Stable transformation of Drosophila Kc cells to antibiotic resistance with the bacterial neomycin resistance gene

By transfection with a plasmid containing the APH(3') gene under control of the HSV I thymidine kinase promoter, independent series of stably transformed Drosophila cells were established and grown for more than one and a half years under highly selective pressure (2 mg G 418/ml). Analysis of transformed Drosophila cell DNAs shows that the APH(3') gene was integrated into the genome. Neomycin phosphotransferase is constitutively expressed in transformed cells. This efficient selective system by a dominant marker makes it possible to introduce, by cotransfection, any DNA sequence of interest into the genome of cultured Drosophila cells.     

Deficient production of lysyl oxidase in cultures of malignantly transformed human cells

Lysyl oxidase activity in the culture medium of eight malignantly transformed human cell lines was very low compared with that in four control fibroblast lines, being 9-16% in five sarcoma cell lines and 7-11% in three other tumour cell lines. The low enzyme activity was probably due to deficient enzyme synthesis rather than impaired secretion into the cell medium, as low activity was also found in urea extracts of the cell pellets. Lysyl oxidase production thus appears to be closely regulated with deficient collagen gene expression in malignant transformation.     

Analysis of karyotype and C-banding pattern ofNicotiana plumbaginifolia using two techniques

The morphological study of the chromosomes ofNicotiana plumbaginifolia was carried out using two karyological techniques referred to as the squash method and the protoplast method. The latter involves isolation of protoplasts from unfixed root meristems and provides mitotic images in which the chromosomes, conveniently dispersed and disposed on the same plane, are significantly longer, and better structured, than those obtained by the squash method. A critical analysis of this technique is carried out. The results of this study led us to propose a karyotype forNicotiana plumbaginifolia and to characterize each chromosome by mere morphological examination. This characterization was further detailed by C-banding.     

Spreading and orientation of epithelial cells on grooved substrata

The spreading and orientation of epithelial (E) cells was studied on titanium-coated grooved substrata by light, transmission (TEM) and scanning electron microscopy (SEM). Vertical-walled grooves and V-shaped grooves, 3-60 microns deep, were produced in silicon wafers by micromachining, a process which was developed for the fabrication of micro-electronic components, and the grooved substrata were replicated in Epon. Photolithography was used to prepare photoresist-based and silicon dioxide-silicon substrata with grooves of approximately 2 and approximately 0.5 micron deep, respectively. Cell clusters were markedly oriented by all the grooved substrata examined, with the orientation index being highest for substrata with grooves of the smallest repeat spacing. Time-lapse cinemicrography showed that the grooves directed the migration of E cells, but the control was not absolute, as some cells crossed over the ridges and descended into the grooves. The 0.5 micron grooves appeared less effective than the deeper grooves in directing cell locomotion. SEM and TEM of E cells spreading on the grooved substrata demonstrated that cell processes, including lamellae and filopodia, were capable of bending around and closely adapting to groove edges. E cells did not flatten as extensively on a substratum with 22 microns deep V-shaped grooves as on a smooth surface, although some cells were markedly elongated. One mechanism proposed to explain contact guidance of fibroblasts is that linear elements of the locomotory system, such as microfilament bundles, are unable to operate when bent. The observed flexibility of epithelial cell processes and the ability of substrata with shallow grooves to orient E cells indicate that contact guidance of E cells on micromachined substrata cannot be explained by the mechanical stiffness of long linear cytoskeletal elements.     

Plasmodium berghei: reaction of sporozoites with chemically and enzymatically modified hepatoma cells

Plasmodium berghei sporozoites were observed to react with human hepatoma (HepG2) target cells which had been fixed with methanol, formaldehyde, or glutaraldehyde. The reaction consisted of attachment of sporozoites to the fixed target cells and the release of circumsporozoite protein which bound to target cell areas adjacent to the attachment sites. Treatment of fixed target cells with 0.1 N H2SO4 at 80 C, neuraminidases, neuraminidase plus galactose oxidase or inclusion of transferrin, orosomucoid, their asialo forms, or various monosaccharides in the incubation medium had no significant effect on target cell reactivity with sporozoites. Fixed cells oxidized with periodate or cells extracted with methanol or chloroform-methanol were reactive but lost activity if allowed to air dry after treatment. Treatment with papain or chymotrypsin at levels producing heavy cell structure damage caused a major loss of activity.     

Plasmodium falciparum: synchronization of cultures with DL-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis

DL-alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum. Asynchronous cultures were pretreated with sorbitol and incubated for 28-30 hr. Then, when cultures consisted of mainly schizont stage parasites, DL-alpha-difluoromethylornithine was added to the growth medium for another 38-47 hr of incubation. Putrescine was added to parasites arrested at the early trophozoite stage. This resulted in a synchronous resumption of growth. After 19 hr, 83% of parasites were at the schizont stage. After 30 hr, more than 98% of the parasites were in the ring form stage. Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia. Synchrony was maintained for 4-5 biological cycles as confirmed also by flow fluorimetry. It appears that this new approach to synchronize P. falciparum cultures is simple, reproducible, and effective.     

Castanospermine: a potent inhibitor of sucrase from the human enterocyte-like cell line Caco-2

The addition of castanospermine (5-50 microM) to a culture medium of Caco-2 cells results in a specific suppression of sucrase activity without modification of the biosynthesis of the enzyme. This effect is due to a direct inhibiting effect of castanospermine on Caco-2 sucrase activity. This inhibition is time-dependent (half-maximum efficiency at 10 min for 100 nM), enhanced by preincubation (suggesting a strong interaction with the enzyme), dose-dependent (ED50 at 4 nM after 1 h preincubation period) and of the fully non-competitive type. The calculated Ki (2.6 nM) suggests that castanospermine is the most potent inhibitor of sucrase so far reported.     

Biochemical differentiation among karyotypic forms of Australian Rattus

Isozyme electrophoresis of up to 55 loci, and microcomplement fixation of albumin were used to assess the extent of structural gene divergence among karyotypic forms of Australian Rattus. The results show that the Australian Rattus are monophyletic with respect to R. rattus or R. norvegicus. Within the Australian Rattus, rates of chromosomal evolution have varied enormously, the highest rates being found among members of the R. sordidus group, where extensive chromosomal repatterning has occurred with little or no structural gene divergence.     

Cloning and characteristics of recA gene in Pseudomonas aeruginosa

A recA-like gene from Pseudomonas aeruginosa was cloned and identified by means of interspecific complementation of gene recA repair defect in Escherichia coli. The gene was mapped in the PvuII-HindIII Ps. aeruginosa chromosome fragment of 1.5 kbp in length. Having been recloned in pUC18 or 19 plasmids in either of possible orientations, this fragment was shown to complement three different defects of E. coli recA mutants: in repair, recombination and SOS functions.     

Improvement of diabetes after treatment with somatostatin analogue SMS 201-995 in an acromegalic patient

The beneficial effects of long acting somatostatin analogue SMS 201-995 in an acromegalic patient affected by severe diabetes mellitus are reported. Neither human insulin alone nor human insulin plus bromocriptine allowed satisfactory metabolic control though, with the latter treatment, virtually normal plasma GH levels were reached. Conversely, addition of SMS 201-995 to insulin treatment led to normalization of blood glucose. This result was obtained with a dose of SMS 201-995 of 400 micrograms/day and only after 3 weeks of therapy.