OPTICAL PROPERTIES OF HUMAN BRAIN

Abstract— Optical properties of the human brain in the wave-length region from blue to near infrared are presented. There are significant variations in the optical penetration depth from the neonatal and to the adult brain. Typical values for the penetration depth in the adult brain are: 0.5 mm for blue/green, 1.5 mm for red and 3.5 mm for near infrared. The values for the neonatal brain are typically 2–3 times larger.     

CHEMICAL DERIVATION OF HUMAN INSULIN SUPERAGONISM

The role of three highly conserved insulin residues Tyr^B26 was studied to better understand the relationship between insulin and receptor from rat adipose tissue plasma membranes, lnsulin analogues with a single amino acid substitution or single N-methylation of the peptide bond in the position B26 were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by the binding to the insulin receptor. From our results, we can deduce several conclusions： （1） the replacement of tyrosine in the position B26 by histidine, [N-MeHis^B26]-des-tetrapeptide-（B27-B30）-insulin-B26-amide and [N-MeGlu^B26]-des-tetrapeptide- （B2-B30）-insulin-B26-amide, have no significant effect on the binding affinity and they show binding affinity 105%, 190% and 208%, respectively, of that of human insulin; （2） [Aad^B26] -des-tetrapeptide-（B27-B30）-insulin-B26-amide and [Phe（4-carboxy^B26）]-des-tetrapeptide- （B27~B30）-insulin-B26-amide affect the potency highly positively in vitro studies; they show binding affinity 529 and 289 %, respectively, of that of human insulin.     

UV-INDUCED ALKALINE LABILE DNA DAMAGE IN HUMAN ADENOVIRUS AND ITS REPAIR AFTER INFECTION OF HUMAN CELLS

Abstract— UV-induced alkaline labile viral DNA damage was detected following irradiation of adenovirus type 2 and found to be repaired following the infection of human KB cells. Human adenovirus type 2 was irradiated with various doses of UV and subsequently used to infect human KB cells in tissue culture at approximately 2 × 10³ particles per cell. Before, and at various times after infection, the viral DNA was examined on alkaline sucrose gradients. Irradiated free virus DNA showed a dose dependent decrease in molecular weight compared to unirradiated virus DNA, indicating the presence of UV-induced alkaline labile lesions. Furthermore, an increase in the molecular weight of the irradiated virus DNA was found after infection indicating that alkaline labile lesions were removed from the viral DNA by a host mediated repair mechanism. After infection, the molecular weight of the irradiated virus DNA reached a value similar to that of unirradiated virus DNA for all the UV doses studied.     

PHOTOPHYSICAL STUDIES ON HUMAN RETINAL LIPOFUSCIN

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age-related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time-resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV-visible absorption of longer-lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady-state and time-resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time-resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime caμs) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age-related decline of RPE function and blue light damage.     

OPTICAL PENETRATION IN HUMAN INTRACRANIAL TUMORS

Abstract— The optical penetration depth has been measured in human intracranial tumors. The penetration depth in highly malignant gliomas is typically twice as large as the penetration depth in the surrounding normal brain tissue. Typical values for different wavelengths are: 1 mm for blue/green, 2.5 mm for red and 6 mm for near infrared. The penetration depth in a metastasis (from lung) is about the same as in the surrounding tissue.     

PYRIMIDINE DIMER FORMATION IN HUMAN SKIN

Cyclobutyl pyrimidine dimers are major photoproducts formed upon irradiation of DNA with ultraviolet light. We have developed a method for detecting as few as one pyrimidine dimer per million bases in about 50 ng of non-radioactive DNA, and have used this method to quantitate dimer yields in human skin DNA exposed in situ to UV. We found that UVA radiation (320–400 nm) produces detectable levels of dimers in the DNA of human skin. We also measured UVB-induced dimer yields in skin of individuals of differing sun sensitivity and found higher yields in individuals with higher UVB minimal erythema doses and greater sun sensitivity. These approaches should provide important information on damage induced in human skin upon exposure to natural or artificial sources of ultraviolet radiation.     

FUNCTIONS OF LIPIDS ON HUMAN SKIN

In order to clarify the roles of lipids in the water-holding property of stratum corneum, the forearm skin of healthy male volunteers was treated with acetone/ether (1/1) or sodium dodecyl sulfate (5%) for 1-30 min. A prolonged treatment period of 5-30 min produced a chapped and scaly appearance of the stratum corneum without any inflammatory reactions. Under these conditions, there was a marked decrease in the water-holding capacity of the stratum corneum accompanied by a selective loss of stratum corneum lipids such as cholesterol, cholesterol esters, and sphingolipids. Two daily applications of the isolated stratum corneum lipids to experimentally induced dry skins caused a significant increase of conductance, accompanied by a marked improvement in the level of scaling. Meanwhile, the isolated sebaceous lipids exhibited no significant recovery in both the conductance value and the scaling. Out of chroma-tographically separated fractions of the stratum corneum lipids, topical applications of ceramide fraction induced the highest increase in the conductance value. Topical applications of synthesized pseudo-ceramides also showed a significant recovery of the water-retaining properties accompanied by an improvement in the scaling only when the polar group has an amide bond in the major linkage. Analysis of the alkyl chain structures has revealed that a structural requirement for the recovery of the water retaining capacity is the presence of saturated-straight alkyl chains, not unsaturated or branched alkyl chains. These structural characteristics required for water-retaining function also paralleled their capacity to form multiconcentric lamellae vesicles in vitro which is also capable of acquiring bound water as shown by DSC thermograms. The present study suggests that ceramides with relatively shorter alkyl chain length serve as a water modulator in the multi-lipid bilayers through the stratum corneum.     

PORPHYRIN-INDUCED PHOTODAMAGE TO ISOLATED HUMAN NEUTROPHILS

Abstract— Human neutrophils were irradiated with light at 340–380 nm in the presence of low concentrations of protoporphyrin or uroporphyrin. At increasing light doses or increasing concentrations of protoporphyrin, the neutrophils rapidly lost the ability of locomotion. Also, neutrophil chemiluminescence and hexose-monophosphate shunt activity rapidly declined. An early event was leakage of endogenous K⁺ followed by lactate dehydrogenase and at a later stage leakage of particle-bound acid phosphatase. A number of cellular enzymes were inactivated, the susceptibility to inactivation decreased in the order: succinate dehydrogenase > lactate dehydrogenase > glutamate dehydrogenase > acid phosphatase. Uroporphyrin had no effect on neutrophil functions, leakage of K⁺, or cellular enzymes. The results suggest that photodamage to the plasma membrane and the mitochondria are earlier events than photodamage to lysosomes.     

THERMODYNAMICS OF THE BINDING OF HEMATOPORPHYRIN ESTER, A HEMATOPORPHYRIN DERIVATIVE-LIKE PHOTOSENSITIZER, AND ITS COMPONENTS TO HUMAN SERUM ALBUMIN, HUMAN HIGH-DENSITY LIPOPROTEIN AND HUMAN LOW-DENSITY LIPOPROTEIN

The phenomena of the high affinity of porphyrins to the human serum proteins, albumin, high-density lipoproteins (HDL) and low-density lipoproteins (LDL) is well established. Yet, evaluation of the activities of these proteins as endogenous porphyrin carriers, especially with respect to receptor-mediated porphyrin uptake into tumor cells, the merits of which are still in dispute, requires more quantitative protein-porphyrin binding data. As a continuation of previous studies on this issue, the binding of several porphyrin systems to each of the three proteins, employing previously developed spectral methodologies, was studied. The specific systems reported here are hematoporphyrin ester (HPE), which is a novel hematoporphyrin derivative (HPD)-like system, two porphyrin trimers (denoted O1 and O2) and a porphyrin dimer (denoted O3) isolated from HPE. Human serum albumin (HSA) was found to have a single high-affinity site for the monomeric components of HPE, with an equilibrium binding constant of 3.6 × 10⁶. The equilibrium parameters determined for the binding of the three HPE-isolated oligomers to each of the serum proteins are: (1) Binding constants (K_b') of 2.3 × 10⁶, 6.9 × 10⁴ and 1.5 × 10⁴ and number of sites per protein molecule (n) of 3, 1 and 5, for the binding of 01, 02 and 03, respectively, to HSA. (2) K_b’values of 15.5 × 10³, 15.3 × 10³ and 6.6 × 10³ and n values of 1, 2 and 2, for the binding of O1, O2 and O3, respectively, to HDL. (3) K_b’values of 3.3 × 10³, 2.28 × 10⁴ and 8.0 × 10³ and n values of 50, 20 and 16 for the binding of O1, O2 and O3, respectively, to LDL. These data are direct and clear support not only for the high affinity of porphyrins to serum proteins but specifically of stable oligomers that have been assigned critical roles in the photodynamic treatment of tumors. Of the three proteins, LDL is clearly the best camer, providing the highest drug payload with a moderate affinity (enough to bind and not too much to prevent release). These data are suggested to be promising for the postulated role of LDL in porphyrin uptake into tumor cells and to be useful in the future as benchmarks for novel porphyrin systems.     

流动注射氢化物发生原子吸收光谱法测定发铅

发样消化液用铁氰化钾预先氧化处理将二价铅氧化为四价形态铅,以硼氢化钾为还原剂,稀盐酸为载液,用流动注射氢化物发生原子吸收光谱法测定铅。方法的检出限为0.26ng·ml-1,线性范围为0.60～35ng·ml-1,相对标准偏差为2.5%～4.7%,样品加标回收率为96.8%～104.4%,对人发标准物质进行测定,结果与标准值符合。方法灵敏度高、选择性好、试剂及样品用量少、操作方便快速,有推广应用价值。     

AMIODARONE PHOTOTOXICITY TO HUMAN ERYTHROCYTES AND LYMPHOCYTES

Amiodarone (AD) therapy for cardiac arrhythmia frequently leads to cutaneous phototoxicity. Amiodarone and its metabolite, desethylamiodarone (DEA), photosensitized hemolysis of red blood cells (RBC) and were phototoxic to lymphocytes. Hemolysis photosensitized by 3.3 μ M AD was partially oxygen dependent and was partially quenched 5 m M sodium azide, 50 m M mannitol, superoxide dismutase (251 U/me e ) and catalase (1500 U/m e ), but was unaffected when H₂O was replaced by D₂O. These results suggest that membrane damage may be important in the in vivo phototoxicity to AD, that both oxygen dependent and oxygen independent mechanisms may operate, and that active oxygen species such as O₂ and hydrogen peroxide may be involved. Photohemolysis was more rapid in the presence of DEA than of AD. However, this may be due to the greater fragility of the cell membrane in the presence of DEA. The greater phototoxicity of DEA than AD towards lymphocytes was not due to greater membrane fragility.     

COLLAGEN ALTERATIONS IN CHRONICALLY SUN-DAMAGED HUMAN SKIN

Abstract The major histological characteristic of sun-damaged skin is the accumulation of an elastotic material that appears to replace collagen. This elastotic material consists primarily of elastin and histological studies suggest a large loss of collagen in the dermis of chronically sun-damaged skin. In this study, we examine the content and distribution of collagen and procollagen in sun-damaged human skin. The total collagen content of sun-damaged skin was 20% less than nonsolar-exposed skin (524 μ g collagen per mg total protein in sun-damaged skin and 667 μg collagen per mg total protein in nonsolar-exposed skin). In addition, there was a 40% decrease in the content of intact amino propeptide moiety of type III procollagen in sun-damaged skin (0.68 U per 50 mg wet weight) as compared to nonsolar-exposed skin (1.12 U per 50 mg wet weight). The data suggest that this change in collagen content is due to increased degradation. The distribution of collagen in sun-damaged skin was examined by indirect immunofluo-rescence. Mild digestion of sun-damaged skin with elastase removed the elastin and revealed the presence of collagen in the elastotic material. Therefore, the elastin appears to mask the presence of collagen fibers in the dermis of sun-damaged skin.     

QUENCHING OF SINGLET OXYGEN BY HUMAN PLASMA

Direct measurements of the decay of singlet oxygen phosphorescence at 1270 nm were made in human plasma diluted with various amounts of deuterium oxide. The Stern-Volmer plot of the singlet oxygen lifetimes was linear up to 15% plasma concentration (vol/vol). Extrapolation of these measurements to 100% plasma content gave a singlet oxygen lifetime of 1.04 +/- 0.03 microseconds in human plasma. Biological molecules accounted for 77% of the total singlet oxygen quenching while water accounted for 23% of the quenching. The contributions of various types of biological molecules to the total singlet oxygen quenching were calculated from their plasma concentrations and their quenching constants. Plasma proteins quenched most of the singlet oxygen. Uric acid also quenched a significant amount of singlet oxygen (12%). Tocopherols, carotenoids, ascorbic acid and bilirubin made only small contributions to the total singlet oxygen quenching (less than or equal to 4%).     

FLUORESCENCE STUDIES WITH HUMAN EPIDERMAL GROWTH FACTOR

Steady-state and time-resolved fluorescence studies have been performed with human epidermal growth factor, a small globular protein having two adjacent tryptophan residues near its C-terminus. Based on the relatively red fluorescence and accessibility to solute quenchers, the two tryptophan residues are found to be exposed to solvent. Anisotropy decay measurements show the dominant depolarizing process to have a sub-nanosecond rotational correlation time indicating the existence of rapid segmental motion of the fluorescing tryptophan residues. From an analysis of the low-temperature excitation anisotropy spectrum of the protein (and in comparison with that of tryptophan, the peptide melittin, and the dipeptide trp-trp), it is concluded that homo-energy transfer and/or exciton interaction occurs between the adjacent tryptophan residues. A thermal transition in the structure of the protein, which is observed by circular dichroism measurements, is not sensed by the steady-state fluorescence of the protein. This result, in conjunction with the anisotropy decay results, indicates that the two tryptophan residues are in a highly flexible C-terminus segment, which is not an integral part of the three-dimensional structure of the protein. Fluorescence measurements with three site-directed mutants also show very little variation.     

FLUORESCENCE PROPERTIES OF PORPHYRIN-GLOBIN FROM HUMAN HEMOGLOBIN

Fluorescence excitation and emission spectra, decays, and quantum yields are reported for the porphyrin-globin of hemoglobin (Hb^desFe) in aqueous solution of pH 8, at 4°C. A very weak fluorescence was observed in the UV (maximum at 334 nm), due to tryptophan and tyrosine residues, in addition to the strong porphyrin emission in the visible (maxima at 624 and 692 nm) reported previously. The absorption and fluorescence properties of the porphyrins of Hb^desFe were compared to those for free porphyrin in organic solvents and in aqueous solution. The close similarity of the fluorescence decays and quantum yields in Hb^desFe and in solution indicate the absence of stronger, specific porphyrin-protein interactions; however, slight spectral shifts point to the existence of water molecules in the Hb^desFe porphyrin environment. The fluorescence study also demonstrates the existence of efficient Trp-porphyrin energy transfer of Förster type. The extent of transfer is in satisfactory agreement with the value expected from crystallographic data for hemoglobin. The results are discussed and compared to previous fluorescence studies of hemoglobin and apohemoglobin. An improved method for the preparation of Hb^desFe is reported.     

LIGHT-DEPENDENT GENERATION OF SUPEROXIDE FROM HUMAN ERYTHROCYTES

Using the cytochrome c reduction method, we investigated light-dependent erythrocytic superoxide production. After 4 h light and dark exposure of erythrocytes from eight healthy human subjects, an average of 18.6% more superoxide was generated by erythrocytes exposed to light. Pretreatment of erythrocytes with the superoxide dismutase inhibitor N,N-diethyldithiocarbamate increased detection of superoxide while pretreatment with the anion channel blocker 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene decreased detection. These findings indicate that substantially more spontaneously generated superoxide is produced and escapes from normal erythrocytes at ambient oxygen tensions on exposure to light. This excess generation and escape of superoxide from erythrocytes exposed to light may result in tissue photosensitization, especially in the retina of the eye, where high oxygen tension, blood and chronic light exposure occur simultaneously.     

FORWARD SCATTERING PROPERTIES OF HUMAN EPIDERMAL LAYERS

Abstract From an optical point of view the outermost skin layers contain numerous structures by which penetrating radiation may be scattered as well as absorbed. The nature and strength of this scattering may strongly influence the extent of penetration. We illuminated samples of stratum corneum and full-thickness epidermis with collimated radiation and measured the angular intensity distribution of the transmitted radiation; we did this in the ultraviolet for several angles of incidence, and in the visible for perpendicular incidence only. Skin samples were obtained from the skin of the lower back and upper leg of Caucasian volunteers. Epidermis and subsequently stratum corneum were separated by chemical methods. In the case of stratum corneum, the angular intensity distribution of the transmitted radiation peaks strongly at all wavelengths, in approximately the direction of the incident radiation, that has been refracted at the surface of the sample. With full-thickness epidermis, the distribution of the transmitted radiation also peaks, though less strongly than with stratum corneum. These features suggest a forward oriented scattering mechanism. Both in the case of stratum corneum and full-thickness epidermis, the angular distribution flattens towards the shorter wavelengths and with increasing thickness. The wavelength dependence suggests that both scattering and absorption increase towards the shorter wavelengths. The existence of a thickness dependence indicates that volume scattering occurs. Hydration of stratum corneum is found to influence its scattering properties. Dry samples scatter less than hydrated samples. The consequences of our findings for modelling skin optics are briefly discussed.