FLUORESCENT LABELLING OF THE CYTOSKELETON IN CERAMIUM STRICTUM (RHODOPHYTA)1,2

Using rhodamine-phalloidin to detect F-actin/microfilaments and indirect immunofluorescence to detect tubulin/microtubules, we studied the cytoskeleton in axial cells of Ceramium strictum Harv., especially microfilaments and microtubules associated with cytoplasmic strands (trabeculae) that extend longitudinally through the central vacuole. As axial cells attained mature size, trabeculae became progressively thinner, branched, and then broke down. An extensive microfilament array was present in peripheral parts of axial cells as well as in trabeculae. Microfilament arrays were highly disrupted by cytochalasin-B; this resulted in small irregular actin structures in axial cell peripheries and occasional dense aggregations at the base of cells. No actin-fluorescence was detected in intact trabeculae after cytochalasin-B treatment. Microtubules formed a primary structural component in trabeculae, which were disrupted by griseofulvin (5 to 0.005 μM) but reformed after two days in griseofulvin-free medium.     

Nerven und Nervenausbreitungen im Subarachnoidalen Balkenwerk und in der Arachnoidea der Cisterna Cerebellomedullaris des Menschen

Zusammenfassung Die Arachnoidea ist, im Gegensatz zur bisherigen Auffassung, nicht nervenfrei. In ihr und im subarachnoidalen Balkenwerk der Cisterna cerebellomedullaris des erwachsenen Menschen kommen Nervenausbreitungen vor.In den subarachnoidalen Trabekeln liegen bis zu 1 mm lange Endformationen mit Ösen, Knöpfen, Varicositäten und Faserkörben.Die Nervenformationen der Spinnwebenhaut sind in ihrer Form variabel (Schlingenkörperchen, Netzformationen, vegetative Netze in der Umgebung der Blutgefäße).Im Gegensatz zur Pia ist in der Arachnoidea kein ausgedehntes nervöses Grundnetz vorhanden. Große Teile der Spinnwebenhaut sind nervenfrei. Ganglienzellen kommen in ihr nicht vor.Zarte subarachnoidale Balken, hyaline Körperchen und Bindegewebskörperchen dürfen nicht mit Nerven oder Nervenzellen verwechselt werden.

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Summary In contrast to current views the arachnoid is not free of nerves. In adult humans nerve endings are found in the arachnoid itself as well as the subarachnoid trabeculae of the cisterna cerebellomedullaris. End formations, which show knobs, loops, varicosities, and fibre baskets up to 1 mm in length, are found in the subarachnoid trabeculae. The shape of the nerve endings in the arachnoid is variable (loops, net-like formations, vegetative nets in the vicinity of blood vessels). In contrast to the situation in the pia, the arachnoid has no extensive nervous network. Large areas of the arachnoid are free of nerves. Ganglion cells are absent in it. Delicate subarachnoid trabeculae, hyaline bodies, and connective tissue elements are not to be taken for nerves or nerve cells.

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Herrn Prof. Dr. Dr. Heinbich v. Hayek zur Vollendung des 65. Lebensjahres.     

DISTRIBUTION OF TRABECULAE AND ELYTRAL SURFACE STRUCTURES OF THE HORNED BEETLE, ALLOMYRINA DICHOTOMA (LINNÉ) (COLEOPTERA: SCARABAEIDAE)

Abstract To clarify the dynamic construction of Allomyrina dichotoma (Linné) elytra, the distribution of trabeculae and surface structures has been investigated using scanning electron microscopy and transmitted light. There are solid trabeculae in the elytron and under penetrating light these can be seen as black dots. It is clear that trabeculae arrangement is almost entirely irregular throughout the elytron, except for some approximately straight alignment near some trachea. This irregular arrangement is different from the longitudinal rows of striations that are well known in other species, and there are no hollowed striae (punctures) on the elytral surface of A. dichotoma . Throughout the internal architecture of the elytron, there are mesh-like (honeycomb) structures. Each honeycomb usually has 1–2 trabeculae mainly distributed at the corners of the honeycomb. The number of trabeculae present on each honeycomb is dependent on the size of the honeycomb.     

不同地区HPV16 E7基因的克隆及序列差异分析

采用PCR扩增、pGEM T载体克隆和核苷酸序列分析的方法对一例武汉地区及两例五峰县高发区宫颈癌患者体内HPV16型的E7基因编码区进行序列分析并与野生型 (德国标准株 )及已发表的HPV16湖北株 (HPVHB)进行了比较。结果发现武汉地区HPV16型E7基因仅第 5 4位出现一个同义突变 ,而高发区HPV16型E7基因存在差异 ,第 77位氨基酸由精氨酸 (Arg)变为半胱氨酸 (Cys) ,第 96位由谷氨酰氨酸 (Gln)变为精氨酸 (Arg) ,E7蛋白的二级结构及亲、疏水性也相应改变 ,与野生型有较大差异     

The sensory cilium of retinal rods is analogous to the transitional zone of motile cilia

The connecting cilium of rat retinal rods was studied by freeze-fracture and thin-sectioning techniques. Transverse strands of intramembranous particles could be observed on fracture face B on the ciliary plasma membrane. The strands were essentially similar to those found at the transitional zone of motile cilia ("ciliary necklace"). The larger number of intramembranous particles obscured the pattern on fracture face A of the membrane. On longitudinal sections of the cilia, beads showing a periodicity similar to the necklace strands were observed. Each bead consisted of two structures apposed to both sides of the plasma membrane. Transverse sections of the cilia revealed radial Y-shaped structures that connected each ciliary doublet with the plasma membrane. Axial tubules, central sheath, radial spokes and dynein arms were missing in the connecting cilium. Comparing the fine structure of the retinal cilia with that of motile cilia it becomes evident that the connecting cilium is analogous in structure with the transitional zone of motile cilia. The present observations suggest that periodic membrane beads along the plasma membrane on thin sections correspond to strands of necklace particles as observed on freeze-fractured membranes. The arrangement of the particles in transverse strands is probably ensured by the radial connecting structures.     

Morphological changes in the junctional complex of cells in the arachnoid layer of the rat after cold injury

Summary The junctional complexes of cells in the outer arachnoid layer overlying the cerebral cortex of 2-week-old rats were examined with freeze-fracture electron microscopy up to 60 min after transcranial cold injury to the dorsal surface of the brain. Within 30 min after injury, areas of gap and tight junctions with morphological features characteristic of junction formation and/or junction disruption were found scattered among normal junctional complexes in some arachnoid cells. Within 60 min after injury, tight junctions with features typical of less leaky zonulae occludentes were present in all arachnoid cells examined. These morphological features include increases in the number of tight junctional strands and the number of strand-to-strand anatomoses. Gap junctions were interspersed among the tight junctional strands, and many were completely encircled by the strands. The increase in the number and complexity of the tight junctional strands in response to brain injury may be the morphological basis for the maintenance of the cerebrospinal fluid-blood dural barrier.This study was supported by the National Institute of Neurological and Communicative Disorders and Stroke Grant NS20590. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23     

5′-Nucleotidase in spinal meningeal compartments in the rat

Summary The distribution of 5-nucleotidase (5-Nu) is reported in spinal meninges of the rat on the basis of an immunohistochemical and enzyme histochemical investigation. Strong immunoreactivity was found in the arachnoid membrane and in the sheaths of the spinal roots as well as in septa subdividing the roots. Also the superficial layer of the ligamentum denticulatum showed enzyme staining. No immunoreactivity could be detected in the pia mater or along the spinal nerve roots outside the subarachnoid space. Within the arachnoid mater the immunoreactivity was concentrated in the basal zone of the arachnoid membrane, thus appearing as a narrow fluorescent band near the border of the dura. An accentuation of immunoreactivity could be observed in areas where small dural blood vessels approach the subarachnoid space. It is well known that adenine nucleotides released from neural and glial cells of the central nervous system finally reach the cerebrospinal fluid. We presume that 5-Nu in the arachnoid membrane and spinal root sheaths is responsible for the conversion of adenine nucleotides into adenosine and that this conversion is associated with the reabsorption process of cerebrospinal fluid which most probably also takes place in spinal meninges. Adenosine, the product of 5-nucleotidase, could play a role in the reabsorption process by its vasodilatatory effect on dural and epidural vessels.     

A thin-section and freeze-fracture study of microfilament-membrane attachments in choroid plexus and intestinal microvilli

Choroid plexus and intestinal microvilli in thin sections have microfilaments in the cytoplasm adjacent to the membranes, and in replicas have broken strands of filaments in both cytoplasm and on E faces of plasm membranes. The microfilaments contain actin as indicated by their binding of heavy meromyosin (HMM). In sections of choroid plexus, the microfilaments are 7-8 nm in diameter and form a loose meshwork which lies parallel to the membrane and which is connected to the membranes both by short, connecting filaments (8 times 30 nm) and dense globules (approximately 15-20 nm). The filamentous strands seen in replicas are approximately 8 nm in diameter. Because they are similar in diameter and are connected to the membrane, these filamentous strands seen in replicas apparently represent the connecting structures, portions of the microfilaments, or both. The filamentous strands attached to the membrane are usually associated with the E face and appear to be pulled through the P half-membrane. In replicas of intestinal brush border microvilli, the connecting strands attaching core microfilaments to the membrane are readily visualized. In contrast, regions of attachment of core microfilaments to dense material at the tips of microvilli are associated with few particles on P faces and with few filamentous strands on the E faces of the membranes. Freeze-fracture replicas suggest a morphologically similar type of connecting strand attachment for microfilament-membrane binding in both choroid plexus and intestinal microvilli, despite the lack of a prominent core bundle of microfilaments in choroid plexus microvilli.     

The segregation of DNA in epithelial stem cells

It has recently been suggested that stem cells may invariably keep, from one division to the next, the daughter DNA molecules that contain the older of the two parental strands—that is, they may retain a complete set of “immortal strands,” through successive cell divisions (Cairns, 1975). We can test this hypothesis by labeling either the old immortal strands at the time the stem cells are created or the newly synthesized strands during subsequent divisions of the stem cells. In the former case, the stem cells should become permanently labeled; in the latter case, they should eliminate their label on their second division.Experiments of this sort have been conducted with the tongue papilla under steady state conditions and with the regenerating small intestinal crypts. The results clearly show that by far most of the multiplying cells in tongue and intestinal epithelium segregate their DNA “randomly” at mitosis. Nevertheless, the results, though far from conclusive, suggest that there are a small number of cells (1–5 in the stem cell region of each crypt and one at the base of each column of cells in the tongue) that selectively segregate their old and new DNA strands in the expected way. Thus in the immortal strand labeling experiments, there are a few labeled cells that retain their label for up to 4 weeks; conversely, in the new strand labeling experiments, a few cells appear to rid themselves of label after intervals equivalent to approximately two cell cycles.     

Evidence for a structural relationship between successive parallel tubules in the SR network and supernumerary striations of Z line material in purkinje fibers of the chicken, sheep, dog and rhesus monkey heart.

The striations and the intervening filaments observed in the present study have been variously designated in the literature as: prodomal pattern, leptomeric myofibril, microladder, leptomeric organelle, leptofibril and zebra body. Electron microscope examinations of Purkinje fibers from the septa, papillaries, trabeculae carneae and small endocardial strands from chicken, sheep, dog and monkey hearts have revealed a close association between densely stained striations of supernumerary Z line material and successive parallel tubules in the network formed by the sarcoplasmic reticulum (SR). The striations appear to be linked together by filaments that somewhat resemble the part of thin filaments attached to Z lines in normal fibrils. The evidence for a close association of striations and SR tubules is derived from a similarity of spacing between striations and successive parallel tubules in the SR network and from a resemblance of striation and SR network patterns. The evidence for a structural relationship between striations and SR tubules is derived from the observation of electron-opaque strands traversing the space between striations and SR tubules.     

Ultrastructure of the ovary and oogenesis in six species of patellid limpets (Gastropoda: Patellogastropoda) from South Africa

Abstract. The ultrastructural features of the ovary and oogenesis have been described in 6 species of patellid limpets from South Africa. The ovary is a complex organ that is divided radially into numerous compartments or lacunae by plate-like, blind-ended, hollow trabeculae that extend from the outer wall of the ovary to its central lumen. Trabeculae are composed of outer epithelial cells, intermittent smooth muscle bands, and extensive connective tissue. Oocytes arise within the walls of the trabeculae and progressively bulge outward into the ovarian lumen during growth while partially surrounded by squamous follicle cells. During early vitellogenesis, the follicle cells lift from the surface of the underlying oocytes and microvilli appear in the perivitelline space. Follicle cells restrict their contact with the oocytes to digitate foot processes that form desmosomes with the oolamina. When vitellogenesis is initiated, the trabecular epithelial cells hypertrophy and become proteosynthetically active. Yolk synthesis involves the direct incorporation of extraoocytic precursors from the lumen of the trabeculae (hemocoel) into yolk granules via receptor-mediated endocytosis. Lipid droplets arise de novo and Golgi complexes synthesize cortical granules that form a thin band beneath the oolamina. A fibrous jelly coat forms between the vitelline envelope and the overlying follicle cells in all species.     

5'-nucleotidase in spinal meningeal compartments in the rat. An immuno and enzyme histochemical study.

The distribution of 5'-nucleotidase (5'-Nu) is reported in spinal meninges of the rat on the basis of an immunohistochemical and enzyme histochemical investigation. Strong immunoreactivity was found in the arachnoid membrane and in the sheaths of the spinal roots as well as in septa subdividing the roots. Also the superficial layer of the ligamentum denticulatum showed enzyme staining. No immunoreactivity could be detected in the pia mater or along the spinal nerve roots outside the subarachnoid space. Within the arachnoid mater the immunoreactivity was concentrated in the basal zone of the arachnoid membrane, thus appearing as a narrow fluorescent band near the border of the dura. An accentuation of immunoreactivity could be observed in areas where small dural blood vessels approach the subarachnoid space. It is well known that adenine nucleotides released from neural and glial cells of the central nervous system finally reach the cerebrospinal fluid. We presume that 5'-Nu in the arachnoid membrane and spinal root sheaths is responsible for the conversion of adenine nucleotides into adenosine and that this conversion is associated with the reabsorption process of cerebrospinal fluid which most probably also takes place in spinal meninges. Adenosine, the product of 5'-nucleotidase, could play a role in the reabsorption process by its vasodilatatory effect on dural and epidural vessels.     

THE CILIARY NECKLACE : A Ciliary Membrane Specialization

Cilia, primarily of the lamellibranch gill (Elliptio and Mytilus), have been examined in freeze-etch replicas. Without etching, cross fractures rarely reveal the 9 + 2 pattern, although suggestions of ninefold symmetry are present. In etched preparations, longitudinal fractures through the matrix show a triplet spoke alignment corresponding to the spoke periodicity seen in thin sections. Dynein rows can be visualized along the peripheral microtubules in some preparations. Fracture faces of the ciliary membrane are smooth with few membrane particles, except in the regions adjacent to the basal plate. In the transition region below the plate, a unique particle arrangement, the ciliary necklace, is found. In the Elliptio gill, on fracture face A the necklace is comprised of three well-defined rows or strands of membrane particles that encircle the ciliary shaft. The rows are scalloped and each scallop corresponds to a peripheral doublet microtubule. In thin sections at the level of these particles, a series of champagne-glass structures link the microtubular doublets to the ciliary membrane. The ciliary necklace and this "membrane-microtubule" complex may be involved in energy transduction or the timing of ciliary beat. Comparative studies show that these features are present in all somatic cilia examined including those of the ameboflagellate Tetramitus, sea urchin embryos, rat trachea, and nonmotile cilia of cultured chick embryo fibroblasts. The number of necklace strands differs with each species. The necklace has not been found in rat or sea urchin sperm.     

Scanning Electron Microscopic Studies on Four Species of the Genus Syphacia (Nematoda, Oxyuridae)

The ultrastructure of the head end and surface structure of the cuticle of Syphacia petrusewiczi, S. nigeriana, S. frederici and S. stroma was studied. These species may be easily separated on the basis of the differences in their morphology: S. frederici possesses longitudinal septa on the body surface, a row of denticles on each of the three main teeth, and cervical alae; S. nigeriana has longitudinal septa and denticles but lack cervical alae; S. petrusewiczi has longitudinal septa and cervical alae but lacks denticles; S. stroma lacks these three characters.     

Electron Microscopic Observations of the Structure of Sieve-connexions in the Phloem of Angiosperms and Gymnosperms

The sieve elements of Pinus silvestris L., Sorbus aucupariaL., Vitis vinifera L., and Cucurbita pepo L. have been examinedelectron microscopically in ultra-thin section, and the structuresof the corresponding sieve areas or sieve plates have been describedand compared. In Pinus the sieve areas contain groups of connectingstrands which enter the wall from the lumen side as individualsbut coalesce within it in the median cavity. This cavity hasdeveloped by wall breakdown in the middle lamella and primarywall region and corresponds to the median nodule visible undera light microscope. Neither in this nor in the other speciesobserved is there any visible closing membrane. Structural differences between the four species are shown tosuggest that the major evolutionary trend in the evolution ofspecialized conducting strands has been the enlargement of theconnecting strands from groups of small separate strands toa smaller number of larger strands as the median cavity becomesenlarged to form a canal through the wall. The connecting strands appear invariably to be dense, highlyosmiophilic and continuous with the cytoplasmic surface of thecell. No signs of micropores or of other tubular structure inthe strands have been found. The structures thus revealed aremore nearly compatible with active transport of materials acrossthe sieve plate than they are with any purely physical mechanism.It is suggested that they are incompatible with any mass flowhypothesis.     

Frequency-dependent excitability of "membrane" slow responses of Rabbit left atrial trabeculae in the presence of Ba2+ and high K+

Small trabeculae of rabbit left atrium immersed in TKBa solution (Tyrode with 10 mM K+ and 1 mM Ba2+) were used to study frequency dependence of "membrane" slow response excitability at long cycle lengths (greater than 1 s). In TKBa, stimuli generate graded, low- amplitude (2-15 mV) subliminal responses of variable long duration (up to 450 ms). A full all-or-none slow response is generated when a subliminal response depolarizes the membrane to about--35 mV. Subliminal response amplitude and rate of rise augment with stimulus intensity-duration product. For a fixed stimulus, the subliminal response is larger and faster at higher frequencies. Sudden changes in stimulus frequency or time course induce changes in subliminal response tha take four to eight cycles to attain steady state. For a fixed stimulus, slow response latency shortens progressively during the first few cycles after a sudden increase in frequency or when a rested preparation is excited (latency adaptation phenomenon, LAP). Slow response threshold stimulus requirements decrease during LAP (excitability hysteresis). The degree of excitability hysteresis is dependent on stimulation frequency and is more pronounced at higher frequencies. Frequency sensitivity of subliminal response (which causes frequency sensitivity of slow response excitability) is explained in terms of a transient state of enhancement set up by each stimulus. The enhanced state decays between stimuli with a half-time of approximately 4 s, thus allowing cumulative effects to become evident at rates above 0.1 Hz.     

Interstrand pairing patterns in beta-barrel membrane proteins: the positive-outside rule, aromatic rescue, and strand registration prediction

beta-Barrel membrane proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Little is known about how residues in membrane beta-barrels interact preferentially with other residues on adjacent strands. We have developed probabilistic models to quantify propensities of residues for different spatial locations and for interstrand pairwise contact interactions involving strong H-bonds, side-chain interactions, and weak H-bonds. Using the reference state of exhaustive permutation of residues within the same beta-strand, the propensity values and p-values measuring statistical significance are calculated exactly by analytical formulae we have developed. Our findings show that there are characteristic preferences of residues for different membrane locations. Contrary to the "positive-inside" rule for helical membrane proteins, beta-barrel membrane proteins follow a significant albeit weaker "positive-outside" rule, in that the basic residues Arg and Lys are disproportionately favored in the extracellular cap region and disfavored in the periplasmic cap region. We find that different residue pairs prefer strong backbone H-bonded interstrand pairings (e.g. Gly-aromatic) or non-H-bonded pairings (e.g. aromatic-aromatic). In addition, we find that Tyr and Phe participate in aromatic rescue by shielding Gly from polar environments. We also show that these propensities can be used to predict the registration of strand pairs, an important task for the structure prediction of beta-barrel membrane proteins. Our accuracy of 44% is considerably better than random (7%). It also significantly outperforms a comparable registration prediction for soluble beta-sheets under similar conditions. Our results imply several experiments that can help to elucidate the mechanisms of in vitro and in vivo folding of beta-barrel membrane proteins. The propensity scales developed in this study will also be useful for computational structure prediction and for folding simulations.     

Basement membrane structure in situ: evidence for lateral associations in the type IV collagen network

To determine molecular architecture of the type IV collagen network in situ, the human amniotic basement membrane has been studied en face in stereo relief by high resolution unidirectional metal shadow casting aided by antibody decoration and morphometry. The appearance of the intact basement membrane is that of a thin sheet in which there are regions of branching strands. Salt extraction further exposes these strands to reveal an extensive irregular polygonal network that can be specifically decorated with gold-conjugated anti-type IV collagen antibody. At high magnification one sees that the network, which contains integral (9-11 nm net diameter) globular domains, is formed in great part by lateral association of monomolecular filaments to form branching strands of variable but narrow diameters. Branch points are variably spaced apart by an average of 45 nm with 4.4 globular domains per micron of strand length. Monomolecular filaments (1.7-nm net diameter) often appear to twist around each other along the strand axis; we propose that super helix formation is an inherent characteristic of lateral assembly. A previous study (Yurchenco, P. D., and H. Furthmayr. 1984. Biochemistry. 23:1839) presented evidence that purified murine type IV collagen dimers polymerize to form polygonal arrays of laterally as well as end-domain-associated molecules. The architecture of this polymer is similar to the network seen in the amnion, with lateral binding a major contributor to each. Thus, to a first approximation, isolated type IV collagen can reconstitute in vitro the polymeric molecular architecture it assumes in vivo.     

Meningeal calcification of the rat pineal organ. Finestructural localization of calcium-ions

The surface of the pineal organ of the rat is covered by a leptomeningeal tissue, the continuation of the corresponding meningeal layers of the diencephalon. The pineal leptomeninx consists of stratified arachnoid and of pia mater cells which follow the vessels into the pineal nervous tissue. The pineal arachnoid contains electron-lucent and electron dense cells differing from each other in their cytoplasmic components. Corpora arenacea of various size and density occur among these arachnoid cells and can grow into the pineal organ alongside pia mater tissue. Acervuli often form groups in circumscribed meningeal "calcification foci". Concrements are absent or rare in the 1- and 2-month-old animal, while they are usually present in the 4- and 6-month-old rats. The electronmicroscopic localization of Ca-ions was studied in 2- and 4-month-old rats by potassium pyroantimonate cytochemistry. In the 4-month-old animals, arachnoid cells containing a varying amount of Ca-pyroantimonate deposits were found first of all around corpora arenacea, but there were also cells free of deposits in the close vicinity of the acervuli. Deposits were preferentially localized to the cytoplasm of electron dense arachnoid cells and to the cell membrane of electron-lucent cells. Most of the precipitates occurred in locally enlarged intercellular spaces. Here, microacervuli were found in 4-month-old animals suggesting that a calcium-rich environment was responsible for the appearance of the concrements. Intermediate stages between the small acervuli and large concentric corpora arenacea may indicate an appositional growth of the acervuli in the calcification foci.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein

We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.