Cytoplasmic effects on the tissue culture response of wheat (Triticum aestivum) callus

Summary Calli were initiated from immature embryos of eight lines of hexaploid wheat (Triticum aestivum L. em. Thell) with different cytoplasms, the euplasmic nuclear donor Chinese Spring and seven alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. The calli were found to differ in their initial growth rates, their sensitivity to 2,4-D and their ability to organise shoot primordia, demonstrating that the cytoplasm can significantly affect the behaviour of tissues in culture. The potential for improving the responses of tissues in culture by cytoplasmic changes is noted.     

Hormonal regulation of berberine production in cell suspension cultures of Thalictrum minus

Effects of auxin and cytokinin on cell growth and alkaloid production in cell suspension cultures of Thalictrum minus were examined in an attempt to increase the productivity of a medicinal compound, berberine. In Linsmaier and Skoog medium containing auxin such as 2,4-D (1 M), the cultured cells grew rapidly, producing little berberine. On the other hand, the berberine-producing activity was remarkably enhanced by simultaneous administration of auxin and cytokinin, although cell growth was inferior. In particular, for the combination of NAA (60 M) and 6-benzylaminopurine (10 M), the yield of berberine was as high as 20 mg/30 ml medium after 2 weeks of culture. Furthermore, most of the berberine produced by the cells was released into the liquid medium, in which an excess of berberine crystallized. The results of the present experiments are suggestive of an advantage in adopting a two-stage culture method for the production of berberine in fermentor systems.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine     

Enzymatic difference between laticifers and cultured cells of papaya

Cell suspension cultures of Carica papaya were found to produce a papain-like enzyme showing an amidase activity similar to papain. Experiments suggested that the enzyme was an SH-enzyme, but an immunological test indicated the absence of papain in cultured cells. The isoelectric point (4.3) of the enzyme of cultured cells was the same as that of the leaf extract, but was different from that of papain or other amidases in the latex of the fruit.     

Heterogeneity of keratin distribution in the oral mucosa and skin of mammals as determined using monoclonal antibodies

Summary The immunohistochemical localization of keratins in the oral epithelia of several mammals was investigated using the monoclonal antibodies to keratins, PKK1 (41–56 kilodaltons) and KL1 (55–57 kilodaltons). The staining patterns obtained in different locations of the oral mucosa and of the skin epidermis were compared. In the papillae on the dorsal surface of the tongue, some areas exhibited marked PKK1 staining, while other area were PKK1 negative. In general, rodent oral epithelia were negative for PKK1 in the basal layer, while comparatively strong PKK1 staining was observed in cells of the upper spinous layer. In the epidermis, positive PKK1 reactions were confined to the basal layer, while KL1 staining was occasionally seen in the basal layer of oral epithelia. In cats, dogs, and monkeys, different PKK1 and KL1 binding patterns were observed in oral epithelia. Also, the distribution in oral epithelia differed from that seen in the epidermis of these animals. In the epidermis, the distribution of PKK1 and KL1 was regular, with PKK1 usually being confined to the basal layer, while KL1 binding was found in the spinous and granular cell layers, and was dependent on the degree of keratinization. In the animals studies, keratin expression as detected by PKK1 and KL1-was different in the skin epidermis and oral epithelia, and the localization of these keratins differed in the various types of oral mucosa.     

Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene.

The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Antiviral activity of the 3''-amino derivative of (E)-5-(2-bromovinyl)-2''-deoxyuridine.

3'-NH2-BV-dUrd, the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3'-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3'-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5'-triphosphate of 3'-NH2-BV-dUrd (3'-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3'-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3'-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5'-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3'-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.     

Formation of cobalt-porphyrin by Corynebacterium simplex grown on hydrocarbon media

A vitamin B₁₂-producing and hydrocarbon-utilizing bacterium, Corynebacterium simplex, accumulated an appreciable amount of cobalt-porphyrin in cultural filtrates when grown on a n-hexadecane medium containing sufficient amounts of cobaltous sulfate and an appropriate detergent. When grown without the detergent, the cobalt-porphyrin was found only in the cells of the organism. In the latter case, the content of cobalt-porphyrin was comparable to that of vitamin B₁₂ and 7 times lower than that of iron-porphyrin. Though the organism required cobaltous sulfate for optimal growth, the requirement could be efficiently replaced by the supplementation of cobalt-porphyrin and partly of vitamin B₁₂. The porphyrin moieties of extra- and intracellular cobalt-porphyrin were identified as coproporphyrin III in both cases.