Radiation inactivation of hamster acrosin reveals that the biologically active unit is of low molecular size

The relationship between structure and activity of acid-extracted and purified acrosin obtained from cauda epididymal hamster spermatozoa was studied. A four-step purification procedure of acrosin was used; it included 1.) acid extraction, 2.) gel filtration over Sephadex G-100 resin, 3.) ion exchange on CM-Sepharose CL-6B, and 4.) affinity chromatography on proflavin-Sepharose 4B. Analysis of the purified enzyme by high-performance liquid chromatography (300 SW + I-125) revealed a molecular weight of 44,000, which was identical to that obtained for acid-extracted acrosin. Slab-gel electrophoresis under nondenaturing conditions showed only one active band, as revealed with a highly sensitive assay using N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester as substrate. The radiation inactivation size of acid extracted acrosin was calculated to be 8400. This small unit could represent the active polypeptide portion of a larger monomer molecule or could represent the size of active subunits. Because acrosin is autocatalytic and highly active during fertilization, it is suggested that the active portion of the completely processed form of the enzyme is of small molecular weight.     

The effect of monofunctional or difunctional platinum adducts and of various other associated DNA damage on the expression of transfected DNA in mammalian cell lines sensitive or resistant to difunctional agents

The effects of introducing various DNA damage into pSV2gpt DNA on the subsequent expression of xanthine guanine phosphoribosyltransferase (XGPRT), after its transfection into two Walker 256 cell lines, one which is inherently sensitive only to difunctional agents while the other shows a normal sensitivity, have been examined. Both the sensitive (WS) and the relatively resistant (WR) cell lines were shown to be equally capable of both ligation of DNA double-strand breaks (although the efficiency varied with the actual site of the break) introduced into pSV2gpt and homologous recombination of pSV2gpt fragments (recombination events are thought to be important in the repair of DNA-DNA interstrand crosslinks). Reacting the plasmid with either the difunctional platinum compound, Cisplatin, or the monofunctional reacting Pt(Dien) caused a dose-dependent decrease in the subsequent expression of XGPRT. This decrease was about the same with either agent in either cell line when expressed as a function of dose of drug. However, when the actual binding of platinum to DNA by these compounds was measured, a large difference (due to the higher specific binding of Pt(Dien) to DNA) in the effects of the difunctional, as opposed to the monofunctional agent, was apparent and this was a reflection of the relative cytotoxicities of these compounds towards mammalian cells. Although at doses of Cisplatin equitoxic to WS and WR cells 20-fold less Pt is bound to the DNA of WS cells, no significant difference was seen on the expression of pSV2gpt, reacted with this agent, between WS or WR cells. Based upon a knowledge of the proportions of adducts formed in DNA reacted with Cisplatin, the lesion that inactivates expression of XGPRT was probably the intrastrand crosslink and it was calculated that due to the size of the plasmid, the interstrand crosslink was unlikely to be present at these inactivating doses. It is suggested that the inherent sensitivity of WS cells only to difunctional agents is due to their response to such relatively rare lesions such as a DNA-DNA interstrand crosslink.     

Different functional domains of the adenovirus E1A gene are involved in regulation of host cell cycle products.

We have analyzed the cell cycle effects that different domains of the adenovirus E1A proteins have on quiescent primary BRK cells. Studies with deletion mutants that in combination removed all but the N-terminal 85 amino acids common to both the 12S and 13S proteins suggest that this region may be sufficient for the induction of synthesis of proliferating cell nuclear antigen and the stimulation of DNA synthesis. A second domain also common to the N-terminal exon of the 12S and 13S proteins was required for the induction of mitosis and stimulation of proliferation of primary BRK cells. A virus containing a mutation in this region was still able to stimulate DNA synthesis efficiently. A third domain, unique to the 13S protein, was required for the accelerated activation of the cellular thymidylate synthase gene in a manner similar to the 13S-dependent stimulation of adenovirus early region genes.     

Assignment of the prealbumin (PALB) gene (familial amyloidotic polyneuropathy) to human chromosome region 18q11.2–q12.1

Summary The assignment of the human prealbumin (PALB) gene to chromosome region 18q11–q12.1 has been achieved using a human genomic probe in the study of human-mouse somatic cell hybrids and by in situ hybridization. Because familial amyloidotic polyneuropathy was reported previously to be due to a mutation in prealbumin, it can be inferred that the gene for this disorder also maps to 18q11.2–q12.1.     

Homozygous paracentric inversion 12 in a mentally retarded boy: a case report and review of the literature

Summary A mentally retarded male was found to be homozygous for a paracentric inversion of the long arm of chromosome 12(inv(12)(q21.1q23.2)). His parents, who are first cousins, and his phenotypically normal younger brother are inversion heterozygotes. Homozygous structural rearrangements are discussed and cases of paracentric inversions, including a further nine previously unpublished, are reviewed.     

A natural hybrid newt, Triturus helveticus×T. vulgaris, from a pond in mid-Wales

The first unequivocal example of a natural Trilurus helvelicus × T. vulgaris hybrid is described. The specimen was a male and discriminant analysis of physical characters indicated that it was morphologically intermediate between the parent species. A karyotype confirmed that the hybrid bore a haploid set of chromosomes from T. helveticus and a haploid set from T. vulgaris. Examination of the sex chromosomes showed that it was the result of mating between a male T. helveticus and a female T. vulgaris. As numerous mature sperm bundles were observed in both testes, the hybrid was therefore potentially fertile.     

The response of murine intestinal crypts to short-range promethium-147 beta irradiation: deductions concerning clonogenic cell numbers and positions

An exteriorized loop of mouse intestine was exposed to 147Pm low-energy electrons, where the dose rate decreased by a factor of 5 from the base of the crypt to the top of the proliferative zone. A crypt survival curve was obtained, expressed in terms of exposure time. The shape of the curve was interpreted in terms of survival parameters for colony-forming cells (clonogens) derived using 137Cs gamma rays and the depth-dose curve measured for 147Pm electrons. It is concluded that the shape of the crypt survival curve using 147Pm electrons is inconsistent with the notion of either the presence of a large number of clonogens or a small number near the top of the proliferative zone. A computer fitting procedure showed that the best agreement between predicted and observed curves was achieved with 2.7 +/- 0.5 clonogens at cell position 5.6 +/- 0.6, in the putative stem-cell zone.     

Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C.

Chromogranin A is a major component of storage granules in many different secretory cell types. After [35S]methionine labelling of proteins from cultured bovine chromaffin cells, chromogranin A was immunoprecipitated with specific antibodies, and the radioactivity incorporated into chromogranin A was determined and used as an index of its synthesis rate. Depolarization of cells with nicotine or high K+ evoked a Ca2+-dependent increase in chromogranin A synthesis, whereas muscarine, which does not evoke significant Ca2+ influx from bovine chromaffin cells, had no effect on chromogranin A synthesis. Forskolin, an activator of adenylate cyclase, affected neither the basal nor the nicotine-stimulated rate of chromogranin A synthesis. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, significantly enhanced the incorporation of radioactivity into chromogranin A. Sphingosine, an inhibitor of protein kinase C, abolished both nicotine-stimulated and TPA-induced chromogranin A synthesis. In addition, long-term treatment of chromaffin cells with TPA decreased protein kinase C activity and inhibited the nicotine-stimulated chromogranin A synthesis. These results suggest that protein kinase C may play an important role in the control of chromogranin A synthesis.