Quantitative immunoassay of total cellular GAP junction protein connexin32 during liver regeneration using antibodies specific to the COOH-terminus.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.     

Substrate effects on the enzymatic activity of alpha-chymotrypsin in reverse micelles

Six different substrates have been used for measuring the activity of alpha-chymotrypsin in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The substrates were glutaryl-Phe p-nitroanilide, succinyl-Phe p-nitroanilide, acetyl-Phe p-nitroanilide, succinyl-Ala-Ala-Phe p-nitroanilide, succinyl-Ala-Ala-Pro-Phe p-nitroanilide and acetyl-Trp methyl ester. It has been shown that the dependence of the kinetic constants (kcat and Km) on the water content of the system, on wo (= [H2O]/[AOT]), is different for the different substrates. This indicates that activity-wo profiles for alpha-chymotrypsin in reverse micelles not only reflect an intrinsic feature of the enzyme alone. For the p-nitroanilides it was found that the lower kcat (and the higher Km) in aqueous solution, the higher kcat as well as Km in reverse micelles. "Superactivity" of alpha-chymotrypsin could only be found with the ester substrate and with relatively "poor" p-nitroanilides. The presence of a negative charge in the substrate molecule is not a prerequisite for alpha-chymotrypsin to show "superactivity".     

Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.     

Improved high-performance liquid chromatography of the new antineoplastic agents bisantrene and mitoxantrone

Bisantrene and mitoxantrone are two new anthracene derivatives which have shown significant antitumor activity against a wide variety of animal tumors and in human phase I and II clinical trials. We have developed a rapid, simple and sensitive sample cleanup procedure and high-performance liquid chromatographic (HPLC) assay for both drugs. This method uses a commercially available mini-cartridge with C₁₈ reversed-phase packing to isolate the drugs from the biological matrix prior to HPLC. For both drugs the average recovery of the assay was 98 ± 6% with a coefficient of variation (C.V.) of less than 7%. Using this new method our assay sensitivity has improved to less than 10 ng/ml for bisantrene and 1 ng/ml for mitoxantrone, allowing us to document a prologned terminal phase plasma half-life for both bisantrene and mitoxantrone. Equilibrium dialysis studies showed that both drugs are highly protein bound. Mitoxantrone appears less stable in human plasma than bisantrene. Recoveries from plasma after a 24-h incubation at 25 and 37°C were 40 and 20% for mitoxantrone and 90 and 85% for bisantrene, respectively. Addition of ascorbic acid prior to incubation of mitoxantrone in human plasma at 37°C resulted in less than a 10% decrease in the latter's concentration over a 24-h period. To maintain sample integrity, all plasma samples should be fortified with ascorbic acid and kept frozen prior to analyses.     

Thymic cell migration in the subnodular spaces of draining lymph nodes of rats

Little is known about the pathway of possible lymphocyte traffic in the nodules (germinal centers) of the node. Observations reported here indicate the involvement of the subnodular spaces. These spaces, which constitute the inner limit of the nodules, are contiguous to the perivascular channels of the postcapillary venules which partially encircle each nodule. Twelve hours after a local transfer of labeled thymic cells, they were observed in the subnodular spaces and in the perivascular channels of the draining nodes. It is proposed that the spaces and the channels provide a pathway for the rapid migration of lymphocytes entering a node via the afferent lymph and, probably, carrying an immunogenic information. The pathway would permit these cells to transmit the information rapidly to the appropriate cell population(s) of a node draining a tissue undergoing an immunological process.     

NEUROSECRETORY CELL PROTEIN METABOLISM IN THE LAND SNAIL,OTALA LACTEA

—Protein synthesis in an identified molluscan neurosecretory cell of the land snail, Otala lactea was examined using three different types of polyacrylamide gel electrophoresis. Cells taken from active snails synthesized specific low molecular weight proteins while those from aestivated snails did not. Most of the newly synthesized low molecular weight proteins in the active snails were lost from the cell body when the preparations was chased for 19 h in label-free enriched medium in the presence of anisomycin, an inhibitor of protein synthesis. If colchicine, a blocker of axonal transport, was included in the chase medium, the proteins present following a pulse were largely replaced by smaller molecular weight species. The results suggest that specific low molecular weight proteins are converted to smaller species and then transported from the cell body.