Aza-peptides. III. Experimental structural analysis of aza-alanme and aza-asparagine-containing peptides

To determine the structural perturbations induced by the CαH→Nα exchange in aza-peptides, we have examined by H NMR and IR spectroscopy various derivatives of the aza-analogues of alanine, aspartic acid and asparagine in different organic solvents with increasing polarity. Their general formulas are: R'-AzXaa-NR²R³, R'-Pro-AzXaa-NR²R³ and R-AzXaa-Pro-NR²R³ (where AzXaa denotes the aza-analogue of the amino acid residue Xaa = Ala, Asp, Asn; R = Boc, Z; R², R³= H, Me, iPr). The aza-analogue of an amino acid residue appears to be a strong p-turn-inducing motif, and the AzAsn carboxamide side-chain is capable of interacting, as a proton donor, with the preceding peptide carbonyl group.     

C-terminal tail of β-adrenergic receptors: immunocytochemical localization within astrocytes and their relation to catecholaminergic neurons in N. tractus solitarii and area postrema

β-Adrenergic receptors (βAR) in the medial nuclei of tractus solitarii (m-NTS) and area postrema (AP) may bind to catecholamines released from neurons, whereas only the AP has fenestrated capillaries allowing access to circulating catecholamines. Since varied autonomic responses are seen following βAR activation of the dorsal vagal complex, including the m-NTS and AP, we hypothesized that there might be a cellular basis for varied responses to βAR stimulation that depends pn the differential access to circulating catecholamines. Therefore, we comparatively examined the ultrastructural localization of the βAR in relation to catecholaminergic neurons in these regions. An antibody directed against the C-terminal tail (amino acids 404–418) of hamster β-adrenergic receptor (βAR404) was used in this study. The localization of βAR404 was achieved by the avidin-biotin peroxidase complex (ABC) technique in combination with a pre-embed immunogold labeling method to localize tyrosine hydroxylase (TH), the catecholamine-synthesizing enzyme. Within m-NTS and at subpostremal border, labeling for βAR404 was evident along the intracellular surface of plasma membranes of small, apparently distal, astrocytic processes. Astrocytic processes with βAR404-immunoreactivity formed multiple, thin lamellae around TH-labeled and non-TH neuronal cell bodies and dendrites. βAR404-immunoreactive astrocytes also extended end-feet around blood vessels and surrounded groups of axon terminals that were directly juxtaposed to each other. Some, but not all, of these axons demonstrated TH-immunoreactivity. Fewer βAR404-immunoreactive astrocytes were detected in AP, regardless of their proximity to catecholaminergic processes or blood vessels. The present astrocytic localization of βAR404, together with the earlier, neuronal localization of βAR's third intracellular loop, suggest that the βAR may be substantially different between neurons and astrocytes. The regional difference in the prevalence of βAR404-immunoreactive astrocytes suggests that these receptive sites may either: (i) be preferentially activated by catecholamines released from terminals rather than circulating catecholamines; or (ii) be down-regulated in AP due to blood-born substances, such as catecholamines. The extensive localization of βAR in the border between m-NTS and AP also suggests that catecholaminergic activation of these astrocytes may dictate the degree of diffusion of catecholamines which are of neuronal or vascular origin. The specific localization of βAR404-immunoreactivity to the more distal portions of astrocytes suggests the possibility that astrocytes have restrictive distributions of βAR and that the β-adrenergic activation lead to morphological or chemical changes that are also localized to the distal portions of astrocytes. Additionally, the detection of βAR404 in astrocytes contacting non-TH-immunoreactive neurons suggests the possibility for catecholaminergic modulation of non-catecholaminergic neurons via the activation of astrocytes.     

The pharmacokinetics of (125I) 3-deoxy-3-iodine glucose on experimental tumor modelsin Vivo

The dynamics of¹²⁵I distribution is studied in rats with induced tumors of the prostate and mammary gland for intravenous administration of¹²⁵I-3D-G. It is found that 80% of the activity is eliminated in the first 24 hours. A relatively high level of¹²⁵I accumulation is found in necrotically altered regions of the tumor. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, № 3, pp. 294–295, March, 1994.     

β2-glycoprotein I, anti-β2-glycoprotein I, and fibrinolysis

β₂-glycoprotein-I (β₂GPI) is a phospholipid-binding plasma protein that consists of five homologous domains. Domain V is distinguished from others by bearing a positively charged lysine cluster and hydrophobic extra C-terminal loop. β₂GPI has been known as a natural anticoagulant regulator. β₂GPI exerts anticoagulant activity by inhibition of phospholipid-dependent coagulation reactions such as prothrombinase, tenase, and factor XII activation. It also binds factor XI and inhibits its activation. On the other hand, β₂GPI inhibits anticoagulant activity of activated protein C. According to the data from knockout mice, β₂GPI may contribute to thrombin generation in vivo. Phospholipid-bound β₂GPI is one of the major target antigens for antiphospholipid antibodies present in patients with antiphospholipid syndrome (APS). Binding of pathogenic anti-β₂GPI antibodies increases the affinity of β₂GPI to the cell surface and disrupts the coagulation/fibrinolysis balance on the cell surface. These pathogenic antibodies activate endothelial cells via signal transduction events in the presence of β₂GPI. Impaired fibrinolysis has been reported in patients with APS. Using a newly developed chromogenic assay, we demonstrated lower activity of intrinsic fibrinolysis in euglobulin fractions from APS patients. Addition of monoclonal anti-β₂GPI antibodies with β₂GPI also decreased fibrinolytic activity in this assay system. β₂GPI is proteolytically cleaved by plasmin in domain V (nicked β₂GPI) and becomes unable to bind to phospholipids, reducing antigenicity against antiphospholipid antibodies. This cleavage occurs in patients with increased fibrinolysis turnover. Nicked β₂GPI binds to plasminogen and suppresses plasmin generation in the presence of fibrin, plasminogen, and tissue plasminogen activator (tPA). Thus, nicked β₂GPI plays a role in the extrinsic fibrinolysis via a negative feedback pathway loop.     

血清CA125检测对子宫内膜异位症的临床诊断价值

目的 :探讨血清CA12 5测定对术前子宫内膜异位症的诊断价值。方法 :99例子宫内膜异位症经腹手术前测定CA12 5水平 ,并与 90例作对照比较。结果 :子宫内膜异位症CA12 5阳性率 >3 5U /ml,为 5 7.5 7% ,对照组为 8.88% (P <0 .0 1)。内膜异位囊肿高于卵巢囊肿 (P <0 .0 1) ,子宫腺肌病高于子宫肌瘤 (P <0 .0 5 )。结论 :血清CA12 5测定对子宫内膜异位症有较理想的诊断价值 ,对异位囊肿与卵巢囊肿 ,子宫腺肌病与子宫肌瘤有鉴别意义     

Crystal structure and conformation of polypeptides: L-leucylglycylglycylglycine

Crystals of L-leucylglycylglycylglycine, LGGG (C₁₂H₂₂N₄O₅), grown from an ethanol-water solution, are orthorhombic, space groups P2₁2₁2₁, with unit cell dimensions (at 22 ± 3°) a = 9.337(1), b = 10.995(1), c = 15.235(1)Å, v = 1563.4 ų, Z = 4 with a density of D_obs= 1.29 g-cm^-3 and D_calc= 1.279 g°cm^-3. The crystal structure was solved by the application of direct methods and refined to an R value of 0.029 for 1018 reflections with I ± 2s?. The molecule exists as a zwitterion in the crystal. The trans peptide backbone takes up a folded conformation at the middle glycylglycyl link accompanied by a significant nonplanarity up to Δω of 8° at the middle peptide and is relatively more extended at the two ends. The molecules are linked together intermolecularly in an infinite sequence of head to tail 1–4′ hydrogen bonds, as is typical of charged peptides. It is interesting to note that while glycylglycylglycine takes up an extended β-sheet conformation, addition of Leu to the N-terminal results in a bent conformation.     

Synthesis of 3-(3-pyridyl)- and 3-(3-benzo[b]thienyl)-D-alanine

The DL-arylamino acid ethyl ester derivatives of β-(3-pyridyl)-DL-alanine, and β-(3-benzo[b]thienyl)-DL-alanine were synthesized by diethyl acetamidomalonate condensation with the respective arylmethyl halides followed by partial hydrolysis to the monoethyl ester and decarboxylation. Each derivative was enzymatically resolved to a separable mixture of the corresponding N-acetyl-L-amino acid and the unchanged D amino acid derivative. Acidic hydrolysis of the latter gave the corresponding D-amino acid, the optical purity of which was established by HPLC analysis of the 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) derivative. The free D amino acids were converted to D-BOC derivatives by reaction with di-tert-butyldicarbonate in tert-butyl alcohol, water and sodium hydroxide.     

Analysis of a decreased Na+ conductance by tumor necrosis factor in identified neurons of Aplysia kurodai.

The ionic mechanisms of the effect of extracellularly ejected recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on the membrane of identified neurons R9 and R10 of Aplysia kurodai was investigated with conventional voltage-clamp, micropressure ejection, and ion substitution techniques. Micropressure-ejected rhTNF caused a marked hyperpolarization in the unclamped neuron. Clamping the same neuron at it resting potential level (-60 mV) and reejecting rhTNF-alpha with the same dose produced a slow outward current [Io (TNF)] associated with a decrease in input membrane conductance. Io (TNF) was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of Io (TNF) was approximately +10 mV. Ion substitution and pharmacological experiments suggest that Io (TNF) in identified neurons R9 and R10 of A. kurodai is due to a decreased Na+ conductance but not due to an activation of the Na(+)-K+ pump. Our results demonstrate that the immunomodulator TNF can act directly on the nervous system as well as on the immune system.     

Platelets as targets of snake venom metalloproteinases

For centuries snake venoms have been known to interfere with haemostasis and this is now known basically due either to toxins activating/inhibiting clotting factors, having effects on blood vessels or interfering with platelet function. In this short review, the interaction of one major group of toxins, the snake venom metalloproteinases, with platelets is considered. This is relevant for understanding the mechanism of haemorrhage induced by these toxins.