A new class of cold shock-induced proteins that may be involved in an adaptive process required for cell viability at low temperatures or may function as antifreeze proteins in Escherichia coli and Saccharomyces cerevisiae has been identified. We purified a small Bacillus subtilis cold shock protein (CspB) and determined its amino-terminal sequence. By using mixed degenerate oligonucleotides, the corresponding gene (cspB) was cloned on two overlapping fragments of 5 and 6 kb. The gene encodes an acidic 67-amino-acid protein (pI 4.31) with a predicted molecular mass of 7,365 Da. Nucleotide and deduced amino acid sequence comparisons revealed 61% identity to the major cold shock protein of E. coli and 43% identity to a family of eukaryotic DNA binding proteins. Northern RNA blot and primer extension studies indicated the presence of one cspB transcript that was initiated 119 bp upstream of the initiation codon and was found to be induced severalfold when exponentially growing B. subtilis cell cultures were transferred from 37 degrees C to 10 degrees C. Consistent with this cold shock induction of cspB mRNA, a six- to eightfold induction of a cspB-directed beta-galactosidase synthesis was observed upon downshift in temperature. To investigate the function of CspB, we inactivated the cold shock protein by replacing the cspB gene in the B. subtilis chromosome with a cat-interrupted copy (cspB::cat) by marker replacement recombination. The viability of cells of this mutant strain, GW1, at freezing temperatures was strongly affected. However, the effect of having no CspB in GW1 could be slightly compensated for when cells were preincubated at 10 degrees C before freezing. These results indicate that CspB belongs to a new type of stress-inducible proteins that might be able to protect B. subtilis cells from damage caused by ice crystal formation during freezing.

Background. The purpose of this study was to investigate the incidence of micrometastases from squamous cell carcinomas of the upper aerodigestive tract in neck dissection specimens, and to determine whether features of the primary tumor might be of prognostic value for metastasizing. Methods. Seventy‐six originally pN0 staged neck dissection specimens from 60 patients were evaluated using serial sectioning in 10‐μm intervals, H&E‐staining and immunostaining with an antibody to pan‐cytokeratin. The influence of the variables pT‐category, cytologic grade, and maximum depth of invasion of the primary tumor on the nodal status was analyzed in 128 patients. Results. The examination of 1020 lymph nodes from 76 neck dissection specimens revealed 8 micrometastases in 6 specimens (7.9%) from 6 patients with oral and pharyngeal primaries, resulting in upstaging. Six micrometastases were located in lymph nodes of 3–6 mm in diameter. Depth of invasion was the only significant risk factor for metastasizing selected in logistic regression. Conclusion. The surgeon should be aware of a relatively high incidence of micrometastases from oral and pharyngeal carcinomas, which are neither detectable preoperatively nor histopathologically by a reasonable effort. The measurement of the maximum depth of invasion of the primary can delineate a group of patients who should be treated by elective neck dissection. © 1995 Jons Wiley & Sons, Inc.

Using immunohistochemical and enzyme biochemical methods we investigated the expression of L- and M2-pyruvate kinase (PK) in normal renal tissue, renal cell carcinomas (RCCs; of clear cell, chromophilic cell and mixed cell type) and RCC metastases. L-PK was expressed in the proximal tubules of normal renal tissue and, to a variable extent, in 23/25 primary RCCs, in 1 RCC recurrence and in 10 RCC metastases. Staining intensity and percentage of stained tissue did not correlate with tumour grade. One renal oncocytoma and all extrarenal malignancies examined lacked L-PK immunoreactivity. M2-PK was mainly expressed in the distal tubules of the normal kidney and was found in all renal tumours as well as extrarenal malignancies. Quantitative biochemical investigations yielded a two- to seventeen-fold increase in PK activity in RCCs compared to the normal renal cortex taken from the same patient, whereas fructose-1,6-bisphosphatase and cytosolic glycerol-3-phosphate dehydrogenase activity was dramatically lower in RCCs. Otherwise, the activity of all other enzymes investigated (glucose-6-phosphate dehydrogenase, enolase and lactate dehydrogenase) was not significantly changed in the RCCs. The immunocytochemical results suggest that L-PK is a useful marker for RCC and its metastases, if acetone-fixed tissue is available. The quantitative changes of the concentration of PK and other enzymes in RCCs when compared with normal renal tissue probably reflect metabolic alterations related to tumour growth.

In order to characterize human notochordal tissue we investigated notochords from 32 human embryos and fetuses ranging between the 5th and 13th gestational week, using immunohistochemistry to detect intermediate filament proteins cytokeratin, vimentin and desmin, the cytokeratin subtypes 7, 8, 18, 19 and 20, epithelial membrane antigen (EMA), and adhesion molecules pan-cadherin and E-cadherin. Strong immunoreactions could be demonstrated for pan-cytokeratin, but not for desmin or EMA. Staining for pan-cadherin and weak staining for E-cadherin was found on cell membranes of notochordal cells. Also it was demonstrated that notochordal cells of all developmental stages contain the cytokeratins 8, 18 and 19, but not 7 or 20. Some cells in the embryonic notochord also contained some vimentin. Vimentin reactivity increased between the 8th and 13th gestational week parallel to morphological changes leading from an epithelial phenotype to the chorda reticulum which represents a mesenchymal tissue within the intervertebral disc anlagen. This coexpression reflects the epithelial-mesenchymal transformation of the notochord, which also loses E-cadherin expression during later stages. Our findings cannot elucidate a histogenetic germ layer origin of the human notochord but demonstrate its epithelial character. Thus, morphogenetic inductive processes between the human notochord and its surrounding vertebral column anlagen can be classified as epithelial-mesenchymal interactions.

The role of conjugating enzymes is best understood by looking at the interaction between phase I (mostly cytochromes P-450) and phase II (conjugation) enzymes of drug metabolism. A balance between phase I and II enzymes of detoxication largely determines the disposition to drug toxicity. Reactive electrophilic metabolites, generated by phase I enzymes, are often controlled by GSH-transferases, whereas nucleophilic metabolites such as phenols are controlled by UDP-glucuronosyltransferases (GT) and sulfotransferases. It is more and more recognized that the control of the more stable and more abundant nucleophiles is as important as the control of electrophiles, since the former can be readily converted to electrophiles. For example, phenols and quinols can undergo quinone/quinol redox-cycles with the generation of reactive oxygen species. In the case of benzo(a)pyrene-3,6-quinol toxicity can be prevented by glucuronidation. Conjugating enzymes consist of families of isoenzymes with distinct but overlapping substrate specificity. Rather than dealing with individual isoenzymes, adaptive programs are emphasized by which gene expression of a battery of phase I and II enzymes is turned on by certain types of inducing agents. Mechanistically best known is the program turned on by 3-methylcholanthrene-type inducers which includes enhanced synthesis of certain isoenzymes of cytochrome P-450, GT and probably GSH-transferase. The program may adapt the organism to efficiently detoxify and eliminate aromatic compounds such as benzo(a)pyrene. Evidence is presented that this program exists in both rodents and humans.(ABSTRACT TRUNCATED AT 250 WORDS)