Spheroidal and ring nucleoli in amphibian oocytes. Patterns of uridine incorporation and fine structural features.

In maturing oocytes of the newt Triturus viridescens, the nucleoli undergo a series of morphological changes that are very similar to those described by Callan for the axolotl, Ambystoma mexicanum. The nucleoli first assume the form of spheroids which then become extended into ring or necklace shapes that are DNase-sensitive; in mature oocytes the nucleoli revert to a spheroidal form. Short term in vitro incorporation studies with uridine-(3)H on both species show that RNA synthesis occurs in a restricted, eccentric portion of the spheroidal nucleoli, thereby producing an asymmetrical pattern of labeling. In the ring forms, however, the localization of the radioactivity suggests that synthesis takes place symmetrically throughout their entire length. The changes in nucleolar morphology apparently reflect the fact that the component DNA has undergone a redistribution from a localized region in the spheroidal nucleoli to an extended circle in the rings; the patterns of uridine-(3)H incorporation, therefore, parallel the distribution of DNA in both the spheroidal and the ring nucleoli. Ultrastructurally, the nucleoli contain a fibrillar component that corresponds in position to that of the DNA. The typical spheroidal nucleolus consists of a fibrillar core situated eccentrically and surrounded by a hull of granular, ribonucleoprotein material. The ring nucleoli are composed of a central fibrous region that is ensheathed all around its circumference by a layer of similar granular material. This granular substance is thicker at intervals along the length of the rings, representing the "beads" of the necklaces.

Distribution of phosphatases in the golgi region and associated structures of the thoracic ganglionic neurons in the grasshopper, Melanoplus differentialis.

The neuronal perikarya of the grasshopper contain sudanophilic lipochondria which exhibit an affinity for vital dyes. These lipochondria are membrane-delimited and display acid phosphatase activity; hence they correspond to lysosomes. Unlike those of most vertebrates, these lysosomes also hydrolyze thiamine pyrophosphate and adenosine triphosphate. Like vertebrate lysosomal "dense bodies," they are electron-opaque and contain granular, vesicular, or lamellar material. Along with several types of smaller dense bodies, they are found in close spatial association with the Golgi apparatus. The Golgi complexes are frequently arranged in concentric configurations within which these dense bodies lie. Some of the smaller dense bodies often lie close to or in association with the periphery of dense multivesicular bodies. Further, bodies occur that display gradations in structure between these multivesicular bodies and the dense lysosomes. Acid phosphatase activity is present in the small as well as the larger dense bodies, in the multivesicular bodies, and in some of the Golgi saccules, associated vesicles, and fenestrated membranes; thiamine pyrophosphatase is found in both the dense bodies and parts of the Golgi complex. The close spatial association of these organelles, together with their enzymatic similarities, suggests the existence of a functional or developmental relationship between them.

Definitive evidence for the existence of tight junctions in invertebrates.

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.

Tight junctions in a fluid-transporting epithelium of an insect.

Occluding junctions have been found between the lateral cell borders at the base of the rectum of Periplaneta americana. They appear as punctate membrane appositions in thin sections, and after incubation in physiological solutions containing lanthanum before fixation the inward penetration of tracer is impeded in this same basal area. Moreover, freeze-fracture studies of this region reveal simple linear ridges on fracture face P and grooves on fracture face E, which are similar to the less complex vertebrate tight junctions. The luminal clefts, which permit free inward diffusion of tracers, present no tight junctions, but do have septate junctions. These results support the contention that, contrary to earlier speculation, arthropods do possess tight junctions; these, rather than septate junctions, appear to form the morphological basis of at least some of the permeability barriers observed in invertebrates.

Certain HLA antigens are associated with specific morphologic and cytogenetic subsets of acute myeloid leukemia.

Although many associations have been found between specific HLA antigens and an increased susceptibility to various diseases, previous attempts to associate class I and II antigens with acute myeloid leukemia (AML) have been inconclusive, probably due in part to the heterogeneity of AML. We subdivided 165 consecutive adults with AML de novo into distinct clinical, morphological, and cytogenetic subsets and then tested for statistically significant associations with specific HLA antigens. Both morphology and cytogenetic pattern identified subsets of patients with important clinical features and different outcomes. Ten statistically significant (P < 0.05) HLA cytogenetic associations were observed: HLA-A11 with t(8;21), A26 with t(15;17), B7 with 11q23 abnormalities, B44 with +8, Cw2 with -20/del(20q), DR3 with t(15;17) and FAB-M3, DR4 with inv(16) and FAB-M4Eo, DQ2 with +8, and DQ6 with +22. HLA-DQ1 had a negative association with -5/del(5q), which was present in 13% of the 165 AML patients overall but in none of the 27 with DQ1. Certain HLA antigens were significantly correlated with more favorable remission rates, remission duration and survival. Possible mechanisms for the association of HLA antigens with particular subtypes of AML include the linkage or co-inheritance of an oncogene, the facilitation of binding of a transforming virus, toxin, or cytokine, or a permissive role involving impaired immune recognition of an emerging neoplasm. Given the heterogeneity of both the HLA system of immune recognition genes and the cytogenetic subtypes of AML, however, larger numbers of patients must be studied to have confidence that biologically important relationships truly exist.

Clinical and prognostic significance of chromosomal abnormalities in childhood acute myeloid leukemia de novo.

We report on the chromosomal pattern of 120 patients with childhood AML de novo. One hundred and fifteen patients (96%) had adequate samples for analysis; 98 (85%) of these showed clonal karyotypic abnormalities. They were classified into cytogenetic subgroups which were closely correlated with FAB subtypes: t(8;21) and M2 (n = 9); t(15;17) and M3 (n = 12); inv(16) and M4Eo (n = 9); t(9;11) and M5a (n = 10); t(11q23) other than t(9;11) and M4-M5 (n = 11); and t(1;22) and M7 (n = 4). In patients with -7/del(7q) (n = 6), leukemia was preceded by MDS in half of the cases, although they had diverse FAB subtypes. Thirty-seven patients had miscellaneous abnormalities. Despite a high CR rate, patients with t(8;21) had a very poor survival: only one child was event-free at 3 years from diagnosis. One third of patients with t(15;17) died during induction. Those eight who achieved CR fared well: only two relapsed, and six were event-free survivors. Patients with inv(16) had a high remission rate and a long survival: five children were in CR 20 to 136 months. Both groups with t(9;11) and t(11q23) had a high remission rate: however, outcome was superior for the t(9;11) group when compared to either the t(11q23) group (EFS at 3 years +/- SE, 56 +/- 17% vs. 11 +/- 10%, p = 0.07) or to the remaining patients (p = 0.06). Both -7/del(7q) and t(1;22) groups had low CR rates (50%) and poor survival. Cytogenetic analysis identifies clinically distinct subsets of childhood AML and is useful in tailoring treatment for these patients. Favorable cytogenetic groups (t(15;17), inv(16), and t(9;11)) may do well with current therapy protocols, whereas unfavorable groups (t(11q23), t(8;21), -7/del(7q), and t(1;22)) require more effective therapies.

Mechanism of neurogenesis during the embryonic development of a tunicate.

Ascidian and vertebrate nervous systems share basic characteristics, such as their origin from a neural plate, a tripartite regionalization of the brain, and the expression of similar genes during development. In ascidians, the larval chordate-like nervous system regresses during metamorphosis, and the adult's neural complex, composed of the cerebral ganglion and the associated neural gland is formed. Classically, the homology of the neural gland with the vertebrate hypophysis has long been debated. We show that in the colonial ascidian Botryllus schlosseri, the primordium of the neural complex consists of the ectodermal neurohypophysial duct, which forms from the left side of the anterior end of the embryonal neural tube. The duct contacts and fuses with the ciliated duct rudiment, a pharyngeal dorsal evagination whose cells exhibit ectodermic markers being covered by a tunic. The neurohypophysial duct then differentiates into the neural gland rudiment whereas its ventral wall begins to proliferate pioneer nerve cells which migrate and converge to make up the cerebral ganglion. The most posterior part of the neural gland differentiates into the dorsal organ, homologous to the dorsal strand. Neurogenetic mechanisms in embryogenesis and vegetative reproduction of B. schlosseri are compared, and the possible homology of the neurohypophysial duct with the olfactory/adenohypophysial/hypothalamic placodes of vertebrates is discussed. In particular, the evidence that neurohypophysial duct cells are able to delaminate and migrate as neuronal cells suggests that the common ancestor of all chordates possessed the precursor of vertebrate neural crest/placode cells.

Neurogenic role of the neural gland in the development of the ascidian, Botryllus schlosseri (Tunicata, Urochordata).

In adult ascidians, the neural complex consists of a cerebral ganglion (the brain) and the associated neural gland. We have studied the development of the neural complex during the vegetative reproduction of the colonial ascidian Botryllus schlosseri, the buds of which arise from the atrial mantle of the parental zooid. Each bud develops into a new organism within which a neural complex becomes differentiated. We found that the presumptive (pioneer) nerve cells that ultimately form the cerebral ganglion of the adult arise as migratory cells from a primordial cluster of rudimentary gland cells. Hence, the neural gland appears to be neurogenic in that it serves as the cellular source of components that differentiate into conventional nerve cells. In the adult, these cells take on the form of a typical invertebrate ganglion with an outer cortex of nerve cell bodies and an internal medulla. This medulla consists of a neuropile of neuronal processes making classical synaptic contacts. The adult neural gland differentiates into a structure with a ciliated duct that opens into the branchial chamber, the body of the gland, and the dorsal organ, which is quite distinct from the dorsal strand of other ascidians. The rudimentary neural gland cells, therefore, differentiate into one of two distinct pathways: the first, glandular, is possibly involved in the evaluation of environmental signals, and the other, nervous, leads to brain formation. This compares with the vertebrate situation in which the olfactory-pituitary placodes are thought to originate from a common cellular source. Thus, these data support the earlier contention of a homology between the tunicate neural gland and the vertebrate adenohypophysis.

Inhibition of leukotriene B4 synthesis does not prevent development of acute renal failure following storage and transplantation.

Compound BW B70C, a selective 5-lipoxygenase inhibitor was tested for its ability to reduce inflammatory damage in an in vivo rabbit model of renal storage and transplantation. Kidneys were stored at 0-2 degrees C for 48 hr prior to autografting. In controls, renal vein LTB4 levels rose significantly after 30 min reperfusion but fell after 2 hr to baseline. TxB2 levels remained at baseline for the 6 hr measured. 6-k-PGF1 alpha levels rose significantly after 1 hr of reperfusion and remained elevated thereafter. Histology after 6 hr reperfusion showed moderate-to-severe cortical edema and mild congestion. Infused colloidal carbon was retained in the perivascular area in a narrow band at the corticomedullary junction, indicating a zone of vascular permeability. At 3 days after transplant, kidneys exhibited widespread tubular necrosis and calcification but little inflammation. Serum creatinine and urea peaked between days 3 and 5. 3/6 rabbits showed no symptoms of renal failure after 3 weeks. Pretreatment with BW B70C prevented the increase in LTB4 but had little effect on TxB2 and 6-k-PGF1 alpha levels. Histology showed no amelioration of cortical edema at 6 hr and congestion and hemorrhage were exacerbated. BW B70C had no effect on either colloidal carbon retention or distribution but did significantly reduce tubular necrosis and calcification at day 3. There was very little inflammatory infiltrate. BW B70C treatment did not improve the long-term viability of transplanted kidneys: 2/6 rabbits showed no symptoms of renal failure after 3 weeks. These data indicate that inhibition of LTB4 synthesis by BW B70C does not prevent the development of acute renal failure following 48 hr hypothermic storage and transplantation.

Effect of mannitol and polyethylene glycol on the action of frusemide during renal storage and transplantation.

Hypoxic injury is a major cause of tubular necrosis in the corticomedullary junction of isolated perfused kidneys, and is ameliorated by inhibitors of active reabsorption, such as frusemide. Our objective was to determine whether frusemide has a similar effect on hypothermically stored transplanted kidneys and whether this effect is modulated by impermeant solutes included in the preservation solution. The effect of frusemide on cytochrome oxidase (cyt aa3) oxidation, renal hemodynamics, and morphology was investigated in the New Zealand White rabbit renal autograft model using near-infrared spectroscopy and light microscopy. A total of 30 kidneys were autografted in six groups. Kidneys were transplanted with or without frusemide either (1) without storage (groups 1 and 2) or after 72 hr of storage in: (2) hypertonic citrate containing mannitol (groups 3 and 4); and (3) hypertonic citrate containing polyethylene glycol (PEG) (groups 5 and 6). In unstored transplanted kidneys, frusemide infusion stimulated a significant (P < 0.05) increase in hemoglobin oxygenation, compared with untreated controls. There was a tendency for cyt aa3 to become reduced, but there were no significant differences between groups 1 and 2. After 72 hr of storage, frusemide infusion stimulated a significant increase in hemoglobin oxygenation in kidneys stored in mannitol (P < 0.005), but there was no significant change in the kidneys stored in PEG. There was a corresponding reduction in cyt aa3 in kidneys stored in mannitol (P < 0.05) but no change in those stored in PEG. These results suggest that frusemide has a significant effect on cortical hemoglobin oxygenation in transplanted kidneys and on active reabsorption in the corticomedullary junction. The selection of impermeant is important and mannitol is significantly superior to PEG in this model.

Hemoglobin oxygenation kinetics and secondary ischemia in renal transplantation.

The significance of poor medullary reperfusion in the etiology of acute tubular necrosis during renal transplantation is poorly understood. Our objective was to determine the kinetics of renal hemoglobin oxygenation using near-infrared spectroscopy during renal transplantation, to provide a framework against which the timing of mitochondrial dysfunction could be considered. New Zealand White rabbit kidneys were flushed with hypertonic citrate solution (0-2 degrees C and autografted immediately (group 1) or stored at 0-2 degrees C for 72 hours before autografting (group 2). Changes in oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) were monitored by near-infrared spectroscopy for 3 hours of reperfusion. Intrarenal perfusion was evaluated separately by barium sulfate angiography. Reperfusion resulted in rapid increases in HbO2 within 1 minute in both groups. Group 1 HbO2 fell sharply to a minimum at 3 minutes but recovered by 20 minutes; group 2 changes were similar, but there was no recovery (P<0.05 by 10 minutes). Hb increased rapidly in both groups upon reperfusion but in group 2 was significantly greater after 10 minutes (P<0.05). Total hemoglobin levels were similar in both groups. Renal hemoglobin saturation was 69% at 1 minute in both groups; there was no significant change in group 1 but a profound desaturation in group 2 to 25% at 10 minute (P<0.005) and no recovery thereafter. Barium sulfate distribution was normal in all group 1 kidneys; cortical distribution was normal in all group 2 kidneys, but medullary perfusion was poor for the first 60 minutes. Renal hemoglobin oxygenation kinetics as determined here do not correlate with the timing of mitochondrial dysfunction previously reported (Thorniley et al., Kidney International, 1994; 45: 1489). We conclude that secondary ischemia during reflow is not the only mechanism leading to acute tubular necrosis.

Ebselen. Antioxidant capacity in renal preservation.

Ebselen (PZ51) was tested for its ability to inhibit oxidative membrane damage and improve outcome of rabbit kidneys rendered cold ischaemic for 72 hr. In view of the rapid metabolism of ebselen, the antioxidant capacities of its two principal metabolites were first compared with that of the parent drug in an in vitro hepatic microsomal lipid peroxidation system initiated by NADPH/Fe(3+)-ADP. The potent antioxidant activity of ebselen was confirmed but metabolite I (2-glucuronylselenobenzanilide) exhibited no antioxidant potential up to a concentration of 50 microM; metabolite II (4-hydroxy-2-methyl-selenobenzanilide) did inhibit lipid peroxidation but was about 80 times less effective than the parent compound. The storage of rabbit kidneys in hypertonic citrate solution at 0 degrees for 72 hr of cold ischaemia resulted in greatly increased susceptibility to oxidative membrane damage in both the cortex and medulla as determined by the subsequent in vitro formation of two markers of lipid peroxidation (Schiff's bases and thiobarbituric acid-reactive material). Inclusion of ebselen (50 microM) in the flush and storage solution led to a highly significant reduction in these oxidative markers in both regions of the kidney. Intracellular and interstitial oedema was noted in organs subjected to 72 hr cold ischaemia and was reduced by ebselen (50 microM in the flush/storage solution). The rate of post-ischaemic lipid peroxidation was found to correlate well with the extent of oedema in the renal medulla (r = 0.84, P less than 0.001) but no such correlation was found in the cortex. Administration of ebselen (5.5 mg/kg i.v. and 100 microM in the flush/storage solution) did not improve the long-term survival of rabbits following autotransplantation of a single kidney stored for 48 or 72 hr. No protective effect of ebselen could be demonstrated either in terms of graded physiological function or histological outcome.

Morphology of glial blood-brain barriers.

Glial cells, in certain situations in the CNS, may become modified to form the structural basis of the blood-brain barrier. This occurs in more primitive vertebrates, such as the elasmobranch fish, and in some higher invertebrates. In the latter, the outermost glial sheath, often called the perineurium in avascular ganglia, substitutes functionally for the vascular endothelium of higher organisms. The intercellular junctions between the lateral borders of these modified glial or perineurial cells may be of several types. In nearly all cases, adhesive and communicating (gap) junctions are found together with an occluding junctional structure. The latter is assumed to be the morphologic basis of the observed blood-brain barrier. It varies in nature and may be one in which the adjacent cell membranes fuse, partially or completely, to form a classical tight junction, or it may be one in which the cell membranes remain separated by a distinct intercellular cleft. If the latter, the cleft may be straddled by columns or septal ribbons, between which a charged matrix substance may be found. Restrictive linker junctions, recently found to be the basis of the interglial barrier in cephalopod CNS, as well as that of myriapods, are characterized by cross-striations or columns which, in combination with charged residues, inherent either in them or in the associated extracellular matrix, slow down the entry of exogenous molecules. Septate junctions, which occur between glial cells in certain other invertebrates, exhibit intercellular septal ribbons, which do not prohibit paracellular transport of all substances but may slow down the passage of some by virtue of charged moieties. There is an association of cytoskeletal components with these septate, linker, and tight junctions; the role of the cytoskeleton in tight junctions, which can be seen by freeze fracture to be based on simple ridges in insects or a more complex network of them in arachnids, may also be important in the regulation of paracellular permeability. The structural details of the junctions in different groups are summarized and their physiologic implications discussed.

A phase I trial of concomitant chemoradiotherapy with cisplatin dose intensification and granulocyte-colony stimulating factor support for advanced malignancies of the chest.

UNLABELLED: Concomitant chemoradiotherapy with cisplatin and combination chemotherapy in the neoadjuvant setting have both shown promising results. PURPOSE: To identify a locally and systemically active concomitant chemoradiotherapy regimen incorporating high-dose cisplatin, interferon alfa-2a (IFN), fluorouracil (5-FU), hydroxyurea (HU) and radiotherapy. METHODS: Phase I cohort design establishing the maximal tolerated dose (MTD) of cisplatin with and without granulocyte colony stimulating factor (GCSF). For the first six dose levels, a 4-week cycle consisted of escalating doses of cisplatin during weeks 1 and 2, IFN (week 1), and 5-FU and HU (week 2) with single daily radiation fractions of 200 cGy during days 1-5 of weeks 1-3 and no treatment in week 4. When dose-limiting neutropenia was encountered. GCSF was added during weeks 1, 3, and 4. Finally, to decrease esophagitis, the radiotherapy schedule was altered to 150 cGy twice daily during weeks 1 and 2, followed by a 2-week break (level 7). RESULTS: Forty-nine patients with refractory chest malignancies were treated. The MTD of this regimen without GCSF was cisplatin 50 mg/m2 in weeks 1 and 2, IFN 5 million Units (MU)/m2 per day on days 1-5 in week 1, 5-FU 800 mg/m2 per day for 5 days by continuous infusion, and HU 500 mg every 12 h for 11 doses during week 2. The addition of GCSF during weeks 1, 3, and 4 allowed for escalation of cisplatin to 100 mg/m2 during weeks 1 and 2, with a decreased dose of IFN at 2.5 MU/m2 per day to avoid renal toxicity. Dose-limiting toxicity (DLT) included severe neutropenia, thrombocytopenia, and esophagitis in 5 of 13 patients. Increased thrombocytopenia in patients receiving GCSF was not observed. During hyperfractionated radiotherapy (level 7) chemotherapy doses were as above except for a reduction of 5-FU to 600 mg/m2 per day. While severe esophagitis was reduced, grade 4 thrombocytopenia became more prevalent and was seen in 6 of 7 patients. In-field tumor responses were observed in 17 of 28 evaluated patients with non-small-cell lung cancer. The median times to progression and survival were 4 and 6 months, respectively. When only patients with all known disease confined to the radiotherapy field were considered the corresponding times were 6 and 15 months, respectively. Most treatment failures occurred outside of the irradiated field. CONCLUSIONS: (1) This intensive multimodality regimen can be given with aggressive supportive care incorporating GCSF. The recommended phase II doses for a 4-week cycle are cisplatin 50 mg/m2 week 1, and 100 mg/m2 week 2, IFN 2.5 MU, HU 500 mg every 12 h x 11 and 5-FU 800 mg/m2 per day with single fraction radiotherapy during weeks 1-3 and GCSF during weeks 1, 3, and 4. (2) GCSF can be safely administered and provides effective support of neutrophils when administered simultaneously with IFN, cisplatin, and chest radiotherapy. (3) There is synergistic renal toxicity when high doses of IFN and cisplatin are given together. (4) Hyperfractionated radiotherapy decreases the severity of esophagitis but increases thrombocytopenia. (5) Although highly toxic, response rates, time to progression and survival figures with this regimen are encouraging and support its investigation in the phase II setting.

Intercellular junctions and the development of the blood-brain barrier in Manduca sexta.

In early embryonic development of the tobacco horn moth no blood-brain barrier is present, as shown by the unimpeded entry of exogenous tracers into the nervous system. However, later on, just before hatching, lanthanum and horseradish peroxidase (HRP) are unable to move inwardly beyond the level of the perineurium, which is the morphological site of the blood--brain barrier in the adult moth, as well as in other insects. Freeze-fracture studies indicate that in the early embryo, 10 nm particles are scattered about in the perineurial membrane PF, either as separate entities or as short linear arrays. By hatching or just before, however, the 10 nm particles have become aligned into lengthy linear aggregates as PF ridges with EF grooves. These would appear to be the simple, arthropod-form of tight junction, and are presumed to be the basis of the perineurial blood-brain barrier. At about the same time, gap junctional elements appear both between adjacent perineurial cells and between glial cells. In both cell types, the gap junctions form from free 13 nm EF particles which gradually become aligned or clumped into strands and aggregates which ultimately coalesce to form first irregular masses and then the macular plaques typical of mature gap junctions. Many of the latter stages are coincident with the hatching of a motile larvae, so that the perineurial and glial cells are by this stage coupled via the channels of the gap junctional particles. They are therefore able to undergo both ionic and metabolic exchange and cooperation during larval life, in addition to being able to respond to hormonal substances in an integrated way. During the 5 larval instars more gap junctions form as the perineurial layer grows thicker. These junctions become more regular in outline and their particles more tightly packed; these larval structures are compared with junctions found in the adult which tend to be more extensive but otherwise similar. Since no septate junctions are apparent during Manduca embryonic or larval life when the blood-brain barrier forms, nor in adults, the results of this report support the contention that it is the tight junctions rather than septate ones which form the basis of permeability barriers in this, and probably other, arthropod systems.

Physiological and ultrastructural evidence for an extracellular anion matrix in the central nervous system of an insect (Periplaneta americana).

The efflux of radiocations (22Na, 2K and 45Ca) and of radiochloride occur as two-stage processes from intact cockroach nerve cords. It is suggested that the initial, fast fraction of efflux comes mainly from the superficial connective tissue layer, the neural lamella, and the clefts between the underlying layer of neuroglia, the perineurium. This is deduced from the lack of effect of a metabolic inhibitor and sodium-transport inhibitors on the fast component of 22Na efflux (which contrast with their effects both on the size an the half-time of the slow component) and from the typically extracellular ratios between the fast components of substantial increase in the fast fractions of 22Na and 45Ca efflux but only a small increase in 36Cl efflux: effects which would be expected if the addition to the fast fraction consisted of ions maintained in Donnan equilibrium with fixed anionic sites within the extracellular system. The presence of such anionic sites is also indicated by lanthanum-binding in the extracellular matrix and by the previous histochemical demonstration of hyaluronic acid in the matrix by Ashhurst and Costin. It is suggested that the anionic glycosaminoglycans provide an extracellular cation reservoir which could serve a role in short-term ionic homeostasis of the brain microenvironment.

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.

Intramembranous particles in the form of ridges, bracelets or assemblies in arthropod tissues.

Non-junctional intramembranous particle arrays in the form of ridges, bracelets or rectilinear assemblies have been found by freeze-fracturing in the cytoplasmic half or P face of the plasma membrane in a variety of arthropod tissues. These tissues include both excitable cells, nerve and muscle, and such other cells as those from the intestinal tract, the tracheal system and the connective tissue. The intramembranous ridges are short rows of fused particles about 10 nm in diameter; comparable particles comprise the bracelets and the rectilinear aggregates, although the former are of lower profile. In cells sending out cytoplasmic projections during migration and development, for example, axons in embryonic, newly hatched or pupal tissues, tracheoles or fibroblasts, the intramembranous ridges are always aligned parallel to the longitudinal axis of the cellular process. The physiological significance of these may be that they play some role in recognition during development, perhaps by contact guidance. The ridges and rectilinear arrays found in the gut could also be involved in recognition and/or adhesion. In muscle, bead-like ridges are intimately associated with the transverse tubular system and may have a receptor function. Irregular and circular low-profile ridges occur in the tissues of the horseshoe crab, Limulus, and 'bracelet' forms are found in the inner membrane of insect pupal tracheae. The latter may play a part in the initiation and development of small tracheoles.