Immunocytochemical localization of the 27K beta-crystallin polypeptide in the mouse lens during development using a specific monoclonal antibody: implications for cataract formation in the Philly mouse

The Philly mouse develops a hereditary cataract about 5 weeks after birth. Although the causative agent is not known, data suggest that there is a correlation between cataract formation and the selective absence of a 27 kilodalton (27K) beta-crystallin lens polypeptide. The ontogeny of the 27K beta-crystallin polypeptide was examined in normal mice in order to evaluate its role in normal development and determine what impact its absence may have on the Philly mouse lens. A monoclonal antibody was used with the PAP method to immunocytochemically localize the 27K polypeptide in lenses of normal mice during development. beta-Crystallins detected with polyclonal antisera were found in differentiated fiber cells throughout the lens. In contrast, the 27K beta-crystallin polypeptide detected with a specific monoclonal antibody was not found in the fiber cells of the inner part of the lens (nucleus), but was specifically localized in the fiber cells of the outer part of the lens called the cortex. The polypeptide was found only in elongating and differentiated fiber cells and not in mitotically active epithelial cells. Although a minor component of the 2-day-old lens, the 27K polypeptide comprised a large portion of the 16-day-old lens including the anterior and posterior poles. These data show that the 27K polypeptide is a minor component of the embryonic lens, but becomes a major contributor to the postnatal lens. The 27K beta-crystallin lens polypeptide is abundant in the fiber cells of the normal postnatal mouse lens. The absence of the 27K polypeptide in the Philly mouse may contribute to the observed failure of fiber cells to differentiate in the Philly mouse after birth or may be deleterious in some other manner to normal lens development. The selective absence of the 27K beta-crystallin polypeptide, a defect which precedes cataract formation in the Philly mouse, is intriguing since it suggests a relationship between this major lens polypeptide and lens clarity.     

Difference in arginine vasopressin responsive cAMP production between apical and basolateral membrane of cultured renal epithelial cells (MDCK)

It has been reported that vasopressin (AVP)-sensitive renal epithelial cell line (MDCK) forms morphologically polarized monolayers when cultured on plates. We studied whether the AVP-responsive cAMP production system would be located solely on the basolateral surface of these cells as has already been shown on the renal tubules. We used two methods to overcome the inaccessibility to the basolateral surface of the cultured cell layer and to study the apical and basolateral surfaces separately. One was culture on collagen sheet and the other was on Millipore filters. Our experiments showed that MDCK cell increased adenosine 3':5'-cyclic monophosphate (cAMP) content prominently only when vasopressin was accessible to the basolateral surface. Accordingly, MDCK cells were shown to have the AVP-responsive cAMP production system predominantly on the basolateral surface of the cell membrane.     

Measurement of urinary steroid profile in patients with adrenal tumor as a screening method for carcinoma

Results of measurement of urinary steroid metabolite profile using gas chromatographic analysis in eight patients with adrenocortical tumors, i.e. 3 adenomas with Cushing's Syndrome, one adenoma with virilization, one adenoma without clinical manifestations, one carcinoma with Cushing's syndrome and virilization, one carcinoma with Cushing's syndrome and feminization, and one carcinoma without endocrinological symptoms, are reported. A unique pattern dominated by 5 beta and 11 beta-hydroxy steroid metabolites was confirmed in five patients with Cushing's syndrome consisting of three cases with adenomas and two with carcinomas. Excessive 3 alpha, 17 alpha, 21-trihydroxy-5 beta-pregnan-20-one (tetrahydro-11-deoxycortisol, THS) and delta 5-pregnene-3 beta, 11 alpha, 20 alpha-triol (delta 5-pregnenetriol) values were found in all three carcinomas including a nonfunctional carcinoma. These findings would strongly suggest the tumor to be a carcinoma, although excessive excretion of THS and delta 5-pregnenetriol was detected in one patient with a large adenoma associated with virilization. One patient with carcinoma was responsive to ACTH stimulation while the remainder show almost no response to exogenous ACTH. Urinary steroid profiling using gas chromatographic analysis, especially the values for THS and delta 5-pregnenetriol, appears to be a useful method to use in detecting these steroid metabolic characteristics in patients with adrenocortical carcinoma.     

Analysis of apolipoproteins in high density lipoproteins by high performance liquid chromatography

A simple and rapid method for the analysis of apolipoproteins in high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) has been developed (Kinoshita et al. (1983) J. Biochem. 94, 615-617). With this method, using a sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS) as an eluent, apolipoproteins can be analyzed from a very small amount of HDL fraction without delipidation using organic solvents. Separation profiles of apolipoproteins by this method were examined using several techniques. The elution pattern monitored by A280 can give precise quantitative as well as qualitative information about size-distribution of apolipoproteins, except for the apo C group. Moreover, separation of apo E from apo A-I was found to be improved by column elongation.     

Specific cytolysis of fresh tumor cells by an autologous killer T cell line derived from an adult T cell leukemia/lymphoma patient

Cytotoxic T cells (Tc) derived from one patient with adult T cell leukemia/lymphoma (ATL) killed fresh autologous lymphoma cells in vitro. The Tc were induced from peripheral blood leukocytes (PBL) of this patient during remission by multiple in vitro stimulations with an autologous ATLV-bearing cell line (ILT) that was previously established by cloning of PBL in the presence of interleukin 2 (IL 2). PBL from eight other ATL patients were stimulated in the same manner, and responder cells from a patient in remission also showed cytotoxicity specific for ATL virus (ATLV)-bearing cells. Fresh lymphoma cells were obtained in relapse and were used as target cells for the autologous Tc induced. They became susceptible to the Tc within 4 hr of in vitro incubation, and their susceptibility increased with incubation time for at least 12 hr. ATLV antigens on the cell surface of these lymphoma cells, however, were not detected by radioimmunoassay during these incubation periods, but were detectable after 16 hr of incubation. In addition, cytotoxicity against lymphoma cells was completely inhibited by autologous ILT cells used as "cold" target competitor cells. These findings indicate that the target antigen of the Tc was expressed on both autologous ILT cells and lymphoma cells, and it may be different from ATLV antigens detected by serologic methods. In addition, the data suggested allogeneic restriction of the Tc in that the preferentially killed allogeneic ATLV-bearing cells share several HLA antigens.     

Phorbol myristate acetate-stimulated release of cyclooxygenase products in rat pleural cells: derivatization of prostaglandins with 9-anthryldiazomethane for fluorometric determination by high performance liquid chromatography

White cells were collected from the wash of rat pleural cavity after exsanguination. The incubation mixture of the pleural cells with 1 microM phorbol myristate acetate (PMA) was extracted with acidified ethanol and purified with a Sep-pak C18. The resultant fraction containing prostaglandins (PG) and thromboxane (TX) was allowed to react with 9-anthryldiazomethane (ADAM). After removing contaminants and degraded reagent by silica gel Sep-pak, samples were applied to reversed phase high performance liquid chromatography of octadecylsilyl silica gel and monitored by a fluorescent detector. ADAM derivatives of the authentic PGD2, PGE2, PGF2 alpha, 6-keto-PGF1 alpha, 6-keto-PGE1, TXB2, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2 alpha and 13,14-dihydro-15-keto-PGE2 showed linear regression lines of peak heights within a range of 0.5-25 ng. By using this method PGD2, 6-keto-PGF1 alpha and TXB2 were detected in the incubation mixture of the rat pleural cells with PMA. The result clarified the origin of these PGs and TX found in the exudate of rat pleurisy induced by PMA. ADAM method for HPLC with a help of clean-up by Sep-pak could be a useful tool for detection of a series of arachidonate metabolites in biological materials.     

Prostaglandin stimulation of adenosine 3',5'-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cyclic AMP level in cultured chondrocytes were examined. Prostaglandin E1 at 0.4 to 30 microM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D2 and E2 also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E1. Prostaglandin A1 had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E1, D2 or E2 was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E1, E2, or D2 on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin and PTH were more than additive. Addition of an inhibitor of cyclic nucleotide phosphodiesterase with prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E1, E2, and D2 increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

Fermentative production of 5′-purine ribonucleotide

Micrococcus sodonensis KY 3765 and Arthrobacter citreus KY 3155 were found capable of accumulating IMP in media supplemented with hypoxanthine as a precursor. High concentrations of phosphate and magnesium salts were required for high yields of IMP. Manganese deficiency in the media was also essential. Excessive Mn²⁺ effects were also seen in the IMP fermentation carried out with an adenineless mutant, of Cornynebacterium glutamicum. In M. sodonensis, R5P-like substances, 5-phosphoribose pyrophosphokinase and IMP pyrophosphorylase, were leaked out, of the cells grown in suboptimal Mn²⁺ levels. This excretion was inhibited by high levels of Mn²⁺. Such a phenomenon was not noted in A. citreus. An adenineless mutant (KY 7208) of Brevibacterium ammoniagenes was found to accumulate an appreciable amount of IMP. The chemical changes in this fermentation showed that, hypoxanthine was first produced de novo, excreted, and then reconverted into IMP by a salvage pathway. When hypoxanthine was added to 7208 culture, IMP yield was increased appreciably. In fact exogenous ¹⁴C-hypoxanthine was incorporated into ¹⁴C-IMP. Subsequent experiments showed that indeed Br. ammoniagenes ATCC 6872, a parent culture of KY 7208, was able to produce IMP, GMP, and AMP, in good yield from hypoxanthine, guanine, and adenine, respectively.