Cell culture and in vitro studies of fresh and cryopreserved human insulinoma

Summary Dispersed cells from both fresh and cryopreserved human insulinoma have been maintained in cell culture. Initial yield of viable cells was 50% for fresh and 25% for cryopreserved tissue. Viability of cells in culture was documented by increasing numbers of cells (doubling time approximately 5 d initially and 2 d at the sixth subculture for both fresh and cryopreserved tissue) and continued release of insulin over time (approximately 100 ng/ml per 10⁵ cells at 10 d and 175 ng/ml per 10⁵ cells at 30 d of culture for both fresh and cryopreserved tissue). Evidence that cells growing in culture were beta cells was provided by: (a) recovery of intracellular and extracellular immunoreactive insulin (IRI), (b) electron microscopic morphology, and (c) immunohistochemical staining. Cells from fresh insulinoma incubated with increasing concentrations of extracellular glucose released increasing amounts of IRI up to approximately 15 mM glucose, which paralleled changes in plasma insulin obtained during a preoperative glucose tolerance test. Under an Intergovernmental Personnel Act Exchange from the Department of Surgery, University of California, Davis, Sacramento Medical Center.     

Use of the cylinder osteotome for cancellous bone grafting

An instrument is described for removing iliac crest cancellous bone grafts under local anesthesia. The device has widespread applications for plastic and orthopedic surgery. The convenience of obtaining bone grafts with this device broadens the use of bone-grafting techniques in patients in whom the need for bone graft is unexpected.     

Differential irreversible insertion of protein kinase C into phospholipid vesicles by phorbol esters and related activators.

Incubation of protein kinase C (PKC) alpha with phorbol 12,13-dibutyrate and phospholipid vesicles promoted a time-dependent irreversible insertion of the enzyme into the vesicles and the generation of a calcium-independent kinase activity. Calcium neither caused insertion nor influenced the insertion induced by the phorbol ester. The effect was strongly dependent on the phosphatidylserine concentration in the vesicle and could also be supported by other anionic phospholipids. An analysis of the structure-activity relations of PKC activators for the calcium-independent kinase activity revealed marked relative differences in potencies for binding and for insertion. Compounds such as phorbol 13-myristate 12-acetate and mezerein were very efficient at inducing insertion. In contrast, 12-deoxyphorbol esters and diacylglycerol were relatively inefficient at inducing insertion, requiring higher concentrations than expected from their binding affinities. The insertion of PKC alpha depended substantially on the length of the aliphatic esters in the 12- and 13-positions of the phorbol derivatives, and once again, potencies for insertion and binding were not directly proportional. Our findings suggest two different sites for ligand interaction on the molecule of PKC alpha with different structure-activity requirements. We speculate that the differential ability of compounds to promote insertion could contribute to the documented marked differences in the biological behavior of PKC activators.     

Corolla wilting facilitates delayed autonomous self-pollination in Pedicularis dunniana (Orobanchaceae)

Structural changes associated with corolla wilting may serve as a mechanism for effecting self-pollination. Low pollinator visitation, high seed production and a corolla that persists after anthesis indicates that Pedicularis dunniana is autogamous. Delayed autonomous self-pollination is facilitated by corolla wilting. Wilting of the upper lip (galea) brought the pollen laden anthers into contact with the stigma resulting in the deposition of self pollen on the stigma. The seed set of flowers either emasculated, or with restrained galeae thus preventing anthers brushing against the stigma, was significantly lower than that of open-pollinated flowers. This demonstrates that autogamy occurs in this species through corolla wilting. Germination experiments indicated that outcross seedlings were more vigorous than selfed seedlings as a result of inbreeding depression. It is likely that autogamy provides reproductive assurance for P. dunniana under conditions of pollinator scarcity.     

Identification of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations

Peptides produced by mild chymotryptic digestion of human erythrocyte protein 4.1 mimic the ability of intact 4.1 to promote the binding of spectrin to F-actin. This complex-promoting activity was found to reside in an 8-kDa peptide which was fully functional when dissociated from other protein 4.1-derived peptides, indicating that noncovalent complexes of multiple peptides were not essential for activity. The 8-kDa peptide was incorporated into a ternary complex with spectrin and F-actin in approximately stoichiometric amounts. Amino acid composition and two-dimensional peptide mapping show that the 8-kDa active peptide is located within the 10-kDa region of protein 4.1 which contains a cAMP-dependent phosphorylated site.     

Purification and characterization of an acid metalloproteinase from human articular cartilage

A metalloprotease that digests cartilage proteoglycan optimally at pH 5.3 has been purified (4400-fold) to homogeneity from 20-g samples of human articular cartilage containing about 100 micrograms of enzyme. This enzyme was cleanly separated from a related neutral metalloprotease with an optimum pH of 7.2. The acid metalloprotease displays 40% of its maximum activity at pH 7.2 and so has significant activity at physiological pH. The protease is calcium-dependent and indirect evidence suggests that it may contain zinc at its active center. It occurs largely in a latent form that can be activated by aminophenylmercuric acetate. The apparent Mr of the latent form is 55,000 and of the active form, 35,000. The isoelectric point is at pH 4.9. The protease activity is inhibited by chelators, Z-phenylalanine, ovostatin, and tissue inhibitor of metalloproteinase from human articular cartilage. It differs from metalloproteinases such as enkephalinase and kidney brush-border protease in its failure to be strongly inhibited by phosphoramidon and Zincov. It cleaves the proteoglycan monomer of bovine nasal cartilage to fragments of approximately 140,000 Da. It cleaves the B chain of insulin at Ala14-Leu15 and Tyr16-Leu17. A survey of 26 cartilage extracts indicates this enzyme is elevated to about 3 times the normal level in human osteoarthritic cartilage and that the tissue inhibitor of metalloproteinase is only slightly diminished. Preliminary evidence points to the presence of a similar acid metalloprotease activity in human polymorphonuclear leukocytes.     

Antibiotics in surgical treatment of acute abscesses.

A four-way, double-blind, prospective trial of treatment of abscesses by incision, curettage, and primary closure with and without antibiotic cover (clindamycin injection before operation or capsules after operation, or both) was conducted. There was no appreciable difference in mean healing time between the patients given both the antibiotic injection and the antibiotic capsules and those given the injection and placebo capsules, whereas healing times in those given the placebo injection and antibiotic capsules or placebo only were appreciably longer. Four of the patients who were not given the antibiotic injection developed bacteraemia; one patient who was given the antibiotic injection also developed a bacteraemia, but this was caused by clindamycin-resistant bacteria. These results show that a single injection of an effective antibiotic before operation is sufficient to protect the patient against bacteraemia and permit optimum healing.     

Imprints of glacial history and current environment on correlations between endemic plant and invertebrate species richness

Aim We evaluate how closely diversity patterns of endemic species of vascular plants, beetles, butterflies, molluscs and spiders are correlated with each other, and to what extent similar environmental requirements or survival in common glacial refugia and comparable dispersal limitations account for their existing congruence. Location Austria. Methods We calculated pairwise correlations among species numbers of the five taxonomic groups in 1405 cells of a 3′ × 5′ raster (c. 35 km²) using the raw data as well as the residuals of regression models that accounted for: (1) environmental variables, (2) environmental variables and the occurrence of potential refugia during the Last Glacial Maximum, or (3) environmental variables, refugia and spatial filters. Results Pairwise cross‐taxonomic group Spearman’s rank correlations in the raw data were significantly positive in most cases, but only moderate (0.3 < ρ < 0.5) to weak (ρ < 0.3) throughout. Correlations were closest between plants and beetles, plants and butterflies, and plants and snails, respectively, whereas the distribution of endemic spiders was largely uncorrelated with those of the other groups. Environmental variables explained only a moderate proportion of the variance in endemic richness patterns, and the response of individual groups to environmental gradients was only partly consistent. The inclusion of refugium locations and the spatial filters increased the goodness of model fit for all five taxonomic groups. Moreover, removing the effects of environmental conditions reduced congruence in endemic richness patterns to a lesser extent than did filtering the influence of refugium locations and spatial autocorrelation, except for spiders, which are probably the least dispersal‐limited of the five groups. Main conclusions The moderate to weak congruence of endemic richness patterns clearly limits the usefulness of a surrogacy approach for designating areas for the protection of regional endemics. On the other hand, our results suggest that dispersal limitations still shape the distributions of many endemic plant, snail, beetle and butterfly species, even at the regional scale; that is, survival in shared refugia and subsequent restricted spread retain a detectable signal in existing correlations. Concentrating conservation efforts on well‐known Pleistocene refugia hence appears to be a reasonable first step towards a strategy for protecting regional endemics of at least the less mobile invertebrate groups.