Stimulation of growth and phospholipase A2 by the peptides mastoparan and melittin and by the auxin 2,4-dichlorophenoxyacetic acid

The peptide mastoparan from wasp venom and the peptide melittin from bee venom stimulated growth in etiolated zucchini (Cucurbita pepo L.) hypocotyls. Both peptides were only effective in hypocotyls with abraded cuticles. At concentrations of 2 g ml^–1 peptide growth was stimulated 72% by mastoparan and 50% by melittin after 2 h as compared to the controls. Mastoparan (5 g ml^–1), melittin (10 g l^–1) and 2,4-dichlorophenoxyacetic acid (5×10^–4 M) stimulated accumulation of ¹⁴C-choline-labeled lysophosphatidylcholine in less than 10 min in cultured soybean cells (Glycine max L.), all to about the same extent. The effects of these peptides are among the first to be reported on plant cells and may be related to important events coupled to growth stimulation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid     

Cytochemical studies on the localization and functional properties of calcium in anterior pituitary cells

Summary The localization of calcium and its functional properties in anterior pituitary cells were studied using a potassium pyroantimonate technique. In all kinds of secretory cells, the precipitates of the calcium-pyroantimonate complex were distributed on the limiting membrane of the secretory granule. They were present also in the cytoplasmic matrix, the mitochondrial matrix, small smooth vesicles, coated vesicles, and in the nuclear euchromatin area. The precipitates were usually seen at the contact region between the limiting membranes of two adjacent secretory granules, or between the granule limiting membrane and the plasma membrane. When the tissues were incubated in the medium containing A23187 (10 M) for 5 min, the deposits on the granule limiting membrane were increased in number and those on the mitochondrial matrix were decreased; the reaction products almost disappeared on the limiting membranes of the secretory granules after membrane fusion following single or multigranular exocytosis induced by A23187-treatment. In addition, small vesicles in the capillary endothelium contained reaction precipitates. Based on these results we propose a hypothetical model for the relationship between the localization of calcium and secretory activity.This study was supported by grants from the Japan Ministry of Education     

Exocytosis in Single Chromaffin Cells: Regulation by a Secretory Granule-Associated Go Protein

1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, Go has beenfound associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells.2. Microinjection of GTPS and mastoparan strongly inhibits the amperometric response to either nicotine or high K⁺.3. The presence of mastoparan in the cell incubation medium had no effect on K⁺-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane.4.Microinjection of anti-Go antibodies potentiates by more than 50% the K⁺-evoked secretion, whereas anti-Gi_1/2 antibodies have no effect.5. Thus an inhibitory Go protein, probably associated with secretory granules, controls exocytosis in chromaffin cells. The intracellular proteins controlling organelle-associated G proteins are currently unknown. The neuronal cytosolicprotein GAP-43 stimulates Go in purified chromaffin granule membranes and inhibits exocytosis in permeabilized cells. We show here that microinjection of a synthetic peptide corresponding to the domain of GAP-43 that interacts with Go inhibits secretion. We suggest that GAP-43 or a related cytosolic protein controls the exocytotic priming step in chromaffin cells by stimulating a granule-associated Go protein.     

Hyperosmolality inhibits exocytosis in sea urchin eggs by formation of a granule-free zone and arrest of pore widening

Summary Hyperosmolality is known to inhibit membrane fusion during exocytosis. In this study cortical granule exocytosis in sea urchin eggs is used as a model system to determine at what step this inhibition occurs.Strongylocentrotus purpuratus eggs were incubated in hyperosmotic seawater (Na₂SO₄, sucrose or sodium HEPES used as osmoticants), the eggs activated with 20 m A23187 to trigger exocytosis, and then quick frozen or chemically fixed for electron microscopy. Thin sections and freeze-fracture replicas show that at high osmolality (2.31 osmol/kg), there is a decrease in cortical granule size, a 90% reduction in granule-plasma membrane fusion, and formation of a granulefree zone between the plasma membrane and cortical granules. This zone averages 0.64 m in thickness and prevents the majority of granules from docking at the plasma membrane. The remaining granules (10%) exhibit early stages of fusion which appear to have been stabilized; the matrix of these granules remains intact. We conclude that exocytosis is blocked by two separate mechanisms. First, the granule-free zone prevents granule-plasma membrane contact required for fusion. Second, in cases where fusion does occur, opening of the pocket and dispersal of the granule contents are slowed in hyperosmotic media.     

Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding,G proteins,phosphoinositide breakdown,cyclic AMP generation,and calcium influx

Summary 1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A₁-adenosine receptors.2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTPS to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins.3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin.N-Ethylmaleimide inhibits the response to both peptides.4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTPS-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects.5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered byN-ethylmaleimide.6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.     

Human non-lineage antigen,CD46 (HuLy-m5): purification and partial sequencing demonstrates structural homology with complement-regulating glycoproteins

CD46, until recently known as HuLy-m5, is a non-lineage restricted surface antigen ubiquitously expressed by almost all human cells except erythrocytes. The CD46 antigen is identified by the E4.3 monoclonal antibody (mAb) and exists at the surface of human peripheral blood lymphocytes (PBLs) as two acidic, non-disulfide bonded chains, and , ofM _r 66 000 and 56 000. Receptor density analysis showed that CD46 was of moderately low abundance on PBLs with 7.5×10³ molecules present on each cell. The two chains of CD46 were purified (144 000-fold) by immunoaffinity-chromatography with E4.3 mAb from the plasma membranes of a human spleen infiltrated with chronic myelogenous leukemia cells. Amino acid sequence analysis of the NH₂-terminal of both and chains yielded the same sequence; XEEPPQ/TFEAMELIGKPKPYYEIGE. Peptide mapping studies confirmed that both CD46 chains were closely related, except for one peptide fragment. This amino acid sequence is identical to that of the NH₂-terminal of the recently cloned membrane co-factor protein (MCP), a membrane protein that binds the C3b and C4b fragments of complement and acts as a co-factor for I protein-mediated decay of the complement convertases. CD46 shares a cross-reactive epitope with some primate retroviruses, and this may indicate that some retroviruses mimic the mechanisms used by autologous human cells to evade complement-mediated immune clearance. Offprint requests to: I. F. C. McKenzie.     

DNA variations in regenerated plants of pea (Pisum sativum L.)

Summary The aim of this study was to determine whether DNA variations could be detected in regenerated pea plants. Two different genotypes were analyzed by cytogenetic and molecular techniques: the Dolce Provenza cultivar and the 5075 experimental line. Dolce Provenza regenerated plants showed a reduction in DNA content, particularly at the level of unique sequences and ribosomal genes. Moreover, regeneration was associated with an increase in DNA methylation of both internal and external cytosines of the CCG sequence. On the other hand, the DNA content of the 5075 line remained stable after regeneration. DNA reduction was found only in 5075 plants regenerated from callus cultures maintained for long incubation periods (about a year). The DNA variations observed are discussed both in relation to the genotype source and the role of tissue-culture stress.     

Ultrastructure of the rat posterior pituitary gland and evidence of hormone release by exocytosis as revealed by freeze-fracturing

Summary Posterior pituitary glands from normal rats, and rats which had been deprived of water for varying periods, were examined by the freeze-fracture method. This technique reveals large areas of the nerve cell membrane. Images consistent with exocytosis as the mechanism of release of the neurohypophysial hormones were observed. These modifications were most numerous after the rat had been starved of water for 2 days.In normal rats, the large number of neurosecretory granules within the nerve fibres caused a bulging of the nerve cell membrane. The bulges disappeared 2 days after removal of drinking water. Regions of the membrane displaying bulges were characterised by the absence of the typical membrane-associated particles.It is postulated that the close proximity of the neurosecretory granules to the nerve cell membrane may result in rapid fusion of the neurosecretory granules on stimulation of the gland. The change in properties of the nerve cell membrane overlying the neurosecretory granules, as suggested by the loss of membrane-associated particles, may represent a change in the structure of the membrane to a form which is more favourable for fusion.This work was supported in part by a grant from the New Zealand Medical Research Council.     

Characterization of Melittin Effects in Synaptosomes

The effects of melittin at increasing concentrations on: [³H]GABA release from mouse brain synaptosomes; on the radioactivity released from [³H]arachidonic acid labeled synaptosomal membranes; on synaptosomes ultrastructure and on the leakage of the cytoplasmic marker, lactate-dehydrogenase (LDH) was investigated. Melittin 0.3, 1, 3, 7, and 10 M progressively increases [³H]GABA release, but the efficacy of melittin is decreased when the amount of tissue exposed to a constant concentration of the toxin increases. The release of [³H]GABA induced by melittin below 3 M is Ca²⁺ dependent, but not that induced by the higher concentrations. The Ca²⁺ dependent fraction of the [³H]GABA released by 0.3 M melittin is selectively inhibited by 10 M quinacrine and 1 M nordihydroguaiaretic acid (NDGA) and facilitated by 3 M indomethacin, whereas the Ca²⁺ independent fraction of the [³H]GABA released by melittin is not. In the presence of Ca²⁺, melittin 0.3, 1 and 10 M progressively increases [³H]arachidonic acid release over control release, but the effectiveness of melittin is also decreased as the amount of tissue increases. No apparent changes in synaptosomes ultrastructure are observed in 0.3 M treated synaptosomes, but a noticeable disorganization is produced in 10 M melittin-treated synaptosomes, independently on the presence of external Ca²⁺. LDH activity only increases over control activity in the supernatant solutions of 10 M melittin treated synaptosomes, also in a Ca²⁺ independent manner. Our interpretation of these results is that the Ca²⁺-dependent, pharmacologic sensitive component of melittin-induced release of [³H]GABA, unmasked when 0.3 M melittin was used, involves the activation of a Ca²⁺-dependent type of membrane PLA₂. The Ca²⁺-independent release of [³H]GABA is in contrast, highly probable to be due to the membrane perturbation produced by complex melittin/lipid interactions.     

Gonadectomy induces laminin biosynthesis and basement membrane assembly in anterior pituitary glands of adult rats

Summary Laminin biosynthesis and basement membrane assembly in anterior pituitary glands of gonadectomized rats were studied by immuno-electron microscopy and radioimmunoassay. Three weeks after gonadectomy, rats received intravenous injections of sheep anti-laminin IgG conjugated to horseradish peroxidase, and glands were fixed and processed for microscopy 1 h later. Peroxidase reaction product uniformly labeled all perivascular and glandular epithelial basement membranes. In addition, reaction product was also found in abnormally multi-layered basement membranes seen especially beneath gonadotrophs, and unusual basement membrane-like structures projecting between gonadotrophs were also labeled. Pituitary sections from gonadectomized rats labeled with pre-embedding immunoperoxidase and post-embedding immungold techniques also localized intracellular laminin within biosynthetic organelles and light body vesicles of gonadotrophs. Neither abnormal basement membrane structures nor intracellular laminin were detected in pituitaries of nongonadectomized, control rats. Radioimmunoassays of pituitary homogenates showed nearly twice as much soluble laminin ( 15 ng/gland) in gonadectomized rats than in controls ( 8 ng/gland), which paralleled gland growth, but serum laminin concentrations did not differ ( 10 ng/ml in both groups). When anterior pituitary glands of gonadectomized rats that received injections of anti-laminin IgG-HRP were fixed 5 days after injection, lengths of unlabeled basement membrane were distributed between labeled lengths. This indicated that new basement membrane was spliced into old by a process similar to that seen in normal development. Supplementation of gonadectomized rats with testosterone, however, arrested laminin biosynthesis and basement membrane assembly and reversed glandular hypertrophy. These results indicate that, in an absence of sex hormone feedback, renewed synthesis of basement membrane components occurs in the anterior pituitary and is probably necessary to support the additional growth and differentiation of gonadotrophs and other pituitary cells.     

Ultrastructural aspects of the effect of calcium ionophore A23187 on incubated anterior pituitary cells of rats

In order to study the fine structural effect of calcium influx on secretory activity of rat anterior pituitary cells, small pieces of anterior pituitary were incubated in Krebs' medium containing the calcium ionophore A23187 (0.15 mM) and were examined electron microscopically. Marked changes were present in all types of secretory cells incubated for 3, 12 and 20 min in the medium containing calcium and A23187. Secretory granules tended to accumulate in the peripheral cytoplasm of the secretory cells, and more numerous images of granule release by exocytosis were observed in somatotroph (STH cell), luteotroph (LTH cell), thyrotroph (TSH cell), corticotroph (ACTH cell), type 1 gonadotroph (Type 1 GTH cell), and type 2 gonadotroph (Type 2 GTH cell). In addition to the increase in the number of exocytosis of single granules, the simultaneous extrusion of multiple granules, "multigranular exocytosis", was often observed in all kinds of secretory cells, especially the ACTH-cells. Large numbers of granule cores were often located in large vacuole-like or channel-like structures, irregular in shape and size, which were open to the intercellular or pericapillary space. Some parts of the membrane of the vacuole-like or channel-like structures were coated. These observations are interpreted to suggest that the calcium influx stimulates the extrusion of the secretory granules by single or multigranular exocytosis.     

Localization and function of an FMRFamide-like substance in the aorta of Helix aspersa

Summary With an antiserum (aFM) against the molluscan cardio-active FMRFamide (Phe-Met-Arg-Phe-NH₂) numerous immunoreactive axons were found in the outer, longitudinal, muscle layer of the anterior aorta of Helix aspersa. Immunoreactive axons were rare in the inner, circular, muscle layer. At the ultrastructural level four types of axons could be distinguished. The granules containing the immunoreactive substance (mean diameter ca. 100 nm) are present in type-2 axons. The effect of synthetic FMRF-amide was tested in vitro on preparations of ring- and tubule-shaped pieces of the anterior aorta. Physiological doses (3 × 10^-7 M) provoked contractions of the circular muscle fibres, but had no effect on the longitudinal muscle cells. Apparently in vivo the FMRF-like substance diffuses from the richly innervated longitudinal muscle layer to the circular muscle layer, where it exerts its effect. This conclusion is sustained by the observation that the contents of the aFM-immunoreactive granules in type-2 axons are released by exocytosis in a non-synaptic fashion.     

Possible sites of release of neurosecretory granules in the sinus gland of the crayfish,Orconectes nais

Summary The sinus gland is a neurohemal organ located in the crayfish eyestalk and represents a storage site for neurohormones prior to their release into the circulation. The sinus gland contains three classes of electron dense, membrane-limited granules. Class 3 granules are the largest and most electron dense of the granules found in the sinus gland. Granules of class 1 are the smallest, while those of class 2 are the most abundant. Generally, all granules undergo similar changes during their release.Release of neurosecretory material may be initiated by a preliminary fragmentation of the parent granules into smaller granules. Following the formation of numerous smaller granules, these move to the plasma membrane and their limiting membrane apparently fuses with it thus releasing its contents into the external lamina which is applied to the sinusoidal surface of the axon terminals. Granule release does not appear to occur along the entire plasma membrane adjacent to the blood sinus but, instead, probably occurs only at specific active sites on the membrane. The active sites are characterized in part by an accumulation of small granules and clear vesicles against the cytoplasmic side of the plasma membrane. At the site of release of the neurohormone, there is often an accumulation of dense homogeneous material beneath the axolemma.Occasionally, axon endings filled with large, electron lucent vesicles are seen. These clear granules vary from 1150–1750 Å in diameter and often exhibit broken limiting membranes. Few small vesicles are seen near the plasma membrane of these endings; however, instances of invaginations of the plasma membrane occur. The significance of endings filled with clear granules is discussed.Supported by a grant from the National Research Council of Canada (No. A-4675).     

Sensory innervation of the tentacles of the polychaete,Sabella pavonina

Summary Following observation of conical groups of stiff, but motile cilia on the tentacles of the branchial crown of Sabella pavonina, these were examined with the electron microscope. The bundles consist of about 40 unenclosed standard cilia supported by one or two primary sense cells with centrally directed axons of 0.1–0.2 diameter. Axons in the distal portions of the branchial crown occur in small bundles surrounded by a basement membrane. More centrally, glial elements appear and the nerves are surrounded by a collagenous sheath. The branchial nerve trunk shows similarities in organisation to other previously investigated annelid central nervous tissue in that the whole nerve is surrounded by a fibrous sheath central to which there is a layer of glial cells with processes penetrating a central neuropile. The 0.1–0.2 axons commonly occur in glial-enveloped groups of < 40 whilst other axons of larger and mixed diameter are found together.Each tentacle has two branchial nerves on the oral side, and each nerve gives rise to two small 75-axon branches running to each pinnule. The branchial nerves fuse to form the branchial nerve trunk running to the supra-oesophageal ganglia.Sections of the branchial nerves of the branchial crown at progressively more central levels show that the branchial nerve trunk contains enough axons of 0.1–0.2 diameter to account for all the sensory cells on the tentacles. This is taken as evidence for the sensory cells having axons terminating within the central nervous system and that there is no peripheral confluence or fusion of these afferent axons.     

Measurements of electrical potential differences across yeast plasma membranes with microelectrodes are consistent with values from steady-state distribution of tetraphenylphosphonium inPichia humboldtii

Summary Electrical potential differences across the plasma membrane () of the yeastPichia humboldtii were measured with microelectrodes (filled with 0.1m KCl) inserted into cells immobilized in microfunnels. The registered signals were reproducible and stable for several minutes. On attainment of stable reading for the specific membrane resistanceR _sp was determined by applying square-current pulses to the preparation. Both andR _sp were pH dependent and displayed equal but opposite deflection, reaching its maximal value of –88±9 mV (n=13) andR _sp its minimal value of 10 k·cm² (maximal conductance) at pH 6.5. Uncouplers and the polyene antibiotic nystatin depolarized the cells, decreasing to –21±15 mV (n=10) with concomitant decrease ofR _sp. Comparison of values from microelectrode measurements with those calculated from the steady-state distribution of tetraphenylphosphonium ions agreed within 10 mV under all physiological conditions tested, except at pH values above 7.0. During microelectrode insertion transient voltage signals (a few msec long) were detected by means of an oscilloscope. These voltage signals were superimposed on the stable recordings described above. These short voltage signals disappeared in uncoupled cells. The closely related values obtained by two independent methods (direct measurements with microelectrodes and calculation from steady-state distribution of a lipophilic cation) provide evidence that these values reffect the true membrane potential of intact cells.     

Free Ia Eα chain expression in the E α + :E β − : recombinant strain A.TFR5

Molecular and biochemical techniques have been used to explore the reasons behind low E chain expression in the E ⁺ E ^– I-region recombinant strain, A.TFR5. A.TFR5 (A ^f E ^k, ap5), a recombinant between A.CA (A ^f E ^f) and A.TL (A ^k E ^k), carries the E ^k subregion. Previous results have shown that it expresses the E chain, but at reduced levels relative to E ⁺ E ⁺ strains. No E chains were detected, which is consistent with the A.TFR5E gene being derived from the A.CA parent, which carries the null E ^f allele. In this paper, the defect in E-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of E, in the region of the large intervening sequence of E. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the E message, but no E message. Two-dimensional gel analysis of 15 min pulse-labeled A.TFR5, A.CA, and A.TL E immunoprecipitates shows decreased levels of the intracellular E chain in A.TFR5 relative to A.TL. However, analysis of total cell extracts shows normal levels of this protein. A glycoprotein fraction isolated from total cell extracts of 5 h labeled cells contains normal amounts of intracellular E, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of E, the E chain (1) takes on an altered conformation that is not as efficiently recognized by alloantibodies, and (2) is found in normal levels as the partially glycosylated intracellular precursor, but is not processed and/or transported efficiently to the cell surface.     

The hypothalamo-hypophysial system in acipenseridae

Summary The neurohypophysis of sexually mature male and female Acipenser güldenstädti Brandt and Acipenser stellatus Pallas was studied light and electron microscopically. The recessus hypophysei lined with ependymal cells of two main types, narrow and wide, are in the center of the neurohypophysial roots. Processes from both cell types run radially to the basement membrane of the connective tissue layers abutting on the hypophysial intermediate lobe. Protrusions penetrating deep into the recessus hypophyseus are found in the apical parts of the wide cells. Pituicytes are rare in the neurohypophysis. The ultrastructure of both ependymal cell types and of the pituicytes is described. Nonmyelinated Gomori-positive (peptidergic) neurosecretory A₁ and A₂ type fibres and their terminals containing elementary neurosecretory granules (1400–1800 Å and 1000–1400 Å respectively) are the main structural elements of the neurohypophysis. Some dark and single myelinated neurosecretory fibres have been found. The adrenergic fibres (type B) were described earlier (Polenov et al., 1972a). The structural peculiarities of the neurohypophysis are discussed in functional and comparative-morphological terms.     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.     

Unique features of secretory granules observed in the pituitary growth hormone-secreting (GH) cells of the musk shrew (Suncus murinus L.)

Summary Unique rod-shaped secretory granules were observed among oval or spherical secretory granules in GH cells of the anterior pituitary gland of musk shrew using the protein A-gold procedure combined with electron microscopy. The rod-shaped and spherical secretory granules were both immunoreactive by the immuno-gold method using antiserum to sheep GH. The rod-shaped secretory granules, which seem to be formed directly from the Golgi vesicles, extend from several hundred to several thousand nm in length. They often touch each other and fuse. The spherical secretory granules are also unique in that they may also fuse with loss of dense contents to leave empty circular membrane profiles in the cytoplasm. Both the rodshaped and spherical secretory granules are secreted from the cell by exocytosis.     

Synthesis of an analogue of the lipoglycopeptide membrane intermediate I of peptidoglycan biosynthesis

-Dihydroheptaprenyl-pyrophosphoryl- N-acetylmuramoyl-L-Ala--D-Glu- meso-diaminopimeloyl(N-dansyl)-D-Ala-D-Ala ( 1), an analogue of lipid I ofpeptidoglycan biosynthesis, was synthesized from natural UDP-N-acetylmuramoyl-pentapeptide in three steps. Compound 1 was shown to be asubstrate for the MurG transferase from Escherichia coli, even in theabsence of membranes. When membranes were present, dansylated peptidoglycanwas also formed.