Maintenance of aPaecilomyces variotii culture in the frozen and Freeze-dried state and in distilled water

The highest number of viable.elements ofPaecilomyces variotii, forming colonies after a 1-year maintenance, was detected in frozen sample. Decreased viability of the frozen culture and culture maintained in distilled water was usually statistically significant after 1 year and decreased further with increasing age of the culture used for sample preparation. Freeze-drying also significantly decreased the strain viability, depending on culture age. In the freeze-dried culture stored in a refrigerator the relative number of viable elements was substantially higher than after storage at room temperature. After a 3-year storage of freeze-dried P.variotii in a refrigerator 14-44% of culture elements survived, as compared with the number detected immediately after freeze-drying.     

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.     

Metabolic incorporation of 9-(2-anthryl)-nonanoic acid, a new fluorescent and photoactivable probe, into the membrane lipids of Chinese hamster ovary cells

9-(2-Anthryl)-nonanoic acid is a new fluorescent and photoactivable probe, which has been designed for studying the lateral diffusion rate and the lateral distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. It is shown that this anthracene fatty acid is metabolically incorporated into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of the eukaryotic Chinese hamster ovary cells in culture. Under our culture conditions (Eagle's minimal essential medium plus delipidized fetal calf serum) this incorporation proceeded with a very good rate (up to 45 mol/100 mol, after two days culture) and could be easily modulated depending on the way the cells were fed with the anthracene fatty acid. It occurred to a similar extent at the sn-1 (55 +/- 5%) or at the sn-2 (45 +/- 5%) position on the phospholipid glycerol backbone, without any degradation or elongation. No double labelling at the sn-1 and sn-2 positions was detected. Although incorporation of the anthracene fatty acid affected the cell growth rate (generation time of 48 h compared to a generation time of 21 h for control cells) it did not bring about cell mortality. This incorporation took place not only into the phospholipids but also into the triglycerides with, as a consequence, the appearance of strongly fluorescent lipid vesicles inside the cells. It affected the whole cell fatty acid composition by slightly increasing the amount of palmitic acid and markedly decreasing the amount of stearic and oleic acids.     

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.     

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.     

Response to low-dose pulsatile cortisol in Addison's disease with suspected corticotropinoma

A 65 year old woman with long-standing Addison's disease treated with oral glucocorticoid and mineralocorticoid replacement had persistently high ACTH levels, inadequate suppression of ACTH on low-dose dexamethasone, sellar enlargement, and pigmentation, and thus resembled patients alleged to develop corticotropinomas while on oral replacement for adrenal insufficiency. Since animal studies suggested that rapid rises of corticosteroids within the physiologic range can inhibit ACTH release, we administered brief infusions of cortisol every three hours with total daily dose equal to her chronic dose. Prompt suppression of ACTH and immunoreactive beta-endorphin occurred during each cortisol dose profiled, suggesting a role for ultradian cortisol fluctuations in tonic inhibition of ACTH secretion in humans, and a possible therapeutic benefit of mimicking ultradian cortisol rhythms during replacement therapy.