Serum Ornithine Carbamoyl Transferase as a Surrogate Marker in Malaria

Ornithine carbamoyl transferase (OCT) activity and other liver function tests were studied in a total of 50 patients of clinical malaria and 15 controls. They were grouped as group I (positive for malarial parasite on peripheral blood smear, n=18), group II (negative for malarial parasite on peripheral blood smear (PBS) but responded to antimalarials, n=17) and group III (peripheral blood smear negative and did not respond to antimalarial therapy, n=15). The mean OCT levels were significantly raised in group I (6.79 ± 1.84 IU/L, p value = 0.006) and group II (5.0 ± 1.15 IU/L, p value = 0.014) as compared to controls (2.5 ± 1.13 IU/L) and returned to normal after treatment In contrast, group III had normal levels except in a case of kala azar and septicemia where OCT levels were high and increased further on treatment. Taking PBS positivity as a gold standard of diagnostic criteria, OCT had a sensitivity of 83% and specificity of 86% with a high positive predictive value of 88% as compared to ALT which had a lower sensitivity of 55% and specificity of 80%. The clinical response rate in PBS negative cases of fever having high OCT level was 83% as compared to 35% in cases with normal OCT level, making OCT a good surrogate marker of malaria. OCT levels could also be of prognostic significance as 2 cases of cerebral malaria had high OCT levels of 11.1 UAL and 10.7 IU/L, respectively.Key Words: Malaria, Ornithine carbamoyl transferase     

Spectrin-alpha I/61: a new structural variant of alpha-spectrin in a double-heterozygous form of hereditary pyropoikilocytosis

Recent biochemical studies have led to the identification of abnormal spectrins in the erythrocytes of patients with hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis (HE). In this report we describe the biochemical characterization of the erythrocytes from a proband with severe HPP who is doubly heterozygous for two mutant spectrins (Sp): Sp alpha I/74 and a new, previously undetected, mutant of alpha-spectrin designated Sp alpha I/61. The proband's erythrocytes are unstable when exposed to 45 degrees C, and her membrane skeletons exhibit instability to shear stress. The content of spectrin in the proband's erythrocyte membranes is decreased to 75% of control values. The amount of spectrin dimers in crude 4 degrees C spectrin extracts is increased (58%) as compared with control values (6% +/- 4%). Limited tryptic digestion reveals a marked decrease in the normal 80,000-dalton alpha I domain, an increase in the 74,000-dalton fragment that is characteristic of Sp alpha I/74, and an increase in a series of new fragments of 61,000, 55,000, 21,000, and 16,000 daltons. Both parents are asymptomatic, but they have increased amounts of spectrin dimers (17% to 25%). Limited tryptic digestion of the father's spectrin demonstrates the presence of a previously identified abnormal spectrin (Sp alpha I/74) that is characterized by a decrease in content of the 80,000-dalton peptide and an increase in concentration of the 74,000-dalton peptide. The mother's spectrin digests show a decrease in the amount of 80,000-dalton peptide and the formation of new peptides of 61,000, 55,000, 21,000, and 16,000 daltons. The data indicate that this severe form of HPP is due to the inheritance of two distinct abnormal spectrins, Sp alpha I/74 and a new spectrin mutant, Sp alpha I/61.     

Exopolysaccharide defects cause hyper-thymineless death in Escherichia coli via massive loss of chromosomal DNA and cell lysis

Thymineless death in Escherichia coli thyA mutants growing in the absence of thymidine (dT) is preceded by a substantial resistance phase, during which the culture titer remains static, as if the chromosome has to accumulate damage before ultimately failing. Significant chromosomal replication and fragmentation during the resistance phase could provide appropriate sources of this damage. Alternatively, the initial chromosomal replication in thymine (T)-starved cells could reflect a considerable endogenous dT source, making the resistance phase a delay of acute starvation, rather than an integral part of thymineless death. Here we identify such a low-molecular-weight (LMW)-dT source as mostly dTDP-glucose and its derivatives, used to synthesize enterobacterial common antigen (ECA). The thyA mutant, in which dTDP-glucose production is blocked by the rfbA rffH mutations, lacks a LMW-dT pool, the initial DNA synthesis during T-starvation and the resistance phase. Remarkably, the thyA mutant that makes dTDP-glucose and initiates ECA synthesis normally yet cannot complete it due to the rffC defect, maintains a regular LMW-dT pool, but cannot recover dTTP from it, and thus suffers T-hyperstarvation, dying precipitously, completely losing chromosomal DNA and eventually lysing, even without chromosomal replication. At the same time, its ECA⁺ thyA parent does not lyse during T-starvation, while both the dramatic killing and chromosomal DNA loss in the ECA-deficient thyA mutants precede cell lysis. We conclude that: 1) the significant pool of dTDP-hexoses delays acute T-starvation; 2) T-starvation destabilizes even nonreplicating chromosomes, while T-hyperstarvation destroys them; and 3) beyond the chromosome, T-hyperstarvation also destabilizes the cell envelope.

Acute starvation for thymidine triphosphate (dTTP), one of the four precursors for DNA synthesis, is lethal in both bacterial and eukaryotic cells (1). Following a short resistance phase, the rapid death of thyA auxotrophs in media lacking thymine or thymidine (“T-starvation”) known as thymineless death (TLD) was first described in Escherichia coli (2, 3) and since then was extensively studied to identify the cause of lethality (1, 4, 5). Because the bulk of thymidine (dT) in any cell is used for chromosomal DNA synthesis, lack of dT was always assumed to cause some form of chromosomal damage, and hence the role of DNA repair pathways during T-starvation was the focus of intense investigation (6–9). These studies revealed that certain pathways, like double-strand break repair initiated by the RecBCD helicase/nuclease, Holliday junction resolution by RuvABC, and antirecombination activity of the UvrD helicase, keep cells alive during the resistance phase of T-starvation. Other events, like attempted single-strand gap repair initiated by the RecFOR complex, the function of the RecQ helicase and RecJ exonuclease, and SOS induction of the cell division inhibitor SulA, are detrimental for T-starved E. coli cells (8, 10–12). However, the thyA mutants of E. coli inactivated for all of the latter “toxic DNA repair pathways” still die by two orders of magnitude during T-starvation (8), indicating some other yet-to-be-identified major lethality factors.Since actively growing cells continuously require a lot of dT to replicate chromosomal DNA, existing replication forks were inferred to be the points of TLD pathology (7, 8, 13–15). Indeed, T-starvation severely inhibits chromosomal DNA replication (15) and is associated with accumulation of single-stranded DNA, suggesting generation of single-strand (ss) gaps by attempted replication in the absence of dT (7, 16). These ss-gaps induce the SOS response (7, 8, 17), which contributes to the pathology of TLD by induction of the SulA cell division inhibitor (8). Also, replication initiation spike in the T-starved cells triggers the destruction of the origin-centered chromosomal subdomain during TLD, suggesting that it is the demise of the nascent replication bubbles, rather than the existing replication forks, that eventually kills the chromosome (15, 17).Although the thyA mutants cannot synthesize dT, they grow normally if supplemented with exogenous dT/T. Upon removal of dT from the growth medium, the E. coli thyA strain has a two-generation-long resistance phase (also called the lag phase) (1), when the colony-forming unit (CFU) titer of the culture stays constant (Fig. 1 A, Top). This is followed by the rapid exponential death (RED) phase, when the CFU titer falls by approximately three orders of magnitude within several hours (Fig. 1 A, Top).Open in a separate windowFig. 1.A significant endogenous pool of LMW dT decreases during T-starvation. (A) The phenomenon of TLD in E. coli suggests accumulation of chromosomal damage during the resistance phase (green) that would later kill cells during the RED phase (red). The data are adapted from ref. 16. Henceforth, the data are means (n ≥ 3) ± SEM. Cultures were grown at 37 °C in the presence of dT, which was removed at time = 0, while incubation in the growth medium continued. Top, cell death begins after 1-h-long resistance phase, during which the culture titer is stable. Bottom, during the same first hour without dT, cells manage to synthesize the amount of DNA equal to half of what they already had before dT removal. However, during the RED phase genomic DNA is gradually lost. (B) Scheme of 50% methanol fractionation of the intracellular thymidine into HMW dT (the dT content of the chromosomal DNA) and LMW dT. (C) A 0.7% agarose gel analysis of the HMW and LMW fractions of the 50% methanol-treated cells, as well as pure LB treated the same way, for DNA and RNA content (staining with ethidium bromide). Inverse images of stained gels are shown, in which the indicated samples were either incubated in the buffer or with the indicated enzyme (DNase I for the top gel, RNase A for the bottom gel). (D) The size of the LMW-dT pool, normalized to the HMW-dT content of the chromosome, either during normal growth in dT-supplemented medium or during T-starvation. Thymidine was removed at time = 0. The strain is KKW58.An obvious explanation for the resistance phase is existence of an intracellular source of dT to support slow replication; however, chromosomal DNA amount was consistently reported to remain flat during TLD (15, 18–20). Besides, the recent systematic test of potential candidates for a source of dT or its analogs supporting the resistance phase returned empty-handed (16). Thus, the mechanisms behind the initial resistance to T-starvation, followed by the sudden shift to the RED phase remain unclear, leading to a reasonable assumption that the resistance phase is an integral part of the TLD phenomenon, during which chromosomal damage accumulates until it becomes irreparable, ushering the RED phase (1, 5). Specific early events during the resistance phase of TLD that would later turn poisonous during the RED phase were proposed to be futile incorporation–excision cycles (1, 21), ss-gap accumulation causing the SOS induction (1, 8, 16), futile fork breakage-repair cycles (16, 22), and overinitiation from the origin (5, 15).Two recent observations, in combination with an old popular TLD explanation, further support the idea of the resistance phase as the TLD period during which chromosomal damage accumulates without affecting viability for the time being. First, the resistance phase coincides with accumulation of double-strand breaks in the chromosome, which then paradoxically disappear during the RED phase (7, 16). Second (and in contrast to the reports mentioned above of constant chromosomal DNA amount during T-starvation) (15, 18–20), we have found that during the resistance phase the amount of the chromosomal DNA actually increases ∼1.5 times over the prestarvation level, but then the chromosomal DNA is apparently destabilized during the RED phase, since it is slowly reduced to the original level (16) (Fig. 1 A, Bottom). Therefore, both the apparent chromosomal replication and the significant chromosome fragmentation during the resistance phase could lead to accumulation of chromosomal damage (SOS induction is an indicator of this accumulation) (7).On the other hand, the early DNA synthesis and the resistance phase in T-starved cells could reflect the existence of a source of dT, available early on during T-starvation, that fuels the initial DNA accumulation and delays viability loss until this pool is exhausted. In other words, the resistance phase could simply postpone TLD, rather than being an integral part of it. Previously, we have tested the two obvious high-molecular-weight (HMW) dT sources, namely, the stable RNAs and the chromosomal DNA, but found that incapacitation of neither one reduced the resistance phase or precluded the early DNA synthesis during T-starvation (16). Thus, the question of whether the resistance phase is a part of the TLD phenomenon remains unresolved.In the current study, we investigated a seemingly remote possibility of a substantial low-molecular-weight (LMW)-dT pool supporting the resistance phase of T-starvation in E. coli. While the bulk of dTTP in E. coli immediately incorporates into the chromosomal DNA, a fraction of dTTP is recruited into the dTDP-hexose pool (23), to participate in the synthesis of the exopolysaccharide (EPS) capsule, made of core lipopolysaccharide (LPS) (24), O-antigen (OA) (25), and enterobacterial common antigen (ECA) in E. coli (26). The first step of this recruitment is to conjugate dTTP with glucose; the hexose moiety of the resulting dTDP-glucose then undergoes several modifications, before eventually incorporating into oligosaccharide precursors of the outer antigens, while the activating dTDP handle is released back into the DNA precursor pools (26). We ignored LMW dT before because, if the total dT content of the chromosomal DNA is taken for 100%, the pool of dTTP constitutes ∼0.7% of it, while dTDP-glucose (unresolved from other dTDP-hexoses?) adds only another 2.4% (27). No more LMW-dT species are known in the cell, so the total expected LMW-dT pool comes to ∼3% of the total dT content of the chromosomal DNA, not nearly enough to support the resistance phase with its ∼50% increase in the chromosomal DNA mass (Fig. 1A).To investigate the role of the LMW-dT pool in TLD, we started by developing a simple protocol to extract the LMW-dT pool from growing cells and to compare it to the (HMW) chromosomal dT content. Here we show that early on during T-starvation the pool of dTDP-sugars becomes the major source of dTTP for the chromosomal DNA replication. This unexpected rebalancing of the dTTP pool with the help of cell envelope metabolism delays TLD and prevents T-hyperstarvation, a significantly more lethal phenomenon accompanied by complete chromosome destruction and cell lysis.     

Cell transplantation in surgical treatment of familial adenomatous polyposis coli

We developed a method of surgical treatment of familial adenomatous polyposis coli giving an opportunity to prevent the growth of new polyps in the preserved part of the rectum and consisting in transplantation of fetal cells of the epithelial origin into the rectum wall after mucosectomy. Since the rectum is partially preserved, ileorectal anastomosis can be formed after colectomy, which preserves natural bowel passage. Complex examination 4 weeks after surgery revealed the formation of normal rectal mucosa. No new polyps were detected in the rectum 1–3 years after surgery.