Localization and distribution of Microccus lysodeikticus membrane ATPase determined by ferritin labeling

Antiserum to Ca²⁺-activated ATP phosphohydrolase (EC 3.6.1.3) isolated and purified from membranes of Micrococcus lysodeikicus was prepared in rabbits and guinea pigs. The γ-globulin fractions of these antisera reacted with and inhibited ATPase activity in isolated membranes but failed to absorb to intact protoplasts or purified mesosome fractions. ATPase activity was not detectable in the purified mesosomal preparations and trypsin treatment and sonication failed to release any activity. Ferritin conjugated to the γ-globulin fractions of the antiserum reacted with the ATPase particles on the membrane as visualized in negatively stained preparations examined in the electron microscope. Labeled membranes showed a distribution of ferritin very similar to the patterns observed for ATPase particles on untreated membranes. No significant labeling occurred when the ferritin conjugate was reacted with intact protoplasts or mesosome fractions. Thin sections of ferritin-labeled membranes established the asymmetric disposition of the ATPase, with the conjugate visible on only one side of the membrane. The results indicate that the ATPase protein occurs on the inner face of the membrane. All labeling experiments were verified immunologically. When ferritin-labeled membranes were subjected to the selective release procedure used in releasing the ATPase-like particles from the membranes, a complex of ferritin-conjugate associated with the ATPase particles was released. The selective release of ferritin-antibody-enzyme complexes from the membrane opens up a new way of studying the molecular architecture of cell membranes.     

Role of the cro gene in bacteriophage lambda development

Previous experiments have shown that the product of the cro gene of baeteriophage λ can exert an anti-repression activity, defined by the capacity of certain “cro-constitutive” defective lysogens to channel a superinfecting λ phage toward lytic development. We have used a combination of biological and biochemical assays to draw two main conclusions concerning this anti-repression activity: (1) after infection of a cro-constitutive cell, the superinfecting phage is unable to establish repression because it is unable to commence synthesis of cI protein (λ repressor) at a substantial rate; (2) the cause of this diminished synthesis of cI protein is the capacity of cro product to repress synthesis of the cII and cIII proteins, which normally activate the cI gene to establish repression in an infected cell. From our experiments and those of others, we suggest that cro product possesses a repression activity which is similar to that of the cI protein itself, but normally exerts a very different physiological role: the turnoff of synthesis of replication, recombination and regulation proteins as the virus enters the late stage of lytic development.     

Effect of time post mortem on the concentration of endotoxin in rat organs: implications for sudden infant death syndrome (SIDS)

The aim of the study was to test the following hypotheses: (i) that endotoxin injected 40 min prior to death can be detected in rat organs post mortem and (ii) that endotoxin levels do not change with increasing time post mortem. Rats were injected with or without endotoxin in buffered saline, 40 min prior to being killed. Endotoxin levels in rat organs were assessed using a Limulus amoebocyte assay. The effect of storage time post mortem was assessed by following various storage regimes at 25 degrees C and 8 degrees C. Significant differences (P = < 0.001) in endotoxin levels of all samples tested were found between rats injected with and without endotoxin. A significant increase in detectable endotoxin was observed between 0 h and 6 h post mortem in rats injected with or without endotoxin. No difference in detectable endotoxin levels in the kidney, liver and spleen was observed from 30 h to 102 h post mortem in rats injected with or without endotoxin. In rats injected with endotoxin, detectable endotoxin levels in the heart were raised between 0 h and 6 h, 6 h and 54 h, and 30 h and 78 h. Endotoxin injected into rats 40 min prior to death can be detected post mortem. For rats injected with saline or endotoxin prior to death levels in the kidney, liver and spleen were not affected by storage at 8 degrees C for 30-102 h, after initial storage at room temperature for 6 h. Levels of endotoxin detected in the hearts of rats injected with saline were not affected by storage up to 102 h. In rats injected with endotoxin prior to death, detectable levels in the heart were significantly affected by increasing time in storage.     

Naturally occurring CCR5 extracellular and transmembrane domain variants affect HIV-1 Co-receptor and ligand binding function.

Analysis of CCR5 variants in human immunodeficiency virus, type 1 (HIV-1), high risk cohorts led to the identification of multiple single amino acid substitutions in the amino-terminal third of the HIV-1 co-receptor CCR5 suggesting the possibility of protective and permissive genotypes; unfortunately, the low frequency of these mutations did not led to correlation with function. Therefore, we used analytical methods to assess the functional and structural significance of six of these variant receptors in vitro. These studies showed three categories of effects on CCR5 function. 1) Mutations in the first extracellular domain of CCR5 severely reduce specific ligand binding and chemokine-induced chemotaxis. 2) An extracellular domain variant, A29S, when co-expressed with CD4, supported HIV-1 infection whereas the others do not. 3) The transmembrane region variants of CCR5 support monotropic HIV-1 infection that is blocked by addition of some receptor agonists. Mutations in the first and second transmembrane domains increase RANTES (regulated on activation normal T-cell expressed) binding affinity but did not affect MIP1beta binding affinity presumably based on differences in ligand-receptor interaction sites. Furthermore, the CCR5 transmembrane mutants do not respond to RANTES with the classical bell-shaped chemotactic response curve, suggesting that they are resistant to RANTES-induced desensitization. These data demonstrate that single amino acid changes in the extracellular domains of CCR5 can have profound effects on both HIV-1 co-receptor and specific ligand-induced functions, whereas mutations in the transmembrane domain only affect the response to chemokine ligands.     

CEP-1347/KT7515 prevents motor neuronal programmed cell death and injury-induced dedifferentiation in vivo

CEP-1347, also known as KT7515, a derivative of a natural product indolocarbazole, inhibited motor neuronal death in vitro, inhibited activation of the stress-activated kinase JNK1 (c-jun NH terminal kinase) in cultured spinal motor neurons, but had no effect on the mitogen-activated protein kinase ERK1 in these cells. Results reported here profile the functional activity of CEP-1347/KT7515 in vivo in models of motor neuronal death or dedifferentiation. Application of CEP-1347/KT7515 to the chorioallantoic membrane of embryonic chicks rescued 40% of the lumbar motor neurons that normally die during the developmental period assessed. Peripheral administration of low doses (0.5 and 1 mg/kg daily) of CEP-1347/KT7515 reduced death of motor neurons of the spinal nucleus of the bulbocavernosus in postnatal female rats, with efficacy comparable to testosterone. Strikingly, daily administration of CEP-1347/KT7515 during the 4-day postnatal window of motor neuronal death resulted in persistent long-term motor neuronal survival in adult animals that received no additional CEP-1347/KT7515. In a model of adult motor neuronal dedifferentiation following axotomy, local application of CEP-1347/KT7515 to the transected hypoglossal nerve substantially reduced the loss of choline acetyl transferase immunoreactivity observed 7 days postaxotomy compared to untreated animals. Results from these experiments demonstrate that a small organic molecule that inhibits a signaling pathway associated with stress and injury also reduces neuronal death and degeneration in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 361–370, 1998     

Androgens rescue avian embryonic lumbar spinal motoneurons from injury‐induced but not naturally occurring cell death

The regulation of survival of spinal motoneurons (MNs) has been shown to depend during development and after injury on a variety of neurotrophic molecules produced by skeletal muscle target tissue. Increasing evidence also suggests that other sources of trophic support prevent MNs from undergoing naturally occurring or injury‐induced death. We have examined the role of endogenous and exogenous androgens on the survival of developing avian lumbar spinal MNs during their period of programmed cell death (PCD) between embryonic day (E)6 and E11 or after axotomy on E12. We found that although treatment with testosterone, dihydrotestosterone (DHT), or the androgen receptor antagonist flutamide (FL) failed to affect the number of these MNs during PCD, administration of DHT from E12 to E15 following axotomy on E12 significantly attenuated injury‐induced MN death. This effect was inhibited by cotreatment with FL, whereas treatment with FL alone did not affect MN survival. Finally, we examined the spinal cord at various times during development and following axotomy on E12 for the expression of androgen receptor using the polyclonal PG‐21 antibody. Our results suggest that exogenously applied androgens are capable of rescuing MNs from injury‐induced cell death and that they act directly on these cells via an androgen receptor‐mediated mechanism. By contrast, endogenous androgens do not appear to be involved in the regulation of normal PCD of developing avian MNs. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 585–595, 1999     

Temporin/VesCP (T/V)-like antibiotic peptides, derived from frogs and wasps, induce migration of human monocytes and neutrophils

Summary  Several naturally occurring antimicrobial peptides, from mammals and insects, have previously been shown to be chemotactic for human inflammatory cells. Based on this evidence, ten synthetic analogs of naturally occurring antibiotic peptides from the skin secretions of three species of Ranid frogs and the venom of one species of Vespid wasp (i.e., T/V-like peptides) were tested for their abilities to induce migration of human neutrophils and monocytes. These included temporin A (TA fromRana temporaria), temporin 1P (T1P fromR. pipens), ranateurin 6 (Rana-6 fromR. catesbeiana)], three TA analogs [all D-amino acids (D-TA), reversed sequence (Rev-TA), and Pro3→Gly (G3-TA)], two frog skin-related T/V-like peptide consensus sequences (I4S10-Con and I4G10-Con), VesCP-M (VCP-M fromVespa mandarinia), and a hybrid peptide composed of portions of the insect antibiotic peptide, cecropin A (CA), and TA (CATA). TA, T1P, Rana-6, VCP-M, G3-TA, I4S10-Con, I4G10-Con, and CATA all induced cell migration at micromolar concentrations. D-TA and Rev-TA did not induce cell migration, suggesting that this process involves a chiral interaction, such as receptor binding, and also depends on the order of amino acids within TA. The results demonstrate, for the first time, that certain T/V-like antibiotic peptides are capable of inducing chemotaxis of human phagocytes and suggest that these peptides are multifunctional molecules with antimicrobial, hemolytic, and chemotactic capabilities.     

Temporin/VesCP (T/V)-like antibiotic peptides,derived from frogs and wasps,induce migration of human monocytes and neutrophils

Summary Several naturally occurring antimicrobial peptides, from mammals and insects, have previously been shown to be chemotactic for human inflammatory cells. Based on this evidence, ten synthetic analogs of naturally occurring antibiotic peptides from the skin secretions of three species of Ranid frogs and the venom of one species of Vespid wasp (i.e., T/V-like peptides) were tested for their abilities to induce migration of human neutrophils and monocytes. These included temporin A (TA fromRana temporaria), temporin 1P (T1P fromR. pipens), ranateurin 6 (Rana-6 fromR. catesbeiana)], three TA analogs [all D-amino acids (D-TA), reversed sequence (Rev-TA), and Pro3→Gly (G3-TA)], two frog skin-related T/V-like peptide consensus sequences (I4S10-Con and I4G10-Con), VesCP-M (VCP-M fromVespa mandarinia), and a hybrid peptide composed of portions of the insect antibiotic peptide, cecropin A (CA), and TA (CATA). TA, T1P, Rana-6, VCP-M, G3-TA, I4S10-Con, I4G10-Con, and CATA all induced cell migration at micromolar concentrations. D-TA and Rev-TA did not induce cell migration, suggesting that this process involves a chiral interaction, such as receptor binding, and also depends on the order of amino acids within TA. The results demonstrate, for the first time, that certain T/V-like antibiotic peptides are capable of inducing chemotaxis of human phagocytes and suggest that these peptides are multifunctional molecules with antimicrobial, hemolytic, and chemotactic capabilities.     

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.Cholinergic neurotransmission has key functions in life, as it regulates several central and peripheral nervous system outputs. Acetylcholine (ACh) is synthesized in the cytoplasm by the enzyme choline acetyltransferase (ChAT) (16). Choline supplied by the high-affinity choline transporter (CHT1) is required to maintain ACh synthesis (52). A lack of ChAT (4, 35) or the high-affinity choline transporter (21) in genetically modified mice is incompatible with life. ACh plays an important role in wiring the neuromuscular junction (NMJ) during development (38, 43). Embryonic synthesis of ACh is fundamental for the development of proper nerve-muscle patterning at the mammalian NMJ, as ChAT-null mice present aberrant nicotinic ACh receptor (nAChR) localization and increased motoneuron (MN) survival, axonal sprouting, and branching (4, 35).The vesicular ACh transporter (VAChT) exchanges cytoplasmic ACh for two vesicular protons (37, 41). Previously reported electrophysiological studies showed that quantal size is decreased by vesamicol, an inhibitor of VAChT, but only in nerve terminals that have been electrically stimulated (19, 59, 60, 63). VAChT overexpression in developing Xenopus MNs increases both the size and frequency of miniature-end-plate currents (54). In Caenorhabditis elegans, mutations in VAChT affect behavior (65). Moreover, a decrease in VAChT expression has functional consequences for mammals, as mutant mice with a 70% reduction in the expression levels of this transporter (VAChT knockdown [KD^HOM] mice) are myasthenic and have cognitive deficits (47). Hence, vesicular transport activity is rate limiting for neurotransmission “in vivo” (18, 47).Exocytosis of synaptic vesicle contents is the predominant mechanism for the regulated secretion of neurotransmitters (55). However, alternative mechanisms of secretion have been proposed (20, 56, 61). Quantal ACh release, comparable to that seen in developing nerve terminals, has been detected in myocytes and fibroblasts in culture, which presumably do not express VAChT (14, 24). More recently, it was found that the correct targeting of Drosophila photoreceptor axons is disrupted in flies with null mutations in ChAT (64). Remarkably, the inactivation of VAChT did not produce the same result (64). The result suggests that the release of ACh during development is not dependent on VAChT, perhaps because it is nonvesicular or because vesicular storage can occur without VAChT.To test if the VAChT-independent secretion of ACh has any physiological role in the mammalian nervous system, we generated a mouse line in which the VAChT gene is deleted. These mice lack the stimulated release of ACh from synaptosomes, die after birth, and show several alterations in neuromuscular wiring consistent with a severe decrease in the cholinergic input to muscles during development. These experiments indicate that VAChT has an important role in maintaining activity-dependent ACh release that supports life and the correct patterning of innervation at the NMJ.