Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.     

Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

Relevance of signaling molecules for apoptosis induction on influenza A virus replication

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.     

Is CD36 gene polymorphism in region encoding lipid-binding domain associated with early onset CAD?

CD36 is a fatty acid translocase in striated muscle cells and cardiomyocytes. Some study suggested that alterations in CD36 gene may be associated with coronary artery disease (CAD) risk. The aim of the current study was to compare the frequency of CD36 variants in region encoding lipid-binding domain in Caucasian patients with early-onset CAD, no-CAD adult controls and neonates. The study group comprised 100 patients with early onset CAD. The genetic control groups were 306 infants and 40 no-CAD adults aged over 70 years. Exons 4, 5 and 6 including fragments of flanking introns were studied using the denaturing high-performance liquid chromatography technique and direct sequencing. Changes detected in analyzed fragment of CD36: IVS3-6 T/C (rs3173798), IVS4-10 G/A (rs3211892), C311T (Thr104Ile, not described so far) in exon 5, G550A (Asp184Asn, rs138897347), C572T (Pro191Leu, rs143150225), G573A (Pro191Pro, rs5956) and A591T (Thr197Thr, rs141680676) in exon 6. No significant differences in the CD36 genotype, allele and haplotype frequencies were found between the three groups. Only borderline differences (p = 0.066) were found between early onset CAD patients and newborns in the frequencies of 591T allele (2.00% vs 0.50%) and CGCGCGT haplotype (2.00% vs 0.50%) with both IVS3-6C and 591T variant alleles. In conclusion, CD36 variants: rs3173798, rs3211892, rs138897347, rs5956, rs143150225 rs141680676 and C311T do not seem to be involved in the risk of early-onset CAD in Caucasian population.     

右美托咪定对脑缺血/再灌注大鼠海马兴奋性氨基酸含量及NMDA受体亚单位NR1表达的影响

目的：观察右美托咪定预处理对全脑缺血/再灌注大鼠海马细胞外谷氨酸（Glu）、天门冬氨酸（Asp）含量及N-甲基-D-天冬氨酸（NMDA）受体1（NR1）表达的影响,探讨右美托咪定脑保护作用及其神经递质机制。方法：雄性Wistar大鼠54只,随机分为3组（n=18）：假手术组、脑缺血/再灌注组和右美托咪定预处理组。用四血管闭塞法建立大鼠全脑缺血模型。收集清醒、缺血15 min及再灌注0~1 h微透析标本。于全脑缺血15 min再灌注1 h后,迅速断头取脑,采用免疫组化法和蛋白免疫印迹法检测海马NMDA受体NR1亚单位的表达情况。结果：与脑缺血/再灌注组相应时点比较,右美托咪定预处理组大鼠海马微透析液中Glu、Asp含量明显降低（P<0.05, 0.01）;免疫组化和Western-blot法检测显示右美托咪定预处理组大鼠海马组织NMDA受体亚单位NR1表达明显受抑制（P<0.05, 0.01）。结论：右美托咪定预处理不仅减少脑缺血/再灌注时兴奋性氨基酸释放,还能抑制NMDA受体亚单位NR1的高表达而产生脑保护作用。     

Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters

A neutral protease with an estimated M_r of about 26 kD and responsible for cleavage of myosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced. Although the hamster protein shared 63-85% identity in nucleotide and amino acid sequences with rat and mouse mast cell proteases, it had a higher degree of specificity for myosin LC2 than mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin because of its unique enzymatic specificities to distinguish it from other mast cell proteases. A polyclonal antibody was raised specific to the hamster muscle and human cardiac muscle mekratins without apparent cross-reaction with rat mast cell proteases. We have earlier demonstrated the presence in excess of a neutral protease that specifically cleaves LC2 in human hearts obtained at end stage idiopathic dilated cardiomyopathy (IDC). Western analyses revealed that heart tissue from patients with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was similar to that seen in myopathic hamster skeletal muscle. No bands were recognized by the antibody when IDC myofibrils were probed due to the removal of soluble proteins during sample preparation. Thus, these results strongly suggest that the anti-mekratin antibody will provide positive identification of IDC in many cases and diagnosis by exclusion may be replaced.     

ADAM10 is the physiologically relevant, constitutive ��-secretase of the amyloid precursor protein in primary neurons

The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called α‐secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid‐β peptide (Aβ). α‐Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel α‐secretase‐cleavage site‐specific antibody, we found that RNAi‐mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP α‐secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of α‐cleavage. This finding was further confirmed by mass‐spectrometric detection of APP‐cleavage fragments. Surprisingly, in different cell lines, the reduction of α‐secretase cleavage was not paralleled by a corresponding increase in the Aβ‐generating β‐secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with γ‐secretase for the cleavage of a C‐terminal APP fragment generated by β‐secretase. We conclude that ADAM10 is the physiologically relevant, constitutive α‐secretase of APP.     

Induction of synchronous differentiation (encystment) in Physarum polycephalum myxamoebae

Encystment of Physarum polycephalum myxamoebae, grown under nearly identical physiological conditions as plasmodia is induced by transfer to a salts medium containing 0.5 M mannitol or mannose. After 24 h induction approximately 50% of amoebae had differentiated to cells which were identified to be young cysts by light and electron microscopy. Several other polyols, sugars, biogenic amines, and a starvation period from 24 h to one week caused no reproducible cyst formation. In contrast to the formation of dormant forms in the plasmodial stage of the life cycle, the induction of cysts and their germination to amoebae are not inhibited neither by actinomycin C nor by cycloheximide. In addition, the isoenzyme spectra of aminopeptidases and acid proteases remain nearly identical in growing and differentiating amoebae.Abbreviations SD semi-defined BSS basal salts solution The investigation is a part of the Ph. D. thesis of A. Haars, Göttingen, 1976