THE VISIBILITY OF MONOCHROMATIC RADIATION AND THE ABSORPTION SPECTRUM OF VISUAL PURPLE

1. After a consideration of the existing data and of the sources of error involved, an arrangement of apparatus, free from these errors, is described for measuring the relative energy necessary in different portions of the spectrum in order to produce a colorless sensation in the eye. 2. Following certain reasoning, it is shown that the reciprocal of this relative energy at any wave-length is proportional to the absorption coefficient of a sensitive substance in the eye. The absorption spectrum of this substance is then mapped out. 3. The curve representing the visibility of the spectrum at very low intensities has exactly the same shape as that for the visibility at high intensities involving color vision. The only difference between them is their position in the spectrum, that at high intensities being 48 µµ farther toward the red. 4. The possibility is considered that the sensitive substances responsible for the two visibility curves are identical, and reasons are developed for the failure to demonstrate optically the presence of a colored substance in the cones. The shift of the high intensity visibility curve toward the red is explained in terms of Kundt''s rule for the progressive shift of the absorption maximum of a substance in solvents of increasing refractive index and density. 5. Assuming Kundt''s rule, it is deduced that the absorption spectrum of visual purple as measured directly in water solution should not coincide with its position in the rods, because of the greater density and refractive index of the rods. It is then shown that, measured by the position of the visibility curve at low intensities, this shift toward the red actually occurs, and is about 7 or 8 µµ in extent. Examination of the older data consistently confirms this difference of position between the curves representing visibility at low intensities and those representing the absorption spectrum of visual purple in water solution. 6. It is therefore held as a possible hypothesis, capable of direct, experimental verification, that the same substance—visual purple—whose absorption maximum in water solution is at 503 µµ, is dissolved in the rods where its absorption maximum is at 511 µµ, and in the cones where its maximum is at 554 µµ (or at 540 µµ, if macular absorption is taken into account, as indeed it must be).     

THE ABSORPTION OF ULTRA-VIOLET RADIATION BY CRYSTALLINE PEPSIN

Determination of the absorption spectra of pure preparations of Northrop''s crystalline pepsin inactivated by irradiation with ultra-violet light shows that the total absorption in the ultra-violet region of the spectrum increases with the degree of inactivation. This increase is especially marked between 2400 and 2750 Å.u. The rate of photoinactivation is shown to be sensitive to changes in pH, increasing with lower values, and evidently bears a one-quantum relationship to the energy flux. Tests of the rate of inactivation of pepsin exposed to several different bands of the ultra-violet spectrum, in relation to the absorbed energy, indicate that the destruction spectrum of the enzyme agrees essentially with its absorption spectrum and is similar to that of urease.     

ISOLATION,CRYSTALLIZATION, AND PROPERTIES OF SWINE PEPSINOGEN

1. A method is described for the preparation of pepsinogen from swine gastric mucosae which consists of extraction and fractional precipitation with ammonium sulfate solutions followed by two precipitations with a copper hydroxide reagent under particular conditions. Crystallization as very thin needles takes place at 10°C., pH 5.0 and from 0.4 saturated ammonium sulfate solution containing 3–5 mg. protein nitrogen per milliliter. 2. Solubility measurements, fractional recrystallization, and fractionation experiments based on separation after partial heat or alkali denaturation and after partial reversal of heat or alkali denaturation failed to reveal the presence of any protein impurity. 3. The properties of the enzymatically inactive pepsinogen were studied and compared with the properties of crystalline pepsin. The properties of pepsinogen which are similar to those of pepsin are: molecular weight, absorption spectrum, tyrosine-tryptophane content, and elementary analysis. The properties in which they differ are: enzymatic activity, crystalline form, amino nitrogen, titration curve, pH stability range, specific optical rotation, isoelectric point, and the reversibility of heat or alkali denaturation. 4. Conversion of pepsinogen into pepsin at pH 4.6 was found to be autocatalytic; i.e., the pepsin formed catalyzes the reaction. Conversion of pepsinogen into pepsin is accompanied by the splitting off of a portion of the molecule containing 15–20 per cent of the pepsinogen nitrogen.     

CONCENTRATION AND PURIFICATION OF BACTERIOPHAGE

1. A method for isolating a nucleoprotein from lysed staphylococci culture is described. 2. It is homogeneous in the ultracentrifuge and has a sedimentation constant of 650 x 10^–13 cm. dyne^–1 sec.^–1, corresponding to a molecular weight of about 300,000,000. 3. The diffusion coefficient varies from about 0.001 cm.²/day in solutions containing more than 0.1 mg. protein/ml. to 0.02 in solutions containing less than 0.001 mg. protein/ml. The rate of sedimentation also decreases as the concentration decreases. It is suggested, therefore, that this protein exists in various sized molecules of from 500,000–300,000,000 molecular weight, the proportion of small molecules increasing as the concentration decreases. 4. This protein is very unstable and is denatured by acidity greater than pH 5.0, by temperature over 50°C. for 5 minutes. It is digested by chymo-trypsin but not by trypsin. 5. The loss in activity by heat, acid, and chymo-trypsin digestion is roughly proportional to the amount of denatured protein formed under these conditions. 6. The rate of diffusion of the protein is the same as that of the active agent. 7. The rate of sedimentation of the protein is the same as that of the active agent. 8. The loss in activity when susceptible living or dead bacteria are added to a solution of the protein is proportional to the loss in protein from the solution. Non-susceptible bacteria remove neither protein nor activity. 9. The relative ultraviolet light absorption, as determined directly, agrees with that calculated from Gates'' inactivation experiments in the range of 2500–3000 Å. u. but is somewhat greater in the range of 2000–2500 Å. u. 10. Solubility determinations showed that most of the preparations contained at least two proteins, one being probably the denatured form of the other. Two preparations were obtained, however, which had about twice the specific activity of the earlier ones and which gave a solubility curve approximating that of a pure substance. 11. It is suggested that the formation of phage may be more simply explained by analogy with the autocatalytic formation of pepsin and trypsin than by analogy with the far more complicated system of living organisms.     

AN ATTEMPT AT PEPTIC SYNTHESIS OF INSULIN

1. Synthesis of plastein from the products of peptic hydrolysis of small quantities of egg albumin can be demonstrated with amorphous or crystalline pepsin. 2. Synthesis of plastein from the products of peptic hydrolysis of amorphous or crystalline insulin can be demonstrated with amorphous or crystalline pepsin. 3. The plastein synthesised by pepsin from the products of peptic hydrolysis of insulin is physiologically inactive. 4. The plastein formed in the insulin experiments could not be crystallised by the methods used for the crystallisation of insulin. 5. The physiological activity of insulin is not destroyed by repeated freezing (at about –50°C.) and melting of an aqueous or an alcoholic solution of this hormone. 6. No marked decrease in the physiological activity of insulin after incubation at 37°C. with pepsin at pH 4.0, in dilute or concentrated solutions, was detected.     

Revised UV extinction coefficients for nucleoside-5′-monophosphates and unpaired DNA and RNA

Ultraviolet absorption provides the nearly universal basis for determining concentrations of nucleic acids. Values for the UV extinction coefficients of DNA and RNA rely on the mononucleotide values determined 30–50 years ago. We show that nearly all of the previously published extinction coefficients for the nucleoside-5′-monophosphates are too large, and in error by as much as 7%. Concentrations based on complete hydrolysis and the older set of values are too low by ~4% for typical RNA and 2–3% for typical DNA samples. We also analyzed data in the literature for the extinction coefficients of unpaired DNA oligomers. Robust prediction of concentrations can be made using 38 µg/A₂₆₀ unit for single-stranded DNA (ssDNA) having non-repetitive sequences and 40–80% GC. This is superior to currently used predictions that account for nearest-neighbor frequency or base composition. The latter result in concentrations that are 10–30% too low for typical ssDNA used as primers for PCR and other similar techniques. Methods are described here to accurately measure concentrations of nucleotides by nuclear magnetic resonance. NMR can be used to accurately determine concentrations (and extinction coefficients) of biomolecules within 1%.     

Direct evidence for an acylated thiol as an intermediate in papain- and ficin-catalysed hydrolyses

1. Methyl thionohippurate was prepared and shown to be a specific substrate for both papain and ficin. 2. The ultraviolet-absorption properties of the acyl-enzyme intermediate for both papain and ficin with methyl thionohippurate was that expected of an acyl-thiol. The possibility that other functional groups present in papain or ficin might be the site of acylation has been excluded. 3. The change in extinction at the absorption maximum with time was as expected for the acyl-enzyme on the basis of the known Michaelis–Menten parameters for methyl thionohippurate. 4. The variation of extinction with initial substrate concentration for both papain and ficin was that expected from the Michaelis–Menten parameters. 5. The extinction of the absorption maximum of the thionohippuryl-enzyme intermediate was suppressed by the addition of methyl hippurate to the extent predicted from the Michaelis–Menten parameters. 6. The decay of the extinction for the acyl-enzyme was arrested by adjusting the pH of the solution to 2·5. 7. These experiments provide compelling evidence that the acylation by substrate of both papain and ficin takes place through a thiol residue.     

Visual Pigments of Goldfish Cones : Spectral Properties and Dichroism

Freshly isolated retinal photoreceptors of goldfish were studied microspectrophotometrically. Absolute absorptance spectra obtained from dark-adapted cone outer segments reaffirm the existence of three spectrally distinct cone types with absorption maxima at 455 ± 3,530 ± 3, and 625 ± 5 nm. These types were found often recognizable by gross cellular morphology. Side-illuminated cone outer segments were dichroic. The measured dichroic ratio for the main absorption band of each type was 2–3:1. Rapidly bleached cells revealed spectral and dichroic transitions in regions near 400–410, 435–455, and 350–360 nm. These photoproducts decay about fivefold as fast as the intermediates in frog rods. The spectral maxima of photoproducts, combined with other evidence, indicate that retinene₂ is the chromophore of all three cone pigments. The average specific optical density for goldfish cone outer segments was found to be 0.0124 ± 0.0015/µm. The spectra of the blue-, and green-absorbing cones appeared to match porphyropsin standards with half-band width Δν = 4,832 ± 100 cm^–1. The red-absorbing spectrum was found narrower, having Δν = 3,625 ± 100 cm^–1. The results are consistent with the notion that visual pigment concentration within the outer segments is about the same for frog rods and goldfish cones, but that the blue-, and green-absorbing pigments possess molar extinctions of 30,000 liter/mol cm. The red-absorbing pigment was found to have extinction of 40,000 liter/mol cm, assuming invariance of oscillator strength among the three cone spectra.     

The stability of rhodopsin and opsin; effects of pH and aging

The stability of cattle rhodopsin and of its protein moiety opsin toward acids and alkalies and on aging was determined by two criteria: maintenance of absorption spectrum, and capacity to regenerate after exposure to light. On storage at 3°C. at pH near neutrality, the absorption spectrum in the visible region may remain unchanged for as long as 6 months; but the regenerability progressively declines, at very different rates in different preparations. The cause of this decline has not been determined. It may involve denaturation at sites other than the retinene-protein bond, which by the evidence of the absorption spectrum remains intact. Cattle rhodopsin maintains its absorption spectrum at any pH from 3.9–9.6 for at least an hour at 25–27°C. To both sides of this pH range the pigment bleaches, the extinction falling to half in 1 hour at pH 3.3 and 10.5. The exposure of rhodopsin to light greatly increases the vulnerability of the product (opsin) to acids and bases. Opsin rapidly loses its capacity to regenerate rhodopsin to both sides of the range of pH 5.5–7.0. Half the regenerability is lost within 45 seconds at pH 3.4 and 9.1; and within 1 hour at pH 5 and 8.     

Environmental Lead Exposure among Preschool Children in Shanghai,China: Blood Lead Levels and Risk Factors

Objective

To determine blood lead levels and to identify related risk factors among children in Shanghai; to explore the lead change trend of children after industrial transformation and to provide data for policy development to control environmental lead pollution in Shanghai.

Methods

A stratified-clustered-random sampling method was used. A tungsten atomizer absorption spectrophotometer was employed to determine blood lead levels.

Results

The arithmetic mean, geometric mean and median of blood lead levels of 0- to 6-year-old children from Shanghai were 22.49 µg/L, 19.65 µg/L and 19.5 µg/L, including 0.26% (6/2291) with concentrations ≥100 µg/L and 2.7% (61/2291) with concentrations ≥50 µg/L. Boys'' levels (23.57 µg/L) were greater than those of girls (21.2 µg/L). The blood lead levels increased with age. This survey showed that the Chongming district was the highest and Yangpu district was the lowest, this result is completely opposite with the earlier survey in Shanghai. Risk factors for lead contamination included housing environment, parents'' education levels, social status, hobbies, and children''s nutritional status.

Conclusions

The blood lead levels of children in Shanghai were lower than the earlier data of Shanghai and those of published studies in China, but higher than the blood lead levels of developed countries. The blood lead levels of urban districts are higher than the central districts with the industrial transformation. Society and the government should take an active interest in childhood lead poisoning of urban areas.     

THE RATE OF CO2 ASSIMILATION BY PURPLE BACTERIA AT VARIOUS WAVE LENGTHS OF LIGHT

1. The relative absorption spectrum of the pigments in their natural state in the photosynthetic bacterium Spirillum rubrum is given from 400 to 900 mµ. The position of the absorption maxima in the live bacteria due to each of the pigments is: green pigment, 420, 590, 880; red pigment, 490, 510, 550. 2. The relative absorption spectrum of the green pigment in methyl alcohol has been determined from 400 to 900 mµ. Bands at 410, 605, and 770 mµ were found. 3. The wave length sensitivity curve of the photosynthetic mechanism has been determined and shows maxima at 590 and about 900 mµ. 4. It is concluded that the green bacteriochlorophyll alone and not the red pigment can act as a light absorber for photochemical CO₂ reduction.     

Distribution of Young's Modulus in Porcine Corneas after Riboflavin/UVA-Induced Collagen Cross-Linking as Measured by Atomic Force Microscopy

Riboflavin/UVA-induced corneal collagen cross-linking has become an effective clinical application to treat keratoconus and other ectatic disorders of the cornea. Its beneficial effects are attributed to a marked stiffening of the unphysiologically weak stroma. Previous studies located the stiffening effect predominantly within the anterior cornea. In this study, we present an atomic force microscopy-derived analysis of the depth-dependent distribution of the Young''s modulus with a depth resolution of 5 µm in 8 cross-linked porcine corneas and 8 contralateral controls. Sagittal cryosections were fabricated from every specimen and subjected to force mapping. The mean stromal depth of the zone with effective cross-linking was found to be 219±67 µm. Within this cross-linked zone, the mean Young''s modulus declined from 49±18 kPa at the corneal surface to 46±17 kPa, 33±11 kPa, 17±5 kPa, 10±4 kPa and 10±4 kPa at stromal depth intervals of 0–50 µm, 50–100 µm, 100–150 µm, 150–200 µm and 200–250 µm, respectively. This corresponded to a stiffening by a factor of 8.1 (corneal surface), 7.6 (0–50 µm), 5.4 (50–100 µm), 3.0 (100–150 µm), 1.6 (150–200 µm), and 1.5 (200–250 µm), when compared to the Young''s modulus of the posterior 100 µm. The mean Young''s modulus within the cross-linked zone was 20±8 kPa (2.9-fold stiffening), while it was 11±4 kPa (1.7-fold stiffening) for the entire stroma. Both values were significantly distinct from the mean Young''s modulus obtained from the posterior 100 µm of the cross-linked corneas and from the contralateral controls. In conclusion, we were able to specify the depth-dependent distribution of the stiffening effect elicited by standard collagen cross-linking in porcine corneas. Apart from determining the depth of the zone with effective corneal cross-linking, we also developed a method that allows for atomic force microscopy-based measurements of gradients of Young''s modulus in soft tissues in general.     

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit ε with the catalytic complex of Escherichia coli ATP synthase. Bacterial F-type ATP synthaseis the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit ε could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of ε undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of ε''s conformations is predominant. The assay measures kinetics of ε''s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound ε to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on ε''s conformational changes.     

WAVE LENGTH OF LIGHT AND PHOTIC INHIBITION OF STEREOTROPISM IN TENEBRIO LARV?

A definite intensity of white light is required (about 136 m.c.) to produce negative phototropic orientation of creeping Tenebrio larvæ away from contact with a vertical glass surface. This gives a measure of stereotropism in terms of phototropism, or reciprocally. The effectiveness of light for the suppression of stereotropism varies with wave length. It is therefore simple to obtain a measure of the relation between wave length and stimulating efficiency in this case of phototropic orientation. By determinations of the minimal energy required to inhibit stereotropism with different regions of the spectrum, it is found that the maximum effectiveness is sharply localized in the neighborhood of 535µµ. The curve connecting stimulating efficiency with wave length, while giving a picture of the effective absorption by the photosensory receptors, probably does not permit accurate characterization of the essential photosensitive material.     

An Investigation of Outpatient Children's Blood Lead Level in Wuhan China

Objective

Blood lead levels (BLLs) and possible influencing factors in children in Wuhan China were investigated in order to understand current lead pollution exposure and provide a scientific basis for prevention and policy making.

Materials and Methods

BLL data were collected from 15,536 out-patients in Wuhan Children Hospital in 2012 full year. All of them were under 18 years of age (Mean ± SD: 4.32±3.2, 64.4% boys). The BLLs were measured by an atomic absorption spectrometry (BH2100).

Results

The geometric mean of BLLs for all the subjects was 44.75 µg/L (95%CI: 44.46 µg/L – 45.05 µg/L), much lower than that reported in previous studies. The prevalence of the elevated BLLs (≥ 100 µg/L) in the children tested was 2% in 2012 and the prevalence of BLLs (≥ 50 µg/L) was 44%. Age and sex could be possible influencing factors for BLLs in the children (p<0.001). In addition, the BLLs in different seasons were different (p<0.001).

Conclusions

These results demonstrate that BLLs have significantly decreased in children in Wuhan during recent years. However, we should continuously pay attention to lead pollution and emphasize that prevention is much more important than treatment for controlling children''s BLLs.     

Application of Polarizing Microscopy to Medical Mycology

The hyphae of 15 species of fungi were cultivated by conventional slide-culture technique and then examined by polarizing microscopy. Extinction angles exhibited by the anisotropic chitin in the cell walls of hyphae were, as a rule, measurable within a minimal growth range of 2 to 3 days after inoculation to slide culture. Of the 15 species examined, 8 saprophytic fungi were found to possess a range of common extinction angles (10° ± 2°). The remaining seven organisms implicated as pathogens were found to possess a range of common extinction angles (20° ± 2°). There appears to be a correlation existing between the pathogenic nature of the examined fungi and their extinction angles, as demonstrated by the polarizing microscope.     

CRYSTALLINE TRYPSIN : II. GENERAL PROPERTIES

A method is described for isolating a crystalline protein of high tryptic activity from beef pancreas. The protein has constant proteolytic activity and optical activity under various conditions and no indication of further fractionation could be obtained. The loss in activity corresponds to the decrease in native protein when the protein is denatured by heat, digested by pepsin, or hydrolyzed in dilute alkali. The enzyme digests casein, gelatin, edestin, and denatured hemoglobin, but not native hemoglobin. It accelerates the coagulation of blood but has little effect on the clotting of milk. It digests peptone prepared by the action of pepsin on casein, edestin or gelatin. The extent of the digestion of gelatin caused by this enzyme is the same as that caused by crystalline pepsin and is approximately equivalent to tripling the number of carboxyl groups present in the solution. The activity of the preparation is not increased by enterokinase. The molecular weight by osmotic pressure measure is about 34,000. The diffusion coefficient in ½ saturated magnesium sulfate at 6°C. is 0.020 ±0.001 cm.² per day, corresponding to a molecular radius of 2.6 x 10^–7 cm. The isoelectric point is probably between pH 7.0 and pH 8.0. The optimum pH for the digestion of casein is from 8.0–9.0. The optimum stability is at pH 1.8.     

Ultraviolet difference spectra of pepsin

A shift of pH of pepsin solutions from 4.6 to 1.0 gives rise to spectral displacements in the ultraviolet. If represented as difference spectra three peaks with maxima at 2770, 2850, and 2930 Ångströms are present which can be attributed to the tyrosine and tryptophan residues in the protein. On mild autolysis of pepsin at pH 2.0 the absorbancy in the ultraviolet further decreases. Although some of these effects can be ascribed to the occurrence of hydrogen bonding between the aromatic residues and a carboxylate ion, those observed on autolysis are caused by charge effects of newly formed polar groups in the vicinity of a chromophore. No direct relation between the optical properties described here and enzymic activity of pepsin has been observed.     

A STUDY OF THE BACTERICIDAL ACTION OF ULTRA VIOLET LIGHT : III. THE ABSORPTION OF ULTRA VIOLET LIGHT BY BACTERIA

The simple conclusion of former investigators that the shorter the wave length of ultra violet light the greater the bactericidal action is in error. A study with measured monochromatic energy reveals a characteristic curve of bactericidal effectiveness with a striking maximum between 260 and 270 m.µ. The reciprocal of this abiotic energy curve suggests its close relation to specific light absorption by some single essential substance in the cell. Methods are described for determining the absorption curve, or absorption coefficients, of intact bacteria. These curves for S. aureus and B. coli have important points of similarity and of difference with the reciprocals of the curves of bactericidal incident energy, and point the way in a further search for the specific substance, or substances, involved in the lethal reaction.     

Plasma plant sterols serve as poor markers of cholesterol absorption in man

The validation of the use of plasma plant sterols as a marker of cholesterol absorption is frail. Nevertheless, plant sterol concentrations are routinely used to describe treatment-induced changes in cholesterol absorption. Their use has also been advocated as a clinical tool to tailor cholesterol-lowering therapy. Prior to wider implementation, however, the validity of plant sterols as absorption markers needs solid evaluation. Therefore, we compared plasma plant sterol concentrations to gold-standard stable isotope-determined cholesterol absorption. Plasma campesterol/TC concentrations (camp/TC) were measured in a population of 175 mildly hypercholesterolemic individuals (age: 59.7 ± 5.6 years; BMI: 25.5 ± 2.9kg/m²; LDL-C: 4.01 ± 0.56 mmol/l). We compared cholesterol absorption according to the plasma dual-isotope method in subjects with the highest camp/TC concentrations (N = 41, camp/TC: 2.14 ± 0.68 μg/mg) and the lowest camp/TC concentrations (N = 39, camp/TC: 0.97 ± 0.22 μg/mg). Fractional cholesterol absorption did not differ between the groups (24 ± 12% versus 25 ± 16%, P = 0.60), nor was it associated with plasma camp/TC concentrations in the total population of 80 individuals (β = 0.13; P = 0.30, adjusted for BMI and plasma triglycerides). Our findings do not support a relation between plasma plant sterol concentrations and true cholesterol absorption and, therefore, do not favor the use of these sterols as markers of cholesterol absorption. This bears direct consequences for the interpretation of earlier studies, as well as for future studies targeting intestinal regulation of cholesterol metabolism.