PREPARATION AND CONFORMATIONAL STUDY OF CLUPEINE FRAGMENTS*

Fragments of clupeines YI, YII, and Z of divergent chain length and different amino acid composition were prepared by digestion with thermolysin and a mixture of carboxypeptidases A and B, and their conformational preferences examined as a function of pH, added salts, presence of a helix-supporting solvent, and temperature. All these highly basic oligopeptides adopt an essentially unordered conformation in aqueous solution. 2-Chloroethanol supports in various amounts the onset of the right-handed α-helical form in the carboxypeptidase fragments.     

Interaction of botulinum type A,B and E derivative toxins with synaptosomes of rat brain

Summary Clostridium botulinum ¹²⁵I-labelled derivative toxin immediately bound to rat synaptosomes. Of the two fragments of type B derivative toxin, the large-molecular-weight fragment (fragment I) inhibited the binding of labelled type B derivative toxin to synaptosomes in the same manner as unlabelled type B toxin did. The inhibition by the small-molecular-weight fragment (fragment II) was less than that by fragment I. These findings suggest that type B toxin binds to synaptosomes mainly with some part of fragment I. The binding of labelled type A and E derivative toxins was inhibited by either of the unlabelled type A or E derivative toxins, but not by type B derivative toxin. It is concluded that synaptosomes of rat brain possess relatively specific binding sites for botulinum toxin types.     

Calcium metabolism and active tension in mechanically disaggregated heart muscle

We have measured the active tension generated by spontaneously beating fragments of mouse ventricular myocardium prepared by mechanical mincing. Tension oscillations were observed only when the ionized calcium concentration in the medium was more than 10^?7m and less than 10^?4m. Sustained relaxation or contraction, respectively, were observed at these concentration limits. In contrast to this sensitivity to calcium, wide variations in $\text{Na}\text{K}$ had almost no effect on contractility. Lanthanum had no effect at low concentrations, but caused contracture at 10^?3m. These observations suggest that the sarcolemma of these spontaneously beating fragments is fully permeable to Na⁺, K⁺, Ca²⁺, and La³⁺, and that trans-sarcolemmal electrical events are not the determinant of beating. These conclusions were further supported by our failure to detect any significant transmembrane potential with intracellular electrodes, and our inability to pace the fragments by field stimulation. The responses to azide, oligomycin, and caffeine were used to determine the roles of mitochondria and SR in control of tension oscillations. These experiments indicated that while mitochondria may control resting tension, SR function is needed for cyclic contraction and relaxation in these myocardial fragments.     

Probing the structure of human growth hormone by limited proteolysis

Digestion of human growth hormone (hGH) with the Glu-specific protease from Staphylococcus aureus V8 was performed at 20-22°C or 37°C at a 1:20 ratio (by weight) at pH 7.8 with or without 0.2% SDS. There are 14 Glu-residues evenly distributed along the polypeptide chain of hGH as possible sites of proteolytic cleavage of V8-protease. The pattern of fragmentation of hGH was analyzed by electrophoresis and reversed-phase HPLC, and the identity of the proteolytic fragments isolated to homogeneity was established by their partial sequencing and amino acid analysis after acid hydrolysis. Kinetic analysis of the proteolytic digestion process allowed to establish that initial nicking of the protein occurs at Glu³³ and subsequently at Glu⁵⁶ and Glu⁶⁶. Much slower cleavages occur at G1u³⁰ and Glu¹⁸⁶. These cleavage sites are located at chain loops in the hGH molecule, and in particular outside the helical segments of the four-helix bundle of the crystal structure of hGH. Fragments 1-33 and 67-191 comprising entirely the N-terminal helix and the three C-terminal helices of hGH, respectively, were isolated to homogeneity in amounts useful for subsequent conformational and functional studies. The results of this study and of previous ones [Li, C.H. (1982) Mol. Cell. Biochem. 46 , 31-41] describing limited proteolysis of hGH by various proteases have been interpreted on the basis of the three-dimensional structure and dynamics of hGH. Overall, it is shown that proteolytic enzymes preferentially cleave hGH at exposed and flexible loops only, thus emphasizing the fact that proteases can be used as reliable probes of protein structure and dynamics. © Munksgaard 1995.     

Digoxin toxicity in a premature infant: Treatment with Fab fragments of digoxin-specific antibodies

Summary The first use of Fab fragments to treat digoxin toxicity in a premature infant with renal failure, 18 h after the onset of severe arrhythmias, is reported with dramatic results. The development of digoxin toxicity in the context of accepted therapeutic dosing to treat heart failure due to a cerebral arteriovenous malformation is discussed.     

Effects of peptides cleaved from human fibrinogen by plasmin on rabbit kidney cells in culture

Low molecular weight fibrinogen degradation products (LMW-FDP) containing a mixture of dialysable peptides cleaved from human fibrinogen by plasmin are cytotoxic to an established line of rabbit kidney cells and to primary cultures of rabbit kidney cells. The presence of LMW-FDP in a concentration of 50 micrograms/ml during the cell cultivation caused a considerable release of 51Cr from prelabelled cells and inhibited 3H-thymidine and 86Rb uptake. Among three isolated peptides of established primary structure only one, 6D: Ser-Gln-Leu-Gln-Lys-Val-Pro-Pro-Glu-Trp-Lys, induced a significant effect, i.e. it enhanced 3H-thymidine incorporation. Two others, 6A: Ala-Arg-Pro-Ala-Lys and 6E: Thr-Ser-Glu-Val-Lys, did not influence the examined parameters. Hence other components of LMW-FDP must be assumed to be responsible for the cytotoxic effect on kidney cell cultures.     

Some effects of an ACTH 4-9 analog (Org 2766) on human performance.

The effects (on human beings) of the ACTH analog Org 2766 were investigated for a range of performance tests: complicated serial reaction task, running memory span, verbal learning and non-verbal mental ability tests (closure flexibility and non-verbal abstraction). Subjective ratings on feelings were taken and heart rate was measured. Only the 30-min reaction task produced significant drug effects. In this task performance tends to deteriorate as a function of time on task. During the test period there is a gradual increase in the number of lapses in performance due to moments of inattention. This deterioration is counteracted by the ACTH 4-9 analog, a result also found in previous study using ACTH 4-10 (Org OI63). Thus ACTH fragments which are devoid of endocrine effects seem to have a beneficial effect on goal-directed motivation oriented on the requirements of the task.     

NSCLC患者血清sB7-H3和CYFRA21-1检测及临床应用

目的 探讨血清可溶性B7-H3（sB7-H3）和细胞角蛋白19片段抗原（CYFRA21-1）检测对非小细胞肺癌（non-small cell lung cancer,NSCLC）的诊断价值.方法 分别采用 ELISA和电化学发光法检测 72 例 NSCLC及 68 例肺部良性病患者血清sB7-H3 和 CYFRA21-1水平.结果 NSCLC 患者血清sB7-H3水平及阳性率[（98.34±35.17）ng/ml,61.11%]均明显高于肺部良性病患者[（70.89±20.62）ng/ml,7.32%];NSCLC 患者血清CYFRA21-1 水平及阳性率[（15.08±7.98）ng/ml,66.67%]均明显高于肺部良性病患者[（3.21±2.65）ng/ml,4.41%];差异均具有统计学意义（P〈0.05）.NSCLC 经TNM分期后,随着肿瘤分级级别的升高,sB7-H3水平[Ⅰ/Ⅱ期、Ⅲ期和Ⅳ期分别为（85.21±36.14）ng/ml、（98.62±＋40.38）ng/ml和（107.17±41.66）ng/ml],CYFRA21-1水平和[Ⅰ/Ⅱ期、Ⅲ期和Ⅳ期分别为（10.23±5.65）ng/ml、（15.52±6.84）ng/ml和（18.08±5.93）ng/ml]也升高.sB7-H3 和 CYFRA21-1联检后,灵敏度和一致性增高,特异性降低.结论 sB7-H3 和 CYFRA21-1对 NSCLC 有一定的辅助诊断价值,另外,或可应用于对TNM的分期,联检可提高对NSCLC的阳性诊断.     

血清CYFRA21-1在食管鳞状细胞癌诊断中的价值

目的：探讨细胞角蛋白19片段（CYFRA21-1）在食管鳞状细胞癌诊断中的应用价值。方法：采用电化学发光法分别检测健康对照组（45例）和初次诊断食管鳞状细胞癌患者（90例）血清中CYFRA21—1和癌胚抗原（CEA）质量浓度,并对患者进行随访,出现淋巴结转移和（或）远处转移者再次检测血清中CYFRA21-1和CEA质量的浓度。结果：食管磷状细胞癌组CYFRA21-1和CEA质量浓度明显高于对照组[分别为（3．81±1．7）μg·L^-1比（1．15±0．68）μg·L^-1,（7．36±3．5）μg·L^-1比（1．34±0．65）μg·L^-1,均P〈0．05]。食管磷状细胞癌组出现淋巴结转移和（或）远处转移者CYFRA21-1和CEA质量浓度高于初次诊断时[分别为（4．93±0．83）μg·L^-1比（2．04±0．29）μg·L^-1,（16±2．96）μg·L^-1。比（7．55±1．23）μg·L^-1,差异具有统计学意义（均P〈0．05）。初次诊断食管鳞癌患者CYFRA21-1阳性率（56．7％,51／90）高于CEA（20．0％,18／90）,差异有统计学意义（X^2=25．59,P〈0．01）;出现淋巴结转移和远处转移的患者CYFRA21-1阳性率（71．1％,32／45）高于CEA（26．7％,12／45）,差异有统计学意义（X^2=17．79,P〈0．01）。结论：血清CYFRA21—1对食管鳞状细胞癌诊断和判断预后方面比CEA更优。