A Golgi and ultrastructural study of a dominant form of Kufs' disease

Summary A cerebral biopsy from a patient with inherited dominant autosomic Kufs' disease was studied with Golgi's method and ultrastructurally. A marked PAS positive, sudanophile, autofluorescent deposit was observed in the cytoplasm and in the proximal region of the axon of neurons from the third layer. Ultrastructurally this is a granular, membrane-bound product, sometimes with a dense, compact rectilinear pattern in which the typical clear component of adult lipofuscin is scanty. Sections stained with Golgi's method show a prominent, sometimes double, axon hillock swelling without dendritic spines. These facts are compared with additional samples of Alzheimer's disease and Huntington's chorea processed in a similar way.

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Zusammenfassung Die Gehirnbiopsie eines Patienten mit einer dominant autosomal erblichen Form der Kufs-Krankheit wurde mit der Golgi-Methode und elektronenmikroskopisch untersucht. PAS-positives, sudanophiles, autofluoreszierendes Material wurde vor allem im Cytoplasma sowie in den proximalen Anteilen des Axons von Neuronen der dritten Schicht nachgewiesen. Elektronenoptisch erwies sich dies als eine granuläre, an Membrane gebundene Substanz. Diese weist stellenweise eine dichte, geradlinige Struktur auf, in welcher die typischen hellen Komponenten des adulten Lipofuszins nur selten vorkommen. Schnitte, die mit der Golgi-Methode gefärbt wurden, zeigten einen gelegentlich doppelten Axonhöcker ohne Dendriten. Diese Tatsache wurde mit Fällen von Alzheimer-Krankheit und Huntington-Chorea, die in gleicher Weise verarbeitet wurden, verglichen.

     

A unique, versatile, real-time software package for controlling a Nicolet 1074 averager from a laboratory minicomputer

The use and operation of a newly-developed real-time software interface between a Nicolet 1074 digital signal averager and PDP-11/34A minicomputer is described. The system was developed for rapid, iterative, simultaneous measurements and correlations between electrical membrane properties of living cells and optically measured intracellular chemical changes. When such hard-wired averagers are interactively interfaced to minicomputers, the combination presents an overwhelmingly powerful experimental tool and enables data manipulations not performable by the hard-wired signal averager alone. Both the necessary hardware modifications to the standard Nicolet C-11 interface, major controlling assembly language software programs, and the versatility and adaptability of the system to a wide variety of laboratory applications are described.     

Laboratory assessment of von Willebrand factor: differential influence of prolonged ambient temperature specimen storage on assay results

Summary. The laboratory assessment of von Willebrand factor (VWF) is typically performed at specialist laboratory sites, particularly when performed as a battery of laboratory tests in a thorough workup for the diagnosis of von Willebrand's disease (VWD). In these cases, specimens could derive from a variety of off-site sources, including smaller laboratories, and general clinical practitioners. Because of the potential for lack of control by the specialist laboratory over the method of specimen handling and transport from these sources, and recent VWF methodological advances, we investigated the effect of prolonged ambient temperature specimen storage on laboratory assay results. Thus, specimens were collected from 10 separate individuals, and each variably processed to provide an ideal (‘control') plasma specimen, and additional specimens that were then stored at ambient temperature for up to 7 days. Specifically, specimens were stored either as whole blood, or as separated plasma, and VWF tested in isolated plasma, post 24 h, post 48 h and post 7 days storage. Three separate laboratory assays for VWF were performed; (i) a standard ‘antigen’ (antisera-ELISA-based) assay (VWF:Ag), (ii) a standard ristocetin-dependent-platelet-agglutination procedure (VWF:RCof) and (iii) a more recently described ELISA-based functional collagen-VWF binding assay (VWF:CBA). Results can be summarized as follows, (i) Plasma storage: there was no (statistically significant) change in VWF:Ag or VWF:RCof assay results over the 7-day storage period; however, there was a small but statistically significant fall (P= 0.009) in VWF:CBA assay results after 7 days storage of plasma. (ii) Whole blood storage: there was no (statistically significant) change in VWF:Ag, VWF:CBA or VWF:RCof assay results over the 7-day storage period, although the data suggested a trend towards increasing VWF:Ag over time. As a result of the change in assayed VWF:CBA following prolonged plasma storage, a similar small but statistically significant (P= 0.009) change (increase) in the VWF:Ag to VWF:CBA ratio was observed. This ratio has previously been determined to be useful in the differential diagnosis of VWD subtypes, with high VWF:Ag to VWF:CBA ratios potentially indicative of Type 2 VWD. Fortunately, the absolute magnitude of the altered ratio following prolonged plasma storage is unlikely in practice to affect the diagnosis of VWD in most testing cases. Nevertheless, there will be occasional borderline normal cases in whom the change in VWF:CBA, or in the calculated VWF:Ag to VWF:CBA ratio, may otherwise influence a clinical diagnosis of VWD. Caution is therefore suggested in the interpretation of laboratory-derived VWF data, particularly if the specimen is derived as an off-site referral.     

Two cases of phosphofructokinase deficiency associated with congenital hemolytic anemia found in Japan

Two kindreds of phosphofructokinase (PFK) deficiency associated with congenital nonspherocytic hemolytic anemia and mild myopathy were found in Japan. Both probands had jaundice, gallstones, and slight to moderate degree of exercise intolerance. They showed decreased level of red cell PFK activity and no increase of blood lactate in forearm ischemic exercise test. We studied these probands' red cell PFKs by partial purification and condensation. Muscle type isozyme of PFK in both cases was not demonstrable in starch gel electrophoresis and DEAE-Sephadex chromatography. The clinical symptoms are considered to be due to a defect of muscle type isozyme.     

Potential Improvement in Shelf Life Using the Prodrug Approach. II. A Systematic Examination of Kinetic Requirements

The utilization time (UT) for a solution of a prodrug that is rapidly and completely converted to drug in the blood may be longer than the time for 10% loss of the initial concentration. The UT for an intravenous prodrug solution is the period during which the total prodrug and drug concentration exceeds 90% of the initial concentration. The influence of the rate of prodrug degradation (k _nc), its conversion (k _c) to drug, and the subsequent drug degradation (k _h) on the UT of a stored solution was examined by simulating the prodrug and drug concentration–time courses. The ratio of the shelf life of a prodrug solution to that of the parent drug (UT_ratio) was calculated using a wide range of values for the three rate constants. Three-dimensional plots relating the UT_ratio to the k _c, k _nc, and k _h values provide a basis for making a priori assessments of kinetic requirements for designing a prodrug to increase storage time. A parenteral prodrug intended to increase storage time may have a larger overall rate of loss than the parent drug, but it must have a smaller degradation rate (k _nc < k _h) to be successful. The UT for an oral prodrug solution depends upon the bioavailability of the prodrug relative to the drug in addition to the values for k_nc, k _c, and k _h. Two ampicillin prodrugs were used as models to calculate actual UT_ratio versus pH profiles. Intravenous solutions showed modest gains in the UT_ratio in the acid region, whereas oral solutions reached a UT_ratio as high as 22 by combining favorable rate constants with increased bioavailability. These actual UT_ratio versus pH profiles were interpreted in terms of the theory established using the simulations.     

大米保鲜的研究—大米陈化指标和精洁米保鲜性能的初步研究

本研究以实验论证了用大米硫化氢含量代替脂肪酸含量作为大米新鲜度指标的合理性。通过对此试验证实珠光精洁米此市售标一粳具有较好的保鲜性能。     

A reliable method for recovering cryopreserved hybridoma cells for cloning

Summary A method is described for the reliable recovery of hybridoma cell lines from cryogenic storage. This procedure provides for (a) the efficient retrieval of cells secreting antibody for rapid confirmation of specific antibody production; (b) cells for cloning; (c) cells for restocking the liquid nitrogen storage banks; and (d) cultures to detect and possibly eliminate contamination.     

Preservation of Pancreas Tissue During Cold Storage Assessed by X-Ray Microanalysis

Clinical transplantation requires cold storage of tissue for several hours. We have examined the elemental content in exocrine and endocrine cells in mouse pancreas after cold storage by X-ray microanalysis, and in parallel carried out morphological studies. Tissue was stored at 4 degrees C for 4-12 h in Normal Krebs-Ringer's (high Na+/K+ ratio), Modified Krebs-Ringer's (low Na+/K+ ratio), Euro-Collins, University of Wisconsin (UW) solution, and seven modified version of UW solution. Incubation in Normal Krebs-Ringer's solution caused significantly increased Na and decreased K concentrations in contrast to incubation in other solutions. The cellular concentration of Na and Cl followed the concentration in the storage solution. Changes in the endocrine cells were similar to, but less pronounced than those in exocrine cells. Calcium was retained best in UW and some variants of UW, and least in Euro-Collins. This may indicate differences in preservation of secretory granules. Also, morphological studies showed that endocrine cells were less affected than exocrine cells. In conclusion, the only factor determining the intracellular concentration of diffusible ions after cold tissue storage is the ionic composition of the extracellular medium. X-ray microanalysis provides an objective method to assess whether the intracellular ionic composition of tissue is maintained during storage.     

Proteolytic activity during storage of platelets in plasma

Proteolytic activity was studied in platelet concentrates (PC) stored in plasma at 22 degrees C. In experiment 1, two PC with a higher (A) and a lower (B) white cell concentration were prepared from each of nine donors by centrifugation. Aliquots of the cell-free plasma, PPP, were stored as a control. Samples for the assay of fibrinopeptide A (FPA), elastase, spontaneous proteolytic activity (SPA), kallikrein-inhibiting activity, thrombin-antithrombin complexes (TAT) and D-dimers were collected initially and on days 1, 3, 5 and 7 of storage. Consumption of glucose, pH and concentrations of lactate dehydrogenase (LDH) and ATP were determined to investigate the metabolic status of the PC. The decrease in pH correlated to the leucocyte count, r = -0.74, P < 0.001 and to the increase in LDH, r = -0.74, P < 0.01. The levels of elastase and the SPA were consistently low in the PPP bags. In the PC elastase had increased by day 5 and the SPA by day 3; the levels in PC A were significantly higher than in PC B, P < 0.01. The leucocyte count correlated with the elastase activity, r = 0.71, P < 0.01, and with the SPA, r = 0.65, P < 0.01. A minor increase in FPA was demonstrated while no TAT and D-dimers could be detected. The cause of the formation of FPA was studied in experiment 2; three bags of PC and four of PPP were prepared from each of 16 donors. To the PC and three of the PPP bags either hirudin, aprotinin or no enzyme inhibitor (control) was added.(ABSTRACT TRUNCATED AT 250 WORDS)     

The empty sack syndrome: a platelet storage pool deficiency associated with empty dense granules

Summary. Two sisters with lifelong bleeding tendencies were examined to determine whether their condition was associated with a platelet defect. Their platelet aggregation in response to epinephrine and collagen was abnormal, and the secretion of serotonin and ATP was markedly reduced. The platelet contents of serotonin, ADP, and ATP were all diminished and the ATP:ADP ratio was increased. Direct enumeration by whole-mount and quinacrine-fluorescence techniques demonstrated that the platelets from both sisters had significantly fewer dense granules than controls. These characteristics are similar to an individual with Hermansky-Pudlak syndrome and are consistent with a platelet dense granule deficiency. In contrast, immunofluorescence studies using an antibody against the dense granule membrane protein granulophysin suggested that both sisters had numbers of granules within the normal range. Evaluation by immunoblotting and ELISA indicated the presence of normal levels of granulophysin in the platelets from both sisters: FACS analysis demonstrated the surface expression of granulophysin under conditions of selective dense granule release. These results are consistent with these sisters having a form of dense granule storage pool deficiency where the granular membranes are present but the granules have reduced contents. This observation represents a novel form of storage pool disease which we have termed the empty sack syndrome.