoskeletal components were investigated during in vitro culture of cattle oocyte cumulus complexes (OCC). Freeze-fracture analysis (FF), microinjections of the fluorescent dye Lucifer Yellow (LY), immunofluorescence, and ultrastructural immunocytochemistry were used. The cumulus cells (CC) remained in close contact via gap junctions (GJ) constituted of connexin43 (Cx43) during the entire culture time. Whereas the GJ decreased in diameter after 24 h of culture, their number was still substantially great at that time. The Cx43-positive GJ, localized between corona radiata cell projections and oolemma, disappeared after 6 h of culture. Concomitantly, the OCC lost the ability to transfer LY from cumulus to oocyte, and connexin32 (Cx32) became detectable in the oocytes. Both the changes in corona-oocyte coupling and cumulus expansion were preceded by the redistribution of F-actin in cytoplasm of CC. These data indicate that functional GJ linked the CC until the second meiotic arrest. However, the removal of Cx43-positive GJ interconnecting cytoplasmic projections of corona radiata cells with the oocyte was temporally correlated with germinal vesicle breakdown. The present results suggest the pivotal role of the cytoskeleton (F-actin) in cumulus expansion.     

Molecular organization and functional regulation of cell to cell junctions in the endothelium

Intercellular junctions are important structural determinants of endothelial permeability. These organelles are formed by a complex network of transmembrane proteins linked to a well developed plasmalemmal undercoat. One of the typical characteristics of endothelial junctions is their dynamic organization. Endothelial cells are able to rapidly change the architecture of the junctions to allow the passage of plasma constituents and circulating cells. This effect, in most of the cases, is quickly reversible and the endothelium is able to disorganize/reorganize the intercellular junctions within minutes. The mechanisms that regulate the opening and the closure of endothelial junctions are still obscure. It is conceivable that inflammatory agents increase permeability by binding to specific receptors generating intracellular signals which in turn cause cytoskeletal reorganization and opening of interendothelial gaps. Endothelial junctions also control leukocyte extravasation. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial clefts occurs. The mechanism by which this takes place is unknown, but it might present characteristics similar to that triggered by soluble mediators.     

The generation of DC potentials in a computational model of the organ of Corti: effects of voltage-dependent K+ channels in the basolateral membrane of the inner hair cell

A computational model of the organ of Corti is described to assist in the interpretation of electrophysiological data concerning the role of the K+ channels residing in the basolateral membrane of cochlear hair cells. Recent in vivo data from Van Emst et al. (Hear. Res. 88, 27-35 (1995); Hear. Res. 102, 70-80 (1996)) about the effects of selective blocking of K+ channels indicate that these channels affect the magnitude of the summating potential. In order to understand the nature of this effect, the model of Dallos (Hear. Res. 14, 281-291 (1984)) was extended to account for the voltage- and time-dependent properties of the K+ channels in the basolateral membrane of the inner hair cell (IHC) (Kros and Crawford, J. Physiol. 421, 262-291 (1990)). The model shows that the K+ channels induce a shift in the mean IHC basolateral conductance when high-frequency stimuli are present. As a result, cochlear transduction shifts to a different electrical operating state and this is the source of a marked decrease in the stimulus-evoked DC response of the IHC. Extracellularly, in contrast, the magnitude of the DC response increases slightly. At low frequencies, the K+ channels respond to the stimulus waveform on a cycle-by-cycle basis. The waveform distortion associated with this dynamic basolateral impedance induces a further decrease in the intracellular stimulus-evoked DC response of the IHC. Thus, K+ channels in the IHC appear to be directly involved in the generation of the DC receptor potential at low frequencies, but at high frequencies they simply modify the size of the DC response.     

The cytoskeleton of the vertebrate smooth muscle cell

Four experiments were conducted to determine the effects of dopamine (DA) antagonists and DA depletions on progressive-ratio responding for food reinforcement. On this schedule, ratio requirement increased by one response after each reinforcer was obtained, and rats were tested in 30-min sessions. Response rates and highest ratio completed were reduced in a dose-related manner by systemic injections of the D1 antagonist SCH 23390, and also by the D2 antagonists haloperidol and raclopride. Drug-treated rats also showed reductions in time to complete the last ratio, demonstrating that they had stopped responding before the end of the session. DA depletions produced by injections of 6-OHDA directly into the nucleus accumbens substantially decreased both the number of responses and the highest ratio completed. The deficits in response number and highest ratio completed induced by DA depletions persisted through the first 3 weeks of postsurgical testing, with some recovery by the fourth week. However, the deficits resulting from dopamine depletions were largely a manifestation of a decrease in response rate; although time to complete the last ratio was significantly reduced by dopamine depletions in the first few days of testing, rats recovered on this measure by the fifth day after surgery. Although previous work has shown that performance on several schedules (e.g., continuous, low value ratios, variable interval) is relatively unaffected by accumbens DA depletions, the present data demonstrate that such depletions do produce a substantial and persistent impairment of progressive ratio response output. Rats with accumbens DA depletions appear to have deficits in maintaining the high work output necessary for responding at large ratio values. The relative sparing of responding on some simple schedules, together with the present progressive ratio results, suggest that rats with accumbens DA depletions remain directed toward the acquisition and consumption of food, but they show deficits in work output for food.     

A novel zinc finger gene preferentially expressed in the retina and the organ of Corti localizes to human chromosome 12q24.3

Substitution of the conserved Gly127 for residues having a side chain markedly changed the substrate specificity of subtilisin E from Bacillus subtilis. The crystallographic findings suggested that Gly127 is responsible for accepting even the large P1 substrates, and the marked change of specificity was attributed to the introduction of a side chain in this position. To test this hypothesis, Gly127 was replaced with 3 non-charged amino acids, Ala, Ser and Val. When assayed with synthetic peptide substrates, all mutants purified from the periplasmic space in Escherichia coli showed a marked preference for small P1 substrate up to 150-fold relative to the wild-type. The kinetic data and molecular modeling analysis suggest that large hydrophobic P1 residues were unable to access the binding pocket due to steric hindrance.