Detailed studies of the maturation of stimulus-secretion coupling of the pancreatic B-cell requires a supply of isolated fetal islets, which has so far been difficult to obtain. To overcome this problem we have maintained minced and mildly collagenase-digested fetal rat pancreatic glands (21.5 days gestational age) in tissue culture to enable degeneration of the acinar part, leaving the endocrine cells in an isolated and surviving state. Indeed, after 1 wk in culture there was a complete separation between acinar and endocrine cells with the appearance of numerous discrete islets and the disappearance or dedifferentiation of the exocrine cells. Isolated islets were either free floating or attached on top of a monolayer of fibroblast-like cells. Their number after 1 wk in culture was estimated as about 90 per explanted fetal pancreas and a total yield of about 5000 isolated islets was easily achieved. Both light arid electron microscopic examinations showed an excellent structural preservation with a marked predominance of well-granulated B-cells. Numerous islets of the same weight as that measured in cultured islets of adult rats were regularly found after 1 wk in culture. The insulin concentration of the cultured fetal islets was related to the glucose concentration of the growth medium. A similar relationship was found with respect to the insulin release in response to glucose. Thus, fetal islets cultured for 8 days in growth media containing 11.1 or 22.2 mM glucose showed a marked and significant insulin response to glucose in batch-type incubations at the end of the culture period. By contrast, the glucose stimulation of insulin release was insignificant in islets cultured at 5.5 or 2.8 mM glucose. When the culture period was confined to 1 day, there were no effects of glucose on the insulin release irrespective of the glucose concentration of the growth medium. It is concluded that the present technique for tissue culture of fetal rat pancreas makes it possible to isolate substantial amounts of fetal islets predominantly composed of B-cells. The transition in vitro from a poor glucose sensitivity to an adult-type insulin response indicates that the technique can be used in further detailed studies of the molecular mechanisms involved in the growth and development of the pancreatic B-cell.

The metazoan cell cycle-regulated histone mRNAs are the only known cellular mRNAs that do not terminate in a poly(A) tall. Instead, mammalian histone mRNAs terminate in a highly conserved stem-loop structure which is required for 3'-end processing and regulates mRNA stability. The poly(A) tail not only regulates translational efficiency and mRNA stability but is required for the function of the cap in translation (m(7)GpppN). We show that the histone terminal stem-loop is functionally similar to a poly(A) tail in that it enhances translational efficiency and is co-dependent on a cap in order to establish an efficient level of translation. The histone stem-loop is sufficient and necessary to increase the translation of reporter mRNA in transfected Chinese hamster ovary cells but must be positioned at the 3'-terminus in order to function optimally. Mutations within the conserved stem or loop regions reduced its ability to facilitate translation. All histone mRNAs in higher plants are polyadenylated. The histone stem-loop did not function to influence translational efficiency or mRNA stability in plant protoplasts. These data demonstrate that the histone stem/loop directs efficient translation and that it is functionally analogous to a poly(A) tail.

A simian virus 40 late replacement vector encoding human immunodeficiency virus type 1 (HIV-1) gp120 (pGP120) was used to define a region within the HIV-2 genome that could work as a rev-responsive element (RRE). Our previous work showed that gp120 expression in this system required a functional RRE in cis and required the rev protein in trans (M.-L. Hammarskjöld, J. Heimer, B. Hammarskjöld, I. Sangwan, L. Albert, and D. Rekosh, J. Virol. 63:1959-1966, 1989). Using pGP120, we first mapped an RRE to a 1,042-base-pair (bp) Sau3a fragment in the env region of HIV-2. Both HIV-1 rev (rev1) and HIV-2 rev (rev2) could work in conjunction with this fragment. Further mapping showed that a 272-bp subfragment within the 1,042-bp region was sufficient as an RRE. Surprisingly, the smaller fragment worked only with the rev1 protein and not with its homologous rev2 protein. In addition, the rev2 protein failed to function together with the RRE from HIV-1. We also utilized this system to examine the ability of the rex genes of human T-cell leukemia virus types I and II to functionally substitute for rev. These experiments showed that complementation by both the rexI and rexII proteins required the presence of an RRE. The rex proteins worked well in conjunction with either the HIV-1 or the HIV-2 RRE (the 1,042-bp as well as the 272-bp fragment).

Whole rat embryos were explanted at head-fold, late pre-somite stage (day 9.5 of gestation) and cultured in rat sera varyingly supplemented with glucose (3, 6, 9, or 12 mg/mL), D,L sodium beta-hydroxybutyrate (2, 4, 8, or 16 mM), or both (6 mg/mL D-glucose plus 8 mM beta-hydroxybutyrate). During 48 h culture, increasing glucose alone or beta-hydroxybutyrate alone effected growth retardation and faulty neural and extraneural organogenesis in dose-dependent fashion. Synergistic dysmorphogenic effects occurred when minimally teratogenic concentrations of glucose and beta-hydroxybutyrate were combined. Sera from diabetic animals containing somatomedin inhibitor bioactivity were also able to produce growth retardation and major developmental lesions in presence of amounts of glucose and ketones which of themselves were not teratogenic. Thus, aberrant fuels and fuel-related products can impair growth and organogenesis in early post-implantation embryo. Such fuel-mediated teratogenesis may be multifactorial and include possibilities for synergistic and additive interactions.

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