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Keyhole limpet hemocyanin (KLH), the large respiratory glycoprotein from the primitive gastropod mollusc, Megathura crenulata, is a potent immunogen used classically as a carrier protein for haptens and more recently in human vaccines and for immunotherapy of bladder cancer. Two KLH isoforms were identified and isolated by high-performance anion exchange chromatography. Subsequent analyses disclosed that these isoforms--designated KLH-A and KLH-B--were composed of distinct subunits that differed in primary structure, molecular weight (KLH-A was 449,000 and KLH-B was 392,000), polymerization/reassociation characteristics, and O2-binding constants (KLH-A had a P50 of 7.32 and KLH-B had a P50 of 2.46). Both subunits appear to be composed of eight oxygen binding domains, and reassociate in solution only with like subunits. These results support the concept that structural and functional heterogeneity is a common feature of molluscan hemocyanins, and provide a rational basis for studying and optimizing the immunostimulatory properties of KLH.

The antitumor activity and potential toxicity of a clinical-grade keyhole limpet hemocyanin preparation (KLH-Immune Activator; KLH-IA) were determined in the MB-49 intravesical murine bladder tumor model. Mice were immunized subcutaneously with KLH-IA two weeks prior to intravesical implantation of MB-49 tumor cells. Treatment consisted of intravesical KLH-IA (10 or 100 micrograms.) 1, 4, 7, 14 and 21 days after implantation. Control animals either were not immunized prior to tumor implantation and KLH-IA treatment, or were immunized with KLH-IA and treated with the vehicle. By 4 weeks after implantation tumor outgrowth in the treated groups was significantly decreased (p < 0.01, Fisher's Exact) relative to the control groups. Prior subcutaneous immunization was required to elicit antitumor activity of KLH-IA; thus, the mechanism of action is immune-mediated and not due to spurious interference with tumor implantation by intravesical instillations. Animals treated with a dissociated form of KLH exhibited decreased tumor outgrowth approaching, but not attaining, significance (p < 0.09, Fisher's Exact). A separate toxicity study in which KLH-IA was given subcutaneously (4 mg./kg.), intraperitoneally (40 mg./kg.), or intravesically (40 mg./kg.) disclosed no significant gross or histopathologic abnormalities except for mild-to-moderate papillary hyperplasia in all catheterized animals. These results establish the efficacy and safety of KLH-IA in mice and suggest that clinical trials for intravesical treatment of superficial bladder cancer may be warranted.

We have developed procedures that permit isolation and propagation of clonal cell cultures from the olfactory epithelium of the 5- to 7-day-old rat that appear to represent the neuroblasts that repopulate the sensory neurons in the olfactory epithelium throughout life. The cell lines we report here synthesize neuron-specific enolase, which is a neuron marker, 43-kDa growth-associated protein, a protein associated with neuronal growth cones, and carnosine, a possible olfactory neurotransmitter. In two of the cell lines we have found dose-dependent cAMP accumulation following exposure to submicromolar concentrations of chemical odorants in the medium. These two cell lines show different patterns of odorant specificity when tested against a panel of six chemicals commonly used as test odorants. We anticipate that these and similarly derived cell lines will prove valuable in studying aspects of neurogenesis and olfaction.

An NADH-linked disulfide reductase specific for disulfides containing pantethine 4',4"-diphosphate moieties was purified 23,000-fold to homogeneity from spores of Bacillus megaterium. The enzyme had a native molecular weight of 122,000 with two apparently identical subunits, contained one molecule of flavin adenine dinucleotide per subunit, and was inhibited by the vicinal dithiol reagent arsenite. The enzyme was active only on disulfides containing pantethine 4',4"-diphosphate moieties, including pantethine 4',4"-diphosphate, oxidized coenzyme A, and coenzyme A in disulfide linkage to acyl carrier protein. However, the Km values for pantethine 4',4"-diphosphate and oxidized coenzyme A were 0.65 and 7.4 mM, respectively. The enzyme was at a low level in log-phase cells but increased up to 10-fold early in the stationary phase and had a similar specific activity in both the mother cell and the forespore compartment; the enzyme activity fell only slowly during spore germination and outgrowth. The enzyme was not detected in several eucaryotic sources and was present in at most a low level in a number of gram-negative bacteria. Surprisingly, the specific activity of this enzyme varied more than 200-fold in extracts from different Bacillus species, with values in B. subtilis being 5- to 6-fold lower and values in B. cereus and B. sphaericus being 8- and 35-fold higher, respectively, than the maximum value in B. megaterium. However, the high specific activity in B. sphaericus did not represent more enzyme protein than in B. megaterium. The possible function of this newly discovered enzyme is discussed.

Two distinct membrane bands were obtained after sucrose velocity gradient centrifugation of crude inner membranes from dormant Bacillus megaterium spores disrupted under conditions which minimized endogenous enzyme action. These two inner membrane fractions (termed LD and HD) contained similar amounts of total and individual phospholipid species. However, LD and HD differed significantly in phospholipid/protein ratios (4.3 and 0.47 mg/mg, respectively), equilibrium densities (1.12 and 1.18 g/cm3), NADH oxidase specific activity (less than 0.01 and 0.13 mumol/min X mg), and content of specific proteins. In contrast, crude membranes prepared in identical fashion from germinated spores gave only a single inner membrane band (termed G) on sucrose velocity gradients. G had a phospholipid/protein ratio of 0.98 mg/mg, an equilibrium density of 1.16 g/cm3, and an NADH oxidase specific activity of 2.1 mumol/min X mg. Essentially all of the proteins present in LD or HD or both were found in G, consistent with the latter membrane being derived from a mixture of LD and HD. No evidence was found suggesting that there is significant degradation of dormant spore inner membrane protein upon spore germination.

Bacillus megaterium contains an NADH-linked disulfide reductase that is specific for disulfides containing pantethine 4',4''-diphosphate moieties. This reductase is at its highest level in cells late in sporulation and in dormant spores, and could be involved in the formation and cleavage of coenzyme A-protein disulfides which take place late in sporulation and early in spore germination, respectively.