The effect of α-factor on the rate of cell-cycle initiation in Saccharomyces cerevisiae : α-Factor modulates transition probability in yeast

The rate of cell-cycle initiation was studied in a-cells of S. cerevisiae in the presence of the synthetic analogue of α-factor [N-Trp, Arg⁷]-α-factor (TA-αF). It was shown that TA-αF lowers the rate constant of cell-cycle initiation (or transition probability) for each separate cell. It was concluded on the basis of these results that the term ‘arrest’ in G1 by α-factor should be interpreted in a quantitative sense as the decrease in the probability of the emergence from ‘start’ per unit time and not as being equivalent to an ‘all-or-none’ response. The dependence of the rate constant of the cell-cycle initiation on the concentration of TA-αF can be described by the Hill equation where n = 1.01 + 0.05 and K = 14.5 ± 2.5 nM (±S.E.). It is demonstrated that after transfer of cells into the medium with a higher or a lower concentration of TA-αF, the rate constant of cell-cycle initiation changes abruptly from one value to another after a lag-period of 30 and 40 min respectively. This suggests a multistep mechanism of action for α-factor. The difference in the lag-periods allows us to suggest that α-factor exerts its action by two independent pathways. Since the Hill coefficient is practically equal to unit, no cooperative interactions are likely to be involved at least in one of these pathways. The inhibition of cell-cycle initiation can be used as a more adequate and sensitive test for biological activity of α-factor as compared to morphological measurements.     

Alpha-factor inhibition of the rate of cell passage through the "start" step of cell division in Saccharomyces cerevisiae yeast: estimation of the division delay per alpha-factor.receptor complex

A highly sensitive, kinetically unambiguous assay for alpha-factor-induced delay of cell passage through the "start" step of cell division in yeast is presented. The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the alpha-factor execution point of start. The t1/2 for cell passage through start by this population of cells is 31 min in the absence of alpha-factor. The inhibition constant, KI, represents the alpha-factor concentration which produces a 50% inhibition of this rate and is equal to 2 X 10(-10) M. A second assay for maximal cell division arrest by alpha-factor on whole populations of cells is presented. This assay shows a maximum cell division arrest time of 125 +/- 5 h at saturating alpha-factor, and a K50 (that is, an alpha-factor concentration which produces a half-maximal response) of 2.5 X 10(-8) M. Both assays were performed in the effective absence of alpha-factor inactivation. Values of the dissociation constant KD and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5 X 10(-8) M and 10(4), respectively. These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor.alpha-factor complex which is present on the cell at equilibrium producing a 43 +/- 10 s delay of cell passage through start. Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to alpha-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 nM alpha-factor to a 125 +/- 5 h maximum arrest at saturating alpha-factor concentrations of greater than 170 nM. The possible significance of this observation toward the mechanism of alpha-factor-induced cell division arrest is discussed.     

“Active monomer” polymerization of δ-benzyl L-α-aminoadipate

The N-carboxyanhydride of δ-benzyl L -α-aminoadipate polymerizes when initiated by strong base through an “active monomer” mechanism. This is shown by the isolation of 6-oxo-L -pipecolic acid from the product mixture, since this byproduct could only arise from an “active monomer” pathway. This study also specifies conditions for preparing high molecular weight poly(δ-benzyl L -α-aminoadipate) in high yield.     

Control of cell division in the yeast Saccharomyces cerevisiae cultured at different growth rates

Saccharomyces cerevisiae has been grown with different generation times by alterations in media richness and by altering the flow rate of the limiting nutrient, glucose in a chemostat. Within the generation time range 2.89-approx. 8.0 h the time from the initiation of DNA synthesis to cell division was independent of generation time and was approx. 2 h. Thus the cell cycle of yeast can be divided into an expandable phase from cell division to the initiation of DNA synthesis, the length of which is dependent on growth rate and a constant phase from the initiation of DNA synthesis to cell division which takes a constant time independent of generation time. In cells growing with generation times longer than 8.6 h this constant phase expands somewhat in time. These results are reminiscent of the observation that in the bacterium Escherichia coliB/R the time from initiation of DNA synthesis to cell division is constant except at very slow growth rates.     

The isolation of nonsense mutations in cell division cycle genes of the yeast Saccharomyces cerevisiae

Summary Nonsense mutations have been isolated in cell division cycle genes in a strain of Saccharomyces cerevisiae that carries a temperature-sensitive amber suppressor. These mutants may be valuable in identifying the products of genes involved in the cycle and in determining the pattern of their synthesis during the cell cycle.     

Low fluences of ultraviolet irradiation stimulate hela cell surface aminopeptidase and candidate “TGFαase” activity

Several forms of perturbation result in the release of bioactive molecules into the microenvironment of injured cells to mediate the inflammatory or reparative reactions which restore normal tissue structure and function. Amongst other products, ultraviolet irradiation (UV) causes the release of the growth factor TGFα from a variety of epithelial cell sources, apparently by a post-translational mechanism. Here we have explored the hypothesis that UV results in the activation of cell surface proteases which may then be capable of excising mature TGFα from its plasma membrane-bound precursor. Using a recently described, sensitive assay of peptidase activity tailored to the substrate requirements for cleavage of the scissile bonds in proTGFα, we have found that nonlethal fluences of UV ( < 12 Jm^?2) to HeLa cell cultures are followed by large increases in cell surface proteolytic activities. Amongst these, endopeptidase activity produces a similar product profile from the nonapeptide substrate to that of human leukocyte elastase, an enzyme previously shown to be capable of releasing a bioactive, mature form of TGFα from its cell-bound precursor. However, in addition to this candidate “TGFase” activity, cell surface aminopeptidase activity was also very significantly increased. The increase in the two classes of peptidase function differed in the timing of their responses. Aminopeptidase activation occurred immediately following UV, peaking after some 15–20 h, whereas the increase in endopeptidase activity lagged 6 h behind, cresting after 20–24 h. No evidence for a role for aminopeptidase in the activation of the endopeptidase could be found. Also, there was no increase in the total proteolytic activity demonstratable in cell extracts following UV. Attempts to interrupt the UV peptidase activation by inhibiting protein synthesis with cycloheximide were unsuccessful; rather, the inhibitor itself caused an increase in both classes of peptidase activity during the first 20 h. Unlike the UV response, both the aminopeptidase and endopeptidase ectoactivities increased simultaneously within a few hours of introducing cycloheximide into the medium of unirradiated cultures. The cycloheximide induced activity peaked after 20 h. Interestingly, cycloheximide alone has previously been shown to potentiate TGFα release from a cell line producing its precursor constitutively. These data suggest that both UV and cycloheximide can initiate reactions in HeLa cells which result in ectopeptidase activation of a global nature. Since both agents result in rapid interruption of DNA synthesis, it is possible that this cell surface proteolytic response may be analogous to, or part of, the “mammalian genetic stress response.” The mechanism of the activation of the cell surface proteases appears to be post-translational, perhaps part of a proteolytic cascade originating from perturbed macromolecular synthesis. © 1993 Wiley-Liss, Inc.     

“Capacity”, “best interests”, “will and preferences” and the UN Convention on the Rights of Persons with Disabilities

The United Nations (UN) Convention on the Rights of Persons with Disabilities (CRPD) is the most up‐to‐date international legal instrument concerning the rights of persons with disabilities. Such persons are taken to include those with serious mental disorders. According to an authoritative interpretation of a crucial Article (Article 12 ‐ Equal recognition before the law) by the UN CRPD Committee, involuntary detention and treatment of people with mental health disabilities are prohibited under the Convention. Both conventional mental health law and “capacity‐based” law are deemed to violate the Convention. However, some other UN bodies are not in full agreement (for example, the UN Human Rights Committee and the Subcommittee on Prevention of Torture and Other Cruel, Inhuman or Degrading Treatment or Punishment), while others are less explicitly absolutist (for example, the Human Rights Council). Furthermore, strong criticisms of the position of the CRPD Committee have been mounted from a number of academic quarters. These criticisms center on whether the role of a person's ability to make a decision can be ignored, no matter the circumstances. Much of the above debate turns on the concept of “legal capacity” and the now often‐repeated precept that one must always respect the “will and preferences” of the person with a disability. However, “will and preferences” remains undefined. In this paper, I offer an analysis of “will and preferences” that can clarify interventions that may be acceptable or non‐acceptable under the terms of the UN Convention.     

A critique of the “ultra‐high risk” and “transition” paradigm

The transdiagnostic expression of psychotic experiences in common mental disorder (anxiety/depression/substance use disorder) is associated with a poorer prognosis, and a small minority of people may indeed develop a clinical picture that meets criteria for schizophrenia. However, it appears neither useful nor valid to observe early states of multidimensional psychopathology in young people through the “schizo”‐prism, and apply misleadingly simple, unnecessary and inefficient binary concepts of “risk” and “transition”. A review of the “ultra‐high risk” (UHR) or “clinical high risk” (CHR) literature indicates that UHR/CHR samples are highly heterogeneous and represent individuals diagnosed with common mental disorder (anxiety/depression/substance use disorder) and a degree of psychotic experiences. Epidemiological research has shown that psychotic experiences are a (possibly non‐causal) marker of the severity of multidimensional psychopathology, driving poor outcome, yet notions of “risk” and “transition” in UHR/CHR research are restrictively defined on the basis of positive psychotic phenomena alone, ignoring how baseline differences in multidimensional psychopathology may differentially impact course and outcome. The concepts of “risk” and “transition” in UHR/CHR research are measured on the same dimensional scale, yet are used to produce artificial diagnostic shifts. In fact, “transition” in UHR/CHR research occurs mainly as a function of variable sample enrichment strategies rather than the UHR/CHR “criteria” themselves. Furthermore, transition rates in UHR/CHR research are inflated as they do not exclude false positives associated with the natural fluctuation of dimensional expression of psychosis. Biological associations with “transition” thus likely represent false positive findings, as was the initial claim of strong effects of omega‐3 polyunsatured fatty acids in UHR samples. A large body of UHR/CHR intervention research has focused on the questionable outcome of “transition”, which shows lack of correlation with functional outcome. It may be more productive to consider the full range of person‐specific psychopathology in all young individuals who seek help for mental health problems, instead of “policing” youngsters for the transdiagnostic dimension of psychosis. Instead of the relatively inefficient medical high‐risk approach, a public health perspective, focusing on improved access to a low‐stigma, high‐hope, small scale and youth‐specific environment with acceptable language and interventions may represent a more useful and efficient strategy.     

Distribution of the Character States “Early Sympetaly” and “Late Sympetaly” Within the “Sympetalae Tetracyclicae” and Presumably Allied Groups*

Within the Asteridae (“Sympetalae Tetracyclicae”) two main developmental patterns can be distinguished as regards the formation of corolla tubes, namely “early” and “late sympetaly”. Since the character “ontogeny of sympetaly”, after intensive studies, proved to be valuable for systematic considerations, we now recognize two blocks of orders within the Asteridae (and related groups): the Asteridae A-block and the Asteridae B-block (Figs. 82 and 83). By and large this bipartition referring mainly to the corolla tube development corresponds well with major lineages indentified by recent molecular data (see, e.g., Chase et al., 1993). Asteridae A-block is characterized predominantely by “late sympetaly” the “early sympetaly” in Rubiales and Oleales can be interpreted as derived within this block or can be linked with that in Asteridae B-block. Asteridae B-block is uniform throughout in its “early sympetaly”. In this block it is a primitive character, as judged from its occasional occurrence in the choripetalous Cornales and Apiales (Apiaceae, Araliaceae, Pittosporaceae), which can be regarded as ancestral for block B.     

“On A Piece of Chalk”-Updated1

ABSTRACT. Many advances have been made in our knowledge of the biology of foraminifera over the past several decades. Fine structural, biophysical, and molecular biological studies have shown that the most prominent components of their distinctive bidirectional granuloreliculopods are bundles of micro tubules linked by crossbridges to each other, as well as to membrane-bound organelles and the plasma membrane. the microtubules ratchet past each other as dynein transduces the free energy of ATP to produce pseudopodal movements. In spite of the fact that there are over 40,000 described species of living and fossil species of foraminifera, there have been many recent exciting discoveries of new species and groups. New casting techniques are providing us with greater understanding of the complexities and functional aspects of form in the group. Significant advances are being made in understanding the distribution and energetics of deep-sea forms. Larger and planktonic foraminifera are the hosts for a particularly diverse range of endosymbiotic algae, including dinoflagellates, chlorophytes, unicellular rhodophytes, and diatoms. Chloroplast husbandry also occurs. Significant research effort has been expended yielding us considerable insight into various aspects of the endosymbiotic phenomenon. A unified conceptual framework has been drawn to help us understand the life cycle options found in foraminifera.     

β-Alanine containing cyclic peptides with turned structure: The “pseudo type II β-turn.” VI

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and nmr solution characterization, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(L -Pro-L -Phe-β-Ala)₂. The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from methanol-dichloro-methane solution. The two identical halves of the molecule adopt in the solid state two different conformations. One β-Ala-L -Pro peptide bond is trans, while the second is cis. The molecule is present in dimethylsulfoxide d₆ solutions as a mixture of conformational families. One of these corresponds to a C₂ symmetrical molecule with both β-Ala-Pro cis peptide bonds, while the second major conformation is very similar to that observed in the solid state. All Pro-Phe segments, both in the solid state and the symmetrical and unsym-metrical solution conformations, display ?,ψ angles close to that of position i + 1 and i + 2 of type II β-turns. In addition, the segments preceeded by a trans β-Ala-Pro peptide bond are characterized by a typical i ← i + 3 hydrogen bond, which is absent in the conformer containing a cis β-Ala-Pro peptide bond. The latter conformation corresponds to a new structural domain we define as the “pseudo type II β-turn.” © 1994 John Wiley & Sons, Inc.     

“Prolonged grief disorder” and “persistent complex bereavement disorder”, but not “complicated grief”, are one and the same diagnostic entity: an analysis of data from the Yale Bereavement Study

There exists a general consensus that prolonged grief disorder (PGD), or some variant of PGD, represents a distinct mental disorder worthy of diagnosis and treatment. Nevertheless, confusion remains over whether different names and proposed symptom criteria for this disorder identify the same or different diagnostic entities. This study aimed to determine whether PGD, complicated grief (CG), and persistent complex bereavement disorder (PCBD) as described by the DSM‐5 are substantively or merely semantically different diagnostic entities. Data were derived from the Yale Bereavement Study, a longitudinal community‐based study of bereaved individuals funded by the US National Institute of Mental Health, designed explicitly to evaluate diagnostic criteria for disordered grief. The results suggested that the difference between PGD and PCBD is only semantic. The level of agreement between the original PGD test, a new version of the PGD test proposed for ICD‐11 and the PCBD test was high (pairwise kappa coefficients = 0.80‐0.84). Their estimates of rate of disorder in this community sample were similarly low (～10%). Their levels of diagnostic specificity were comparably high (95.0‐98.3%). Their predictive validity was comparable. In contrast, the test for CG had only moderate agreement with those for PGD and PCBD; its estimate of rate of disorder was three‐fold higher (～30%); its diagnostic specificity was poorer, and it had no predictive validity. We conclude that PGD, PCBD and proposed ICD‐11, but not CG, symptom‐diagnostic tests identify a single diagnostic entity. Ultimately, brief symptom‐diagnostic tests, such as the one proposed here for ICD‐11, may have the greatest clinical utility.     

Kinetics of product inhibition in alcohol fermentation-temperature effect in the “sake” brewing

One of the kinteic equations derived previously from a series of sophisticated batch and continuous alcohol fermentations by using a respiration-deficient mutant of baker's yeast is as follows: where dp/dt = ethanol production rate, v0 = specific rate of ethanol production at p = 0, k₂ = empirical constant, K′_s = saturation constant, S = glucose concentration, and X = cell mass concentration. The above equation was confirmed in the previous paper to fit, the brewing of “sake.” The temperature of the specific brewing is not always constant (10 to 18°C). The effect of temperature on v0 was assessed from the Arrhenius plot, assuming that k₂ was independent of temperature. Values of dp/dt taken from the “sake” brewing data were rearranged, taking the temperature change into account. These datu, corrected for the temperature, were found to follow quite favorably the kinetic equation mentioned above. So far, a prediction of the ethanol production rate in practice was rectified to the extent of p = 19%.