Immunodetection of hemocytes, peneidins and α2-macroglobulin in the lymphoid organ of white spot syndrome virus infected shrimp

Viral diseases restrict the development of the world shrimp industry and there are few studies on cell response to the presence of viral infections. We performed immunohistochemistry assays to characterize hemocytes subpopulations involved in the immune process occurring in the LO of Litopenaeus vannamei shrimp. Tissue sections of animals that increased their LO spheroids and hemocytes infiltration after WSSV induced infection, were used. Three MABs namely, 40E10 (recognizing small granule hemocytes), 40E2 (recognizing large granule hemocytes), and 41B12, which recognize α(2)-macroglobulin were used. Additionally one polyclonal antibody was used against the penaeidins antimicrobial peptides, and to detect WSSV a commercial immunohistochemistry kit (DiagXotics) was used. Numerous small granule hemocytes were detected in the stromal matrix of LO tubules, whereas large granule hemocytes were less numerous and located mainly in hemal sinuses. The exocytosis of two molecules, which have been related to the phagocytosis process, i.e. penaeidins, and α(2)-macroglobulin, was detected in the external stromal matrix and the outer tubule walls. α(2) -macroglobulin inhibits phenoloxidase activity and its strong release in LO tissue may explain the absence of melanization in the immune processes occurring in it. The immunolabeling of vesicles within the LO spheroids with MABs 41B12 40E10 and antipenaedin antibody suggests that LOS are formed by phagocytic cells derived from small granule and hyaline hemocytes, with a possible role of peneidins and α(2)-macroglobulin acting as opsonines.     

Nicotine-evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage-gated calcium channels.

It has previously been shown that nicotine-evoked dopamine release from rat striatal synaptosomes and nicotine-evoked norepinephrine release from hippocampal synaptosomes are mediated by distinct nicotinic acetylcholine receptor (nAChR) subtypes. In the present study, the functional association of these nicotinic receptors with specific subtypes of voltage-gated calcium channels was examined. Cd(2+) (200 microM), as well as omega-conotoxin MVIIC (5 microM), blocks approximately 85% of nicotine-evoked dopamine release from striatal synaptosomes, indicating a major involvement of calcium channels. Furthermore, the toxin-susceptibility suggests that these calcium channels contain alpha(1A) and/or alpha(1B) subunits. Inhibition of nicotine-evoked dopamine release by conotoxins alpha-MII and omega-GVIA is additive and indicates that presynaptic alpha3beta2 nAChRs are functionally coupled to alpha(1A), but not alpha(1B), calcium channel subtypes. Conversely, insensitivity to alpha-AuIB and sensitivity to omega-MVIIC indicate that non-alpha3beta2/alpha3beta4-containing nAChRs are functionally coupled to alpha(1B)-containing calcium channels. In contrast, Cd(2+) blocks only 65% of nicotine-evoked norepinephrine release from hippocampal synaptosomes, indicating that a substantial fraction of this release occurs through mechanisms not involving calcium channels. This Cd(2+)-insensitive component of release is blocked by alpha-AuIB and therefore appears to be triggered by Ca(2+) flowing directly through the channels of presynaptic alpha3beta4 nAChRs. Thus, these data indicate that different presynaptic termini can have distinctive functional associations of specific nAChRs and voltage-gated calcium channels.     

A new active vitamin D analog,ED-71, causes increase in bone mass with preferential effects on bone in osteoporotic patients

As a candidate for active vitamin D analogs that have selective effects on bone, 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71) has been synthesized and is currently under clinical trials. In ovariectomized rat model for osteoporosis, ED-71 caused an increase bone mass at the lumbar vertebra to a greater extent than 1alpha-hydroxyvitamin D3 (alfacalcidol), while enhancing calcium absorption and decreasing serum parathyroid hormone levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers.An early phase II clinical trial was conducted with 109 primary osteoporotic patients. The results indicate that oral daily administration of ED-71 (0.25, 0.5, 0.75, and 1.0 microgram) for 6 months increased lumbar bone mineral density in a dose-dependent manner without causing hypercalcemia and hypercalciuria. ED-71 also exhibited a dose-dependent suppression of urinary deoxypyridinoline with no significant reduction in serum osteocalcin. These results demonstrate that ED-71 has preferential effects on bone with diminished effects on intestinal calcium absorption. ED-71 offers potentially a new modality of therapy for osteoporosis with selective effects on bone.     

Studies on intramolecular hydrogen bonding between the pyridine nitrogen and the amide hydrogen of the peptide: synthesis and conformational analysis of tripeptides containing novel amino acids with a pyridine ring.

For the first time tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1) and AA(3) = Gly or Aib, Xaa = 2Pmg and 2Pyg) were prepared containing alpha-methyl-alpha-(2-pyridyl)glycine (2Pmg) and alpha-(2-pyridyl)glycine (2Pyg) by solid-phase Ugi reaction. These results clearly indicate that for the preparation of tripeptides containing an amino acid with a pyridine ring, the solid-phase Ugi reaction is very useful.NMR analysis clarified that 2Pmg-containing tripeptides adopt a unique conformation with an intramolecular hydrogen bond between 2Pmg-NH and the pyridine nitrogen. However, in the case of Z-Gly-2Pyg-Gly-OMe, the intramolecular hydrogen bonding between 2Pyg-NH and the pyridine nitrogen was not observed, whereas Z-Aib-2Pyg-Aib-OMe adopts a unique conformation with an intramolecular hydrogen bond between 2Pyg-NH and a pyridine nitrogen. Conformational analysis of the tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1), AA(3) = Gly or Aib, Xaa = alpha,alpha-di(2-pyridyl)glycine (2Dpy), alpha-phenyl-alpha-(2-pyridyl)glycine (2Ppg), 2Pmg and 2Pyg), clarified that when an alpha,alpha-disubstituted glycine with a 2-pyridyl group at an alpha-carbon atom is introduced into any peptide, an intramolecular hydrogen bond between a pyridine nitrogen and an amide proton is formed and conformational mobility of the peptide backbone is restricted.     

Inhibition of α-synuclein aggregation by small heat shock proteins

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.     

Chirality transferring [3,3] sigmatropic rearrangement of (1-Acyloxy-2-alkenyl)trianlkylsilane synthesis of optically active vinylsilane-containing alpha-amino acid

A vinylsilane-containing alpha-amino acid and alpha,alpha-disubstituted alpha-amino acid 2 having two contiguous asymmetric carbon centers at their alpha and beta positions were synthesized in an optically active form by ester-enolate Claisen rearrangement of the alpha-acyloxysilane 1 as the key step, where the chirality of an alpha-acyloxy-TBDMS group was completely transferred to the rearranged product.