Microrheological aspects of adhesion of Escherichia coli on glass

The adhesion of both live and fixed bacteria (Escherichia coli) on glass has been studied under well-defined hydrodynamic conditions, created in an impinging jet apparatus. With this technique one can accurately measure the initial deposition rate jo on the surface, the average lifetime of a bacterium on the surface, tau esc, and the surface area blocked per deposited bacterium, normalized by its projected area, gamma. The experimental results are compared to theoretical results for equivalent spheres. It is found that near the stagnation point the deposition rate jo is mainly controlled by convective diffusive transport which, for rod-shaped Eschericia coli, with an axis ratio of about 2, is found to be equal to that for spheres. No differences in jo and tau esc were found between live and fixed bacteria at low flow rates. At high flow rates fixed bacteria adhered to the surface at a slower rate. In both systems jo was found to decrease suddenly at a distance of about 150 microns from the stagnation point, in contrast to systems of spherical particles for which jo is uniform over the surface. Most likely this is due to the rotation of the rod-shaped particles, which vary their distance to the surface periodically with time. The main difference between live and fixed bacteria, besides different deposition rates in strong flows, is that gamma is about 30% larger for fixed bacteria than for live ones, resulting in a much lower final coverage for fixed bacteria. These results imply a larger repulsion between fixed bacteria than between living ones. From detachment experiments we can conclude that not all bacteria stick to the surface with the same bond strength. The variation in the bond strength is due to the aging of the bonds between the bacteria and the surface. The average bond strength corresponds to an energy of about 13-15 kT.     

Hepatic glycogen metabolism in the db/db mouse

Summary Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2–15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen synthase activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in V_max, and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios. Its use should clarify the regulation of hepatic glycogen metabolism and other metabolic processes known to be controlled by these hormones. The other animal models of type 2 diabetes, ob/ob mouse and fatty Zucker (fa/fa) rat, show similar impairment of hepatic glycogen metabolism. The concentrations of glycogen metabolizing enzymes are high and in vitro studies indicate enhanced rate of glycogen synthesis and breakdown. However, streptozotocin-induced diabetic animals and BB rats which resemble insulin-deficient type 1 diabetes are characterized by decreased glycogen turnover as a result of reduction in the levels of glycogen metabolizing enzymes.     

Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1×10⁶ insect cells produced up to 3 g opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.Abbreviations dpi days post infection - pfu plaque forming units - AcNPV Autographa californica nuclear polyhedrosis virus     

A missense mutation (Asp250----Asn) in exon 6 of the human lipoprotein lipase gene causes chylomicronemia in patients of different ancestries.

We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency.     

Human breast milk stimulates prostaglandin synthesis in cultured human skin fibroblasts

Effect of human breast milk or its fractions on prostaglandin synthesis was investigated in cultured human skin fibroblasts. Prostaglandins released into the media were measured by radioimmunoassay. Incorporation of breast milk (2% level) into 10% fetal calf serum media (for 48 hours) stimulated the synthesis of 6-keto-PGF1 alpha (stable product of prostacyclin) by 800%. This stimulating effect of milk persisted after cold acetone extraction to remove phospholipids and potentiated further after dialysis. Stimulation by one of the commercial formulas (Similac) was less than 50% of the milk effect. Milk also stimulated PGE2 synthesis, although to a much lesser degree. These studies show for the first time that a) human breast milk contains potent factor(s) capable of influencing prostaglandin synthesis and suggest that b) these factors might have a role in the development of lipid synthetic pathways during early life.     

Ontogeny of guinea pig liver cholesterol 7 alpha-hydroxylase: unexpected inverse correlation of enzyme activity with expanding bile acid pool

Changes in the activity of guinea pig liver cholesterol 7 alpha-hydroxylase (rate limiting enzyme of cholesterol catabolism) from birth to adult life was investigated using a microsomal acetone extraction method (to remove endogenous cholesterol). Contrary to the previously held notion, it was noted that while the total bile acid pool increased progressively with age after birth, hepatic cholesterol 7 alpha-hydroxylase specific activity declined. Neonatal hepatic cholesterol 7 alpha-hydroxylase showed an increase in enzyme activity in response to cell supernatant factors (100,000 xg supernatant) from neonatal livers, but not from adult livers.     

Human c-fms proto-oncogene: comparative analysis with an abnormal allele.

The organization of the human c-fms proto-oncogene has been determined and compared with an abnormal allele. The human v-fms homologous genetic sequences are dispersed discontinuously and colinearly with the viral oncogene over a DNA region of ca. 32 kilobase pairs. The abnormal c-fms locus contains a small deletion in its 3' portion. DNA sequencing analysis indicated that it was 426 base pairs in size and located in close proximity to a putative c-fms exon.     

Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics

The objective of this study was to develop a relatively simple test to evaluate the functional integrity of the membranes of human spermatozoa. As in some other species, human spermatozoa 'swell' under hypo-osmotic conditions due to the influx of water and the expansion of the membranes. A mixture of equal parts of fructose and sodium citrate (150 mosmol) with calculated ionic strength of 0.15 resulted in a maximal number of clearly identifiable swollen spermatozoa. Only small variations were seen when different aliquants of the same semen samples were separately evaluated. A high correlation (r = 0.94) was obtained between expected and observed values of swollen spermatozoa when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. A good correlation (r = 0.90) was also observed between the % spermatozoa in a semen sample that were capable of undergoing swelling and the % of denuded hamster oocytes that were penetrated by capacitated spermatozoa from the same semen sample. By contrast, the correlations between % sperm swelling in ejaculates and % normal sperm forms, % motile spermatozoa and % spermatozoa that do not stain with eosin-Y (supravital stain) in the same ejaculates were 0.30, 0.61 and 0.52, respectively. Therefore, the hypoosmotic swelling technique to evaluate the functional integrity of the sperm membrane appears to give high repeatability and accuracy and is closely correlated to the in-vitro fertilizing ability of spermatozoa. It may be a useful addition to the standard semen analysis.