Hyperosmotic injury in mammalian cells. Volume and alkali cation alterations of CHO cells in unprotected and DMSO-treated cultures.

Correlated studies on volume distributions and cation (Na⁺ and K⁺) content of CHO cells in suspension were carried out after various exposures to hypertonic NaCl or sucrose (500–7550 mOsm in both the presence and absence of DMSO (5–20%; $\text{w}\text{v}$). The effects superimposed by ouabain (10^?2–10^?4m), amphotericin B (6–18 μg/ml), and glutaraldehyde (1.25%) on the above-mentioned parameters were also investigated. Volumetric analysis of CHO cells with the Coulter Channelyzer indicated a biphasic dose-dependent response to hypertonic media, the duration of the

TABLE 2. Correlation between Volume, Survival, and Cation Content of CHO Cells Exposed to Hypertonic Media in Suspension

     

2.

A simple system for extending refrigerated, nonfrozen preservation of biological material using pressure.     

S E Charm  H E Longmaid  J Carver 《Cryobiology》1977,14(5):625-636

High pressure (above 238 atmg) substantially extends the refrigerated storage of highly perishable biological material in a nonfrozen state.

5. Calculated Equivalent Values of Pressure and Temperature for Reducing Reaction Rates of Horseradish Peroxidase

Osmolality mOsm	Exposure (min)								Hypertonic agent
					NaCl				Sucrose
		$\text{V}{}^{\mathrm{a}}$	Na+	K+	$\text{S}{}^{\mathrm{b}}$	V	Na+	K+	S
1000	60 or less	Small	High	High	High	Normal	Low	High	High
1500–2000	60 or less	Small	High	Low	Low	Normal	Low	High	High
2000 or over	60 or more	Small or largec	High	Very low	Very low	Small	Very low	Very low	Very low

Pressure (atmg)	Temperature (°K)	$\text{T}\text{T}{}_0$a
0	296	0.999
272	289	0.975
340	287	0.969
408	285	0.963

a

$\text{T}{}_0\text{= 296 °}\text{K}$It has been known for some time that high pressure stops microbial growth. The effect of high pressure is to reduce further the enzyme activity at refrigerated temperatures. Two enzymes studied, peroxidase and crude trypsin from red crab intestine, demonstrated this effect.A number of food materials such as fish, beef, and chicken were tested for microbial growth and organoleptic qualities after high-pressure storage in a simple 14-liter pressure chamber. Pressure was generated by a hand pump. The results indicated that after 30 days those items held in a non-frozen state at ?3 °C and 238 atmg were not significantly different microbiologically and organoleptically from frozen controls at atmospheric pressure and ?20 °C.This system should be useful for the preservation of biological materials where freezing or thawing effects are undesirable or unknown.The energy saved compared to freezing should also be considered. Only 62% of the energy is required for storage at ?3 °C as compared with frozen storage at ?20 °C, and about 28 cal/g must be removed in cooling to ?3 °C as compared with 120 cal/g in cooling to ?20 °C.

     

A simple system for extending refrigerated, nonfrozen preservation of biological material using pressure.

High pressure (above 238 atmg) substantially extends the refrigerated storage of highly perishable biological material in a nonfrozen state.

5. Calculated Equivalent Values of Pressure and Temperature for Reducing Reaction Rates of Horseradish Peroxidase

Hemopoietic activities of cryopreserved murine fresh and postmortem bone marrow cells

The percentage of preservation of erythropoietic and granulopoietic precursor cells in the murine bone marrow was studied using in vitro methylcellulose clonal cell culture assays and in vivo murine spleen colony assays. This study clearly demonstrates

a. Type of Spleen Colonies Induced by 6-hr Postmortem Murine Bone Marrow Cells^a

	Mean (%)
Type of colonies	t Score	P Value	Unfrozen	Frozen
			Erythrocytic	26.283	14.100	2.09	0.059
Granulocytic	23.741	32.917	1.45	0.173
Mixed	49.321	52.700	0.55	0.59

a

$\text{N = 92}$. the presence of pluripotent hemopoietic precursor cells in cryopreserved 0-, 3-, 6-, 9-, and 12-hr postmortem murine bone marrow cells. Apparently, the erythropoietic precursor cells are more sensitive to freezing injury as compared to granulopoietic precursor cells.

     

Integrity of lysosomes in the isolated perfused rat liver before and after exposure to dimethylsulphoxide.

We have studied the integrity of lysosomes in isolated rat livers perfused for 3, 4, or 6 hr at 35 °C with BSA (40 g/l) in Krebs Ringer bicarbonate buffer. The latency and sedimentability of β-glucuronidase in homogenates of these livers was well maintained even after 6 hr. The latency and sedimentability of acid phosphatase remained at about control levels during the first 4 hr of perfusion but decreased between 4 and 6 hr. These decreases in latency and sedimentability correlated with a decrease in bile production and an increase in the rate of release of GOT into the perfusate and could indicate either intracellular disruption of lysosomes

Latencies, Sedimentabilities, and Specific Activities of Acid Phosphatase and β-Glucuronidase in Homogenates of Rat Liver Prepared before or at Various Times after Exposure to 1.4 m Me₂SO for 1 hr

     

5.

An experimental study using tissue culture medium as preservation and perfusion fluid     

T Nilsson  E Schueller  G P Murphy  W J Staubitz 《Cryobiology》1973,10(3):191-196

A commercially available tissue culture medium has been proven capable of preserving dog kidney function for at least 24 hr after simple cooling. The advantages of using tissue culture medium as preservation fluid instead of plasma or albumin solutions from the infectious and immunological points of view are obvious. An in vitro study was completed using the tissue

1.

Time (hr)	0	3	4	6
Acid phosphatase
Latency (%)	83.2 ± 0.8	64.7 ± 5.1	62.4 ± 7.9	68.9 ± 5.4
Sedimentability (%)	81.7 ± 0.6	77.6 ± 3.1	81.0 ± 3.4	79.4 ± 5.1
Specific activity (mIU/mg protein)	2.8 ± 0.4	2.8 ± 0.2	2.4 ± 0.4	1.6 ± 0.2
β-Glucuronidase
Latency (%)	06.8 ± 1.5	71.3 ± 4.3	74.2 ± 2.5	63.3 ± 4.5
Sedimentability (%)	69.5 ± 0.3	75.4 ± 3.2	75.0 ± 1.1	74.6 ± 2.0
Specific activity (mIU/mg protein)	1.8 ± 0.1	1.6 ± 0.1	1.6 ± 0.2	1.6 ± 0.2

     

6.

A digital thermocouple thermometer     

A R Hayes 《Cryobiology》1974,11(4):378-381

Measurements of the reproducibility of a random selection of copper/constantan thermocouples were made and it was found that they agreed within 1 ° C. Based on this finding, a digital thermocouple thermometer was designed and constructed incorporating a thermocouple linearizer and cold junction compensation. The instrument

Accuracy of the Completed Digital Thermometer

Code (animal No.)	Perfusion time (br)	Perfusion pressure mm/Hg	Flow ml/min	Weight gain	pH	pO2 mm/Hg	Histological appearance
1	24	70-60 systolic	96	35	7.3	150–180	Grossly normal
2	24		45-40 diastolic	108	30
3	24		96	30
4	48	70-60 systolic	80	35	7.3	150–180	Grossly normal
5	48	45-40 diastolic	120	40	7.4
6	48		100	40
7	72	70-60 systolic	115	40	7.4	150–180	Slight vacuolization of the tubular cells
8	72	45-40 diastolic	96	40
9	72		80	40
10	24	70-60 systolic	110	35	7.3	150–180	Used for transplantation
11	24	45-40 diastolic	120	35
12	24		140	40
13	24		100	30
14	24		96	30

     

7.

DNA synthesis catalysed by endogenous templates and DNA-dependent DNA polymerases in spermatogenic cells from rat     

Norman B. Hecht  Martti Parvinen 《Experimental cell research》1981,135(1):103-114

The activity levels of DNA polymerases α and β have been measured by autoradiography in squash preparations from rat testis of sexually mature animals. Similar results were obtained with ‘fixed’ samples (dipped in acetone: ethanol for 5 min at 25 °C) or ‘unfixed’ samples (frozen in liquid nitrogen and freeze-dried). The activities of DNA polymerases α and β in situ were distinguished by differential assay conditions and by selective inhibition with compounds such as N-ethylmaleimide and aphidicolin. Using the endogenous chromatin as template, maximal activity for both enzymes was obtained in the presence of all four deoxyribonucleoside triphosphates, MgCl₂ and ethylene glycol. When DNA polymerase activities in several predominant testicular cell types (pre-leptotene primary spermatocytes, pachytene primary spermatocytes, round spermatids and elongated spermatids) were quantitatively compared, on a per cell basis, the following percentage distribution was observed:

Temperature	Indicated	Error
(°C)	temperature	(°C)
	(°C)
?1.95.75	?195	0.75
?77.02	?78	0.38
0	0	0
52.49	53	0.51
Mean		0.413

     

8.

Cell Biology: Paper alert     

《Current opinion in cell biology》2000,12(5):525-535

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in cell biology.

	Pre-leptotene primary spermatocyte %	Pachytene primary spermatocyte %	Round spermatid %	Elongated spermatid %
DNA polymerase α	25	42	30	3
DNA polymerase β	29	34	36	1

     

9.

Heat exchange in the cryosurgery of Menière's disease: Experimental and clinical studies     

S. Harly  J.E. Aastrup  O. Elbrønd 《Cryobiology》1977,14(5):609-613

The temperature course in the lateral semicircular canal and in the facial canal was studied in experiments during freezing of the semicircular canal. The course of the temperature was measured with thermocouples. Concurrently, the heat flow was measured, and also the total heat exchange was measured throughout the freezing period by a thermoelectric heat flowmeter incorporated in the cryotip. The measurements showed correlation between the total amount of heat exchanged, the freezing time, and the temperature in the semicircular canal. This correlation was utilized to assess and calculate (the temperature of the lateral semicircular canal) the course of the cryoprocess in vivo, where it is possible to measure the heat flow and the total heat exchange during the freezing period only.

2. Results upon Vertigo

Contents (chosen by)
525	Cytoskeleton (Desai and Holleran)
526	Cell regulation (Roche, Servant and Weiner)
528	Nucleus and gene expression (Aasland and Weinzierl)
529	Membranes and sorting (Ponnambalam)
530	Membrane permeability (Slesinger)
531	Cell-to-cell contact and extracellular matrix (Pfaff)
533	Cell differentiation (van Roessel, Kaltschmidt, Tsang and Huckriede)
534	Cell multiplication (Sclafani)

     

10.

Clinical challenges     

《Clinical eye and vision care》1997,9(3):165-167

Glaucomatocyclitic crisis should be considered in the differential diagnosis of unilaterally increased IOP. A careful history, slit-lamp examination and gonioscopic examination will assist the examiner in making an accurate diagnosis.The etiology of this syndrome remains unknown. If indeed it is a disturbance of the anterior intraocular vasculature secondary to an autonomic abnormality, any of the proposed etiologies might possibly trigger an attack (Table 2).Treatment at this time should consist of 1 gH. 0.5% apraclonidine given in office or a topical steroid, cycloplegic, and ocular hypotensive agent combination. Follow-up should be within 24 h to monitor for IOP reduction. If treatment with apraclonidine is initiated, additional drops should be instilled as needed to lower the IOP to acceptable levels. Prophylactic treatment with topical steroids has been considered in this mostly benign syndrome [14]. However, the potential side-effects of long-term steroid use and the unpredictable frequency of glaucomatocyclitic crises may preclude steroid use in these cases. Table 2. Proposed etiological factors and diseases related to glaucomatocyclitis crisis.

	No Vertigo	Improved	Unchanged
Number of patients	7	5	3

     

11.

Introducing Titratable Water to All-Atom Molecular Dynamics at Constant?pH     

Wei Chen  Jason?A. Wallace  Zhi Yue  Jana?K. Shen 《Biophysical journal》2013,105(4):L15-L17

Recent development of titratable coions has paved the way for realizing all-atom molecular dynamics at constant pH. To further improve physical realism, here we describe a technique in which proton titration of the solute is directly coupled to the interconversion between water and hydroxide or hydronium. We test the new method in replica-exchange continuous constant pH molecular dynamics simulations of three proteins, HP36, BBL, and HEWL. The calculated pK_a values based on 10-ns sampling per replica have the average absolute and root-mean-square errors of 0.7 and 0.9 pH units, respectively. Introducing titratable water in molecular dynamics offers a means to model proton exchange between solute and solvent, thus opening a door to gaining new insights into the intricate details of biological phenomena involving proton translocation.Solution pH is an important factor in biology. Although neutral pH in extracellular medium accounts for balanced electrostatics and proper folding of protein structures, pH gradients across cell membranes induce large conformational changes that are necessary for biological functions, such as ATP synthesis and efflux of small molecules out of the cell. To gain detailed insights into pH-dependent conformational phenomena, several constant pH molecular dynamics (pHMD) methods, based on either discrete or continuous titration coordinates, have been developed in the last decade (1–4). In the continuous pHMD (CpHMD) framework (2,4), a set of titration coordinates {λ_i} are simultaneously propagated along with the conformational degrees of freedom. Although the original CpHMD method based on the generalized Born (GB) implicit-solvent models (2,4) offers quantitative prediction of pK_a values and pH dependence of folding and conformational dynamics of proteins (5), its accuracy and applicability to highly charged systems and those with dominantly hydrophobic regions are limited due to the approximate nature of the underlying implicit-solvent models.Motivated by the above-mentioned need, three groups have made efforts to develop a CpHMD method using exclusively the explicit-solvent models (6–8). In our development, the titration of acidic and basic sites is coupled with that of coions to level the total charge of the system (8). To further improve physical realism, here we replace the coions by titratable water molecules, which not only absorb the excess charge but also enable direct modeling of solute-solvent proton exchange in classical molecular dynamics simulations.To illustrate the utility of the new methodology, we applied it to the titration simulations of three proteins that were previously used to benchmark the GB-based CpHMD. Although this work does not explore specific interactions between titratable waters and proteins, the methodology can be further tested or improved to provide a rigorous way for modeling proton transfer in molecular dynamics, which is a computationally efficient alternative to the empirical valence-bond theory-based methodologies (9,10).We define titration of water as:

• 1.Loss of a proton to give a negatively charged hydroxide,

H₂O ? OH^? + H⁺, (1)or

• 2.Gain of a proton to give a positively charged hydronium,

H₂O + H⁺ ? H₃O⁺.(2)We now couple the titration of hydroxide (Eq. 1) with that of an acidic site of the solute in the CpHMD simulation,HA+OH−⇌KaA−+H2O.(3)The use of hydronium is avoided here to prevent a potential artifact due to prolonged attraction with A⁻. Analogously, we couple the titration of hydronium (Eq. 2) with that of a basic site,BH++H2O⇌KbH3O++B.(4)Thus, effectively, a proton is transferred between the solute and solvent. However, we should note that in CpHMD simulations, titratable protons are represented by covalently attached dummies (2,4). Through varying the atomic charges and van der Waals interactions, they are seen by other atoms in the protonated state but not in the unprotonated state (see Table S1 in the Supporting Material). Furthermore, the solution proton concentration is implicitly modeled through a free energy term (2,4).In CpHMD, the reference potential of mean force (PMF) for titration is that of the model compound (blocked single amino acid in water) along λ (2,4). In the presence of cotitrating water molecules, it is necessary to add the PMF for the conversion of water to hydroxide or hydronium. One-nanosecond NPT simulations at ambient pressure and temperature were performed to calculate the average force, 〈dU/d,θ〉 at given θ-values, which are related to λ by λ = sin² θ (see Fig. S1 in the Supporting Material). Thermodynamic integration was then applied to calculate the PMF. We found that the average force can be accurately fit when assuming the PMF is quadratic in λ (Fig. 1). The same applies to the PMFs for titration of models Asp, Glu, and His. After testing on the titration of model compounds (see Table S2), we performed 10-ns all-atom CpHMD simulations with the pH replica-exchange protocol for three proteins: HP36, BBL and HEWL (see the Supporting Material for details). Most of the calculated pK_a values were converged in 10 ns per replica (see Fig. S3). Results are summarized in Fig. S4. Based on the 10-ns data, the root-mean-square (RMS) and average absolute errors are 0.9 and 0.7 pH units, respectively, while the largest absolute error is 2.5 (Glu³⁵ of HEWL). Linear regression of the calculation versus experiment gives R² of 0.8 and slope of 1.2.Open in a separate windowFigure 1Average force and potential of mean force for converting a water molecule to hydroxide (A) and hydronium. (B) (Data points) Average forces. (Dashed curves) Best fits using a linear function, 2A(λ − B). (Solid curves) Corresponding potential of mean force.

Table 1

Calculated and experimental pK_a values of three proteins

Form of developmental glaucoma

Autonomic imbalance

Allergy, peptic ulcer, stress

Herpes simplex virus

Cytomegalovirus

Varicella-zoster virus

Mesodermal dysgenesis

Residue	Experimenta	GBa	All-atom CpHMD
	Time (ns)b	0–1	0–5	5–10	0–10
HP36
Asp44	3.10 (0.01)	3.2 (0.1)	2.0	3.0	2.6 (0.5)
Glu45	3.95 (0.01)	3.5 (0.1)	4.3	4.5	4.4 (0.1)
Asp46	3.45 (0.12)	3.5 (0.1)	2.4	3.7	3.1 (0.6)
Glu72	4.37 (0.03)	3.5 (0.1)	4.4	4.4	4.4 (0.0)
BBL
Asp129	3.88 (0.02)	3.2 (0.0)	2.2	3.2	2.7 (0.5)
Glu141	4.46 (0.04)	4.3 (0.0)	4.0	4.4	4.2 (0.2)
His142	6.47 (0.04)	7.1 (0.0)	5.9	5.8	5.8 (0.0)
Asp145	3.65 (0.04)	2.8 (0.2)	3.0	3.1	3.1 (0.0)
Glu161	3.72 (0.05)	3.6 (0.3)	4.2	3.9	4.0 (0.2)
Asp162	3.18 (0.04)	3.4 (0.3)	2.9	3.5	3.2 (0.3)
Glu164	4.50 (0.03)	4.5 (0.1)	5.7	4.6	5.2 (0.6)
His166	5.39 (0.02)	5.4 (0.1)	4.4	4.4	4.4 (0.0)
HEWL
Glu7	2.6 (0.2)	2.6 (0.1)	3.6	3.4	3.5 (0.1)
His15	5.5 (0.2)	5.3 (0.5)	5.1	5.1	5.1 (0.0)
Asp18	2.8 (0.3)	2.9 (0.0)	2.5	3.3	2.9 (0.4)
Glu35	6.1 (0.4)	4.4 (0.2)	8.5	8.7	8.6 (0.1)
Asp48	1.4 (0.2)	2.8 (0.2)	−0.1	1.1	0.6 (0.6)
Asp52	3.6 (0.3)	4.6 (0.0)	5.4	5.6	5.5 (0.1)
Asp66	1.2 (0.2)	1.2 (0.4)	−0.6	0.8	0.3 (0.7)
Asp87	2.2 (0.1)	2.0 (0.1)	0.8	2.1	1.5 (0.7)
Asp101	4.5 (0.1)	3.3 (0.3)	6.1	5.7	5.9 (0.2)
Asp119	3.5 (0.3)	2.5 (0.1)	3.0	3.3	3.2 (0.1)
Maximum absolute deviation		1.8	2.4	2.6	2.5
Average absolute deviation (RMS deviation)		0.5 (0.7)	1.0 (1.2)	0.6 (0.9)	0.7 (0.9)
Linear fit R2 (slope)		0.7 (0.8)	0.8 (1.4)	0.7 (1.1)	0.8 (1.2)

Open in a separate window^aTaken from Wallace and Shen (12). The pK_a''s of BBL were recalculated.^bSampling time per pH replica.Breaking the simulations in two halves, we noticed that the second 5-ns sampling gave better agreement with experiment. The RMS deviation is reduced from 1.2 to 0.9 pH units, while the average absolute deviation is reduced from 1.0 to 0.6 pH units. The linear regression against experimental data is also improved, with the slope decreasing from 1.4 to 1.1 although R₂ remains the same. Comparing these second-half results with the GB-based simulations, we find that the RMS and average absolute deviations are about the same as the GB-CpHMD results; however, the all-atom simulations show a small systematic overestimation (regression slope >1), whereas GB simulations show a systematic underestimation (regression slope <1).The improvement in the second halves of the simulations are seen mainly for residues involved in attractive electrostatic interactions, including Asp⁴⁴ and Asp⁴⁶ of HP36, Asp¹²⁹ of BBL, and Asp⁴⁸, Asp⁶⁶, and Asp⁸⁷ of HEWL. These residues are initially locked in salt-bridges or hydrogen bonds. However, in the second 5 ns, the attractive interactions weakened, leading to a decrease in the calculated pK_a shifts relative to the model values and better agreement with experiment. For instance, Asp⁴⁴ was initially in a salt-bridge distance from Arg⁵⁵. However, the salt-bridge positions were sampled less often in the second 5 ns (see Fig. S5), which explains the 1-unit reduction in the calculated pK_a shift. Significant fluctuation in ion-pair interactions was also observed in the work by Alexov (11). The carboxyl oxygen of Asp⁴⁶ was a hydrogen-bond acceptor with both the backbone amide and hydroxyl of Ser⁴³. These hydrogen bonds were less frequently sampled in the second 5 ns (see Fig. S6), leading to a decrease of the pK_a shift for Asp⁴⁶ by 1.3 units. These results indicate that extensive conformational sampling is necessary to give an accurate estimate of the ratio between the charged and neutral populations.Limited conformational sampling is also a contributing factor to the overestimation of the pK_a shifts for buried residues (Fig. S7 and Fig. S8). The increase in SASA is correlated with the more frequent sampling of the states with λ close to 1, i.e., the deprotonated form (see Fig. S9). However, because Glu³⁵ was buried in the starting conformation and the transition between buried and exposed states is slow compared to the simulation length, the exposed state may not be sufficiently sampled, leading to overestimation of the pK_a shift.In contrast to Glu³⁵, the SASA of Asp⁵² in HEWL is almost identical for both protonation states. The lack of conformational fluctuation is due to the strong hydrogen bonding with the side-chain amino group of Asn⁴⁶ and Asn⁵⁹ (data not shown). Overestimation of the pK_a shifts for buried residues can also be attributed to the limitation of the additive force field which underestimates dielectric response in protein environment (more discussion see Supporting Material) of the pK_a shifts for buried residues.Finally, to ascertain if the presence of hydroxide/hydronium introduces artifacts, we studied the interaction between hydroxide/hydronium and the titratable sites/ions. Comparing the hydroxide/hydronium with respective chloride/sodium ions, we find that the spatial distributions are nearly identical (see plots of distance distributions and radial distribution functions in Figs. S10–S13). However, the relative occupancy of the hydroxide around the neutral Asp/Glu, positive histidine, or sodium ion is 2–3 times as that of a chloride. The water-bridged interaction between sodium and chloride ions becomes much weaker when chloride is replaced by hydroxide or sodium is replaced by hydronium. By contrast, the occupancy of the hydronium around the solute is similar to that of the sodium. Furthermore, similar pK_a results for these proteins were obtained when coions were used instead of titratable waters (data not shown). Thus, we believe that potential artifacts related to the ionized forms of water are negligible. Work is underway to further understand the limitations of the methodology and to explore applications to protein dynamics coupled to proton transfer.In summary, we have developed and tested titratable water models for use in all-atom CpHMD simulations. Although the benchmark pK_a calculations indicate a comparable accuracy as the GB-CpHMD method, the all-atom method offers physical rigor and most importantly, it is applicable to systems that cannot be studied with GB-based simulations such as lipids and nucleic acids. We anticipate that the accuracy of this methodology can be further improved by incorporating the new-generation force fields that account for polarization. The coupling between proton titration of water and solute offers a computationally efficient way to model proton transfer in molecular mechanics simulations.     

An experimental study using tissue culture medium as preservation and perfusion fluid

A commercially available tissue culture medium has been proven capable of preserving dog kidney function for at least 24 hr after simple cooling. The advantages of using tissue culture medium as preservation fluid instead of plasma or albumin solutions from the infectious and immunological points of view are obvious. An in vitro study was completed using the tissue

1.

A digital thermocouple thermometer

Measurements of the reproducibility of a random selection of copper/constantan thermocouples were made and it was found that they agreed within 1 ° C. Based on this finding, a digital thermocouple thermometer was designed and constructed incorporating a thermocouple linearizer and cold junction compensation. The instrument

Accuracy of the Completed Digital Thermometer

DNA synthesis catalysed by endogenous templates and DNA-dependent DNA polymerases in spermatogenic cells from rat

The activity levels of DNA polymerases α and β have been measured by autoradiography in squash preparations from rat testis of sexually mature animals. Similar results were obtained with ‘fixed’ samples (dipped in acetone: ethanol for 5 min at 25 °C) or ‘unfixed’ samples (frozen in liquid nitrogen and freeze-dried). The activities of DNA polymerases α and β in situ were distinguished by differential assay conditions and by selective inhibition with compounds such as N-ethylmaleimide and aphidicolin. Using the endogenous chromatin as template, maximal activity for both enzymes was obtained in the presence of all four deoxyribonucleoside triphosphates, MgCl₂ and ethylene glycol. When DNA polymerase activities in several predominant testicular cell types (pre-leptotene primary spermatocytes, pachytene primary spermatocytes, round spermatids and elongated spermatids) were quantitatively compared, on a per cell basis, the following percentage distribution was observed:

Cell Biology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in cell biology.

Heat exchange in the cryosurgery of Menière's disease: Experimental and clinical studies

The temperature course in the lateral semicircular canal and in the facial canal was studied in experiments during freezing of the semicircular canal. The course of the temperature was measured with thermocouples. Concurrently, the heat flow was measured, and also the total heat exchange was measured throughout the freezing period by a thermoelectric heat flowmeter incorporated in the cryotip. The measurements showed correlation between the total amount of heat exchanged, the freezing time, and the temperature in the semicircular canal. This correlation was utilized to assess and calculate (the temperature of the lateral semicircular canal) the course of the cryoprocess in vivo, where it is possible to measure the heat flow and the total heat exchange during the freezing period only.

2. Results upon Vertigo

Clinical challenges

Glaucomatocyclitic crisis should be considered in the differential diagnosis of unilaterally increased IOP. A careful history, slit-lamp examination and gonioscopic examination will assist the examiner in making an accurate diagnosis.The etiology of this syndrome remains unknown. If indeed it is a disturbance of the anterior intraocular vasculature secondary to an autonomic abnormality, any of the proposed etiologies might possibly trigger an attack (Table 2).Treatment at this time should consist of 1 gH. 0.5% apraclonidine given in office or a topical steroid, cycloplegic, and ocular hypotensive agent combination. Follow-up should be within 24 h to monitor for IOP reduction. If treatment with apraclonidine is initiated, additional drops should be instilled as needed to lower the IOP to acceptable levels. Prophylactic treatment with topical steroids has been considered in this mostly benign syndrome [14]. However, the potential side-effects of long-term steroid use and the unpredictable frequency of glaucomatocyclitic crises may preclude steroid use in these cases. Table 2. Proposed etiological factors and diseases related to glaucomatocyclitis crisis.

Introducing Titratable Water to All-Atom Molecular Dynamics at Constant?pH

Recent development of titratable coions has paved the way for realizing all-atom molecular dynamics at constant pH. To further improve physical realism, here we describe a technique in which proton titration of the solute is directly coupled to the interconversion between water and hydroxide or hydronium. We test the new method in replica-exchange continuous constant pH molecular dynamics simulations of three proteins, HP36, BBL, and HEWL. The calculated pK_a values based on 10-ns sampling per replica have the average absolute and root-mean-square errors of 0.7 and 0.9 pH units, respectively. Introducing titratable water in molecular dynamics offers a means to model proton exchange between solute and solvent, thus opening a door to gaining new insights into the intricate details of biological phenomena involving proton translocation.Solution pH is an important factor in biology. Although neutral pH in extracellular medium accounts for balanced electrostatics and proper folding of protein structures, pH gradients across cell membranes induce large conformational changes that are necessary for biological functions, such as ATP synthesis and efflux of small molecules out of the cell. To gain detailed insights into pH-dependent conformational phenomena, several constant pH molecular dynamics (pHMD) methods, based on either discrete or continuous titration coordinates, have been developed in the last decade (1–4). In the continuous pHMD (CpHMD) framework (2,4), a set of titration coordinates {λ_i} are simultaneously propagated along with the conformational degrees of freedom. Although the original CpHMD method based on the generalized Born (GB) implicit-solvent models (2,4) offers quantitative prediction of pK_a values and pH dependence of folding and conformational dynamics of proteins (5), its accuracy and applicability to highly charged systems and those with dominantly hydrophobic regions are limited due to the approximate nature of the underlying implicit-solvent models.Motivated by the above-mentioned need, three groups have made efforts to develop a CpHMD method using exclusively the explicit-solvent models (6–8). In our development, the titration of acidic and basic sites is coupled with that of coions to level the total charge of the system (8). To further improve physical realism, here we replace the coions by titratable water molecules, which not only absorb the excess charge but also enable direct modeling of solute-solvent proton exchange in classical molecular dynamics simulations.To illustrate the utility of the new methodology, we applied it to the titration simulations of three proteins that were previously used to benchmark the GB-based CpHMD. Although this work does not explore specific interactions between titratable waters and proteins, the methodology can be further tested or improved to provide a rigorous way for modeling proton transfer in molecular dynamics, which is a computationally efficient alternative to the empirical valence-bond theory-based methodologies (9,10).We define titration of water as:

• 1.Loss of a proton to give a negatively charged hydroxide,

H₂O ? OH^? + H⁺, (1)or

• 2.Gain of a proton to give a positively charged hydronium,

H₂O + H⁺ ? H₃O⁺.(2)We now couple the titration of hydroxide (Eq. 1) with that of an acidic site of the solute in the CpHMD simulation,HA+OH−⇌KaA−+H2O.(3)The use of hydronium is avoided here to prevent a potential artifact due to prolonged attraction with A⁻. Analogously, we couple the titration of hydronium (Eq. 2) with that of a basic site,BH++H2O⇌KbH3O++B.(4)Thus, effectively, a proton is transferred between the solute and solvent. However, we should note that in CpHMD simulations, titratable protons are represented by covalently attached dummies (2,4). Through varying the atomic charges and van der Waals interactions, they are seen by other atoms in the protonated state but not in the unprotonated state (see Table S1 in the Supporting Material). Furthermore, the solution proton concentration is implicitly modeled through a free energy term (2,4).In CpHMD, the reference potential of mean force (PMF) for titration is that of the model compound (blocked single amino acid in water) along λ (2,4). In the presence of cotitrating water molecules, it is necessary to add the PMF for the conversion of water to hydroxide or hydronium. One-nanosecond NPT simulations at ambient pressure and temperature were performed to calculate the average force, 〈dU/d,θ〉 at given θ-values, which are related to λ by λ = sin² θ (see Fig. S1 in the Supporting Material). Thermodynamic integration was then applied to calculate the PMF. We found that the average force can be accurately fit when assuming the PMF is quadratic in λ (Fig. 1). The same applies to the PMFs for titration of models Asp, Glu, and His. After testing on the titration of model compounds (see Table S2), we performed 10-ns all-atom CpHMD simulations with the pH replica-exchange protocol for three proteins: HP36, BBL and HEWL (see the Supporting Material for details). Most of the calculated pK_a values were converged in 10 ns per replica (see Fig. S3). Results are summarized in Fig. S4. Based on the 10-ns data, the root-mean-square (RMS) and average absolute errors are 0.9 and 0.7 pH units, respectively, while the largest absolute error is 2.5 (Glu³⁵ of HEWL). Linear regression of the calculation versus experiment gives R² of 0.8 and slope of 1.2.Open in a separate windowFigure 1Average force and potential of mean force for converting a water molecule to hydroxide (A) and hydronium. (B) (Data points) Average forces. (Dashed curves) Best fits using a linear function, 2A(λ − B). (Solid curves) Corresponding potential of mean force.

Table 1

Calculated and experimental pK_a values of three proteins

Posters. P9 A case of carcinosarcoma presenting in a cervical ThinPrep® sample

Introduction Direct endometrial sampling with cytology and or histology is used at our hospital as part of the investigation of abnormal uterine bleeding. It is used in cases where there is a low clinical suspicion of malignancy. The advantage of the technique is that it can be done as an outpatient procedure with minimal patient discomfort. Reports in the literature give mixed results. We present a 3‐year retrospective of our experience with follow‐up.

ALGAL RESPONSE TO NUTRIENT ENRICHMENT IN FORESTED OLIGOTROPHIC STREAM1

Nutrient input in streams alters the density and species composition of attached algal communities in open systems. However, in forested streams, the light reaching the streambed (rather than the local nutrient levels) may limit the growth of these communities. A nutrient‐enrichment experiment in a forested oligotrophic stream was performed to test the hypothesis that nutrient addition has only minor effects on the community composition of attached algae and cyanobacteria under light limitation. Moderate nutrient addition consisted of increasing basal phosphorus (P) concentrations 3‐fold and basal nitrogen (N) concentrations 2‐fold. Two upstream control reaches were compared to a downstream reach before and after nutrient addition. Nutrients were added continuously to the downstream reach for 1 year. Algal biofilms growing on ceramic tiles were sampled and identified for more than a year before nutrient addition to 12 months after. Diatoms were the most abundant taxonomic group in the three stream reaches. Nutrient enrichment caused significant variations in the composition of the diatom community. While some taxa showed significant decreases (e.g., Achnanthes minutissima, Gomphonema angustum), increases for other taxa (such as Rhoicosphenia abbreviata and Amphora ovalis) were detected in the enriched reach (for taxonomic authors, see Table 2 ). Epiphytic and adnate taxa of large size were enhanced, particularly during periods of favorable growth conditions (spring). Nutrients also caused a change in the algal chl a, which increased from 0.5–5.8 to 2.1–10.7 μg chl · cm^?2. Our results indicate that in oligotrophic forested streams, long‐term nutrient addition has significant effects on the algal biomass and community composition, which are detectable despite the low light availability caused by the tree canopy. Low light availability moderates but does not detain the long‐term tendency toward a nutrient‐tolerant community. Furthermore, the effects of nutrient addition on the algal community occur in spite of seasonal variations in light, water flow, and water chemical characteristics, which may confound the observations.

Table 2. Percent abundances of the most frequent taxa in three reaches of the Fuirosos stream. U1 and U2 untreated; E, enriched both in the periods before (bef) and after (aft) the enrichment of the E reach. Acronyms identifying the taxa are indicated.

Economic Benefits of Investing in Women’s Health: A Systematic Review

Background

Globally, the status of women’s health falls short of its potential. In addition to the deleterious ethical and human rights implications of this deficit, the negative economic impact may also be consequential, but these mechanisms are poorly understood. Building on the literature that highlights health as a driver of economic growth and poverty alleviation, we aim to systematically investigate the broader economic benefits of investing in women’s health.

Methods

Using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, we systematically reviewed health, gender, and economic literature to identify studies that investigate the impact of women’s health on micro- and macroeconomic outcomes. We developed an extensive search algorithm and conducted searches using 10 unique databases spanning the timeframe 01/01/1970 to 01/04/2013. Articles were included if they reported on economic impacts stemming from changes in women’s health (table of outcome measures included in full review,

Widespread Distribution of Cell Defense against d-Aminoacyl-tRNAs

Several l-aminoacyl-tRNA synthetases can transfer a d-amino acid onto their cognate tRNA(s). This harmful reaction is counteracted by the enzyme d-aminoacyl-tRNA deacylase. Two distinct deacylases were already identified in bacteria (DTD1) and in archaea (DTD2), respectively. Evidence was given that DTD1 homologs also exist in nearly all eukaryotes, whereas DTD2 homologs occur in plants. On the other hand, several bacteria, including most cyanobacteria, lack genes encoding a DTD1 homolog. Here we show that Synechocystis sp. PCC6803 produces a third type of deacylase (DTD3). Inactivation of the corresponding gene (dtd3) renders the growth of Synechocystis sp. hypersensitive to the presence of d-tyrosine. Based on the available genomes, DTD3-like proteins are predicted to occur in all cyanobacteria. Moreover, one or several dtd3-like genes can be recognized in all cellular types, arguing in favor of the nearubiquity of an enzymatic function involved in the defense of translational systems against invasion by d-amino acids.Although they are detected in various living organisms (reviewed in Ref. 1), d-amino acids are thought not to be incorporated into proteins, because of the stereospecificity of aminoacyl-tRNA synthetases and of the translational machinery, including EF-Tu and the ribosome (2). However, the discrimination between l- and d-amino acids by aminoacyl-tRNA synthetases is not equal to 100%. Significant d-aminoacylation of their cognate tRNAs by Escherichia coli tyrosyl-, tryptophanyl-, aspartyl-, lysyl-, and histidyl-tRNA synthetases has been characterized in vitro (3–9). Recently, using a bacterium, transfer of d-tyrosine onto tRNA^Tyr was shown to occur in vivo (10).With such misacylation reactions, the resulting d-aminoacyl-tRNAs form a pool of metabolically inactive molecules, at best. At worst, d-aminoacylated tRNAs infiltrate the protein synthesis machinery. Although the latter harmful possibility has not yet been firmly established, several cells were shown to possess a d-tyrosyl-tRNA deacylase, or DTD, that should help them counteract the accumulation of d-aminoacyl-tRNAs. This enzyme shows a broad specificity, being able to remove various d-aminoacyl moieties from the 3′-end of a tRNA (4–6, 11). Such a function makes the deacylase a member of the family of enzymes capable of editing in trans mis-aminoacylated tRNAs. This family includes several homologs of aminoacyl-tRNA synthetase editing domains (12), as well as peptidyl-tRNA hydrolase (13, 14).Two distinct deacylases have already been discovered. The first one, called DTD1, is predicted to occur in most bacteria and eukaryotes (see d-amino acids, including d-tyrosine (6). In fact, in an E. coli Δdtd strain grown in the presence of 2.4 mm d-tyrosine, as much as 40% of the cellular tRNA^Tyr pool becomes esterified with d-tyrosine (10).

TABLE 1

Distribution of DTD1 and DTD2 homologs in various phylogenetic groupsHomologs of DTD1 and DTD2 were searched for using a genomic Blast analysis against complete genomes in the NCBI Database (www.ncbi.nlm.nih.gov). Values in the table are number of species. For instance, E. coli is counted only once in γ-proteobacteria despite the fact that several E. coli strains have been sequenced.

A conifer genome spruces up plant phylogenomics

The Norway spruce genome provides key insights into the evolution of plant genomes, leading to testable new hypotheses about conifer, gymnosperm, and vascular plant evolution.In the past year a burst of plant genome sequences have been published, providing enhanced phylogenetic coverage of green plants (Figure ​(Figure1)1) and inclusion of new agricultural, ecological, and evolutionary models. Collectively, these sequences are revealing some extraordinary structural and evolutionary attributes in plant genomes. Perhaps most surprising is the exceptionally high frequency of whole-genome duplication (WGD): nearly every genome that has been analyzed has borne the signature of one or more WGDs, with particularly notable events having occurred in the common ancestors of seed plants, of angiosperms, and of core eudicots (the latter ''WGD'' represents two WGDs in close succession) [1,2]. Given this tendency for plant genomes to duplicate and then return to an essentially diploid genetic system (an example is the cotton genomes, which have accumulated the effects of perhaps 15 WGDs [3]), the conservation of genomes in terms of gene number, chromosomal organization, and gene content is astonishing. From the publication of the first plant genome, Arabidopsis thaliana [4], the number of inferred genes has been between 25,000 and 30,000, with many gene families shared across all land plants, although the number of members and patterns of expansion and contraction vary. Furthermore, conserved synteny has been detected across the genomes of diverse angiosperms, despite WGDs, diploidization, and millions of years of evolution.Open in a separate windowFigure 1Simplified phylogeny of land plants, showing major clades and their component lineages. Asterisks indicate species (or lineage) for which whole-genome sequence (or sequences) is (are) available. Increases and decreases in genome size are shown by arrows.Despite the proliferation of genome sequences available for angiosperms, genome-level data for both ferns (and their relatives, collectively termed monilophytes; Figure ​Figure1)1) and gymnosperms have been conspicuously lacking - until recently, with the publication of the genome sequence of the gymnosperm Norway spruce (Picea abies) [5]. The large genome sizes for both monilophytes and gymnosperms have discouraged attempts at genome sequencing and assembly, whereas the smaller genome size of angiosperms has resulted in more genome sequences being available (Table ​(Table1)1) [6]. Because of this limited phylogenetic sample, our understanding of the timing and phylogenetic positions of WGDs, the core number of plant genes, possible conserved syntenic regions, and patterns of expansion and contraction of gene families across both tracheophytes (vascular plants) and across all land plants is imperfect. This sampling problem is particularly acute in analyses of the genes and genomes of seed plants; many hundreds of genes are present in angiosperms that are not present in mosses or lycophytes, but whether these genes arose in the common ancestor of seed plants or of angiosperms cannot be determined without a gymnosperm genome sequence. The Norway spruce genome therefore offers tremendous power, not only for understanding the structure and evolution of conifer genomes, but also as a reference for interpreting gene and genome evolution in angiosperms.

Table 1

Genome sizes in land plants

Proffered Papers 10.30–11.15 Monday 15 September 2003 3 5 year outcome of women with borderline and mild dyskaryosis smears 3 5 year outcome of women with borderline and mild dyskaryosis smears

Aims

• 1 To identify the outcome status of women with borderline and mild dyskaryosis smears.
• 2 To determine whether the presence or absence of koilocytosis influences the outcome status.
• 3 To identify the proportion of women with borderline smears showing koilocytosis.

Materials and methods Borderline and mild dyskaryosis cervical smears diagnosed during January to March 1997 were identified from the laboratory database. Each slide was reviewed by two researchers independently, who then agreed a final consensus diagnosis. All slides were classified according to the presence or absence of koilocytosis. Slides were excluded from the study if the review diagnosis was negative, inadequate or high‐grade dyskaryosis. The outcome status was classified according to the worst lesion identified histologically and/or cytologically during the 5‐year follow‐up period. Results 1974 women were identified with borderline or mild dyskaryosis cervical smears of which 1597 were included in the study. Table 1 shows the outcome status of these women.

Table 1. . The outcome status of these women

Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study