A new bacteriocin, termed lactococcin A (LCN-A), from Lactococcus lactis subsp. cremoris LMG 2130 was purified and sequenced. The polypeptide contained no unusual amino acids and showed no significant sequence similarity to other known proteins. Only lactococci were killed by the bacteriocin. Of more than 120 L. lactis strains tested, only 1 was found resistant to LCN-A. The most sensitive strain tested, L. lactis subsp. cremoris NCDO 1198, was inhibited by 7 pM LCN-A. By use of a synthetic DNA probe, kcnA was found to be located on a 55-kb plasmid. The lcnA gene was cloned and sequenced. The sequence data revealed that LCN-A is ribosomally synthesized as a 75-amino-acid precursor including a 21-amino-acid N-terminal extension. An open reading frame encoding a 98-amino-acid polypeptide was found downstream of and in the same operon as kcnA. We propose that this open reading frame encodes an immunity function for LCN-A. In Escherichia coli IcnA did not cause an LCN-A+ phenotype. L. lacfis subsp. lactis IL 1403 produced small amounts of the bacteriocin and became resistant to LCN-A after transformation with a recombinant plasmid carrying kcnA. The other lactococcal strains transformed with the same recombinant plasmid became resistant to LCN-A but did not produce any detectable amount of the bacteriocin.A number of strains of Lactococcus lactis produce bacteriocins. In an extensive survey by Geis et al. (15), it was found that about 5% of the 280 lactococcal strains tested were bacteriocin producers. These workers divided the bacteriocins into eight different classes, but none of them was characterized in detail. Despite numerous reports of lactococcal bacteriocins, little is known about their chemical composition and structure, mode of action, or genetics. Nisin is the only bacteriocinlike compound from L. lactis that has been studied in detail. The molecular structure and genetic determinant of nisin have been identified and, to some extent, its mode of action has been elucidated (5,11,16,21,22,36). Another bacteriocin, termed diplococcin, produced by L. lactis subsp. cremoris, has also been purified and its amino acid composition has been determined (10). Davey (9) showed that the gene coding for diplococcin was located on a 54-MDa conjugative plasmid. Conjugal transfer of bacteriocin plasmids in L. lactis has also been observed by others (30,38). Harmon and McKay (19) identified a Bcll DNA fragment carrying a bacteriocin determinant from a conjugative plasmid. Recently, two bacteriocin genes from another conjugative lactococcal plasmid, previously described by Neve et al. (30), were cloned by van Belkum et al. (46). The clones also carried the immunity factors of the two bacteriocins. The inhibitory spectra of the different lactococcal bacteriocins described vary but are generally more narrow than that of nisin (15). It is therefore plausible that many of the lactococcal bacteriocins described are very different from nisin and thus do not belong to the lanthibiotic family (39) of bacteriocinlike compounds....

Alpha-mannosidosis is an inherited lysosomal storage disorder characterized by immune deficiency, facial and skeletal abnormalities, hearing impairment, and intellectual disability. It occurs in approximately 1 of 500,000 live births. The children are often born apparently normal, and their condition worsens progressively. Some children are born with ankle equinus or develop hydrocephalus in the first year of life. Main features are immune deficiency (manifested by recurrent infections, especially in the first decade of life), skeletal abnormalities (mild-to-moderate dysostosis multiplex, scoliosis and deformation of the sternum), hearing impairment (moderate-to-severe sensorineural hearing loss), gradual impairment of mental functions and speech, and often, periods of psychosis. Associated motor function disturbances include muscular weakness, joint abnormalities and ataxia. The facial trait include large head with prominent forehead, rounded eyebrows, flattened nasal bridge, macroglossia, widely spaced teeth, and prognathism. Slight strabismus is common. The clinical variability is significant, representing a continuum in severity. The disorder is caused by lysosomal alpha-mannosidase deficiency. Alpha-mannosidosis is inherited in an autosomal recessive fashion and is caused by mutations in the MAN2B1 gene located on chromosome 19 (19 p13.2-q12). Diagnosis is made by measuring acid alpha-mannosidase activity in leukocytes or other nucleated cells and can be confirmed by genetic testing. Elevated urinary secretion of mannose-rich oligosaccharides is suggestive, but not diagnostic. Differential diagnoses are mainly the other lysosomal storage diseases like the mucopolysaccharidoses. Genetic counseling should be given to explain the nature of the disease and to detect carriers. Antenatal diagnosis is possible, based on both biochemical and genetic methods. The management should be pro-active, preventing complications and treating manifestations. Infections must be treated frequently. Otolaryngological treatment of fluid in the middle ear is often required and use of hearing aids is invariably required. Early educational intervention for development of social skills is needed and physiotherapy is important to improve bodily function. Orthopedic surgery may be necessary. The long-term prognosis is poor. There is an insidiously slow progression of neuromuscular and skeletal deterioration over several decades, making most patients wheel-chair dependent. No patients manage to be completely socially independent. Many patients are over 50 years of age.

Abstract. Collagen VII is the major structural constituent of anchoring fibrils in the skin. It is synthesized as a procollagen that is larger than the collagen deposited in the tissue. In this study, we investigated the conversion of procollagen VII to collagen VII in human skin and in cutaneous cells in vitro and identified the propeptide using domain-specific antibodies. For this purpose, two bacterial fusion proteins containing unique sequences of the carboxy-terminal globular NC-2 domain of procollagen VII were prepared, and polyclonal antibodies raised against them. Immunoblotting showed that the anti-NC-2 antibodies reacted with procollagen VII isolated from cultured keratinocytes, but not with collagen VII extracted from the skin. Immunohistochemical experiments with the NC-2 antibodies revealed a strong reaction in cultured keratinocytes, but the basement membrane zone of normal skin remained negative. The staining could not be rendered positive by chemical or enzymatic unmasking of potential hidden epitopes in the skin, indicating that most of the NC-2 domain is absent from normal skin. In contrast, a positive staining with NC-2 antibodies was observed in the skin of a patient with dystrophic epidermolysis bullosa, who carried a 14-bp deletion at one of the intron-exon junctions of the collagen VII gene. This aberration led to an in-frame skipping of exon 115 from the mRNA and eliminated 29 amino acids from the NC-2 domain which include the putative cleavage site for the physiological processing enzyme, procollagen C-proteinase. The resuits indicate that in normal human skin, the removal of the NC-2 domain from procollagen VII precedes its deposition at the dermal-epidermal junction. Furthermore, they suggest that an aberration in the procollagen VII cleavage interferes with the normal fibrillogenesis of the anchoring fibrils.CHORIN6 fibrils extend from the lamina densa of the epidermal basement membrane to the papillary connective tissue, thus ensuring tight cohesion between the basement membrane and the dermis in the skin. The main structural protein of the fibrils is collagen VII, a homotrimeric collagen containing three identical al(VII) chains (4,5,31). This collagen is synthesized mainly by epidermal keratinocytes (20,30). The primary synthetic product is larger than the collagen deposited in the tissue, and has been called procollagen VII in analogy to other members of the collagen protein family (5,18,20). repeat sequence, a large globular NH2-terminal NC-1 domain, and a small globular COOH-terminal NC-2 domain (24,25). Based on the cDNA-derived amino acid sequence, the following molecular masses have been calculated for the three procollagen VII domains: NC-1, 133 kD; collagenous domain, 145 kD; and NC-2, ~18 kD (7,12). Rotary shadowing electron micrographs of anchoring fibrils from tissue extracts suggested that NC-2 cleavage is necessary for correct assembly of collagen VII (24,25). During fibrillogenesis, collagen VII molecules form antiparallel tail-to-tail dimers with a small carboxy-term...

a-Mannosidosis (MIM 248500) is an autosomal recessive lysosomal storage disorder resulting from deficient activity of lysosomal alpha-mannosidase (LAMAN) (EC 3.2.1.24). The disease is characterized by massive intracellular accumulation of mannose-rich oligosaccharides with resulting mental retardation, hearing loss, immune deficiency and skeletal changes. We report here the purification and characterization of human placenta LAMAN. The enzyme is synthesized as a single-chain precursor which is processed into three glycopeptides of 70, 42 and 15 kDa. The 70 kDa peptide is further partially proteolysed into three more peptides that are joined by disulfide bridges. The laman cDNA sequence was assembled from overlapping fragments obtained by PCR on human fibroblast and human lung cDNA. The deduced amino acid sequence contains a putative signal peptide of 48 amino acids followed by a polypeptide sequence of 962 amino acids. Northern blot analyses revealed a single transcript of approximately 3.5 kb present in all tissues examined but at varying levels. Two affected siblings of Palestinian origin were homozygous for a mutation that causes a His-->Leu replacement at a position which is conserved among class 2 alpha-mannosidases from several species.