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Purpose. To test whether the cystatin-like functional domain in tear specific lipocalin (TSL) is functionally active in tears during the normal diurnal cycle and during external ocular infections. Methods. Capillary tube collected reflex (RTF), open (OTF) and closed (CTF) eye tear samples were recovered from six normals and semi-quantitatively western blot assayed for cystatin C and TSL. CTF samples were immunoprecipitated with antibodies raised against TSL, cystatin C and other antiproteases and screened for the co-precipitation of proteases by casein and gelatin zymography. OTF samples recovered from individuals with viral, fungal and bacterial keratitis were similarly screened for TSL-bound proteases. Human tissue was subjected to immunohistochemical study. Results. Western blot analysis reveals a progressive increase in cystatin C in going from RTF to OTF to CTF samples (∼3, 7 and 30 ng μl−1, respectively). In contrast, the concentration of TSL remains constant (∼1500 ng μl−1). Immunocytochemistry data show staining of the apical surface of the human conjunctiva and some intra-cellular staining for cystatin C, but not for cystatin A. Zymography confirms earlier data that CTF contains exceptionally high levels of proteases bound to a wide range of specific inhibitors. However, only trace amounts of proteases are complexed with cystatin C and no protease can be detected bound to TSL in either the pathological or CTF samples. Conclusion. Although TSL contains a functional cystatin-like domain, it is not physiologically active during the normal diurnal cycle or during external ocular infections. Reactive proteases in CTF are most likely controlled by the presence of excess levels of more reactive cystatins, especially cystatin C, which accumulates during prolonged eye closure. Immunohistochemical data suggest that the apical conjunctiva may be a contributing source for the accumulating cystatin C.

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.

A highly selective and potentially general methodology for the synthesis of conjugated E , E - and E , Z -dienes, E -enynes, and E -1,2,3-butatrienes via hydroboration of alkynes is reported. The observed stereosclectivity was >98–99%.

Viable cells have been isolated, by enzyme digestion, from mouse preputial glands and have been separated, by isopycnic centrifugation, into populations of different buoyant densities. The separated cells have been evaluated by morphological and biochemical criteria to assess the success of the separation procedure in terms of the stage of differentiation of each cell population. Use of Ficoll as gradient medium for the isopycnic centrifugation proved unsuccessful, but good separations were obtained when Metrizamide was used. The separated cells from the Metrizamide gradient were shown to be in different stages of differentiation. Techniques are described for the special handling of these cells as well as suitable assay procedures. Some of the properties of the separated cells are described.

The reaction of 1-halo-1-alkynes with dialkylboranes followed by treatment with either lithium triethylborohydride or potassium tri-s-butylborohydride produces cis-alkenylboranes in high yields in a highly stereoselective manner.

Analytical separation of serum ultrafiltrates by high-performance liquid chromatography produces a distinctive peak with a retention time of 18.5-21 min (subfraction 18.5) from cystic fibrosis serum ultrafiltrates and obligate heterozygote serum ultrafiltrates, but not in significant concentrations from control or asthmatic serum ultrafiltrates. Semipreparative separation of control serum ultrafiltrates produced a small peak with similar retention time that was approximately 1% of the arbitrary absorbance units found in this cystic fibrosis subfraction. Subfraction 18.5 had biological activity only when separated from cystic fibrosis serum ultrafiltrate, but did not contain measurable amounts of C3a des-arginine and C4a des-arginine. Subfraction 18.5 is a low-molecular-weight material (1000-1400 daltons) that contains 14.9 micrograms orcinol positive material per 50 micrograms protein. The spectrum of subfraction 18.5 indicates that it has to be purified to homogeneity.

Cimetidine is a well known inhibitor of the heme-containing enzyme cytochrome P-450. We have found that it also inhibits delta-aminolevulinic acid synthase (ALA-S) and microsomal heme oxygenase, the rate-limiting enzymes for heme synthesis and heme degradation respectively. Cytochrome P-450 content was decreased but microsomal heme concentration remained unaltered for a period of 30 min after in vivo cimetidine administration to rats. In vitro incubation of cimetidine with each of the above enzymes revealed no direct effect of cimetidine on ALA-S but about 50% inhibition of heme oxygenase and 20% reduction in cytochrome P-450 content. This suggests that a metabolite of cimetidine inhibits ALA-S activity in vivo, while the drug itself or a metabolite inhibits heme oxygenase both in vivo and in vitro. A rise in ALA-S activity seen after its early inhibition and its return to approximate control values after 60 min suggest a reversible inhibition of ALA-S by a metabolite of cimetidine and may correspond to its clearance from the animal. An elevation in microsomal heme content paralleled the rise in ALA-S activity while microsomal heme oxygenase activity returned to only 65% of control value 60 min after cimetidine treatment. Cytochrome P-450 content did not change after its initial decrease, suggesting that irreversible alteration had occurred.