Fast atom bombardment: A new mass spectrometric method for peptide sequence analysis

We have studied a selection of peptides using a new mass spectrometric ionisation technique - fast atom bombardment (FAB). We define the fragmentation pathways observed and comment on the utility in sequence analysis. A simple acetylation experiment is shown to aid rapid sequence assignment.     

Individual variation of nucleoside triphosphate pyrophosphohydrolase activity in human erythrocytes,granulocytes, lymphocytes,and platelets

Analysis of the nucleoside triphosphate pyrophosphohydrolase specific activity of red cells obtained from a random Caucasian population indicated at least two subclasses. The specific activity of 18% of the population ranged from undetectable activity to 27.5 nmol ITP cleaved/20 min/mg hemoglobin. The remainder of the population had higher activity, 27.5–125 nmoles ITP cleaved/20 min/mg hemoglobin. The variation of NTPH activity evident in the red cells of an individual is reflected in granulocytes, lymphocytes, and platelets of that individual. Erythrocyte activity ranges from 0.7 to 21 units (nmol of ITP cleaved in 20 min)/10⁷ cells, granulocytes have 17–201 units/10⁷ cells, lymphocytes have 91–462 units/10⁷ cells, and platelets have 1.1–7.1 units/10⁷ platelets. These cell differences are discussed with respect to the hypothesis that NTPH prevents incorporation of ITP or dITP into nucleic acids.This work was supported by funds allocated by the Agricultural Experiment Station, Michigan State University. Michigan Agricultural Experiment Station Journal No. 8727.     

Relationships between cell-substratum interactions and the distribution of concanavalin A receptors of mouse embryo fibroblasts

The mobility of concanavalin A (ConA) receptors on the surfaces of mouse embryo cells are affected by cell-substratum interactions. When the cells are grown on a substratum from which they are easily detached by EDTA, their receptors have an increased mobility and are redistributed into patches after incubation with ConA at 37 °C.     

The terminal structures of feather keratin mRNA

Terminal labeling of embryonic feather keratin mRNA with [³H]KBH₄ followed by digestion with ribonuclease T₁ and T₂, alkaline phosphatase, snake venom phosphodiesterase, and nucleotide pyrophosphatase and analysis of the products by high voltage paper electrophoresis, indicated the presence of the sequence m⁷G(5)ppp(5)N at the 5-end of the mRNA. Ribonuclease T₁ and A digests of the terminally labeled, and also of unlabeled mRNA followed by fractionation on denaturing polyacrylamide gels indicated the presence of polyadenylate tracts ranging in size from 45 to 165 nucleotides at the 3-end of the mRNA.     

Properties of the activation by pepsin of inactive renin in human amniotic fluid.

1. The renin present in human amniotic fluid was found to have an apparent Mr of 58 000 by gel filtration and is thus bigger than renin in untreated kidney extracts and plasma (Mr approximately 40 000). 2. Treatment with pepsin (40 microgram/ml pH 4.8, 2 h, 22 degrees C) caused a 6-fold increase in activity of this renin species, although Mr was not very different (57 000). 3. Unlike renal renin, renin in human amniotic fluid was not a glycoprotein and behaved similarly on concanavalin A-Sepharose before and after activation by pepsin. 4. Ion-exchange chromatography demonstrated a small change in the ionization properties of human amniotic fluid renin after activation by pepsin. 5. Pepsin-mediated activation resulted in a five-fold increase in V, but only a small decrease in the Km of renin to 39% of normal, so that the increase in activity observed was not due to an increase in the affinity of the enzyme for its substrate. The kinetic data were consistent with the theory of noncompetitive inhibition. 6. The activation of human amniotic fluid renin by pepsin may be caused by a change in the tertiary structure of the molecule subsequent to a proteolytic action that does not remove detectable polypeptide components.     

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.     

Measuring the unknown: An estimator and simulation study for assessing case reporting during epidemics

The fraction of cases reported, known as ‘reporting’, is a key performance indicator in an outbreak response, and an essential factor to consider when modelling epidemics and assessing their impact on populations. Unfortunately, its estimation is inherently difficult, as it relates to the part of an epidemic which is, by definition, not observed. We introduce a simple statistical method for estimating reporting, initially developed for the response to Ebola in Eastern Democratic Republic of the Congo (DRC), 2018–2020. This approach uses transmission chain data typically gathered through case investigation and contact tracing, and uses the proportion of investigated cases with a known, reported infector as a proxy for reporting. Using simulated epidemics, we study how this method performs for different outbreak sizes and reporting levels. Results suggest that our method has low bias, reasonable precision, and despite sub-optimal coverage, usually provides estimates within close range (5–10%) of the true value. Being fast and simple, this method could be useful for estimating reporting in real-time in settings where person-to-person transmission is the main driver of the epidemic, and where case investigation is routinely performed as part of surveillance and contact tracing activities.