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Biomedical subjects

E Martegani

Publications and source records attributed to E Martegani.

At least 73 records · Page 4Linked to original sources
PubMed1984

Dansylalanyllysylchloromethyl ketone as a fluorescent probe for localization of acrosin activity in boar and human spermatozoa.

The localization of acrosin (EC 3.4.21.10) activity in mammalian spermatozoa was investigated by use of the fluorescent site-directed acrosin inhibitor, dansylalanyllysylchloromethyl ketone (DALCK). Fluorescence microscope preparations revealed, after the spermatozoa were subjected to a specific treatment, that acrosin activity is confined specifically to the inner acrosomal membrane (IAM). Spectrofluorometric and fluorescence polarization investigations verified that the fluorescent probe, once it is specifically bound to the treated spermatozoa, lies in a very hydrophobic environment and shows a remarkable reduction of rotational freedom. These results are compatible with the hypothesis that, under the experimental conditions used, active acrosin is tightly bound to the IAM and that the "specificity site" of the acrosin-active center is probably of a highly hydrophobic nature.

Acrosin↗
PubMed1983

Control points in Neurospora crassa nuclear division cycle: different effects of the inhibition of protein accumulation.

The correlation between protein synthesis and the nuclear division cycle in Neurospora crassa hyphae was studied by inhibiting protein accumulation by two different experimental procedures: (1) starvation for lysine in a lysine-requiring mutant (lys-1); and (2) addition of cycloheximide. Lysine starvation in a lys-1 strain of N. crassa quickly blocked the nuclear division cycle and nuclei accumulated in G1 phase, as judged by their DNA content. After re-addition of lysine to starved cultures, a discontinuous pattern of uridine incorporation into DNA can be seen, showing that the nuclei were well synchronized. On the other hand, treatment with cycloheximide caused the arrest of a large proportion of the nuclei, also, in the G2 phase of the cell cycle. These results indicate that inhibition of protein synthesis may have multiple effects on the cell cycle in N. crassa and that, while moderate inhibition specifically blocks nuclei at a regulatory point in late G1, strong or complete inhibition demonstrates requirement for protein synthesis at other points in the cycle that are not necessarily regulatory points.

Cell Division↗
PubMed1982

Cytochemical demonstration that acrosin is unavailable in intact ejaculated boar and bull spermatozoa.

The cytochemical localization of acrosin (EC 3.4.21.10) was investigated using a fluorescent technique, based on the use of fluorogenic substrates, derivatives of 4-methoxy-beta-naphthylamine, which liberate a fluorescent tag that, if coupled to 5-nitrosalicylaldehyde, generates an insoluble, fluorescent product. From the data obtained from boar and bull spermatozoa that had been subjected to different treatments, we have found that the acrosomal, trypsinlike proteinase is practically inaccessible in intact, freshly ejaculated spermatozoa. Acrosin activity was detected after subjecting the spermatozoa to a treatment that simulates the process they undergo at the moment of fertilization, showing a particulate localization in the acrosome of some of the spermatozoa.

Acrosin↗
PubMed1982

Cell cycle analysis in a human cell line (EUE cells).

A new procedure to determine the durations of cell cycle phases is based on th autoradiography of labeled nuclei and a standard chemical determination of DNA content per cell. It has been used to study the cell cycle timing of a human established cell line, EUE cells, so far poorly characterized in its kinetic aspects. The cycle phase durations obtained with this method are compared with the DNA frequency profiles determined by flow cytometry and found to agree closely. The relevance of the growth conditions of the exponentially growing cultures for a correct estimation of the cycle phase duration is indicated. Cell size distribution has been shown to vary according to growth condition.

Autoradiography↗
PubMed1981

Inhibition of RNA synthesis in Neurospora crassa hyphae treated with picolinic acid.

Picolinic acid, a metal-chelating agent, blocks the nuclei of germinating conidia of Neurospora crassa in the G1 phase. The addition of picolinic acid to exponentially growing cultures of N. crassa in glucose medium causes an immediate inhibition of growth. Concentrations higher than 50 mM completely inhibit growth of mycelia, whereas lower concentrations (20-30 mM) give only a partial inhibition. Picolinic acid, added at the concentration of 20 mM, immediately blocks the accumulation of stable RNA, while protein accumulation continues at an unchanged rate for at least 30 min and only thereafter is slightly inhibited. DNA accumulation rapidly slows down after the addition of the drug. The level of polyadenylated RNA decreases quickly after the addition of picolinic acid, and the rate of its synthesis also appears to be inhibited. Picolinic acid greatly affects the metabolism of stable RNA in N. crassa: both processing and transcription of rRNA are sequentially inhibited with a moderate accumulation of precursor rRNA. It is concluded that the effects of picolinic acid on the nuclear division cycle in Neurospora may be related to the requirement of specific gene products to enter the S phase, whose synthesis is inhibited following the reduction of polyadenylated RNA synthesis.

Centrifugation, Density Gradient↗
PubMed1981

Timing of nuclear division cycle in Neurospora crassa.

The timing of the nuclear division cycle in Neurospora crassa was studied by inhibiting DNA synthesis with hydroxyurea added to exponentially growing cultures at concentrations that do not inhibit cytoplasmic growth. After the addition of hydroxyurea only the nuclei that have passed the S-phase divide, and therefore by counting the increase in the average number of nuclei per hypha, the stage in the cycle in which completion of S phase occurred was determined in different nutritional conditions of exponential growth. The stage at which DNA replication terminates is different in different conditions of exponential growth; however, when the length of G2+M phases is calculated a constancy of these phases is shown. Our data indicate that in N. crassa the coordination between cytoplasmic growth and the nuclear division cycle is achieved mainly through a growth-rate-dependent expansion of the G1 phase, according to a sizer-plus-timer model of control of the nuclear division cycle.

Cell Cycle↗
PubMed1980

Reduction of ribosome activity and synthesis of stable RNA in Neurospora crassa.

The addition of cycloheximide (0.02 micrograms/ml) to exponentially growing cultures of Neurospora crassa causes a reduction in growth rate and a decrease in the rate of protein accumulation, due to a partial inhibition of protein synthesis, while RNA accumulation is unaffected for about 1 h. Thus, an increased RNA:protein ratio is established in the presence of the inhibitor. RNA that accumulates during treatment with cycloheximide has the same characteristics as that of the control cultures and this, together with the enhancement of the relative rate of synthesis of ribosomal proteins induced by cycloheximide, seems to indicate that more mature ribosomes are present in cycloheximide-treated cultures. The endocellular level of several amino acids begins to increase significantly only 60 min after cycloheximide addition. A possible explanation of the stimulation of ribosome production induced by cycloheximide is given in terms of the existence of a feed-back mechanism controlling ribosome synthesis.

Cell Division↗
PubMed1980

Nuclear division cycle in Neurospora crassa hyphae under different growth conditions.

Treatment with picolinic acid blocked Neurospora crassa nuclei in G1, and recovery from the treatment allowed a synchronous wave of deoxyribonucleic acid synthesis to occur. Nuclei, which appeared as compact globular bodies during the period of blockage, assumed a ring shape during the following S phase, which was also maintained in the G2 phase. The proportion of compact globular nuclei was much higher in hyphae growing at lower rates, whereas that of ring nuclei increased when the hyphae were growing at higher rates. Horseshoe nuclei (probably mitotic nuclei) and double ring nuclei were also observed in growing hyphae, but their frequencies were low and fairly independent of the rate of growth. The length of the S phase of the Neurospora nuclear division cycle was determined to be about 30 min. From the frequencies of the phase-specific nuclear shapes, the durations of the G1 phase and the combined S plus G2 phases were calculated. The results showed that variations in the growth rates of the mycelia were mainly coupled with variations in the G1 phase of the nuclear division cycle. For mycelia growing in minimal sucrose, the lengths of all of the phases of the nuclear division cycle were estimated.

Cell Division↗
PubMed1979

Level and turnover of polyadenylate-containing ribonucleic acid in Neurospora crassa in different steady states of growth.

Mycelia of Neurospora crassa in a steady state of growth in different media have a ribosomal content proportional to the rate of growth. Moreover, both the percentage of polysomes and the average ribosomal activity are about the same at all different growth rates. The content of polyadenylated RNA was determined in three different conditions of exponential growth, which allowed growth rates that ranged from 0.26 to 0.51 duplications/h, and was found to constitute about the same fraction of total RNA (4.5--5.2%). Using a kinetic approach, an equation was derived which allowed determination of the average half-lives of polyadenylated RNA: in each medium the cultures were labeled from the moment of the inoculation with [32P]orthophosphate and were then given a 10-min pulse with [5-3H]uridine when they were in the exponential phase. It was found that the determined half-lives of polyadenylated RNA vary, depending on the growth medium, between 30 and 60 min, but with no direct correlation with the growth rate. Moreover, the rate of synthesis of polyadenylated RNA relative to that of stable RNA decreased with the growth rate. On the basis of previous data on the rates of synthesis of stable RNA, it was possible to make an evaluation of the absolute rate of synthesis of polyadenylated RNA. Whereas the rate of synthesis of stable ribosomal RNA increases as a function of the square of the number of duplications per hour, the increase in the rate of synthesis of polyadenylated RNA with the growth rate is much less consistent. It is concluded that in Neurospora the growth rate does not depend on the rate of synthesis of mRNA but rather on the rate of synthesis of rRNA, which sets both the ribosomal level and the steady-state level of mRNA.

Acetates↗
PubMed1979

Intracellular protein degradation in Neurospora crassa.

In exponentially growing cultures of Neurospora crassa, the basal rate of protein degradation increases as the constant of the rate of growth decreases, so that in slow growing cells (mu = 0.13) the rate of protein degradation is about 25% of the rate of protein accumulation. During glucose starvation and shift-down transition of growth, the rate of protein degradation is greatly enhanced, and a moderate reduction (about 30%) of the ATP level is observed. Treatment of glucose-starved cells with 2-deoxyglucose reduces the ATP content by 70% and blocks protein degradation. The addition of cycloheximide, given at the onset of glucose starvation, prevents the enhancement of protein degradation; instead cycloheximide is without effect if added when proteolysis has already started. At a supraoptimal temperature (42 degrees C) the basal rate of protein degradation is not stimulated, contrary to the behavior observed in bacteria. Guanosine nucleotides, which appear to have a regulatory role for protein degradation in bacteria, are not found in N. crassa.

Amino Acids↗
PubMedDate not supplied

The sequence of 8.8 kb of yeast chromosome III cloned in lambda PM3270 contains an unusual long ORF (YCR601).

We have determined the nucleotide sequence of a segment of chromosome III contained in the right part of the lambda PM3270 clone, for a total of 8824 bp. This sequence contains an unusual long open reading frame, YCR601, of 6501 bp that encodes for a protein of 2167 amino acids that show no homology with other known proteins. YCR601 was disrupted by internal deletion and insertion of LEU2 gene and is a non-essential gene, however, it is transcribed during vegetative growth yielding a polyadenylated mRNA of approximately 7 kb.

Amino Acid Sequence↗
PubMedDate not supplied

A bimolecular mechanism for the cell size control of the cell cycle.

A molecular model for the control of cell size has been developed. It is based on two molecules, one (I) acts as an inhibitor of the entrance into S phase, and it is synthetised just after cell separation in a fixed amount per nucleus. The other (A) is an activator of the S phase, and it is synthetised at a ratio proportional to the overall protein accumulation. The activator reacts stoichiometrically with (I), and after all the (I) molecules have been titrated, (A) begins to accumulate. When it reaches a threshold value, it triggers the onset of DNA replication. This model was tested by simulation and when applied to the case of unequal division explains a number of features of an exponentially growing yeast cell population: (a) the lengths of TP (cycle time of parent cells) and TD (cycle time of daughter cells) verify the condition exp(- KTP ) + exp(- KTD ) = 1; (b) the changes of the average cell size of populations at different growth rates; (c) the frequency of parents and daughters at various growth rates; (d) the increase of cell size at bud initiation for cells of increasing genealogical age; (e) the existence of a TP - TB period (difference between the cycle time of parents and the length of budded phase) that depends linearly upon the doubling time of the population.

Cell Cycle↗
PubMedDate not supplied

Identification of different daughter and parent subpopulations in an asynchronously growing Saccharomyces cerevisiae population.

Under all growth conditions, a growing Saccharomyces cerevisiae yeast population is extremely heterogeneous, since individual cells differ in their cell size; this is due to their position in the cell division cycle and their genealogical age. To gain insight into the structure of a growing yeast population, we used a recently developed flow cytometric approach which enables, in asynchronously growing S. cerevisiae populations, tagging of both the cell age and the protein content of individual cells. This approach enabled the identification of daughter cells belonging to different cell cycle positions (i.e. newborn, G1, S + G2 + M + G1*, and dividing), thus yielding information about the relative fraction in the whole population, cell size and variability. More limited information could be obtained for the parent subpopulation; however, we were able to identify and characterize the dividing parent cells. The coefficient of variation (CV) of the protein content distribution for the dividing parents (27) was much higher than the CV of dividing daughters (18). Further findings obtained indicated a large overlap between the cell protein content distributions of daughter and parent cells as well as between the protein content of cells of the same subpopulation but belonging to different stages of the cell division cycle. The analysis of these differences enables a better understanding of the complex structure of an asynchronously growing yeast population.

Cell Cycle↗